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Immunological analysis of plasminogen activators from normal and transformed hamster cells. Evidence that the plasminogen activators produced by SV40 virus-transformed hamster embryo cells and normal hamster lung cells are antigenically identical.

Rabbits were immunized against the plasminogen activator released by SV4- virus-transformed hamster embryo cells. The resulting antiplasminogen activator immunoglobulin (APA-IgG) inhibited the enzymatic activity of the plasminogen activator produced by SV40-transformed hamster cells, and the plasmin-catalyzed release of these cells from the tissue culture dish. APA-IgG was not cytotoxic for these cells even in the presence of complement and did not inhibit their release of plasminogen activator. APA-IgG formed a single precipitin line in immunodiffusion plates using highly purified plasminogen activator as antigen. APA-IgG inhibited the plasminogen activator produced by newborn hamster lung cells and by an established diploid line (DON) of hamster lung cells, but did not inhibit plasminogen activators produced by normal or transformed hamster kidney cells or by cells of other species (mouse and human). We derive three major conclusions from these data: (a) There are several immunologically distinguishable forms (isozymes) of plasminogen activators in normal hamster tissues. (b) The plasminogen activators produced by normal hamster lung cells and by SV40 virus-transformed hamster embryo cells share antigenic determinants and are presumably the same isozyme. (c) The plasminogen activators produced by different hamster tumor cells do not share antigenic determinants and are presumably different isozymes.

Protein patterns of cerebrospinal fluid in hereditary ataxias and hereditary spastic paraplegia.

The CSF findings in hereditary ataxias and allief disorders have hitherto mostly been reported as normal if one excludes Refsum's syndrome. The CSF-protein patterns found on isoelectric focusing and quantitative paper electrophoresis were studied in 12 patients with hereditary ataxias and hereditary spastic paraplegia. Using a recently-developed technique of isoelectric focusing of CSF-proteins in flat beds of polyacrylamide gel, the authors could show abnormal CSF-protein patterns in all but 1 of the present cases. The aberrant CSF-protein patterns found showed differences between the syndromes studied. Two unique patterns with conspicuous fractions in the acid range were observed in patients with Marie-Sanger-Brown's ataxia (mother and daughter) and Holmes' ataxia. A third CSF-protein pattern was found in a sibship with Friedreich's ataxia including a double fraction in the acid region (pI 5.9-6.1) in all 4 subjects and a highly alkaline fraction (HAF) with pI about 9.3, in 3 of them. Similar acid fractions (pI 5.9-6.1) were also detected in 3 of 4 patients with hereditary spastic paraplegia, a brother and sister showing a very similar CSF-protein pattern. Double fractions with pI 5.9-6.1 and/or HAF may also occur in other neurological diseases, mostly, however, associated with other distinctive features of their CSF-protein patterns. A possibility in the future of distinguishing hereditary CNS-diseases by examination of the CSF-protein pattern is suggested.

[The advantages and limitations of an automatic ultrasound determination of the cerebral midline--a study of 1889 cases].

The examination of the brain with ultrasound has become an indispensible diagnostic tool in all cases of intracranial pathology. Valuable information is obtained both in emergencies and in follow-up studies. Since long practice is necessary before the results of echoencephalograms can be interpreted accurately, a need was felt for a standardization of the examination which would eliminate the subjective influence of the investigator. The automatic midline computer, called Midliner, which has recently become available, makes fewer demands on the experience of the investigator although the diagnostic possibilities are not as broad. The position of the midline echo is of primary interest, although the width of the third ventricle can be successfully determined in occasional cases. this study, which presents the results of the Midliner examinations in three clinics, is grouped according to diagnosis. Through a comparison with conventional A-scan echoencephalograms, as well as with neuroradiological and operative findings in 1889 cases, the reliability of this method is clearly demonstrated. The advantages and disadvantages of the examination are discussed and the field of application indicated.

Elicidation by a H-2-receptor antagonist of the significance of mucosal histamine mobilization in exciting acid secretion.

1. The consequence of H-2-receptor blockade for the secretory responses of the gastric mucosa to hormonal or cholinergic stimulation was studied in conscious rats with Heindenhain pouches or Pavlov pouches with the antrum retained or resected. 2. Metiamide almost completely abolished acid secretion induced by pentagastrin without altering significantly the amount of histamine excreted in the urine. Histamine mobilization on pentagastrin infusion determined in vitro, seemed to be larger during H-2-receptor blockade than with pentagastrin alone. 3. CCK-PZ mobilized mucosal histamine to a considerable extent; the secretory response to this hormone was completely abolished by H-2-receptor blockade. 4. Acid secretion in response to 2-deoxy-D-glucose was inhibited by H-2-receptor blockade in the presence or absence of the antrum; however the inhibition was less complete than with hormone-induced secretion. 5. The acid secretory response to 100 mg/kg of 2-deoxy-D-glucose appeared to be less susceptible to H-2-receptor blockade than that of 50-mg/kg of 2-deoxy-D-glucose. 6. Feeding induced a secretory response in the Pavlov pouch which initially was more effectively inhibited by H-2-receptor blockade than the response to 2-deoxy-D-glucose. In the absence of antral gastrin secretion by either stimulus was equally inhibited. 7. Methacholine-induced acid secretion was inhibited by infusion of the H-2-receptor antagonist, an inhibition that was absent when pentagastrin was concomitantly infused. 8. Although acid secretion induced by cholinergic stimuli was readily inhibited by the H-2-receptor antagonist, slight or nor inhibition was noted on pepsin secretion. 9. The role of histamine as a physiological stimulus for the parietal cell is discussed in view of the fact that the secretory effect of natural stimuli, known or demonstrated to mobilize mucosal histamine, is restrained by H-2-receptor blockade.

Biological and physical modifications of a murine oncornavirus by 2-deoxy-D-glucose.

2-Deoxy-D-glucose (2-DG) inhibited the release of transforming Kirsten murine sarcoma-leukemia virus [KiMSV(KiMuLV)] from transformed rat kidney (NRK-K) cells. At a concentration of 30 mM 2-DG, RNA synthesis in NRK-K cells was inhibited by approximately 30 percent and protein synthesis was inhibited by as much as 80 percent of control levels. RNA synthesis was not inhibited in nontransformed normal rat kidney (NRK) cells, although protein synthesis was equally suppressed in NRK and NRK-K cells. After treatment with 2-DG, the release of physical particles of KiMSV(KiMuLV) from NRK-K cels was not reduced as determined by equilibrium density gradient centrifugation and assays for RNA-dependent DNA polymerase of culture fluids. The ability to detect virion-associated radioactivity in equilibrium density gradients was dependent on the conditions of labeling. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of KiMSV(KiMulLV) proteins revealed marked structural alterations after propagation of the virus in 30 mM 2-DG. These alterations may account for the observed loss of transforming ability of KiMSV(KiMuLV).

Quantitative nucleotide sequence relationships of mammalian RNA tumor viruses.

A molecular hybridization technique has been used to quantitatively measure the nucleotide sequence relationships of selected mammalian RNA tumor viruses. Reciprocal cross-hybridization tests were done in which a given radioactively labeled, viral genomic RNA species was annealed with an excess of unlabeled, complementary DNA product synthesized in endogenously instructed reverse transcriptase reactions. Hybrid formation was measured with pancreatic RNase A. Three representative mammalian RNA tumor virus groups were examined: murine viruses, simian viruses, and feline viruses. The results of reciprocal cross-hybridization testing have revealed that the murine viruses consist of four distinctly related subgroups: (i) the Friend leukemia virus/Rauscher leukemia virus subgroup, (ii) the Gross leukemia virus subgroup, (iii) the Moloney sarcoma virus subgroup, and (iv) the Kirsten sarcoma virus subgroup. Simian sarcoma virus, the only simian virus examined, appeared to share limited interspecies sequence relationships with members of the other virus groups and in particular with Kirsten sarcoma virus. Of the two members of the feline virus group tested, Rickard feline sarcoma virus and RD-114, each was placed in a separate, unrelated subgroup. Rickard feline sarcoma virus exhibited limited sequence relatedness with members of the other virus groups, whereas RD-114 exhibited none.

Plasma-lipids and glucose/insulin relationship in non-insulin-requiring diabetics with and without retinopathy.

Serum-lipid concentrations and their relationship to blood-glucose and serum-insulin were examined in non-insulin-requiring diabetics, 62 with and 45 without retinopathy. The age, sex-body-weight, and duration of known diabetes was comparable in the two groups. All were treated by diet only or diet and oral hypoglycaemic agents. Patients with retinopathy had higher fasting serumtriglyceride and serum--cholesterol levels than those without. Compared with a non-diabetic population, significantly more diabetics with retinopathy had raised derum-lipids. The lipid concentrations did not correlate with body-weight, serum-thyroid-stimulating-hormone levels, renal involvement, or fasting blood-sugar. While the blood-sugar concentrations were similiar in the two groups the absolute insulin increment and the relative insulin response to a 50 g. oral glucose load were significantly lower in those with retinopathy than in those without. The impairment of insulin response correlated significantly with the frequency of hyperlipidaemia. It is suggested that insulin deficiency with secondary hyperlipidaemia is characteristic of diabetic patients with retinopathy.

Role of glucagon and other hormones in development of diabetic ketoacidosis.

Blood concentrations of pancreatic glucagon, cortisol, noradrenaline, adrenaline, and growth hormone have been measured during the first 41 hours of insulin deprivation in six insulin-dependent diabetics to assess the importance of these hormones in the pathogenesis of diabetic ketoacidosis. Plasma-glucagon showed an early small significant rise and thereafter a slow increase to a plateau during the remaining experimental period. Plasma-cortisol increased only at the end of the insulin-deprivation period, while plasma-catecholamines and serum-growth-hormone concentrations did not change. In the three of the six patients who developed significant ketosis, plasma-glucagon showed a close correlation with blood-ketones and plasma-free-fatty-acids while for the whole group the change in glucagon concentration correlated significantly with the rise in ketone-body concentration. It is suggested that the excess of glucagon in addition to the insulin lack may be an important factor in determining the degree of hyperglycaemia had hyperketonaemia in the early stages of insulin deprivation.

A day-surgery programme for children incorporating anaesthetic outpatient clinic.

A day-surgery programme for children incorporating an outpatient anaesthetic assessment clinic was welcomed by parents and general practitioners and resulted in better deployment of hospital resources. It is suggested that the difficulties usually associated with day-case surgery can be reduced by holding an outpatient assessment clinic a few days before operation.

Fetal blood drawing.

A small sample of fetal blood suitable for studies of haemoglobin synthesis was obtained from a placental vessel under endoscopic visualisation in 23 of 26 patients in whom the procedure was attempted prior to second-trimester abortion. Fetal blood loss, calculated in 23 cases, was between 0-2 ml. and 2-5 ml., and fetal blood-volume depletion varied from 0-5% to 15%. No short-term ill-effects were demonstrated in mother or fetus in any of 16 patients in whom the injection of aborti-facient was postponed for between 16 and 24 hours after the procedure.

Early detection of false-positive acid-fast smears. An epidemiological approach.

An unusual series of positive acid-fast smears, including positive stains of a commercial saline solution, has been studied. Short of waiting 2--6 weeks for culture results, a laboratory investigation of these findings appeared hopeless: repeat staining failed to confirm the origin smear results, and a review of laboratory techniques and supplies failed to pinpoint any source of specimen or smear contamination. An alternative epidemiological approach was adopted. A review of the laboratory records indicated that, during the time the unusual series occurred, there had been a distinct change in the distribution of positive smears by type of specimen submitted and by ward of origin. The change in distribution indicated that the unusual series was probably part of a larger episode of false-positive acid-fast smears caused by random specimen or smear contamination in the hospital laboratory. Culture results eventually confirmed this and showed that the period of random laboratory contamination had ended just before the present investigation (thus explaining the failure of the initial approach). This experience suggests that ongoing analysis of specimen and ward distribution of positive acid-fast smear results will enable hospitals to detect episodes of false-positive smears earlier, thus reducing erroneous diagnoses and permitting prompt evaluation of sources of specimen and smear contamination.

[Hepatic reaction in ulcerative colitis and Crohn's disease (author's transl)].

Within the framework of a prospective study on the course and prognosis of ulcerative colitis and Crohn's disease begun in 1968, serial blind needle biopsies of the liver were carried out for the early establishment of liver involvement. In 201 needle biopsies taken in 114 patients with ulcerative colitis, normal findings were observed in 51, fatty infiltration in 24, and accompanying inflammation in 23, fatty infiltration together with a mesenchymal reaction in 8, fatty liver in 6 and sclerosing cholangitis and granulomatous hepatitis in 1 patient each. Of 69 needle biopsies taken in 45 patients with Crohn's disease we established normal findings in 13, an insignificant accompanying inflammation in 19, fatty infiltration in 5, granulomatous hepatitis in 3, fatty liver in 2, fatty liver together with a mesenchymal reaction in 2 and serum hepatitis in 1. Laboratory tests (alkaline phosphatase, SGOT, SGPT, BSP excretion) are not particularly suitable as screening tests. The diagnostic yield of serial blind needle biopsies of the liver is low and, despite the low-risk nature of the method, often dispensable. Laparoscopy, or at least blind needle biopsy of the liver, should be retained for the further clarification of serious deviations of laboratory values from the normal range, or of suspicious palpation findings.

[Activity of the hypophyseal-thyroid gland system in relation to the funcitonal state of the sex glands. Report I].

Experiments were conducted on intact female rats; it was revealed that the content of protein-bound iodine (PBI) in the blood depended on the stage of the estral cycle. It was decreased at the metestrus and diestrus stages. Castration produced an even greater reduction in the blood PBI content. The blood PBI content proved to elevate in administration of estradiol dipropionate (EDP) to castrated rats. The TSH content in the hypophysis increased at the metestrus and diestrus stages and decreased at the proestrus and estrus stages. The relationship was reverse in the case with the blood TTH content. Castration was followed by a marked increase in the TSH content in the hypophysis accompanied by a reduction in the blood hormone level. The TSH concentration in the hypophysis decreased and in the blood increased under the effect of EDP in castrated animals. The data obtained indicated that the interrelationship between the thyroid gland and the sex glands was realized at the level of the hypophysis, and possibly, of the hypothalamus.

Induction of histamine release and densensitization in human leukocytes.

Protein A from Staphylococcus aureus has been found to react with all human leukocyte preparations tested. In 70 percent of the experiments the reaction leads to histamine release. Furthermore, protein A treatment of cells at 37 degrees C, both in complete and Ca-2+-free medium, results in the inhibition of anti-IgE-induced histamine release in all cell preparations, indicating that protein A and anti-IgE antibodies release histamine from the same cells. This inhibition seems to be due to the blocking or exhaustion of a step in the biochemical pathway, leading to histamine release activated by both protein A and anti-IgE. In some cell preparations desensitization but no histamine liberation is induced by protein A. No inhibition occurs if the protein A treatment is performed at 4 degrees C. It is concluded that protein A elicits histamine liberation and desensitization by acting on IgG present on the surface of the basophil granulocytes. Treatment of leukocytes at 37 degrees C with anti-IgE antibodies, or F(ab)2 fragments from such antibodies, also results in an inhibition of a subsequent anti-IgE-induced histamine release. Desensitization with low doses of anti-IgE results in an inhibition of the same type as that obtained with protein A. Supraoptimum amounts of anti-IgE or high amounts of monovalent Fab fragments from anti-IgE immunoglobulin G give an inhibition that could be due to a competition between the sensitizing and the challenging agents for combining with cell fixed IgE molecules. This inhibition is independent of temperature and calcium concentration.

Tannic acid-iron alum reactions: stain of choice for macroscopic sections of brain to be embedded in plastic.

As a macroscopic stain for gross brain sections to be embedded in plastic, tannic acid-iron alum is superior to the generally recommended LeMasurier's variation of the Berlin blue technique because of its greater permanency in plastic. However, as originally adopted for use with brain tissue by Mulligan, the intense black staining of gray matter is too dark for plastic embedded specimens. A modification of this method designed to overcome this difficulty is described. Staining procedure: Wash formalin-fixed brain slices overnight in running water. Wash in distilled water, 2 changes, 30 minutes each. Place slices individually in Mulligan's solution at a temperature of 60-65 C for 4 minutes. Rinse in ice water for 10 seconds. Mordant in 0.4% tannic acid in distilled water for 1 minute. Wash in running tap water for 1 minute. Develop in 0.08% ferric ammonium sulfate in distilled water until gray matter is light gray, about 10-15 seconds. Wash in lukewarm running water for 1 hour, then gently hand-rub whitish film from myelinated surfaces. Store briefly in 3% formalin or 25% glycerine if necessary depending on plastic embedding procedure to be followed.

A systematic study of testosterone metabolism in benign prostatic hypertrophy (BPH): in vitro results.

An in vitro system for testing steroids which might be effective in treating benign prostatic hypertrophy (BPH) has been developed based upon the transformation of H-3-testosterone into the 5-alpha-reduction products dihydrotestosterone and 3-alpha-androstanediol. In scrutinizing the influence of the amount of BPH-tissue, time, and pH, 300 mg of tissue incubated for 2 h at the physiological pH of 7.4 were used in the standard experiment. -The H-3-testosterone concentration was varied from 0.17-100 times 10-8 M. Plotting the resulting 5-alpha-reduction products as a function of testosterone concentration a hyperbolic pattern of enzyme kinetics ensued. Performing a double reciprocal plot of 4 experiments with double determination of each value regression lines could be computed. Those two regression lines most different in their slopes were considered "normal" limits. The rate of H-3-testosterone metabolism could not be enhanced after the endogenous testosterone content within the prostate glands had been used up by means of a preincubation. Scrutinizing the effect of heparin, a weak non-specific enzyme inhibitor, no suppression of the appearance of 5-alpha-reduction products was found. Damaging the BPH-cells, however, by repetitive freezing and thawing lead to an almost complete inhibition of H-3-testosterone turnover.

[Investigations of some metachrome-yellow-preparations as an basic ingredient for metachromgelb-wasserblau-laktose-agar (Gassners medium) (author's transl)].

Experiments with 5 commercial- and 4 testpreparations of Metachrome Yellow have been conducted. Results of this investigations show that the value of Gassners Medium is depending on the quality of the inhibitory substance. The microbiologically active substance (inhibition of grampositive bacteria and prevention of swarming of Proteus) was chemically identified as "Beizengelb GT Color Index 14025" correlating with CI Mordant Yellow I. Test sample II of CHROMA-GESELL-SCHAFT, STUTTGART is recommended as the best "Metachrom-Yellow for preparation of Gassners-Medium Now. Presumable this medium was modified repeatedly after its introduction in bacteriology in 1918 by Gassner. This can be an explanation for the different evaluations of Gassners medium and also for the numerous experiments which have been conducted to modify the medium. Indentity control of chemicals used in microbiology is done by thin layer and paper chromatography. This control should be done in cooperation with chemists more frequently than before.

Unusual ultrastructural features of leiomyosarcoma of the lung.

The electron microscopic features of a leiomyosarcoma occurring in the lung, a rare site, were studied. Unusual was the finding of extremely electron-dense bands that interrupted the longitudinally-arranged microfilaments and dense bodies of many of the tumor cells. The bands superficially resembled anomalous Z-bands seen in nemalin myopathy and aging rat cardiac muscle, but have not been described to occur in smooth muscle tumors. They are thought to represent condensations of dense bodies in degenerating tumor cells.

Supernumerary bisatellited chromosome in a family ascertained through a patient with Sturge-Weber syndrome.

The presence of a structurally abnormal extra chromosome in a patient with Sturge-Weber syndrome and several members of her family is described. With routine techniques the abnormal chromosome is slightly submetacentric, of the size of a G group chromosome and shows satellites on both arms. C-banding suggested the presence of 2 centromeric regions rather than one, and to explain this finding, in addition to the segregation of the abnormal chromosome through 3 generations and why only one centromere is visible with the usual cytogenetic technique, an hypothesis is advanced suggesting that it resulted from an unusual type of Robertsonian translocation, in which one of the breacks involved directly the centromere of an acrocentric producing a partially dicentric bisate-lited chromosome. The association of Sturge-Weber syndrome with the chromosome abnormality is thought to be fortuitous and the lack of clinical manifestations of all members of this family with the abnormal chromosome, including one with two extra ones, is explained by the fact that it was almost entirely formed by heterochromatic material. The usefulness of C-banding in the study of this patient is strongly emphasized.

[Correlation of in vitro testing and therapeutical results in animal transplanted tumors after cytostatic treatment].

The antineoplastic activity of 5 substances was tested in vivo and in vitro on four different tumours (plasmocytoma and melanoma Fortner III of the Syrian golden hamster, the Walker carcinosarkoma 256 and an adenocarcinoma of the rat). The substances involved were 2,3,5-triethyleniminobenzoquinone-(1,4) (triaziquone), actinomycin D, podophyllinic ethylhydrazide (mitopodozide), bleomycin and adriamycin hydrochloride. The effect of the substances in vivo was measured on the size of the tumour, and in vitro on the incorporation of 3-H-thymidine and 3-H-uridine in short-term incubations of tumour-cell suspensions. No correlation was observed between the 3-H-thymidine incorporation in vitro and the response of the tumours in vivo. On the other hand, the 3-H-uridine incorporation in the tumour-cell suspensions in vitro was in good agreement with the results of therapy in the animal experiments. This is compatible with the results of earlier experiments using other substances to investigate the possible correlation between tumour therapy and in vitro tests.

Human myeloma IgG half-molecules. Structural and antigenic analyses,.

The structure and antigenic characteristics of a human k, IgG myeloma protein that formed half-molecules were analyzed. Most of the myeloma protein found in the patient's serum and urine consisted to two chain 4.3S half-molecules. A small amount of four chain 7S myeloma protein was, however, found in the serum and was apparently formed by the same clone of tumor cells. Polyacrylamide gel electrophoresis in 8 M urea and 1% sodium dodecyl sulfate and analytical ultracentrifugation in 6 M guanidine of the fully reduced and alkylated half-molecule indicated that this myeloma protein had a heavy chain of a smaller molecular weight (approximately 45,000) than that of normal gamma chains, Except for this apparent deletion, the heavy chain resembled gamma1 chains. The amino acid composition of the peptides containing the half-cysteine residues forming the interchain disulfide bonds, the glycopeptide of the Fc fragment and the COOH-terminal structure were similar if not identical with the analogous structures of gamma1 chains. No Fc fragment could be prepared because the Fc portion of the heavy chain of the myeloma protein was extremely susceptible to degradation with papain. After mild reduction and alkylation, the 7S myeloma protein dissociated into half-molecules, indicating a lack of noncovalent interactions in the Fc fragment that are present in all classes of human immunoglogulins and are responsible for the formation ofFc dimers. The half-molecule was antigenically deficient in the Fc fragment. It failed to precipitate with anti-Fc fragment antisera in double gel diffusion tests and inhibited a Fc-anti-Fc fragment binding reaction weakly and incompletely. The half-molecule and the 7S protein had the same genetic markers on the first and second homology region of the gamma chain. The half-molecule lacked, however, the corresponding markers on the third homology region, These findings suggest that this myeloma protein had a deletion in the gamma chain which was probably located in third homology region and was likely the structural abnormality responsible for the lack of noncovalent interaction in the Fc fragment and absence of most of the antigenic determinants characteristic of gamma chains.

IgM deficiency.

Primary isolated IgM deficiency accounted for 0.1% of hospital admissions; secondary IgM deficiency for 2.0%. Although 19% were asymptomatic the rest of 89 subjects (4M:1F) suffered infection (60%), septicemia (36%), atopy (22%), splenomegaly (11%), neoplasia(7%) and autoimmune disorders (3%) with a mortality of 10%. Serious early treatment is needed to avert death from unopposed spread of organisms throughout the blood. Qualitative IgM deficiency (absence of isohemagglutinins) and delayed maturation of IgM can result in similar symptomatology.

Specificity of retrograde transport of nerve growth factor (NGF) in sensory neurons: a biochemical and morphological study.

In previous studies it has been shown that nerve growth factor (NGF) is taken up with a high selectivity by adrenergic nerve terminals and is transported retrogradely to the perikaryon11,22. It was the aim of the present experiments to investigate whether the sensory neurons exhibit the same high degree of selectivity for retrograde transport throughout the whole life cycle, although it is known that their dramatic response to NGF is confined to a short period of ontogenetic development. Unilateral injection of [125I]NGF into the forepaw of adult rats was followed by a preferential accumulation of radioactivity in the sensory ganglia (C6-C7) of the injected side. However, this preferential accumulation was not detectable earlier than 6 h after injection and reached a maximum (ratio between injected and non-injected side, 5:1) after 11-16 h. Transection of the plexus brachialis abolished and local administration of colchicine prior to that of [125I]NGF greatly reduced the preferential accumulation of radioactivity in the ganglia of the injected side. The rate of retrograde transport of NGF in sensory neurons was calculated to be 13 mm/h which is about 5 times faster than that in adrenergic neurons. The selectivity of this retrograde transport was demonstrated by the fact that injection of 125I-labeled bovine serum albumin and cytochrome c did not result in a preferential accumulation of radioactivity in the sensory ganglia of the injected side. Light microscopic autoradiography revealed heavily labeled cells in the sensory ganglia (C6-C7) of the injected side after administration of [125I]NGF into the forepaw. Only cells belonging to the large cell type were labeled. Prolonged (7 mug/g/day over 5 days) injection of NGF into the forepaw of 10-day-old rats did not result in a hypertropic response of the sensory neurons as far as can be judged from morphometric studies at the light microscopic level.

Kinetics of both leukemic and normal cell population reduction following 5-azacytidine.

The cytotoxic effect of 5-azacytidine (AzaCR) on normal hematopoietic colony-forming units (NCFU) and L1210 leukemic colony-forming units (LCFU) in the femoral marrow of BALB/c x DBA/2 F1 mice was studied using the spleen colony assay. Dose-survival curves for LCFU and NCFU were biphasic. Repopulation of LCFU was rapid at a low dose of AzaCR (0.1 mg/mouse) but was delayed for greater than 6 days at higher doses (0.25 mg/mouse and above). Of the agents tested in this system, only AzaCR exhibited these properties. Survival of mice with L1210 leukemia following AzaCR administration was prolonged beyond that predicted by the degree of LCFU reduction alone, and reflected the delay in LCFU repopulation. In contrast, repopulation of NCFU in normal mice was not delayed at a high dose of AzaCR (0.5 mg/mouse). AzaCR produced a nine-fold greater reduction of NCFU in leukemic mice than in normal mice, measured 5 days after AzaCR injection. While divided doses of AzaCR produced LCFU cytotoxicity equivalent to a single dose, 24-hr infusions of high doses were inferior to single infections.

Antibody in cats to mammalian RNA tumor virus interspecies antigens.

The studies were undertaken to determine whether the cat, a mammalian species that carries xenotropic endogenous C-type virus(es) and in addition undergoes horizontally transmitted oncogenic C-type RNA tumor virus infections, responds immunologically to the mammalian C-type virus interspecies antigens. Sera from normal cats and from cats with spontaneous or virus-induced neoplasms were examined for antibodies to interspecies antigen antigen by complement-fixation inhibition, by inhibition of the paired radioiodine-labeled antibody technique (PRILAT inhibition), and by two-step radioimmunoelectrophoresis. Using three separate complement-fixation inhibition systems designed to detect antibodies to interspecies antigen(s), 23 of 23 sera from tumor-bearing cats and 24 of 31 sera from normal cats were positive in both systems. The negative sera were from germ-free cats. Among the 49 positive sera, 47 yielded titers of 1:16 or greater by one or more complement-fixation inhibition tests. Of these 47 sera, 42 were positive by the paired radioiodine-labeled antibody technique inhibition test; the 5 paired radioiodine-labeled antibody technique-negative sera were from normal specific-pathogen-free cats. Direct reaction with the interspecies determinant on the p30 protein from Rauscher murine leukemia virus by immunoglobulin from cats immunized with feline leukemia virus was shown by two-step radioimmunoelectrophoresis. The feline antibody was also identified as an immunoglobulin by column chromatography and two-step radioimmunoelectrophoresis. These antibodies did not fix guinea pig complement during reaction with the interspecies antigen. That other mammals may produce similar noncomplement-fixing (guinea pig) antibodies to RNA tumor virus antigens is likely.

An ultrastructural study of the microfilaments in rat brain by means of heavy meromyosin labeling. I. The perikaryon, the dendrites and the axon.

Nervous tissue pieces from the caudate nucleus and the substantia nigra of the rat were incubated in cold glycerol solutions of decreasing concentrations and then transferred into standard phosphate buffer (pH 7.0) or into tris-K+-Mg++-Ca++ buffer (pH 7.9) containing HMM, prepared from rabbit skeletal muscle by tryptic digestion. As controls, pieces were immersed for an identical period in the same buffers (1) without HMM or (2) with HMM to which had been added 2.5 mM Na+ pyrophosphate or 5 mM ATP. In control neurons smooth-surfaced microfilaments, about 50 A in diameter, were observed. After reaction with HMM, the microfilaments were increased in number and density and in width to 180-200 A. A meshwork was formed. Arrowheads pointing in the same direction were spaced at regular intervals (300-350 A) among short segments of the surfaces of the microfilaments, depending upon the plane of section. More often, however, typical arrowheads were not observed, and the surfaces of the microfilaments were seen coated with polarized side-arms cross-bridging the spaces between adjacent elements at more or less regular intervals. When cross-sectioned, the microfilaments appeared as dense dots from which a material of lesser electron density radiated. Following incubation in HMM solutions containing Na+ pyrophosphate or ATP, no arrowhead structures were seen. Of particular interest was the structural relation of the actin-like filaments with occasional, tapered myosin-like filaments, and with the plasma membrane, which served as anchor points. Mitochondria and smooth ER membranes were observed to be attached to the actin-like filaments or enmeshed in the network. The microtubules, as well as most of the neurofilaments, were disrupted by the glycerination procedure at 4 degrees, and thus no precision about the structural relationship of the actin-like filaments with the latter elements could be added. The role of the actin-like filaments in the transport of material, by a mechanism of chemomechanical transduction, throughout the neuron from sites of synthesis to functional locations, and between several functional locations, is discussed.

The differences in atrioventricular conduction of premature beats in young and adult goats.

Atrioventricular (AV) conduction was studied in anesthetized, open-chest, 1-35-day-old goats. Atrial, bundle of His, and ventricular bipolar electrograms were recorded, and the functional refractory periods of the AV conduction system and the ventricles were determined. Supraventricular premature excitation invaded the ventricles during their vulnerable period. This AV conduction property is different from that of the adult goat heart; thus, the existence of a lateral accessory bypass tract was investigated. Electrocardiographic leads I, aVF, and V10 revealed no delta waves indicative of ventricular preexcitation. Bundle of His electrograms showed that: (1) bundle of His excitation always preceded the onset of ventricular depolarization, (2) no shortening occurred in the bundle of His to ventricular activation time following early atrial premature beats, and (3) the functional refractory period of the AV node was less than that of the ventricle. The ventricular epicardial excitation sequence indicated no involvement of a lateral bypass tract in the AV conduction of basic or premature beats. Interruption of the bundle of His caused complete AV block. Therefore, no functional lateral accessory bypass tracts are present in the young goat heart, and the AV node and the ventricular specialized conduction system of the young goat are capable of conducting premature atrial excitation to the ventricles within plus or minus 10 msec of the expiration of the ventricular functional refractory period.

Molecular basis of chromosome banding. I. The effect of mouse DNA fractions on two fluorescent dyes in vitro.

The effects of mouse satellite, main band and total DNA on the fluorescence intensity of quinacrine and of the bibenzimidazole derivative Hoechst 33258 were tested in solution. No significant differences were noticed between the double-stranded DNAs in spite of the 5% difference in AT-content between satellite and main band DNA. Single-stranded DNAs enhanced the fluorescence intensity of Hoechst 33258 far less than double-stranded DNAs. Having been denaturated and then reassociated the DNA fractions were intermediate in their enhancing effects on the fluorescence intensity of Hoechst 33258, the differences presumably being due to different degrees of reassociation. The effect of denatured and subsequently reassociated satellite DNA on the fluorescence intensity of quinacrine was similar to that of the native DNAs. Main band and total DNA quenched the fluorescence intensity of quinacrine more after denaturation-reassociation than it did when native. In the discussion the results are related to known cytological data.

[Electrophysiologic semiology of daytime sleep in 7 to 9-year-old children].

The afternoon sleep of 12 children aged 7-9 was studied; its electrophysiological indices and sequential organization were described and compared to those of afternoon sleep of adults and to those of night sleep of adults and children. The EEG indices which differentiate sleep patterns of children from adults' were the following: abundance of slow rhythms from onset of sleep; absence of low voltage fast activity at sleep onset and during REM sleep; early appearance of a large amount of transitory potentials in the form of sharp rolandic waves and sharp occipital waves. Moreover, either focal or generalized paroxysmal discharges occasionally occurred. Even when it covers a complete sleep cycle, the afternoon sleep of children appears shorter than both that of adults and the first cycle of night sleep in children of the same age. The organization of sleep components does not allow identification of the classical stages defined in adults nor to describe homogeneous stages which are specific to children. The very atypical character of REM sleep also makes it difficult to differentiate unequivocally the classical slow and paradoxical sleeps. The significance of rolandic and occipital sharp transients is discussed; the role of maturation and the influence of the time of occurrence within the circadian rhythm are considered, in order to explain the phenomenological and temporal characteristics of day sleep in children.

EEG in brain abscess: its value in localization compared to other diagnostic tests.

The EEG records of 13 cases of brain abscess were reviewed for their value in localization. Of the 11 cases of supratentorial abscesses localization was achieved in 10, and correct lateralization in the remaining 1 case. Two other cases were cerebellar abscesses; 1 of them showed a false cerebral localization; the other showed no focal abnormalities. The most consistent EEG localizing sign was focal arrhythmic delta waves. These were as slow as 0.5 c/sec in 6 of the 13 cases. Epileptiform discharges in the pre-operative EEG were seen in 4 of the 13 cases. Asymmetry of beta activity correctly lateralized the abscess in 7 of the 11 supratentorial abscesses. Generalized abnormalities when present correlated with depression of consciousness rather than with the duration of illness or the presence of raised intracranial pressure. Indium brain scans were done in 6 supratentorial abscesses and provided correct or approximate localization in 5. Carotid angiograms were also done in 7 cases and localized the abscess in 6. Our findings show that the EEG is comparable to brain scans and contrast radiological studies in localizing supratentorial abscesses. We feel that the combination of EEGs and brain scanslization in suspected brain abscess.

Changes in frequency and amplitude of delta activity during sleep.

Modified period analysis was applied to all-night sleep recordings from 14 young adult males. The modifications involved addition of measures of integrated amplitude and of time in frequency band to the zero crossings and zero counts of the first derivative. The analysis was directed toward changes in the characteristics of delta activity (0--3 c/sec) across the night. Delta shifted toward lower frequencies and decreased in amplitude as sleep progressed. These trends were apparent in mean values for successive periods of slow-wave sleep (SSWPs). For epochs of record classified as stage 4 EEG, these trends were seen both within and across SSWPs. The physiological significance of these changes is unknown. We suggest that they may reflect the kinetics of the metabolic processes underlying sleep. Whatever their theoretical significance, the present results provide new, normative features of the sleep EEG which are not evident in sleep stage classification. The trends we observed appear sufficiently reliable to merit empirical investigation as a function of age, clinical state, or the administration of drugs in addition to further studies aimed at determining their biological significance.

Snoring.

Eight heavy snorers underwent nocturnal polygraphic recordings. The principal results are the following: 1. Snoring is an inspiratory (or primarily inspiratory) noise linked to subobstruction of the upper airways. It appears with falling asleep (stage 1) and intensified progressively through the deepening of slow sleep; in REM sleep it becomes discontinuous and is comparable to stage 2 snowing in intensity. 2. In heavy snorers, obstructive apneas are always present and particularly abundant during light sleep (state 2) and REM sleep. 3. In heavy snorers during sleep the systemic arterial pressure reaches and remains at levels higher than those of wakefulness instead of diminishing normally. 4. Some degree of alveolar hypoventilation is associated with snoring when the apneas are especially abundant. These findings confirm the existence of significant polygraphic analogies between snoring and hypersomnia with periodic apneas and indicate that snoring may represent the first phase in the development of this syndrome. Moreover, the effects of snoring on alveolar ventilation and the systemic pressure during sleep suggest that heavy, constant snoring has physio-pathological implications for the cardio-circulatory apparatus.

Stimulus novelty, task relevance and the visual evoked potential in man.

Visual evoked potentials (VEPs) were recorded from normal adult subjects performing in a visual discrimination task. Subjects counted the number of presentations of the numeral 4 which was interposed rarely and randomly within a sequence of tachistoscopically flashed background stimuli (numeral 2s). Intrusive, task-irrelevant (not counted) stimuli were also interspersed rarely and randomly in the sequence of 2s; these stimuli were of two types: simples, which were easily recognizable (e.g., geometric figures), and novels, which were completely unrecognizable (i.e., complex, colorful patterns). It was found that the simples and the counted 4s evoked posteriorly distributed P3 waves (latency 380-430 msec) while the irrelevant novels evoked large, frontally distributed P3 waves (latency 360-380 msec). These large, frontal P3 waves to novels were also found to be preceded by large N2 waves (latency 278 msec). These findings indicate that "the P3" wave is not a unitary phenomenon but should be considered in terms of a family of waves, differing in their brain generators and in their psychological correlates. These late positive components are discussed in terms of task-relevance, recognition and Pavlov's "what is it" response.

Analysis of motor conduction velocity in the human median nerve by computer simulation of compound muscle action potentials.

A digital computer was used to reconstruct compound muscle action potentials recorded from the human thenar eminence after stimulation of the median nerve. The programme allowed the following parameters to be varied: (1) the dimensions of a representative single motor unit potential; (2) the number of motor units in the muscle and the range and distribution of conduction velocities in their nerve fibres; and (3) the distance along the nerve from the point of stimulation to the muscle. The reconstructed compound muscle action potentials were similar to real compound potentials recorded from normal subjects. The number of single motor units and the range of conduction velocities required for the reconstruction correlated with quantitative histologic studies of the recurrent branch of the median nerve to the thenar muscles. By altering the distribution of conduction velocities it was possible to study the effect of abnormal patterns of nerve conduction on the configuration of the simulated compound muscle action potentials. It was found that abnormally slow conduction caused an increased discrepancy between the main parameters of compound potentials corresponding to stimulation of the nerve at proximal and distal sites. These observations suggest that a careful analysis of the differences between pairs of compound muscle action potentials may provide a method for more detailed assessment of conduction velocity in clinical studies of peripheral nerve disorders.

The number and duration of the episodes of the various EEG stages of sleep in young and older people.

Episodes of any one electrophysiological sleep stage and of intervening wakefulness were examined as to their numbers and durations in sleep of ten young normal subjects (mean age 22) and in significantly more disturbed sleep of fourteen late middle age normal people (mean age 55). No significant difference between the two age groups was found for any of the stages in the mean number of episodes taken from 4 nights. For any of the stages, the individual numbers of episodes accumulated over the 4 nights varied in a wide range and showed significant consistency with similar data obtained in another set of 4 nights. There were significant differences between the two age groups in episode duration. The sleep of the older group contained significantly higher proportions of longer episodes of wakefulness and drowsiness and significantly lower proportions of longer episodes of slow wave sleep and REM sleep. Thus, the initiation of episodes of EEG stages was on average similar in the better sleep of young and impaired sleep of older subjects, but the maintaining or termination of the episodes were different.

Antigenic determinants in the disulfide regions of bovine fibrinogen.

Rabbit antibodies to bovine fibrinogen were used to study the antigenic activity of four cyanogen bromide peptides containing the disulfide regions of this molecule. In precipitation tests the highest activity was associated with the peptide F-CB3 which is exclusively derived from the alpha-chain. Reduction of the single disulfide bridge in peptide F-CB3 did not influence its serologic activity. Weaker reactions were observed with the N-terminal multichain peptide F-CB1. The antigenicity of peptide F-CB1 was not affected by removal of fibrinopeptides A and B but it was lost after reduction. The immunological activity of the multichain peptide F-CB2 was even less than that of peptide F-CB1 and the antigenic determinants were destroyed by reduction. A large fragment essentially composed of peptides F-CB1 and F-CB2 could be obtained by limited cyanogen bromide cleavage and showed considerably better immunological activity than peptides F-CB1 and F-CB2 together. Apparently no activity was associated with a mixture of small hydrophobic, disulfide-loop peptides tentatively called peptide F-CB4. The large loss of antigenic activity in cyanogen bromide digests of fibrinogen suggests that the disulfide-stabilized regions do not have an important role in maintaining conformational antigenic determinants of fibroinogen. Changes in noncovalently stabilized conformation requiring uncleaved chains is considered as a possible reason for the findings observed.

Problems raised by potency evaluation of allergen preparations.

Wide variations are observed in the potency of allergen preparations available for use on humans. There are numerous methods of in vivo or in vitro testing: skin testing, passive anaphylaxis testing on animals, study of precipitins, histamine release tests on human leukocytes, radio-immunological assay test (RAST), blasticlymphocyte transformation test (TTB). The first investigations seemed to show that the allergenic potency of a large number of inducing substances was due to proteic activity; this gave rise to the theory that potency could be evaluated by protein nitrogen units (PNU). However, the protein content of an allergenic extract is not always active. In addition, natural specific proteins are altered during the extraction process or subsequently during preservation. Where the active antigen is known, comparison of different testing with various preparations may be made. The results show excellent correlation between the two biological results (cutaneous reaction and histamine release) and the content of E antigen. No relation with PNU has been observed. Certain authors propose the following: skin testing, PNU titration, dosage of certain elements (polysaccharides, lipids, phosphates, etc.), RAST. The great differences observed between the value of skin testing and the RAST may be due to the large number of active antigens in different 'dusts' and to the fact that the RAST method measures only IgE whereas allergic activity of these allergens is no doubt associated with other types of antibodies. The complexity of the problem shows that a great many studies are still necessary to provide a satisfactory solution.

Clinical observations during a relatively early stage of hepatocellular carcinoma, with special reference to serum alpha-fetoprotein levels.

Five cases of hepatocellular carcinoma in whom diagnosis was made when the tumor was relatively small, are described. In 2 cases, serum alpha-fetoprotein (AFP) strted to rise sharply, which enabled early detection and surgical removal of the tumor. Serum AFP was below 100 ng per ml, but above the upper normal limit by radioimmunoassay, and was unfluctuating for a considerable period of time before it began to rise in 2 cases. It was negative throughout in 1 case, who lived more than 4 years after the tumor had reached a detectable size. In 4 of 5 cases, the tumor seemed to have evolved during a stage of chronic hepatitis or its transition to cirrhosis. In 1 case with chronic schistosomiasis and advanced mixed macro- and micronodular cirrhosis, a 1.5-cm tumor was detected by celiac angiography. These observations on time relationship of oncogenesis may be generalized to modify the cirrhotic liver. Necessity is emphasized for the early detection of this type of carcinoma to monitor serum AFP in chronic hepatitis patients, particularly in those with unfluctuating, mildly abnormal levels of AFP.

[On the role of pentachlorocyclohexene in the metabolism and action of hexachlorocyclohexane. I. Synthesis of beta-pentachlorocyclohexene and its identification as the monodehydrochlorination product of alpha-hexachlorocyclohexane (author's transl)].

It is the aim of a series of investigations to test whether or not beta-pentachloro-1-cyclohexene is an intermediate in the biodegradation of alpha-hexachlorocyclohexane. This paper describes attempts to synthesize this intermediate by chemical methods. 1) Pentachlorocyclohexene was synthesized by partial additive chlorination of chlorobenzene. Combined gas chromatography-mass spectrometry revealed that at least five different isomers of pentachlorocyclohexene had been formed. 2) Treatment of alpha-hexachlorocyclohexane with alkaline buffer (pH 8) produced trichlorobenzenes and, in small yield (4%), a pentachlorocyclohexene. This was isolated and identified as the beta-isomer by melting point (71.8 - 72.6 degrees C, uncorr.), IR- and mass spectrum. Dehydrochlorination of beta-pentachlorocyclohexene produced the trichlorobenzene isomers in a pattern which is characteristic of alpha-hexachlorocyclohexane. The position of the chlorine substituents in the beta-pentachlorocyclohexene molecule as judged from NMR studies is e-aeee. This confirms that it is the monodehydrochlorination product of alpha-hexachlorocyclohexane. The configurations of gamma- and delta-pentachlorocyclohexene, determined for comparison, are e-eeaa and e-eeee, respectively. The kinetics of dehydrochlorination of both alpha-hexachlorocyclohexane and beta-pentachlorocyclohexene in alkaline acetone/water (3 + 2) was studied by means of conductometry. Both reactions are of second order: kappa alpha-HCH 0.0495 [1 times mol- minus 1 times s- minus 1[; kappa beta-PCH 0.905 [1 times mol- minus 1 times s- minus 1] (3.6 degrees C). 3) Dehydrochlorination of alpha-hexachlorocyclohexane in pyridine/xylene (3 + 4) was also studied. An earlier report claiming that gamma-pentachlorocyclohexene (and not the beta isomer) is produced in this medium was confirmed, if the reaction was performed at high temperature (120 - 140 degrees C). Moreover, the ratio of trichlorobenzene isomers formed from alpha-hexachlorocyclohexane shifted to a pattern characteristic of the gamma (or gamma) isomer. However, at temperatures of 90 degrees C or less, beta-pentachlorocyclohexene was the main product. The results strongly suggest that in pyridine/xylene, the same isomer is primarily produced from alpha-hexachlorocyclohexane and is isomerized to the gamma, delta and at least two other isomers of pentachlorocyclohexene before further dehydrochlorination ensues. A simple method for the synthesis of beta-pentachlorocyclohexene is presented.

A study of idiotypic suppression in adult rabbits immunized with Salmonella abortus-equi.

It is possible to induce idiotypic suppression in adult rabbits immunized with Salmonella abortus-equi (S.a.e.). Ten months after priming we injected the rabbit with anti-idiotypic serum prepared against its own antibodies to S.a.e. and, 3 weeks later, gave it a booster injection of bacteria. A new anti-idiotypic serum was preparedwith the serum to S.a.e. collected after this boost and was used for the following isiotypic suppression attempt made 10 months after the first one. Using this procedurewe succeeded in two successive idiotypic suppression attempts in the same rabbit. Inthe three attempts we carried out, idiotypic suppression was totally effective, i.e. idiotypes detected by the serum used for the suppression totally disappeared after thesuppression, and the suppression lasted during the life of the rabbits (maximum 10 months). This observation is consistent with a suppression resulti-g from an interaction of anti-idiotypic antibodies with the complementary receptors at the surface of memory cells. This suppression was without effect on antibody to S.a.e. titre and on IgG concentration. Idiotypes detected by the anti-idiotypic serum prepared withthe serum to S.a.e. collected after the suppression. These idiotypes were different from those detected by the anti-idiotypic serum used for the suppression. This observation confirms that idiotypic recognition is confined to a limited number of clonal products, despite the fact that a very heterogeneous antibody population was used forthe anti-idiotypic immunization. Thus we did not observe the appearance of new idiotypes produced previously silent cell clones. All the different idiotypes we detected during the successive idiotypic suppression attempts carried determinants shich remained peculiar to each individual rabbit.

Antibody response in the parotid fluid and serum of Irus monkeys (Macaca fascicularis) after local immunization with Streptococcus mutans.

The antibody response of Macaca fascicularis in parotid saliva and serum to local immunization by two routes with Streptococcus mutans was studied and compared over 1 year. Antibodies were titrated and classified by indirect immunofluorescent staining using specific antiglobulin conjugates. Antiglucosyltransferase activity was assayed by an enzyme inhibition test. Animals were immunized first by injecting formalin-killed bacterial cells and cell products subcutaneously into the vicinity of the four major salivary glands. The monkeys were next immunized by retrograde instillation of antigen into the parotid duct. Extensive subcutaneous local immunization gave a serum response only. After parotid duct immunization, high titers of immunoglobulin A (IgA) antibody, along with traces of immunoglobulin G (IgG) immunoglobulin M (IgM) antibody, appeared in the parotid saliva, and in the serum high titers of IgG antibody were present along with lower titers of IgA and IgM. IgA antibodies in parotid fluid were shown by double immunofluorescent staining to be associated with antigenic determinants which cross-reacted with an antiserum directed to human secretory component. Titers in parotid fluids and sera fell sharply when immunization was stopped. This response pattern was reproducible. High concentrations of antibody capable of inhibiting glucosyltransferase prepared from S. mutans were found in the sera, but relatively little was detected in the parotid fluids. Extensive immunization via the parotid duct resulted in transient functional impairment of the gland, as evidenced by diminished salivary flow rates. We conclude that parotid ductal immunization can be an effective method for stimulating a salivary secretory IgA antibacterial antibody response.

A fetuin-like antigen from human nephroblastoma.

An antigen was detected in pooled human nephroblastomas using antiserum prepared in rabbits against an ethylemediaminetetra acetic acid (EDTA) extract of the tumors. This antigen was not found in normal human plasma or kidney extracts, and was not related to the ABO or Forssman blood groups. The antigen was detected in extracts of cultured nephroblastoma cells, but was not present in extracts of normal human fetal kidney cell cultures. The antigen is believed to be present at the cell surface, as cell viability was not significantly lowered during the extraction procedure. A reaction of complete identity was demonstrated by Ouchterlony double diffusion experiments with this antigen and purified bovine fetuin. The antigen was not found in extracts of human fetal spleen, thymus or kidney, nor in human fetal serum. Furthermore, the antigen does not possess determinants in common with the human alpha-fetoprotein of hepatomas, nor was it detected in human renal clear cell carcinoma. Initial characterization of the antigen showed it to be nondialysable, not sedimentable at 100,000 times g for 2 h, stable to repeated freeze-thawing and to incubation at 56 degrees C for 1 h, and water soluble over a wide pH range. The antigen was susceptible to digestion with pronase and trypsin and possibly hyaluronidase, but not to ribonuclease or neuraminidase. The protein portion is therefore of major importance to the structural integrity of this antigen. The relationship between this antigen and other abnormal materials reported previously in nephroblastoma patients is being studied.

Storage granules of thyroid C cells in the dog: a cytochemical and ultrastructural study, in relation to the masked metachromasia reaction.

Masked metachromasia can be demonstrated in thyroid C cells, and other cells of the APUD series, by staining with a metachromatic basic dye after hydrolysis of suitably fixed tissue. The reaction is thought to be due to the presence of polypeptides with a high concentration of side-chain acidic groups. Since most APUD cells possess storage granules, presumed to contain a polypeptide hormone, it has been assumed that the masked metachromasia reaction gives information concerning the contents of these granules. However, there has been an increasing suspicion that the reaction might actually be due to the membrane bounding these granules, rather than to the contents. We have examined, cytochemically and ultrastructurally, dog thyroid tissue which has been subjected to fixation and hydrolysis as in the usual method for masked metachromasia. We found that the membrane surrounding the C cell granules is removed by hydrolysis, confirming the hypothesis that the reaction is due to the contents (hormone and/or matrix)rather than to the membrane. Tissues were fixed in an aqueous mixture containing glutaraldehyde (6 25% v/v), picric acid (three-quarters saturation) and sodium acetate (I% W/V)adjusted to PH 7 with sodium hydroxide. This was found to be a very satisfactory fixative for electron microscopy Some morphological details of C cells were noted, such as the richness of desmosomes between C cells in this species, and frequent direct contact with the colloid.

Involvement of cytosol proteins in oleate activation of rabbit liver fructose-1,6-diphosphatase.

Dialyzed rabbit liver cytosol was specifically freed of endogenous fructose-1,6-diphosphatase by immunoadsorption on a column of Sepharose-immobilized anti-fructose-1,6-diphosphatase. This material increased the specific activity of homogeneous enzyme to the maximal rate observed with EDTA and shifted the pH optimum from 8.4 to 7.4. With oleate or other fatty acids as activators, the hydrolysis of fructose-1,6-diphosphatase by enzyme, at neutral pH, showed nonlinear initial rates dropping to lower linear rates. Cytosol activator acted synergistically with oleate both to increase neutral enzyme activity and to maintain the high initial catalytic rates. After sucrose density centrifugation or gel filtration, the cytosol had no effect by itself, but still potentiated oleate activation. The factor was destroyed by treatment with subtilisin or trypsin, but all attempts to identify a unique protein component in cytosol were unsuccessful. The presence of Na dodecyl-SOJ, deoxycholate, or urea did not improve the resolution of the factor, but these compounds did lower the K50 for activation by cytosol. Since fatty acids are the only unique compounds which have been isolated from cytosol which activated fructose-1,6-diphosphatase, it appears that soluble proteins can act as natural carriers for the fatty acids. This was supported by the fact that both dialyzed rabbit alpha-globulins and muscle phosphofructokinase also acted synergistically with oleate in a manner similar to cytosol. Phosphatidic acid and phosphatidylserine activated fructose-1,6-diphosphatase, and their action was synergistic with oleate. Glutathione (1 mM) activated the enzyme 5-fold at pH 7.3 and its effects were additive with oleate and cytosol or alpha-globulins.

Autoradiographic studies of the projections of the midbrain reticular formation: descending projections of nucleus cuneiformis.

The descending projections of nucleus cuneiformis in the cat were traced by autoradiography in the transverse and sagittal planes following stereotaxically placed injections of 3H-leucine. Many descending axons are organized into distinct fiber systems, of which the largest and most well-defined crosses directly in the midbrain and descends through the ventromedial tegmentum of the brain stem. This fiber system first terminates profusely in n. reticularis tegmenti pontis and then proceeds through the rhombencephalic tegmentum emitting transversely oriented branches to n. reticularis pontis caudalis and gigantocellularis, the raphe magnus and the facial nucleus...

Golgi studies in the substantia gelatinosa neurons in the spinal trigeminal nucleus.

This Golgi study identifies three neuronal cell types in the substantia gelatinosa (SG) layer of the spinal trigeminal nucleus. The SG neurons are distinguished from each other based on: (1) dendritic branching pattern, (2) denritic spine distribution, (3) geometric shape of the denritic tree, (4) laminar distribution of the dendrites, (5) axonal branching pattern and (6) laminar distribution of the axonal arbor. The islet cell is found in small clusters and its dendrites and axonal arbor are confined within the SG layer. Its dendrites span the full width of the SG layer and extend up to 500 mum in the long axis of the layer. Dendritic spines are generally sparse with small clusters of spines found on the higher order dendritic branches. The islet cell axon extends for at least 1 mm in the long axis of the layer. Each of its collaterals divide every 50-100 mum with one branch doubling back in the direction of the cell body and the other branch continuing on in the direction of its parent. In this manner each islet cell generates a profuse axonal plexus in the SG layer. The stalked cell is found individually within the SG layer. Its cell body is usually found in the inner half of the SG layer and its sinuous dendrites cross the SG layer and enter the marginal layer. The stalked cell dendrites emit numerous fine stalk-like branches and dentritic spines. Its axon emits branches in the SG and marginal layers. The spiny cell is found singly between groups of islet cells. Its extensive dendritic tree spans up to 500 mum rostrocaudally and mediolaterally crossing into both the marginal and magnocellular layers. Spiny cells have evenly distributed dendritic spines along their dendrites in the SG layer. The spiny cell axon sends branches into all three layers of nucleus caudalis. Numerous branches enter the outer 300 mum of the magnocellular layer where they undergo further branching with some branches returning in recurrent fashion toward the SG layer. The three neuronal cell types of the SG layer satisfy all of the morphological criteria for Golgi type II interneurons. Their highly branched axons generate many collaterals within the confines of their dendritic trees and do not project out of nucleus caudalis. The SG neurons are considered to be inhibitory interneurons interposed between V nerve primary afferent axons which arborize in the SG layer and second order neurons of nucleus caudalis.

Long-lasting in vitro immune response to a distinct antigenic determinant of a bacterial protein. Cyclic changes of antibody titer and affinity.

Long-lasting (60 days or more) antibody responses in vitro by rabbit lymph node fragments to a distinct determinant of Escherichia coli beta-D-galactosidase were obtained by supplementing culture medium with fetal calf and horse serum. Antibodies released in the supernatant were removed every 3rd to 5th day together with the spent medium, without pooling to minimize intermixing of molecules synthesized far apart in time. Antibody titer, association constant, and heterogeneity index were measured in medium samples collected throughout the response in order to draw profiles of their changes under conditions whereby a limited number of clones synthesize antibodies in a closed system without connection to antigen depots, central lymphoid organs, and circulating cell and antibody pools. It was found that antibody affinity changes cyclically and that such cycles may be repeated. Cycles are composed of an ascendant limb with a gradual increase in affinity and a parallel diminution of heterogeneity. A descendant limb follows with the opposite modifications. High affinity antibodies predominate at the peak of the cycles, whereas low affinity molecules take over at the end of the cycles until the next ascendant limb begins; these persist after the last cycle has waned.

Identification of the cell population involved in viral-specific cell-mediated cytotoxicity in man: evidence for T cell specificity.

The nature of the cell population involved in lymphocyte mediated cytotoxicity to baby-hamster-kidney (BHK-21) target cells persistently infected with rubella virus was investigated by a 51Cr-release microassay. After depletion of the T cell population with an antiserum to human0thymus-lymphoid tissue antigen (HTLA), the purified B cell population showed a decrease in E-rosette formation (9.0 +/- 2.2% compared to 69.6 +/- 9.1% before treatment) and an insignificant degree of cytotoxic activity against rubella-infected target cells (specific immune release of 51Cr was 0.9 +/- 2.6% compared to 24.2 +/- 3.8 before treatment). A purified T cell population, prepared by depletion of B cells with an anti-human immunoglobulin serum and complement, was found to show no alteration in E-rosette formation (85.2 +/- 6.2%) or cytotoxicity (30.3 +/- 4.4% SIR) but showed decreased EA- and EAC-rosette formation (2.7 +/- 1.5% and 10.5 +/- 3.2%, respectively, compared to 19.4 +/- 2.9% and 28.0 +/- 4.1% before treatment). A monocyte-depleted population prepared by removal of the plastic adherent mononuclear cells showed no significant alteration of rosette formation or cytotoxicity. These experiments suggest that the predominant lymphoid population responsible for direct cell-mediated cytotoxicity to virus infected target cells in the 51Cr-release microassay appears to be effected by a thymus-dependent lymphocyte population.

Immunochemical characterization of the anti-RNA antibodies found in scleroderma and systemic lupus erythematosus. I. Differences in reactivity with Poly (U) and Poly-(A) Poly (U).

In a previous study, all 40 sera from patients with scleroderma, 20 of 40 sera from SLE patients, but none of 40 sera from normal controls, were found to have antibodies to ssRNA. All scleroderma sera were also found to react with HSA-coupled uridine and UMP and their reaction with HSA-coupled uridine and UMP and their reaction with ssRNA could be inhibited by uracil, uridine, and UMP. To characterize further these uracil-specific anti-RNA antibodies found in scleroderma and compare them with the anti-RNA antibodies found in SLE, we tested their reactivity with Poly (U) and with Poly (A)-Poly (U) and all but one failed to react with Poly (A)-Poly (U). This same serum was the only one in which the reaction with Poly (U) could not be inhibited with uracil. Reactivity of SLE sera was strikingly different from that found in scleroderma sera. Seventeen of 34 SLE sera studied reacted with ssRNA but only four of these reacted with Poly (U). Conversely, two SLE sera that reacted with Poly (U) did not react with ssRNA. Fifteen reacted with Poly (A)-Poly (U) and only two of these failed to react with ssRNA. Five SLE sera which were reactive with ssRNA did not precipitate with Poly (A)-Poly (U). All SLE sera which reacted with Poly (U) could be inhibited with uracil, although less effectively than in scleroderma. Reactivity with Poly (A)-Poly )U) was not inhibited with uracil nor with adenosine. These findings confirm that antibodies to RNA that are found in scleroderma are directed to uracil and thus specific to ssRNA, whereas RNA antibodies found in SLE sera are heterogeneous and directed to either the base, to the site of union of the base and sugar moiety to the ribose backbone, or to the helical structure of double stranded RNA. These differences and the respective antigenic specificities of these anti-RNA antibodies found in scleroderma and SLE may be theoretically important.

Class, amounts, and affinities of anti-dinitrophenyl antibodies in chickens. II. Production of a restricted population of high affinity 7S antibodies by injection of antigen emulsified in adjuvant.

The response of chickens given a single intramuscular injection of maximally coupled dinitrophenylated-gamma-bovine beta-globulin in either Freund's complete (FCA) or incomplete (FIA) adjuvants was characterized by an initial synthesis of 7S and 17S antibodies followed by the exclusive and persistent production of 7S antibodies. The 17S antibodies were not detected either 3 to 4 weeks after a single injection or after an intravenous boost 16 months later. Injections of low doses of antigen in FCA induced the synthesis of 7S antibodies of high affinity at least by 4 months. Analyses of the Sips plots generated from equilibrium dialysis data indicated that a shift in the distribution of 7S antibody affinities occurred because of the production of a restricted population of high affinity antibodies. The changes in the binding properties of antibody during the immune response from chickens given antigen in FIA were less apparent, although qualitatively similar, to those found in birds given antigen in FCA. Three possibilities were presented to explain the effect of adjuvant on the class and affinity of the antibody: a) the requirement of a second signal for B cell differentiation, b) the presence of subpopulation of B cells, and c) somatic mutation events.

Bovine keratohyalin: anatomical, histochemical, ultrastructural, immunologic, and biochemical studies.

Salt extraction studies showed that keratohyalin (KH) could be solubilized and extracted from fresh bovine hoof epidermis. The solubility of KH varied in relation to the molarity of the salt solution used for extraction. Using this information, the extracted KH was aggregated in vitro by dialyzing the high salt extract against distilled water. Histochemical, ultrastructural, and immunologic studies of the resultant particles or macroaggregates showed that the latter had the same properties and immunogenicity as the KH granule in situ and produced antibodies against it. Fractionation of the macroaggregates by polyacrylamide gel electrophoresis demonstrated that the macroaggregates were compsed of sets of 20 polymers whose subunits or monomers had a molecular weight of 16,900. Amino acid analyses showed that the macroaggregates and the various fractionated polymers were similar and that the protein ahd 116 amino acid residues. Serine, arginine, glycine, glutamic acid, and histidine constituted 78% of all residues, and serine alone represented 27%. The molecular weight by amino acid analyses was 16,150 after correction for the 8% ribonucleic acid which appears to be complexed to the protein.

Ultrastructural and immunohistochemical studies in five cases of Argentine hemorrhagic fever.

Ultrastructural and immunohistochemical studies on tissues from five patients with Argentine hemorrhagic fever revealed previously undetected lesions caused by the viral infection. Two types of particle were seen in the cells of all organs examined. The particles had some characteristics similar to those described for arenaviruses. However, the virus-like particles were intracellular, had a single membrane, and apparently originated by a process of budding into the endoplasmic reticulum cisternae. Intranuclear bodies and three types of cytopolasmic change were observed in conjunction with the virus-like particles; Antigenic determinants of Junin virus were demonstrated in cells of all organs examined. Immunohistochemical experiments also indicated alterations in the cellular mechanisms of protein synthesis. Until now the pathogenesis of human diseases produced by arenaviruses has not been established. The results of this study suggest that in Argentine hemorrhagic fever the virus is responsible for a direct pathogenic action.

Shared idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants. I. Demonstration of similar or identical idiotypes on IgG molecules and T-cell receptors with specificity for the same alloantigens.

Antigen-binding receptors on T lymphocytes and IgG antibodies with the same antigen-binding specificity as the T-cell receptors display shared or identical idiotypes. This was shown using a system where adult F1 hybrid rats between two inbred strains were inoculated with T lymphocytes from one parental strain. Such F1 hybrid rats produce antibodies directed against idiotypic determinants present on IgG alloantibodies, produced in the T donor genotype strain and with specificity for the alloantigens of the other parental strain. The idiotypic nature of the F1 antialloantibody serum against the parental alloantibodies was demonstrated both by indirect hemagglutination tests or by gel diffusion using alloantisera with different specificity as targets. Furthermore, the F1 anti-T-lymphocyte sera could be shown to contain antibodies against idiotypic parental T lymphocytes as well. This was shown by the capacity of the antisera, in the presence of complement, to wipe out the relevant parental T-cell reactivity against the other parental strain (as measured in MLC or GVH) whilst leaving the T-lymphocyte reactivity against a third, unrelated allogeneic strain intact. These findings demonstrate that F1 hybrid rats inoculated with parental T lymphocytes make anti-idiotypic antibodies directed against both the T cell receptors and IgG alloantibodies of that parental strain with specificity for alloantigens of the other parental strain. In order to prove identity between the anti-idiotypic antibodies against the B and T-cell antigen-binding molecules the following experiments were carried out; highly purified IgG from relevant alloantibody-containing serum in immunosorbent from could be shown to selectively remove both anti-idiotypic activities from the F1 antiserum. Further more, parental normal T lymphocytes could be shown capable of removing from the anti-idiotypic antisera all those antibodies that would cause agglutination of the relevant alloantibody-coated erythrocytes in the indirect agglutination assay. We would thus conclude that T and B lymphocytes reactive against a given antigenic determinant use receptors with antigen-binding areas coded for by the same variable gene subset(s).

Histological subtypes and prognostic problems in meningiomas.

The incidence of the various histological subtypes of meningiomas was examined in 1238 patients with surgically treated meningiomas, about 80% arising within the cranial cavity. The histological classification used was that of Courville (1950) and Rubinstein (1972), but "angioblastic" meningiomas were segregated into 3 groups: highly vascularized meningiomas, hemangioblastomas, and hemangiopericytomas. Endotheliomatous and transitional forms constituted 85% of the total (71.5% of intracranial tumors), fibroblastic forms 6.6 and 7.5%, respectively, and highly vascularized (endotheliomatous or transitional) meningiomas 5.2% of the intracranial tumors, while true "angioblastic" meningiomas (hemangioblastomas and hemangiopericytomas) amounted to 2.8% of the total (3.1% of the intracranial tumors). 1.2% were "atypical" (so-called malignant) meningiomas; true meningeal sarcomas were excluded. The incidence of recurrence in patients surviving at least 5 years after apparently complete removal of the tumor was 13% for all sites, and 14.2% for intracranial tumors, but almost twice as high after partial removal. There were no significant differences in the recurrence rate and intervals between first and second operation according to the various histological subtypes of meningiomas, except for hemangiopericytomas which recurred with significantly higher frequency and, together with atypical meningiomas, at much shorter intervals than the others. The prognostic significance of some histological criteria in "non-angiomatous" meningiomas was examined in 211 patients surviving at least 5 years after apparently complete removal of the tumor. Among the recurrences, there was a significantly higher degree of cellularity and increased mitotic rate and, probably, of cortical invasion, while nuclear pleomorphism, increased vascularity, and focal necroses showed no definite differences. The presence of mitotic figures alone appeared to be of no prognostic value. While most recurrent meningiomas did not change their basic morphological type significantly, about 12.5% of the recurrences appeared to have a different rate of growth as suggested by increased cellularity and mitotic rates. In 2 cases an isomorphic (benign) meningioma became a true spindle cell sarcoma.

Determination of dicumarol metabolites in bile of rats.

After intravenous administration of dicumarol-14C to rats, the bile excreted over the next 24 hr contained from 32 to 46% of the administered radioactivity. At least three primary metabolites and a small amount of unchanged dicumarol were present in the bile. Over 91% of the primary metabolites was converted to dicumarol and 7-hydroxydicumarol by hydrolysis with beta-glucuronidase. Some primary metabolites were hydrolyzed simply by acidification to pH 3 or by treatment under the acidic conditions utilized in the enzymatic hydrolysis. The three primary metabolites contain carboxylic acid groups, as indicated by their electrophoretic mobility-pH profiles, and some are simple glucuronides of dicumarol and 7-hydroxydicumarol. The possibility that others are derivatives of these compounds in which a coumarin lactone ring is opened cannot be ruled out. When the metabolites released by either acidification or enzymatic hydrolysis were chromatographed in n-butanol-3 M ammonia, artifacts were produced, presumably as a result of decomposition of 7-hydroxydicumarol. The question is raised whether a previously reported metabolite (B055) is an artifact.

Nuclear division in the ameboflagellate Adelphamoeba galeacystis.

Nuclear division is synchronized cultures of the ameoboflagellate Adelphamoeba galecystis has been described. Division in this organism is typically promitotic. It occurs within an intact nuclear membrane and is characterized by the persistence of the nucleolus and its transformation into 2 polar masses. The nucleolus is stained with pyronin-Y by the methyl green pyronin-Y technic, and with Heidenhain's hematoxylin, but is unstained by the Feulgen reaction. The reaction with these stsins is removed after digestion of the nucleolus by ribonuclease. During mitosis the nucleolus undergoes an orderly series of vacuolizations before forming the polar masses. The chromatin is Feulgen positive, stains with methyl green by the methyl green pyronin-Y technic and undergoes a series of characteristic changes during the division process. Synchronizationof amebae grown on coverglasses was accomplished by transfer of cells from 30 to 38.5 C for a period of 100 min. A temporal sequence of nucleolar and chromatin participation in the nuclear division of this organism is suggested.

A golgi study of the optic tectum of the tegu lizard, Tupinambis nigropunctatus.

The dendritic patterns of cells in the optic tectum of the tegu lizard, Tupinambis nigropunctatus, were analyzed with the Ramon-Moliner modification of the Golgi-Cox technique. Cell types were compared with those described by other authors in the tectum of other reptiles; particular comparisons of our results were made with the description of cell types in the chameleon (Ramń, 1896), as the latter is the most complete analysis in the literature. The periventricular gray layers 3 and 5 consist primarily of two cell types--piriform or pyramidal shaped cells and horizontal cells. Cells in the medial portion of the tectum, in an area coextensive with the bilateral spinal projection zone, possess dendrites that extend across the midline. The latter cells have either fusiform or pyramidal shaped somas. The central white zone, layer 6, contains fibers, large fusiform or pyramidal shaped cells, fusiform cells, and small horizontal cells. The central gray zone, layer 7, is composed predominately of fusiform cells which have dendrites extending to the superficial optic layers, large polygonal cells, and horizontal cells. The superficial gray and white layers, layers 8-13, contain polygonal, fusiform, stellate, and horizontal elements. Layer 14 is composed solely of afferent optic tract fibers. Several differences in the occurrence and distribution of cell types between the tegu and the other reptiles studied are noted. Additionally, the laminar distribution of retinal, tectotectal, telencephalic, and spinal projections in the tegutectum can be related to the distribution of cell types, and those cells which may be postsynaptic to specific inputs can be identified. The highly differentiated laminar structure of the reptilian optic tectum, both in regard to cell type and to afferent and efferent connections, may serve as a model for studying some functional properties of lamination common to cortical structures.

Long-time observations on the Blalock-Taussig operation VIII. 20 to 28 year follow-up on patients with a tetralogy of Fallot.

This study extends our previous observation on patients with a tetralogy of Fallot, operated on by the late Dr; Alfred Blalock and his associates between 1945 and 1951, from a 15 year follow-up to 20 years and assesses the final status of these patients 20 to 28 years after their first operation. This study is mainly concerned with the 432 patients known to be alive at the beginning of the 15th postoperative year. At the beginning of the 20th year, 376 patients were alive, 24 had died, 32 had been lost to follow-up. Review of the final status of those 432 patients showed that 169 had no further cardiac surgery after their initial operation, 36 had further palliative surgery, and 227 had total correction. Thirty-seven percent of the first group, and 79.3% of those with total correction were doing well. These two groups, however, are not comparable. Approximately 250 patients have married; 161 have one or more children. Thirty-five percent have graduated from college and 68.7% are earning substantial incomes. The high scholastic achievement of many of these patients is strong evidence that low oxygen saturation of arterial blood is not a prime cause of mental retardation. The occupations of the patients indicate that the quality of their lives is extremely good and that a cardiac handicap in childhood does not preclude success in adult life. Approximately 69% of these patients have repaid in taxes the cost to society of their rehabilitation.

Age-related refractoriness of PHA-induced lymphocyte transformation. II. 125-I-PHA binding to spleen cells from young and old mice.

Using 125-I-labelled red kidney bean phytohemagglutinin (125-I-PHA), we have found that spleen cells from old BC3F1 mice bind this plant mitogen equally well, if not better, than spleen cells from young BC3F1 mice, although PHA-induced blastogenesis of spleen cells from old mice is sharply reduced. Analyes demonstrated that there is neither significant alteration of binding affinity nor decreased total number of membrane receptor sites for PHA in senescing mouse spleen cells. The amount of PHA which was initially bound to spleen cells in serum-free medium appeared to be insufficient for a subsequent full stimulation of blastogenesis ([3-H]thymidine incorporation) in either young or old mouse spleen cells; when washed free of unbound extracellular PHA and upon clutivation in serum-containing culture medium, spleen cells rapidly released more than 90% of the bound PHA. Also, temperatures which change cell membrane morphology played a significant role in the binding and retention of PHA. However, no difference was observed between young and old mouse spleen cells in all these phenomena of PHA-cell membrane interaction.

[Fine needle aspiration biopsy of abdominal and retroperitoneal tumours under ultasonic guidance (author's transl)].

In 94 patients with tumours of the abdomen or the retroperitoneal space detected by ultrasonography fine needle aspiration puncture was performed under ultrasonic control. The aspirated material was stained with Pappenheim's solution for cytological examination. In 86 patients the diagnosis was definitely established. Of 49 patients with malignant growths (liver 35, kidney 9, colon 2, subcutaneous tissue 2, pancreas 1) tumour cells were gained in 40. In 4 other pathients the cytological findings were suspicious of malignancy. In 37 patients with benign processes (liver 35, subcutaneous tissue 2) no false-positive diagnosis was made. Complications induced by the puncture, especially hemorrhage were not encountered. The combined ultrasonic-cytological examination is simple, fast, almost riskless and well-tolerated. It allows to avoid other more expensive methods to obtain tumour material which usually carry some risk. It is especially valuable as a screening method for malignant growth in the abdominal cavity and the retroperitoneum.

Posttranslational protein modifications, with special attention to collagen and elastin.

It is apparent that significant progress has been made in our understanding of the biosynthesis, modifications, and maturation of collagen and elastin. We now recognize and partially understand special reactions involved in hydroxylations within the cell and complex cross-linking processes occurring outside the cell. Recent experiments (191) have shown that in human diploid fibroblast cultures of limited doubling potential (191) the hydroxylation of collagen prolyl residues appears to be "age" or passage-level dependent. With increasing passage level of these cultures, both the ascorbate requirements and the extent of collagen hydroxylation decrease. "Young" cell cultures have a strong requirement for complete hydroxylation and without ascorbate there is only about 50% of the normal level. "Middle-aged" cultures show higher hydroxylation without and full hydroxylation with ascorbate, whereas "old" (or cultures close to "senescence") are incapable of full hydroxylation with or without ascorbic acid. Although the overall system may show some deterioration with increasing passage levels, it appears that with increasing passage levels other components in the cell replace the ascorbate dependence of the hydroxylase system to a greater exten. In some ways, aging WI-38 cultures begin to resemble some transformed cells in their biochemical reactions, although they continue to remain diploid and eventually lose the ability to replicate. It is not yet known whether old animals can produce collagen, which may now be underhydroxylated, perhaps contributing to certain senescent changes. Careful examination of the hydroxylation index of collagen produced in organoid cultures of tissue biopsies as a function of donor age might be informative, particularly if one looks at the quality of collagen by employing collagenase and other proteolytic digests with collagen (191). One could comare the levels of frequent and characteristic peptide triplet sequences such as Gly-Pro-Hyp to Gly-Pro-Pro, Gly-Ala-Hyp to Gly-Ala-Pro, or Gly-Pro-Hyl to Gly-Pro-Lys and others for evaluation of hydroxylation throughout the entire molecule or at selected sequences.

Symphysial changes in rheumatoid arthritis. A microradiographical and histological study.

Autopsied pubic symphyses of 10 male and 10 female non-rheumatoid subjects and of 11 subjects with RA were studied. Osteo-arthritic changes were common in all elderly symphyses. This caused changes in the structure of the end plates and trabeculae. Some non-rheumatoid samples showed changes suggestive of erosion, but no evidence of inflammation. At microradiography the erosions seen in rheumatoid symphyses differed from the controls due to a lack of reactive sclerosis in the surrounding bone, although true inflammation was seen histologically in only two cases.

Third HL-A segregant series: genetic analysis and molecular independence on lymphocyte surface.

Five sera which detect two alleles of the third HL-A locus (T2 and T4) are described. Statistical analysis of a panel of 220 donors gave gene frequencies in a French Parisian population which are comparable to the results of Scandinavian authors. Segregation observed in 50 families emphasized the linkage disequilibrium with SD2 alleles. T1 was found to be associated with one part of W22 (Da30) and T3 with the other part non-Da30). No recombination was observed between SD2 and SD3, and the distance between these two loci can be estimated at less than 0.0042 U. Differential capping experiments showed that redistribution of SD3 antigens did not affect the SD2 or SD1 products, indicating that they are borne on the cell surface by independent structures. Moreover, capping of SD1, SD2 and SD3 antigens did not provoke complete redistribution of beta2microglobulin which suggests that not all beta2M molecules are linked to HL-A antigens.

Allogeneic radiation chimeras. Long-term studies.

Lethally irradiated mice protected with allogeneic fetal liver cells or with syngeneic or allogeneic marrow and spleen cells treated with antisera to mouse immunoglobulins or to the T cell-associated theta antigen and their controls were observed for up to 750 days. The best survival rates were found in the large groups given syngeneic marrow and spleen or allogeneic fetal liver cells (70-85% 700-day survival); in contrast, 43% of the group injected with allogeneic cells treated with anti-theta serum and 19% of those given antiimmunoglobulin-treated cells were alive 700 days postradiation. Pulmonary infection was the most frequent cause of death of long-term survivors in all groups. Tumor incidence was increased in recipients of allogeneic cells (13% versus 4% among syngeneic chimeras), but the renal pathology seen in these groups was no greater than that noted in the syngeneic controls. Beginning 600 days after irradiation, mice from experimental and control groups were killed and their spleens were cultured with thymus-dependent antigens and the mitogens concanavalin A and lipopolysaccharide, Escherichia coli. The most frequent finding in all groups was mild to moderate impairment of T cell-dependent responses.

[Early and late results after cryosurgical treatment of bladder neck obstructions (author's transl)].

We report on late results of 100 risk patients, who were operated by cryosurgical technique because of bladder neck obstructions. Detailed representation of complications. The primary mortality was 5%. Controls after one year showed good functional results on 80% of the patients. 60% of the patients had sterile urine, whereas only 17% of the male patients had no infection before the operation. As to the anatomical results we could talk of a cryoprostatectomy in 25% of the cases. Because of a 20% failure cryosurgery remains a palliative operation, which can only be carried out on risk patients. Because of little hemorrhagic diathesis and the possibility to perform this operation under local anesthesia cryosurgical operations on the other hand are suitable for patients, who cannot be anesthetised. Otherwise these patients had needed a permanent catheter. We prefer shorter freezing times of 3--4 min. Essentially we could show the same functional results as other authors, who preferred longer freezing times, until they reached a certain temperature in the prostatic capsule. By shorter freezing times on the contrary we could avoid secondary transurethral resections of larger necroses. New disturbed micturition after cryosurgical treatment of the prostate is unusual. The question of a later recidivation is of secondary importance because of the primary high morbidity rate of the selected number of cases.

[Effect of gamma globulin on reactivity of the organism. V. Types of biological activity of gamma globulin preparations].

Biological activity of 110 series of commercial gamma-globulin preparations was studied; they were found to contain placental antigens, group-specific blood substances, gonadotropic hormones and antibodies to them. Placental antigens were found in 12% of placental and abortive gamma-globulin batches in titres of 1 : 2--1 : 16; no placental protein was revealed in donor gamma-globulin. There were group-specific blood substances in all the batches of placental and abortive gamma-globulin studied (in titres of 1 : 138--A, 1 : 112 B in the placental gamma-globulin and in titres of 1 : 48.9--A, 1 : 32--B in the abortive gamma-globulin). In the preparations from the venous blood group-specific substances were either absent or present in lowe titres only (1 : 2). The value of gonadotropic hormones in the placental gamma-globulin batches constituted 873+/-157, and in the abortive--991.4+/-147 IU/l; no gonadotropins were revealed in donor gamma-globulin. The mean titres of antibodies to gonadotropin hormone in the gamma-globulin preparations made of placental blood constituted 1 : 236+/-32, of abortive--1 : 131+/-16.6, and of the venous blood--1 : 46+/-24.7. The presence of biologically-active substances in the gamma-globulin preparations pointed to the necessity of increased requirement of their quality; additional requirements to its standardization proved to be also necessary.

Warty dyskeratoma.

A case is reported of a 53-year-old woman, who had had for one year a wart-like papillomatus lesion on the alveolar process in the region corresponding to m The remaining mucosa exhibited a normal clinical picture. The patient's general health was satisfactory and no skin manifestations of interest were apparent. The lesion was extirpated and examined histologically and microradiographically and was found to have histopathological characteristic of the same kind as in warty dyskeratoma. The discussion is concerned with aetiological factors and with problems of differential diagnosis.

Studies on maturity in newborn infants. VIII. Alpha-foetoprotein and albumin.

Cord blood of 125 newborn infants of various gestational ages has been analysed for the amounts of alpha-foetoprotein and albumin using an electroimmunoassay. The quotient alpha-foetoprotein/birth weight was recorded as well. All the three measureements correlate fairly well with the gestational age of newborn infants. Measuring alpha-foetoprotein is not of the same value, however, in estimating gestational age as is the simple scoring of external characteristics.

Hepatitis B surface antigen subtypes in Malaysia.

One hundred and ninety hepatitis B surface antigen positive (HBsAG+) sera were subtyped, belonging to : blood donors, hepatitis patients, patients and staff in a hemodialysis unit, all from Kuala Lumpur; Malaysian aborigines from three jungle locations in Peninsular Malaysia; and East Malaysians from Sarawak, East Malaysia; Three subtypes adr, adw and ayw were present in Malaysia in the following frequencies: 44%, 29%, and 27%, respectively; In Kuala Lumpur 87% had subdeterminant d and 13 per cent y, whereas in the deep jungle aborigines of Perak and Pahang, the y subdeterminant was present in 87% and the d in 13%. A similar pattern of preponderance of y prevailed in Sarawak, East Malaysia. In Kuala Lumpur the two main ethnic groups, Malays and Chinese, differed in subtype distribution, in that adr predominated in the Malays (61%), while the adw predominated in the Chinese (51%); Subtype distribution was not related to age or sex of carriers of the antigen, or to whether they had hepatitis, or asymptomatic antigenemia.

Factors responsible for ADP-induced release reaction of human platelets.

Extensive aggregation of human platelets can be induced by ADP without secondaryaggregation or release of granule contents. This occurs with washed platelets in Tyrode solution containing 0.35% albumin, human fibrinogen, and apyrase, and in platelet-rich, heparin- or hirudin-plasma. Conditions that caused release during ADP-inducedaggregation were-citrate as the anticoagulant in platelet-rich plasma; addition of citrate (11-15 mM) to a suspension of washed platelets, or to hirudin-plasma or heparin-plasma; suspension of platelets in a medium containing magnesium but no calcium;and the presence of trace amounts of thrombin or aggregated gamma globulin in the platelet suspensions. Acetylsalicylic acid, phenylbutazone, or sulfinpyrazone inhibited secondary aggregation and release in all these circumstances. Heparin or hirudin inhibited ADP-INDUCED SECONDARY AGGREGATION AND RELEASE PROMOTED BY TRACES OF THROMBIN. Although fibrinogen is required for ADP-induced primary aggregation, it does not support secondary aggregation and release, provided that it has no clot-promoting activity. The main agent responsible for ADP-induced secondary aggregation and release in human, citrated, platelet-rich plasma appears to be sodium citrate. Suspending washed human platelets in a medium without calcium mimics the effect of citrate.

Tyrosinase activity in three types of the malignant melanoma: superficial spreading melanoma, lentigo maligna melanoma and nodular melanoma.

Quantitative differences in the tyrosinase activity are found at the three types of malignant melanoma of Clark and Mihm by the combined 3,4-dihydroxyphenylalanine-premelanin-reaction. Only a very small activity is present in the junction nevus. In the superficial spreading melanoma the tyrosinase activity is clear, but limited. The lentigo maligna melanoma shows an increased pigmentation. The topmost activity after incubation however is present in the nodular melanoma.

Inhibition of potassium uptake by low concentrations of norepinephrine and dibutyryl cyclic AMP.

(1) The inhibition of potassium uptake by low concentration of norepinephrine (3 X 10-8 M) and of dibutyryl cyclic AMP (DBcAMP, 10 minus5 M) was studied in cardiac Purkynĕ fibres. (2) The inhibitory action of DBcAMP on K uptake was abolished by the alpha blocker phentolamine. (3) Norepinephrine alone decreased K uptake and such inhibition was somewhat larger when DBcAMP was added. DBcAMP alone caused the usual decrease in K uptake but addition of norepinephrine abolished it. (4) The inhibition caused by norepinephrine reduced the increase in uptake caused by a high concentration (10 minus 3 M) of DBcAMP. (5) The inhibitory effect of norepinephrine was reversed in the presence of high concentration of magnesium (5.25 mM). (6) The inhibitory effect of norepinephrine was reversed by aminophylline and abolished by caffeine. (7) The inhibitory action of norepinephrine and BCcAMP was reversed or abolished, respectively, by imidazole. (8) It is concluded that the inhibition of potassium uptake by low concentration of DBcAMP is mediated by an alpha receptor mechanism and that possibly the "receptors" for this effect of norepinephrine and DBcAMP are located at different sites. Also it appears that DBcAMP may be acting at the membrane and that the action of methylxanthines and imidazole is not necessarily mediated only by a modification of phosphodiesterase activity.

[Bleomycin effect on DNA replication and proliferation kinetics using Ehrlich ascites tumor as a model].

The effect of the cytostatic agent bleomycin isolated from Streptomyces verticillus on DNA-synthesis and cell cycle of hyperidiploid Ehrlich-Lettré ascites tumor was studied in vivo and in vitro by means of liquid scintillation counting and pulse cytophotometry. A highly significant and dose dependent inhibition of 3H-thymidine incorporation into the tumor cells in vitro could be found. This inhibition of DNA-synthesis results in vivo in an initial decrease of cells in the S and (G9 plus M) phase in the DNA histogram. A partial synchronization of the tumor can be shown using 12.5, 25.0 and 50.0 mg/kg body weight of bleomycin. At a dose of 12.5 mg/kg body weight bleomycin, two subpopulations which pass through the cell cycle partially synchronized at a time interval of four hours, can be discriminated. This effect declines after the passage through one cell cycle. After 120 h most of the cells are in G1-area, another compartment in the late S-area and the smallest compartment in the (G2 plus M)-area of the DNA histogram. The arrest in the late S-area probably is the consequence of damage to the DNA synthetizing apparatus: DNA-synthesis cannot be completed and G2 cannot be reached. The significance of investigations concerning proliferation kinetics for tumor therapy is pointed out.

Studies on mouse Moloney virus induced tumours: I. The detection of p30 as a cytotoxic target on murine Moloney leukaemic spleen cells, and on an in vitro Moloney sarcoma line by antibody mediated cytotoxicity.

Antigenic determinants of p30, the most abundant internal virion protein of C type RNA viruses, were detected on the surface of spleen cells from mice bearing Moloney leukaemia and on an in vitro line of Moloney sarcoma, MSC. On both cell types, these determinants on the p30 molecules served as cytotoxic targets in a xenogenic complement dependent antibody mediated 51Cr release assay. Two antisera were used: a rat anti MLV -M induced lymphoma serum, and an antiserum raised in goats to either disrupted FeLV. The cytotoxic target antigens of these antisera were analysed by inhibition of cytotoxicity with viral and cellular proteins.

Studies on the cerebellar projections from the main and external cuneate nuclei in the cat by means of retrograde axonal transport of horseradish peroxidase.

The cerebellar projections from the main and external cuneate nuclei in the cat have been studied by means of retrograde axonal transport of horseradish peroxidase. The main projection from the external cuneate nucleus (ECN) is to the intermediate and, possibly, the small lateral part of lobule V and to the paramedian lobule on the ipsilateral side. The projection from the ECN to the cerebellar regions mentioned is topographically organized. Cells in the caudal part of the ECN send their axons to the caudal parts of lobule V and to the rostral part of the paramedian lobule. Cells in the rostral part of the ECN project to the rostralmost part of lobule V and to the folia in the caudal part of the paramedian lobule. The experimental study also shows that cells in the main cuneate nucleus (MCN) send their axons to the cerebellum. These axons, like those from the ECN, terminate in the intermediate part of lobule V of the anterior lobe and in the paramedian lobule. However, the axons of the cells in the MCN terminate only in the superficial parts of the folia, whereas those from the ECN terminate in the depth of the folia in these two cerebellar areas. The present study also gives evidence that cells in the ventral part of the gracile nucleus send their axons to lobules I and II of the anterior lobe vermis. The observations referred to here are to our knowledge the first anatomical findings demonstrating a projection from the main cuneate and gracile nuclei onto the cerebellar cortex. The observations confirm previous physiological studies.

The role of chemotherapy in the management of cancer of the head and neck: a review.

Although experience with drug therapy of advanced or recurrent squamous cell carcinoma of the head and neck is limited, several agents have produced convincing and reproducible tumor regression in these patients. Methotrexate has had the widest usage, and produces 30-50% response rates; bleomycin, hydroxyurea, and adriamycin appear to be somewhat less effective. Location of the malignancy and previous x-ray treatment appear to be important determinants of responsiveness to methotrexate, while degree of differentiation has not yet been shown to be an important factor for response to this drug. Attempts to improve the response rate and duration of chemotherapeutic response by utilizing combinations of drugs, or use of drugs to sensitize the tumor to x-ray treatment, or to reduce the bulk of tumor before x-ray treatment, are reviewed; they have been only moderately encouraging. Intra-arterial chemotherapy appears to have a therapeutic advantage over intravenous treatment; however, the morbidity associated with the former approach limits its usefulness for routine usage. The use of drugs as adjuncts following surgery and/or radiation therapy or immunotherapy are newer approaches that have not been investigated sufficiently, but are promising areas for investigation.

In vitro biochemical and cytotoxicity studies with 1-beta-D-arabinofuranosylcytosine and 5-azacytidine in combination.

The effect of 1-beta-D-arabinofuranosylcytosine (ara-C) and 5-azacytidine (5-aza-C), alone and in combination, on DNA synthesis and cytotoxicity in hamster fibrosarcoma cells has been studied. After a 2-hr exposure of S-phase cells to ara-C at concentrations of 2 to 200 muM, the cells required about 4 to 6 hr to recover from inhibition of DNA synthesis. When 2 exposures to ara-C were used, maximal cytotoxicity occurred when the 2nd dose of ara-C was administered at the time when the cells recovered from the inhibition of DNA synthesis. When the S-phase cells were exposed to ara-C, the maximal killing effect of 5-aza-C occurred when this agent was administered 6 hr later, at the time when the cells had recovered from the inhibition of DNA synthesis. When S-phase cells were exposed to 5-aza-C, the maximal cell kill produced by ara-C also occurred 5 to 6 hr later. When the S-phase cells were exposed simultaneously to both ara-C and 5-aza-C, significant antagonism with respect to cytotoxicity was observed between these 2 agents. When cells in G1 were exposed to 5-aza-C, the cytotoxicity produced by ara-C on these cells when they entered S phase was additive with respect to the cytotoxicity produced by 5-aza-C exposure alone.

Endogenous primate and feline type C viruses.

1. Endogenous type C viruses have been detected in a wide variety of mammalian species. Multiple copies of related, but not identical, virogene sequences can be found in the DNA of these species. 2. The endogenous type C virogenes are subject to the pressures of natural selection, and closely related species possess related virogene sequences. These genes evolve as cellular entities diverging from one another in a manner which correlates well with taxonomic relatedness of the species. 3. The endogenous type C viruses of baboons and domestic cats are related, but they can be distinguished by biologic and immunologic criteria and by partial nucleic acid sequence homology. Virogene sequences in the DNA of Old World monkeys and domestic cats also show a degree of relatedness not shared by the unique sequence DNA of these species. The data suggest that progenitors of domestic cats were exogenously infected by a type C virus that also gave rise to present-day endogenous type C viruses of Old World monkeys. 4. The genomes of exogenously infectious viruses replicating in permissive host cells appear to evolve much more rapidly than endogenous virogenes which replicate as cellular genes. Laboratory strains of efficiently oncogenic type C viruses are presumed to be derived from activated endogenous viruses which have been selected for virulence and which, in certain cases, have acquired the capacity to replicate in the host's own cells. 5. The ubiquitous presence of endogenous type C viruses among vertegrates and their preservation throughout millions of years of evolution suggests that these genes express normal physiologic functions which provide a selective advantage to the species.

Interactions of platinum metals and their complexes in biological systems.

Platinum-metal oxidation catalysts are to be introduced in exhaust systems of many 1975 model-year automobiles in the U.S. to meet Clean Air Act standards. Small quantities of finely divided catalyst have been found issuing from prototype systems; platinum and palladium compounds may be found also. Although platinum exhibits a remarkable resistance to oxidation and chemical attack, it reacts chemically under some conditions producing coordination complex compounds. Palladium reacts more readily than platinum. Some platinum-metal complexes interact with biological systems as bacteriostatic, bacteriocidal, viricidal, and immunosuppressive agents. Workers chronically exposed to platinum complexes often develop asthma-like respiratory distress and skin reactions called platinosis. Platinum complexes used alone and in combination therapy with other drugs have recently emerged as effective agents in cancer chemotherapy. Understanding toxic and favorable interactions of metal species with living organisms requires basic information on quantities and chemical characteristics of complexes at trace concentrations in biological materials. Some basic chemical kinetic and thermodynamic data are presented to characterize the chemical behavior of the complex cis-[Pt(NH3)2Cl2] used therapeutically. A brief discussion of platinum at manogram levels in biological tissue is discussed.

Studies on the toxicity of lindane on Colisa fasciatus (part I: TLm measurements and histopathological changes in certain tissues).

1. In the present investigation static bioassay studies and histopathological changes in some tissues (gills, liver and kidney) induced by Lindane 20% E.C. in the fish Colisa fasciatus have been taken under consideration. 2. With the help of bioassay studies, the TLm were noted. The TLm values were 0.87, 0.78, 0.68 and 0.64 mg/l for the temperature range 17 degrees C to 20 degrees C and 0.60, 0.56, 0.46 and 0.41 mg/l for the temperature range 32 degrees C to 35 degrees C at 12, 24, 48 and 96 h respectively. 3. The statistical analysis of different factors such as concentrations, time and temperature range based on the observed data gave the following results: a) At both temperature ranges, survival rates at different concentrations and time are significant at 1% and 5% level. b) The difference in survival rates at different temperature range are insignificant both at 1% and 5pected survival numbers. The standard errors were also calculated for these two lines which gave the insignificant results and there was no much change in the shape of experimental and expected lines. 4. The histopathological changes in the tissues (gills, liver and kidney) shows the following results: a) In the case of the gills there was a loss of different types of cells i.e. respiratory cells and blood cells. Blood vessels were atrophied and the outer membrane of gill lamellae was ruptured. The erosion of the tips of the gill filaments were also observed. b) In the case of liver the outer membrane was ruptured. Some hepatic cells were vacuolated and some completely degenerated. The central areas were much affected. Due to the toxic effect, large splitting of tissues were found inside the liver. c) In the case of kidney the outer epithelium, the parenchymatous cells of renal tubules, cuboidal cells of uriniferous tubules were much affected. Due to the degeneration of the cells large spaces were formed inside the tissue. Much affection was observed in the central areas.

[Liberation of anti-heparin activity during platelet aggregation in patients with blastic leukosis and blastic crisis of chronic myelosis].

The activity of platelet factor 4 (FP4) was examined in 37 patients affected with blastic leukaemia, in 16 patients with blastic crisis in chronic myeloid leukaemia and in 2 patients with Willebrand's syndrome. The real activity of FP4 determined by modifying the method according to NIEWIAROWSKI after a complete lysis of granular membrances by means of triton X-100 was found to be lowered in 9 patients affected with blastic leukaemia, in 5 patients with blastic crisis and in two patients with Willebrand's syndrome. Presuming that a maximal FP4 release of the irreversible platelet aggregation must be obtained, which corresponds to the real activity, the author has examined the apparent activity of FP4 released from the aggregated platelets with the help of her own method. In this way the quality of the release reaction from the platelets can indirectly be characterized with their extremely important role for haemostasis. Whereas in exacerbated myeloid leukaemia and Willebrand's syndrome the apparent activity will correspond to the real one, there is a severe specific disturbance of the platelet release response in blastic leukaemia. The platelet aggregation is incomplete caused by the derailment of the energy metabolism and the disturbance of the adenine nucleotides, thus causing an apparent as well as a real FP4 deficiency which can be brought into the same line with pathogenesis of thrombopathy in blastic leukaemia.

Inhibition of IgE and compound 48/80-induced histamine release by lectins.

Lectins from Ricinus communis and Glycine max, as well as wheat germ agglutinin and concanavalin A, caused a dose-dependent release of histamine from mast cells present in the mixed peritoneal cells from the rat. In addition, histamine release in an IgE-mediated and a compound 48/80-mediated reaction was inhibited in cells which had been pretreated with these lectins. With concanavalin A and the R. communis lectin both effect were prevented by the addition of the appropriate monosaccharides to the incubations. However, the lectin-induced histamine release and the lectin-induced inhibition of subsequent IgE-mediated histamine release could be dissociated: thus L-rhamnose, a hexose not ordinarily found on mammalian cell membranes, a specifically inhibited histamine release which was caused by the lectin from R. communis without affecting the inhibition of IgE-mediated histamine release. Conversely, D-fucose, which also is not a constituent of cell membrane glycolipids or glycoproteins prevented the inhibition of IgE-mediated histamine release by this lectin without affecting the lectin-induced histamine release. Furthermore, the nominally galactose-specific lectins from Sophora japonica and Ulex europeus inhibited IgE-mediated histamine release while causing little if any histamine release themselves. High concentrations of the lectin from Lotus tetragonolobus failed to cause histamine release or to affect the IgE-mediated histamine release reaction. Based on the known structural specificity of these lectins and the amounts of the lectins which were required to demonstrate an effect, it was concluded that D-galactose, alpha-linked, intrachain D-glucose (or mannose), and N-acetylglucosamine residues but probably not N-acetyl-galactosamine or L-fucose residues in the glycolipids or glycoproteins of the mast cell membrane can play a role in the initiation of histamine release and in the desensitization of the cells to subsequent histamine release-inducing stimuli.

Stimulation of adenylate cyclase activity in retro-orbital tissue membranes by thyrotropin and an exophthalmogenic factor derived from thyrotropin.

Retro-orbital tissue membranes have been shown to have adenylate cyclase activity which can be stimulated by thyrotropin and by an exophthalmogenic factor derived from the thyrotropin molecule by partial pepsin digestion. This stimulable activity is maximal after 15 min and is optimal in the presence of 3 mM magnesium and 1.5 mM ATP. Calcium salts are exquisitely inhibitory to the hormonal stimulation; sodium, lithium, and ammonium salts are significantly less inhibitory. Thyrotropin and the exophthalmogenic factor induce similar maximal levels of stimulation but a 4- to 5-fold higher concentration of exophthalmogenic factor is required to achieve this level. Fluoride stimulates adenylate cyclase activity 2- to 3-fold higher than either thyrotropin or the exophthalmogenic factor; thyrotropin, luteinizing hormone, the beta subunit of thyrotropin, and the alpha subunit of thyrotropin have relative activities for stimulation of cyclase activity of 100:2:2 less than 0.5. Several other polypeptide and glycoprotein hormones have no effect. The gamma-globulin from patients with malignant exophthalmos has no significant effect on cyclase activity either alone or in the presence of maximal levels of thyrotropin or the exophthalmogenic factor; this gamma-globulin does, however, stimulate cyclase activity at submaximal hormone levels. Trypsin not only destroys the hormone-stimulable adenylate cyclase activity on retro-orbital tissue plasma membranes, but also destroys it on the 15,000 to 30,000 molecular weight receptor fragment released from the membranes by the tryptic action.

Selective subcellular localization of cations with variants of the potassium (pyro)antimonate technique.

Fixation of rat parotid with an unbuffered osmium tetroxide solution containing nearly saturated potassium (pyro)antimonate resulted in abundant deposition of cation-antimonate precipatates in acinar cells. Altering the antimonate concentration, including buffers or chelators in the solution or changing the primary fixative resulted in an altered intensity and distribution of the precipitates formed in the tissue, apparently reflecting a degree of selectivity in ion localization. Decreasing the concentration of pyroantimonate to about half-saturation preserved predominantly the less soluble antimonate salts (e.g., Na+, Ca++) and resulted in preferential retention of deposits along the plasmalemma and in mitochondrial "dense bodies," with loss of most cytoplasmic and nuclear precipitates. A similar pattern was seen if fixation with the high concentration antimonate-osmium procedure was followed by a prolonged rinse. Adding phosphate or collidine buffers markedly decreased precipitates in the nuclei and on granular reticulum as well. Phosphate buffer or ehtyleneglycoltetraacetate inhibited in vitro precipitation of calcium and sodium and decreased or abolished plasmalemmal deposits. Glutaraldehyde fixation, either in the presence of antimonate or prior to antimonate-containing osmium tetroxide, abolished heterochromatin deposits. Mitochondrial dense bodies were of two types, one containing precipitate and the other inherently osmiophilic. The latter were also observed in pyrophosphate-osmium controls. Results from in vitro titrations of cations with the various antimonate methods and from neutron activation analyses of fixed tissues supported conclusions drawn from fine structural distribution patterns and were interpreted as follows. In rat parotid acinar cells, deposits in heterochromatin and on granular reticulum probably arose from precipitation in sites of high K+ and H+ as well as--NH3+-rich histones. Plasmalemmal antimonate deposits demonstrated sites of sodium and/or calcium accumulation. Some mitochondrial dense bodies contained Ca++ whereas others were inherently osmiophilic. Large, extracellular deposits were probably predominantly sodium precipitates.

[Subacute myelo-optic-neuropathy (S.M.O.N.) following treatment with clioquinol (author's transl)].

2 patients, who were treated with clioquinol after radical resection of carcinoma of the rectum and colostomy, developed symmetrical sensorimotor polyneuropathy, mild posterior tract ataxia, bilateral pyramidal tract lesions and optic neuropathy, a clinical picture compatible with subacute myelo-optic-neuropathy (S.M.O.N.). One patient had neurological symptoms after having received 750 g of clioquinol, 3 years after treatment started, and impairment of vision was noted after having received 1200 g. The other patient had neurological symptoms 6 weeks after clioquinol was first given, having received 65 g, the average daily dose being 1.5 g, and vision was impaired after 765 g had been administered. On examination 12 and 14 months after clioquinol had been discontinued, the first patient's vision was slightly improved, but he was otherwise unchanged, while the vision of the other patient was unchanged, but she had otherwise deteriorated slightly neurologically. Electrophysiological examinations confirmed the clinical observations. A multifactor etiology of the syndrome: neurotoxicity of clioquinol, paraneoplastic neuropathy and malabsorption, is discussed.

DC potentials of temporal lobe seizures in the monkey.

In 8 monkeys, made epileptic by alum or penicillin injection into temporal lobe structures, 40 seizures were studied by both DC cortical potential and subcortical EEG recordings. Eighteen seizures of lateral temporal origin had an abrupt negative DC potential shift of 0.5 to 2.0 mV in and around the focus. The frontal, parietal and occipital cortices did not develop DC potential changes, perhaps due to the limited propagation of the neocortical seizures. Twenty-two seizures of medial temporal origin showed a negative shift of the anterior, inferior or lateral temporal cortex in 85% of seizures. The other 15% had a positive or no shift. In hippocampal seizures, a positive displacement was sometimes seen prior to the main negative shift in the lateral temporal cortex. The remote cortex developed only a minimal positive shift in 30% of the mediotemporal seizures. A marked negative shift in the frontocentral cortex was the first sign of impending generalization, which may result from a series of chain reactions with seizure propagation, involving more and more structures of the brain. Registration of DC potentials in temporal lobe seizures may give insight into the nature of abnormal EEG activities and to some extent into the origin of seizures.

The internal auditory artery: (embryology, anatomy, angiography, pathology).

A review of the literature on the embryology, anatomy and angiography of the internal auditory artery has shown that there may be considerable variation as to the origin and number of internal auditory arteries. The present study, based on serial magnification angiographies of the internal auditory artery, has demonstrated 7 variants of the origin of this artery although in 45.4% of cases the internal auditory artery arose from the anterior inferior cerebellar artery. For the diagnosis of pathological processes in the cerebellopontine angle (tumors, sudden deafness, vascular abnormalities) magnification angiography is of special importance. Acoustic neurinomas in particular can be diagnosed early on the basis of vascular dislocations and possible tumor staining. Magnification angiography is the method of choice for the demonstration of vascular malformations. The etiology of sudden deafness can not be determined by angiographic studies alone, due to the fact that the internal auditory artery can show considerable variations as regards length and caliber. Magnification angiography of this region should be carried out in all cases with the clinical picture of a cerebellopontine angle lesion in order to achieve diagnostic clarification.

[The diagnostic value of the F wave latency (author's transl)].

The N. medianus was stimulated electrically in 44 healthy persons and in 30 unselected diabetics. Latencies of the F wave were measured, corrected for different lengths of the upper extremity, and used for computation of nerve conduction velocity. In comparison with conventional motor nerve conduction velocities the F wave latencies showed a more pronounced difference between the normal and the pathological (diabetic) range. The F wave nerve conduction velocities also varied less than conventional nerve conduction velocities. The explanation for this might be that the F wave recordings are the result of a full anti- and orthodromic impulse propagation which travels 5 to 6 times further than the impulse measured with conventional nerve conduction velocity methods. Therefore small delays of nerve conduction will be amplified and can be better recognized by F wave latency measurements. The present findings suggest that F wave latencies may be more sensitive than conventional nerve conduction velocities in evaluating mild cases and in identifying the initial phases of neuropathies.

[The problem of early diagnosis of brain tumours causing seizures only (author's transl)].

The diagnosis of brain tumour could not be made in 91 cases at the first investigation in a group of 1155 brain tumours. Slowly growing gliomas causing only epileptic fits and no other symptoms are especially difficult to diagnose. Of 21 personal observations of tumour seizures, in which the diagnosis of the neoplasm was missed at the first investigation in hospital, 9 were oligodendrogliomas, 5 astrocytomas, 3 glioblastomas, 2 spongioblastomas, 1 gangliocytoma and 1 a metastasis. They were all located in the frontal or centroparietal region. In most cases the seizures appeared during the third or fourth decade. The average interval between the first epileptic fit and the tumour diagnosis was 8.2 years in cases of oligodendrogliomas and 2.2 years in astrocytomas. 5 patients had major seizures, 2 had psychomotor attacks and all the others suffered from partial epilepsy. Anticonvulsive therapy was often successfull; either the frequency of the fits diminished or, in 2 cases, the character of the seizures changed. 18 patients had a normal neurostatus at time of the first investigation. Only 3 patients had a slight difference of physiological reflexes, but no other pathological signs. In none of the patients did investigation of the CSF, skull X-rays, brain scanning, pneumencephalography or cerebral angiography first lead to the diagnosis of a brain tumour. The EEG alone showed focal signs corresponding to the location of the tumour in about 50% of the cases.

Effects of bleomycin on mouse bone-marrow stem cells.

The mouse hematopoietic stem-cell population was tested by the spleen colony technique for effects of the antineoplastic agent bleomycin (BLM). The time response of normal bone marrow was investigated by a single dose of BLM (400 mg/kg) between 0 and 72 hours. The dose response was studied over a wide range of doses (from 40 to 1,600 mg/kg) at a 4-hour exposure. Additional experiments concerned 1) the fraction of colony-forming units in the S phase after BLM administration (by means of pulse hydroxyurea treatment), 2) the response of bone marrow stimulated by endotoxin, and 3) the effects of split-dose treatments. The relatively low toxicity of BLM on both the differentiated and stem-cell populations of unstimulated bone marrow was confirmed and detailed. This drug exhibited peculiar, proliferation-dependent cell inactivation kinetics. Furthermore, BLM induced parasynchronous behavior in the unstimulated stem-cell population. The various aspects of BLM action are discussed with regard to its use in cancer chemotherapy.

Immunoprophylaxis and cytotoxic effector cells against EL 4 leukemia induced in syngeneic C57BL/6J mice by use of irradiated EL 4 cells.

Tumor-specific immunoprophylaxis was achieved in C57BL/6J mice against EL 4 leukosis cell challenge by sensitization of the syngeneic host with multiple ip injections of irradiated EL 4 cells. A minimal radiation dose was used to replication-block EL 4 cells before inoculation, as defined by dose-response analysis of irradiated EL 4 cells. Multiple ip injections of irradiated EL 4 cells stimulated development of significant, yet relatively low, levels of cytotoxic lymphoid activity (CLA) in lymphoid cells of the peritoneal exudate as measured by in vitro 51Cr-release cytotoxicity assays. The specific temporal and frequency dependencies of the inoculation regimen for achieving immunoprophylaxis indicated that, in addition to CLA, other, short-lived, immune processes were important in the tumor rejection. These observations showed the capacity of the C57BL/6J host for tumor-specific immune recognition and rejection of the syngeneic EL 4 leukemia. The tumor rejection could be elicited solely by inoculations of irradiated EL 4 cells and did not require exogenous amplifiers, such as immunoadjuvants, chemical modifiers, and/or allogeneic immune information transfer.

Interferon-directed inhibition of chronic murine leukemia virus production in cell cultures: lack of effect on intracellular viral markers.

Extracellular murine leukemia virus (MLV) reverse transcriptase activity was decreased by interferon treatment in four interferon-sensitive mouse cell lines which were chronic MLV producers. In three cell lines which were relatively insensitive to interferon, extracellular enzyme activity remained unchanged by interferon treatment. The concentrations of interferon used had no effect on DNA synthesis or cell replication of AKR,C+ cells which were chronic producers of AKR-MLV. In AKR,C+ cultures interferon treatment also had no effect on the level of intracellular viral reverse transcriptase activity in spite of an inhibition of extracellular enzyme activity. Treatment of AKRC+ cultures with interferon for 9 days inhibited extracellular viral reverse transcriptase levels throughout the period of treatment; however, the intracellular enzyme activity remained unchanged, and concentrations of viral p30 (gs) antigen were increased in the interferon-treated cells. When the cells were washed to remove interferon, however, virus production rapidly rose and intracellular p30 antigen fell to the levels of untreated AKR,C+ cells. These and previously reported results suggested that in interferon-treated AKR,C+ cells virus production is inhibited at a late step in the MLV replication cycle, either directly or through the inhibition of the production of a protein required for virus assembly.

Isolation and electrophoretic study on Mallory bodies from the livers of alcoholic cirrhosis.

Mallory bodies (MBs) were isolated from the livers of eight alcoholic patients. The method consisted of homogenization, velocity sedimentation in 60 per cent sucrose solution and two-phase polymer centrifugation using a polyethylene glycol-dextran system. MBs were isolated in large quantity and high purity (more than 95 per cent in some cases). Isolated MBs maintained the characteristic filamentous structures. They were solubilized in 6 M guanidine hydrochloride in 10 per cent or 1 per cent acetic acid by sonication or in 1 per cent sodium dodecyl sulfate with 5 per cent 2-mercaptoethanol and 8 M urea. The solubilized MB protein produced three peaks on Sephadex G-100 chromatography and at least five intense protein bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The fractions from control livers contained several bands which were identical with the major components of isolated MB protein. These findings suggest that MBs are not homogeneous protein, but that they are probably complexes of various proteins, and that some components of MBs are present in normal hepatocytes. The exact nature of MBs has yet to be established.

Renin-angiotensin system in essential hypertension.

In a random sample of normotensive and hypertensive fifty-year-old men plasma-renin-activity (P.R.A.), plasma-renin-concentration (P.R.C.), and renin substrate were measured using radioimmunoassay for angiotensin I. P.R.A. in normotensives and untreated hypertensives were normally distributed with slight skewness to the right. The mean P.R.A. for untreated hypertensives (0.65 ng. per ml. per hour) was slightly, but not significantly, lower than that of the normotensive reference group (0.78 ng. per ml. per hour). Previously untreated hypertensives who had been off treatment for four weeks had either high or low P.R.A. depending on the previous treatment. No differences in the angiotensin-generation rate were noted as judged from the P.R.A./P.R.C. ratio. No differences in the renin-substrate concentration between the groups were found. The findings suggest that renin changes in essential hypertenion are secondary to pressure changes. Thus, the renin-angiotensin system may not be of primary pathogenetic importance in the development of essential hypertension.

Hyperuricaemic acute renal failure after epileptic seizures.

Seven patients admitted to hospital during or immediately after status epilepticus or recurrent episodes of grand-mal seizures had very high concentrations of uric acid in their blood at a time when the blood-urea was normal in five of them. The blood-lactic-acid was high in the five patients in whom it was measured. All of the patients developed reversible renal failure, and two required haemodialysis. The blood-uric-acid should be measured in patients who have had prolonged seizures, and the measures which might be taken in hyperuricaemic patients to prevent the development of acute renal failure include rehydration, alkalinisation of urine, and, where alkalinisation is impossible, haemodialysis.

Inhaled corticosteroids compared with oral prednisone in patients starting long-term corticosteroid therapy for asthma. A controlled trial by the British Thoracic and Tuberculosis Association.

Inhaled beclomethasone dipropionate and inhaled betamethasone valerate have been compared with oral prednisone in the treatment of 75 patients with asthma who were starting long-term corticosteroids for the first time. Both of the inhaled corticosteroids controlled asthma as well as did oral prednisone in those who had responded to therapy in the initial period of the trial. A daily dose of 400 mug of inhaled drug was approximately equivalent to 7-5 mg daily of prednisone. Prednisone suppressed the adrenal response to tetracosactrin, whereas the mean responses in the groups receiving inhaled corticosteroids did not change significantly from pre-trial values. The 30% incidence of other systemic unwanted effects of prednisone contrasted sharply with the low incidence (5%) of symptomatic oropharyngeal candidiasis in the patients receiving inhaled corticosteroids. In a sample of 19 patients no change in exfoliative cytology was detected over the period of the trial nor was there any evidence of fungal colonisation of the bronchial tree. There was no difference between the three treatment groups in the number of antibiotic courses prescribed. The persistent production of sputum made no difference to the response to inhaled corticosteroids. Patients not on sodium cromoglycate did as well in the trial as those receiving sodium cromoglycate. Both inhaled beclomethasone dipropionate and inhaled betamethasone valerate have advantages over oral prednisone in the maintenance treatment of patients with asthma, but in the management of exacerbations systemic corticosteroids will usually be needed as a supplement to inhaled therapy.

Thyroid function in the long-term follow-up of patients treated with iodine-131 for thyrotoxicosis.

In February, 1972, 58% of patients euthyroid after iodine-131 therapy for thyrotoxicosis between 1954 and 1966 had a raised plasma thyroid-stimulating-hormone (T.S.H.) (greater than 7-4 mU/l) and 42% a normal T.S.H. level. A group of 69 of the euthyroid patients with a raised plasma T.S.H. (25-0 +/- 2-0 mU/l) in 1972 was re-examined annually for three years. There was no apparent change in the mean plasma T.S.H. level between 1972 and 1975 in the patients remaining euthyroid, but overt hypothyroidism developed in 3 patients in 1973, in a further 3 patients in 1974, and in 1 patient in 1975. In contrast, none of a group of 61 patients, euthyroid with a normal plasma T.S.H. (4-0 +/- 0-2 mU/l) in 1972, developed overt hypothyroidism over the next three years, although slightly raised T.S.H. levels were recorded in 3 patients in 1974 and in a further 6 patients in 1975. Both the mean serum T-4 and T-3 in the euthyroid patients with a raised plasma T.S.H. were significantly lower, but still in the respective normal ranges, than those in the euthyroid patients with a normal plasma T.S.H. No significant difference in the fasting serum-cholesterol or triglyceride levels could be demonstrated between the two groups. Since no patient with a normal plasma T.S.H. after iodine-131 treatment for thyrotoxicosis six to eighteen years earlier developed overt hypothyroidism over a three-year period, the follow-up of such patients need not be so frequent as that of similarly treated euthyroid patients with a raised plasma T.S.H. in whom overt hypothyroidism develops at the rate of 2-5% per year.

Biodegradation of alpha-hexachlorocyclohexane. V. Characterization of the major urinary metabolites.

1. Free 2,4,5- and 2,4,6-trichlorophenol (TCP) have been identified by co-crystallization with authentic carrier as constituents of the urine of rats given 3-H-labelled alpha-hexachlorocyclohexane (alpha-HCH). The two TCP's constituted 7% and 63%, respectively, of the free phenol fraction which was found to account for less than 5% of all urinary metabolites. 2. 75% of the label contained in the urine of rats collected for four weeks after an i.p. dose of 14-C-alpha-HCH could be extracted into an organic solvent after alkaline and acid hydrolysis. The radioactive material thus extracted was examined by TLC and GLC. It is shown to consist mainly of chlorophenols and chlorothiophenols. 3. The extracts' major constituent was 2,4,6-TCP. Its amount was determined by GLC and was found to account for, on average, 45% of the total urinary metabolites. In conjunction with other evidence, this is considered to establish 2,4,6-TCP as the major product of alpha-HCH-biodegradation in the rat. 4. Chromatographic evidence is presented for 2,3,4,5-tetrachlorophenol and 2,4,5-TCP being other components of the chlorophenol fraction isolated from hydrolysed urine. 5. The amount of label associated with the chlorothiophenol fraction suggests a pathway involving endogenous thiol to be of significance in alpha-HCH-biodegradation in rats. Pretreatment with the drug itself increased the proportion of label associated with the fraction.

[Drug therapy of myocardial infarct in ambulatory practice].

For the practicing physician the medicamentous treatment of the patients with infarction is the main problem of the secondary prevention in the prehospital phase as well as in the after-treatment. In these cases in the acute phase not the myocardial insufficiency is in the centre of the out-patient care, but the therapy of the disturbances of cardiac rhythm, which mainly cause the high lethality in the early phase. Therefore, uncomplicated infarctions, in whch care must be taken only for a sedation of sympathico-adrenergic reactions and a volume reduction of the heart, should be differed from complicated cases. However, an immediate transport to the hospital must be guaranteed. If there appear a contraction insufficiency of the left ventricle or threatening disturbances of the rhythm, additionally glycosides and saluretics must be administered as well as an aimed antiarrhythmic therapy must be initiated. The necessary medicamentous measures are described dependent upon the diagnosis of brady- and tachycardiac disturbances of the rhythm. The author enters briefly the problems of volume substitution, treatment of acidosis as well as the administration of beta-sympathicolytics and gluco-corticoids. - In the after-treatment of infarctions anticoagulants are the only medicaments to be prescribed, when findings completely without complications are present. If, however, there are signs of activity of the coronary heart disease in the post-infarction phase, a basic therapy with a glycoside and anticoagulants as well as an individually to be varied additive therapy with nitro-preparations, beta-sympathicolytics, saluretics, anti-hypertensive agents and antiarrhythmic agents are necessary.

[Sensitization against the antigens of the brain after experimental vaccinia infection. II. Humoral anti-brain antibodies and morphological changes in the CNS (author's transl)].

29 guinea pigs, strain Pirbright, were infected with vaccinia virus, strain Elstree, by the dermal route. The observation period was 14 days. Thereafter, the animals were killed and their central nervous systems (CNS) histologically and immunohistologically, the blood fluorescence-serologically examined. Histological examination revealed meningitis, ependymitis or disseminated meningoencephalitis with slight perivascular cuffing in 72% of the animals. The viral antigen was found in 3 animals (10%). It was present most often in the cytoplasma of the arachnoidal and/or ependymal cells, as well as in the cells of the vessel walls and less often in the glial and/or nerve cells. The infected cells showed no severe degenerative changes. The blood-brain-barrier displayed localized disturbances. The examination of the myelin sheaths revealed disseminated foci of disappearance of myelin fluorescence in the perivascular, paraventricular and subcortical regions. Antibodies directed against myelin sheaths, or nerve cells could be detected in the sera of 48% of the animals. The results give evidence that the vaccinia infection is capable to induce a potentially pathogenic autoimmune reaction directed against brain. Such an immunomechanism can be triggered without any signs of acute lytic infection of the CNS. The mechanism and significance of this reaction are discussed.