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Protracted treatment of C57B1 mice with Zn-DTPA after 241Am injection reduces the long-term radiation effects. Repeated intraperitoneal injections of ZnNa3-diethylenetriaminepenta-acetate (ZnDTPA), once a week, during 8 successive weeks, and starting 4 days after injection of 58 and 373 kBq 241Am/kg to C57B1 mice, were an effective protection against long-term radiation damage. At both dose levels of 241Am, Zn-DTPA reduced the 241Am concentration in bones by between 33% and 45%, and in the liver by 97%. Mean survival with 241Am was shortened in Zn-DTPA-treated mice, by 17% at the lower dose level and by 70% at the higher dose level. After treatment with DTPA at the lower 241Am level, survival became equal to that of control mice without 241Am, while at the higher level life span was still shortened. After the lower 241Am dose the incidence of bone tumours, liver carcinomas and the total number of all malignant tumours were significantly reduced by chelation therapy. The decrease in bone tumour incidence was proportional to the decrease in 241Am concentration and reduction in cumulative radiation dose in bone after chelation therapy. The incidence of liver carcinomas was reduced to that in non-241Am-injected mice and the reduction was thus proportional to the 97% reduction in 241Am concentration of the liver at the end of chelation therapy. After the higher 241Am dose no tumours showed up in sham-treated mice, probably due to the overkill effect on the cells at risk. In the corresponding Zn-DTPA-treated mice, bone tumours and a few other malignant tumours were observed.

In vitro assessment of cardiac performance after irradiation using an isolated working rat heart preparation. The effect of irradiation on cardiac function was assessed using an isolated working rat heart preparation. The animals were given single doses of X-rays in the range 15-30 Gy to their hearts. Cardiac output (CO = aortic flow + coronary flow), heart weight and body weight were followed for a period of 10 months after treatment. Irradiation led to a decrease in cardiac function. This reduction was dose-dependent and progressive with time after treatment. The shape of the Frank-Starling curves constructed for irradiated hearts suggests a loss of contractile function of the myocardium. Coronary flow rates measured in 'working' hearts and in 'Langendorff' hearts were not significantly changed by the irradiation treatment. The isolated working rat heart preparation proved to be a simple and suitable animal model for the investigation of irradiation-induced cardiotoxicity.

Protection of pig epidermis against radiation-induced damage by the infusion of BW12C. BW12C, which was developed as an agent for the treatment of sickle cell anaemia, increases the binding of oxygen to haemoglobin and hence reduces the availability of oxygen to tissues. Due to these changes in oxygen availability BW12C could act as a protector against radiation-induced injury to normal tissues. In this study the potential value of BW12C, as a radioprotector, was studied in the irradiated epidermis of the pig. The infusion of BW12C caused an instant left shift of the oxygen dissociation curve, an effect that lasted for approximately 1.5 h. This left shift in the oxygen dissociation curves increased with increasing dose of the drug. There appeared to be no long-term systemic effects produced by doses of 20-100 mg/kg of BW12C. In the first 90 min after the infusion of BW12C skin fields were irradiated with single doses of beta-rays from strontium-90 plaques. The incidence of moist desquamation was used as an endpoint for assessing the severity of the radiation response. With animals breathing approximately 70% oxygen in the anaesthetic gas mixture, the ED50 values for moist desquamation were 30-31 Gy after a dose of 30 and 50 mg/kg, and 37-38 Gy for 75 and 100 mg/kg doses of BW12C. These ED50 values were significantly higher than the value of 27.3 Gy for radiation alone. This indicated dose modification factors (DMF) with mean values of approximately 1.13 and approximately 1.40 for irradiation following the infusion of low (30-50 mg/kg) and high (75-100 mg/kg) doses of the drug, respectively. With the animals breathing air (approximately 21% of oxygen) in the 2% halothane anaesthesia gas mixture, irradiation in the presence of 30 and 50 mg/kg of BW12C resulted in ED50 values of approximately 39 Gy for moist desquamation, which was significantly higher than the value of 31.2 Gy for radiation alone. Surprisingly, a higher dose of 75 mg/kg of BW12C resulted in a lower ED50 value for moist desquamation of 34.38 Gy. Irradiation in the presence of a dose of 100 mg/kg of BW12C produced an ED50 value which was not significantly different from that for radiation alone. In the situation where animals were breathing air (approximately 21% oxygen) during irradiation a DMF of 1.14 was obtained for irradiation alone, when the results were compared with those for irradiation alone with approximately 70% oxygen in the anaesthetic gas mixture.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological indicators for radiation damage. Methods for estimating radiation dose using biological indicators have made rapid progress during recent years. Chromosome analysis in lymphocytes still plays a central role, but it is no longer the only quantitative system in biological dosimetry. The best approach seems to be to combine several of the assays exploiting their specific advantages: the high sensitivity in the case of dicentrics in lymphocytes (starting at about 0.05 Gy low-LET radiation), the broad dose range covered by the electron spin resonance technique (0.5-100 Gy), the possibility of identifying the localization of partial-body exposure when determining hair diameter, and the individual prognostic information obtained from changes in the frequency of blood cells after exposures exceeding about 1 Gy. In specific situations other methods may replace or supplement these indicators for radiation damage.

Characterization of free radicals in gamma-irradiated polycrystalline uridine 5'-monophosphate: a study combining ESR, spin-trapping and HPLC. Free radicals generated in gamma-irradiated polycrystalline uridine 5'-monophosphate (5'-UMP) were studied by ESR, spin-trapping and high-performance liquid chromatography (HPLC). After gamma-irradiation at 0 degree C (70kGy), poly-crystalline 5'-UMP was dissolved in an anaerobic aqueous solution of 2-methyl-2-nitrosopropane as a spin trap at room temperature. Since an ESR spectrum consisting of several components was observed immediately after irradiation, these components were separated with reverse-phase HPLC in the ion-suppression mode and again analysed by ESR spectrometry. Although HPLC ultimately gave four spin-adducts, one component that was originally present disappeared during HPLC. Spin adducts due to two types of C6 radicals were identified. One of these was thought to be formed by electron addition and subsequent protonation at the C6 position, and the other was presumed to be produced by electron addition and subsequent protonation at the O4 position. The spin adducts derived from the C5 and C5' radicals were also identified. The spin adduct that disappeared during HPLC was thought to correspond to the C4'-centred radical. Computer simulation of ESR spectra was carried out to estimate the hyperfine splitting constants.

Rates for repair of pBR 322 DNA radicals by thiols as measured by the gas explosion technique: evidence that counter-ion condensation and co-ion depletion are significant at physiological ionic strength. Rates of repair of pBR 322 plasmid DNA radicals by thiols of varying net charge (Z) at pH 7 and physiological ionic strength were measured using the oxygen explosion technique. The extent of conversion of supercoiled to relaxed circular plasmid was measured by HPLC as a function of the time of oxygen exposure before or after irradiation, the time-courses being fitted by a pseudo-first-order kinetic expression with k1 = k2[RSH]. Values of k2 (M-1 S-1) were: 2.1 x 10(5) (GSH, Z = -1), 1.4 x 10(6) (2-mercaptoethanol, Z = 0), 1.2 x 10(7) (cysteamine, Z = +1), 6.6 x 10(7) (WR-1065 or N-(2-mercaptoethyl)-1,3-diaminopropane, Z = +2). The approximately 6-fold increase in rate with each unit increase in Z is attributed to concentration of cationic thiols near DNA as a consequence of counter-ion condensation and reduced levels of anionic thiols near DNA owing to co-ion depletion. The results are quantitatively consistent with chemical repair as a significant mechanism for radioprotection of cells by neutral and cationic thiols under aerobic conditions, but indicate that repair by GSH will compete effectively with oxygen only at low oxygen tension.

The effects of counter-ion condensation and co-ion depletion upon the rates of chemical repair of poly(U) radicals by thiols. Bimolecular rate constants for reactions of poly(U) radicals with a series of thiols of varying net charge (Z) were measured by pulse radiolysis with conductivity detection at low ionic strength. At pH 7 and 18 degrees C the values of k2 (M-1s-1) were: reduced glutathione (Z = -1), less than 500; 2-mercaptoethanesulphonic acid (Z = -1), 1.5 x 10(3); 2-mercaptoethanol (Z = 0), 1.8 x 10(5); cysteine (Z = 0), 2.0 x 10(5); cysteamine (Z = +1), 4.1 x 10(7). Values determined at pH 4 were: 2-mercaptoethanol, 6.1 x 10(5); cysteamine 2.2 x 10(8); N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065, Z = +2), 4.6 x 10(8). The variation in rate with structure could not reasonably be attributed to inherent reactivity differences in the thiols and was ascribed to inhomogeneous distributions of the thiols in solution resulting from electrostatic interactions. Thus, cationic thiols are concentrated approximately 100-fold near poly(U), relative to neutral thiols, as a consequence of counter-ion condensation, whereas anionic thiols have approximately 100-fold lower concentration near poly(U) than neutral thiols as a result of co-ion depletion. These results show that the ability of a thiol to repair radical sites in a polyanion is dramatically influenced by its net charge as a consequence of the counter-ion condensation and co-ion depletion phenomena.

Size distribution of DNA molecules recovered from non-denaturing filter elution. DNA fragments removed from the filter during non-denaturing filter elution were collected and loaded on top of neutral sucrose gradients. Their size distribution was determined by low-speed centrifugation in neutral sucrose gradients. The average size of eluted DNA was found to be approximately 110 S, yet the average size of DNA collected after short elution times was found to be slightly larger than that after long elution times. It is concluded that the size of eluted DNA fragments is not correlated with their elution rate, and it is proposed that shear forces generated at the pores of the filter cause degradation of the DNA. A comparison of the sedimentation profiles of carefully prepared cellular DNA before and after elution revealed that the generated shear forces during elution break down the DNA to an extent equivalent to around 20,000 DNA double-strand breaks (dsb) per G1 cell. The size of DNA fragments decreased with increasing radiation dose; however, five times more dsb were found than expected after exposure to radiation alone. It is proposed that this excess of dsb may derive from the transformation of other radiation-induced lesions to dsb under the action of the shear forces generated during elution.

Negative supercoiling increases the sensitivity of plasmid DNA to single-strand break induction by X-rays. Negatively supercoiled topoisomers of the plasmid pIBI 30 were irradiated with 250 kV X-rays and assayed for strand scission by agarose gel electrophoresis. The survival of supercoiled molecules (Form I) decreased exponentially with increasing X-ray exposure and the dose required to reduce the fraction of DNA in Form I to 37% of its value in unirradiated controls (D37) decreased with increasing negative superhelicity. This enhanced radiation sensitivity of underwound DNA is tentatively attributed to the transient denaturation of the double helix that increases the susceptibility of individual strands to free radical attack.

Comparison of DNA double-strand break rejoining as measured by pulsed field gel electrophoresis, neutral sucrose gradient centrifugation and non-unwinding filter elution in irradiated plateau-phase CHO cells. The initial (up to 30 min) rate of DNA double-strand break (dsb) rejoining was measured in irradiated plateau-phase CHO cells, in a set of parallel experiments using the same cell suspension, by means of non-unwinding filter elution, neutral sucrose gradient centrifugation, and two pulsed-field gel electrophoresis assays: asymmetric field inversion gel electrophoresis (AFIGE) and clamped homogeneous electric field (CHEF) gel electrophoresis. The rate of DNA dsb rejoining was compared to the rate of rejoining of chromatin breaks measured, also in the same cell population, using the technique of premature chromosome condensation (PCC). Two radiation exposures, 25 Gy and/or 50 Gy, were used and applied to the individual parts of the experiments according to the sensitivity of the assay under investigation. Similar values for the initial rate of DNA dsb rejoining were obtained with all assays used, with t 1/2 ranging between 10 and 12 min after exposure to 25 Gy and between 15 and 20 min after exposure to 50 Gy. The initial rate of rejoining of chromatin breaks was slower than that of DNA dsb and occurred with t 1/2 of 87 min. The results suggest that all major techniques currently used for assaying rejoining of DNA dsb give similar results despite their widely different biophysical basis, and indicate that more information is required before a direct correlation between rejoining of DNA dsb and rejoining of chromatin breaks can be established.

Nucleoid sedimentation analysis of DNA superstructure, gamma-radiation-induced damage and repair in human and chacma baboon (Papia ursinus) peripheral lymphocytes. The use of the chacma baboon (Papio ursinus) in radiobiological investigations justifies special attention because it answers many of the criteria of parallelism to the human. The present study was undertaken to establish whether in vitro gamma-radiation effects in chacma baboon and human lymphocytes are comparable. The sensitive and rapid nucleoid sedimentation technique was employed to evaluate in vitro DNA superstructure, damage and repair in readily obtainable radiosensitive peripheral lymphocytes. Dose-response curves after 60Co gamma-irradiation were obtained, and by applying single-hit kinetics of the target theory, an estimation of molecular masses of the supercoiled domains was made. The baboon and human lymphocytes produced analogous results, while an ethidium bromide intercalation study also revealed a similarity in average DNA superhelical density. Lymphocyte DNA repair after 0.5-4.0 Gy gamma-irradiation and repair times from 0.5 to 5.0 h were evaluated. The repair data obtained from baboon and human cells after 2.0 Gy irradiation compared favourably in extent of DNA repair as well as the profiles of the kinetic curves. These findings indicate that the chacma baboon would be a useful and relevant model for further in vivo radiobiological studies on lymphocytes. The effects of sedimentation conditions and advantages of using vertical-tube rotors in the nucleoid sedimentation technique are also discussed.

Repair of potentially lethal damage by introduction of T4 DNA ligase in eucaryotic cells. The bacterial enzyme PvuII, which generates blunt-ended DNA double-strand breaks, and T4 DNA ligase, which seals adjacent DNA fragments in coupling to ATP cleavage, were introduced in mouse C3H10T1/2 fibroblasts using osmolytic shock of pinocytic vesicles. Cells were then assayed for their clonogenic ability. In agreement with previous studies by others, we find that the PvuII restriction endonuclease simulates ionizing radiation effects by causing a dose-dependent loss of reproductive capacity. Here we show that the concomitant treatment with DNA ligase considerably increases cell survival. Survival curves were shown to be dependent on the ligase enzyme dose and on ATP concentration in the hypertonic medium. We conclude that T4 DNA ligase is able to repair some of the potentially lethal damage produced by restriction endonucleases in eucaryotic cells.

Modification of X-ray induced chromosome aberration frequency by pre- and postirradiation hyperthermia of human peripheral lymphocytes. Human peripheral lymphocytes (HPL) were used to study the synergistic effects of hyperthermia and X-irradiation. The effects on the chromosome aberration frequencies of different combinations of heat and radiation exposure at different cell cycle phases were analysed at the first mitosis after irradiation. When unstimulated HPL were heated after irradiation with 2 and 3 Gy, respectively, the chromosomal aberration frequencies were significantly higher than following radiation exposure alone. Heat treatment during different phases of the cell cycle and irradiation during the G2 phase led to an increase in the aberration frequencies when the cells were heat-treated not later than 16 h before radiation. Furthermore we show that the number of breaks increased linearly with the duration of hyperthermia when HPL were heated for periods of 1-15 min at 45 degrees C 32 h before irradiation at 48 h after stimulation. Heat treatment alone during any phase of the cell cycle did not induce chromosome aberrations. The increased aberration frequencies are probably the result of inhibited repair of radiation-induced DNA strand breaks.

Synchrotron-produced ultrasoft X-rays: a tool for testing biophysical models of radiation action. Ultrasoft X-rays are useful for mechanistic studies of ionizing radiation damage in living cells due to the localized nature of their energy depositions. To date radiobiology experiments in this energy region have relied on characteristic X-rays (mainly Alk and Ck) from X-ray tubes. However, limitations in the photon intensity and the available energies from X-ray tube sources prevent a definitive characterization of the relationship between photon energy and biological damage. Synchrotron radiation has the potential to avoid these limitations, since it produces X-rays with high intensity over a continuous spectrum. We have established a synchrotron-based system for radiation biology studies using the ES-0 exposure station of the Center for X-ray Lithography at the University of Wisconsin Synchrotron Radiation Center storage ring, Aladdin. A characterization of the system including spectral and intensity properties of the photon beam is presented. The first mammalian cell survival curve for synchrotron-produced ultrasoft X-rays was generated and is presented. Cell survival curves of C3H/10T 1/2 cells using synchrotron radiation of 1.48 keV agree with previous data using Alk X-rays (1.49 keV). An RBE of 1.47 +/- 0.30 at the 10% survival level was measured with reference to 250 kVp X-rays.

Desensitization studies using perifused rat pituitary cells show that growth hormone-releasing hormone and His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 stimulate growth hormone release through distinct receptor sites. The hexapeptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6) and GH-releasing factor (GHRH) produced a rapid release of GH upon perifusion of dispersed rat pituitary cells. In contrast to the native hormone GHRH, GHRP-6 elicited a response of short duration. When perifusion of each secretagogue was continued until the cells no longer released GH, a challenge by the alternative secretagogue immediately resulted in a secondary release of GH. These results are consistent with each secretagogue causing desensitization of discrete receptor-linked second messenger pathways. Cells which were perifused for 1 min with GHRP-6 required continued perifusion with culture medium alone for 60 min before they completely regained responsiveness to a subsequent challenge with GHRP-6. Somatostatin (SRIF) was able to inhibit the action of either secretagogue completely. However, when both GHRH and GHRP-6 were perifused together, SRIF attenuated but did not block GH secretion. These perifusion data add support to conclusions derived from static cell culture studies, that GHRH and GHRP-6 act through different receptor sites and that through discrete signalling pathways their individual effects on GH release are amplified.

Cortisol reduces plasticity in the kitten visual cortex. We investigated the effect of elevated levels of cortisol on plasticity in the visual cortex of the cat. Animals were given daily injections of cortisol i.m. for 20 days starting around 35 days of age. After 10 days they were monocularly deprived, and after an additional 10 days recordings were made from the visual cortex to construct an ocular dominance histogram. The results were compared with those from normal animals of the same age, and with animals monocularly deprived for the same period but not treated with cortisol. Cortisol reduced the ocular dominance shift in a dose-dependent manner, but did not totally abolish it even at the highest doses used. Two other series of animals were recorded, one slightly later in the critical period and one slightly earlier, with care taken to give cortisol before the animals were exposed to light in the morning. In both cases, cortisol reduced the ocular dominance shift but did not abolish it. To interpret these results, we measured levels of plasma cortisol in normal cats of various ages. Average levels were fairly constant between birth and 12 months of age (0.5-1 microgram/dl), and increased slightly after that, but there was a large variation between animals. Thus elevated levels of cortisol can have a substantial effect on plasticity in the visual cortex of the cat, but the decline of the critical period for plasticity between 6 weeks and 3-5 months of age does not seem to be due to a rise in cortisol levels during this time.

An epidemiologically valuable typing method for Neisseria meningitidis by analysis of restriction fragment length polymorphisms. A restriction fragment length polymorphism (RFLP) typing method was developed for Neisseria meningitidis. A cloned EcoRI fragment from a Neisseria meningitidis Group B serotype 15P1.16 sulphonamide-resistant strain was used to probe Southern blots of total chromosomal DNA restriction fragments (enzyme AvaI). A group of 75 apparently unrelated organisms gave rise to 26 different restriction fragment length patterns and two different groups of epidemiologically related strains had RFLP patterns that were distinct for each group. The technique was highly reproducible and discriminatory. The RFLP data were compared with the results of serotyping and subtyping and isoenzyme electrophoretotyping. The RFLP data were consistent with those from the alternative typing methods; clones defined by isoenzyme analysis were subdivided by this technique. The use of RFLP typing by cloned probes should be of considerable epidemiological value.

Determination of the functional dissociation constant of a partial agonist by comparison with a higher efficacy agonist. A formula derived by Gero and Tallarida (1977) relates the equilibrium dissociation constant of a partial agonist (P) and that of a second agonist (A) of greater efficacy that acts on the same receptor. The second agonist may or may not be a strong agonist. Accordingly, if the dissociation constant (K) of one of the compounds is known, say from the method of partial irreversible receptor blockade, then the dissociation constant for the other may be determined from the complete concentration-effect curves of the compounds and the derived formula: kp = KA (Ap-Ai)Pi/(Ap + KA)Ai, where Pi and Ai are equieffective concentrations of P and A, Ap = the concentration of A that gives an effect = the maximum effect of P. The practical use of this formula is illustrated here for several agonists, and for each, the value of K obtained is compared to that obtained by partial irreversible receptor blockade. In all cases tested, the agreement is quite good, thus suggesting that this method may be a practical alternative.

[Detection of the new tumor marker MUSE 11 antigen in sera of pancreatic cancer patients: a comparison with sialyl SSEA-I antigen]. Using sera from patients with pancreatic cancer and chronic pancreatitis, we measured the level of the adenocarcinoma-associated antigen MUSE 11. A comparative study between levels of MUSE 11 and levels of sialyl SSEA-1 antigen (SLX) was also carried out. With respect to the MUSE 11 antigen, positive incidence was found in 17 out of 26 pancreatic cancer patients (65%), and in 1 out of 13 chronic pancreatitis patients (8%). Similar results were obtained from the assay of SLX levels. However, no correlation was found between the two markers. Out of 9 samples which showed MUSE 11 negative, three were positive for SLX. Out of 13 samples showing SLX negative, seven were positive for MUSE 11. Twenty out of 26 (77%) cases showed positive results for either antigen. These data suggest that the MUSE 11 antigen may be useful for the diagnosis of pancreatic cancers.

Expression of CD15 as predictor of relapse in children with acute lymphoblastic leukemia of the pre-B type. Pre-B acute lymphoblastic leukemia (ALL) was diagnosed in 37 children morphologically, histochemically, and by immunophenotyping by flow cytometry. In all patients the leukemic blasts expressed HLA-DR and CD19 (Leu-12). In 10 patients 20% or more of the blast cells expressed a myeloid antigen: CD15 (Leu-M1) in seven, CD33 (My9) in two and CD13 (My7) in one patient. All 37 children achieved complete remission, but eight relapsed. Relapse occurred in six of seven patients with CD15-positive blasts, but in only two of 27 patients with CD15-negative blasts (p = 0.0003). Thus, the occurrence of CD15 on the blasts of children with pre-B ALL shows a remarkable association with a high risk of relapse, and these patients should therefore be considered to belong to the high-risk group regardless of other prognostic factors.

6-mercaptopurine therapy in selected cases of corticosteroid-dependent Crohn's disease. Nineteen patients (12 children and 7 adults) with severe Crohn's disease, all of whom were dependent on corticosteroids, were treated with 6-mercaptopurine. All patients received a daily dose of 6-mercaptopurine of 50 mg; in two pediatric patients with a poor response after 2 months, the dosage was increased to 75 mg/day. A complete or partial response to 6-mercaptopurine therapy was noted in 47% of patients, and therapy failed in 53%. The age of the patients, prior resection, or initial symptoms did not influence the response. The clinical response was better in male than in female patients and in patients with involvement of both the small intestine and the colon than in those with only enteritis. 6-Mercaptopurine is a possible alternative to long-term corticosteroid therapy or surgical treatment in selected patients with severe Crohn's disease.

Intramuscular desferrioxamine in patients with Alzheimer's disease. Although epidemiological and biochemical evidence suggests that aluminium may be associated with Alzheimer's disease (AD), there is no convincing proof of a causal link for aluminium in disease progression. We have completed a two year, single-blind study to investigate whether the progression of dementia could be slowed by the trivalent ion chelator, desferrioxamine. 48 patients with probable AD were randomly assigned to receive desferrioxamine (125 mg intramuscularly twice daily, 5 days per week, for 24 months), oral placebo (lecithin), or no treatment. No significant differences in baseline measures of intelligence, memory, or speech ability existed between groups. Activities of daily living were assessed and videorecorded at 6, 12, 18, and 24 month intervals. There were no differences in the rate of deterioration of patients receiving either placebo or no treatment. Desferrioxamine treatment led to significant reduction in the rate of decline of daily living skills as assessed by both group means (p = 0.03) and variances (p less than 0.04). The mean rate of decline was twice as rapid for the no-treatment group. Appetite (n = 4) and weight (n = 1) loss were the only reported side-effects. We conclude that sustained administration of desferrioxamine may slow the clinical progression of the dementia associated with AD.

Prospective, randomised, multicentre trial of effect of protein restriction on progression of chronic renal insufficiency. Northern Italian Cooperative Study Group. A multicentre, prospective trial was organised to clarify the role of protein restriction in the progression of chronic renal insufficiency (CRI). 456 adult patients were assigned either a low-protein diet (0.6 g/kg body weight daily; n = 226) or a "normal" controlled-protein diet (1.0 g/kg daily; n = 230) and were stratified into three groups (A-C) with increasing baseline plasma creatinine concentrations. Each patient was followed up for 2 years or until an endpoint (a doubling of the baseline plasma creatinine or a need for dialysis) was reached. The difference between the diet groups in cumulative renal survival defined by these endpoints (27 low-protein, 42 controlled-protein) was of borderline significance (p less than 0.06). The difference in renal survival between the low-protein and controlled-protein diet groups was of borderline significance in group A (0 vs 4 endpoints), significant in group B (10 vs 21 endpoints; p less than 0.025), and not significant in group C. There were no differences among the diet groups or subgroups in mean plasma creatinine concentrations, creatinine clearance, the slope of the plasma creatinine reciprocal, or mean blood pressures. Compliance was good in the controlled-protein group but poor for the low-protein diet: the difference in protein intake between the groups was substantially less than that required by the protocol. However, there was no correlation between the progression of renal failure and protein catabolic rate. These findings offer little, if any, support to the hypothesis that protein restriction retards CRI progression: careful medical care and a "normal" controlled protein intake also allow very slow progression of CRI.

Mitochondrial encephalopathies: molecular genetic diagnosis from blood samples. Point mutations of mitochondrial DNA have been described in the muscle of patients with syndromes of myoclonic epilepsy and ragged red fibres (MERRF) and of mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS). We have found the MERRF mutation in members of 6 British kindreds; 2 of these had unusual phenotypes but all index patients had myoclonus. The MELAS mutation was detected in 17 patients from 16 families, who had a wide range of clinical features that particularly affected the central nervous system; stroke-like episodes were observed in 10.3 patients with mitochondrial DNA mutations did not have ragged red fibres on muscle biopsy, generally considered to be the morphological hallmark of mitochondrial diseases. In all 6 patients with the MERRF mutation, and 10 of 11 with the MELAS mutation, the genetic defect was easily detected in blood cells as well as muscle (blood samples were not available in 6 patients with MELAS mutations in muscle). Molecular genetic analysis of blood samples represents an inexpensive and reliable screening test for mitochondrial encephalopathies, and use of such techniques could influence diagnosis and genetic counselling in patients with seizure disorders and young-onset stroke.

Allergic reactions to long-term benzathine penicillin prophylaxis for rheumatic fever. International Rheumatic Fever Study Group. 1790 patients from 11 countries were enrolled in a prospective international study to determine the incidence of allergic reactions to monthly intramuscular benzathine penicillin (penicillin G benzathine) injections to prevent recurrences of rheumatic fever. After 32,430 injections during 2736 patient years of observation, 57 of the 1790 patients (3.2%) had an allergic reaction. 4 had anaphylaxis, an incidence of 0.2% (1.2/10,000 injections), all in patients over 12 years of age, and 1 patient died, a fatality incidence of 0.05% (0.31/10,000 injections). These rates are similar to those described for patients without rheumatic fever who receive short-term treatment with parenteral penicillin. Rheumatic fever recurred in 8 of 1790 patients (0.45%) who received benzathine penicillin prophylaxis compared with 11 of 96 (11.5%) who did not comply with treatment. Life-threatening allergic reactions are rare in patients on long-term parenteral benzathine penicillin to prevent recurrences of rheumatic fever; the long-term benefits of such prophylaxis by far outweigh the risk of a serious allergic reaction.

Antibodies to gp41 and nef in otherwise HIV-negative homosexual man with Kaposi's sarcoma. A homosexual man with histologically confirmed Kaposi's sarcoma remained seronegative for HIV-1, HIV-2, and HTLV-1 on conventional tests over a 4-year period. HIV cultures were also negative on thirteen separate occasions. However, serum antibodies to synthetic peptide analogues of the gp41 and nef regions of HIV-1 were consistently detected on an enzyme immunoassay. Tests with the polymerase chain reaction with primers directed to the gag and env regions were negative. The antigens to which the antibodies were produced might have come from a defective HIV mutant, another retrovirus, or a hitherto unknown "agent of Kaposi's sarcoma" with similar antigenic epitopes.

Field evaluation of alternative HIV testing strategy with a rapid immunobinding assay and an agglutination assay. A rapid immunobinding assay ('HIVCHEK', Ortho) and an agglutination assay ('Serodia-HIV', Fujirebio) were evaluated as an alternative to enzyme-linked immunosorbent assay (ELISA) and western blot under field conditions in Africa for detection of antibody to human immunodeficiency virus (HIV). 7106 specimens were tested at 25 laboratories in Kenya, Ghana, Senegal, and Zaire. HIVCHEK was used as a screening test, and serodia-HIV as a supplemental test to evaluate these assays in an alternative testing strategy to the standard ELISA/western blot testing procedure. In each country, HIVCHEK was more sensitive and specific than ELISA when compared with western blot. The sensitivity of HIVCHEK ranged from 87.0 to 96.3% and the specificity from 99.0 to 100%. The sensitivity and specificity of serodia-HIV ranged from 85 to 98% and from 88 to 98%, respectively. The sensitivity and specificity were affected by the presence of HIV-2 in Ghana and Senegal. Overall, with an HIV-1 prevalence of 14.8% in Kenya and 22.5% in Zaire, the sensitivities of the alternative strategy were 96.4% and 91.4%, the specificities 99.6% and 100%, the positive predictive values 97.6% and 100%, and the negative predictive values 99.3% and 97.9% for Kenya and Zaire, respectively. With this testing format there was an estimated average cost saving of up to 82% over the conventional strategy with ELISA/western blot. This procedure constitutes a reasonable alternative to the standard ELISA/western blot combination.

Cloning of hippocampal poly(A) RNA sequences that increase after entorhinal cortex lesion in adult rat. Evidence is given for altered gene expression in the hippocampus in response to entorhinal cortex lesioning. Three RNA markers encoding glial fibrillary acidic protein, apolipoprotein E and alpha-tubulin were isolated from a rat hippocampal cDNA library by differential screening with cDNA probes from entorhinal cortex lesioned and control rat hippocampus RNA. By Northern blot analysis, mRNA for apolipoprotein E and alpha-tubulin increased to peak around 6 days after the lesion and returned to near control level at 30 days. The increased synthesis of both mRNAs coincides with the acute phase of synaptogenesis, protein synthesis, and polyribosomes accumulation in the deafferented hippocampal area.

Cloning and functional characterization of the rat glutamine synthetase gene. Glutamine synthetase catalyzes the formation of glutamine from glutamate and ammonia. It plays a central role in both amino acid neurotransmitter metabolism and ammonia detoxification in the central nervous system. Glutamine synthetase expression is regulated in developmental, hormonal, and in tissue- and cell-specific manners. We have cloned a full-length cDNA coding for rat glutamine synthetase, and have found an AT-rich area of conservation in the 3' untranslated regions between rat, mouse, and chicken, which may play a part in the regulation of the stability of the glutamine synthetase message. We have also cloned and mapped the gene coding for rat glutamine synthetase, and identified, by sequence analysis, areas potentially important for the regulation of glutamine synthetase transcription. Transient transfection of a variety of cell lines with deletion constructs of the glutamine synthetase promoter driving a chloramphenicol acetyltransferase reporter gene functionally demonstrates regions of the promoter containing elements important for transcriptional regulation.

[Prenatal DNA-diagnosis of Duchenne muscular dystrophy]. Two prenatal diagnoses were carried out by the technique of intragenic polymorphous marker detecting heterozygosity in pregnant women in the families with cases of Duchenne muscular dystrophy. In both cases the DNA fragment from pERT87-15 region was amplified. This fragment includes a polymorphous site in BamHI region of recognition. DNA analyses of the families members have been made and the genetical risk has been calculated by the Bayes method. The prognoses for both fetuses are good.

A bacterial position effect: when the F factor in E. coli K12 is integrated in cis to a chromosomal gene that is flanked by IS1 repeats the elements are activated so that amplification and other regulatory changes that affect the gene can occur. In Escherichia coli K12 the argF gene is located within Tn2901, a genomic unit of approx. 12 kb that is flanked by IS1 elements in direct repeat. When strains in which the F factor is integrated in cis to Tn2901 are subjected to the appropriate selection, regulatory changes that result in over-production of the argF-encoded ornithine transcarbamylase occur at relatively high frequencies. When amplification occurs, the F factor is required only for the initial exchange between the IS1 elements, homologous recombination between tandem repeats of Tn2901 being independent of the F status of the cell. While amplification of Tn2901 is frequent in some Hfr strains, in others regulatory changes that do not involve amplification are predominant. The transfer region of the F factor does not contribute to the position effect.

Biochemical and behavioural effects of isamoltane, a beta-adrenoceptor antagonist with affinity for the 5-HT1B receptor of rat brain. The biochemical and behavioural effects of isamoltane, a beta-adrenoceptor and 5-HT1B receptor antagonist that has higher affinity for 5-HT1B receptors than for 5-HT1A receptors, on 5-HT neurotransmission in the rat brain were examined. In binding experiments isamoltane was found to be about five times more potent as a ligand for the 5-HT1B receptor than for the 5-HT1A receptor (Ki values 21 and 112 nmol/l, respectively). Isamoltane increased the K(+)-evoked overflow of 3H from 3H-5-HT loaded slices of rat occipital cortex at 0.1 mumol/l, consistent with inhibition of the terminal 5-HT autoreceptor. In vivo, isamoltane significantly increased the concentration of 5-hydroxyindoleacetic acid in hypothalamus and hippocampus indicating an increased 5-HT turnover with a maximal effect at 3 mg/kg s.c. A higher dose produced a less pronounced effect. This effect did not seem to be due to the beta-adrenoceptor blocking action of isamoltane since the beta-adrenoceptor antagonists. (-)-alprenolol, betaxolol or ICI 118.551 had no significant effects on 5-HT turnover at 5 mg/kg s.c. Isamoltane at 3 mg/kg s.c. induced the wet-dog shake response which was blocked by the tryptophan hydroxylase inhibitor p-chlorophenylalanine. In contrast, the same response induced by the 5-HT2 receptor agonist quipazine was not blocked by pretreatment with p-chlorophenylalanine. The wet-dog shakes evoked by isamoltane and quipazine were blocked by ritanserin, which indicates that 5-HT2 receptors are involved in their expression. These observations indicate that isamoltane, by inhibiting the terminal 5-HT autoreceptors, increased the synaptic concentration of 5-HT to a level that induced a behavioural response.

Ipsilateral but not contralateral blockade of excitatory amino acid receptors in the caudal ventrolateral medulla inhibits aortic baroreceptor reflex in rats. The caudal ventrolateral medulla (CVLM) contains vasodepressor neurons which, when activated, decrease vasomotor tone. To investigate whether excitatory amino acid receptors in the CVLM of the rat are involved in mediation of the aortic baroreceptor reflex, we microinjected amino acid antagonists unilaterally into the CVLM and examined their effects on the depressor response to electrical stimulation of the aortic nerve which contains mainly baroreceptor afferent fibers in rats. Male Wistar rats were anaesthetized with urethane, paralyzed and artificially ventilated. To block reflex vagal effects, methylatropine (1 mg/kg) was given intravenously. Kynurenate (227 ng), an excitatory amino acid antagonist, injected ipsilaterally but not contralaterally into the CVLM markedly inhibited the depressor response to aortic nerve stimulation, while both injections produced a similar small increase in basal blood pressure. Muscimol (1 ng), a GABA receptor agonist, injected ipsilaterally into the CVLM partly inhibited the baroreflex response, while it produced a moderate increase in basal blood pressure. 2-Amino-5-phosphonovalerate (APV) (10 ng), a N-methyl-D-aspartate (NMDA) receptor antagonist, and MK-801 (30 ng), a NMDA receptor channel blocker, partly inhibited the baroreflex response. MK-801 (30 ng) injected into the CVLM reduced the depressor response to the NMDA receptor agonist NMDA (0.3 ng) but not to the quisqualate receptor agonist quisqualate (0.1 ng) and the kainate receptor agonist kainate (0.1 ng), while kynurenate (227 ng) inhibited the depressor response to all three excitatory amino acid receptor agonists. These findings provide further evidence for the presence of excitatory amino acid receptors involved in mediating the aortic baroreceptor reflex in the rat CVLM.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical, autoradiographic and pharmacological evidence for the involvement of tubular DA-1 receptors in the natriuretic response to dopexamine hydrochloride. Dopexamine hydrochloride (DPX) is a dopamine analog and it possesses agonistic action at DA-1 receptors and beta 2-adrenoceptors. It also is a weak agonist at DA-2 receptors. In the present study, we have examined the anatomical localization of DPX binding sites in rat kidney and their functional significance in terms of the renal effects of this compound. In receptor-ligand binding studies, [3H]-DPX was found to bind specifically to sections of rat kidney in a time (maximum binding at 60 min), temperature (optimal temperature 25 degrees C) and concentration (highest specific/non-specific ratio at 2 nmol/l) dependent manner. Autoradiographic studies revealed the presence of [3H]-DPX binding sites in renal tubules, glomerulus and various layers of small and large blood vessels. Inhibition studies with SCH 23390, ICI 118.551 and 1-sulpiride showed that DPX binds primarily to DA-1 receptors in tubules, only to beta 2-adrenoceptors in glomerulus and to beta 2-adrenoceptors, DA-1 and DA-2 receptors in blood vessels. Also, DPX caused concentration related increases in cyclic AMP levels in rat kidney membrane particles, which could be completely abolished by a combined presence of SCH 23390 and propranolol suggesting that both binding sites of DPX are linked to adenylate cyclase. In functional studies DPX (1 microgram/kg.min for 30 min) produced a modest fall in blood pressure, pronounced tachycardia and slight but significant increase in renal blood flow (11%). These responses were accompanied by increases in urine output (97%), urinary sodium excretion (89%), and fractional excretion of sodium (132%). There was no change in glomerular filtration rate. Propranolol pretreatment abolished DPX-induced hypotension and tachycardia but seemed to potentiate the natriuretic responses to DPX. On the other hand, SCH 23390, a DA-1 receptor antagonist completely abolished DPX-induced hypotension, natriuresis and diuresis without affecting tachycardia. These results indicate that (1) DPX binds predominantly to DA-1 receptors in renal tubules, to beta 2-adrenoceptors in glomerulus and to beta 2-adrenoceptors, as well as DA-1 and DA-2 receptors in renal blood vessels (2) DPX stimulates cAMP formation in the kidney by activating both DA-1 and beta 2-adrenoceptors and (3) DPX produces natriuresis and diuresis by selectively activating DA-1 receptors located on renal tubules.

Addition of misoprostol to ranitidine in non-responders to H2-blockers and pirenzepine. In the present study, which comprised 14 patients in whom 300 mg and 900 mg of ranitidine as well as in combination with pirenzepine had failed to suppress acid secretion, we tested the effect of adding misoprostol. Night-time intragastric pH was continuously monitored, a rise in the intragastric pH above 4.0 h more than 6 h following the oral dose at 18.00 h being considered a response. In only 1 of the 7 patients with cirrhosis, and in 1 of the 7 control patients, did the combination of 300 mg ranitidine and 400 micrograms of misoprostol result in sufficient acid suppression. Mean pH profiles during the four study periods were not significantly different and the effect of misoprostol was similar in cirrhotics and controls. The inefficacy of the addition of misoprostol in this selected group of patients who did not respond even high doses of ranitidine (+/- pirenzepine) does not favour the hypothesis that gastric mucosal prostaglandin efficiency may be an important factor in the non-response to H2-receptor antagonists. It appears unlikely that the cause is localized solely at the site of H2-receptors.

Coronary vascular responses to nicotine in the anaesthetized dog. The effect of the intra-coronary (i.c.) injection of nicotine on large coronary artery diameter and coronary blood flow was examined in anaesthetized dogs. In sixteen untreated dogs nicotine (20 micrograms i.c.) had a biphasic effect on arterial pressure (initial increase, 7 +/- 2 mmHg; secondary decrease, -8 +/- 3 mmHg) which was accompanied by small and variable effects on heart rate and an increase in LV dP/dt. Nicotine increased large coronary artery diameter by 5.8 +/- 0.8% but had a biphasic effect on coronary blood flow (initial increase, 41 +/- 7 ml/min; secondary decrease, -10 +/- 2 ml/min). Bilateral vagotomy or muscarinic receptor blockade with atropine (0.1 mg/kg i.v.) did not significantly affect the nicotine-induced changes in coronary artery diameter or coronary blood flow. The additional antagonism of beta-adrenoceptors with propranolol (1 mg/kg i.v.) abolished the effect of nicotine in coronary artery diameter (delta CD = 0.2 +/- 0.2%) and the initial increase in coronary blood flow (delta CBF = 1 +/- 1 ml/min) but enhanced the secondary decrease in flow (delta CBF = -25 +/- 3 ml/min). The nicotine-induced decrease in coronary blood flow observed after muscarinic and beta-adrenoceptor blockade was attenuated by antagonism of alpha 1-adrenoceptors with prazosin (10 micrograms/kg i.c., delta CBF = -15 +/- 3 ml/min) and abolished after additional antagonism of alpha 2-adrenoceptors with idazoxan (50 micrograms/kg i.c., delta CBF = -2 +/- 1 ml/min). These results indicate that in the anaesthetized dog intra-coronary injection of nicotine results in beta-adrenoceptor mediated dilatation of both large and small coronary arteries.(ABSTRACT TRUNCATED AT 250 WORDS)

c-met is amplified but not mutated in a cell line with an activated met tyrosine kinase. The putative tyrosine kinase receptor encoded by the oncogene c-met is activated (tyrosine-phosphorylated in vivo) in the human gastric carcinoma cell line GTL-16. The corresponding gene is amplified and over-expressed. In this study we show that c-met is part of an amplification unit measuring more than 3000 kb. The multiple copies of the amplicon are located on a novel chromosome different from chromosome 7. We have previously shown that the c-met protein present in GTL-16 cells is indistinguishable from that found in other cells. Kinase activation could be due to over-expression of the normal c-met protein or to the presence of activating mutation(s). To verify the primary structure of the c-met protein in GTL-16 cells we sequenced a series of overlapping cDNAs obtained from GTL-16 cell RNA by reverse transcription and polymerase chain reaction. Two differences were found in the c-met coding region with respect to the published human c-met cDNA: (1) the lack of 54 nucleotides corresponding to a stretch of 18 amino acids located in the extracellular domain of the receptor, and (2) the substitution of the codon specifying alanine 1209 (located in the tyrosine kinase domain) with one coding for glycine. However, we also obtained cDNAs identical to that just described from a number of control cell lines. These results suggest: (1) that the present c-met cDNA presumably reflects the sequence of the most abundant transcript in several cell types, and (2) that over-expression of the normal c-met protein, alone or in combination with an autocrine loop, is most probably responsible for the activation of the c-met kinase in GTL-16 cells.

Chronic exposure to anxiolytic drugs, working by different mechanisms causes up-regulation of dihydropyridine binding sites on cultured bovine adrenal chromaffin cells. Exposure of bovine adrenal chromaffin cells to ethanol [50 mM], alprazolam [10(-7) M] and buspirone [10(-7) M] inhibited basal and carbachol-induced release of catecholamines from these cells. The inhibition produced by alprazolam was prevented, and that produced by ethanol inhibited, by the presence of the benzodiazepine receptor antagonist, flumazenil [10(-8) M]. The inhibition produced by buspirone was unaffected by flumazenil, but was mimicked by the selective 5-HT1A receptor agonist, 8-OH DPAT and prevented by the 5-HT receptor antagonist spiperone [10(-6) M]. These results suggest that bovine adrenal chromaffin cells express GABAA receptors, containing a benzodiazepine recognition site and also 5-HT1A receptors. Ethanol and alprazolam appear to inhibit the excitability of bovine adrenal chromaffin cells by an action related to the former, while buspirone probably inhibits these cells through the latter. Maintaining bovine adrenal chromaffin cells for several days in culture medium, containing inhibitory concentrations of ethanol alprazolam or buspirone, produced a marked increase in binding sites for a [3H]dihydropyridine [DHP] calcium channel antagonist, on cell membranes. The increase in binding sites produced by alprazolam was greater than that produced by the other two agents and was almost completely prevented by the concomitant presence of flumazenil. The effects of ethanol and buspirone on the binding of DHP were not prevented by flumazenil. The results suggest that drugs which decrease excitability of bovine adrenal chromaffin cells by different mechanisms, may evoke a similar adaptive response involving an increase in DHP-sensitive calcium channels.

A soluble protein related to the HER-2 proto-oncogene product is released from human breast carcinoma cells. Amplified expression of p185HER-2, the protein product of the HER-2/neu proto-oncogene, appears to be involved in carcinogenesis of human mammary epithelial cells. Our data suggest that an extracellular 130 Kda glycoprotein released from breast carcinoma cells may be related to the ectodomain of p185HER-2: (1) Both cellular p185HER-2 and extracellular p130, when reduced and alkylated, reacted with antipeptide antibody against the N-terminus of the HER-2 protein [alpha N(HER-21)] and reactivity was blocked by cognate peptide; (2) Neither p130 nor other extracellular proteins reacted with antiserum against the C-terminus of p185HER-2 [alpha C(HER-2)]; (3) Partial proteolysis of p185HER-2 generated an immunoreactive fragment of 130 kDa that was similar to extracellular p130; (4) Both p130 and p185HER-2 contained carbohydrate that bound to Con-A Sepharose; (5) The amount of p130 released from breast cells corresponded to the levels of expression of cellular p185HER-2. Release of the ectodomain of p185HER-2 from breast carcinoma cells may be involved in their malignant growth and may be a useful marker for assessing the expression of p185HER-2 in human tumors.

Facs analysis of myeloid differentiation stages in epiphyseal bone marrow, adjacent to joints affected with rheumatoid arthritis. To analyze the differentiation stages of myeloids statistically, we adopted a two-color FACS system and used appropriate monoclonal antibodies belonging to CD15, CD16 and CD11b. By using HL60 treated with DMSO or human bone marrow MNCs from patients with rheumatoid arthritis, it was proved that with this system, myeloids could be clearly separated according to differentiation stages. Furthermore, the number of myeloids at certain stages of differentiation in the epiphyseal bone marrow of patients with RA or OA was measured. Nine of 15 samples from RA patients showed immature and relatively mature myeloids, while none of the 8 OA samples did. When the proportions of myeloids in epiphyseal bone marrow MNCs were compared with the clinical features, disease subsets in RA and the degree of synovitis, seemed to be important factors for abnormal myelopoiesis.

[Adrenogenital syndrome. II. Molecular biology]. The adrenogenital syndrome (AGS) is usually caused by steroid 21-hydroxylase deficiency. Two steroid 21-hydroxylase genes are present within the major histocompatibility complex (MHC) on chromosome 6: an active gene (CYP21) and a pseudogene (CYP21P). Several types of mutations have been described; these mutations can be categorized as gene deletions, gene duplications, gene conversions and smaller mutations inside the gene. Some of these cause a defect in the CYP21 gene, possibly resulting in 21-hydroxylase deficiency. Apart from the intrinsic scientific value of these results, the methods applied become increasingly important in diagnostics.

Effectiveness of N-acetylcysteine in protecting against mercuric chloride-induced nephrotoxicity. Mercuric chloride (HgCl2)-induced nephrotoxicity, as measured by functional and biochemical parameters was evaluated in rats at different kidney non-protein sulfhydryls (NPS) levels. Diethylmaleate (DEM) induced a 75% of NPS diminution 1 h after the administration. Renal function (clearance) and biochemical measurements (gamma-glutamyltranspeptidase activity in urine, and lipoperoxides in kidney tissue) were impaired when the animals were HgCl2-treated. Values were highly impaired when the kidneys were NPS-depleted and were improved when NPS pools were previously increased although they were not similar to control values. DEM treatment promoted a higher accumulation of HgCl2 in both kidney and liver while NAC-treatment reduced significantly the metal content in these organs. These data are in favour of a positive relationship among mercury content and organ injury. On the other hand, mercury content increased while NPS levels diminished. NPS might play a role in the HgCl2 detoxification and thus avoids mercury accumulation and mercury effects.

Persistence of bcr-able gene expression following bone marrow transplantation for chronic myelogenous leukemia in chronic phase. The bcr-abl RNA transcript is the molecular counterpart of the Philadelphia chromosome and is detectable by an extremely sensitive polymerase chain reaction assay in most patients with chronic myelogenous leukemia. To determine the effectiveness of ablative radiochemotherapy and bone marrow transplantation in eradicating molecular evidence of the malignant clone, we assayed for bcr-abl RNA expression in specimens from 19 patients with CML in chronic phase (CP) who have survived for at least one year post-BMT. We correlated these results with the patients' remission status based on cytogenetic analysis and BM morphology, and with evidence of mixed hematopoietic chimerism by analysis of RBC antigen and DNA restriction fragment length polymorphism patterns. Thirteen of the 19 patients had detectable bcr-abl RNA at some time following BMT. Twelve of these patients have remained in remission by morphologic and karyotypic criteria from 16.6 to 63.7 months following BMT. One of these 13 patients relapsed both by cytogenetic and clinical criteria at 28.1 months after BMT. Six of these 13 patients are still positive at the time of their most recent analysis. Only two patients have evidence for mixed chimerism of normal hematopoietic elements by either RBC antigen or DNA RFLP patterns. These results suggest that, in some patients transplanted for CML in CP, small numbers of residual leukemic cells may persist or reappear transiently without leading to clinical relapse. The definition of complete remission in CML may need to be revised in light of the enhanced ability to detect minimal residual disease by PCR technology.

The effect of partial in vivo depletion of CD4 T cells by monoclonal antibody. Evidence that incomplete depletion increases IgG production and augments in vitro thymic-dependent antibody responses. In vivo depletion or inactivation of CD4 T cells by monoclonal antibody inhibits of T-cell-dependent immune responses and, in some cases, ameliorates clinical autoimmune disease. Impairment of T cell function occurs in situations where mice are treated with relatively large doses of anti-CD4 antibody. When adult (C57BL/6xDBA/2)F1 mice were treated with a low dose of anti-CD4 antibody augmentation of certain thymic-dependent responses occurred. Twice-weekly injections of 50 micrograms of monoclonal antibody GK1.5 for a period of three weeks resulted in a 50% reduction of splenic CD4 T cells. Mice that were partially depleted of CD4 T cells exhibited a 55% increase in serum IgG levels with a 165% increase in serum IgG1. Simulation of spleen cells from these mice with LPS resulted in a significant increase in differentiation of IgG secretion. When spleen cells from partially CD4-depleted mice were challenged in vitro with SRBC, they mounted a direct PFC response that was more than four times the observed PFC response of mice that received either saline or rat IgG. These findings indicate that partial depletion/inactivation of CD4 T cells by in vivo administration of anti-CD4 monoclonal antibody results in a significant augmentation of certain T-cell-dependent humoral responses.

Alpha adrenergic antagonism by cyclosporine. Cyclosporine has been shown to exert antiadrenergic actions in previous studies from this laboratory. Interactions of CsA with the alpha-one and alpha-two receptor were studied in rabbits. Blood pressure studies revealed that CsA exerts a competitive, reversible antagonism against norepinephrine, epinephrine, and phenylephrine. Further studies of heart rate depression by clonidine also demonstrated that CsA antagonizes this action. In conclusion, cyclosporine appears to act as a nonspecific alpha antagonist.

Application of HLA-DR oligotyping to 110 kidney transplant patients with doubtful serological typing. In renal transplantation a good HLA-DR match is associated with a higher success rate of graft outcome. However, due to a number of technical problems, reliable serological DR typing cannot always be obtained, and the very large number of HLA-DR alleles now discovered renders such DR matching more difficult. In view of the medical importance of HLA class II polymorphism in transplantation immunology, we have developed a simple HLA-DR oligotyping procedure on PCR-amplified DNA, by hybridization with 14 synthetic oligonucleotide probes able to recognize all major HLA-DR specificities. In particular, the probes used in this study allow the unambiguous discrimination of the DRw11, w12, w13, w14-Dw9 specificities or of rare alleles such as DR-Br or DRw13-DwHAG, which are very often difficult or impossible to identify by serology. In order to explore the potential of this methodology, we have analyzed by oligotyping 110 kidney transplant patients with doubtful or unreliable serological assignment, or with DR blank alleles. Comparison between serology and oligotyping shows that in 66.3% of the patients we observed an excellent correlation. About half these patients are homozygotes, as ultimately verified by RFLP typing. In 26.4% of the patients however, at least one HLA-DR antigen was discrepant, and in 7.3% of the cases oligotyping resolved uninterpretable serology. Almost all of the discrepancies were due to errors in allele assignment within the DRw52 group, mostly in the case of DRw13 alleles. This study confirms the expected qualitative advantage of the oligotyping technique and its simplicity as compared with the RFLP DNA typing procedure. Large scale application of the oligotyping methodology will therefore be beneficial to optimize HLA-DR matching in organ transplantation, particularly in high responders with first kidney graft rejection.

[The role of prostaglandins in the effect of beta-blockers on stimulated blood platelets]. Atenolol, a selective beta-blocker, slightly potentiates the stimulated aggregation of platelets against dose-dependence. The drug potentiates similarly stimulated release of arachidonic acid, it does not influence the formation of malondialdehyde and the thromboxane B2 production in stimulated platelets. The non-selective beta-blocker propranolol inhibits in a dose-dependent way stimulated aggregation, the release of arachidonic acid, it reduces thromboxane B2 production in stimulated platelets. The authors revealed that the anti-aggregating action of beta-blockers is closely related to their ability to influence prostaglandin synthesis in stimulated platelets.

[Kinetics of metipranolol in patients with chronic kidney failure and during hemodialysis]. The authors investigated the metipranolol plasma concentration in seven patients with renal failure after a single dose of 20 mg of the preparation (2 tablets of Trimepranol) by the oral route during haemodialysis and on days without dialysis, and evaluated also the effect of dialysis on selected pharmacokinetic constants. Although there was a great individual variability of plasma concentrations of metipranolol, these concentrations were at the end of dialysis lower than during the corresponding interval between dialyses. The mean values of the area beneath the concentration curve, the velocity elimination constants, the half-life of elimination and total clearance indicate a certain dialysability of metipranolol. During the practical application of these conclusions it is, however, important to consider the effect on blood pressure and cardiac rhythm resp. by changes of volume and the composition of the extracellular fluid caused by haemodialysis.

[The use of highly polymorphic DNA systems in the demonstration of mixed chimerism following bone marrow transplantation]. The demonstration of restriction fragment length polymorphism (RFLP) of the highly polymorphic systems MS1, MS31, g3, and MS43 to detect mixed chimerism after bone marrow transplantation is discussed. Degree of heterozygosity, somatic stability and sensitivity are the parameters investigated to demonstrate the practicability of this method. Examples of mixed chimerism after bone marrow transplantation are shown.

[Identification of an old frozen alcohol blood sample using a genetic probe technique]. Determination of ethanol concentration in a blood sample drawn from a person who caused a serious car crash showed a level which was markedly above the upper limit tolerated legally i.e. 0.08%. At the court hearing the accused car driver challenged the drunken driving charge and claimed that there might have been a mix up of the blood samples, whereby his was replaced by another blood sample, since the tube containing his blood was not marked with his name. The blood sample had been stored without anticoagulants for about 6 months at -20 degrees C. Due to haemolysis it was impossible to determine conventional haemogenetic marker systems. We therefore tried to extract DNA from the blood sample and to determine the restriction fragment length polymorphism (RFLP) by means of five DNA probes recognizing highly polymorphic single-locus systems as described by Jeffreys et al. We analyzed the RFLP's of both the old blood sample and of fresh blood drawn from the accused car driver and we were able to identify the blood sample as certainly having been taken from the accused.

Effects of a single oral administration of Epanolol on exercise tolerance in patients with stable effort angina pectoris. A single oral dosage of 100, 200 or 300 mg Epanolol or placebo was administered in a randomized double-blind, cross-over fashion to 12 patients with stable effort angina. Symptom limited bicycle ergometer tests were performed before and 2 and 8 hours after each drug administration. Spontaneous diurnal and day to day differences in exercise tolerance and related analyzed variables were not present; 48 hours after drug intake a carry-over effect of the previous Epanolol administration could not be demonstrated. At each dose level of Epanolol, 2 hours after drug intake, heart rate and rate pressure product at peak exercise fell while exercise duration rose significantly. After 8 hours these effects were less marked. No significant differences between the 3 dose levels were found. In the pooled data of the 3 Epanolol doses, systolic blood pressure at peak exercise and maximal ischemic ST segment depression during exercise were also significantly reduced after 2 hours, while total performed external work rose. The percentage increase in heart rate during exercise after Epanolol administration was similar to the control tests at lower exercise levels but was less marked during the later stages of the test. Heart rate after Epanolol intake was percentage wise more reduced at higher exercise levels. Plasma levels of Epanolol were lower 8 hours after drug administration than after 2 hours. There was no correlation between plasma levels of Epanolol and the observed changes of any of the exercise variables 2 hours after drug intake.

Alterations in copy number of c-erbB-2 and c-myc proto-oncogenes in advanced stage of human breast cancer. In our previous study, amplification of c-erb B-2 and c-myc proto-oncogenes in DNA of human breast cancer occurred in 16% and 4% of cases, respectively, and increased copy number of these genes is suggested to be associated with aggressive primary tumors. We examined change in the copy number of c-erb B-2 and c-myc proto-oncogenes between primary and multiple metastatic tumors in 10 patients with breast cancer, who underwent breast surgery and were later autopsied, by using DNAs isolated from formalin-fixed paraffin-embedded tissues and the dot blot-hybridization method. In primary tumors, amplification of c-erb B-2 and c-myc was detected in three and two cases, whereas at the stage with systemic metastasis, it was detected in four and three cases, respectively. In all four cases with amplified c-erb B-2 gene and in one of the three with amplified c-myc gene, the copy number was clearly increased in the metastatic tumors in comparison with the primary. Microscopically, more than five mitotic figures per high power field were detected in metastatic tumors of five cases including three with amplified c-erb B-2, but in only two primary. These results suggested that the aggressive nature of breast cancer is frequently enhanced in accordance with cancer metastasis.

Histopathological study of mucosal and submucosal cysts of stomach. To clarify the characteristics of mucosal and submucosal gastric cysts, five stomal cancers of the remnant stomach, two cancers with a diffuse cystic distribution and three ordinarily resected cancers were used to study mucinous patterns, proliferative cells and humoral defensive factors in the cystic epithelia. Most cysts showed a gastric appearance with relatively dominant ConA III (+) pyloric gland-like features, and were positively stained with high-iron diamine-alcian blue. Although a few cells positive for proliferative cell nuclear antigen (PCNA) were seen in epithelia, cysts generally revealed a low degree of proliferation, less than 2% of epithelial cells being PCNA (+). Cysts were formed from the isthmus or from ordinary glands, and showed an increase of IgA and secretory component in epithelia of the supranuclear, luminal or basolateral region and increased lysozyme (Ly) activity. Some cysts contained cells with strong positivity for Ly which could not be confirmed as Paneth cells by HE staining. Mucosal and submucosal cysts are considered to arise as a reaction to inflammation, and have a tendency to show both gastric and intestinal characteristics.

Patterns of care among people with long-term functional psychosis in three different areas of Stockholm County. This study of long-term functionally psychotic people in Stockholm County describes the psychiatric and somatic care provided as well as social welfare support and medication in a total cohort. This group included all non-organic cases of psychosis aged 18-64 years. The group was found still to be very dependent on institutional care, with an average of 75 d of psychiatric inpatient care. Males spent twice as long as females as inpatients, and people from the urban area spent a longer time than those from the other areas. Antipsychotic medication increased from the rural to the urban area. The diagnosis of schizophrenia and early age at onset were each per se associated with higher likelihood of inpatient treatment and depot medication. Contrary to expectations, medication with antipsychotic drugs was shown to increase with illness duration.

Tardive dyskinesia in the mentally retarded: comparison of prevalence, risk factors and topography with a schizophrenic population. Thirty mentally retarded patients treated with neuroleptics for aberrant behavior were compared with 30 neuroleptic-treated schizophrenics for the presence, topography and risk factors associated with tardive dyskinesia (TD). In the total sample (n = 60), female sex, schizophrenic diagnosis and increasing age were associated with TD. The length of neuroleptic treatment and current neuroleptic dose were not significantly associated with TD. The only topographical difference in TD presentation was that the mentally retarded group had significantly more tongue involvement.

Constituents of Bidens pilosa L.: do the components found so far explain the use of this plant in traditional medicine? The dried aerial parts of Bidens pilosa L. were extracted with petrol ether, chloroform, methanol, and methanol/water. The petrol ether and the methanol/water extracts showed some antimicrobial activity. Fractionation of the extracts yielded well known substances, most of which have, however, not yet been described as constituents of Bidens pilosa. Several of these substances have previously been shown to be biologically active. Thus, phenylheptatriyne, linolic acid and linolenic acid have antimicrobial activities. On the other hand, friedelin and friedelan-3 beta-ol, as well as several of the flavonoids found are anti-inflammatory agents. The detection of these compounds in extracts from B. pilosa may rationalize the use of this plant in traditional medicine in the treatment of wounds, against inflammations and against bacterial infections of the gastrointestinal tract.

Erythrocyte survival in severe falciparum malaria. Erythrocyte survival was studied in 17 Thai patients (10 males, 7 females; aged 13-57 years) with severe falciparum malaria. To ensure radioisotopic labelling of cells before bone marrow recovery and survival analysis under near-steady state conditions, 51Cr labelling of autologous erythrocytes was performed at the time of admission (0 h) and calculation of mean cell lifespan (MCL) was based on semilogarithmic plots of corrected counts from 60 h onwards. Five patients received blood transfusions, all within 48 h of admission. The overall mean (+/- S.D.) MCL was short (44.1 +/- 21.7 days). Nontransfused patients had similar MCL values (43.6 +/- 20.4) to those of transfused patients (45.5 +/- 27.3 days, p greater than 0.8). Patients with and without palpable splenomegaly had MCL values which were not significantly different (54.1 +/- 28.8 vs. 37.2 +/- 12.3 days respectively, p greater than 0.1). There was no association between admission haematocrit or peripheral parasitaemia and MCL (p greater than 0.2 in each case), but there was an inverse correlation between total serum bilirubin and MCL (r = -0.49, p less than 0.025). There is accelerated destruction of non-parasitised erythrocytes in severe malaria resulting in a mean MCL that is half that found previously in healthy Thai volunteers (89.6 +/- 13.1 days, p less than 0.001) and significantly shorter than that reported previously in Thai patients with uncomplicated P. falciparum infections studied after parasite clearance (56.8 +/- 10.2 days, p less than 0.05).

Genetic aspects of control of anaemia development in trypanotolerant N'Dama cattle. 148 one-year-old N'Dama cattle, progeny of 29 sires, were exposed for 92 days to a medium natural tsetse-trypanosome challenge in Gabon, Central Africa. Matching health and performance data were recorded on 11 occasions. Average packed red cell volume percent (PCV) and lowest PCV reached during the period were evaluated as measures of ability to control the development of anaemia. Attempts were made to systematically control other possible causes of anaemia. In animals detected as parasitaemic, those with above average average PCV values or above average lowest PCV reached had 34% and 35% respectively higher daily weight gains than those with below average. Even when not detected as parasitaemic, those with above average average PCV values or above average lowest PCV reached had 14% and 12% respectively higher gain indicating that a proportion of these animals actually were parasitaemic. When all environmental and parasitaemia information was taken into account, the heritability of growth, average PCV and lowest PCV reached was 0.39 +/- 0.31, 0.64 +/- 0.33 and 0.50 +/- 0.32 respectively. The genetic correlation between average PCV and growth was 0.70 +/- 0.42 and between lowest PCV reached and growth was 0.28 +/- 0.55. While the standard errors are large, the higher heritabilities of PCV measures compared to animal growth and the positive genetic correlations between PCV and growth do indicate an opportunity for selection on PCV when animals can be detected as parasitaemic. All heritabilities and genetic correlations increased in size when parasitaemia information was utilized in the analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

The reproductive lifespan of Onchocerca volvulus in West African savanna. The epidemiological model ONCHOSIM--a model and computer simulation program for the transmission and control of onchocerciasis--has been used to determine the range of plausible values for the reproductive lifespan of Onchocerca volvulus. Model predictions based on different lifespan quantifications were compared with the results of longitudinal skin-snip surveys undertaken in 4 reference villages during 13 to 14 years of successful vector control in the Onchocerciasis Control Programme in West Africa. Good fits between predicted and observed trends in skin microfilarial loads could be obtained for all villages. It is concluded that the reproductive lifespan of the savanna strain of O. volvulus lies between 9 and 11 years, and that 95% of the parasites reach the end of reproduction before the age of 13 to 14 years.

The conditions required for the maintenance of Onchocerca lienalis microfilariae in vitro. Microfilariae of the bovine parasite Onchocerca lienalis were maintained in vitro using a tick cell line as a feeder layer, and under the conditions provided would develop to the sausage stage. A number of media supported this achievement with CMRL 1066 producing the highest yields, particularly when used in conjunction with either medium 199, HAM's F-12, RPMI 1640 or Mark's M20. The addition of inactivated foetal calf serum (iFCS) suppressed development; on the other hand, the addition of tryptose phosphate broth (TPB) enhanced it. The pH range of the medium preferred by the developing worms was 7.35-7.85, and the most favourable osmolality lay in the range 360 to 390 mosM/kg. Insect-derived hormones did not improve yields of developed larvae nor promote moulting to the second larval stage. The culture conditions described in this work, which favour parasite survival and development, provide further insights into the physiological requirements of filariae as well as guide lines to achieving successful in vitro maintenance of Onchocerca sp. A morphological examination of the developing larvae was carried out at electron microscopical level.

Humoral immune response to infective larval antigen in Brugia malayi infected Mastomys natalensis. The sequential changes in the humoral immune response against infective larval antigens during the course of Brugia malayi infection in Mastomys natalensis have been studied using enzyme linked immunosorbent assay. IgM antibody against B. malayi infective larval excretory secretory (ES) antigen was detected in the peripheral circulation within a week of infection, whereas IgM antibody against B. malayi infective larval somatic antigen and IgG antibody against both somatic and ES antigens were detected on day 20 post-inoculation. Thereafter, the antibody levels showed a steady increase until day 150. A gradual decrease of IgM antibody level was observed upto day 360, whereas IgG antibody level was decreased upto day 250 and then maintained almost the same level upto day 360. Wuchereria bancrofti cross reactive antigen as well as B. malayi infective larval ES antigen were detected in blood circulation on day 20, the level increased upto day 150 and then remained almost the same upto day 360 with slight variations. Studies of antigen and antibody levels in microfilaraemic and amicrofilaraemic animals show that there is no significant difference in antibody level whereas elevated antigen titre was observed in active infection with microfilaraemia.

Symptomatic C-cell hyperplasia associated with chronic lymphocytic thyroiditis. A 58-year-old euthyroid man with episodic flushing and a 2-year history of progressive wheezing was found to have a hypoechoic lesion in one lobe of his thyroid and hypercalcitoninemia in response to pentagastrin stimulation. Thyroidectomy revealed bilateral C-cell hyperplasia unexpectedly associated with chronic lymphocytic thyroiditis. The C-cells exhibited positive immunohistochemical staining for calcitonin and polyclonal carcinoembryonic antigen (CEA). Postoperatively, the wheezing and flushing subsided and the serum calcitonin level was not elevated with pentagastrin stimulation. The substance or substances responsible for the wheezing and flushing were not specifically identified. Nine other specimens of chronic lymphocytic thyroiditis were examined for C-cell hyperplasia and two had small hyperplastic foci, but of a lesser degree than the index case. These patients did not exhibit wheezing and flushing. The development of C-cell hyperplasia in chronic lymphocytic thyroiditis is uncommon and the mechanism for its occurrence is unexplained. This patient appears to be the first reported case of symptomatic C-cell hyperplasia associated with chronic lymphocytic thyroiditis. The substance or substances responsible for the clinical symptoms remain to be identified.

Expression of Her-2/neu oncogene protein product and epidermal growth factor receptors in surgical specimens of human breast cancers. Her-2/neu protein product was immunocytochemically analyzed in 139 breast cancers. Epidermal growth factor receptors were similarly analyzed in 74 breast cancers from the same patient pool. These results were also separated on the basis of estrogen receptor proteins and of combined aneuploidy with elevated S-phase from flow cytometry. Invasive breast cancer yielded a positive label for Her-2/neu protein (26%) and for epidermal growth factor receptor (25%), with no significant difference. Correlations with estrogen receptor labeling yielded differences significant inversely for both Her-2/neu protein (p less than 0.02) and epidermal growth factor receptor (p less than 0.01). Positive Her-2/neu protein labels correlated with a positive combination of aneuploidy and elevated S-phase (37%) and a negative combination of aneuploidy and elevated S-phase (21%), with a statistically nonsignificant difference. Positive epidermal growth factor receptor cases with aneuploidy and an elevated S-phase (75%) and without aneuploidy and elevated S-phase (42%) did differ with significance at p less than 0.05. There were eight cases positive for both Her-2/neu protein and epidermal growth factor receptor, four of six cases with negative estrogen receptor, four of six cases with negative estrogen receptor, six of six cases aneuploid, and five of six cases with an elevated S-phase. All eight cases had threatening disease--either stage III or stage IV, with one case of extensive ductal carcinoma in situ (comedo). Correlation of negative Her-2/neu protein with negative epidermal growth factor receptor was significant (p less than 0.05) in 74 cases. However, positive Her-2/neu protein did not correlate with positive epidermal growth factor receptor; there was a trend toward inverse correlation. We conclude that epidermal growth factor receptor labeling results show similarities to Her-2/neu protein results, but epidermal growth factor receptor tended to correlate with unfavorable ploidy and S-phase. Epidermal growth factor receptor labeling might be useful in breast cancers with macrocysts reported to show high epidermal growth factor activity.

Neurotransmitters in nociceptive modulatory circuits. Significant advances have been made in our understanding of nociceptive modulation from RVM. Among the most useful conceptually has been the discovery that there are two classes of modulatory neurons in the RVM that are likely to have opposing actions on nociception: on-cells, which may facilitate nociceptive transmission, and off-cells, which probably have a net inhibitory effect on nociception. The similarity in response properties among the members of each class, their large, somatic "receptive fields," and the wide distribution of the terminal fields of axons of individual neurons to the trigeminal sensory complex and to multiple spinal segments indicate that these neurons exert a global influence over nociceptive responsiveness. Drug microinjections into the RVM presumably shift the balance between states of on- or off-cell firing and also produce measurable changes in the threshold for nocifensor reflexes. The meaningful unit of function in the RVM nociceptive modulatory system therefore probably consists of large ensembles of physiologically and pharmacologically similar neurons. The strong coordination of activity of the two classes of RVM neuron may depend largely upon intranuclear projections from RVM off-cells that excite other off-cells and inhibit on-cells. The off-cell pause is GABA-mediated, and it is likely that there is a subset of GABA-containing RVM on-cells that directly inhibit off-cells. Furthermore, the available evidence indicates that exogenous opiates activate off-cells by inhibiting GABAergic release. Presumably, enkephalinergic cells in the RVM disinhibit off-cells in a similar way. Although non-serotonin-containing off-cells certainly exist, we propose that some off-cells contain serotonin. Other possible connections are based on more limited data; however, ACh, neurotensin, NE, and EAAs are present in neurons that project to the RVM, and each of these compounds, when microinjected into the RVM, has a modulating effect on nociceptive transmission. The local circuits in the RVM that underlie these actions remain to be elucidated. At the level of the dorsal horn, there is good evidence for each of three inhibitory mechanisms: direct inhibition of nociceptive projection neurons, inhibition of excitatory relay interneurons, and excitation of an inhibitory interneuron. The relative contribution made by each of these circuits is unknown.(ABSTRACT TRUNCATED AT 400 WORDS)

[Restriction fragment study (RFLP) of DNA polymorphism in criminology: quantitative and qualitative analyses beginning with dried blood and semen on various supports]. Deoxyribonucleic acid (DNA) purified from various forensic samples was characterized by RFLP (Restriction Fragments Length Polymorphism). DNA isolated from dried bloodstains and dried semen stains was digested with restriction endonucleases (HinfI, PvuII, TaqI) and fractionated by electrophoresis on 0.7% agarose gels. After transfer to a filter, DNA was hybridized with different radioactively labeled recombinant probes: with HinfI, G3, MS1, MS8, MS31, MS43, cloned from 33.6 and 33.15 Jeffreys probes, with PvuII 3'HVR, with TaqI MR24/1. This probes recognize polymorphic DNA regions, so variation between individuals at fundamental level of their DNA can be used to discriminate them. Therefore, pattern of RFLP detected by this test was used to determine the probable identity of different samples. DNA was isolated and detected by Southern blot from since 50 microliters dried bloodstains (HinfI, G3, MS1, MS31), from various specimen bloodstains and from dried semen stains. Highly variable minisatellites cloned from Jeffreys probes took more satisfaction for sensitivity than others. However, DNA polymorphism analysis can be used with caution because method limits can be perturbed interpretation of results.

[DNA polymorphism: comparison of allelic frequencies obtained with two HVR probes in a population from Northeastern Brazil and in two other ethnic groups]. DNAS from 325 individuals representing 2 different populations (234 Brazilians and 91 Asiatics) were analysed for Restriction Fragment Length Polymorphisms (RFLPs). These DNAs were digested with Pvu II enzyme and successively hybridized to two HVR probes: alpha globin-3'HVR and Mucin-HVR. An allele frequency distribution was determined for each couple probe/enzyme and each ethnic groupe studied. The results were compared with frequencies observed in french population and we showed that there is no statistic significant difference between allele frequencies obtained with the couple probe/enzyme 3'HVR/Pvu II in Brazilian and French populations. We also showed that both populations studied (Brazilian and Asiatic) do not follow a Hardy-Weinberg balance.

Applications of a computer simulation model of the natural history of CD4 T-cell number in HIV-infected individuals. Data from the Multicenter AIDS Cohort Study (MACS) of 1637 gay men, recruited in 1984 and 1985 in Los Angeles and followed at 6-monthly intervals, are used to develop a computer simulation model of the typical pattern of CD4 T-cell number changes in HIV-infected AIDS-free subjects. The empirical model incorporates the following features: (1) within-person and between-person variability in CD4 measurements; (2) variation in the rates of decline of CD4 values; (3) variation in the level of CD4 at which clinical AIDS is diagnosed, and (4) greater absolute variation in CD4 values in men with high CD4 levels compared with men with low CD4 values. Three applications of the model to assist in the design and interpretation of clinical trials are given. Further applications to clinical trials and to estimate the current and future spectrum of HIV-mediated immunological disease in the USA, as measured by the CD4 values, are discussed.

Negative correlation between blood cell counts and serum neopterin concentration in patients with HIV-1 infection. Hematopoietic disturbances are common in patients with HIV-1 infection. Recent studies on immune activation markers such as neopterin demonstrate that HIV-1 infection is associated with chronic immune activation. We investigated a possible association between serum neopterin concentrations and blood cell counts (CD4+ T cells, white blood cells, platelets, red blood cells) and hemoglobin and hematocrit in 94 HIV-1-seropositive individuals [52 Walter Reed (WR) stage 1, 31 WR2, one WR5, and 10 WR6]. There were significant negative correlations between neopterin concentrations and CD4+ T cells, hemoglobin, hematocrit and platelets. These correlations were also significant if either only WR1 and WR2 patients or the entire set of data were considered for calculations. Thus, hematological abnormalities are associated with chronic immune activation in patients with HIV-1 infection. Large amounts of neopterin are released by human macrophages on stimulation with interferon-gamma (IFN gamma), and tumor necrosis factor alpha (TNF alpha) further enhances the effect of IFN gamma. Therefore, our data suggest that activated immune cells and specific cytokines such as IFN gamma and TNF alpha are involved inhibiting hematopoiesis.

Antihypertensive effects of chronic 5-hydroxytryptamine (5-HT2) receptor blockade with irindalone in the spontaneously hypertensive rat. The present study was conducted to evaluate the antihypertensive effects of irindalone, a potent 5-HT2-receptor blocking agent with weak alpha 1-adrenoceptorblocking properties. Young spontaneously hypertensive rats (SHR) received irindalone in the food (daily intake estimated to 10 mg/kg) for 10 weeks and older SHR received irindalone subcutaneously for 2 weeks (3 mg/kg/day by osmotic pump). The indirect systolic blood pressure was measured each week (tail plethysmography) and at the end of the intervention periods the direct intraarterial blood pressure was measured in conscious rats. Subsequently a dose-response curve of pressor responses to phenylephrine was constructed in pithed rats. Chronic oral treatment with irindalone reduced the development of hypertension but influenced neither body weight nor heart weight. The subcutaneous treatment with irindalone reduced the blood pressure in relatively older SHR compared with controls. Pressor responses to phenylephrine were antagonized in rats receiving oral treatment. However, during subcutaneous treatment with irindalone the dose-response curve to phenylephrine was not influenced, suggesting that the blood pressure reduction was not directly related to a concomitant alpha 1-adrenoceptor blockade. These results demonstrate that irindalone effectively reduces the blood pressure in SHR and that no tolerance develops to its antihypertensive effects. Since the blood pressure reduction, at least following subcutaneous treatment, was not directly related to a concomitant alpha 1-blockade, it is suggested that the 5-HT2-receptor blockade may be of relevance for the antihypertensive effect.

Diagnostic significance of angiographically observed visceral aneurysms with regard to polyarteritis nodosa. During a 10-year period, intraparenchymal aneurysms were found in 38 of 748 patients at selective abdominal angiography with magnification technique. According to strict criteria, 17 patients were classified as suffering from necrotizing vasculitis of the polyarteritis nodosa group (PAN), 7 from severe arterial hypertension, and 3 from rheumatoid arthritis. The diagnoses of 5 patients remained to be confirmed, and each of the remaining 6 patients suffered from various other diseases. PAN was diagnosed histopathologically in 2 patients without angiographic aneurysms. Based on the 156 patients in whom the indication for angiography was suspicion of arteritis, the angiographic diagnosis of PAN had a sensitivity of 89 percent and a specificity of 90 percent, a positive predictive value of 55 percent and a negative predictive value of 98 percent. The mean number of both renal and hepatic aneurysms was higher in patients with PAN than in the other patients (p less than 0.01 and p less than 0.05, respectively). Five PAN patients had numerous and large aneurysms, whereas the aneurysms of the other 12 PAN patients did not differ from those of patients with other diseases. Patients with PAN had renal infarcts more often than the other patients (p less than 0.05). Our findings suggest that visceral angiography is useful in establishing the diagnosis of PAN, but the angiographic finding of aneurysms is not pathognomonic.

[Electron microscopic studies on the glycocalyx of the utricular macula following amikacin in guinea pigs]. Twelve guinea pigs were randomly separated into 4 groups, one group as control, the other groups were given different dosages of Amikacin for 5 days. Ten days later, the animals were killed by decapitation and the right utricular maculae removed. After staining with reuthenium red, the glycocalyx was observed under TEM. In the control group, the glycocalyx was found covering the surface of both cells and sensory hairs and forming cross links with all the sensory hairs in the same hair band. The main changes in the experimental groups were: 1. decrease or disappearance of glycocalyx on the surface of cells and hairs; 2. fusion of sensory hairs and large blebs formed at the side of hairs. The results of the present study suggest that the decrease or disappearance of glycocalyx may be the first change of the damages caused by Amikacin.

Effects of prolonged cysteamine administration on the rat adrenal cortex: evidence that endogenous somatostatin is involved in the control of the growth and steroidogenic capacity of zona glomerulosa. A week daily administration of cysteamine (CYS, 300 mg kg-1) lowered plasma aldosterone concentration in rats, without affecting PRA, kalaemia and the plasma levels of ACTH and corticosterone. Prolonged CYS treatment caused a notable hypertrophy of adrenal zona glomerulosa (ZG) and its parenchymal cells, without inducing any apparent change in zona fasciculata morphology. Isolated ZG cells from CYS-treated rats evidenced a notable enhancement in their basal and maximally-stimulated productions of aldosterone and corticosterone. All these effects of chronic CYS administration were completely reversed by the simultaneous infusion of rats with somatostatin (SRIF, 12 micrograms kg-1 h-1). CYS exposure was not found to directly affect the secretory activity of isolated ZG cells from normal rats. Since CYS is known to be a specific depletor of SRIF in different organs of rats, these findings suggest that endogenous SRIF may be involved in the modulation of ZG function.

Surgical correction of abdominal testes after Fowler-Stephens using the neodymium: YAG laser for preliminary vessel dissection. Two boys with high undescended testes were submitted to laparoscopic laser transsection of the internal spermatic vessels. Subsequently strong collaterals developed. Definitive scrotal transfer was performed six weeks later through an inguinal incision. Two and a half years later, the testes had recovered very well. Their volume had increased markedly, and further development was normal.

Fractionation of renal brush border membrane proteins with Triton X-114 phase partitioning. Analysis of brush border membrane proteins by gel electrophoresis has revealed a complex polypeptide composition. We have investigated the use of Triton X-114 phase partitioning to fractionate such proteins on the basis of their degree of hydrophobicity. Each of the fractions was composed of a complex but distinct set of proteins. Most proteins were solubilized by Triton X-114 and partitioned into the detergent-poor fraction. Trehalase, gamma-glutamyl transpeptidase, and leucine aminopeptidase were well solubilized (greater than 80%) and enriched 5.1-, 3.9-, and 2.5-fold in the detergent-rich fraction. In contrast, alkaline phosphatase and 5'-nucleotidase were poorly solubilized. The specific activities of these enzymes were increased 2.7- and 2.3-fold in the insoluble protein fraction. Maltase was almost completely solubilized and partitioned into the detergent-poor fraction with a small enrichment factor (1.3). These results suggest that Triton X-114 phase partitioning could be useful as a first step in the purification of many brush border membrane proteins.

Krox-20: a candidate gene for the regulation of pattern formation in the hindbrain. The mouse gene Krox-20 was isolated on the basis of cross-hybridization with Krüppel, a Drosophila segmentation gene. During recent years, an accumulation of structural, biochemical and expression data has indicated that Krox-20 encodes a transcription factor which may play a key role within a regulatory network involved in pattern formation in the developing hindbrain. The DNA-binding domain of Krox-20 consists of 3 zinc fingers and the protein recognizes a specific GC-rich nucleotide sequence. It can activate the transcription of genes located in the vicinity of this sequence. Krox-20 transcription itself is modulated by growth factors. During formation of the vertebrate central nervous system (CNS), the hindbrain is organized into segmental units, called rhombomeres. Prior to the appearance of morphological segmentation, Krox-20 is expressed in 2 stripes within the hindbrain. Later on, the Krox-20 expression domains match with 2 alternate rhombomeres. These data suggest that Krox-20 may regulate aspects of the segmentation process. The observation of segment-specific expression boundaries for homeobox containing genes in the hindbrain suggests that these genes may also be part of the regulatory network governing pattern formation in this region of the CNS. The possible interactions between Krox-20 and homeobox containing genes as well as the search for other members of the network will be discussed.

Magnetic resonance spectroscopy and metabolism. Applications of proton and 13C NMR to the study of glutamate metabolism in cultured glial cells and human brain in vivo. Nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of cells from the central nervous system both in vitro on perchloric acid extracts obtained either from cultured tumoral cells (C6 rat glioma) or rat astrocytes in primary culture, and in vivo within the human brain. Analysis of carbon 13 NMR spectra of perchloric acid extracts prepared from cultured cells in the presence of NMR [1-13C] glucose as substrate allowed determination of the glutamate and glutamine enrichments in both normal and tumoral cells. Preliminary results indicated large changes in the metabolism of these amino acids (and also of aspartate and alanine) in the C6 cell as compared to its normal counterpart. Localized proton NMR spectra of the human brain in vivo were obtained at 1.5 T, in order to evaluate the content of various metabolites, including glutamate, in peritumoral edema from a selected volume of 2 x 2 x 2 cm3. N-acetyl aspartate, glutamate, phosphocreatine, creatine, choline and inositol derivative resonances were observed in 15 min spectra. N-acetyl-aspartate was found to be at a lower level in contrast to glutamate which was detected at a higher level in the injured area as compared to the contralateral unaffected side.

Progress in the DNA diagnosis of hemophilias. DNA diagnosis of hemophilias has received much attention in recent years, and different methods are presently available to analyze the coagulation factor IX and VIII genes. This review is intended to serve as a brief guide to current and emerging diagnostic strategies.

High performance liquid chromatographic determination of Picumast and two active metabolites in plasma using on-line sample preparation. A method for determining Picumast, an antiallergic drug, in plasma by HPLC and column switching has been developed. The system consisted of two precolumns, an analytical column, three pumps, an autosampler and a fluorescence detector. The precolumns (17 x 4.6 mm i.d.) were packed with LiChroprep RPR (a moderately polar reversed phase) and the analytical column with Nucleosil ODS (RP 18, 5 microns). The columns were connected according to the alternating precolumn technique. The mobile phase consisted of 30% CH3CN/70% 0.05 M KH2PO4, pH 2.5, with a flow gradient. Detection wavelengths were 333 nm for excitation and 383 nm for emission. The retention times of Picumast, M1 and M2 were 12, 3.6 and 4.0 min, respectively. Total run time was 15 min. The limit of detection was 3 ng/mL for M1 and 1 ng/mL for M2 and Picumast using an injection volume of 150 microL. The recoveries vary between 89% and 97% with standard deviations between 2.4 and 3.3%.

Multidrug resistance: clinical relevance in haematological malignancies. Multidrug resistance describes an experimental observation which appears to explain cross-resistance to certain structurally unrelated cytotoxic agents, including anthracyclines, vinca alkaloids and podophyllotoxins. It is now clear that a major factor responsible for its development is increased expression of a membrane glycoprotein--P-glycoprotein, which functions as an energy-dependent efflux pump. Recent data, particularly in haematological malignancies such as acute non-lymphocytic leukaemia, myeloma and non-Hodgkin's lymphoma, indicate that P-glycoprotein may be involved in the development of clinical drug resistance. The potential therefore exists for new therapeutic studies aimed at circumventing resistance which develops through this mechanism, by using modulators, such as verapamil, quinidine and several others, which prevent cellular drug efflux by competitive binding to P-glycoprotein.

The molecular genetics of familial venous thrombosis. Inherited defects of antithrombin III, protein C, protein S, heparin cofactor II, plasminogen and the fibrinogens are thought to be responsible for between 10 and 15% of all patients presenting with recurrent venous thrombosis. The structure, function and expression of these genes and the nature of the gene lesions underlying the deficiency states are reviewed in detail.

Usefulness of T-cell phenotype characterization in endomyocardial biopsy fragments from human cardiac allografts. The mean numbers of cytotoxic/suppressor (CD8+) and helper/inducer (CD4+) T cells were determined in 111 successive endomyocardial biopsy fragments from eight cardiac allograft patients in an attempt to define their significance in the rejection process. Endomyocardial fragments from autopsy or donor hearts without myocarditis were evaluated as controls. The mean numbers of CD8+ and CD4+ T cells in the control group were 0.8 and 0.5 cells/field at x400 magnification, respectively. The mean numbers of CD8+ T cells per field in the cardiac allograft biopsies were 2.4, no rejection group; 5.4 mild rejection group; 11.1, moderate rejection group; and 4.9, resolving rejection group. The mean numbers of CD4+ T cells per field for the same groups were slightly lower than those of the CD8+ T cells. The number of CD8+ T cells per field reliably indicated the severity of rejection. Patients with normal numbers of CD8+ T cells and no evidence of rejection had better long-term outcomes (two or fewer moderate rejection episodes) than those with higher numbers. Analysis of the data suggests that the presence of two or fewer CD8+ T cells/field may be considered normal in the myocardial interstitium. The diagnosis of no evidence of rejection should be coupled to the presence of a normal number of CD8+ T cells. High numbers (greater than 10) of CD8+ T cells, even in absence of myocytolysis, should be treated more assertively, including the use of high doses of prednisone, because all our cases with high numbers showed a worse histologic picture at the subsequent biopsy.(ABSTRACT TRUNCATED AT 250 WORDS)

Multidrug resistance in heart transplant patients: a preliminary communication on a possible mechanism of therapy-resistant rejection. Multidrug resistance refers to a complex cellular phenotype, the hallmark of which is cross-resistance to multiple drugs, for example, chemotherapeutic agents, that are unrelated to the selecting agent in structure, cellular target, and mode of action. The expression of this multidrug resistance is connected with the overexpression of P-glycoprotein. By applying the method of immunocytochemical assay, we have demonstrated the appearance of the multidrug-resistant phenotype (P-glycoprotein+ cells, multidrug-resistant cells) in mononuclear cells of the peripheral blood from 32/49 patients receiving triple-drug (azathioprine, steroids, cyclosporine) immunosuppressive therapy after heart transplantation. In the group of patients showing not only the presence of cells with multidrug-resistant phenotype in the peripheral blood, but also a significant increase in the number of these cells during the interval of observation (0 to 767 days)-16/32/49 cases--a significantly increased incidence of acute rejection episodes could be demonstrated. This supports the hypothesis of a possible existence of a therapy-resistant form of acute rejection, with an involvement of mechanisms of multidrug-resistance playing a role in its causal development.

[Clinical application of V-Y flap with subcutaneous pedicle]. From March 1987 to October 1989, 27 wounds in 27 cases have been repaired successfully by a V-Y flap with a subcutaneous pedicle beside the flap. Results were satisfactory. 16 cases of them have been followed-up for 2 months to 2 years, and the appearance has been satisfactory. It seems that V-Y flap with subcutaneous pedicle possesses the following advantages: (1) the advancing distance is large; (2) it has a wide range of application; (3) there is little damage to the blood supply and sensation of the flap.

Distinctive glycolipid patterns in Wilms' tumor and renal cell carcinoma. Glycolipid patterns were analysed chromatographically in Wilms' tumor and renal cell carcinoma tissues and compared with those of uninvolved tissue. Ganglioside GM3 was found to be increased in both cancer tissues, whereas sulfatides accumulated only in renal cell carcinoma, as reported earlier. Neolactotetraosylceramide was detected in both cancer tissues, but not in the uninvolved kidney tissues. In four cases of Wilms' tumors, only a low level of sulfotransferase towards galactosylceramide was found in one case, while no activity was detected in the three other cases. Present results show that the increased sulfatide(s) in the renal cell carcinoma and the deficiency of the sulfatides in Wilms' tumors appear to be biochemical characteristics of histologically different carcinomas.

Expression of dipeptidyl aminopeptidase IV activity in thyroid carcinoma. Dipeptidyl aminopeptidase IV activity staining was performed in various thyroid tissues to evaluate this enzyme activity as a thyroid tumor marker. A total of 195 thyroid tissues were tested for their enzyme activity expression. All papillary and follicular carcinomas, 40 cases and 3 cases, respectively, showed enzyme activity, although two other carcinomas, one medullary and one anaplastic, were not stained. Follicular adenoma expressed enzyme activity in 4 of 26 cases. Fifty-two cases with adenomatous goiter, 54 with Graves' disease and 13 with chronic thyroiditis were judged to be negative. Five normal thyroids expressed no activity except for occasional positive staining of capillary endothelia. These data suggest that dipeptidyl aminopeptidase IV activity staining is very useful for pathological diagnosis of thyroid tumors.

The truncated glucocorticoid receptor in the P1798 mouse lymphosarcoma is associated with resistance to glucocorticoid lysis but not to other glucocorticoid-induced functions. Glucocorticoid receptors in lines of the P1798 mouse lymphosarcoma either sensitive or resistant to glucocorticoid-induced lysis have been characterized and their functional significance determined. The glucocorticoid receptor from the cortisol-sensitive tumor is an Mr approximately 98,000 protein with a Stokes radius of 7.4 nm in the oligomeric, non-DNA-binding state and 5.6 nm in the transformed, DNA-binding state. This receptor binds glucocorticoid and reacts with the BUGR-2 monoclonal antibody. In contrast, two abnormal receptor species were identified in the cortisol-resistant tumor. One is an Mr approximately 98,000 non-steroid-binding but immunologically reactive protein. The other is an Mr approximately 45,000 species which contains both steroid- and DNA-binding sites but exhibits little or no reactivity with BUGR-2, suggesting that its NH2 terminus is truncated in a region within or adjacent to the BUGR epitope. This species had Stokes radii of 5.8 and 3.5 nm in nontransformed and transformed states, respectively. In both tumor lines, glucocorticoids stimulated the activities of glutamine synthetase and 5'-nucleotidase and the synthesis of glucocortin. However, glucocorticoid-induced tumor regression occurred only in the cortisol-sensitive tumor. Additionally, the glucocorticoid inducibility of a specific protein in the sensitive, but not in the resistant, tumor was demonstrated, as well as the presence of a protein specific to the resistant line. Taken together, these results suggest that the truncated glucocorticoid receptor in the P1798 lymphosarcoma is functional, although possibly in a more restricted gene-specific manner, and that the lysis defect, while possibly resulting from a truncated receptor, may also result from the inability of glucocorticoids to induce a critical protein in the pathway of programmed cell death and/or from the presence of a protein which inhibits the lytic response.

A phenotype conferring selective resistance to lipophilic antifolates in Chinese hamster ovary cells. Trimetrexate, a lipid-soluble analogue of methotrexate, appears to enter mammalian cells by passive diffusion, thus circumventing the methotrexate transport system which is frequently a subject for alterations leading to methotrexate resistance. Using a single-step selection protocol with trimetrexate, we have isolated 45 clonal variants and found the majority of them to be selectively resistant to lipophilic antifolates while retaining their sensitivity to methotrexate and drugs involved in multidrug resistance. The majority of spontaneously induced trimetrexate-resistant clones showed a change in neither the mRNA levels of dihydrofolate reductase (24 of 30) and P-glycoprotein (26 of 30) nor their gene copy numbers, whereas a small fraction of clones (4 of 30) showed multidrug resistance gene amplification and P-glycoprotein mRNA overexpression. gamma-Irradiation prior to selection markedly enhanced the frequency of trimetrexate resistance (100-fold after 1000 rads). None of the gamma-ray-induced trimetrexate-resistant clones (0 of 15) had evidence of dihydrofolate reductase and multidrug resistance gene amplification and/or overexpression. Flow cytometry data on trimetrexate-resistant clones showed no defect in the transport of trimetrexate. Verapamil, a modulator of the multidrug resistance phenotype, had no cytotoxic effect on parental and trimetrexate-resistant clones. However, when present with trimetrexate, verapamil (0.3-0.6 microM) reversed the lipophilic antifolate-resistant phenotype in clones that had invariant levels of P-glycoprotein and dihydrofolate reductase. This selective resistance to lipid-soluble antifolates was initially unstable but became stable after continued drug-selective growth. Two-dimensional gel electrophoresis showed some differences in protein(s) that may potentially be associated with this phenotype of selective resistance to lipophilic antifolates. We conclude that a gamma-radiation-enhanceable, verapamil-reversible, stable phenotype of selective resistance to lipid-soluble antifolates frequently emerges which requires neither the amplification nor the overexpression of dihydrofolate reductase or multidrug resistance genes.

Polymorphism and altered methylation of the lactoferrin gene in normal leukocytes, leukemic cells, and breast cancer. Human lactoferrin has been found to be decreased or absent in most breast cancer and leukemia cells. In order to examine the lactoferrin gene for both structural alterations and the degree of methylation, we isolated a 2117-kilobase complementary DNA from human breast tissue. This complementary DNA was used to probe DNA extracted from normal peripheral blood, leukemia cells from patients, leukemia cell lines, and breast cancer cell lines. Immunocytochemical staining of these cells confirmed the decreased production of lactoferrin in malignancy. MspI restriction enzyme fragment patterns demonstrated genetic polymorphism which occurred in DNA from both normal and malignant cells. Polymorphism was also noted with XbaI. In this case, there were two fragment patterns that were only found in DNA from malignant cells. The degree of DNA methylation was also evaluated. The methylation pattern of DNA extracted from malignant cells was highly variable and generally less methylated than DNA extracted from normal WBCs. It is possible that the decrease in lactoferrin associated with cancer is multifactorial and includes gene structural changes as well as altered regulation. Further study is needed to determine whether the changes found in this study are the result of the malignancy or contribute to its onset or maintenance.

Sex-specific splicing and polyadenylation of dsx pre-mRNA requires a sequence that binds specifically to tra-2 protein in vitro. Somatic sex determination in Drosophila involves a hierarchy of regulated alternative pre-mRNA processing. Female-specific splicing and/or polyadenylation of doublesex (dsx) pre-mRNA, the final gene in this pathway, requires transformer (tra) and transformer-2 (tra-2) proteins. The mechanisms by which these proteins regulate RNA processing has not been characterized. In this paper we show that tra-2 produced in Escherichia coli binds specifically to a site within the female-specific exon of dsx pre-mRNA. This site, which contains six copies of a 13 nucleotide repeat, is required not only for female-specific splicing, but also for female-specific polyadenylation. These observations suggest that tra-2 is a positive regulator of dsx pre-mRNA processing.

Different constructs for the expression of mammalian gamma-glutamyltransferase cDNAs in Escherichia coli and in Saccharomyces cerevisiae. To prepare a reference material for gamma-glutamyltransferase (GGT; EC 2.3.2.2) measurements in clinical chemistry, we constructed different vectors containing either the rat kidney or the human hepatoma Hep G2 GGT cDNA downstream from an inducible promoter for expression in Escherichia coli and Saccharomyces cerevisiae. Transformed bacterial and yeast cells were tested for GGT production by use of Western blot analysis and enzymatic activity measurements. Both rat renal and Hep G2 GGT cDNAs were expressed in E. coli, producing active and nonglycosylated enzymes localized in the periplasmic space. Recombinant Hep G2 GGT was synthesized as a single-chain protein, unlike rat renal GGT, which presented two polypeptides of 62 and 30 kDa, identified as the precursor and a GGT heavy-subunit-like peptide, respectively. Rat renal GGT was produced in S. cerevisiae as two polypeptides, 55 and 30 kDa, detected by antisera against rat renal GGT. These results suggest maturation mechanisms such as glycosylation and cleavage steps, enhancing the interest of S. cerevisiae as a useful expression system for producing active mammalian proteins as reference materials.

Body mass index and liver enzyme activity in serum. The association between body mass index (BMI) and serum liver enzyme activity [gamma-glutamyltransferase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST)] was studied in 3167 subjects, 2373 men and 794 women. The subjects were managers and employees, ages 18-64 years, who were examined during a program of preventive medicine. Analysis of covariance was used to compare the serum liver enzyme activities (expressed as natural logarithms) of the subjects, who were subdivided according to BMI, while also considering age, alcohol and cigarette consumption, and physical activity. In men, the percentage increase in the geometric mean of liver enzyme activity of the obese subjects (BMI greater than 30 kg/m2) compared with that of the normal subjects (BMI less than or equal to 25 kg/m2) was 47.7% (P less than 0.001) for GGT, 55.3% (P less than 0.001) for ALT, and 19.7% (P less than 0.001) for AST; in women, the increase was 63.2% (P less than 0.01) for GGT, 58.4% (P less than 0.001) for ALT, and 7.3% (P greater than 0.05) for AST. Thus, our observations demonstrate a relation between BMI and serum liver enzyme activity.

Cardiovascular functions of central noradrenergic neurons in rabbits. 1. The cardiovascular actions of central noradrenergic (NA) neurons was examined using the acute neurotransmitter releasing actions of intracisternal 6-hydroxydopamine in conscious rabbits. 2. The predominant actions of NA pathways in the brain-stem are to inhibit vasomotor and cardiac sympathetic activity and to facilitate cardiac vagal responses. 3. NA projections to the spinal cord tonically inhibit vasoconstrictor tone and form part of a high gain control system for responding to changes in specific afferent information. 4. The forebrain NA projections influence heart rate through modulation of the baroreflex gain of the cardiac vagus. 5. Central antihypertensive drugs such as clonidine and alpha-methyldopa mimic most of the brainstem actions of the central NA neurons. They also utilize the ascending NA pathways influencing the baroreflex vagal gain to minimize the baroreflex effects of reducing mean arterial pressure. 6. Thus, NA neurons influence the circulatory system through actions at all levels of the central nervous system and provide a comprehensive framework for integrating cardiovascular information from multiple inputs. This provides a number of targets for future development of useful pharmacological agents.

Thrombolytic therapy in refractory unstable angina: the role of Holter monitoring. We tested the safety and the usefulness of intravenous urokinase (2 million units administered over 30 min) in 44 patients with refractory unstable angina, defined as persistence of ischemic episodes during 48-h Holter monitoring (Phase 1) despite maximal medical therapy. After thrombolysis, recurrence of ischemia was observed during a week of observation in the CCU, including two 24-h Holter monitorings at the beginning and the end of the week (Phase 2). Seventeen patients completed the observation period without either symptomatic or asymptomatic ischemic episodes (Group A); the remaining 27 continued to manifest ischemia (Group B). No bleeding complications occurred. Within a 6-month follow-up, 2 patients of Group A had recurrence of unstable angina while in Group B, 19 patients had refractory angina or a major cardiac event [10 patients underwent coronary artery bypass surgery (CABG) or percutaneous transluminal coronary angioplasty (PTCA) for refractory angina (p less than 0.001), 6 other patients with refractory angina continued medical therapy, one patient had a myocardial infarction, and two patients died]. In Phase 1 the duration of total ischemia (min/24 h) was a relevant prognostic marker: higher duration correlated with adverse clinical outcome (p less than 0.01). In comparison to Phase 1, duration of total ischemia in Phase 2 was significantly reduced in both groups (16.9 +/- 19.6 vs. 25.4 +/- 17.7; p less than .001). A percent value expressing this variation was calculated for each patient: the variation thus obtained again gave information on the clinical outcome--the greater the reduction, the lower the risk of cardiac events (p less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse fetal kidneys in serum-free organ culture: effects of epidermal growth factor and hydrocortisone. 1. The present study was undertaken to determine whether epidermal growth factor (EGF, 100 ng/ml) or hydrocortisone (HC, 10(-8)-10(-5) M) directly influence proliferation and differentiation of mouse fetal kidney maturing in serum-free organ culture. 2. Addition of EGF to the medium significantly stimulated DNA synthesis after 2 and 5 days of culture. Labelled nuclei were mainly localized in the mesenchymal tissue. Protein synthesis remained unchanged. Activities of three hydrolases, markers of brush border differentiation, were reduced. 3. Hydrocortisone (HC), at all concentrations used, significantly inhibited DNA synthesis. Labelled nuclei were distributed in various cell populations of both control and treated explants. Protein synthesis was stimulated by 10(-7) M after 5 days of culture. Hydrolase activities were slightly modified by HC treatment. 4. The present results indicate that EGF stimulates whereas HC decreases proliferation. Both factors have regulatory effects on brush border maturation. 5. Thus, this culture model is a valuable tool for the study of nephrogenesis.

Symptomatic bradycardia induced by the combination of oral diltiazem and beta blockers. Ten patients, who were admitted to the Intensive Coronary Care Unit during a one year period with symptomatic bradycardia while on combination therapy with oral diltiazem and beta-blocker agents, are described. The important features of this adverse reaction to drug combination were that it appeared mainly in a relatively elderly age group and with presenting symptoms of lethargy, dizziness, syncope, chest pain, and (in one patient with poor left ventricular function) pulmonary edema. It was not dose dependent and occurred even in very low doses of each drug. Electrophysiologic abnormalities were localized to the sinus node in all 10 patients and the primary rhythm disorders were junctional escape rhythm, sinus bradycardia, and sinus pause. These rhythm abnormalities resolved within 24 h following withdrawal of the offending drugs. Temporary pacemaker insertion was necessary in four patients. The duration of drug combination used before the acute episode range from within hours to up to 2 years. In conclusion, although combination diltiazem/beta blocker therapy is very effective in ischemic syndrome, caution is advised when this combination is used especially in the elderly or in patients with left ventricular dysfunction or antecedent sinoatrial or atrioventricular conduction abnormality.

Binding properties of Paracentrotus lividus (Echinoidea) hemolysin. 1. Paracentrotus lividus hemolysin binds erythrocytes, zymosan particles, lipopolysaccharide and laminarin surfaces but not auto and allogeneic cell membranes. 2. The binding could, at least for erythrocytes, involve phospholipids and cholesterol. 3. The protease activity of the coelomic fluid is not related to hemolysis. 4. The finding that very low concentrations of Zn2+ inactivate the hemolysin suggests a possible regulative function of the ion in the hemolytic reaction. 5. Ultrastructural observations on rabbit erythrocyte membranes indicate that most likely the transmembrane pores are induced by the lytic molecules.

Influence of pregnancy and lactation on diurnal and seasonal changes in lactic acid and pyruvic acid levels and in values of pH, pCO2 and pO2 in the mare blood. 1. The diurnal changes in the levels of lactic (LA) and pyruvic (PA) acids and in values of pH, pO2 and pCO2 were studied in the blood of barren and later on in pregnant and lactating mares, throughout three subsequent years. 2. Blood samples were taken every 4 hr, for one day, each month, throughout 3 years. 3. The mares were kept and fed in the same conditions, lighting was natural. 4. In barren mares, diurnal rhythm in LA, PA, pO2 and pCO2 was found. 5. The pregnancy as well as lactation masked diurnal rhythms in parameters studied, except the LA level during lactation but then the acrophase was shifted by 3-4 hr. 6. Seasonal cyclicity was found in the values of LA, PA and pCO2 in barren mares. The pregnancy abolished cyclicity in LA level and modified the behaviour of PA and pCO2 values causing a shift of acrophases and lowering the amplitudes of the indices. 7. In the pO2 tensions no seasonal cycles were observed. 8. In the values of pH neither diurnal rhythms nor seasonal cycles throughout study years were observed.

Insulin stimulatory action on amino acid uptake in bovine adrenal cortex or glomerulosa zone. 1. Insulin stimulated the [1-14C] methylaminoisobutyric acid and [1-14C] aminoisobutyric acid uptake in the bovine adrenal cortex or in the glomerulosa zone through the A system. 2. Verapamil nullified the insulin stimulatory action indicating that this hormonal action is probably related to the voltage-dependent Ca2+ channels.

Milk secretion in the rat: progressive changes in milk composition during lactation and weaning and the effect of diet. 1. Progressive changes in the composition of milk from rats has been studied from day 0 to 20 of lactation and for 3 days following separation of the dams and pups at day 20 post partum. 2. The changes in concentration of Na, K and lactose suggested that secretion both prepartum and following weaning occurred by a paracellular mechanism whereas a transcellular pathway existed during established lactation. 3. The concentration of total protein and casein increased gradually throughout lactation. In contrast, the concentration of serum albumin increased and transferrin decreased markedly during early lactation. The fat content of milk declined 3-fold within 5 days of birth but the concentration of Ca, Mg and inorganic P increased. The concentration of each of these milk constituents remained constant during established lactation. 4. Following weaning the pronounced decline in lactose, K and inorganic P was negatively correlated with an increase in all other milk constituents except fat. 5. Rats fed a low energy diet produced milk with a lower fat content but with an unaltered concentration of protein and carbohydrate. The growth rate of these litters was similar for the first 5 days of lactation when compared to litters from dams fed a high energy diet. The growth rate of litters thereafter and following weaning was greater for rats fed a high energy diet.

The effect of protein intake on the potential activity of the lysosomal vacuolar system in the cat. 1. Experiments were conducted to examine the relationship between protein intake and protein degradation in the liver of cats. 2. The cats were fed either a low protein/high carbohydrate diet (LP) or a high protein diet devoid of carbohydrate (HP). 3. The potential proteolytic activity of the lysosomal vacuolar system in the liver was assessed by both indirect (osmotic fragility of hepatic lysosomes) and direct (stereological measurement of lysosomal volume) methods. 4. The results from both tests indicated a significantly lower autophagic activity of the lysosomal system in the LP fed animals than in the HP fed cats. 5. This suppression of lysosomal protein degradation may represent an important mechanism for the conservation of proteins by the cat when low protein diets are fed.