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Possible association between mutant frequency in peripheral lymphocytes and domestic radon concentrations. To investigate whether previously found geographical correlations between leukaemia incidence and exposure to radon are reflected in a detectable mutagenic effect on individuals, the frequency of mutations in the hypoxanthine guanine phosphoribosyl transferase gene (hprt) in peripheral blood T lymphocytes was measured in subjects with known domestic radon concentrations. These concentrations were measured in December, 1989, in houses in Street, Somerset, UK, by passive alpha-track radon detectors. 20 non-smoking subjects aged 36-55 years were selected from the patient list at the local health centre on the basis of the radon concentrations in their homes--the range selected varied by a factor of ten. Blood samples for preparation of T lymphocytes were taken in July, 1990. There was a significant association between the log mutant frequency and radon concentration (t = 3.47, p less than 0.01). A second analysis of a further set of radon measurements (October, 1990, to January, 1991), in both living rooms and bedrooms, and repeated mutant frequency determinations also showed a significant relation, which remained significant even after exclusion of the highest frequency and adjustment for subject's age and cloning efficiency. These data must be regarded as preliminary and further more extensive studies should be done to determine whether the observed association is causal.

Chromosome translocation and c-MYC activation by Epstein-Barr virus and Euphorbia tirucalli in B lymphocytes. Dual exposure to Epstein-Barr virus and purified 4-deoxyphorbol ester derived from the plant Euphorbia tirucalli induced a high frequency of chromosomal rearrangements in human B lymphocytes in vitro. Rearrangements most commonly affected chromosome 8, the chromosome most often showing structural changes in Burkitt's lymphoma (BL) cells. E tirucalli is indigenous in parts of Africa where BL is endemic and may be an important risk factor for the disease.

Dietary effects on breast-cancer risk in Singapore. It is suspected that diet influences the risk of getting breast cancer. A study of diet and breast cancer was done among 200 Singapore Chinese women with histologically confirmed disease and 420 matched controls. A quantitative food-frequency questionnaire was used to assess intakes of selected nutrients and foods 1 year before interview. Daily intakes were computed and risk analysed after adjustment for concomitant risk factors. In premenopausal women, high intakes of animal proteins and red meat were associated with increased risk. Decreased risk was associated with high intakes of polyunsaturated fatty acids (PUFA), beta-carotene, soya proteins, total soya products, a high PUFA to saturated fatty acid ratio, and a high proportion of soya to total protein. In multiple analysis, the variables which were significant after adjustment for each other were red meat (p less than 0.001) as a predisposing factor, and PUFA (p = 0.02), beta-carotene (p = 0.003), and soya protein (p = 0.02) as protective factors. The analysis of dietary variables in postmenopausal women gave uniformly non-significant results. Our finding that soya products may protect against breast cancer in younger women is of interest since these foods are rich in phyto-oestrogens.

Waterborne outbreak of Norwalk virus gastroenteritis at a southwest US resort: role of geological formations in contamination of well water. From April 17 to May 1, 1989, gastroenteritis developed in about 900 people during a visit to a new resort in Arizona, USA. Of 240 guests surveyed, 110 had a gastrointestinal illness that was significantly associated with the drinking of tap water from the resort's well (relative risk = 16.1, 95% confidence interval 14.5 to 17.8) and this risk increased significantly with the number of glasses of water consumed (p less than 0.005). Three of seven paired sera tested for antibodies to the Norwalk agent had a four-fold or greater rise in titre. Water contaminated with faecal coliforms was traced back to the deep water well, which remained contaminated even after prolonged pumping. Effluent from the resort's sewage treatment facility seeped through fractures in the subsurface rock (with little filtration) directly into the resort's deep well. Although the latest technology was used to design the resort's water and sewage treatment plants, the region's unique geological conditions posed unexpected problems that may trouble developers faced with similar subsurface geological formations and arid climatic conditions in many parts of the world. In these areas, novel solutions are needed to provide adequate facilities for the treatment of sewage and supply of pure drinking water.

[99Tcm]MAG3 gamma camera nephrography in epidemic nephritis. There is a lack of systematic nephrographic studies on epidemic nephritis (EN). We studied 10 patients with EN using [99Tcm]MAG3 gamma camera nephrography and followed up 9 of them 22-68 days later when they had clinically recovered. Variables for renal clearance of [99Tcm]MAG3 and the retention of radioactivity in the kidneys and blood were calculated. In all patients renal function was impaired acutely. There was marked reconstitution of renal function in the control studies. [99Tcm]MAG3 clearance was inversely related to serum creatinine. On visual inspection the nephrograms showed no focal changes. Nephrography was more sensitive than sonography at identifying renal impairment. [99Tcm]MAG3 nephrography is a sensitive method for identifying renal involvement and reconstitution of renal function in EN. It may be a valuable adjunct to the diagnostic arsenal, especially in nonendemic areas where EN occurs only sporadically and where there may be diagnostic uncertainty in patients presenting acutely with EN.

Flow cytometry analysis of T cell subsets in skin of patients with specific cutaneous manifestation of B non-Hodgkin lymphoma. Cutaneous lesions may be the atypical initial manifestation of blood disorder. Monoclonal antibodies identify surface phenotype of lymphocytes present in specific cutaneous lesions. We studied cutaneous infiltrates with flow cytometry in 10 cases of non-Hodgkin lymphoma. We characterized T cell subset in skin immune system. The percentage of T cells was compared to percentage observed in healthy specimens from same patient. T4 cells predominate in cutaneous infiltrates, with an increase in T4/T8 ratio. There was also an increase in T6+ cells, Langerhans cell markers in the skin. HLA-Dr+, T6 cells increased in all cases tested, indicative of dendritic cells. These data showed a modification of T lymphocytes only in skin infiltrate of lymphoma.

Research on the differentiation of human and murine neuroblastoma cells. In vitro, we were able to induce a differentiation of human (SK-N-MC, IMR-32, Leo-2) and murine neuroblastoma cells (NA-2, C-1300, NIE-115) with dibutyryl cyclic 3'5'-adenosine monophosphate (dbcAMP), hypothalamic factor (HF), and somatostatin. As morphological criteria of cellular differentiation we used the decrease in cell proliferation and the formation of neurites. Functional parameters were the increase of A cholinesterase activity, cAMP level, and protein content, and the decrease of cGMP level. After application of dbcAMP and HF, the effects were stronger than after somatostatin. We believe that the action of HF and somatostatin is caused by an increase in cAMP levels. In the in vivo experiments, human and murine neuroblastoma cells (NA-2, C-1300, and Leo-2) were transplanted into nude/nude mice. After HF treatment of 14 mice with NA-2 tumors, 4 of the mice were tumor-free, and mean tumor weight was reduced to one-third of the controls. Of the animals with C-1300 and Leo-2 tumors, half became tumor-free, and mean tumor weight was reduced to one-fourth. The results indicate that the induction of cellular differentiation by factors and hormones may in future become a method of therapy for human neuroblastoma.

[Treatment of arterial hypertension in insulin-treated diabetic patients. Change over 3 years (1985-1988)]. In 1985, an assessment of arterial hypertension treatment in insulin-treated diabetic patient gave disappointing results. In 1988, we carried out another study in order to assess the impact of new antihypertensive drugs (angiotensin converting enzyme inhibitors and calcium antagonists) on the management of arterial hypertension and to identify patients in whom strict normal blood pressure control is mandatory. Seven hundred and fifty four patients were selected. The prevalence of arterial hypertension was 38.4 p. 100 (n = 290). Two hundred and thirty five patients (31.2 p. 100) were on antihypertensive treatment: monotherapy: 60.4 p. 100 (n = 142), bitherapy: 30.6 p. 100 (n = 72), tritherapy: 9 p. 100 (n = 21). In descending order of frequency, the following drugs were used: angiotensin converting enzyme inhibitors, calcium antagonists, diuretics, cardio-selective beta-blockers, central acting agents. Blood pressure values significantly decreased (148/83 mmHg, in 1988, vs 157/85 mmHg, in 1985, p less than 0.05). However, 20 p. 100 of the patients still had blood pressure values greater than or equal to 160 and/or 95 mmHg with or without antihypertensive treatment, and on average, blood pressure values remained higher in patients with antihypertensive treatment than in those without (148/83 mmHg vs 131/77 mmHg, p less than 0.001). Patients with urinary albumin excretion above or equal 30 mg/24 h compared to those with normal albuminuria had significant higher values of blood pressure, glycosylated haemoglobin and blood lipids (p less than 0.01). Only 51 p. 100 of these patients, received an antihypertensive treatment. This study emphasizes the difficulty of antihypertensive treatment in insulin-treated diabetic patients and the necessity to improve education in patients with high risk for widespread angiopathy, and particularly those with increased urinary albumin excretion.

[Prevention of duodenal ulcer recurrence. A new prospect]. Prevention of recurrence is now the main therapeutic problem in the management of duodenal ulcer disease. It has been shown that in the absence of maintenance treatment the mean recurrence rate within 6 to 12 months from healing obtained with drugs other than H2-receptor antagonists (H2A) is lower than when healing is obtained with H2A. Possible reasons for this discrepancy are examined. When the recurrence rates observed with each of the non-H2A drugs were compared individually with those observed with H2A, the only important and constant difference was in favour of colloidal bismuth subcitrate (CBS). This beneficial effect of CBS is due to its bactericidal action on Helicobacter pylori. Unlike healing, prevention of recurrent ulcer is primarily, if not exclusively, related to eradication of this organism. Although the ideal treatment providing both healing of duodenal ulcers and eradication of Helicobacter pylori has not yet been determined (CBS administered alone eradicates this organism in only 10 to 25 percent of cases), it will soon be possible to modify dramatically the natural course of duodenal ulcer disease and even to obtain its cure.

A novel TRH analog, YM14673, stimulates intracellular signaling systems in the brain more potently than predicted by its pituitary actions. YM14673, a novel analog of TRH, substituting the (oxo-azetidinyl)carbonyl moiety for the pyroglutamyl moiety of TRH, was synthesized and its brain action was evaluated using rat brain slices in vitro. The addition of YM14673 engendered significant and dose-dependent increases in cyclic AMP formation in the hypothalamus and inositol phosphates (IP) accumulation in the cerebellum. The actions of YM14673 did not statistically differ from those of TRH on brain tissues. However, YM14673 did not show a direct interference with TRH binding in the brain membranes. [3H]YM14673 underwent to the same extent the enzymatic degradation by type II pyroglutamyl aminopeptidase as did TRH. As compared with TRH, YM14673 had a lower activity with respect to stimulation of thyrotropin secretion and accumulation of IP in the anterior pituitaries. The data imply that YM14673 possessed potent stimulatory actions on brain intracellular signaling systems with weak hypophyseal activities. These actions of YM14673 were not due to a direct interference with brain TRH binding or a nature impervious to a TRH-degrading enzyme. The present study indicates that the larger doses of YM14673 than TRH can be used for medical treatment of neurological and neuromuscular disorders without strong influences on pituitary functions.

Linkage of a cardiac arrhythmia, the long QT syndrome, and the Harvey ras-1 gene. Genetic factors contribute to heart disease. In this study, linkage analyses have been performed in a family that is predisposed to sudden death from cardiac arrhythmias, the long QT syndrome (LQT). A DNA marker at the Harvey ras-1 locus (H-ras-1) was linked to LQT with a logarithm of the likelihood ratio for linkage (lod score) of 16.44 at theta = 0, which confirms the genetic basis of this trait and localizes this gene to the short arm of chromosome 11. As no recombination was observed between LQT and H-ras-1, and there is a physiological rationale for its involvement in this disease, ras becomes a candidate for the disease locus.

Tranquillisers and health care in crisis. This paper addresses the issue of the crisis of therapeutic efficacy in Britain through a case study of benzodiazepine tranquilliser dependence. The paper traces the rise of tranquillisers and the crisis of legitimacy in prescribing behaviour in the 1980s. It documents growing concern with dependence and the claims made about it by experts and consumer groups. The paper goes on to analyse the importance of the television in these claims-making activity and its influence in shaping perceptions. Finally, we consider the implications of these events for the future of benzodiazepine tranquillisers as a form of treatment.

[Akathisia. A frequent adverse effect in treatment with neuroleptics]. Neuroleptics are employed in the treatment of psychotic states but also in the treatment of anxiety states, as anti-emetics, in the treatment of pain and for anaesthesia. In approximately 20% of the patients, side effect in the form of akathisia are observed. Akathisia caused by neuroleptics may be difficult to diagnose as the symptoms are non-specific with anxiety, restlessness and agitation. Treatment consisted previously of anticholinergics but investigations have been published which demonstrate good effects of non-specifically acting beta-receptor blocking agents. On the basis of a review of the literature, the development of akathisia caused by neuroleptics is described, the symptoms and differential diagnoses involved and the therapeutic possibilities, are described.

Laparoscopic evaluation of nonpalpable testes. Laparoscopy was utilized in the evaluation of 27 nonpalpable testes in 21 patients prior to exploration and necessary definitive surgery. Laparoscopic prediction of the location of the nonpalpable testes was accurate in all cases. Laparoscopic findings can help in determining the appropriate technique of orchiopexy expected to yield better testicular salvage. It can also prevent unnecessary exploration of children with anorchia as evidenced by blind-ending vas deferens and spermatic vessels.

The genomic diversity and stability of field strains of suid herpesvirus 1 (Aujeszky's disease virus). The genomic diversity among isolates of suid herpesvirus 1 (SHV-1) collected in the same herd and among clones from the same isolate was studied by restriction fragment pattern (RFP) analysis using BamHI. Tentatively defining a field strain as a transmissible entity, it was concluded that strains of SHV-1 commonly comprise distinguishable genomic variants. Contrary to the hypothesis of genomic lability, it is suggested that the pool of variants is sufficiently stable to specifically characterize a strain. The impact of the results on epizootiological methods based on identification of field isolates is discussed in connection with Aujeszky's disease in Denmark.

Southern blot analysis of strain variation in Campylobacter mucosalis. A panel of three DNA probes were derived at random from a genomic DNA library of Campylobacter mucosalis strain E8384-4. Each probe hybridized specifically to C. mucosalis DNA from bacteria fixed to nylon membranes. The probes did not hybridize to DNA from other Campylobacter species or to other bacteria even at 100-fold higher amounts. Each probe hybridized to all of 24 isolates of C. mucosalis which had been collected over time from different geographic locations. Southern blot analysis of selected C. mucosalis isolates was carried out to determine if the probes would be useful for differentiating among various isolates. It indicated that restriction fragment length polymorphisms (RFLPs) exist at the loci identified by our probes. These differences were used to characterize seven C. mucosalis isolates recovered from pigs in Minnesota. The results suggest that RFLP analysis may be a useful tool for epidemiological studies of C. mucosalis.

Dermal dendrocytes and photochemotherapy. We studied the fate of dermal dendrocytes in patients treated with psoralens and ultraviolet light by combining immunohistochemistry and computerized image analysis. Factor-XIIIa-positive dermal dendrocytes were found to be altered in these patients. When compared with controls, dermal dendrocytes were often increased in number and had an uneven size and tissue distribution. Their cytoplasm was occasionally fragmented. These changes were more pronounced when early photosclerosis was present. The alterations described probably reflect vascular changes, and may be responsible for immunomodulating actions and disorder of the connective tissue structure induced by ultraviolet light.

A deletion and a rearrangement distinguish between the intracisternal A-particle of Hox-2.4 and that of interleukin-3 in the same leukemic cells. Two intracisternal A-particle (IAP) insertions have been identified in WEHI-3B myeloid leukemic cells, one at the interleukin-3 (IL-3) gene and another at the homeobox gene Hox-2.4. In contrast to the 5-kb IL-3-IAP, the Hox-2.4-IAP is only 2.1 kb in size and contains a rearrangement. The homology throughout the remaining sequences suggests that both IAPs originated from a common progenitor molecule. Both proviral insertions have resulted in transcriptional activation of the adjacent genes, which appears to be a significant step in the leukemogenic process in these leukemic cells.

Infectious RNA transcripts from Ross River virus cDNA clones and the construction and characterization of defined chimeras with Sindbis virus. We have constructed a full-length cDNA clone of the virulent T48 strain of Ross River virus, a member of the alphavirus genus. Infectious RNA can be transcribed from this clone using SP6 or T7 RNA polymerase. The rescued virus has properties indistinguishable from those of the T48 strain of Ross River virus. We have used this clone, together with a full-length cDNA clone of Sindbis virus, to construct chimeric plasmids in which the 5' and the 3' nontranslated regions of the Sindbis and Ross River genomes were exchanged. The nontranslated regions of the two viral genomes differ in both size and sequence although they maintain specific conserved sequence elements. Virus was recovered from all four chimeras. Chimeras containing heterologous 3' nontranslated regions had replicative efficiencies equal to those of the parents. In contrast, the chimeras containing heterologous 5' nontranslated regions were defective in RNA synthesis and virus production, and the severity of the defect was dependent upon the host. Replication of a virus containing a heterologous 5' nontranslated region may be inefficient due to the formation of defective protein-RNA complexes, whereas, the presumptive complexes formed between host or virus proteins and the 3' nontranslated region to promote RNA synthesis appear to function normally in the chimeras.

Structure of the Ockelbo virus genome and its relationship to other Sindbis viruses. Ockelbo virus was first isolated in 1982 in Sweden. It is the causal agent of disease in humans characterized by arthritis, rash, and fever and is antigenically very closely related to Sindbis virus. We have determined the nucleotide and translated amino acid sequences of the prototype Ockelbo virus isolate (82-5) to determine the relatedness of Ockelbo virus to Sindbis virus at the genomic level and clarify the taxonomic position of Ockelbo virus within the alphavirus genus. The numbers of nucleotides and of translated amino acids in each region of the Ockelbo virus genome were exactly the same as those for the prototype AR339 strain of Sindbis virus except for three small deletions and insertions in the C-terminal half of nsP3 and for three single nucleotide insertions and deletions in the 3' untranslated region. Overall there were 672 nucleotide differences (5.7% divergence), resulting in 97 amino acid changes (2.6% divergence), between the two viruses: more than 85% of the nucleotide changes were silent. Only the C-terminal domain of nsP3 and the E2 glycoprotein showed a higher degree of amino acid substitution than the overall average. The former domain is not conserved among alphaviruses, and the latter is primarily responsible for antigenic variation. Sequence analysis of 420 nucleotides at the 3' end of a number of other Sindbis-like alphaviruses, including Karelian fever virus and South African, Indian, and Australian isolates of Sindbis virus, demonstrated that Ockelbo virus is more closely related to South African strains of Sindbis virus than it is to the prototypic Egyptian AR339 strain. Thus the South African strains, which have caused epidemic disease in humans, may have been introduced into Northern Europe by man or by migratory birds to establish Ockelbo disease there. The Indian and Australian strains form a distinct branch of the evolutionary tree and differ from prototypic AR339 Sindbis virus in 17% of the nucleotides sequenced.

[Effect of sulfasalazine and its metabolites on prostaglandin and leukotriene liberation from human synovial tissue]. The effects of sulfasalazine (SASP) and its metabolites sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA) were investigated on release of prostaglandins (PG) and leukotrienes (LT) from synovial tissue of 37 patients with osteoarthritis, chondrocalcinosis and rheumatoid arthritis. Calcium ionophore A23187 significantly increased the release of PGE2, 6-keto-PGF1 alpha, LTB4, and LTC4 from human synovial tissue irrespective of the underlying joint disease. SASP inhibited release of LTC4 and increased release of PGE2. On the other hand, 5-ASA and SP inhibited the release of all eicosanoids measured. The effective concentrations of SASP and SP were found to be in the range which can be reached during SASP therapy. On the other hand, blood and synovial fluid levels of 5-ASA are considerably lower than those which inhibit eicosanoid synthesis in vitro. While nonsteroidal anti-inflammatory drugs, which are used for symptomatic therapy of rheumatoid arthritis, inhibit cyclooxygenase only, SP, the active metabolite of the second line anti-rheumatic drug SASP, inhibits both PG and LT release. Inhibition of LT synthesis by SASP and SP could contribute to the second line efficacy of SASP therapy in rheumatoid arthritis.

Certain mutations observed in the 5' sequences of the G gamma- and A gamma-globin genes of beta S chromosomes are specific for chromosomes with major haplotypes. In this paper we describe the distribution of some specific sequence differences in the 5' flanking regions of the A gamma- and G gamma-globin genes from 100 Black adult and 57 newborn SS patients from the southeastern United States, from 76 individuals with AS, S-beta-thal, SC, AC, or A-beta-thal, and from 31 normal individuals. Haplotypes for all adult individuals have been previously determined using various restriction endonucleases. The DNA samples were amplified, dot blotted, and hybridized with 32P-labeled specific oligonucleotide probes. All 134 chromosomes with haplotype 19 were positive for the G----T substitution at position -657 (A gamma), while 132 were also positive for the C----G mutation at -369 (G gamma). The three specific changes for the chromosome with haplotype 20 were found on all 54 chromosomes with this haplotype. The C----T mutation at -158 5' to G gamma was present on all 41 chromosomes with haplotype 3, and on two chromosomes with a related atypical haplotype. Normal and beta-thal chromosomes with each of these substitutions had the same 5' subhaplotype as beta S haplotypes 19 or 20, respectively. The close relationship between the occurrence of specific mutations and the haplotype of beta S chromosomes makes the determination of these haplotypes with specific oligonucleotide probes attractive with respect to time and expense.

Comparison of ephedrine and etilefrine for the treatment of arterial hypotension during spinal anaesthesia in elderly patients. Thirty ASA II-III patients (greater than 65 years) undergoing hip surgery under bupivacaine spinal anaesthesia, and presenting a 25% reduction in mean arterial pressure (MAP), were included in the study. The patients were randomly allocated to receive, under double-blind conditions, either ephedrine 0.07 mg kg-1 or etilefrine 0.03 mg kg-1 boluses i.v. when MAP decreased by 25% from the preanaesthetic reference value. There were no significant differences (mean +/- s.d.) in the time interval between the sympathomimetics administered (ephedrine 20.2 +/- 17.1 min, etilefrine 16.6 +/- 7.0 min) or the number of sympathomimetics needed (ephedrine 3.8 +/- 1.9, etilefrine 3.6 +/- 2.2). In regard to MAP and diastolic arterial pressure (DAP), ephedrine was slightly more potent in restoring arterial blood pressure compared with etilefrine. The increases in systolic arterial pressure (SAP) and heart rate were similar in both groups. The administration of repeated doses of either sympathomimetic showed the same tendency to increase SAP, DAP, and MAP values as the first dose.

Serum beta-hexosaminidase and alpha-mannosidase activities as markers of alcohol abuse. In 25-alcohol-dependent patients in whom detoxication treatment has been introduced serum total beta-hexosaminidase, thermostable beta-hexosaminidase, alpha-mannosidase and gamma glutamylotransferase (GGT) and serum high density lipoprotein (HDL) cholesterol were determined during alcohol withdrawal. The assays were also performed in a group of dependent individuals after a 1 month or longer period of abstinence. Marked increase in beta-hexosaminidase activity was observed in intoxicated patients. The activity decreased rapidly after the cessation of drinking, resembling the decrease in HDL cholesterol level in its dynamics. The alpha-mannosidase activity rise was less pronounced and its normalization was slow, similar to the GGT activity normalization rate. The rise of beta-hexosaminidase activity was mostly due to the thermostable component of the enzyme. Total beta-hexosaminidase or thermostable beta-hexosaminidase activity determinations appear to be simple and reliable markers of alcohol abuse.

Immunoelectron microscopic study of tyrosine hydroxylase immunoreactive nerve fibers and ganglion cells in the rat adrenal gland. The present immunocytochemical study used an antiserum to tyrosine hydroxylase (TH), the first enzyme in the biosynthetic pathway of catecholamines, and revealed TH immunoreactivity in the ganglion cells and in the varicose nerve fibers of the cortex and medulla in the rat adrenal gland. TH immunoreactive nerve fibers in the cortex and medulla contained large and small granular vesicles, and also small clear vesicles. The immunoreactive nerve fibers were in close apposition to cortical cells in the cortex and in apposition to smooth muscle cells of blood vessels in both the cortex and medulla. Furthermore, TH immunoreactive nerve fibers were sometimes in close apposition to pericytes of blood vessels in the cortex and chromaffin cells in the medulla. The present results suggest that the catecholaminergic nerve fibers in the rat adrenal gland may be both intrinsic and extrinsic in origin.

Restriction fragment length polymorphism of ovine casein genes: close linkage between the alpha s1-, alpha s2-, beta- and kappa-casein loci. Restriction fragment length polymorphism (RFLP) of ovine casein genes was investigated. Genomic DNA from 56 rams was digested with 10 restriction endonucleases and Southern blots probed with the four ovine casein cDNAs (alpha s1-, beta-, alpha s2- and kappa-Cn). Five enzymes, namely, BglI, PvuII, RsaI, TaqI and HindIII revealed nine different RFLPs. The inheritance of six of these polymorphisms was studied by segregation analysis of gametes in nine rams' families, and each of them could be related to the existence of alleles at the relevant casein locus. A close linkage between the four ovine casein genes was demonstrated since no recombination within the four pairs of loci examined, alpha s1-beta-Cn, alpha s1-kappa-Cn, beta-kappa-Cn and alpha s2-kappa-Cn, was observed in the progeny of double heterozygous rams. The casein genes are thus clustered in the ovine species as in the case of other mammals.

Analysis of polymorphism in the bovine casein genes by use of the polymerase chain reaction. Methods have been devised for detecting polymorphisms in the bovine beta- and kappa-casein genes using the polymerase chain reaction (PCR) followed either by restriction enzyme digestion (to reveal a restriction fragment length polymorphism (RFLP] or by hybridization of an allele-specific oligonucleotide. These methods, as well as being faster and more sensitive than traditional RFLP methods, are of more general applicability since they can detect any change in DNA sequence. They require only a small sample of blood or semen and are applicable to animals of any age or sex. These methods make possible large-scale screening and thus selection for alleles at these loci. Typing of blood DNA can give erroneous results when the animal concerned is a twin; however, this can be overcome by retesting using milk or semen. Analysis of the kappa-casein genotype of Holstein-Friesian bulls gives frequencies for the A and B alleles of 0.80 and 0.20 respectively. Selection in favour of the B allele, which is superior for cheese production, could thus have a large effect. The A3 and B alleles at the beta-casein locus have been shown to be rare in the Holstein-Friesian population. Linkage disequilibrium exists between beta-casein B and kappa-casein B.

Restriction fragment length polymorphism of the caprine beta-globin gene cluster associated with the Hb beta-globin variants in Norwegian dairy goats. Restriction fragment length polymorphism (RFLP) analysis was used to characterize the beta-globin cluster in Norwegian dairy goats. In 122 individuals, different RFLP patterns were detected after BglII and PstI digestion and hybridization with a caprine beta-globin probe. The location of the polymorphic BglII and PstI sites were determined. The different RFLPs were in linkage disequilibrium with each other and with the beta-globin locus as studied at the protein level. A preferential association between certain RFLP haplotypes and beta-globin variants was observed. Polymorphic DNA fragments in the epsilon-globin genes were detected after MspI digestion and hybridization with a caprine epsilon-probe, and eight different band patterns were observed. Correlation between different MspI haplotypes and the beta-globin variants was determined.

DNA polymorphism at the casein loci in sheep. By using seven endonucleases and four bovine cDNA probes specific for alpha S1-, alpha S2-, beta-, and kappa-casein genes, nine restriction fragment length polymorphisms (RFLPs) have been found in the sheep orthologous DNA regions. In contrast to the low level of variation observed at the protein level, these DNA polymorphisms determine a high level of heterozygosity and, therefore, represent useful tools for genetic analyses since they can also be obtained without the need for gene expression. In fact, informative matings suggest that in sheep, as in cattle, the four loci are linked.

The golden hours of the myocardial infarction: nonthrombolytic interventions. Emergency care of patients with acute myocardial infarction requires active decision making to use agents that may improve morbidity and mortality. Thrombolysis remains the primary tool to accomplish this goal. Other pharmacologic agents, including lidocaine, nitrates, calcium channel blockers, beta-blockers, and aspirin, have been used acutely in myocardial infarction in the hopes of preventing death and salvaging myocardium. The decision to select one or all of these agents requires a knowledge of the clinical evidence of their efficacy and risk-to-benefit ratios. The clinical studies of the use of these agents acutely in the management of myocardial infarction are reviewed.

Prolonged protection against methacholine-induced bronchoconstriction by the inhaled beta 2-agonist formoterol. Formoterol fumarate is a new, high-affinity, beta 2-adrenergic receptor agonist that causes bronchodilation for at least 12 h. The purpose of this study was to compare the magnitude and duration of the effect of inhalation of formoterol (12 and 24 micrograms), albuterol (200 micrograms), and placebo in terms of protection against methacholine-induced bronchoconstriction. The 16 stable adult asthmatic subjects were studied on four separate study days. On each study day the subjects inhaled 2 puffs of the study medication, and methacholine tests were performed at 30 min and 4, 8, and 12 h later. Results were expressed as the provocative concentration of methacholine required to cause a 20% fall in FEV1 (PC20). At 30 min, 12 and 24 micrograms formoterol caused a mean 14- to 20-fold decrease in responsiveness, and albuterol a 12-fold decrease. However, albuterol had no significant protective effect at 4 h or thereafter, whereas there was still an 8-fold decrease in responsiveness for 24 micrograms formoterol and a 6-fold decrease for 12 micrograms formoterol at 12 h. This study has shown that both doses of formoterol were still actively protective against induced bronchoconstriction 12 h after inhalation, with minimal side effects. This suggests that formoterol may prove to be a very useful bronchodilator for the treatment of patients with asthma who have significant airway hyperresponsiveness or nocturnal symptoms and who require inhaled beta 2-agonists at least twice daily.

Assembly of fibronectin into extracellular matrix. The results summarized above suggest that assembly of fibronectin is a fundamental biological process and that knowledge of the process of assembly may reveal new ways by which cells interact with extracellular molecules. Deposition of a fibronectin matrix seems to be regulated as tightly as synthesis of fibronectin or expression of adhesion receptors for fibronectin and is influenced profoundly by two products of blood coagulation--TGF-beta released from platelets and factor XIII activated by thrombin. Fibronectin assembly may be important in all sorts of physiological and pathophysiological processes. Cell A--for instance, a stromal cell--can influence the behavior of cell B--for instance, a lymphocyte--by assembling fibronectin made by cell C--for instance, a hepatocyte. We hope that the testable models of assembly presented in this paper will lead to new understanding of the process of assembly and suggest new modalities for treatment of diseases that result in fibrosis, damaged tissues, and neoplastic growth.

Effect of ototoxic drug administration to the endolymphatic sac. Aminoglycosides were applied to the endolymphatic sac of guinea pigs, and the inner ears were studied by light microscopy. In group 1 gentamicin sulfate application on the lateral surface of the endolymphatic sac produced sensory cell atrophy limited to the macula sacculi. In group 2 gentamicin injection into the sac by extradural and intradural approaches often showed lesions in the maculae sacculi and to the sac from a lateral opening resulted in a similar pattern of degeneration but with less severity in comparison to the injection method. Mild to severe endolymphatic hydrops was sometimes observed. The study shows diffusion of drugs taking place against the longitudinal flow of endolymph. This procedure may be applicable for the treatment of Meniere's disease.

Long-term safety and efficacy of zidovudine in patients with advanced human immunodeficiency virus disease. Zidovudine Epidemiology Study Group. An epidemiologic study was initiated in 1987 to evaluate the long-term safety and efficacy of zidovudine in patients with advanced human immunodeficiency virus disease. Data from 886 patients with the acquired immunodeficiency syndrome (AIDS) or AIDS-related complex and CD4+ lymphocyte count less than 0.25 x 10(9)/L are reported. Eighteen-month survival was 67% for the cohort. Pretreatment factors associated with increased survival time included index diagnosis of AIDS-related complex, hematocrit of 0.35 or greater, CD4+ lymphocyte count of 0.15 x 10(9)/L or greater, high functional status, and time from diagnosis of AIDS to treatment of less than 60 days. By proportional hazards analysis, development of serious anemia was the most significant factor associated with early death. Receiving zidovudine for a high proportion of time significantly improved chances of survival even if anemia developed. Serious leukopenia occurring in 37% and serious anemia occurring in 32% of patients. Nonhematologic adverse events were uncommon and no previously unreported adverse events were seen.

Retinoids induce tissue transglutaminase in NIH-3T3 cells. We report that all-trans and 13-cis-retinoic acid as well as the synthetic compound CH-55 enhance tissue transglutaminase activity as they increase NIH-3T3 cell adhesiveness. The 4-hydroxyphenylretinamide (4-HPR) with low activity in inducing attachment, lectin binding and growth inhibition also fails to induce transglutaminase. Thyroxine (Thy), a compound with a response element common to RA, is inactive. The tumor promoter 12-tetradecanoyl-phorbol-13-acetate (TPA), which increases adhesiveness with different kinetics than RA, failed to enhance tranglutaminase. We conclude that retinoids with biological activity in inducing adhesion, inhibition of growth and increase of lectin binding, are also active in inducing transglutaminase activity.

Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Monolayers of a well differentiated human intestinal epithelial cell line, Caco-2, were used as a model to study passive drug absorption across the intestinal epithelium. Absorption rate constants (expressed as apparent permeability coefficients) were determined for 20 drugs and peptides with different structural properties. The permeability coefficients ranged from approximately 5 x 10(-8) to 5 x 10(-5) cm/s. A good correlation was obtained between data on oral absorption in humans and the results in the Caco-2 model. Drugs that are completely absorbed in humans had permeability coefficients greater than 1 x 10(-6) cm/s. Drugs that are absorbed to greater than 1% but less than 100% had permeability coefficients of 0.1-1.0 x 10(-6) cm/s while drugs and peptides that are absorbed to less than 1% had permeability coefficients of less than or equal to 1 x 10(-7) cm/s. The results indicate that Caco-2 monolayers can be used as a model for studies on intestinal drug absorption.

Molecular cloning of human epidermal transglutaminase cDNA from keratinocytes in culture. We have isolated a cDNA encoding human epidermal transglutaminase, a key enzyme of terminal differentiation of keratinocytes. A cDNA library from cultured human keratinocytes was screened by a PCR-amplified partial cDNA fragment of the enzyme with oligonucleotide primers based on the homology of the transglutaminase family. The cDNA is 2734 bp coding a protein of 817 amino acids. The several regions including the active site cysteine residue are highly conserved among the transglutaminase family. However, the charged N-terminal domain is unique to the epidermal transglutaminse, suggesting that the region is involved in the function of the enzyme in keratinocytes.

Coenzyme Q10 increases T4/T8 ratios of lymphocytes in ordinary subjects and relevance to patients having the AIDS related complex. Coenzyme Q10 (CoQ10) is indispensable to biochemical mechanisms of bioenergetics, and it has a non-specific role as an antioxidant. CoQ10 has shown a hematological activity for the human and has shown an influence on the host defense system. The T4/T8 ratios of lymphocytes are known to be low in patients with AIDS, ARC and malignancies. Our two patients with ARC have survived four-five years without any symptoms of adenopathy or infection on continuous treatment with CoQ10. We have newly found that 14 ordinary subjects responded to CoQ10 by increases in the T4/T8 ratios and an increase in blood levels of CoQ10; both by p less than 0.001. This knowledge and survival of two ARC patients for four-five years on CoQ10 without symptoms, and new data on increasing ratios of T4/T8 lymphocytes in the human by treatment with CoQ10 constitute a rationale for new double blind clinical trials on treating patients with AIDS, ARC and diverse malignancies with CoQ10.

1 alpha,25-dihydroxyvitamin D3 inhibits productive infection of human monocytes by HIV-1. Pretreatment of freshly isolated human peripheral blood monocytes with the steroid hormone, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)D), markedly reduced (by 95%) productive infection of human monocytes by HIV-1. Equivalent concentrations (10nM) of 25-hydroxyvitamin D3 (25(OH)D), the biologic precursor of 1,25(OH)D, were ineffective at reducing either CD4 expression or HIV-1 production. Pretreatment was required for modulation of HIV-1 infection by 1,25(OH)D. Interestingly, 1,25(OH)D-mediated decreases in p24 antigen production were observed prior to any observed reduction in CD4 expression, suggesting that 1,25(OH)D treatment may modulate HIV-1 infection of monocytes through additional factors besides decreased HIV-1 binding. These data raise the possibility that 1,25(OH)D compounds may be important in host resistance to HIV-1.

Defective CD2 pathway T cell activation in systemic lupus erythematosus. CD2 (T11; sheep erythrocyte receptor) is the surface component of an alternative, antigen-independent pathway of human T cell activation. The response to certain anti-CD2 antibodies is relatively independent of accessory cell signals and therefore provides a direct measurement of T cell function. The CD2 pathway may be important in the differentiation of thymocytes, on which the expression of CD2 precedes the appearance of the CD3-T cell receptor complex. In view of the impaired T cell regulation of immune responses in patients with systemic lupus erythematosus (SLE), we examined the activation of peripheral blood lymphocytes by anti-CD2 antibodies in 57 SLE patients and 32 normal control subjects. The CD2 pathway response was lower in the SLE patients (P less than 0.0001); 18 of the 57 SLE patients had a lower response than any of the control subjects. The SLE low-responder patients did not differ from the normal-responder patients in terms of disease activity or use of antiinflammatory and immunosuppressive medications. Low responses to anti-CD2 were corrected to normal by the coaddition of a submitogenic amount of phorbol myristate acetate (1 ng/ml). In some low-responder patients, the responses were normalized by the removal of non-T cells. The data indicate that some SLE patients have impaired responses to CD2 pathway activation and that this may reflect intrinsic T cell defects and/or regulatory influences of non-T cells.

Lack of efficacy of d-propranolol in neuroleptic-induced akathisia. d-Propranolol lacks clinically significant beta-adrenergic receptor blocking properties, but has the same membrane stabilizing effects as racemic (d,l) propranolol. To assess the role of beta-blockade versus membrane stabilization or other shared nonspecific effects in the therapeutic action of propranolol in neuroleptic-induced akathisia (NIA) we treated 11 patients with NIA in a crossover, double-blind study of d-propranolol versus placebo. Akathisia scores were unchanged after both d-propranolol and placebo. Eight patients were subsequently treated in a nonblind manner with racemic propranolol, with a significant reduction in akathisia scores. These findings suggest that beta-blockade, not membrane stabilization or other shared nonspecific effects, contributes to the efficacy of propranolol in NIA.

Neuroleptics improve sustained attention in schizophrenia. A study using signal detection theory. To test the hypothesis that antipsychotic drugs improve attentional processes in schizophrenia, we used a computer-controlled, perceptually degraded continuous performance test (CPT), based on signal detection theory. CPT stimuli were degraded (blurred) to reduce discriminability so that signal detection analysis could be used to distinguish specific attentional processes, as measured by A', from nonspecific factors, as measured by B". Thirteen medicated and 12 neuroleptic-withdrawn schizophrenics visually monitored digits to detect a target under perceptually undegraded and degraded conditions. The principal result was that the neuroleptic-withdrawn patients showed a significant decline in the attention-specific measure of A' over time on task only for the degraded targets, independent of changes in the nonspecific index of B". These results demonstrate that neuroleptic withdrawal may compromise specific attentional processes, namely the ability to sustain attention, as measured by a precise performance task which controlled for nonspecific factors.

N-methyl-D-aspartate receptor antagonist APV blocks acquisition but not expression of fear conditioning. The role of N-methyl-D-aspartate (NMDA) receptors in Pavlovian fear conditioning was examined using the NMDA antagonist DL-2-amino-5-phosphonovaleric acid (APV). Either APV (5 micrograms/rat) or saline was administered before the training phase, the testing phase, or both. APV completely blocked acquisition but not expression of fear conditioning. The L enantiomer of APV did not affect the acquisition of conditional fear. To separate encoding from consolidation processes, APV was administered either before or immediately after the footshock unconditional stimulus (US) during the training phase. The results indicate that APV must be present during the US to produce its effects on fear conditioning. The behavioral effect of the drug is not due to analgesic action because APV did not alter pain sensitivity. The data suggest that NMDA receptors are critical for the acquisition but not expression of fear conditioning. These effects on fear conditioning are parallel to the in vitro effects of APV on the acquisition but not expression of long-term potentiation (LTP) and suggest that endogenously generated NMDA-dependent LTP participates in the neural plasticity underlying fear conditioning.

Effects of centrally administered anxiolytic agents on classically conditioned bradycardia. Rats received infusions of the opioid peptide D-Ala2-Met-enkephalinamide (DALA, 10 micrograms), the alpha 2-noradrenergic agonist clonidine (CLON, 3 micrograms), UK14,304 (UK, 5 micrograms), the corticotropin-releasing factor (CRF) antagonist alpha-helical CRF (9-41) (alpha-HEL, 25 micrograms), or saline in the rostral fourth ventricle. The DALA, CLON, and UK groups showed no evidence of a heart rate (HR) conditioned response (CR) during conditioning, after antagonist administration, or on a nondrug test 48 hr after conditioning. These three groups showed the development of normal CRs when later retrained without drugs. The alpha-HEL group showed an enhanced CR. During a subsequent startle test, the presence of a conditioned stimulus resulted in a pronounced suppression of startle in the SAL and alpha-HEL groups but had no effects on startle in the DALA, CLON, and UK groups. The results indicate an important role for fourth ventricle structures containing opioid and alpha 2 receptors in the learning of an HR CR.

Target-dependent structural changes in sensory neurons of Aplysia accompany long-term heterosynaptic inhibition. FMRFamide evokes both short-term and long-term inhibition of synapses between mechanosensory and motor neurons in Aplysia. We report here, using dissociated cell culture and low-light epifluorescence video microscopy, that depression lasting 24 hr of sensorimotor synapses evoked by four brief applications of FMRFamide is accompanied by a significant loss of sensory cell varicosities and neurites. These structural changes in the sensory cells require the presence of the target motor cell L7. Because the loss of structures known to contain transmitter release sites correlates significantly with the changes in the amplitude of the excitatory postsynaptic potential in L7, our results suggest that the structural changes evoked by FMRFamide reflect a loss of synaptic contacts. Thus, long-term depression parallels long-term facilitation of the sensorimotor synapse produced by serotonin in that both forms of heterosynaptic plasticity involve target-dependent modulation of the number of presynaptic varicosities.

Kinetic analysis of interactions between kainate and AMPA: evidence for activation of a single receptor in mouse hippocampal neurons. AMPA but not kainate produces a rapidly desensitizing response in mouse hippocampal neurons. The characteristic action of these agonists appears to arise from activation of a single receptor with active and desensitized states, for which AMPA and kainate have different relative affinity. The equilibrium potency of a series of five agonists that produce rapidly desensitizing responses at non-NMDA receptors (EC50 1 microM to 4 mM) was similar to their equilibrium potency for block of kainate responses. Increasing the concentration of kainate overcame such block, but in the presence of AMPA the rate of activation of responses to kainate was slowed. Conversely, in the presence of kainate the amplitude of rapidly desensitizing responses evoked by AMPA was reduced, and the rate of onset of desensitization was slowed.

Glutamate-operated channels: developmentally early and mature forms arise by alternative splicing. The expression of two alternative splice variants, Flip and Flop, in mRNAs encoding the four AMPA-selective glutamate receptors (GluR-A, -B, -C, and -D) was studied in the developing brain by in situ hybridization. These receptors are expressed prominently before birth, and patterns of distribution for Flip versions remain largely invariant during postnatal brain development. In contrast, the Flop versions are expressed at low levels prior to postnatal day 8. Around this time, the expression of Flop mRNAs increases throughout the brain, reaching adult levels by postnatal day 14. Thus, receptors carrying the Flop module appear to participate in mature receptor forms.

Expression of P-glycoprotein in breast cancer tissue and in vitro resistance to doxorubicin and vincristine. Expression of P-glycoprotein was evaluated by C219 monoclonal antibody immunoblots in 34 previously untreated and 14 pretreated breast cancers and in benign breast lesions or histologically normal breast glands. P-glycoprotein was not detectable in the few cases of normal or benign tissue. P-glycoprotein was expressed in the 170 kD areas of 29% (10/34) of untreated and 64% (9/14) of previously treated tumours (P = 0.02). In treated tumours, high intensity expression was observed more frequently than in untreated breast cancer (40% vs. 9%). Moreover, there was a significant association between P-glycoprotein expression and in vitro resistance to doxorubicin and vincristine. Simultaneous resistance was observed in all of the P-glycoprotein positive and in only 56% of the P-glycoprotein negative tissues (P less than 0.01). Some aspects of the typical multidrug resistant phenotype, such as P-glycoprotein expression and simultaneous resistance to doxorubicin and vincristine, could be detected in small subsets of breast cancer patients. No relation between P-glycoprotein expression and the type of previous clinical treatment was observed.

Ganglioside antigens in tissue sections of skin, naevi, and melanoma--implications for treatment of melanoma. The ganglioside GD3 was distributed widely on melanocytes, naevi, and practically all melanomas. Not all the cells in melanoma appeared to express GD3, so that treatment with MAbs to GD3 could be expected to leave foci of tumor cells resistant to the effects of the MAbs. GM3 had a similar distribution of GD3 on melanoma, but was expressed on a lower percentage of cells in individual tumors. Expression of GM3 appeared to be suppressed on melanoma and naevus cells in the epidermis. Addition of MAbs to GM3 to those against GD3 in the treatment of melanoma may increase the lytic effect against cells coexpressing both gangliosides, but as GM3 did not appear to be expressed on GM3 -ve cells, the percentage of resistant cells may not be decreased. GD2 was expressed on only approximately 25% of primaries and less than 50% of metastases. In individual tumors there was some evidence of reciprocal expression of GD3 and GD2, so the combination of MAbs to GD3 and GD2 may decrease the percentage of melanoma cells that are resistant to either MAb alone. Both GD3 and GD2, but not GM3, was expressed on lymphocytes around melanoma metastases in LNs and around melanomas in skin. GD2 was detected on a large percentage of lymphocytes around metastases in lymph nodes, but not in the skin, suggesting that the gangliosides GD2 and GD3 may be expressed on different subsets of T-lymphocytes. These findings, together with previous studies showing that the MAbs can enhance lymphocyte responses to a variety of stimuli, provide support for the hypothesis that the clinical effects of the MAbs may reflect activation of host responses against the tumor. Further analysis of the role of gangliosides in lymphocyte function is needed.

Cytogenetic analysis in melanoma and nevi. We have presented data from the cytogenetic analysis of melanocytes derived from four dysplastic nevi and 14 melanomas. We have confirmed previous results from melanomas, including the loss of chromosome 6q and duplication of 1q and 7p. We did not observe an excess of rearrangements involving chromosomes 2 and 3, but we found frequent deletion of chromosomes 9p, 10p, 10q, and 11q. We observed the loss of chromosome 9 in 2 of 4 dysplastic nevi, and combining this observation with that from the melanomas suggests that deletion or inactivation of a gene on 9p may be a primary event in melanocyte transformation. Other tumor suppressor genes are likely to be involved in the transformation process, and these are most likely located on 10p, 10q, 11q, and 6q. We observed many rearrangement involving chromosome band 1p13 and suggest that activation of a gene in that band may also contribute to the transformation process.

Management of severe hypertension in childhood Takayasu's arteritis. Six children presented with severe hypertension caused by Takayasu's arteritis (TA), of whom four had bilateral renal artery narrowing and two coarctation syndrome. Two presented with hypertensive encephalopathy and four with congestive cardiac failure. All had a strongly positive skin reactions to purified protein derivative of mycobacterium tuberculosis. Bilateral renal arterial bypass grafts performed in two children resulted in prolonged normalization of their blood pressures, but the grafts clotted 12-18 months later. Primary renal autotransplantation was unsuccessful in two children, one with bilateral renal arterial narrowing and iliac vessel involvement and one with a long coarctation. Secondary renal autotransplantation was successful in a third child with localized aortitis. A successful aortic patch graft was performed in one child with coarctation of the aorta. Angiotensin-converting-enzyme inhibitors should be used with caution in treating the hypertension caused by TA, since bilateral renal arterial narrowing is common and their administration may result in renal insufficiency. The long-term prognosis is guarded in severely hypertensive children with extensive vascular disease due to TA.

The expression of the multidrug resistance-associated glycoprotein in B-cell chronic lymphocytic leukaemia. The expression of the multidrug resistance (MDR)-associated 170 kDa glycoprotein (p170) was investigated in 63 cases of B-cell chronic lymphocytic leukaemia (CLL), with two monoclonal antibodies (MRK-16 and C-219). By immunocytochemistry with MRK-16 (63 cases), the great majority of the cells was positive, with a weak reaction in 61% of cases and a strong reaction in 39% of cases. By flow cytometry, the proportion of positive cells was 39 +/- 25% with MRK-16 (63 cases), and 23 +/- 22% with C-219 (36 cases). The expression of p170 in leukaemic B-lymphocytes suggests that also in B-CLL the development of MDR can have some therapeutic relevance. By either method the proportion of positive cells was not related to prior treatment, time from diagnosis, absolute lymphocyte count, and clinical stage (Rai's and workshop classifications), but 12 patients who were under treatment with alpha-interferon had more positive cells than the other ones.

In vitro expression of bovine opsin using recombinant baculovirus: the role of glutamic acid (134) in opsin biosynthesis and glycosylation. Expression levels of functional bovine opsin in the insect cell line IPLB-Sf9 using recombinant baculovirus were shown not to depend on the use of novel transfer vectors (pAcRP23, pAcDZ1) that were reported to improve biosynthesis levels of other proteins in this system. A production of 5 micrograms opsin per 10(6) cells (approx. 1.5% of total cell protein) was achieved by batch fermentation of infected cells in spinner cultures. Infection of the cells in the presence of the glycosyltransferase inhibitor tunicamycin led to the synthesis of the complete protein, which, however, now migrated with a substantially lower Mr. This demonstrates that opsin in insect cells also undergoes N-linked glycosylation and allowed partial purification (10-fold) of the resulting rhodopsin by affinity chromatography over Concanavalin A-Sepharose. Through site-directed mutagenesis (rhod)opsin mutants have been obtained allowing dissection of functional domains of opsin. Amino acid substitutions that involved Glu-134 and/or Arg-135 affected the normal biosynthetic process leading in part to nonglycosylated, to a small extent even incomplete, protein. A number of mutations, that involve other charged residues within the second and third transmembrane domain of the protein, had no effect on the biosynthetic processing of the protein. We therefore suggest that the charge-pair Glu-134-Arg-135 is part of an important internal signal sequence and that alterations in this region may result in incorrect membrane translocation and/or folding of the protein.

Adipofascial turn-over flap for reconstruction of the dorsum of the foot. Local adipofascial turn-over flaps with skin grafts were successfully used to reconstruct complicated skin defects over the dorsum of the foot in 7 patients. The blood supply of the flap comes from perforators in the base and from the surrounding subcutaneous and fascial plexuses. A new concept of base-to-flap area ratio is proposed to predict the survival of the turn-over flaps in addition to the conventional base-to-length ratio. This appears to be a valuable technique for difficult wounds of the dorsum of the foot.

Post-traumatic compartment syndrome of the foot. Post-traumatic compartment syndrome in the foot is an unusual but well-recognized entity. If there is an awareness of the condition at the time of presentation it can be diagnosed and treated effectively. A case of compartment syndrome in the foot of a 17-year-old boy after massive lower-extremity trauma is described. The simple and readily available diagnostic techniques are emphasized and an alternative approach to plantar decompression, which avoids exposure of and potential damage to the posterior tibial neurovascular bundle, is presented.

Expression of c-erbB-2 oncoprotein in gastric carcinoma. Immunoreactivity for c-erbB-2 protein is an independent indicator of poor short-term prognosis in patients with gastric carcinoma. Correlations of c-erbB-2 protein expression with clinical outcomes of gastric carcinomas were studied in 189 gastric carcinomas. There were 23 (12.2%) carcinomas with evidence of c-erbB-2 protein in which the reaction was localized to the cell membrane. There was no significant association between c-erbB-2 staining and the macroscopic or histologic type of the carcinomas. c-erbB-2-stained tumors were more likely to be associated with serosal invasion, nodal involvement, and peritoneal metastasis, than c-erbB-2-unstained ones. In addition, c-erbB-2 was stained in none of early gastric carcinomas. The 5-year survival rates of the c-erbB-2 protein-positive and the protein-negative group were 11% and 50%, respectively. When the c-erbB-2 tissue status and seven clinicopathologic variables as conventional prognostic factors were entered simultaneously into the Cox regression model, serosal invasion, hepatic metastasis, peritoneal metastasis, nodal status, and c-erbB-2 tissue status emerged as independent prognostic variables. The results suggested that c-erbB-2 protein expression might be enhanced in advanced stages during the progression of gastric carcinoma. In this particular group of patients, immunoreactivity for c-erbB-2 protein is an indicator of poor short-term prognosis.

Screen of five alkyl carbamates for initiating and promoting activity in rat liver. Five alkyl carbamates, methyl, ethyl, propyl, hydroxypropyl and ethylhexyl, were tested for initiating and promoting activity in rat liver. The test for initiating activity consisted of administering the carbamate by gavage to male Sprague--Dawley rats at either 6 or 18 h after a 2/3 partial hepatectomy. One week later, the rats received 500 ppm sodium phenobarbital in their drinking water until killed 10 weeks later. None of the carbamates at 1/5 the LD50 induced gamma-glutamyltranspeptidase (GGT)-positive foci, indicating the lack of initiating activity. The tumor promoting activity test consisted of initiation with 80 mmol/kg diethylnitrosamine administered 18 h after a partial hepatectomy. One week later, the rats received one of the the carbamates at either 1/10 or 1/20 the LD50 5 days per week until sacrificed 10 weeks later. The alkyl carbamates increased the volume of the foci, the percent of the liver occupied by the foci, the number of foci/cm3 (except ethylhexyl carbamate), and the number of foci/liver (except ethylhexyl carbamate). These results suggest that the alkyl carbamates are tumor promoters and not tumor initiators in rat liver.

Comparison of liver cancer and nodules induced in rats by deoxycholic acid diet with or without prior initiation. The neoplastic responses of liver to prolonged continuous exposures of deoxycholic acid (DCA) in the diet with or without prior initiation was examined in Fischer 344 (F-344) and Sprague-Dawley (SD) male rats. In both rat strains, 7 months of continuous feeding of 0.5% (F-344 rats) or 1.0% (SD rats) of deoxycholic acid in chow diet generated hepatic nodules which persisted up to 8 months after deoxycholic acid was stopped. In one group of Sprague-Dawley male rats, 7 months of deoxycholic acid was preceded by an initiating dose of diethylnitrosamine. In 10 out of 15 of these rats, not only persistent nodules but also 26 liver cancers (large, multifocal, invasive, lung metastases) become evident by 8 months after cessation of deoxycholic acid.

Reversal of multidrug resistance in HL-60 cells by verapamil and liposome-encapsulated doxorubicin. The means of circumventing multidrug resistance was investigated in HL-60 and HL-60R (a drug resistance variant) cell lines. The HL-60R cell line was developed from the parent line by serial exposure to increasing concentrations of doxorubicin over a 4-month period. This drug resistant cell line expressed P-glycoprotein in its cell surface and is 80-fold more resistant than the parent cell line. Multidrug resistance, as evaluated by a cell cytotoxicity assay using doxorubicin, can be overcome by use of verapamil. Multidrug resistance can also be circumvented when doxorubicin is encapsulated in liposomes. The combination of verapamil and doxorubicin-encapsulated liposomes does enhance circumvention of multidrug resistance beyond the effect of each agent alone, implying a synergistic effect. The lipid composition of the liposomes does affect the rate of drug uptake but not the overall cytotoxic effect of doxorubicin. The synergistic reversal of multidrug resistance by doxorubicin-encapsulated liposomes and verapamil suggests a multifactorial basis for drug resistance in this cell line.

[A simple method for detection of P fimbriae in uropathogenic strains of E. coli]. The PF test of the Finnish firm Orion is used for detection of P fimbriae in E. coli strains. Its principle involves the establishment of a chemically defined receptor for these fimbriae on latex particles which can then be agglutinated by the appropriate strains. By means of this test the authors examined 453 uropathogenic E. coli strains. P fimbriae were detected in 121 strains, i.e. in 26.7%. Twenty-five strains agglutinated not only latex particles coated with receptor but also non-coated control particles. The test was thus falsely positive in 5.5% of the cases. Fifty-five strains, where by means of the PF test fimbriae were detected, were examined by the method of mannoresistent haemagglutination of human erythrocytes on a slide. The erythrocytes agglutinated in the presence of d-mannose of all 55 strains, and thus the results agreed in 100%. This suggests that P fimbriae can be detected by the haemagglutination method, but it must be taken into account that this method can, contrary to the PF test, detect also other adhesins. The PF test is very quick, simple and its principle ensures a high reliability.

Achlorhydria-induced hypergastrinaemia: the role of bacteria. 1. Studies of Helicobacter pylori show that microbes can alter gastrin release. Lack of gastric acid (achlorhydria) causes hypergastrinaemia and allows bacteria to grow within the stomach. We speculated that the bacteria contribute to the rise in gastrin seen after acid inhibition, and tested the idea by comparing plasma gastrin levels during inhibition of acid secretion between germ-free and conventional rats. 2. Matched germ-free and conventional rats (n = 8 per group) received either vehicle (saline) or one of two doses of the histamine-H2-receptor antagonist loxtidine for 1 week. Gastrin was measured in cardiac blood by a specific r.i.a. 3. Plasma gastrin concentrations in germ-free and conventional rats were 59 +/- 11 pmol/l (mean +/- SEM) and 36 +/- 8 pmol/l, respectively, after vehicle, and 153 +/- 30 pmol/l and 181 +/- 27 pmol/l, respectively, after loxtidine at a dose of 10 mg day -1 kg -1, which partially inhibits acid secretion. Administration of loxtidine at a dose of 70 mg day -1 kg-1, which completely inhibits acid secretion, did not produce a significant extra rise in plasma gastrin concentration in germ-free rats (178 +/- 11 pmol/l), but further elevated plasma gastrin concentrations to 278 +/- 26 pmol/l in conventional rats (P less than 0.005 compared with germ-free rats). 4. Loxtidine produced a dose-dependent rise in the number of eosinophils in the gastric mucosa of conventional rats. 5. We conclude that partial inhibition of gastric acid secretion increases gastrin release independently of bacteria, but that bacteria are involved in the further rise in gastrin which occurs on more profound inhibition of gastric acid secretion.

Pharmacokinetic and pharmacodynamic properties of histamine H2-receptor antagonists. Relationship between intrinsic potency and effective plasma concentrations. Histamine H2-receptor antagonists are a unique class of compounds. Pharmacologically they are characterised as a family by their ability to inhibit the secretion of gastric acid, and kinetically they are classified as a family by their similarity in absorption, distribution and elimination. All the H2-receptor antagonists exhibit classical competitive drug-receptor interactions, with Schild slope parameters not significantly different from unity. Comparison of the values of the negative logarithm of the molar concentration of antagonist in the presence of which the potency of the agonist is reduced 2-fold (PA2) indicates that famotidine is about 20 to 50 times more potent than cimetidine and 6 to 10 times more potent than ranitidine. To date, famotidine is the most potent among marketed H2-receptor antagonists. Oral absorption of all the H2-receptor antagonists under clinical investigation is fairly rapid. Peak plasma concentrations are usually attained within 1 to 3h after the dose, but a second peak after oral administration has been observed with cimetidine, ranitidine, famotidine, ramixotidine and etintidine. The mean oral bioavailability for the H2-antagonists ranges from 50 to 70%. Reports on the plasma profiles after intravenous administration are available only for cimetidine, ranitidine, famotidine and nizatidine: plasma concentrations of all 4 decline in a biexponential manner. All of the H2-antagonists are eliminated quite rapidly, with a terminal half-life of 1 to 3h and a total body clearance of 24 to 48 L/h. Elimination is mainly attributable to renal excretion, with renal clearances ranging from 13.8 to 30 L/h. As the values for renal clearance greatly exceed the glomerular filtration rate (6 to 7.2 L/h), it is apparent that renal tubular secretion plays an important role. There is a simple, direct correlation between plasma concentrations of H2-receptor antagonists and the inhibition of gastric acid secretion. This implies a rapid equilibration between drug concentration in plasma and at the site of action, and a reversible drug-receptor interaction. Success in correlating the plasma concentration of H2-receptor antagonists and their pharmacological effects stems from reliable and precise measurement of both items. Despite the heterogeneous nature of data sources, 50% inhibitory concentration (IC50) values for cimetidine, ranitidine, famotidine, nizatidine, etintidine and roxatidine obtained in vitro appear to be in good agreement with those determined in vivo. These results suggest that at an early stage of development of an H2-receptor antagonist, IC50 determined from in vitro studies may be useful as a first approximation to predict the clinically effective concentration of the new agent.

Doxepin therapy for postprandial symptomatic hypoglycaemic patients: neurochemical, hormonal and metabolic disturbances. 1. Three oral glucose tolerance tests were performed in each of 32 symptomatic postprandial hypoglycaemic patients (before placebo, before doxepin therapy and after doxepin therapy). Plasma neurotransmitters were determined in parallel with assays of plasma insulin and glucose levels. 2. Three different types of patients were distinguished. Type I showed a low noradrenaline/adrenaline ratio, high dopamine levels and low platelet 5-hydroxytryptamine (serotonin) levels during basal periods. After a glucose load, late peaks of dopamine and free 5-hydroxytryptamine, which coincided with the symptoms but not with the nadir of plasma glucose, were observed. Type II showed a low basal plasma noradrenaline/adrenaline ratio. After a glucose load, progressive increases in adrenaline and decreases in glucose were seen. Adrenergic symptoms coincided with the nadir of glucose. Although type III patients showed hyperinsulinaemia after a glucose load similar to the other types of patient, they did not show hyperglycaemia, but rather exhibited a sustained and progressive reduction in plasma glucose. These patients were characterized by a high basal plasma noradrenaline/adrenaline ratio, high basal plasma levels of 4-hydroxy-3-methoxyphenylethyleneglycol and high basal levels of platelet 5-hydroxytryptamine, all of which increased after a glucose load. Systolic and diastolic blood pressure decreases paralleled reductions in heart rate and glucose. The nadir of plasma glucose occurred simultaneously with the appearance of symptoms (weakness, heartburn, oppressive chest pain, tension headache, abdominal cramps, dizziness, etc.). Therapy with doxepin led to disappearance of the symptoms within 3-4 weeks. Normalization of all other disordered variables (cardiovascular, metabolic and neurochemical, and the clonidine test) paralleled the disappearance of the symptoms. 3. Symptoms varied in the three types of patients and we conclude that they are related to hypoglycaemia-induced disorders of plasma neurotransmitters, rather than to hypoglycaemia per se. We postulate that an uncoping stress situation (type I and II patients) and depression (type III patients) underlie the physiopathological mechanisms.

[Effects of prolonged administration of dapiprazole in rats]. In this paper we have studied the rat under chronic treatment with dapiprazole ip. (a drug with alpha-adrenergic blocking properties). For a 4 week period we have recorded, once a week, weight, rectal temperature, tail-flick, general activity and explorative behaviour on rotarod and one-arm radial maze. Surface (SEEG) and deep (DEEG) EEGrams were also recorded at the end of the experiments (4th week). SEEG and DEEG underwent the Fourier analysis (Fast Fourier Transform--see text). The findings were: 1) No change was observed in weight, rectal temperature and tail-flick time, glycemia and haematocrit. 2) Motor coordination on rotaroad dropped in 1st and 3rd week of treatment. 3) The treated rats, when in a one arm radial maze, showed signs of sedation (no grooming) from 2nd week of treatment. 4) SEEG and DEEG recordings showed a slowing activity; Fourier analysis confirmed these results. The slowing of activity (4) well agrees with the signs of sedation and behaviour (2, 3). It seems thus confirmed that alpha-adrenergic blockade is a feature of the drug together with the psychotropic effects previously described.

Comparison of exercise effects on the hemodynamics of the Bio 14.6 cardiomyopathic hamster. 1. Comparisons of the effects of 4 and 16 weeks of exercise were made on; cardiac output, stroke volume, heart rate, left intraventricular systolic and diastolic pressures, dP/dt, and heart calcium in the Bio 14.6 cardiomyopathic and F1 B hamsters. 2. In the cardiomyopathic hamster the cardiac output, stroke volume, left intraventricular systolic pressure and dP/dt, which were all depressed in the age related sedentary animals, were increased by both periods of exercise. The left intraventricular diastolic pressure which was elevated was likewise decreased by both exercise periods. Only the 16 week exercise period decreased the resting heart rate. 3. In the normal F1 B hamster, both periods of exercise increased the cardiac output and stroke volume while the left intraventricular systolic pressure was decreased. Only the 16 week exercise decreased the resting heart rate and left intraventricular diastolic pressure and increased the left ventricular dP/dt. 4. Both periods of exercise increased the total heart calcium in the Bio 14.6 hamster while the heart calcium in the F1 B was increased only by the 16 week exercise period.

Energetic output of subadult polar bears (Ursus maritimus): resting, disturbance and locomotion. 1. Indirect calorimetry was used to determine metabolic rates in subadult polar bears at rest after human-controlled disturbance and at four rates of locomotion. 2. Disturbance factors that do not result in locomotion would only have a significant effect on energy expenditure if they occurred over an extended period of time. 3. Human disturbance resulting in locomotion would have a relatively high energetic cost to individual animals. 4. Polar bears may require a relatively high energetic output to initiate walking.

Modulation of plasma biotin in the fowl (Gallus domesticus) by oestrogen and ambient temperature. 1. Exposure of laying hens to elevated ambient temperatures (30-40 degrees C) resulted in a significant increase in the biotin concentrations in plasma and egg yolk. 2. Exogenous oestrogen administration to immature pullets increased plasma biotin three-fold. The increase was six-fold when the birds were simultaneously exposed to an ambient temperature of 35 degrees C. 3. It is proposed that ambient temperature affects the balance between thyroid and ovarian hormones resulting in increased circulating levels of biotin and deposition of the vitamin in egg yolk.

The time course in development of hypocalcemia and plasma lactate accumulation in mature and immature hyperthermic domestic fowl (Gallus domesticus). 1. During hyperthermia-induced thermal panting, there was a progressive development of alkalosis, hypocapnia, hypocalcemia and plasma lactate accumulation in both mature and immature domestic fowl. 2. The results of this study further demonstrate that hypocalcemia is a general consequence of respiratory alkalosis in animals. 3. The significant (P less than 0.05) regression analysis of plasma lactate vs arterial blood pH, suggest a relationship between these two variables. 4. Plasma lactate accumulation may therefore play an important role as an extrarenal buffering component in the normal defense against severe alkalosis.

Mammary gland blood flow and plasma concentrations of 6-keto-prostaglandin F1 alpha in the goat. 1. Mammary blood flow and concentrations of the stable metabolite of prostacyclin, 6-keto-prostaglandin F1 alpha, in mixed venous and mammary venous blood plasma have been measured in five lactating goats at various times during the day. 2. Natural variation in blood flow was not associated with any local release of 6-keto-prostaglandin F1 alpha into the mammary venous circulation.

Effect of folic acid deficiency upon lymphocyte subsets from lymphoid organs in mice. 1. Three groups of weanling C57BL/6 female mice were fed one of two folate-deficient diets (0 and 0.1 mg folic acid/kg diet) or a normal folate-containing diet (2 mg folic acid/kg diet) for 8 weeks. A control pair-fed group was introduced with the most severe folate-deficient diet. Seven mice were fed the 0 mg folic acid/kg diet for 8 weeks, then rehabilitated (R) on the 2 mg folic acid/kg diet for 10 days. 2. Mice fed 0 mg folic acid/kg diet were severely folate-deficient (SFD), whereas mice fed 0.1 mg folic acid/kg diet were moderately folate-deficient (MFD), as shown by their folate status parameters. 3. Thymus weight, thymocyte content and positive immature CD4+8+ cells were decreased in SFD mice compared to controls. These values were normalized after 10 days of rehabilitation. 4. Mesenteric lymph node cells were apparently not affected by folate deficiency. 5. The proportion of Thy-1+ splenocytes was mildly lower in SFD mice than in controls. In R mice, mean spleen weight and spleen cellularity were increased compared to the other groups, but the proportions of Thy-1+, CD4+8- and CD4-8+ cells were markedly lower than control values.

Seasonal change in irradiance: a zeitgeber for circannual rhythms in ground squirrels. 1. Zeitgebers for circannual rhythms have been elusive. Demonstration that an external factor is a zeitgeber requires proof of a phase-shift that endures for several years. 2. The California ground squirrel (Spermophilus beecheyi) is an ideal subject. Many features of behavior have circannual rhythms of which change in mass is the easiest to measure. The squirrels thrive in captivity for up to 10 years. The squirrels were kept in individual cages in an air conditioned room, fed lab chow ad lib, and weighed twice a month. They were exposed to a 6-month phase shift of (a) length of day (b) seasonal change in temperature, (c) both, (d) seasonal cycle of irradiance. 3. The squirrels maintained circannual rhythms for up to 9 years. Entrainment was evident only by squirrels exposed to seasonal change in irradiance.

Seasonal changes in cation transport in red blood cells of grey squirrel (Sciurus carolinensis) in relation to thermogenesis and cellular adaptation to cold. 1. Unidirectional influx of 42K was measured in red cells of grey squirrels at seasonal intervals over two years. 2. Na/K pump-related (i.e. ouabain-sensitive) K influx at 37 degrees C was maximal in cells collected in January and was more than three times greater than cells collected in summer. Na/K pump activity, maximized by loading the cells with Na, exhibited a similar difference. 3. At 5 degrees C in fresh cells, ouabain-sensitive K influx, expressed as per cent of that at 37 degrees C, was highest in March. In Na-loaded cells it was lowest in summer. 4. Passive "leak" K influx (i.e., the residual influx remaining in presence of ouabain and bumetanide) was highest in October, and declined progressively to the summer months, when it was only 27% of that in October. 5. Cotransport (i.e., bumetanide-sensitive K influx) exhibited the same seasonal pattern as Na/K pump activity in fresh cells. 6. Net gain of Na in cells stored at 5 degrees C for three days in March was less than half of that in January or summer. 7. High transport activity in January may correlate with a requirement for increased non-shivering thermogenesis. However, red cells of grey squirrels exhibit maximum resistance to low temperature in March and at this time resemble the red cells of hibernating mammals.

Effects of oral, intraperitoneal and intrajugular rehydrations on water retention, rumen volume, kidney function and thirst satiation in goats. 1. In order to test the hypothesis that peripheral receptors are involved in the control of fluid re-distribution following acute dehydration and rapid rehydration, peripheral rehydrations (oral or intraperitoneal) were compared with central (intrajugular) rehydration. 2. The experiments were carried out with four goats dehydrated to about 20% of their initial mass. 3. Following peripheral rehydration, a higher proportion of water was retained in comparison with central rehydration, and this was related to a more effective kidney retention mechanism, i.e. lower GFR and higher tubular reabsorption. 4. Higher proportions of water were retained in the rumen in the peripheral rehydrations in comparison with the central one apparently due to increased saliva secretion. 5. Thirst saturation was more effective with the peripheral rehydration in comparison with the central one and was related to the amount retained in the rumen and to peripheral blood expansion (or dilution).

The effect of ionizing radiation on the viability of Trichomonas vaginalis. 1. The effects of continuous gamma radiation on the viability of Trichomonas vaginalis (ATCC 30001) were assessed by a colony count technique. 2. A triphasic survival curve showed an initial shoulder (Dq) of 3 Gy followed by three linear curves with D0 values of 34, 300, and 90 Gy. 3. Sterilization of 10(6) cells/ml occurred from 1600 to 1800 Gy of radiation. 4. Population growth, subsequent to radiation exposure of 17-100 Gy, showed an increased lag time followed by a faster rate of growth, compared with unirradiated cells. 5. Trichomonas vaginalis is more sensitive to ionizing radiation than free-living protozoa and appears as radiosensitive as those parasitic protozoa examined in radioattenuation experiments.

Taurine uptake in chicken leukocytes and erythrocytes. 1. The intracellular taurine concentration and rate of taurine uptake of chicken erythrocytes and two leukocyte populations were determined from one to six weeks of age. 2. Plasma taurine concentrations increased significantly from the time of hatching to week 2 and remained constant thereafter. 3. Intracellular taurine concentrations in both leukocyte populations increased significantly with age without any significant change in the erythrocytes. 4. Taurine uptake rate for erythrocytes was significantly higher at weeks 1-3 while both leukocyte populations showed no significant change during the six week period studied.

Increased GDP binding and thermogenic activity in brown adipose tissue mitochondria during arousal of the hibernating garden dormouse (Eliomys quercinus L.). 1. The thermogenic activity of brown adipose tissue in hibernating garden dormice during hypothermic torpor and at different states of arousal were studied. High levels of GDP binding were observed on isolated brown fat mitochondria, indicating that the thermogenic proton conductance pathway is very active in brown fat during arousal. 2. In order to investigate this phenomenon, the uncoupling protein was assessed by immunological assay and the mRNA for UCP was analysed. 3. Animals during arousal exhibited neither increase in UCPmRNA nor an increase in UCP. 4. Our results suggest that during the rewarming of garden dormice there is an acute unmasking of GDP binding sites on the protein.

Lipoprotein status in Sprague-Dawley and LA/N-corpulent rats as affected by dietary carbohydrates. 1. To compare the impact of type of carbohydrate, genotype and phenotype on the synthesis and levels of plasma lipoprotein protein. Sprague-Dawley rats and carbohydrate-sensitive LA/N-corpulent (cp) rats were fasted (2 days) and then fed diets containing 54% carbohydrate as either sucrose, fructose or cooked cornstarch for 2 days. 2. The amount of 3H-protein present in the VLDL + chylomicron fraction of Sprague-Dawley rats 2 hr after injection of 3H-leucine was affected by type of dietary carbohydrate: sucrose greater than fructose greater than starch. 3. Obese and lean LA/N-cp rats fed diets containing sucrose or fructose had lower concentrations of HDL protein and higher levels of 3H-protein in VLDL + chylomicron fraction than those fed starch. 4. Obese LA/N-cp rats had more HDL protein and higher levels of 3H-protein in VLDL + chylomicron fraction than their lean littermates.

Butyrylcholinesterase activity in plasma of rats and rabbits fed high-fat diets. 1. Comparative studies with rats and rabbits were carried out to address the question as to whether the amount of dietary fat affects butyrylcholinesterase (EC 3.1.1.8.) activity in plasma. 2. Plasma butyrylcholinesterase activities were about 5-fold higher in rabbits than rats. 3. Ad libitum feeding of diets enriched with corn oil caused increased body weights in rabbits but not in rats 4. Plasma butyrylcholinesterase activities of rats were increased with increasing intakes of corn oil. In rabbits, such an effect could not be demonstrated conclusively. 5. Evidence is presented that in rats the substitution of dietary corn oil for isocaloric amounts of either carbohydrates or protein produces similar increases in plasma butyrylcholinesterase activity. 6. This suggests that among macronutrients the amount of fat primarily determines butyrylcholinesterase activity in the plasma of rats.

Relative effects of feeding saturated fats and cholesterol on fluidity of rabbit lipoproteins. 1. The effects of saturated fat and cholesterol on lipoprotein fluidity were tested in New Zealand white rabbits fed diets containing corn oil (CO) or cocoa butter (CB) with and without added 0.2% cholesterol. 2. Saturated fats had little effect on fluidity in any lipoprotein fraction. 3. Cholesterol feeding dramatically reduced fluidity in VLDL and LDL, but minimal change was noted in HDL. 4. Cholesterol-fed rabbits were hypercholesteroloemic throughout the 10-month study. 5. The rabbits became adapted to cholesterol feeding as VLDL became more fluid with time.

Dietary carbohydrate level and glucose metabolism in turkey poults. 1. Feeding British United turkeys (BUT) and Nicholas turkeys (NT) diets with varying carbohydrate levels for 24 hr post-hatch resulted in lower hepatic glucose-6-phosphatase activity and higher plasma glucose levels as dietary carbohydrate level was increased. 2. There were no differences between the strains in liver weight or glucose-6-phosphatase activity, but BUT exhibited higher plasma glucose values than did NT at the two highest levels of carbohydrate. Plasma glucose did not differ between strains at the lowest level of carbohydrate or in fasted poults. 3. Blood glucose values were consistently higher in both strains when sampled 1 hr after initial sampling of fasted poults. 4. Both strains were able to maintain the 1 hr blood glucose levels through 24 hr when kept at approximately 37 degrees C. 5. When held at approximately 21 C for the first hour and at approximately 37 degrees C through 24 hr fasted NT were able to maintain the initial blood glucose rise while BUT were not.

Identification and properties of an alpha-amylase from a strain of Eubacterium sp. isolated from the rat intestinal tract. 1. A bacterial amylase was isolated from the intestinal content of monoxenic rats inoculated with Eubacterium sp. B86. 2. Affinity chromatography on cross-linked starch allowed its separation from rat endogenous amylases. 3. The bacterial enzyme was characterized by its pI, molecular weight and action pattern. It behaves as a typical endo-amylase (alpha-amylase).

Effects of diabetes and dietary manipulation on rat parotid gland secretory response to sympathetic nerve stimulation. 1. The effects of streptozotocin-diabetes, bulk-diet, low protein diet (8%) and protein-calorie malnutrition on parotid gland response to sympathetic nerve stimulation were studied in male Wistar rats. 2. Mean body weights were considerably less in diabetic and protein-calorie malnourished rats than in the other groups, but parotid gland weight was reduced only in animals placed on a low-protein diet. 3. Salivary flow rate (microliter/min/g tissue) and total protein output (mg secreted/g tissue) were reduced in diabetic rats. 4. Salivary composition was altered in diabetes and protein-calorie restriction, and the specific changes were unique to each condition. 5. Thus, with the possible exception of gland weight the effects of diabetes on parotid gland structure and function are not related to either hyperphagia or nutritional status.

History of neurophysiology in Japan. The progress of the neurophysiological research in Japan during the past 45 years is related. Modern Japanese neurophysiology started immediately after the end of World War 2. The introduction of microelectrode techniques contributed greatly to most fields of Japanese neurophysiology. These techniques were used to study most neurophysiological phenomena: sensory physiology including vision, audition, chemical sensitivity, and other modalities; learning and memory. These techniques plus lesions, transplants, and behavioral physiology were used to study circadian rhythm, posture and motor control, and sex. These and other techniques were used to study neural plasticity, immunity, membrane excitability, pain and other psychophysiological functions. The disciplines advanced quickly into multidiscipline approaches into not only electrophysiological, but biophysical, biochemical and immunological research fields. From the past research results our neurophysiologists can be expected to advance rapidly toward further development in the future of Japanese neurophysiology.

Membrane currents and pharmacology of retinal bipolar cells: a comparative study on goldfish and mouse. We obtained solitary bipolar cells using enzymatic (papain) dissociation of the goldfish and mouse (C57BL/6J, adult) retinae and measured the membrane currents of these cells by whole-cell patch clamp. Bipolar cells of these two species showed two main differences. A. Ca current 1. In the mouse, depolarization evoked a transient Ca current that had maximal amplitude at about -30 mV. 2. The Ca conductance was activated by voltage steps to potentials greater than -60 mV and inactivated fully at potentials greater than -20 mV. 3. The mouse Ca current was insensitive to Cd2+ or dihydropyridine. 4. Contrary to mouse, goldfish bipolar cells had a sustained Ca current, which was activated over a more positive potential range (greater than -30 mV), blocked by either 50 microM Cd2+ or 10 microM nifedipine, and markedly augmented by 10 microM Bay K8644. 5. The transient character of the Ca current in mouse bipolar cells may help to shape phasic responses of ganglion cells, while in goldfish the sustained nature of Ca current may contribute to shape tonic responses of ganglion cells. B. Pharmacology 1. We examined the effects of the inhibitory transmitters, glycine and GABA, on bipolar cells. 2. GABA produced strong inhibitory effects on bipolar cells of both goldfish and mouse. 3. The highest GABA sensitivity was found at the bipolar cell axon terminal, the site of reciprocal connection with amacrine cells. 4. GABA increased the Cl conductance. 5. Unlike GABA, glycine was effective only on the mouse bipolar cells. Axon terminals showed the highest glycine sensitivity. 6. Glycine-induced currents were also carried by Cl ions. 7. Since ECl in intact cells is assumed to be -55 mV, both GABA and glycine are thought to generate hyperpolarizing responses in cells maintained at their resting potential (ca. -45 mV). 8. The present study suggests that inhibition from amacrine cells to bipolar cells, found in both species, is mediated by different transmitters.

Neurotransmitters and neuromodulators controlling the anterior byssus retractor muscle of Mytilus edulis. 1. The anterior byssus retractor muscle (ABRM) of Mytilus edulis is innervated by at least two kinds of nerves, excitatory and relaxing nerves. The principal neurotransmitters released from these nerves have been shown to be acetylcholine and serotonin, respectively. 2. Some other monoamines, such as dopamine and octopamine, and various peptides, such as FMRFamide-related peptides, Mytilus inhibitory peptides, SCP-related peptides and a catch-relaxing peptide, may also be involved in the regulation of the muscle as neurotransmitters or neuromodulators. 3. The ABRM seems to be typical of invertebrate muscles controlled by multiple neurotransmitters and neuromodulators.

Phosphoinositides and synaptic function in NG108-15 neuroblastoma x glioma hybrid cells. 1. The second-messengers system of bradykinin (BK) receptors was examined in NG108-15 neuroblastoma x glioma hybrid cells. 2. An application of BK induced an immediate outward (K+) current and acetylcholine (ACh) release, which are generated through inositol 1,4,5-trisphosphate (InsP3)-dependent Ca2+ ions. 3. Application of phorbol dibutyrate (a protein kinase C activator) produced a voltage-dependent inward current and inhibited another K+ (M)-current. 4. A similar current response has been produced by ACh in NG108-15 cells transfected with rodent muscarinic ACh receptor I and III subtype genes. 5. These results suggest a dual and time-dependent role for these two intracellular messengers in the control of neuronal signalling by BK and ACh.

Effects of astroglia on the development of cultured neurons from embryonic rat cerebral cortex. 1. Previous studies from our laboratory demonstrated that subcultured astroglia enhance neurite outgrowth and survival of cultured neurons from embryonic rat cerebral cortex, but suppress proliferation of neuroblasts. 2. In the present study, the mechanisms of these three effects were further investigated. 3. Dissociated neurons were seeded on poly-L-lysine-coated coverslips which were plated on subcultured astroglia, and the survival, proliferation and neurite outgrowth of the neurons were investigated. Under these conditions, survival and antimitotic effects were also observed, while neurite extension was not stimulated. 4. The results clearly indicate that neuronal survival and proliferation are regulated by soluble factors produced by astroglia. 5. We also postulated that the neurite-promoting effect of astroglia is mediated by cell-cell contact. 6. This idea was confirmed by the finding that neurite extension was enhanced when the neurons were cultured directly on heat-treated astroglia. 7. The neurite-promoting effect was found to be specific to astroglia. 8. We preliminarily characterized the astroglial surface neurite-promoting factors (ASNPFs). 9. The relationship of laminin to ASNPFs was examined by using antibody to laminin. Laminin antibody did not inhibit the ASNPF activity. 10. The effect of digestion of heat-treated astroglia with enzymes (sialidase and endo-beta-galactosidase) on the ASNPF activity was also examined. 11. These enzyme treatments did not inhibit the ASNPF activity. 12. These results suggest that enhancement of the neurite-promoting activity is not associated with the sugar moiety of ASNPFs.

Multiple opioid receptors and GTP-binding proteins. We believed that GTP-binding protein (G-protein)-coupling receptor always transduces stimulatory signals to G-proteins. From our recent experiments using reconstitution techniques, however, it was revealed that some receptors transduce an inhibitory or no signal to G-proteins in specific tissues, despite some interaction between them. Here we discuss the molecular basis of mechanisms of such diverse modes of functional coupling between different subtypes of opioid receptors and G-proteins.

Monoaminergic interaction in the central nervous system: a morphological analysis in the locus coeruleus of the rat. The locus coeruleus of the rat is richly innervated by many aminergic neurons varying in amine content and in site of origin. There are adrenergic and noradrenergic neurons originating in the medulla oblongata, dopaminergic from the hypothalamus, serotonergic from the mesencephalon and also intrinsic noradrenergic neurons in the locus coeruleus complex. Of these, adrenergic and dopaminergic inputs appear relatively specific and powerful.

Comparative studies on the structure of human tyrosine hydroxylase with those of the enzyme of various mammals. 1. Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in catecholamine biosynthesis. 2. The structures of TH from various species have been elucidated. 3. We have cloned and determined the sequences of four types of human TH cDNA and human TH genomic DNA. 4. We have compared the amino acid sequences of TH from various species. 5. The results indicate that the amino acid sequences of TH are highly conserved among various species, and that TH consists of the regulatory domain containing serine residues which are phosphorylated by protein kinases and of the catalytic domain where the substrates, tyrosine and oxygen, and the cofactor, tetrahydrobiopterin, are bound. 6. Comparison of amino acid sequences among TH from various species can give us useful information on the functional importance of each amino acid residue.

Grafting of catecholaminergic cells in the mammalian brain and reconstruction of disturbed function: basic problems to be solved. 1. Catecholaminergic cells were grafted in the caudate nucleus of hemi-Parkinsonian model rats, and the following problems were discussed. 2. Phenotypic plasticity and development of catecholamine-producing cells. 3. Mechanism of motor recovery. 4. Trophic factor. 5. Regulation of immunological responses. 6. Long-term survival and security.

Comparative aspects of horizontal ocular reflexes and their cerebellar adaptive control in vertebrates. 1. Vestibuloocular reflex and optokinetic eye movement response are ocular reflexes conjointly subserving stability of retinal images. 2. These reflexes exhibit marked adaptability under sustained imposition of retinal errors. 3. Comparative studies in various vertebrate species reveal the importance of this adaptability as fundamental neural function based on plasticity of cerebellar synapses.

Cerebral cortical and brainstem areas related to the central control of lens accommodation in cat and monkey. 1. Studies on the central nervous system related to lens accommodation in cat and monkey were reviewed. 2. During the last decade, a considerable amount of neurophysiological data on the peripheral innervation of the ciliary muscle, properties of parasympathetic oculomotor neurons and mesencephalic reticular neurons have accumulated. 3. Neurophysiological and anatomical evidence supporting the contribution of the cerebellum to lens accommodation has been obtained. 4. Recently, cerebral cortical neurons in the parieto-occipital cortex with activities related to lens accommodation were found in cat and monkey.

Electrophysiological analysis of structural aspects of voltage-dependent SR K+ channel. This article presents a brief review on the electrophysiological analysis of the structural aspects of the voltage-dependent SR (sarcoplasmic reticulum) K+ channel. In the first half, early attempts to determine the physical dimensions of the ion conducting mechanism such as the mouth, narrow tunnel, or ion selective filter of the channel, are reviewed. The depicted cartoon of the SR K+ channel, as an extremely short, busy district with a big mouth on each side, is quite similar to the recently-obtained reconstructed structural image of the acetylcholine receptor channel. In the latter half, we introduce our recent attempts to draw a physical image of the gating mechanism of the SR K+ channel. We examined, for example, the location of the gate and the voltage sensor, and the relationship between them. It is suggested that the gate and the sensor are connected tightly and that the sensor would be exposed to the surface of the lumen side of SR when the gate opens. Finally, the issue of substates in SR K+ channel is discussed. It is implied that the substrate-conductances reflect a partial occlusion of the pore by an intermediate-open gate.

Retarded cerebral growth of hormone-deficient mice. 1. Both the Snell dwarf mouse (dw) and the growth hormone-deficient Little mouse (lit) exhibit a microcephalic cerebrum with hypomyelination, retarded neuronal growth, and underdevelopment of axons and dendrites. 2. The hypomyelination is due to arrested glial proliferation, suggesting that the action of growth hormone on the proliferation and maturation of both glial and neuronal cells is a necessary precondition of myelin formation, apart from the complementary or synergistic actions of thyroxine. 3. In contrast, the cerebral hypomyelination present in the inherited hypothyroid mouse (hyt) is not related to arrested glial proliferation, demonstrating that thyroid hormones can act independently on myelinogenesis.

Cellular physiology of epileptogenic phenomena and its application to therapy against intractable epilepsy. 1. During pentylenetetrazol-induced bursting activity which is characteristic intracellular potential change of seizure discharge, intracellular stored calcium is released and moved toward the inner surface of the cell membrane. Calcium is released from lysosome-like granules with morphological changes. During bursting activity, the intracellular free calcium level was higher than the normal state. During bursting activity, intracellular protein of 5 kDa and 15 kDa was changed qualitatively and quantitatively. 2. Primary cultured cerebral cortical neurons of rats and mice showed spontaneous regular firing, and by PTZ application, showed bursting activity. A single potassium channel showed the random open-close state in the normal state and showed burst type open-close state after PTZ application. 3. Primary cultured cerebral cortical neurons of the E1 mouse, the epilepsy animal model, showed developmental defects in neurite extension and content of gangliosides, in addition to their very high susceptibility to convulsions. 4. A new antiepileptic drug, TJ-960, which originates from a mixture of nine herbal drugs, normalized the above-mentioned seizure-related, calcium-related intracellular pathological phenomena. TJ-960 normalized cytochalasin-B-induced looping phenomena and protected the neuron damage induced by cytochalasin B in addition to anticonvulsant action. TJ-960 also completely normalized the cobalt-induced EEG changes and also protected against neuron damage in the hippocampus induced by cobalt application to the cerebral cortex. TJ-960 normalized the developmental defects of the E1 mouse neuron. 5. For better therapy of epilepsy, it is probably necessary to normalize the developmental defects and to protect against neuron damage in addition to inhibition of seizure activity.

T-type calcium channel in mammalian CNS neurones. 1. In all neurones freshly isolated from various brain regions of newborn, adult and aged rats, the T-type Ca2+ currents were elicited by step depolarizations to potentials more positive than -60 mV from a holding potential of -100 mV, and reached a peak in the current-voltage relationship around -30 mV. 2. The activation and inactivation processes were highly potential-dependent, and the latter was fitted by a single exponential function. 3. It was concluded that mammalian brain neurones possess a definite class of T-type Ca2+ channel characterized by both current kinetics and ion selectivity for Ca2+, Ba2+ and Sr2+. However, the pharmacological nature of the T-type Ca2+ channel differed from that in other tissues such as cardiac and smooth muscle cells, peripheral neurones, and cultured cells.

Localization and regulation of mRNAs in the nervous tissue as revealed by in situ hybridization. 1. In situ hybridization histochemistry permits the study of specific mRNAs of neuropeptides, enzymes involved in the synthesis of neurotransmitters, receptors and proteins associated with glial cells in nervous tissue. 2. The central and peripheral nervous systems are composed of heterogeneous elements and specific regulatory mechanisms occur in specific cells. 3. This review will focus on the localization and regulation of different mRNAs in the nervous system from Drosophila to human, as revealed by in situ hybridization histochemistry.

Spider toxin and the glutamate receptors. A neurotoxin (JSTX) was isolated from the venom of spider (Nephila clavata). JSTX blocked both the excitatory postsynaptic (EPSPs) and glutamate-induced potentials in lobster neuromuscular synapse and squid giant synapse. In mammalian central nervous system, JSTX blocked the EPSPs in CA1 pyramidal neurons resulting from stimulation of Schaffer collateral/commissure input. Pharmacological investigation showed that JSTX preferentially suppressed quisqualate/kainate receptor subtypes but was much less effective on NMDA receptor. Using synthesized spider toxins we studied the structure-activity relationship and found that the 2,4 dihydroxyphenylacetyl asparagine in the toxin structure was responsible for suppressive action, while the remaining part containing a polyamine was related to the agonist binding site with the polycationic part enhancing the toxic activity. Labeling of synthesized JSTX was used for histochemical as well as biochemical studies. Using autoradiography, 125I-JSTX-3 was found to bind at the lobster neuromuscular synapse. Histochemical study utilizing the interaction of biotinylated JSTX-3 with avidin showed specific binding of the toxin in rat cerebellum and hippocampus. JSTX-3-binding protein was purified from rat brain by affinity chromatography. SDS-PAGE of the affinity purified protein showed at least 4 bands ranging from 40 to 70 kDa.

Isolation of achatin-I, a neuroactive tetrapeptide having a D-phenylalanine residue, from Achatina ganglia, and its effects on Achatina giant neurones. 1. We have isolated a neuroexcitatory tetrapeptide having a D-phenylalanine (Gly-D-Phe-L-Ala-L-Asp) from the ganglia of Achatina fulica Férussac. This peptide was termed achatin-I (Kamatani et al., 1989). In the present report, we shall present highlights from the original paper concerning the process of peptide isolation and the examination of its effects. 2. From the ganglia of about 30,000 animals, we obtained 50 micrograms of achatin-I and 17 micrograms of its stereoisomer consisting of only L-amino acid residues (Gly-L-Phe-L-Ala-L-Asp) which was termed achatin-II. The data of instrumental analyses (1H-NMR, SIMS, CD and HPLC) of isolated achatin-I and achatin-II were identical to those of synthetic ones. 3. Achatin-I showed marked excitatory effects on the three Achatina giant neurones, PON (periodically oscillating neurone), TAN (tonically autoactive neurone) and v-RCDN (ventral-right cerebral distinct neurone), whereas achatin-II had no effect. Among their stereoisomers, [D-Ala3]-achatin-I (Gly-D-Phe-D-Ala-L-Asp) had slight excitatory effects on the Achatina neurones tested. Amide derivatives of achatin-I and achatin-II were ineffective. 4. Dose-response curves of achatin-I and [D-Ala3]-achatin-I for producing the inward current of PON were measured under voltage clamp at a holding membrane voltage (Vh) of -50 mV.(ABSTRACT TRUNCATED AT 250 WORDS)