Steepness of the clinical dose-control curve and variation in the in vitro radiosensitivity of head and neck squamous cell carcinoma. Inter-tumour heterogeneity in radiobiological parameters has been proposed as an explanation for the quite shallow dose-response curves for local tumour control after radiotherapy observed in clinical data. Variability in the intrinsic radiosensitivity is potentially a very strong source of variation in local control. A method is presented for forcing such variability into a direct analysis (maximum-likelihood estimation) of tumour control data. The method is used to reanalyse a series of local tumour control data in 181 patients with squamous cell carcinoma of the oropharynx taking the distribution of in vitro radiosensitivities from an independent series of patients into account. It is concluded that direct application of the in vitro radiosensitivities leads to an unrealistically high estimate for the number of target cells per cm3. A more realistic fit is obtained after including a dose-modifying factor to correct for the apparent difference between in vitro and clinical radiosensitivities. The value of this factor is estimated at 2.4 with approximate 95% confidence interval (CI) (1.3, 5.9). It is suggested that hypoxia plays a role in reducing the radiosensitivity of tumours in clinical radiotherapy. Using this method provides more biologically reasonable estimates of other radiobiological parameters. The target-cell doubling time during treatment is estimated at 3.2 days with 95% CI (1.7, 8.7) days. Estimates of the target cell density in typical patients vary between 1.8 x 10(-6) and 6.6 x 10(-4) when the delay before accelerated tumour growth is assumed to vary between 0 and 28 days. Using the method presented here, the shallow clinical dose-control curve is interpreted as a superposition of quite steep dose-response relationships in individual patients. The steepness of the dose-control curve for a typical patient is characterized by a normalized dose-response gradient (the percentage change in tumour control for a 1% change in total dose) of 7.3 after stratification for intrinsic radiosensitivity as compared with 1.6 if such stratification is not performed.


Heat resistance and thermotolerance in a radiation-resistant cell line. Exponentially growing TN-368 lepidopteran insect cells have a normal growth temperature of 28 degrees C. These cells were heated in water baths at various temperatures between 33 and 44 degrees C under conditions of constant or fractionated heating. Determinations of cell survival using colony formation as well as measurements of DNA and protein synthesis were performed to assess relative heat resistance and development of thermotolerance. The results demonstrate a marked heat resistance over previously reported findings from the same laboratory for dipteran Drosophila cells in culture. The degree of heat resistance is remarkable, especially when compared to the heat resistance of mammalian cells, i.e. TN-368 cell survival at 41.5 and 44 degrees C was somewhat similar to mammalian cell survival, even though these temperatures are 13.5 and 16 degrees C above the normal growth temperature for TN-368 cells and 4.5 and 7 degrees C above the growth temperature of mammalian cells. Furthermore, the lepidopteran cells maintain the ability to develop a notable amount of thermotolerance in addition to this heat resistance. Thermotolerance development alone is capable of enhancing survival by an additional 10,000-fold. Thermotolerance could also be detected at the level of protein synthesis as a more rapid recovery following heat treatment. In contrast, DNA synthesis inhibition was prolonged even further in cells receiving a prior heat treatment to induce thermotolerance. In summary, it appears that, in addition to their pronounced radiation resistance, the TN-368 cells are also quite resistant to heat. It remains to be seen whether a single mechanism could be responsible for resistance to these agents which act very differently.


Changes in the paramagnetic centres in irradiated and heated dental enamel studied using electron paramagnetic resonance. The EPR signals in dental enamel produced by radiation and by heat were studied. The inherent background signal at g = 2.005, and a radiation-produced signal at g = 2.002 have different saturation behaviour with microwave power, and this affords a method of signal optimization. Heating enamel at temperatures from 100 degrees C to 450 degrees C produces a range of radical species from g = 2.002 to g = 2.005, which have been characterized by their g-values, line widths and saturation behaviour. Standard dental drilling produces a range of radicals which appear to be similar to those produced by heat.


Latent equid herpesviruses 1 and 4: detection and distinction using the polymerase chain reaction and co-cultivation from lymphoid tissues. The polymerase chain reaction (PCR) and co-cultivation were used to identify the lymphoreticular system as the site of latency of equid herpesvirus I (EHV-1). Primers for PCR were designed from aligned nucleotide sequences of the glycoprotein gB genes to amplify the same region of both the EHV-1 and EHV-4 genomes. Subsequent restriction digests using specific enzymes distinguished the amplified fragments of the EHV-1 genome from those of the EHV-4 genome. Ten weeks following an experimental infection of five ponies with EHV-1, latent virus was detected by PCR and recovered by co-cultivation, predominantly from lymphoid tissues draining the respiratory tract. Significantly, latent EHV-1 also persisted in peripheral blood leukocytes (PBL). Latent EHV-4, presumably from a preceding natural infection, was also detected in some tissues, including PBL, from all animals. Of additional interest was the recovery of EHV-1 and -4 only in the presence of the ubiquitous EHV-2.


Thy-1 in hippocampus: normal anatomy and neuritic growth in Alzheimer's disease. Abnormal neuritic sprouting is a prominent feature of Alzheimer's disease (AD), and the Thy-1 glycoprotein has a role in neurite growth in culture. We therefore investigated the distribution of Thy-1 immunoreactivity in the hippocampus of normal elderly patients and of AD patients. Normally, Thy-1 immunoreactivity, which was more prominent in CA1 than elsewhere in the hippocampus, was located mainly in irregular patches on the perikarya of pyramidal cells, their dendrites and axons. In AD, Thy-1-immunoreactive neurons were reduced in number in CA1, and there was diffuse staining of neurofibrillary tangle-bearing pyramidal cells, but neurofibrillary tangles themselves were not immunoreactive. There was also staining of disorganized arrays of dystrophic neurites, some with spiny processes and bizarre filopodial endings. Some Thy-1-immunoreactive dystrophic neurites entered senile plaques. The data confirm that there is extensive growth of abnormal neurites in AD and suggest that Thy-1 is involved in this process.


Immunohistochemical demonstration of glutathione S-transferases in primary human breast carcinomas. The expression of cytosolic glutathione S-transferase (GST) isoenzymes has been assessed in a series of 74 primary human breast carcinomas using an immunohistochemical method. GST pi was detected in sections from all 74 tumours; it was expressed by non-epithelial (stromal and inflammatory) cells in 62 tumours (84 per cent), but by tumour epithelium in only 35 (47 per cent). Non-neoplastic mammary epithelium was uniformly positive for GST pi. Expression of GST alpha and mu was observed in 19 and 42 per cent of the tumours, respectively, and was largely confined to the neoplastic component. Lack of staining of tumour epithelium for GST pi was significantly associated with poorer tumour differentiation (higher grade). There was no association between expression of any of the three isoenzymes and either menopausal status or expression of c-erbB-2 oncogene protein product. Immunohistochemistry is a useful method for the investigation of expression and cellular localization of GSTs within tumours; such data are needed to improve our understanding of the role of these enzymes in neoplasia and in resistance to cytotoxic drug therapy.


A study of cell proliferation in formalin-fixed, wax-embedded bone marrow trephine biopsies using the monoclonal antibody PC10, reactive with proliferating cell nuclear antigen (PCNA). We have investigated proliferation in bone marrow trephine biopsies from 32 patients with normal or abnormal haemopoiesis, using the monoclonal antibody PC10, which detects proliferating cell nuclear antigen (PCNA), together with immunohistochemical markers of haemopoietic cell lineage. PCNA immunostaining revealed the pattern of proliferation within individual haemopoietic lineages in normal marrow. Two unexpected observations were made: of erythroid cells, only pro-erythroblasts and occasional early normoblasts reacted, and positivity of megakaryocytes was unrelated to nuclear lobulation or CD61 expression. The pathological cases represented conditions in which haemopoiesis is increased (reactive hyperplasia, chronic granulocytic leukaemia, myeloproliferative and myelodysplastic syndromes, megaloblastic anaemia). Increases in the number, and disturbances of the spatial organization, of PCNA-expressing cells were present to a variable extent in all cases. Sheets of PCNA-positive megaloblastoid erythrocytes were frequently found in myelodysplastic and myeloproliferative tissue, associated with marked disturbances in the spatial organization of all haemopoietic lineages. Cases of megaloblastic anaemia due to vitamin B12/folate deficiency also demonstrated greatly increased erythroid PCNA expression, with positivity in some giant metamyelocytes. In addition to reflecting increased proliferation, elevated PCNA expression in some bone marrow pathologies may be due to altered kinetics of the protein induced by disturbances in growth factor production.


Familial neurofibromatosis type 1: clinical experience with DNA testing. To determine how DNA testing for familial neurofibromatosis type 1 (NF-1) would be used in a clinical setting by patients and physicians, we performed confirmatory DNA testing on 24 individuals with a family history of NF-1 and on nine couples who requested DNA testing for current or future prenatal diagnosis. A further eight families were unsuitable for DNA linkage testing because of their pedigree structure. For the majority of persons the certainty of the test result was 95% to 99%. In five individuals, only one of whom was less than 6 years of age, the DNA-based diagnosis was discrepant with the clinical diagnosis at the time of referral. In all five cases, results of subsequent clinical re-examinations were consistent with the DNA diagnosis. We conclude that DNA testing by linkage analysis may be most useful as an adjunct to the clinical diagnosis of familial NF-1 (1) in children less than 6 years of age in whom the full manifestations may not yet be apparent, (2) in NF-1 families interested in prenatal testing, and (3) when the resources available for a complete clinical examination are limited.


Alterations of the P53 gene are associated with the progression of a human prostate carcinoma. P53 is a tumor suppressor gene that has been implicated in the molecular genetics of many human malignancies. Nucleotide alterations, most commonly single point mutations, have been shown not only to abrogate the p53 suppressor function but also to contribute to the transformed phenotype. We report the detection of a p53 gene mutation in clinical specimens of a patient with relapsing prostate adenocarcinoma 14 years after definitive external beam radiation. The techniques of single strand conformation polymorphism analysis and direct sequencing of polymerase chain reaction generated products were used for this study. Analysis of tissue from different locations of the primary tumor revealed intratumoral molecular heterogeneity; the mutation was absent in 1 area but present in another. Tumor from a regional lymph node metastasis harbored the identical p53 mutation. Furthermore, an additional genetic alteration, an allelic loss on chromosome 17p but not including the p53 gene, was observed only in the metastatic tissue. These observations in clinical specimens of primary and metastatic sites provide evidence for the association of the p53 gene in the progression of human prostate carcinoma.


Lymphocyte subsets in healthy children during the first 5 years of life. To assess whether relative and absolute values of CD4 and CD8 lymphocytes and CD4/CD8 ratio change in relation to age, and to estimate the fifth and 95th percentiles for these values in children of various ages. Phenotypic analysis of lymphocyte subsets was performed on blood samples from 208 healthy children, aged 1 through 59 months, using standard flow cytometric techniques. Regression analysis demonstrated that CD4 and CD8 lymphocyte counts declined significantly with advancing age (P less than .000001 and P = .03, respectively). Since CD4 and CD8 counts depend on total lymphocyte count, the percentage of total lymphocytes of each phenotype was also analyzed and demonstrated that the CD4 percentage was highly age dependent (P less than .000001). The CD8 percentage increased with age (P = .0001) but not as much as the CD4 percentage decreased. Median CD4 counts (fifth and 95th percentiles) for children 2 through 3, 4 through 8, 12 through 23, and 24 through 59 months of age were 2.83 (1.46 to 5.11), 2.95 (1.69 to 4.61), 2.07 (1.02 to 3.60), and 1.80 (0.90 to 2.86) x 10(9)/L, respectively. Healthy children's CD4 lymphocyte counts are considerably higher than previously established adult values. These data demonstrate that age is an important consideration in interpretation of lymphocyte subsets in children. This may be especially relevant in children who are infected with the human immunodeficiency virus, where CD4 lymphocyte values play a central role in monitoring disease progression and determining thresholds for medical interventions.


Screening for von Hippel-Lindau disease by DNA polymorphism analysis. Von Hippel-Lindau (VHL) disease is a rare, inherited multisystem neoplastic disorder. There is no biochemical test available to distinguish VHL disease gene carriers from their healthy siblings. We evaluated DNA polymorphism analysis as a method for identifying disease gene carriers. Prospective comparison of the results of DNA analysis with a comprehensive clinical screening examination. The Clinical Center of the National Institutes of Health. Blood was collected from 182 members of 16 families with VHL disease. Forty-eight asymptomatic individuals, at risk of developing this hereditary illness (with an affected parent or sibling), were examined for occult disease at the Clinical Center of the National Institutes of Health and tested by DNA polymorphism analysis. DNA polymorphism analysis predicted nine disease gene carriers and 33 individuals with the wild-type (normal) allele among the 48 individuals at risk of developing VHL disease; the test was not informative in six individuals. All nine individuals predicted to carry the VHL gene had evidence of occult disease on clinical examination. There was no clinical evidence of VHL disease in 32 of 33 individuals predicted to carry the wild-type allele. DNA polymorphism analysis can identify individuals likely to carry the VHL disease gene among asymptomatic members of disease families. This technique serves to focus attention on those individuals who require periodic medical examination and may help to alleviate the morbidity and mortality associated with this disease.


Effects of long-term xamoterol in idiopathic dilated cardiomyopathy. In prospective study, the beta 1-partial agonist xamoterol (200 mg daily) was given to 26 patients with idiopathic dilated cardiomyopathy (DCM) in addition to conventional therapy with digitalis, diuretics and vasodilators. The patients were followed for 35 +/- 15 months (6-53 months). Cardiothoracic ratio (CTR), left ventricular end-diastolic dimension (LVDD) and exercise heart rate decreased, and exercise duration, fractional shortening (FS) and ejection fraction (EF) increased after xamoterol therapy. Twenty-one patients survived and one patient dropped out at 7 months. Twelve of the 20 patients improved their NYHA functional class. Blood norepinephrine concentration (NE), LVDD, FS, EF and pulmonary capillary wedge pressure (PCWP) after xamoterol were significantly better in survivors than in non-survivors. Survival rate at 3 years was 83%. The results suggest that adjunctive xamoterol therapy in DCM has a beneficial effect on hemodynamics and symptoms. Prognosis will be satisfactory if improvement in parameters such as NE, LVDD, FS, EF and PCWP is seen during xamoterol therapy.


Effects on coronary artery disease of lipid-lowering diet, or diet plus cholestyramine, in the St Thomas' Atherosclerosis Regression Study (STARS). To assess the effect of dietary reduction of plasma cholesterol concentrations on coronary atherosclerosis, we set up a randomised, controlled, end-point-blinded trial based on quantitative image analysis of coronary angiograms in patients with angina or past myocardial infarction. Another intervention group received diet and cholestyramine, to determine the effect of a greater reduction in circulating cholesterol concentrations. 90 men with coronary heart disease (CHD), who had a mean (SD) plasma cholesterol of 7.23 (0.77) mmol/l were randomised to receive usual care (U, controls), dietary intervention (D), or diet plus cholestyramine (DC), with angiography at baseline and at 39 (SD 3.5) months. Mean plasma cholesterol during the trial period was 6.93 (U), 6.17 (D), and 5.56 (DC) mmol/l. The proportion of patients who showed overall progression of coronary narrowing was significantly reduced by both interventions (U 46%, D 15%, DC 12%), whereas the proportion who showed an increase in luminal diameter rose significantly (U 4%, D 38%, DC 33%). The mean absolute width of the coronary segments (MAWS) studied decreased by 0.201 mm in controls, increased by 0.003 mm in group D, and increased by 0.103 mm in group DC (p less than 0.05), with improvement also seen in the minimum width of segments, percentage diameter stenosis, and edge-irregularity index in intervention groups. The change in MAWS was independently and significantly correlated with LDL cholesterol concentration and LDL/HDL cholesterol ratio during the trial period. Both interventions significantly reduced the frequency of total cardiovascular events. Dietary change alone retarded overall progression and increased overall regression of coronary artery disease, and diet plus cholestyramine was additionally associated with a net increase in coronary lumen diameter. These findings support the use of a lipid-lowering diet, and if necessary of appropriate drug treatment, in men with CHD who have even mildly raised serum cholesterol concentrations.


Mechanism of grass-pollen-induced asthma. Many asthmatics are sensitive to rye-grass pollen, but pollen grains are too large to penetrate the lower airways. Our aim was to investigate the mechanism by which rye-grass pollen causes asthma. A major allergen of rye-grass pollen, Lol pIX, is located in intracellular starch granules within pollen grains. In-vitro tests showed that pollen grains are ruptured in rainwater by osmotic shock, each grain releasing about 700 starch granules into the environment. These granules are small enough to enter the airways (less than 3 microns in diameter). The starch granules were present in atmospheric samples taken during the pollen season, and showed a 50-fold increase in atmospheric concentration on days following rainfall. Isolated granules elicited IgE-mediated responses in asthmatic patients, and 4 patients with rainfall-associated asthma who underwent an inhalation challenge test had striking bronchial constriction after exposure to starch granules. Starch granules released from rye-grass pollen seem to be capable of causing asthma.


Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Nitric oxide (NO), synthesised from L-arginine, contributes to the regulation of blood pressure and to host defence. We describe in-vitro and in-vivo evidence that NO synthesis can be inhibited by an endogenous compound, NG,NG-dimethylarginine (asymmetrical dimethylarginine, ADMA). In man, this inhibitor is found in plasma and more than 10 mg is excreted in urine over 24 h. However, in patients with end-stage chronic renal failure, who have little or no urine output, elimination is blocked and circulating concentrations of the inhibitor rise sufficiently to inhibit NO synthesis. Accumulation of endogenous ADMA, leading to impaired NO synthesis, might contribute to the hypertension and immune dysfunction associated with chronic renal failure.


Mutations of p53 and ras genes in radon-associated lung cancer from uranium miners. Radon increases the risk of lung cancer in smoking and non-smoking underground miners. To investigate the mutational spectrum associated with exposure to high levels of radon, we sequenced exons 5-9 of the p53 tumour suppressor gene and codons 12-13 of the Ki-ras protooncogene in 19 lung cancers from uranium miners exposed to radon and tobacco smoke. Mutations were not found in Ki-ras, but 9 p53 mutations, including 2 deletions, were found in 7 patients by direct DNA sequencing after polymerase chain reaction amplification of DNA from formalin-fixed, paraffin-embedded tissue. In tumours from 5 patients, the mutation produced an aminoacid change and an increased nuclear content of p53 protein. The tumours with either a stop codon or frame-shift deletion in the p53 gene were negative by immunohistochemistry. None of the mutations were G:C to T:A transversions in the coding strand of the p53 gene, which are the most frequent base substitutions associated with tobacco smoking, and none were found at the hotspot codons described in lung cancer. The observed differences from the usual lung cancer mutational spectrum may reflect the genotoxic effects of radon.


Liver regeneration in recipients and donors after transplantation. Reduced-size liver grafts from related donors may not be of an optimal size for adequate function in the recipient. Therefore, liver-graft regeneration is clinically important. We evaluated liver regeneration by liver-volume determinations with serial computed tomography scans in four recipients (aged 9 months to 12 years) and their donors (all fathers of the recipients) after living-related liver transplantation. Standard liver volume was calculated from the recipient's body-surface area. In each recipient, the size of the transplanted liver tended to converge to the standard liver volume with time, regardless of whether initial liver-graft volume was smaller or larger than standard liver volume. In addition, transplanted liver in the recipient regenerated much faster than remnant liver in the donor, even though both consisted of the same hepatocytes, which suggests that regeneration is regulated mainly by factors other than the hepatocytes themselves.


Location of gene for Gorlin syndrome.

Location of gene for Gorlin syndrome. The Gorlin (naevoid-basal-cell-carcinoma) syndrome is an autosomal dominant disorder characterised by multiple naevoid basal-cell carcinomas, recurrent odontogenic keratocysts, skeletal anomalies, intracranial calcification, and developmental malformations. Characterisation of the gene that causes the syndrome may improve our understanding of the pathogenesis of other basal-cell carcinomas. By linkage analysis, we have shown that the gene is located on chromosome 9q22.3-q31; the most likely position is between DNA markers D9S12 and D9S53. Location of the gene for Gorlin syndrome offers the possibility that DNA markers can be used in risk estimation and presymptomatic identification of patients for surveillance.


Incidence of development of factor VIII and factor IX inhibitors in haemophiliacs. The development of factor VIII:C inhibitors remains one of the most serious complications of repeated transfusion in patients with haemophilia A. The proportion of patients affected has been reported to range from 3.6% to 25%, but these figures have been derived mainly from retrospective data and from total numbers of known haemophiliacs instead of number at true risk. The assessment here is based on a prospective study, started in 1976, on the incidence of inhibitor development in haemophiliacs born after 1970 whose FVIII or FIX activity was 5% or less, and who had received replacement therapy at least once. 46 of 63 children with haemophilia A and 13 of 17 with haemophilia B fulfilled the enrollment criteria. Inhibitors developed only in haemophilia A patients who had previously been treated with FVIII products--inhibitor concentrations were high in 12 and low in 3. Inhibitors developed in 24% (15/63) of all haemophilia A patients, and in 52% (14/27) of those with severe disease. The incidence of inhibitor development for all haemophilia patients was 39.1 per 1000 patient-years of observation. All inhibitors were first detected when patients were aged 0.08-5.2 years. The cumulative risk was 33% at age 6 years. The findings indicate that previous reports have underestimated the risk of acquiring FVIII inhibitors. Prospective, standardised studies, especially in children, are needed for the assessment of the true risk of this complication.


Hypocholesterolaemic effects of lovastatin in familial defective apolipoprotein B-100. Familial defective apolipoprotein B-100 (FDB) is an autosomal dominant disorder associated with hypercholesterolaemia in which an aminoacid substitution in apoprotein B-100 leads to low-density lipoprotein (LDL) particles which have defective binding to the LDL receptor. All known patients are heterozygous, and their plasma contains normal and poorly binding LDL particles. 12 hypercholesterolaemic patients from 10 unrelated families with FDB were treated with lovastatin. In 6 patients treated with 20 mg lovastatin daily, LDL cholesterol decreased by 21.5% from 6.23 to 4.89 mmol/l (95% confidence interval 0.74, 1.96 mmol/l), whereas it fell by 32.1%, from 6.99 to 4.81 mmol/l (95% CI 1.55, 2.70 mmol/l), in 9 patients who received 40 mg daily. These results indicate that the hypercholesterolaemia of FDB may respond to treatment with statins.


Regulation of glucose kinetics in trauma patients by insulin and glucagon. The current study was undertaken to evaluate the contribution of insulin and glucagon to regulation of glucose metabolism in man following severe, traumatic injury by manipulating concentrations of insulin and glucagon with infusions of somatostatin. Glucose kinetics were assessed with [U-14C, 6-(3)H]glucose in severely injured patients and compared with data obtained from patients recovering from minor, elective operative procedures. Glucose production was significantly increased in subjects with traumatic injury compared with control subjects (13.0 +/- 0.63 mumol/kg/min v 8.6 +/- 0.27 mumol/kg/min). There was no impairment in glucose oxidation by the injured patients. Modulation of insulin and glucagon with somatostatin indicated that non-insulin-mediated glucose uptake (NIMGU) was significantly elevated in injured patients (12.2 +/- 0.94 mumol/kg/min v 7.4 +/- 0.61 mumol/kg/min). Hepatic glucose output (HGO) in the absence of glucagon was also significantly elevated in injured patients (12.2 +/- 1.20 mumol/kg/min v 5.8 +/- 1.08 mumol/kg/min). Indirect calorimetry showed a 27% increase in resting energy expenditure (REE). Increased protein oxidation accounted for 56% of the increase in REE. Changes in carbohydrate and lipid oxidation accounted for 28% and 15% of the increase in REE. There was no correlation between the injury severity score of the injured patient and the degree of metabolic abnormality. It is concluded from these studies that (1) injured patients have a high rate of glucose turnover in the absence of glucagon and insulin; (2) the reliance on glucose as a source of energy is not diminished in injured subjects; and (3) increases in protein oxidation account for the majority of the increased REE found in injured patients.


Characterization and genetic mapping of a short, highly repeated, interspersed DNA sequence from rice (Oryza sativa L.). A short, highly repeated, interspersed DNA sequence from rice was characterized using a combination of techniques and genetically mapped to rice chromosomes by restriction fragment length polymorphism (RFLP) analysis. A consensus sequence (GGC)n, where n varies from 13-16, for the repeated sequence family was deduced from sequence analysis. Southern blot analysis, restriction mapping of repeat element-containing genomic clones, and DNA sequence analysis indicated that the repeated sequence is interspersed in the rice genome, and is heterogeneous and divergent. About 200,000 copies are present in the rice genome. Single copy sequences flanking the repeat element were used as RFLP markers to map individual repeat elements. Eleven such repeat elements were mapped to seven different chromosomes. The strategy for characterization of highly dispersed repeated DNA and its uses in genetic mapping, DNA fingerprinting, and evolutionary studies are discussed.


Sequence of a cDNA encoding nitrite reductase from the tree Betula pendula and identification of conserved protein regions. The sequence of an mRNA encoding nitrite reductase (NiR, EC 1.7.7.1.) from the tree Betula pendula was determined. A cDNA library constructed from leaf poly(A)+ mRNA was screened with an oligonucleotide probe deduced from NiR sequences from spinach and maize. A 2.5 kb cDNA was isolated that hybridized to an mRNA, the steady-state level of which increased markedly upon induction with nitrate. The nucleotide sequence of the cDNA contains a reading frame encoding a protein of 583 amino acids that reveals 79% identity with NiR from spinach. The transit peptide of the NiR precursor from birch was determined to be 22 amino acids in size by sequence comparison with NiR from spinach and maize and is the shortest transit peptide reported so far. A graphical evaluation of identities found in the NiR sequence alignment revealed nine well conserved sections each exceeding ten amino acids in size. Sequence comparisons with related redox proteins identified essential residues involved in cofactor binding. A putative binding site for ferredoxin was found in the N-terminal half of the protein.


Stimulation of N-methyl-D-aspartate receptor-mediated calcium entry into dissociated neurons by reduced and oxidized glutathione. The effects of GSH (gamma-glutamylcysteinylglycine) and GSSG on intracellular calcium levels ([Ca2+]i) were investigated using fura-2-loaded dissociated brain cells from newborn rat pups. Both produced concentration-dependent increases in [Ca2+]i (EC50 values of 914.3 +/- 190.5 and 583.0 +/- 97.2 microM for GSH and GSSG, respectively), similar to that observed with N-methyl-D-aspartate (NMDA) and other agonists at the NMDA receptor. Maximum response (expressed as percentage change in [Ca2+]i relative to basal) was significantly greater for GSSG (37.5 +/- 1.6%) than for GSH (25.3 +/- 1.6%). The response to both agents was prevented or reversed by competitive (100 microM) (-)-2-amino-5- phosphonovalerate and noncompetitive (400 nM) MK-801 or 1.0 mM Mg2+ antagonists of NMDA receptor-mediated calcium entry, even at concentrations of GSH and GSSG normally producing maximal response. The idea that these effects are mediated, at least in part, by interaction with the NMDA receptor was supported by the effects of GSH and GSSG on the binding of the NMDA receptor ligand [3H]CGP-39653 to membranes isolated from hippocampal and cortical homogenates. Both GSH and GSSG displaced bound [3H]CGP-39653, with IC50 values of 0.93 +/- 0.18 and 11.02 +/- 1.22 microM, respectively, and produced an increase in the apparent Kd of binding (control, 8.92 +/- 0.83 nM, and GSH, 13.31 +/- 1.19 nM; control, 11.59 +/- 0.35 nM, and GSSG, 18.73 +/- 0.66 nM). However, both also produced modest reductions in Bmax (control, 1265 +/- 69 fmol/mg of protein, and GSH, 901 +/- 73 fmol/mg of protein; control, 1068 +/- 30 fmol/mg of protein, and GSSG, 730 +/- 18 fmol/mg of protein) and Hill slopes (GSH, 0.66 +/- 0.02; GSSG, 0.62 +/- 0.04). This suggests complex kinetics for the interaction of GSH and GSSG with the NMDA receptor. Taken together, the results suggest the potential for modulation of the NMDA receptor complex by GSH and GSSG.


Isoproterenol-initiated beta-adrenergic receptor diacytosis in cultured cells. The kinetics of the return of internalized beta-adrenergic receptors to the plasma membrane were measured in human astrocytoma cells. The movement of [125I]iodopindolol-labeled receptors back to the plasma membrane was measured directly and was shown to occur with a t1+2 of 3-4 min. Unlabeled receptors appeared to exhibit the same kinetics of externalization. The process was not inhibited by low concentrations (1-10 microM) of propranolol or even high concentrations of isoproterenol (0.1-1.0 mM). Higher concentrations of propranolol (0.1-1.0 mM) and other lipophilic amines inhibited externalization. The results are consistent with the proposal that catecholamine-induced beta-adrenergic receptor internalization and externalization (diacytosis) occur via the clathrin-coated pit/endosome pathway.


Waardenburg's syndrome patients have mutations in the human homologue of the Pax-3 paired box gene. Waardenburg's syndrome (WS) is an autosomal dominant combination of deafness and pigmentary disturbances, probably caused by defective function of the embryonic neural crest. We have mapped one gene for WS to the distal part of chromosome 2. On the basis of their homologous chromosomal location, their close linkage to an alkaline phosphatase gene, and their related phenotype, we suggested that WS and the mouse mutant Splotch might be homologous. Splotch is caused by mutation in the mouse Pax-3 gene. This gene is one of a family of eight Pax genes known in mice which are involved in regulating embryonic development; each contains a highly conserved transcription control sequence, the paired box. Here we show that some families with WS have mutations in the human homologue of Pax-3. Mutations in a related gene, Pax-6, which, like Pax-3, has both a paired box and a paired-type homeobox sequence, cause the Small-eye mutation in mice and aniridia in man. Thus mutations in the Pax genes are important causes of human developmental defects.


An exonic mutation in the HuP2 paired domain gene causes Waardenburg's syndrome. Here we report the identification and characterization of a gene defect causing Waardenburg's syndrome with hearing loss in a large Brazilian family. This demonstrates a mutation causing Waardenburg's syndrome as well as a mutation causing a form of congenital deafness. The mutation was found in the HuP2 gene, a member of the paired domain family of proteins that bind DNA and regulate gene expression. The mutation occurred in 100% of the cases with the disease in this family and was absent in a random sample of 50 unrelated control subjects. Identification of the Waardenburg's syndrome gene and future characterization of its gene product is likely to increase our understanding of the pathogenesis of this disorder and may allow prevention of deafness of this type.


C. elegans unc-4 gene encodes a homeodomain protein that determines the pattern of synaptic input to specific motor neurons. The creation of neural circuits depends on the formation of synapses between specific sets of neurons. Little is known, however, of the molecular mechanisms governing synaptic choice. A mutation in the unc-4 gene alters the pattern of synaptic input to one class of motor neurons in the Caenorhabditis elegans ventral nerve cord. In unc-4(e120), the presynaptic partners of VA motor neurons are replaced with interneurons appropriate to motor neurons of the VB class. This change in neural specificity is not accompanied by any detectable effects on neuronal morphology or process extension. We show that the absence of a functional unc-4 gene product accounts for the mutant phenotype. The unc-4 gene encodes a homeodomain protein and thus is likely to function as a transcription factor. The limited effect of the unc-4 null mutation on cell fate may mean that unc-4 regulates the expression of a small number of target genes and that the products of these genes are directly involved in the choice of synaptic partners.


Linkage of regression and malignant conversion of rabbit viral papillomas to MHC class II genes. Human papillomaviruses associated with cutaneous and anogenital cancers induce intraepithelial precursor lesions which may regress spontaneously or progress into invasive carcinomas. Cell-mediated immune responses are probably involved in regression of precancerous lesions and the polymorphism of the genes responsible may thus have a key role in the variability of the host response. Skin warts and cancers induced in rabbits by Shope papillomavirus provide a model to test this hypothesis. We analysed a restriction-fragment-length polymorphism of major histocompatibility complex class I and class II genes and T-cell receptor beta-chain genes in infected domestic rabbits. We found a strong linkage between wart regression and a DR alpha EcoRI fragment, and an increased relative risk of malignant transformation associated with a DQ alpha PvuII fragment. This indicates a genetic control of wart evolution, involving genes in the class II region of the major histocompatibility complex.


Activation of dopamine D1 receptors does not affect D2 receptor-mediated inhibition of acetylcholine release in rabbit striatum. The possible involvement of dopamine D1 receptors in the regulation of acetylcholine release in the rabbit caudate nucleus was investigated. Caudate slices, preincubated with [3H]choline, were superfused continuously and subjected to electrical field stimulation with only a single pulse. In agreement with the view that the release of acetylcholine evoked by a single electrical pulse is not influenced by endogenous transmitters, atropine and domperidone failed to increase the evoked release of [3H]acetylcholine, whereas oxotremorine and quinpirole caused a concentration-dependent inhibition of transmitter release. Neither the dopamine D1 receptor antagonist SCH 23390 nor the D1 agonist SKF 38393 in a concentration range of 0.01-1 mumol/l changed the evoked [3H]acetylcholine release. The inhibitory effect of the dopamine D2 receptor agonist quinpirole was virtually abolished in the presence of 0.1 mumol/l domperidone and diminished in the presence of 1 mumol/l SCH 23390. It remained unchanged in the presence of 1 mumol/l SKF 38393. It is concluded that the inhibition of acetylcholine release by dopamine is mediated exclusively via presynaptic dopamine D2 receptors and that the antagonistic effect of SCH 23390 on the inhibition of acetylcholine release by quinpirole is due to its interaction with dopamine D2 rather than D1 receptors located on cholinergic nerve terminals.


Activation of beta 2-adrenoceptors by isoprenaline and adrenaline enhances noradrenaline release in cortical kidney slices of young spontaneously hypertensive rats. The aim of the present study was to investigate beta-adrenoceptor modulation of noradrenaline release from sympathetic nerves in superfused cortical kidney slices of 4-week-old spontaneously hypertensive rats (SHR) and age-matched controls (WKY). After preincubation with 3H-noradrenaline the kidney slices were electrically stimulated in superfusion chambers. The stimulation induced (S-I) outflow of radioactivity was mainly composed of unmetabolized 3H-noradrenaline in both strains and thus taken as an index of noradrenaline release. There was a frequency-dependent (1.25-20 Hz) increase in the S-I outflow of radioactivity. At all stimulation frequencies tested S-I outflow of radioactivity was similar or even slightly lower in SHR than in WKY kidney slices in either the absence or presence of cocaine (10 mumol/l). The non-selective beta-adrenoceptor agonists isoprenaline (0.1 mumol/l) and adrenaline (0.01 and 0.1 mumol/l) enhanced S-I outflow of radioactivity. The facilitatory effects of isoprenaline (0.1 mumol/l) and adrenaline (0.1 mumol/l) were blocked by the selective beta 2-adrenoceptor antagonist ICI 118551 (0.1 mumol/l) but not by the selective beta 1-adrenoceptor antagonist atenolol (0.3 mumol/l). The cell-permeable cAMP analogue 8-bromo-cAMP (300 mumol/l) enhanced S-I outflow of radioactivity to a similar extent in both SHR and WKY kidney slices. A combination of 8-bromo-cAMP (300 mumol/l) and adrenaline (0.1 mumol/l) did not enhance S-I outflow of radioactivity to a greater extent than 8-bromo cAMP (300 mumol/l) alone in both strains.(ABSTRACT TRUNCATED AT 250 WORDS)


Presynaptic effects of dopexamine hydrochloride in the canine kidney. The effects of dopexamine on the vasoconstrictor response to renal nerve stimulation and noradrenaline were determined in anesthetized dogs. Renal vasoconstriction was repeated during the sequential administration of iso-osmotic saline, dopexamine, dopexamine plus the DA1-dopamine receptor antagonist, SCH 23390, and dopexamine plus SCH 23390 and the DA2-dopamine receptor antagonist, domperidone. Renal nerve stimulation-induced vasoconstriction did not change with dopexamine or dopexamine plus SCH 23390, but increased with the addition of domperidone. Dopexamine also potentiated noradrenaline-induced vasoconstriction. Antagonists alone had no affect on the vasoconstrictor response to either stimulus. The findings suggest that the absence of an effect of dopexamine on neuronally-induced renal vasoconstriction was a consequence of a balance between its actions at presynaptic DA2-dopamine receptors (attenuated vasoconstriction) and its ability to inhibit the uptake1 transporter (enhanced vasoconstriction).


Prejunctional opioid mu-receptors and adenosine A1-receptors on the sympathetic nerve endings of the rat tail artery interact with the alpha 2-adrenoceptors. Experiments were designed to study the interaction between prejunctional alpha 2-adrenoceptors and both adenosine and opioid receptors at the postganglionic sympathetic nerve endings innervating the tail artery of the rat. Segments of this vessel were preincubated with [3H]-noradrenaline and then perfused/superfused with [3H]-noradrenaline-free medium. Their perivascular nerves were field stimulated with standard stimulation parameters: 24 pulses at 0.4 Hz, 0.3 ms, 200 mA. In some experiments, the stimulation parameters were adjusted in order to obtain similar reference release values despite the presence of a first release-modulating drug. The adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA; 0.3-10 mumol/l) and [D-Ala2,MePhe4,Glyol5]enkephalin (DAGO; 0.3-10 mumol/l) depressed the stimulation-evoked overflow of tritium in a concentration dependent manner. The release-inhibiting effect of both NECA and DAGO was enhanced in the presence of the alpha 2-adrenoceptor antagonist rauwolscine (3 mumol/1) while it was attenuated in the presence of the alpha 2-adrenoceptor agonist 5-bromo-6-[2-imidazolin-2yl-amino]-quinoxaline (UK-14,304; 0.1 mumol/l). These changes occurred both at standard and adjusted stimulation parameters. These results demonstrate that the prejunctional adenosine A1- and opioid mu-receptors interact with the prejunctional alpha 2-adrenoceptors. The level at which these interactions take place (receptors themselves or transduction mechanisms) as well as the physiological significance of the phenomenon remain to be determined.


Comparison of a cloned ANF-sensitive guanylate cyclase (GC-A) with particulate guanylate cyclase from adrenal cortex. Recently, an ANF-sensitive guanylate cyclase (GC-A) has been cloned from a rat brain cDNA library. Here we studied the stimulation of cyclic GMP accumulation in response to atrial natriuretic factor (ANF), urodilatin and atriopeptin I (AP-1) in a rat glioma C6 cell line permanently transfected with GC-A as well as GC-A activity in membranes from these C6 cells and in membranes from COS-7 cells that were transiently transfected with GC-A. We also measured binding affinities for these natriuretic peptides in the membrane preparations. These characteristics of GC-A were compared to those of membrane preparations from adrenal cortex of bovine and human origin. The order of potency of stimulation of cyclic GMP accumulation in permanently transfected glioma cells was ANF greater than urodilatin greater than AP I; AP I stimulated cyclic GMP accumulation. A similar order of potency was obtained for stimulation of guanylate cyclase activity in membranes from permanently transfected glioma cells as well as from transiently transfected COS-7 cells. In contrast, AP-1 was uneffective to stimulate guanylate cyclase in membrane preparations from adrenal cortex from bovine as well as from human origin. Furthermore, urodilatin was equipotent to ANF in these preparations. Binding affinities were comparable for ANF and urodilatin in membranes from cells transfected with GC-A and in membranes from adrenal cortex of both sources, whereas AP-1 had a weaker affinity in all preparations studied.(ABSTRACT TRUNCATED AT 250 WORDS)


[Parkinsonism following addition of fluoxetine to the treatment with neuroleptics or carbamazepine]. This article describes three patients who developed parkinsonism when fluoxetine was added to their existing medication (neuroleptics or carbamazepine). Based on published pharmacological and neuroanatomical research we postulate a serotonin-dopamine antagonism to be operative in the development of drug-induced parkinsonism. Alternatively, the possibility of a pharmacokinetic interaction remains.


Isolation and characterization of endogenous modulators for GABA system. Pig brain extracts from both soluble and membrane fractions were found to contain potent inhibitors for GABA synthesizing enzyme, GAD, referred to as endogenous GAD inhibitors (EGIs) and for the binding of GABA agonist, muscimol, referred to as muscimol binding inhibitors (MBIs). EGIs and MBIs were first purified through gel-filtration Bio-Gel P-2 columns, in which multiple activity peaks were observed. One of them appears to be co-eluted with either L-glutamate or GABA. However, others are clearly separated from L-glutamate or GABA. EGIs were found to be low MW (less than 1,800 dalton), heat and acid-base stable, negatively charged, non hydrophobic substances. MBIs were found to be low MW (less than 1,800 dalton) neutral or positively charged substances. MBIs had no effect on [3H]flunitrazepam (FNZP) binding, indicating that they are not endogenous benzodiazepine receptor ligands and they may act specifically on GABA binding site.


Electrochemical monitoring of brain ascorbic acid changes associated with hypoxia, spreading depression, and seizure activity. In vivo electrochemistry has been a valuable tool in detecting real time neurochemical changes in extracellular fluid. Absolute selectivity has been difficult to achieve previously, but we report here a carbon fiber electrode and measurement technique which is specific for one oxidizable species: ascorbic acid. Ascorbic acid is highly concentrated in extra- as well as intracellular brain spaces, and appears to undergo dynamic changes in response to a variety of physiological and pathophysiological circumstances. Recent studies have implicated glutamatergic mechanisms which give rise to extracellular changes in brain ascorbate, and we confirm and extend these observations. Preliminary studies, directed towards examining ascorbic acid as an index and/or result of hypoxia, spreading depression, and seizure activity, have been undertaken and the results are reported herein.


Development of the glutamate system in rabbit retina. We have investigated two characteristics of the glutamate system in the developing rabbit retina. 1) Glutamate immunoreactivity was observed at birth within developing processes of four cell types; two of which, photoreceptors and ganglion cells, are known to be glutamatergic in the adult. Two other cell types, type A horizontal cells and amacrine cells, are immunoreactive to both glutamate and GABA at birth, suggesting that endogenous pools of glutamate in GABAergic neurons serve as precursor for GABA synthesis. Thus it appears that endogenous glutamate pools are present within neurons prior to synaptogenesis as part of the early expression of either the glutamate or GABA transmitter phenotype. 2) Analysis of 3H-glutamate metabolism during retinal development showed that rapid conversion of glutamate to glutamine does not occur until the second postnatal week, coincident with the expression of Muller (glial) cell activity. In the absence of glial metabolism in the neonate, extracellular concentrations of glutamate remain relatively high and are likely to have major effects on neuronal maturation.


An adenosine uptake blocker, propentofylline, reduces glutamate release in gerbil hippocampus following transient forebrain ischemia. In the present study, the effect of the adenosine uptake blocker, propentofylline (HWA 285) on the extracellular concentration of several amino acids including glutamate, glycine and taurine following 10 min of forebrain ischemia in gerbil hippocampus was investigated using in vivo microdialysis. Pretreatment with HWA 285 (20 mg/kg i.p.) significantly reduced the extracellular concentration of glutamate following ischemia but did not significantly alter levels of other amino acids such as glycine and taurine. These findings suggest that the neuroprotective effect of HWA 285 may be associated with inhibition of glutamate release in the gerbil hippocampus.


Mitochondrial enzymes related to glutamate and GABA metabolism in the hippocampus of young and aged rats: a quantitative histochemical study. Quantitative histochemistry (scanning microphotometry) was used to determine the activities of the mitochondrial enzymes NAD-linked isocitrate dehydrogenase (EC 1.1.1.41), L-glutamate dehydrogenase (EC 1.4.1.3) and GABA transaminase (EC 2.6.1.19) in various layers of the hippocampus (middle one third) of young (3-4 months old) and memory-impaired aged rats (28-30 months old). For comparison, determinations of cytochrome c oxidase (EC 1.9.3.1) as a marker for mitochondria and energy metabolism were also performed. The study showed that there was a layered reaction pattern in the hippocampus and that the cellular distribution and the levels of enzyme activity were different. However, the activities of the different enzymes (excepting GABA transaminase and cytochrome c oxidase) were significantly correlated in the hippocampus in both age groups. Age-dependent changes were only observed for NAD-linked isocitrate dehydrogenase and GABA transaminase (significant increases of activities in some layers of the hippocampus, preferentially in the terminal field of the perforant path). From the present study it is concluded that, 1. the enzymatic complement of mitochondria in neurons and glia depends upon layer specific metabolic processes of the hippocampus (also with respect to glutamatergic and GABAergic terminal fields) indicating a layer specific interaction of the enzymes studied to produce or catabolize glutamate and GABA, and 2. the age dependent changes of the studied enzymes are very restricted.


The endogenous retroviral ev21 locus in commercial chicken lines and its relationship with the slow-feathering phenotype (K). Provirus ev21 was found in both K- and k(+)-feathering Rhode Island Red commercial layers. Probe EV21-int revealed the presence of two distinct but similar regions, US (unoccupied site) and OS (occupied site). Restriction analysis showed that these regions had at least 19 kb structural homology but were distinguishable by ev21 proviral sequences, OS, and possibly three polymorphics US. The loci OS and US were both located on Chromosome Z. The k(+)-feathering birds were found to have only one site (either OS or US) per individual Z chromosome, whereas K-feathering birds had at least one Z chromosome with both regions in cis configuration. It has been possible to show that the reversion to the k(+)-feathering phenotype is accompanied by the loss of either a US or OS region that disrupts the cis configuration in K-feathering birds.


Sodium-sensitive, probenecid-insensitive p-aminohippuric acid uptake in cultured renal proximal tubule cells of the rabbit. In the intact kidney, renal proximal tubule cells accumulate p-aminohippurate (PAH) via a basolateral, probenecid- and sodium-sensitive transport system. Primary cultures of rabbit proximal tubule cells retain sodium-glucose co-transport in culture, but little is known about PAH transport in this system. Purified proximal tubule cells from a rabbit were grown in culture and assessed for PAH and alpha-methyl-D-glucoside uptake capacities as well as proximal tubule marker enzyme activities. Control PAH uptake on collagen-coated filters (20 +/- 3 pmol/mg protein.min; n = 8) was not significantly different from uptake in the presence of 1 mM probenecid (19 +/- 4 pmol/mg protein.min; n = 8). Uptake from the basal side of the cell was 3.9 +/- 0.7 times greater than that from the apical side. In multi-well plate studies, the uptake was significantly reduced by removing sodium from the medium and stimulated by coating the wells with collagen. Glutarate (10 mM) had no effect on the uptake of PAH. Other differentiated proximal tubule characteristics were retained in culture, including the ability to form domes and to transport glucose by a phlorizin-sensitive system. Phlorizin-sensitive 1 mM alpha-methyl-D-glucoside uptake was 134 +/- 42 pmol/mg protein.min (n = 7; P less than 0.02). The proximal tubule marker enzymes alkaline phosphatase and gamma-glutamyltranspeptidase, increased in activity in the cultures after confluence. It was concluded that whereas some differentiated properties were retained during primary culture of rabbit proximal tubule cells, the PAH transport system was selectively lost or modified from that present in the intact kidney.


Capsaicin-sensitive nerves modulate reactive hyperemia in rat gut. Reactive hyperemia (RH) is a local, vascular response that occurs following release from mechanical occlusion of an artery, with restoration of intra-arterial pressure. The mechanism of this postocclusion hyperemia in the gut has not been identified, although metabolic, myogenic, and neurogenic mediators of this response have been proposed. The present study was conducted to evaluate a possible modulatory role for sensory innervation of the intestinal vasculature in RH, using acute and chronic treatment with capsaicin applied in different ways. In anesthetized rats, the velocity of flowing blood in the gut was determined continuously with a pulsed Doppler velocimeter, and arterial pressure was determined with a transducer. The increase in calculated intestinal vascular conductance at the height of RH (Ch), the excess volume of blood accumulating during RH, and the duration of the hyperemia were also used to quantify RH after occluding the anterior mesenteric artery for 30, 60, and 120 sec. In the initial control group of rats, the maximal increases in the velocity of flowing blood during RH were 61 +/- 4%, 90 +/- 7%, and 129 +/- 10% of control, conductances were increased to 192 +/- 5%, 222 +/- 12%, and 267 +/- 15% of control, volumes were 3.5 +/- 0.6 ml, 7.2 +/- 0.4 ml, and 16.2 +/- 1.8 ml, and durations of hyperemia were 78 +/- 5 sec, 93 +/- 6 sec, and 178 +/- 7 sec, respectively, after each elapsed period of occlusion. Acute treatment with periarterial capsaicin significantly decreased peak conductances in RH by 15-35% for all occlusions tested and reduced both volume and duration values. Rats treated with capsaicin in neonatal life exhibited reduced Ch values, as did adult rats treated chronically with capsaicin. Both periarterial and intrajejunal treatment with capsaicin decreased the duration of RH. Hexamethonium increased both Ch and the duration of RH and tended to reverse reductions in these parameters caused by capsaicin. These results suggest that sensory innervation of the intestinal vasculature exerts a modulatory influence in the regulation of intestinal RH.


Neuroleptic-induced changes in the anxiolytic and myorelaxant properties of diazepam in the rat. Diazepam (2.0 mg/kg) was injected (IP) into rats 30 min before chlorpromazine (2.5, 5.0, or 10.0 mg/kg) on ten occasions. All doses of chlorpromazine enhanced the capacity of diazepam to increase rats' exploration of the exposed arms of an elevated plus-maze, an animal screening test for anxiolytic and anxiogenic substances. When maze testing occurred during each of the ten diazepam----chlorpromazine trials (after diazepam but before chlorpromazine), this enhancement effect appeared on Trial 6 and persisted thereafter. Haloperidol (3.0 mg/kg, IP) changed diazepam-elicited plus-maze activity in the same manner as chlorpromazine; however, thioridazine (10.0 mg/kg) and pimozide (2.0 mg/kg) were ineffective. Additionally, haloperidol, like chlorpromazine, was found to reduce diazepam's muscle relaxation effect (inclined plane test) as a consequence of diazepam----haloperidol pairings; once again, thioridazine and pimozide proved ineffective. These results suggested that not all neuroleptics will alter diazepam activity, and also that dopamine blockade per se is not sufficient to induce such changes. While the reasons for the enhanced plus-maze effects of diazepam induced by haloperidol and chlorpromazine remain elusive, the diminished myorelaxant effect may be linked to a neuroleptic's capacity to induce muscular side effects: thioridazine and pimozide are far less likely to yield such effects than are chlorpromazine and haloperidol. Haloperidol administered chronically by itself was found to have an effect on diazepam-induced myorelaxation. Administration of this butyrophenone either orally (2.0 mg/kg daily for 22 days) or in depot form (haloperidol decanoate, 60.0 mg/kg IM once a month for four months) caused a diminished effect of diazepam in rats subjected to the inclined plane test.(ABSTRACT TRUNCATED AT 250 WORDS)


Locomotor bias produced by intra-accumbens injection of dopamine agonists and antagonists. Several experiments have shown that the dopamine (DA) receptors in the nucleus accumbens control the intensity of locomotor activity; however, there are several contradictory results concerning the role of the accumbens in the regulation of the direction of locomotion. To further evaluate the contribution of dopaminergic function in the accumbens to the direction of locomotion, we first compared the effect on the direction of locomotor activity of unilateral intra-accumbens injections of the nonspecific DA antagonist haloperidol, the specific D-1 antagonist SCH-23390, the specific D-2 antagonist metoclopramide. In the second part of the experiment, we examined the effect on the direction of locomotor activity of unilateral intra-accumbens injections of the non-specific DA agonist apomorphine, the specific D-1 agonist SKF-38393, the specific D-2 agonist LY-171555, and the combination of SKF-38393 and LY-171555. Haloperidol, metoclopramide and to a lesser extent, SCH-23393 together with peripheral amphetamine injections produced a locomotor bias that resulted in ipsilateral turning. Apomorphine, LY-171555 or the combination of SKF-38393 and LY-171555 (but not SKF-38393 alone) produced a locomotor bias that resulted in contralateral turning. No significant locomotor bias was produced by intra-accumbens injection of the various vehicles. These results suggest that the bilateral DA organization thought to exist in the nigro-striatal pathway for the control of locomotion may also be true for the mesolimbic dopamine system.


Sigma ligand-induced emesis in the pigeon. Pigeons were fed a fixed amount of grain-based feed and behavior was observed after administration of doses of ditolyguanidine (DTG), (+)-3-(3-hydroxyphenyl)-N-(1-propyl)-piperidine [(+)-3-PPP], dextromethorphan, haloperidol, (+)-N-allylnormetazocine (NANM), alpha-(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazine-butanol (BMY-14802) apomorphine, pentobarbital, propranolol, and MK-801. Of the drugs tested, DTG, dextromethorphan, and (+)-3-PPP each produced dose-related increases in the percentage of pigeons exhibiting an emetic response. The emetic response produced by DTG was antagonized by haloperidol and BMY-14802 but not by propranolol. These observations suggest that the emetic response in the pigeon may be mediated by sigma sites and is unlikely to be mediated by phencyclidine receptors.


Remoxipride, a specific D2 dopamine antagonist: an examination of its self-administration liability and its effects on d-amphetamine self-administration. The self-administration liability of remoxipride, a specific dopamine D2 antagonist, by laboratory rats was evaluated using an intravenous self-administration paradigm. It was observed that remoxipride failed to support self-administration behavior across the three doses tested. In addition, remoxipride pretreatment attenuated d-amphetamine self-administration. The findings of the present study provide support for the notion that remoxipride appears to have functional similarity in self-administration paradigms as other D2 antagonists.


A lever-release version of the conditioned avoidance response paradigm: effects of haloperidol, clozapine, sulpiride, and BMY-14802. Rats trained on a lever-release version of the conditioned avoidance response (CAR) task were used to test the behavioral effects of established and putative antipsychotic drugs. Baseline CAR latencies decreased as the conditioned-unconditioned stimulus interval was shortened from 500 to 250 ms. Haloperidol, clozapine, and BMY-14802 decreased successful avoidance responses and increased avoidance latencies in a dose-dependent manner without affecting the latency of escape responses. In contrast, sulpiride failed to affect either successful avoidance response rates or avoidance latency. Sulpiride, however, significantly attenuated d-amphetamine-induced locomotion and rearing compared to vehicle-treated controls. Similar effects of these antipsychotics have been reported on shuttlebox avoidance, and these results now are confirmed in a CAR paradigm that achieves greater control over behavior. Because this paradigm elicits a discrete forelimb response without activating numerous muscle groups, it is potentially useful as a tool for examining neuronal mechanisms underlying the behavioral effects of antipsychotic drugs.


Effect of altering dopamine or serotonin neurotransmitters upon cathinone discrimination. Rats were trained to discriminate between the stimulus properties of 0.8 mg/kg l-cathinone and its vehicle in a two-lever, food-motivated operant task. Once trained, rats showed a dose-related decrease in discriminative performance when tested with lower cathinone doses. An analysis of the dose-response curve indicated an ED50 value of 0.23 mg/kg. Pretreatment with CGS 10746B (5-20 mg/kg) resulted in a dose-related decrease in cathinone discrimination with the highest dose blocking cathinone discrimination. In contrast to the ability of this dopamine release inhibitor to decrease cathinone discrimination, pretreatment with three doses of the calcium channel blocker isradipine (2.5-10 mg/kg) or with the 5-HT3 antagonist MDL 72222 (0.1-0.4 mg/kg) had no effect upon cathinone discrimination. The results suggest that cathinone controls differential responding in a discriminative stimulus task by a mechanism involving presynaptic release of dopamine, which may not be regulated by either neuronal calcium influx through L-type calcium channels or by serotonergic neurons.


Chronic effects of nicotine on mesolimbic dopaminergic system in rats. Rats were pretreated with saline or nicotine (1.5 mg/kg/day) by subcutaneously implanting each animal with an Alzet osmotic minipump which continuously released saline or nicotine (1.5 mg/kg/day) for 14 days. The behavioral and biochemical effects of nicotine on the dopaminergic neuronal system in rat nucleus accumbens were examined. It was found that chronic nicotine treatment increased the affinity of L-[3H]nicotine binding site in the nucleus accumbens. This treatment also potentiated the ability of (+)-amphetamine, but not high potassium, to stimulate formation and release of [3H]dopamine in tissue slices from rat nucleus accumbens. Chronic nicotine treatment did not alter the characteristics of [3H]spiperone binding site, the rate of dopamine turnover and the concentrations of gamma-aminobutyric acid in the nucleus accumbens.


Evidence of hyperglycemic hyperalgesia by quinpirole. Male albino rats were tested for antinociception following injections (IP) with saline, quinpirole (Quin) (1 mg/kg), morphine sulfate (M.S.) (5 mg/kg), or both Quin and M.S. (1 mg/kg and 5 mg/kg, respectively). Quin reduced and M.S. increased tail-flick latency as compared to controls. Tail-flick latencies of the animals injected with both drugs were significantly reduced as compared M.S. alone. Quin increased blood glucose levels by 96 percent, as compared to saline controls. In competitive binding studies Quin displaced 3H-DAGO (IC50 = 29.8 microM). CD-1 mice demonstrated a naloxone-reversible analgesia following ICV Quin (100 micrograms). These data are consistent with the hypothesis that the hyperglycemic effects of Quin attenuate M.S. analgesia while the antinociceptive effects of Quin may be mediated through opioid receptors.


Ontogeny of the distribution and colocalization of calbindin D28K within neural and endocrine cells of the gastrointestinal tract of fetal and neonatal sheep. Using immunocytochemical techniques we have demonstrated that Calbindin D28K (CaBP) is present in the gastrointestinal tract of ovine fetuses early in development (by day 45). At day 45, CaBP was limited to neuronal elements in the developing intestine. By day 100, CaBP immunoreactivity was abundant in both epithelial endocrine cells and nerves of the submucous and myenteric ganglia. The location of CaBP containing cells and fibers was similar in duodenal sections taken from day 100 and term (145 days), as well as those taken from 24-48 h postnatal lambs. CaBP is colocalized in endocrine cells containing gastrin, glucagon, somatostatin and neurotensin, but not glucose dependent insulinotrophic peptide (GIP). Furthermore, it is extensively colocalized in nerve fibers and cells containing neurotensin but not somatostatin or vasoactive intestinal peptide. The colocalization of CaBP within various endocrine and nerve cells does not change in fetal sheep over the last one-third of gestation and there is no difference between fetal and neonatal sheep.


Paraquat damage of rat liver mitochondria by superoxide production depends on extramitochondrial NADH. Pure rat liver heavy mitochondrial fractions, in which the absence of significant microsomal contamination was confirmed by electron microscopy and by the lack of glucose-6-phosphatase activity, were used to demonstrate the effect of paraquat on mitochondrial ultrastructure in the presence of external NADH. Starved mitochondria (orthodox conformation) did not show O2 uptake or structural injury from either paraquat alone or NADH alone. Marked O2 uptake and structural breakage occurred only when paraquat and NADH were added in combination. These alterations were resistant to rotenone and malate plus glutamate or NADPH could not substitute for NADH. Paraquat was reduced anaerobically by the mitochondria in the presence of NADH, but not of NADPH. The addition of superoxide dismutase, ferricytochrome c or p-benzoquinone protected against the breakage of mitochondria caused by paraquat plus NADH. These results demonstrate that mitochondria may produce paraquat radicals in the presence of extramitochondrial NADH and thus generate superoxide anion radicals, resulting in structural injury to the mitochondria, by mechanisms that may involve the mitochondrial outer membrane rather than the electron transfer chain. These mitochondrial mechanisms in paraquat toxicity seemed to be more probable in vivo than are microsomal mechanisms; the latter are postulated to function in detoxication because phenobarbital diminished paraquat toxicity and SKF 525-A or cobaltous ions enhanced the toxicity.


Effects of low-level lead exposure on hypothalamic hormones and serum progesterone levels in pregnant guinea pigs. Pregnant guinea pigs were given a daily oral dose of 0, 5.5, or 11 mg lead (as lead acetate) per kg body weight during days 22-52 or 22-62 of gestation. Maternal serum progesterone levels were measured at the end of treatment, as well as hypothalamic levels of gonadotropin-releasing hormone (GnRH) and somatostatin (SRIF) in both the mothers and fetuses. Lead-treated dams had lower serum concentrations of progesterone at the end of treatment than did vehicle-treated animals. This effect was statistically significant for the higher Pb dose only. Hypothalamic levels of GnRH and SRIF were reduced in a dose-dependent manner by lead treatment in both dams and fetuses. The reduction of SRIF levels in 52-day-old fetuses was particularly severe (92%) in the 11 mg group. However, neither litter size nor body and organ weights, including placental weight, of the dams and fetuses was significantly affected. The relevance of these hormonal decreases is unknown, but could include decreased reproductive capacity in both the dams and fetuses that does not become apparent until later in the life-cycle.


Spindle cell haemangioendothelioma: probably a benign vascular lesion not a low-grade angiosarcoma. A clinicopathological, ultrastructural and immunohistochemical study. Ten cases of spindle cell haemangioendothelioma (SCH) were analysed clinicopathologically, including an immunohistochemical survey of seven cases and ultrastructural observations on one. There were seven females and three males, ranging from 16 to 76 years of age. All but one lesion developed on the extremities, predominantly on the hands and feet. Six of the ten patients presented multiple nodules or papules which gradually increased in size and number over a long duration. Among them, four patients had undergone operations twice or more, but no metastatic foci were recognized. Histologically, the lesions were composed of dilated vascular spaces and a proliferation of bland-appearing spindle cells and interspersed epithelioid endothelial cells. Ultrastructural and immunohistochemical studies demonstrated that the spindle cells were mainly made up of fibroblastic cells admixed with pericyte-like cells and macrophages. Smooth muscle cells and primitive mesenchymal cells were also present. The clinical and microscopic features suggest that SCH may be a benign vasoformative lesion of a heterochronological multicentric origin.


Somatostatin cells in human somatotropic adenomas. Data from our group have shown that the human adenomatous and normal anterior pituitary may be the source of somatostatin (SRIH). SRIH-producing cells were identified in two somatotropic adenomas. Immunoreactive SRIH cells were present in both cases. In case 2, material was available for RNA studies, in situ hybridization and electron microscopy. The size of the transcript identified by Northern blot analysis was identical to that of hypothalamic SRIH mRNA. In situ hybridization showed that the SRIH gene was expressed in a cell subset superimposable to that identified by immunocytochemistry. Co-localization studies revealed that SRIH and growth hormone (GH) immunoreactivities were not present in the same cells. Ultrastructural immunogold labelling showed that SRIH cells had features distinct from those of the somatotropes. The results confirm that the somatotropic adenomas have the ability to synthesize SRIH, indicate that SRIH expression is restricted to a subset of adenoma cells different from GH-producing cells, and imply that SRIH cells are involved in paracrine regulation of neighbouring somatotropes.


Preparation of candidate vaccinia-vectored vaccines for haemorrhagic fever with renal syndrome. Two vaccinia-vectored candidate vaccines for haemorrhagic fever with renal syndrome were prepared by inserting cDNA, representing the medium (M) genome segment, or the M and the small (S) genome segments of Hantaan virus into the thymidine kinase gene of the Connaught vaccine strain of vaccinia virus. In the single recombinant, the M segment was placed under control of the vaccinia virus 7.5 kDa promoter. In the double recombinant, the M and S segments were placed under control of the vaccinia virus 7.5 kDa and 11 kDa promoters, respectively. An immunoplaque assay technique was developed to select recombinants without the need for expression of irrelevant genes or use of potential mutagens. Proteins indistinguishable from authentic viral envelope glycoproteins and nucleocapsid protein were observed by immunoprecipitation with antibodies to Hantaan virus. The recombinant expressing both the M and the S segments was selected for further development and testing as a human vaccine.


The age-dependent risk of postvaccination complications in vaccinees with smallpox vaccine. The use of vaccinia virus in recombinant systems may result in the occurrence of complications observed following smallpox vaccination. The results are presented of a large survey carried out in the USSR between 1968 and 1979. High complication rates are reported, particularly in older primary vaccinees; for example, there were 312.5 cases of neurological complications per million in those aged greater than or equal to 5 years. The author concludes that this factor will limit the use of vaccinia virus in recombinant technology.


Epidural bupivacaine, sufentanil or the combination for post-thoracotomy pain. Analgesia with epidural bupivacaine, sufentanil or the combination was studied in 50 patients who had undergone thoracotomy. During operation all patients received an initial dose of bupivacaine 0.5% with adrenaline 5 micrograms.ml-1 (5-10 ml) by thoracic epidural catheter. One hour later the patients were divided into three groups: the bupivacaine group (bupivacaine 0.125%), the sufentanil group (50 micrograms sufentanil in 60 ml normal saline) and the combination group (50 micrograms sufentanil in 60 ml bupivacaine 0.125%). Analgesia in the three groups was provided by a continuous epidural infusion (5-10 ml.h-1) for 3 days. The mean dose of bupivacaine was significantly higher (P less than 0.05) in the bupivacaine group (12.07 mg.h-1 (s.e.mean 0.97 mg.h-1)), compared with the combination group (9.82 mg.h-1 (s.e.mean 0.43 mg.h-1)). The mean dose of sufentanil in the sufentanil group was similar to the combination group (6.37 micrograms.h-1 (s.e.mean 0.23 micrograms.h-1) and 6.52 micrograms.h-1 (s.e.mean 0.28 micrograms.h-1), respectively. The pain scores on the inverse visual analogue scale of most patients in the bupivacaine group were unacceptably low. The sufentanil group had much better pain scores, but on exercise these patients experienced more pain than the combination group. The combination group had, overall, better pain scores. In the combination group, there were better respiratory results.


The meiotic stage of nondisjunction in trisomy 21: determination by using DNA polymorphisms. We have studied DNA polymorphisms at loci in the pericentromeric region on the long arm of chromosome 21 in 200 families with trisomy 21, in order to determine the meiotic origin of nondisjunction. Maintenance of heterozygosity for parental markers in the individual with trisomy 21 was interpreted as resulting from a meiosis I error, while reduction to homozygosity was attributed to a meiosis II error. Nondisjunction was paternal in 9 cases and was maternal in 188 cases, as reported earlier. Among the 188 maternal cases, nondisjunction occurred in meiosis I in 128 cases and in meiosis II in 38 cases; in 22 cases the DNA markers used were uninformative. Therefore meiosis I was responsible for 77.1% and meiosis II for 22.9% of maternal nondisjunction. Among the 9 paternal nondisjunction cases the error occurred in meiosis I in 2 cases (22.2%) and in meiosis II in 7 (77.8%) cases. Since there was no significant difference in the distribution of maternal ages between maternal I error versus maternal II error, it is unlikely that an error at a particular of maternal ages between maternal I error versus maternal II error, it is unlikely that an error at a particular meiotic stage contributes significantly to the increasing incidence of Down syndrome with advancing maternal age. Although the DNA polymorphisms used were at loci which map close to the centromere, it is likely that rare errors in meiotic-origin assignments may have occurred because of a small number of crossovers between the markers and the centromere.(ABSTRACT TRUNCATED AT 250 WORDS)


A genetic linkage map of human chromosome 21: analysis of recombination as a function of sex and age. A genetic linkage map of human chromosome 21 has been constructed using 22 anonymous DNA markers and five complementary DNAs (cDNAs) encoding the amyloid beta protein precursor (APP), superoxide dismutase 1 (SOD1), the ets-2 proto-oncogene (ETS2), the estrogen inducible breast cancer locus (BCEI), and the leukocyte antigen, CD18 (CD18). Segregation of RFLPs detected by these DNA markers was traced in the Venezuelan Reference Pedigree (VRP). A comprehensive genetic linkage map consisting of the 27 DNA markers spans 102 cM on the long arm of chromosome 21. We have confirmed our initial findings of a dramatically increased rate of recombination at the telomere in both females and males and of significantly higher recombination in females in the pericentromeric region. By comparing patterns of recombination in specific regions of chromosome 21 with regard to both parental sex and age, we have now identified a statistically significant downward trend in the frequency of crossovers in the most telomeric portion of chromosome 21 with increasing maternal age. A less significant decrease in recombination with increasing maternal age was observed in the pericentromeric region of the chromosome. These results may help in ultimately understanding the physical relationship between recombination and nondisjunction in the occurrence of trisomy 21.


Friedreich ataxia in Louisiana Acadians: demonstration of a founder effect by analysis of microsatellite-generated extended haplotypes. Eleven Acadian families with Friedreich ataxia (FA) who were from southwest Louisiana were studied with a series of polymorphic markers spanning 310 kb in the D9S5-D9S15 region previously shown to be tightly linked to the disease locus. In particular, three very informative microsatellites were tested. Evidence for a strong founder effect was found, since a specific extended haplotype spanning 230 kb from 26P (D9S5) to MCT112 (D9S15) was present on 70% of independent FA chromosomes and only once (6%) on the normal ones. There was no evident correlation between haplotypes and clinical expression. The typing of an additional microsatellite (GS4) located 80 kb from MCT112 created a divergence of the main FA-linked haplotype, generating four minor and one major haplotype. A similar split was observed with GS4 in a patient homozygous for a rare 26P-to-MCT112 haplotype. These results suggest that GS4 is flanking marker for the disease locus, although other interpretations are possible.


Assignment of a gene (NEMI) for autosomal dominant nemaline myopathy to chromosome I. Nemaline myopathy (NEM) is a neuromuscular disorder characterized by the presence, in skeletal muscle, of nemaline rods composed at least in part of alpha-actinin. A candidate gene and linkage approach was used to localize the gene (NEM1) for an autosomal dominant form (MIM 161800) in one large kindred with 10 living affected family members. Markers on chromosome 19 that were linked to the central core disease gene, a marker at the complement 3 locus, and a marker on chromosome 1 at the alpha-actinin locus exclude these three candidate genes. The family was fully informative for APOA2, which is localized to 1q21-q23. NEM1 was assigned to chromosome 1 by close linkage for APOA2, which is localized to 1q21-q23. NEM1 was assigned to chromosome 1 by close linkage to APOA2, with a lod score of 3.8 at a recombination fraction of 0. Recombinants with NGFB (1p13) and AT3 (1q23-25.1) indicate that NEM1 lies between 1p13 and 1q25.1. In total, 47 loci were investigated on chromosomes 1, 2, 4, 5, 7-11, 14, 16, 17, and 19, with no indications of significant linkage other than to markers on chromosome 1.


Involvement of multiple chromosome 17p loci in medulloblastoma tumorigenesis. Loss of heterozygosity for sequences located on chromosome 17p in several tumor types is often associated with mutations in the tumor suppressor gene p53. We previously showed consistent deletion of chromosome 17p12-13.1 in medulloblastoma, a common childhood brain tumor. Using denaturing gradient gel electrophoresis and direct sequencing, we have detected p53 mutations in only two of 20 medulloblastoma specimens. Moreover, additional RFLP studies of these 20 specimens showed loss of heterozygosity at a more distal and distinct site, 17p13.3. Deletion of 17p almost invariably signified a negative prognosis. Our results suggest that p53 mutations may contribute to the pathogenesis of medulloblastoma in relatively few cases. The consistent deletion of other discrete loci on 17p suggests that additional or alternative tumor suppressor genes may contribute to the tumor's phenotype.


Strong allelic association between the torsion dystonia gene (DYT1) andloci on chromosome 9q34 in Ashkenazi Jews. The DYT1 gene responsible for early-onset, idiopathic torsion dystonia (ITD) in the Ashkenazi Jewish population, as well as in one large non-Jewish family, has been mapped to chromosome 9q32-34. Using (GT)n and RFLP markers in this region, we have identified obligate recombination events in some of these Jewish families, which further delineate the area containing the DYT1 gene to a 6-cM region bounded by loci AK1 and ASS. In 52 unrelated, affected Ashkenazi Jewish individuals, we have found highly significant linkage disequilibrium between a particular extended haplotype at the ABL-ASS loci and the DYT1 gene. The 4/A12 haplotype for ABL-ASS is present on 69% of the disease-bearing chromosomes among affected Jewish individuals and on only 1% of control Jewish chromosomes (chi 2 = 91.07, P much less than .001). The allelic association between this extended haplotype and DYT1 predicts that these three genes lie within 1-2 cM of each other; on the basis of obligate recombination events, the DYT1 gene is centromeric to ASS. Furthermore, this allelic association supports the idea that a single mutation event is responsible for most hereditary cases of dystonia in the Jewish population. Of the 53 definitely affected typed, 13 appear to be sporadic, with no family history of dystonia. However, the proportion of sporadic cases which potentially carry the A12 haplotype at ASS (8/13 [62%]) is similar to the proportion of familial cases with A12 (28/40 [70%]). This suggests that many sporadic cases are hereditary, that the disease gene frequency is greater than 1/15,000, and that the penetrance is lower than 30%, as previously estimated in this population. Most affected individuals were heterozygous for the ABL-ASS haplotype, a finding supporting autosomal dominant inheritance of the DYT1 gene. The ABL-ASS extended-haplotype status will provide predictive value for carrier status in Jewish individuals. This information can be used for molecular diagnosis, evaluation of subclinical expression of the disease, and elucidation of environmental factors which may modify clinical symptoms.


Discriminatory effects of protein kinase inhibitors and calcium ionophore on endothelial ICAM-1 induction. Intercellular adhesion molecule 1 (ICAM-1) is a proinflammatory adhesion glycoprotein induced by cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) as well as lipopolysaccharide (LPS). Little is known, however, concerning the intracellular regulatory mechanisms that modulate ICAM-1 expression in endothelial cells. We probed the involvement of protein kinase function and intracellular calcium ion upon ICAM-1 expression of human umbilical vein endothelial cells activated alternatively by TNF-alpha, IL-1 beta, LPS, or phorbol 12-myristate 13-acetate (PMA). Methodologies for the detection of ICAM-1 included both enzyme-linked immunosorbent assay and immunoprecipitation from biosynthetically labeled cells. The protein kinase inhibitor H-7 blocked induction of ICAM-1 by all of the activators; nonlinear regression analysis revealed 50% inhibitory concentration (IC50) values of 6-10 microM. Another kinase inhibitor, HA1004, did not block expression of the adhesion molecule at concentrations up to 50 microM. In contrast, the kinase inhibitor staurosporine dose dependently inhibited ICAM-1 expression triggered by PMA (IC50 67 +/- 4 nM) but, at similar concentrations, did not inhibit ICAM-1 expression induced by the other inflammatory stimuli. The divalent cation ionophore ionomycin (0.5 microM) interacted synergistically with PMA but not with cytokines or LPS in upregulating ICAM-1. We conclude from these data that although PMA-induced ICAM-1 expression may be triggered through activation of protein kinase C, ICAM-1 induction by IL-1 beta, TNF-alpha, or LPS may involve distinct regulatory pathway(s).


Effect of somatostatin on mesenteric vascular resistance in normal and portal hypertensive rats. Vasoactive effects of natural somatostatin (SRIF-14) and its analogue octreotide were studied in in vitro perfused superior mesenteric arterial beds of normal (Sham) and portal hypertensive (PVL) rats. Tested concentrations covered the whole range used in clinical settings (10(-10) to 10(-5) M for SRIF-14 and 10(-11) to 10(-6) M for octreotide, respectively). Vessel resistances only minimally changed to infusions of SRIF-14 (from 3.5 +/- 0.4 to 3.7 +/- 0.5 mmHg.ml-1.min and 3.8 +/- 0.3 to 3.9 +/- 0.4 mmHg.ml-1.min for PVL and Sham) and octreotide (from 3.3 +/- 0.2 to 3.4 +/- 0.4 mmHg.ml-1.min and 3.8 +/- 0.4 to 4.0 42- 0.4 mmHg.ml-1.min for PVL and Sham). The same was true for bolus injections. In contrast, norepinephrine induced significant increases in vessel resistance (up to 110.6 +/- 20.1 mmHg.ml-1.min). In conclusion, SRIF-14 and octreotide exert no direct effect on vascular smooth muscle tone in splanchnic resistance vessels of Sham and PVL rats. The vasoconstriction reported in vivo seems therefore probably mediated by the ability of these peptides to inhibit the secretion of vasodilatatory substances.


Alpha 1-adrenergic signaling in human airway epithelial cells involves inositol lipid and phosphate metabolism. A role for phospholipase C (PLC) hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) as a mechanism of alpha 1-adrenergic signal transduction in human airway epithelial cells (AEC) was investigated in isolated normal tracheal and cystic fibrosis (CF) nasal epithelial cells grown in in vitro culture and prelabeled with 3 muCi myo-[3H]inositol/ml for 72 h. Breakdown of polyphosphoinositides was measured using thin-layer chromatography to detect phosphatidylinositol, phosphatidylinositol 4-phosphate (PIP), and PIP2. Inositol phosphates were separated by ion-exchange column chromatography. In normal AEC, the addition of the endogenous catecholamine l-epinephrine produced a rapid, transient accumulation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2) and breakdown of PIP and PIP2. IP3 increased 1.7-fold and IP2 1.6-fold after 20 and 40 s, respectively. A maximal decrease of 35% PIP2 and 30% PIP is observed after 20 and 40 s, respectively. The effects of l-epinephrine were not blocked by the beta-adrenergic antagonist dl-propranolol but were mimicked by the alpha 1-adrenergic agonist methoxamine. Prazosin, an alpha 1-adrenergic antagonist, and pertussis toxin (PTX) blocked the effects of l-epinephrine and methoxamine. Addition of l-epinephrine and methoxamine to CF nasal epithelial cells also induced prazosin-sensitive polyphosphoinositide breakdown and inositol phosphate accumulation. A 2.2-fold accumulation of IP3 was observed after 10 s and 2.0-fold increase in IP2 after 20 s. Maximal decreases of 32% PIP2 and 23% PIP levels were observed after 20-s incubation with l-epinephrine. PTX reduced the effects of l-epinephrine and significantly blocked the effects of methoxamine.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of coronary vasoconstriction produced by rostral ventrolateral medulla stimulation in rats. Previous studies have demonstrated that coronary vasoconstriction can be produced by activation of specific central nervous system sites in the cat. The present study was undertaken 1) to develop a rat model for studying central influences on coronary circulation and 2) to utilize this model for characterization of the changes in coronary blood flow (CBF) produced by stimulation of rostral ventrolateral medulla (RVLM). Electrical stimulation of right RVLM in chloralose-anesthetized rats with bilateral vagotomy produced a transient decrease in CBF followed by an increase in CBF concomitant with a decrease in hindquarter blood flow, a pressor response, and tachycardia. After atenolol the tachycardia and increase in CBF were abolished, whereas the decrease in CBF was enhanced and prolonged. Phentolamine (1 mg/kg iv) or removal of the stellate ganglia inhibited the decrease in CBF but did not totally abolish the increase in coronary vascular resistance. Inhibition of nitric oxide synthesis with N-nitro-L-arginine (10 microM/kg iv) enhanced the decrease in CBF produced by stimulation in RVLM. These results indicate that, in rat model, the centrally induced decrease in CBF is 1) mediated by cardiac sympathetic innervation but only partially through alpha-adrenoceptors and 2) enhanced by removal of the inhibitory effect of the endothelium.


Effects of alpha 1-adrenergic blockade on intrarenal hemodynamics in heart failure rats. To investigate intrarenal hemodynamics and effects of alpha 1-adrenergic blockade on glomerular functions in congestive heart failure (CHF), micropuncture studies were performed before and after intravenous injection of terazosin (1 microgram/100 g body wt iv) in eight myocardial infarction (MI) and in nine sham-operated rats after intraperitoneal injection of Inactin (70 mg/kg). CHF was characterized by elevated left ventricular end-diastolic pressure and increased total heart weight. In CHF rats, single nephron glomerular filtration rate (SNGFR), single nephron plasma flow (SNPF), and ultrafiltration coefficient (Kf) were decreased compared with sham-operated rats (SNGFR, 21.9 +/- 1.6 vs. 39.2 +/- 2.9 nl.min-1.g-1, P less than 0.01; SNPF, 66.6 +/- 6.1 vs. 133.8 +/- 10.5 nl.min-1.g-1, P less than 0.01; Kf, 0.019 +/- 0.001 vs. 0.041 +/- 0.004 nl.s-1.mmHg-1.g-1, P less than 0.01). Single nephron filtration fraction (SNFF), glomerular pressure (Pg), and efferent arteriolar resistance (Re) were higher in the CHF-MI rats than in the sham-operated rats (SNFF, 33.4 +/- 1.3 vs. 27.7 +/- 1.0, P less than 0.05; Pg, 60.2 +/- 1.6 vs. 53.3 +/- 0.8 mmHg, P less than 0.01; Re, 3.92 +/- 0.66 vs. 1.62 +/- 0.15 x 10(10) dyn.s.cm-5.g, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Area postrema stimulation induces differential renal hemodynamics with two anesthetics. The effect of area postrema stimulation (APS) on blood pressure, renal blood flow (RBF), and renal vascular resistance was compared in urethan-anesthetized and pentobarbital sodium-anesthetized Sprague-Dawley rats. Mean arterial pressure (MAP) increased in a frequency-dependent manner during APS in both urethan- and pentobarbital-anesthetized rats. Although no significant differences occurred in the maximum percent change in MAP between groups, marked differences occurred in RBF and calculated renal vascular resistance (RVR) changes. In urethan-anesthetized rats, RBF and MAP increased during APS, but RVR did not change. In contrast, APS significantly decreased RBF (-26.5 +/- 1.9%) while increasing RVR (+99.4 +/- 11.4%) in pentobarbital-anesthetized rats. The increase in RVR in the pentobarbital-anesthetized group during APS was eliminated by prior ganglionic blockade. It was concluded that the anesthetic agent employed can significantly alter the degree to which APS can activate renal vasomotion in the rat.


Pipecuronium versus high dose vecuronium. I. A comparison of speed of onset and cumulation during isoflurane anaesthesia. The onset time and tendency to cumulation of pipecuronium and high-dose vecuronium were studied during nitrous oxide anaesthesia supplemented with isoflurane. Pipecuronium 0.06 mg.kg-1 had a similar duration of action to vecuronium 0.015 mg.kg-1 (42 vs 49 min). Patients who received vecuronium had a shorter onset time of neuromuscular blockade (p less than 0.01). The use of pipecuronium was associated with marked cumulation.


Pipecuronium versus high dose vecuronium. II. A comparison of speed of onset and cumulation during propofol anaesthesia. The onset time and tendency to cumulation of pipecuronium and high-dose vecuronium were compared during nitrous oxide anaesthesia supplemented with a propofol infusion. Pipecuronium 0.06 mg.kg-1 had a similar duration of action to vecuronium 0.2 mg.kg-1 (49 vs 43). Patients who received vecuronium had a shorter onset time of neuromuscular blockade (p less than 0.01). Neither pipecuronium nor vecuronium showed marked cumulation.


Changes in vitreous humor associated with postmortem interval in rabbits. Concentrations of serum and vitreous humor constituents at time of death, and concentrations of vitreous humor constituents at time of death and at 7 postmortem intervals were compared in 70 domestic, female New Zealand White rabbits (Oryctolagus cuniculus). Urea nitrogen concentration was significantly (P = 0.0094) different, but was linearly correlated in serum and vitreous humor at time of death and at the 4- and 8-hour postmortem intervals. Concentrations of gamma-glutamyltransferase were not significantly different in serum and vitreous humor at time of death, nor were concentrations significantly different in vitreous humor at time of death and at the 4-hour postmortem interval. The vitreous humor concentrations of glucose, triglycerides, sodium, potassium, cholesterol, total protein, albumin, lactate dehydrogenase, creatine kinase, aspartate transaminase, bilirubin, cortisol, and IgG were neither similar to nor predictive of serum constituents. Vitreous humor can be used as a source for estimates of serum urea nitrogen and gamma-glutamyltransferase up to 8 and 4 hours after death, respectively.


A follow-up study on short-term treatment of agoraphobia. The differential effectiveness of three treatment packages for agoraphobia was tested. Patients received one of three short-term treatments: Breathing Retraining and Cognitive Restructuring, graded Self-Exposure in vivo, or a combination of both. No differential effects were found between the treatment conditions at posttest and at an 18 months follow-up. Improvement at follow-up assessment was associated with whether patients had further treatment during the follow-up period. No relationship was found between further improvement and demographic variables, pre- and posttest scores on psychological questionnaires or the use of medication at follow-up. Implications of these findings are examined.


Vasoconstrictor agonists activate G-protein-dependent receptor-operated calcium channels in pig aortic microsomes. Receptor-operated Ca2+ channels were characterized by their ability to decrease steady-state ATP-dependent Ca2+ accumulation into pig aortic microsomes. The vasoconstrictor agents noradrenaline, angiotensin II and adenosine 5'-[alpha beta-methylene]triphosphate (pp[CH2]pA) all decreased Ca2+ accumulation only when sonicated into vesicles (to allow access to receptor sites) and in the presence of guanosine 5'-[beta gamma-imido]triphosphate to activate transducing G-proteins. The effect of noradrenaline was inhibited by the alpha 2 antagonist yohimbine, but not by the alpha 1 antagonist prazosin. The effect of none of the agonists was reversed by diltiazem. SK&F 96365 (an inhibitor of receptor-mediated Ca2+ influx into intact cells) reversed the effect of noradrenaline, but not that of pp[CH2]pA, which suggests that at least two receptor-operated channels may be present in this preparation.


Effects of activin A and somatostatin on intact FSH secretion and intracellular Ca2+ concentration in human FSH-secreting pituitary adenoma cells. Activin A stimulated synthesis and secretion of intact FSH in dispersed human FSH-secreting adenoma cells. Significant stimulation was observed after 24 hr. Activin A caused an increase in Ca2+ concentration ([Ca2+]i). This response occurred soon after the activin A action. These effects were blocked in Ca(2+)-deficient medium and by nitrendipine (5 microM). Somatostatin inhibited the activin A-induced intact FSH secretion and the [Ca2+]i response. These findings indicated that Ca2+ influx through voltage-gated Ca2+ channel was involved in the activin A induced synthesis and secretion of intact FSH.


Differential inhibition of long-chain acyl-CoA hydrolases by hypolipidemic drugs in vitro. The effect of in vitro addition of three hypolipidemic drugs (clofibric acid, bezafibrate and gemfibrozil) on rat palmitoyl-CoA hydrolases has been studied, by using a spectrophotometric method (Berge RK, Biochim Biophys Acta 574: 321-333, 1979) optimized for valoration of crude enzyme preparations. Mitochondrial and microsomal hepatic palmitoyl-CoA hydrolase activities were inhibited by the three drugs in a concentration-dependent fashion. The order of inhibitory potency was gemfibrozil greater than bezafibrate greater than clofibric acid, irrespective of the enzyme activity tested. Cytosolic rat brain palmitoyl-CoA hydrolase activity was not affected. Kinetic studies with gemfibrozil on the solubilized microsomal palmitoyl-CoA hydrolase activity point to a mixed non-competitive type of inhibition.