Effect of ionizing radiation on neuromuscular junctions in mouse tongues. Radiation damage to the neuromuscular junctions (NMJs) in mouse tongues was studied using local x-irradiation of the tongues with the rest of the body shielded. Transmission electron microscopy (TEM) revealed no significant morphological changes in the fine structures and organelles of the NMJs given 4 Gy. A dose of 8 Gy produced degenevative morphological changes associated with oxon terminal sprouting as early as 2 and 7 days following irradiation. Subsequently, 1-11 weeks later, severe degenerative changes were observed. The number of mitochondria was significantly decreased with increased occurrence of degenerative membranal features. The number of synaptic footplates without terminals or with multiple small terminals within one groove increased gradually with time. Most of these pathological changes persisted for at least 3 months after irradiation. However, the myofibres, blood vessels and interstitial cells appeared to be unaffected throughout the period of follow-up. The present study substantiates our previous reports of ageing-like changes in the tongues' NMJs induced by their excessive exposure to free radicals.


Simultaneous measurement of cell cycle phase position and ionizing radiation-induced DNA strand breakage in single human tumour cells using laser scanning confocal imaging. Techniques for the assessment of DNA damage and repair in individual cells are pertinent to several areas of research, in particular the study of the heterogeneity of tumour cell populations in response to anticancer agents. We describe an adaptation of an in situ alkaline denaturation assay performed on individual nuclei of lysed cells, termed nucleoids, trapped within an agarose film. A novel aspect of the technique described in the application of confocal laser scanning fluorescence microscopy for the measurement of nucleoid relaxation in response to DNA damage. The volumes of spherical nucleoids and their relative DNA contents were determined by ethidium bromide staining and the analysis of confocal sections through the equatorial planes of the nucleoids. Mean nucleoid volume increased as a linear function of X-ray dose (0.5-8 Gy) administered to intact cells prior to lysis. We provide evidence of heterogeneity, in asynchronous cultures, in the DNA unfolding/unwinding characteristics of cells irrespective of cell cycle age. Bivariate plots of relative DNA content versus nucleoid volume allowed the direct assessment of cellular repair capacity with respect to cell cycle position.


Comparison of hepatitis B virus subtyping of d/y determinants by radioimmunoprecipitation assay and the polymerase chain reaction. Using a double polymerase chain reaction a method was devised for detecting and subtyping hepatitis B virus DNA in serum samples. Primers from the S-gene were selected from the sequence analyses of five HBV HBsAg subtypes, to amplify HBV DNA and subtype for y specific DNA. Thirty-eight samples were subtyped for d and y determinants by radioimmunoprecipitation assay (RIPA) and the polymerase chain reaction (PCR). Subtyping by PCR and RIPA was in agreement in 100% of subtype y samples and 83.3% of subtype d, giving an overall correlation of 92.1%. As a third comparison, 12 amplified samples were digested by the restriction enzyme Sau 3A, which differentiates between subtypes y and d. The digest results agreed with PCR in 83.3% of the samples. In addition, we compared our standard phenol/chloroform extraction against a rapid one step method. The phenol/chloroform stage was found to be essential for the removal of nucleases and polymerase inhibitors present in sera.


Antibodies to Epstein-Barr virus and cytomegalovirus in relation to CD4 cell number in human immunodeficiency virus 1 infection. Interaction between herpesviruses and human immunodeficiency virus (HIV)1 is postulated in the progression of HIV disease. In order to evaluate the specific antibody responses directed to Epstein-Barr virus (EBV) and cytomegalovirus (CMV) and to provide serological evidence suggesting reactivation of these viruses able to accelerate the immunodeficiency, we studied IgA and IgG titres to EBV and CMV in the serum of HIV positive patients in relation to the CD4 cell number. The titres of IgG antibodies to EBV and the prevalence of IgG to CMV were significantly higher in HIV positive patients compared to control high risk HIV negative subjects. In HIV infected patients, anti-VCA IgG antibodies increased and anti-EBNA IgG antibodies decreased progressively in relation to the decline of CD4 cell number whereas anti-CMV IgG antibodies did not varied significantly at the same time. Anti-VCA IgA and anti-EA IgG antibodies were found uncommonly and with low titres. IgA antibodies to EA and CMV were not detected in any patient. The variations in EBV antibody response that we describe in HIV infection were previously reported in other immunodeficiency states and could be distinctive of these diseases.


Sensitization of dopamine-stimulated adenylyl cyclase in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rhesus monkeys and patients with idiopathic Parkinson's disease. Dopamine-stimulated adenylyl cyclase activity was measured in striatal homogenates of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated rhesus monkeys and humans with idiopathic Parkinson's disease and compared with the activity in control tissue. No differences between parkinsonian and control tissue were found in the presence of 20 mM NaCl. However, when 120 mM NaCl was included in the assay medium, a significantly higher increase in the Vmax of dopamine-stimulated adenylyl cyclase activity was observed in the caudate of MPTP-parkinsonian rhesus monkeys and the putamen of patients with idiopathic Parkinson's disease. No such sensitization was seen in the MPTP-treated rhesus putamen or human Parkinson's disease caudate tissue. A role of D2 receptors in this sensitization could be ruled out by the concomitant use of the D2 antagonist l-sulpiride and by [3H]spiperone saturation analysis of the D2 receptor density, which was found at control level in the caudate tissue of MPTP-treated rhesus monkeys. Similarly, on the basis of saturation binding with the D1 selective ligand 125I-SCH 23982, there was no difference in caudate nucleus D1 receptor densities between control and MPTP-treated monkeys. Our results point to a region-specific functional sensitization of D1 receptors as a consequence of severe dopaminergic denervation of the striatum and suggest the possibility of a therapeutic potential of a D1 agonist with full intrinsic activity in Parkinson's disease.


Sequences that direct rat tyrosine hydroxylase gene expression. Investigation of neuroendocrine genes has revealed that transcription is regulated via multiple DNA binding sites, including the cyclic AMP response element (CRE). We show here that for the neuronal and chromaffin-specific gene tyrosine hydroxylase (TH), a 70-bp region (-229 to -160) lacking the CRE is sufficient, in either orientation, to confer levels of chloramphenicol acetyltransferase reporter expression equivalent to or greater than that conferred by 4.8 kb of the rat TH enhancer/promoter region. The 70-bp region contains potential binding sites for AP2, AP1, E2A/MyoD, and POU transcription factors, and functions when linked to the TH promoter, but not when joined to a heterologous RSV promoter. This demonstrates that promoter as well as enhancer elements are important for TH expression. In gel-shift assays, the 70-bp fragment forms a cell type-specific complex with nuclear extracts from TH-expressing cells. which is effectively competed by an oligonucleotide containing AP2, AP1, and E2A/MyoD (E box) sites, but not by one containing the POU site. These data suggest that the AP2, AP1, and/or E box sites may be involved in forming the cell-specific complex. Although it lacks an authentic CRE, the 70-bp region also mediated a twofold transcriptional response to forskolin, equivalent to that found with the endogenous gene. A different region (-60 to -29) bearing a consensus CRE mediated a sixfold increase in transcription in response to forskolin, but only minimally activated basal transcription from the TH promoter in the absence of forskolin.


Inhibition of tyrosine hydroxylase by R and S enantiomers of salsolinol, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline. Salsolinol is one of the dopamine-derived tetrahydroisoquinolines and is synthesized from pyruvate or acetaldehyde and dopamine. As it cannot cross the blood-brain barrier, salsolinol as the R enantiomer in the brain is considered to be synthesized in situ in dopaminergic neurons. Effects of R and S enantiomers of salsolinol on kinetic properties of tyrosine hydroxylase [tyrosine, tetrahydrobiopterin:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2], the rate-limiting enzyme of catecholamine biosynthesis, were examined. The naturally occurring cofactor of tyrosine hydroxylase, L-erythro-5,6,7,8-tetrahydrobiopterin, was found to induce allostery to the enzyme polymers and to change the affinity to the biopterin itself. Using L-erythro-5,6,7,8-tetrahydrobiopterin, tyrosine hydroxylase recognized the stereochemical structures of the salsolinols differently. The asymmetric center of salsolinol at C-1 played an important role in changing the affinity to L-tyrosine. The allostery of tyrosine hydroxylase toward biopterin cofactors disappeared, and at low concentrations of biopterin such as in brain tissue, the affinity to the cofactor changed markedly. A new type of inhibition of tyrosine hydroxylase, by depleting the allosteric effect of the endogenous biopterin, was found. It is suggested that under physiological conditions, such a conformational change may alter the regulation of DOPA biosynthesis in the brain.


Regulation of tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels in rat adrenals by a single and repeated immobilization stress. Adrenal catecholamines are known to mediate many of the physiological consequences of the "fight or flight" response to stress. However, the mechanisms by which the long-term responses to repeated stress are mediated are less well understood and possibly involve alterations in gene expression. In this study the effects of a single and repeated immobilization stress on mRNA levels of the adrenal catecholamine biosynthetic enzymes, tyrosine hydroxylase and dopamine beta-hydroxylase, were examined. A repeated 2-hr daily immobilization for 7 consecutive days markedly elevated both tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels (about six- and fourfold, respectively). In contrast, tyrosine hydroxylase but not dopamine beta-hydroxylase mRNA levels were elevated immediately following a single immobilization. The elevation in tyrosine hydroxylase mRNA with a single immobilization was as high as with seven daily repeated immobilizations. This elevation was not sustained and returned toward control values 24 hr later. Both tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels were elevated immediately following two daily immobilizations to levels similar to those observed after seven immobilizations and were maintained 24 hr later. The results indicate that both tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels are elevated by stress; however, the mechanism and/or timing of their regulation are not identical.


Uptake, metabolism, and release of N-[3H]acetylaspartylglutamate by the avian retina. N-Acetylaspartylglutamate (NAAG) is a nervous system-specific dipeptide that is released from retinal neurons on depolarization. In the present study, extracellular metabolism, uptake, and release of [3H]NAAG were examined in the chick retina. After in vitro incubation with NAAG radiolabeled in the glutamate moiety, [3H]glutamate and [3H]NAAG increased in retinal cells through time- and temperature-dependent processes, which were reduced in the absence of extracellular sodium. Coincubation of cells with [3H]NAAG and aspartylglutamate or phosphate resulted in the decreased extracellular appearance of [3H]glutamate, produced by hydrolysis of radiolabeled NAAG, and a consequent increased availability of [3H]NAAG for transport into the retinal cells. When this tissue was incubated with radiolabeled NAAG, glutamate, glutamine, or aspartate under similar conditions, only [3H]NAAG served as a significant source for the appearance of intracellular [3H]NAAG. These data support the conclusion that [3H]NAAG can be transported into retinal cells, whereas [3H]glutamate transport is the predominant process after release of this amino acid from NAAG by extracellular peptidase activities. After uptake, [3H]NAAG entered a cellular pool, from which the peptide was secreted under depolarizing conditions and in a calcium-dependent manner.


Amino acid neurotransmitters and dopamine in brain and pituitary of the goldfish: involvement in the regulation of gonadotropin secretion. An isocratic high-performance liquid chromatographic technique was developed to measure levels of gamma-aminobutyric acid (GABA), glutamate, and taurine in the brain and pituitary of goldfish. Accuracy of this procedure for quantification of these compounds was established by evaluating anesthetic and postmortem effects and by selectively manipulating GABA concentrations by intraperitoneal administration of the glutamic acid decarboxylase (GAD) inhibitor 3-mercaptopropionic acid or the GABA transaminase inhibitor gamma-vinyl GABA. The technique provided a simple, rapid, and reliable method for evaluating the concentrations of these amino acids without the use of complex gradient chromatographic systems. To investigate the relationship between neurotransmitter amino acids and the control of pituitary secretion of gonadotropin, the effects of injection of taurine, GABA, or monosodium glutamate on GABA, glutamate, taurine, and, in some instances, monoamine concentrations in the brain and pituitary were evaluated and related to serum gonadotropin levels. Injection of taurine caused an elevation in serum gonadotropin concentrations. In addition, injection of the taurine precursor hypotaurine but not the taurine catabolite isethionic acid elevated serum gonadotropin levels. Intracerebroventricular injection of either GABA or taurine also elevated serum gonadotropin concentrations. Pretreatment of recrudescent fish with alpha-methyl-p-tyrosine reduced pituitary dopamine concentrations and also potentiated the serum gonadotropin response to taurine. Injection of monosodium glutamate caused an increase of glutamate content in the pituitary at 24 h; this was followed by a decrease at 72 h after administration. Pituitary GABA, taurine, and dopamine concentrations underwent a transient depletion after monosodium glutamate administration, and this was associated with an elevation of serum gonadotropin content.(ABSTRACT TRUNCATED AT 250 WORDS)


Amperozide, a novel antipsychotic drug, inhibits the ability of d-amphetamine to increase dopamine release in vivo in rat striatum and nucleus accumbens. The in vivo effects of amperozide, a novel atypical antipsychotic drug, on the release of dopamine (DA) and the output of its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), were investigated in the striatum and the nucleus accumbens of awake, freely moving rats using microdialysis. Amperozide (2-10 mg/kg, s.c.) significantly increased extracellular levels of DA in both the striatum and nucleus accumbens in a dose-dependent manner. It had a similar but lesser effect on extracellular DOPAC levels in both regions. d-Amphetamine (2 mg/kg, s.c.) alone produced a very large (43-fold) increase in DA release, together with a 70% decrease in DOPAC levels in both the striatum and the nucleus accumbens. Amperozide (1-5 mg/kg, s.c.) 30 min before d-amphetamine (2 mg/kg) dose-dependently attenuated d-amphetamine-induced DA release but had no effect on the d-amphetamine-induced decrease in extracellular DOPAC levels in both regions. The effect of amperozide on d-amphetamine-induced DA release in the nucleus accumbens may explain the inhibitory effect of amperozide on amphetamine-induced locomotor activity. However, the failure of amperozide to block amphetamine-induced stereotypy, despite marked inhibition of striatal DA release, suggests the need to reexamine the importance of striatal DA for amphetamine-induced stereotypy.


Vascular compromise prior to intestinal manifestations of Crohn's disease in a 14-year-old girl. Vascular manifestations as extraintestinal symptoms of Crohn's disease are rare and only occasionally reported in children. A 14-year-old girl with vascular compromise prior to intestinal manifestations of Crohn's disease is described. The vascular symptoms were due to segmental narrowing of several major arteries as shown by angiography. This kind of vascular involvement in our patient is different from the pattern described in Crohn's disease and resembles Takayasu's disease. Recently, it has been suggested that Crohn's disease could be mediated by multifocal gastrointestinal infarction due to chronic focal mesenteric arteritis at the level of the muscularis propria of the gut. In Takayasu's disease, a granulomatous inflammation of the vasa vasorum of affected vessels is frequently found. An intramural arteritis, granulomatous in nature, could be the common pathway in both Crohn's and Takayasu's diseases. Until the etiologies of both diseases are uncovered, the interrelation between them will remain subject to speculation.


Transport of acidic amino acids in Candida albicans. In Candida albicans ATCC 10261, two kinetically different amino acid transport systems with a high (S1) and a low (S2) affinity for aspartic acid (asp) and glutamic acid (glu) were identified. The S1 for the two acidic amino acids was characterized by low Kt values while Kt values of S2 were 30 to 40 times higher. Based on competitive studies of both systems, S1 was found to be specific and common to both asp and glu while S2 was relatively less specific. The S1 and S2 systems were also different in their sensitivity to respiratory inhibitors, mercurials and a K+ channel blocker. Both systems, however, showed maximum transport rates during the mid-exponential growth phase.


Analogues of the dioxolanes dexoxadrol and etoxadrol as potential phencyclidine-like agents. Synthesis and structure-activity relationships. A series of dioxolane analogues based on dexoxadrol ((4S,6S)-2,2-diphenyl-4-(2-piperidyl)-1,3-dioxolane) and etoxadrol ((2S,4S,6S)-2-ethyl-2-phenyl-4-(2-piperidyl)-1,3-dioxolane) were prepared and tested for their ability to displace [3H]TCP (1-[1-(2-thienyl)cyclohexyl]piperidine) from PCP (1-(1-phenylcyclohexyl)piperidine) binding sites in rat brain tissue homogenates. Qualitative structure-activity relationships within this series were explored through modifications of the three major structural units of dexoxadrol, the piperidine, 1,3-dioxolane, and aromatic rings of the molecule. N-Alkyl derivatives of dexoxadrol were found to be inactive, as were those analogues where the dioxolane ring was modified. Phenyl-substituted etoxadrol analogues were compared to similarly substituted PCP analogues and distinct differences were found in their structure-activity relationships suggesting that the aromatic rings in these two drug classes interact differently with the PCP binding sites. The replacement of the phenyl ring in etoxadrol by either a 2- or 3-thienyl ring led to compounds with affinity comparable to etoxadrol, and the replacement of the ethyl moiety on etoxadrol's dioxolane ring with propyl (7) or isopropyl (8) led to compounds which were more potent than etoxadrol or PCP. The most potent compound was (2S,4S,6S)-2-ethyl- 2-(1-chlorophenyl)-4-(2-piperidyl)-1,3-dioxolane (11), where a chlorine moiety was placed in the ortho position in the aromatic ring of etoxadrol. Its potency was comparable with TCP in vitro.


Direct photoaffinity labeling of tubulin with taxol. Taxol is a potent inhibitor of the replication of eukaryotic cells and has significant antitumor activity in human malignancies. The drug induces the formation of bundles of stable microtubules and blocks cells in the mitotic phase of the cell cycle. In vitro, taxol enhances the polymerization of tubulin to microtubules that are resistant to depolymerization. Although it is evident that taxol interacts with the tubulin-microtubule system, no information has been available on the binding site for the drug on the microtubule. Our purpose was to determine if taxol binds to one or both of the tubulin subunits. In the absence of a photoaffinity-labeled analogue of taxol, [3H]taxol was used directly to photolabel tubulin. A complex of microtubule protein and [3H]taxol was irradiated by ultraviolet light and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radiolabeled drug preferentially binds covalently to the beta-subunit of tubulin, and the binding can be competed with unlabeled taxol. This observation is the first step in a study to determine the binding site for taxol on the microtubule.


Cellular basis for interactions between catecholaminergic afferents and neurons containing Leu-enkephalin-like immunoreactivity in rat caudate-putamen nuclei. Dopaminergic afferents to the dorsal striatum, caudate-putamen nuclei, are known to modulate the levels and synthesis of endogenous opiate peptides (Leu5 and Met5-enkephalins). We examined the dual immunocytochemical localization of antisera raised against Leu5-enkephalin and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), to determine the cellular substrates for these and/or other functional interactions. The antisera were identified by combined immunogold-silver and immunoperoxidase labeling in single coronal sections through the caudate-putamen nuclei of adult rats. These animals were given intraventricular injections of colchicine, and the brains were fixed by acrolein perfusion prior to immunocytochemical labeling. By light microscopy, perikarya and processes containing enkephalin-like immunoreactivity (ELI) were seen in close proximity to varicose processes immunoreactive for TH. Electron microscopy further demonstrated that the ELI was localized to perikarya, dendrites, and axon terminals, whereas the TH was exclusively in axons and terminals. The dendrites containing ELI were postsynaptic to terminals that were either (1) without detectable immunoreactivity, or (2) immunoreactive for TH or enkephalin. Nonsynaptic portions of the dendrites containing ELI were covered with astrocytic processes or were in direct apposition to unlabeled dendrites. Terminals containing ELI were densely immunoreactive and were in direct contact with (1) unlabeled and occasionally enkephalin-labeled proximal dendrites, and (2) TH-labeled and unlabeled terminals. In comparison with the opiate terminals, most catecholaminergic terminals were lightly immunoreactive for TH and usually contacted more distal unlabeled dendrites or spines and, more rarely, dendrites containing ELI. In a few favorable planes of section, the terminals containing ELI and those containing TH (1) converged on common unlabeled dendrites, or (2) formed dual contacts on two different labeled or unlabeled targets. Junctions formed by terminals containing ELI and TH were sometimes characterized by symmetric synaptic densities. However, numerous other dendritic and all axonal appositions were without recognized membrane densities. The findings of the study provide anatomical substrates for multilevel interactions between catecholamines, mostly dopamine, and enkephalin in rat dorsal striatum. These include (1) monosynaptic input from dopaminergic terminals to neurons containing enkephalin, (2) presynaptic modulation of transmitter release through axonal appositions, and (3) dual regulation of common targets through convergent input. In addition, the findings suggest that both enkephalin and dopamine may have similar modulatory roles in synchronizing the activity of dual targets postsynaptic to individual axon terminals. Alterations in any one of these multiple types of interactions could account for noted motor or sensory symptoms in neurological disorders characterized by depletion of dopamine or endogenous opiate peptides, or both.


GnRH-associated peptide (GAP) is present in the rat striatum and affects the synthesis and release of dopamine. A possible interference of the GnRH-associated peptide (GAP) with the metabolism of dopamine in the rat striatum was investigated. The presence of the precursor of the peptide in this central region of dopaminergic terminals was first evidenced using specific RIA. The action of GAP on dopamine release was investigated in the caudate nucleus using the local superfusion with a push-pull cannula supplied with an artificial CSF containing the tritiated precursor of dopamine [( 3H]tyrosine). Addition of GAP (1 microM) to the superfusing fluid resulted in an increase of the release of the newly synthesized dopamine without a significant modification of the total amine release. In situ neutralization of GAP by addition in the CSF of a rabbit serum containing antibodies directed against the GAP produced opposite effects evidencing a tonic function for this peptide. In addition to the increased specific activity of the dopamine released during GAP treatment, the alterations observed in the efflux (and the specific activity) of dihydroxyphenyl acetic acid and the activation of dopamine synthesis obtained in vitro in striatal slices in the presence of GAP led us to conclude that the GAP system could be considered as a positive control of dopamine synthesis.


Relationship of human papillomavirus type to grade of cervical intraepithelial neoplasia. OBJECTIVE--To determine the relationship of human papillomavirus (HPV) type to grade of cervical intraepithelial neoplasia (CIN) in a large series of cases. DESIGN--A survey of HPV types in CIN lesions detected using a new, highly accurate method for typing HPV that is based on restriction fragment length polymorphism analysis of amplimers produced during polymerase chain amplification of the conserved L1 region of HPV using consensus primers. SETTING--Private gynecologists' offices and inner-city colposcopy clinics. PATIENTS--A convenience sample of 276 HPV DNA-positive cervical biopsy specimens or samples from patients undergoing colposcopy for abnormal Papanicolaou smears. INTERVENTION--None. MAIN OUTCOME MEASURE(S)--Human papillomavirus type(s). RESULTS--Cervical intraepithelial neoplasia 1 lesions were relatively heterogeneous with regard to associated HPV types. Nineteen percent of CIN 1 lesions were associated with HPV types 6 or 11; 29% contained HPV types 16, 18, or 33; and 19% were associated with "novel types" of HPV. It was also found that 22% of CIN 1 lesions were associated with more than one HPV type. In contrast to CIN 1, both CIN 2 and CIN 3 were relatively homogeneous with regard to associated HPV types. Eighty-eight percent of CIN 2 and 3 lesions contained HPV types 16, 18, or 33. Unlike CIN 1 lesions, which often contained multiple types of HPV, only 7% of CIN 2 and 3 lesions were associated with multiple HPV types. CONCLUSIONS--Cervical intraepithelial neoplasia should be classified into two separate categories--low-grade and high-grade CIN. Since only 29% of low-grade lesions are associated with HPV types 16, 18, or 33, HPV type could potentially play a role in determining the most appropriate clinical management of patients with low-grade CIN. However, prospective follow-up studies of lesional behavior based on HPV type are required before clinical recommendations can be made.


Unstable DNA sequence in myotonic dystrophy. A variable DNA sequence has been detected in patients with myotonic dystrophy. We set out to determine whether identification of this specific molecular defect would improve clinical management of patients and families with myotonic dystrophy. 127 affected patients who were studied had an expanded DNA fragment not seen in 73 normal controls. The increase in length of the fragment correlated broadly with disease severity, and we noted expansion of the sequence in successive generations of the same family. Progressive expansion of the affected gene provides a molecular explanation for an apparently earlier onset in successive generations (anticipation) in myotonic dystrophy and supports the role of an unstable repeat sequence as the basis of the defect. The specificity of this finding will assist in accurate diagnosis of myotonic dystrophy and genetic counselling of affected families.


Insulin resistance and cigarette smoking. Cigarette smoking is associated with increases in plasma triglycerides and decreases in plasma high density-lipoprotein-cholesterol concentration. These changes not only increase risk of coronary heart disease but also are secondary to resistance to insulin-stimulated glucose uptake or hyperinsulinaemia. To see whether there is a relation between cigarette smoking and insulin-mediated glucose uptake we measured plasma lipid and lipoprotein concentrations, plasma glucose and insulin response to an oral glucose challenge, and insulin-mediated glucose uptake in 40 matched healthy volunteers (20 non-smokers, 20 smokers). Smokers had significantly higher mean (SEM) very-low-density-lipoprotein triglycerides (0.66 [0.10] vs 0.39 [0.03] mmol/l, p less than 0.02) and cholesterol (0.45 [0.06] vs 0.23 [0.04] mmol/l, p less than 0.005) concentrations and lower high-density-lipoprotein cholesterol concentrations (1.16 [0.05] vs 1.51 [0.08] mmol/l, p less than 0.001). Although plasma glucose concentrations in response to the oral glucose load were similar in the two groups, plasma insulin response of the smokers was significantly higher (p less than 0.001). Finally, smokers had higher steady-state plasma glucose concentrations in response to a continuous infusion of glucose, insulin, and somatostatin (8.4 [0.2] vs 5.0 [0.3] mmol/l, p less than 0.001), despite similar steady-state plasma insulin concentrations. The findings show that chronic cigarette smokers are insulin resistant, hyperinsulinaemic, and dyslipidaemic compared with a matched group of non-smokers, and may help to explain why smoking increases risk of coronary heart disease.


Effect of vegetarian soy diet on hyperlipidaemia in nephrotic syndrome. Nephrotic patients with persistent proteinuria also have various lipid abnormalities that may promote atherosclerosis and more rapid progression of renal disease. We aimed to find out whether dietary manipulation can correct the hyperlipidaemia found in these patients. After a baseline control period of 8 weeks on their usual diets, 20 untreated patients with chronic glomerular diseases, stable long-lasting severe proteinuria (5.9 [SD 3.4] g/24 h) and hyperlipidaemia (mean serum cholesterol 8.69 [3.34] mmol/l) ate a vegetarian soy diet for 8 weeks. The diet was low in fat (28% of total calories) and protein (0.71 [0.36] g/kg ideal body weight daily), cholesterol free, and rich in monounsaturated and polyunsaturated fatty acids (polyunsaturated/saturated ratio 2.5) and in fibre (40 g/day). After the diet period the patients resumed their usual diets for 8 weeks (washout period). During the soy-diet period there were significant falls in serum cholesterol (total, low-density lipoprotein, and high-density lipoprotein) and apolipoproteins A and B, but serum triglyceride concentrations did not change. Urinary protein excretion fell significantly. The concentrations of all lipid fractions and the amount of proteinuria tended to return towards baseline values during the washout period. We do not know whether the favourable effect of this dietary manipulation on proteinuria was due to the qualitative or quantitative modifications of dietary protein intake or was a direct consequence of the manipulation of dietary lipid intake.


Betel-nut chewing and asthma.

Betel-nut chewing and asthma. Two Asian patients admitted to hospital with acute severe asthma had been chewing betel nut immediately before the attacks. Arecoline, a cholinergic alkaloid, is a major constituent of Areca catechu (betel) nut and causes the euphoric effects. We sought an association between betel-nut chewing and bronchoconstriction in asthmatic patients. In vitro, arecoline caused dose-related contraction of human bronchial smooth-muscle strips, with one-tenth the potency of methacholine. In a double-blind challenge study, inhalation of arecoline caused bronchoconstriction in six of seven asthmatic patients and one of six healthy subjects; methacholine caused bronchoconstriction in all the asthmatic patients and in five controls. The geometric mean concentrations of arecoline and methacholine that caused 20% falls in the forced expiratory volume in 1 s (PC20 FEV1) in the asthmatic subjects were 5.2 mg/ml and 1.6 mg/ml, respectively. We then studied four Bengali asthmatic patients, regular users of betel nut, during a betel-nut challenge. Three showed no adverse effects, but one showed a 30% fall in FEV1 by 150 min after chewing; the effect was reproducible. In the UK, the rate of hospital admission for acute asthma is higher among Asians than among other groups in the population; betel-nut chewing may be one of several factors that affect asthma control and severity of attacks.


Does pralidoxime affect outcome of management in acute organophosphorus poisoning? Acute organophosphorus (OP) poisoning is usually treated with atropine plus cholinesterase reactivators such as oximes, but controlled trials to assess the efficacy of oximes in OP poisoning have not been done. A period when the acetyl cholinesterase reactivator pralidoxime chloride was not available in Sri Lanka gave us the opportunity to compare atropine alone for treatment of moderate to severe OP poisoning (21 patients) with atropine plus pralixodime (24 patients). Outcome, as assessed clinically, was similar in the two groups. These results cast doubt on the necessity of cholinesterase reactivators for treatment of acute OP poisoning.


Velo-cardio-facial syndrome associated with chromosome 22 deletions encompassing the DiGeorge locus. The large clinical overlap between DiGeorge syndrome and velo-cardio-facial syndrome suggests an aetiological connection. DiGeorge syndrome is associated with microdeletions of chromosome 22q11 and is therefore likely to be caused by reduced dosage of genes within this region. We present preliminary data that velocardiofacial syndrome patients have similar chromosome deletions, a finding consistent with the hypothesis that these disorders represent part of a spectrum of abnormalities seen with monosomy for 22q11.


Viral-associated haemophagocytosis with parvovirus-B19-related pancytopenia. Viral-associated haemophagocyte syndrome in response to infection with human parvovirus B19 was seen in 2 patients with hereditary spherocytosis. Depressed reticulocyte response during acute parvovirus infection is a known cause of hypoproliferative crises in patients with reduced erythrocyte lifespan; the observation of parvovirus-associated haemophagocytosis could account for the pancytopenia that may accompany human parvovirus B19 infection.


Controlled trial of hyposensitisation in children with food-induced hyperkinetic syndrome. Food intolerance seems to be an important cause of the hyperkinetic syndrome, but restricted diets are expensive, socially disruptive, and often nutritionally inadequate. Enzyme-potentiated desensitisation (EPD) may overcome some of these difficulties. EPD was tested in a double-blind placebo-controlled trial among 40 children with food-induced hyperkinetic behaviour disorder. A total of 185 children with established hyperkinetic syndrome underwent oligoantigenic dietary treatment for four weeks. 116 whose behaviour responded had provoking foods identified by sequential reintroduction. Foods that reproducibly provoked overactivity were avoided. 40 patients who were then invited to take part in the hyposensitisation trial were randomly assigned to treated and control groups. Treated patients received three doses of EPD (beta-glucuronidase and small quantities of food antigens) intradermally at two-monthly intervals. Controls received buffer only. Thereafter, patients were allowed to eat known provoking foods. Of 20 patients who received active treatment, 16 became tolerant towards provoking foods compared with 4 of 20 who received placebo (p less than 0.001). Our results show that EPD permits children with food-induced hyperkinetic syndrome to eat foods that had previously been identified as responsible for their symptoms. These results also support the notion that food allergy is a possible mechanism of the hyperkinetic syndrome.


Cholesterol inhibition, cancer, and chemotherapy. An important feature of malignant transformation is loss of the cholesterol feedback inhibition mechanism that regulates cholesterol synthesis. Cancer cells seem to require an increase in the concentrations of cholesterol and of cholesterol precursors. Therefore, a reasonable assumption is that prevention of tumour-cell growth can be achieved by restricting either cholesterol availability or cholesterol synthesis. In-vivo and cell-culture experiments have shown that lowering the plasma cholesterol concentration or intervening in the mevalonate pathway with 3-hydroxy-3-methylglutaryl (HMG) CoA reductase inhibitors decreases tumour growth. Currently prescribed doses of HMG-CoA reductase inhibitors given orally or continuously by an implantable infusion pump could achieve tumour therapeutic tissue concentrations of these agents. My hypothesis is that cholesterol inhibition can inhibit tumour cell growth, can act as an adjuvant to cancer chemotherapy, and, possibly, can prevent carcinogenesis.


A special irrigation liquid to increase the reliability of laser-induced shockwave lithotripsy. For the laser-induced shockwave lithotripsy (LISL) the laser-pulses of a Q-switched Nd:YAG laser produce an optical breakdown in the irrigation liquid surrounding the urinary stone. Subsequently high-pressure shockwaves are emitted causing stone fragmentation. Since the LISL is an endoscopic technique, problems arise from the transmission of the laser pulses through optical fibers. The intensity threshold for an optical breakdown in commonly used saline solution amounts to 21 GW/cm2, in optical silica fibers, to about 3 GW/cm2. Therefore bare fibers cannot be used without being destroyed by a breakdown. So we have developed an irrigation liquid by adding small quantities of metal ions to saline solution to lower the threshold intensity. The most suitable ion was Fe3+ in a concentration of 0.02 mmol/l, which shows a lowering to 5 GW/cm2. In combination with a spherically shaped fiber exit the intensities that have to be transmitted are below the threshold of the fiber material. Using this irrigation liquid the overall reliability of the method could be significantly increased and several stone fragmentations can be performed with a single optical fiber.


Comparison of a pulsed dye laser and electrohydraulic lithotripsy on porcine gallbladder and common bile duct in vitro. With the advent of minimal access biliary procedures there is a need for a safe intracorporal lithotripsy technique that can be used through small flexible endoscopes. Currently, the two techniques available are electrohydraulic lithotripsy and laser induced shock wave lithotripsy. In this study we compare the effect of a 504 nm coumarin pulsed dye laser and electrohydraulic lithotripsy on in vitro porcine gallbladder and common bile duct. Electrohydraulic lithotripsy at the lowest energy the generator would deliver caused perforation of both tissues in only a few pulses when a 1.9-F probe was placed in direct contact with the tissue. Energy from a 504 nm coumarin pulsed dye laser delivered through a 320-microns fiber placed in light contact with the tissue caused an energy-dependent perforation after 50 pulses in from none to 44% of tissues. It was also found that there was a higher incidence of perforation in more vascular than non-vascular tissue. When the EHL probe and the laser fiber were held 1-2 mm from the tissue surface, discharge of each resulted in no perforation. On histological examination of the tissues, the perforations were found to be very small with laser lithotripsy and considerably larger with the electrohydraulic lithotripsy. It was felt that laser lithotripsy in the clinical situation was likely to be much safer than electrohydraulic lithotripsy.


Alpha 2-adrenoceptor antagonists block the stimulant effects of cocaine in mice. In the present study we have investigated the effects of the alpha 2-adrenoceptor antagonist idazoxan and its 2-ethoxy derivative RX811059 on the locomotor activity induced by cocaine in mice. The stimulant effects of cocaine (15 mg/kg i.p.) were significantly antagonised by idazoxan (3 mg/kg i.p.) and RX811059 (1 mg/kg i.p.) and also initially suppressed by idazoxan (1 mg/kg i.p.) and RX811059 (0.3 mg/kg i.p.). The alpha 2-adrenoceptor antagonists had no effect on locomotion when given alone. These results suggest that noradrenergic mechanisms may play a role in the stimulant effects of cocaine and that alpha 2-adrenoceptor antagonists like idazoxan may be of some benefit in the clinical management of cocaine abuse.


A key role for type 1 pili in enterobacterial communicability. Up to 80% of faecal Escherichia coli strains are able to produce type 1 pili. These filamentous bacterial surface organelles, which mediate mannose-sensitive attachment to mammalian epithelial cells, are also conserved throughout the Enterobacteriaceae. As a potential explanation for their prevalence among intestinal isolates of enteric bacteria, it has been widely speculated that type 1 pili are important for adherence to the host's intestinal mucosa. However, conclusive evidence for this idea is lacking, and there are reasonable grounds for doubting such an effect. Permanent interruption of type 1 piliation in previously pil+ E. coli (by directed mutagenesis of pilA, the gene coding for the major structural subunit of type 1 pili) does not diminish the density of intestinal colonization in individual animals. Rather, as we demonstrate here, this lesion results in a dramatic decrease in transmission of E. coli K1 from experimentally colonized neonatal rats to their littermates. The enhanced communicability associated with type 1 piliation suggests a heretofore unrecognized explanation for the prevalence of type 1 pili among intestinal E. coli; one that does not necessarily require the direct action of these organelles at the intestinal mucosa.


A leptomycin B resistance gene of Schizosaccharomyces pombe encodes a protein similar to the mammalian P-glycoproteins. Screening for leptomycin B (LMB)-resistant transformants in a gene library constructed in Schizosaccharomyces pombe with the chromosomal DNA of an LMB-resistant mutant of S. pombe and with multicopy plasmid pDB248' as the vector led to the isolation of a gene, named pmd1+, encoding a 1362-amino-acid protein. This protein showed great similarity in amino acid sequence to the mammalian P-glycoprotein encoded by the multidrug resistance gene, mdr, and the Saccharomyces cerevisiae a-factor transporter encoded by STE6. In addition, computer analyses predicted that the protein encoded by pmd1+ formed an intramolecular duplicated structure and each of the halves contained six transmembrane regions as well as two ATP-binding domains, as observed with the P-glycoproteins and the STE6 product. Consistent with this was that S. pombe cells containing the pmd1+ gene on a multicopy plasmid showed resistance not only to LMB but also to several cytotoxic agents. The pmd1 null mutants derived by gene disruption were viable and hypersensitive to these agents. All these data suggest that the pmd1+ gene encodes a protein that is a structural and functional counterpart of mammalian mdr proteins.


[Mannose-resistant hemagglutination (MRHA), type 1 fimbriae and hemolysin production of urinary and fecal Escherichia coli strains]. The prevalence E.coli bearing Mannose-Resistant Hemagglutinating (MRHA) adhesin, Type 1 fimbriae and hemolytic activity was assessed in 103 E.coli strains isolated from 43 male and 60 female adult patients with urinary tract infection. For comparison 83 faecal E.coli strains were also examined for the same properties. MRHA adhesins were present in 58% (60/103) of the urinary strains, and 31% (26/83) of the faecal strains of E.coli. All faecal and 95% (98/103) of the urinary strains of E.coli expressed Type 1 fimbriae. Hemolytic activity was more frequent among urinary strains than among faecal strains (21%, 12%, respectively). The presence of MRHA adhesin and hemolytic activity was found correlated in 13.5% (14/103) of urinary strains whereas only one (1.2%) strain showed these properties together among the faecal isolates.


Trisomy and chromosome size changes in hybrid trypanosomes from a genetic cross between Trypanosoma brucei rhodesiense and T. b. brucei. Further analysis of hybrid clones from an experimental cross of Trypanosoma brucei rhodesiense 058 and T. b. brucei 196 shows 2 of the hybrid clones to have DNA contents about 1.5 times parental values. This represents over 40,000 kb of extra DNA. Comparison of the molecular karyotypes of parental and progeny trypanosomes shows that the bulk of the extra DNA constitutes chromosomes greater than 1 Mb in size, although a small proportion can be accounted for by an increased number of mini-chromosomes. The 2 hybrid clones have 3 alleles at several loci for housekeeping genes as shown by RFLP and isoenzyme analysis. Trisomy of the chromosome carrying phosphoglycerate kinase and tubulin genes and that carrying the phospholipase C gene was demonstrated by analysis of molecular karyotypes. These chromosomes appear prone to substantial size alterations associated with genetic exchange. Our results for one of the hybrid clones are completely consistent with it being triploid and the product of fusion of haploid and diploid nuclei.


An RFLP map of the Plasmodium falciparum genome, recombination rates and favored linkage groups in a genetic cross. We report a genetic linkage map of the Plasmodium falciparum genome, using the inheritance patterns of nearly 90 RFLP markers in a genetic cross. Markers were assigned to polymorphic loci on all 14 nuclear chromosomes. Genetic recombination between parental markers was detected in each of the progeny, indicating that progeny from cross-fertilization events were favored over progeny from self-fertilization of either parent alone. Inheritance patterns among the markers suggested that certain parental linkage groups on chromosomes 2, 3, 12 and 13 were favored in the cross. Recombination frequencies on five chromosomes indicated an approximate map unit size of 15-30 kb per centiMorgan for P. falciparum.


Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis (ALS) is a chronic degenerative neurologic disorder characterized by the death of motor neurons in the cerebral cortex and spinal cord. Recent studies have suggested that the metabolism of glutamate, a potentially neurotoxic amino acid, is abnormal in patients with ALS. We hypothesized that the high-affinity glutamate transporter is the site of the defect. We measured high-affinity, sodium-dependent glutamate transport in synaptosomes from neural tissue obtained from 13 patients with ALS, 17 patients with no neurologic disease, and 27 patients with other neuro-degenerative diseases (Alzheimer's disease in 15 patients and Huntington's disease in 12 patients). The groups were comparable with respect to age and the interval between death and autopsy. Synaptosomes were prepared from spinal cord, motor cortex, sensory cortex, visual cortex, striatum, and hippocampus. We also measured sodium-dependent transport of gamma-aminobutyric acid and phenylalanine in the synaptosomal preparations. In patients with ALS, there was a marked decrease in the maximal velocity of transport for high-affinity glutamate uptake in synaptosomes from spinal cord (-59 percent, P less than 0.001), motor cortex (-70 percent, P less than 0.001), and somatosensory cortex (-39 percent, P less than 0.05), but not in those from visual cortex, striatum, or hippocampus. The affinity of the transporter for glutamate was not altered. No abnormalities in glutamate transport were found in synaptosomes from patients with other chronic neurodegenerative disorders. The transport of gamma-aminobutyric acid and phenylalanine was normal in patients with ALS. ALS is associated with a defect in high-affinity glutamate transport that has disease, region, and chemical specificity. Defects in the clearance of extracellular glutamate because of a faulty transporter could lead to neurotoxic levels of extracellular glutamate and thus be pathogenic in ALS.


[Coma due to an overdose of valnoctamide]. We report a 27-year-old man, who became comatose after autopoisoning with a high dose of valnoctamide. He was mechanically ventilated for 12 hours and survived without serious side effects. Valnoctamide blood levels were monitored in order to study the pharmacokinetics of oral overdosing of this drug. Serum half-time levels appeared to be approximately 15 hours.


Amplification of ERBB-2 (HER-2/NEU) oncogene in different neoplasms of patients from USSR. 203 tumor specimens from 175 patients were studied. Amplification of ERBB-2 was detected in 14 out of 63 (22%) cases of breast carcinoma, in 1 out of 23 patients with ovarian cancers, in 1 out of 19 cases of colon carcinoma and in 1 out of 27 patients with thyroid cancer. We failed to find more than one copy of ERBB-2 in 34 patients with lung cancers, 6 with sarcomas and 3 with melanomas. There was tendency toward correlation between ERBB-2 amplification and lymph node involvement in patients with breast carcinoma. Thus, the oncogene ERBB-2 is often amplified in human tumors, but breast cancer is characterized by an especially high frequency of ERBB-2 amplification.


Differential localization of alpha 2-adrenergic receptor subtypes in brain. The pharmacological identification and characterization of subtypes of alpha 2-adrenergic receptors have been confirmed by molecular biological investigations. Using receptor autoradiographic techniques, it has been possible to show regions of the brain where alpha 2 agonist binding ([3H]para-aminoclonidine) is preferentially labeling the presumed guaninenucleotide-sensitive, high-affinity conformations of the alpha 2 receptor. Careful examination of autoradiograms generated using the tritiated antagonists yohimbine, idazoxan, and rauwolscine also indicates some disparity in the regions occupied by these radiolabeled ligands. Inhibition of [3H]rauwolscine binding with the subtype selective compounds, ARC-239, or oxymetazoline demonstrates that there are discrete regions of the brain where one receptor subtype predominates over the other. These studies indicate that previous investigations utilizing the agonist para-aminoclonidine as the ligand for obtaining labeling of alpha 2 receptors have overlooked some regions of binding due to the subtype selectivity of this ligand. A more complete localization of alpha 2-adrenergic receptors can be obtained using the tritiated antagonist rauwolscine, and the differential distribution of at least two subtypes of the alpha 2 receptor can be obtained by selective inhibition of this binding.


Comparative studies of sulpiride and classical neuroleptics on induction of catalepsy, locomotor activity, and brain dopamine metabolism in mice. The effects of the peripheral administration of sulpiride on the induction of catalepsy, the vertical (VMA) and horizontal (HMA) locomotor activities, and on the dopamine metabolism in the limbic system, striatum, and nucleus accumbens were examined using mice up to 7.5 h after administration of drugs. These effects were compared to those of pimozide and haloperidol. Sulpiride (1.25-160 mg/kg, IP) clearly induced catalepsy similar to pimozide (0.0625-4 mg/kg, IP) and haloperidol (0.0375-0.3 mg/kg, IP). During the induction of catalepsy, the intensity was the strongest at 4.5 h after administration and the ED50 value showed 11.5 mg/kg at that time. However, a moderate dose of sulpiride at 10-20 mg/kg did not show the induction of a dose- and time-dependent catalepsy. During the locomotor activity, the VMA was significantly inhibited at 1.5 h and 6 h after administration of pimozide (0.25 mg/kg, IP) and haloperidol (0.075 mg/kg, IP) as compared to the control group, while the HMA was significantly inhibited at 1.5 h after administration of sulpiride (40 mg/kg, IP) and pimozide (0.25 mg/kg, IP). Subsequently, in the dopamine metabolism, sulpiride, pimozide, and haloperidol: 1) considerably accelerated the turnover in the dopamine metabolism in three distinct brain areas; 2) increased the levels of the DOPAC, HVA, and 3-MT even 6 h after administration, as well as at 1.5 h after administration; and 3) decreased the levels of dopamine in the nucleus accumbens at 1.5 h after administration. These results indicate that sulpiride displays a mode of action different from pimozide and haloperidol with regard to the induction of catalepsy, but not with regard to the locomotion and brain dopamine metabolism.


Effects of chronic SCH 23390 or acute EEDQ on the discriminative stimulus effects of SKF 38393. Three groups of rats (n = 8/group) were trained in a two-lever, food-reinforced drug discrimination paradigm to discriminate the D1 agonist SKF 38393 (SKF; 8.0 mg/kg, IP) from saline. After acquisition of the discrimination, the dose-response function for SKF (2.0-16 mg/kg, IP) was determined using a cumulative dosing procedure. In one group, the SKF dose-response function was redetermined 1 week after a regimen of 0.25 mg/kg of the D1 antagonist SCH 23390 (SCH), IP, once/day for 10 days, again 1 week after a second regimen of 0.5 mg/kg SCH, IP, twice/day for 10 days, and a third time after a regimen of 1.0 mg/kg SCH, IP, twice/day for 21 days. SKF dose-response functions were redetermined in a group of control rats after identical injection regimens of saline. In the third group of rats, SKF dose-response functions were redetermined 24 h after an injection of N-ethoxycarboxyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (irreversible antagonist) vehicle; again 24 h after an injection of 3.0 mg/kg; again 48 h after 6.0 mg/kg EEDQ; and finally 48 h after two consecutive daily injections of 6.0 mg/kg EEDQ (12 mg/kg total). The dose-response function for the percentage of responses that occurred on the SKF lever (%DL) shifted significantly to the left following the second regimen of SCH; there was no further shift after the third regimen. The effects of SKF on response rate were unchanged by SCH administration. Repeated administration of saline did not alter the SKF dose-response function for %DL or response rate.(ABSTRACT TRUNCATED AT 250 WORDS)


Potentiation of 2-deoxy-D-glucose antinociception, but not hyperphagia by zolantidine, a histamine (H2) receptor antagonist. Antagonism of the histamine (H2) receptor reduces antinociception induced by naloxone-resistant foot-shock, naloxone-sensitive foot-shock, and morphine with a rank-order potency similar to their H2 antagonism. The antimetabolic glucose analog 2-deoxy-D-glucose (2DG) produces antinociceptive and hyperphagic responses that dissociate from each other and are in part mediated by opioid systems. The present study determined the effects of the brain-penetrating H2 receptor antagonist zolantidine (ZOL) on 2DG antinociception on the tail-flick and jump tests, as well as on 2DG hyperphagia, in rats. ZOL (0.01-1 mg/kg) potentiated the antinociceptive responses induced by a moderate (450 mg/kg) dose of 2DG, but had lesser effects upon antinociception induced by a lower (100 mg/kg) 2DG dose. ZOL itself slightly increased jump thresholds, but not tail-flick latencies. Combinations of ZOL and 2DG produced supraadditive antinociception, even though ZOL failed to significantly shift the 2DG dose-response curve to the left. In contrast, ZOL failed to alter basal intake or 2DG hyperphagia, supporting previous evidence implicating the H1 but not the H2 receptor in these effects. These results further dissociate the antinociceptive and hyperphagic effects of 2DG, and also support previous results indicating both pro- and antinociceptive roles for H2 receptors.


Combined effects of diazepam and melatonin in two tests for anxiolytic activity in the mouse. The effects of behaviorally nonactive doses of melatonin and diazepam were investigated in two test models for anxiolytics in mice to see whether mutual enhancement could be observed when the two treatments were combined. The test models used were the four plates test and the tail suspension test. In the former test anxiolytics increase the number of punished crossings and in the latter increase the duration of immobility of mice suspended by the tail. In the four plates test combined treatment with melatonin (128 and 256 mg/kg IP) and diazepam (0.5 mg/kg PO) caused a significant increase in the number of punished crossings, whereas each treatment alone was without effect. Similarly, in the tail suspension test, a clear increase in the duration of immobility was observed after combined treatment (256 mg/kg IP melatonin + 0.5 mg/kg PO diazepam), whereas no effects were observed with the individual treatments alone. These results suggest that melatonin can enhance the anxiolytic actions of diazepam.


Memory impairment in schizophrenia: its extent, affiliations and neuropsychological character. In a sample of 60 schizophrenic patients encompassing all grades of severity and chronicity memory impairment was found to be prevalent, often substantial, and disproportionate to the overall level of intellectual impairment. The deficits were not easily attributable to poor cooperation, attention or motivation; nor were they related to neuroleptic or anticholinergic medication. Memory impairment was significantly associated with severity and chronicity of illness and also with negative symptoms and formal thought disorder. There was evidence from the sample as a whole, and from a more detailed examination of five patients with relatively isolated deficits, that schizophrenic memory impairment conformed to the pattern seen in the classical amnesic syndrome. Additionally, there was preliminary evidence for a marked deficit in semantic memory.


Movement disorders and psychological tests of frontal lobe function in schizophrenic patients. Neuropsychological tests of frontal lobe functions were undertaken in 46 chronic schizophrenic patients who were also rated for movement disorders. Tardive dyskinesia was found to have significant associations with most of these psychological tests. The possible mechanisms are discussed within the context of known neostriatal psychological functions.


Neurosteroids: endogenous bimodal modulators of the GABAA receptor. Mechanism of action and physiological significance. The abundant CNS cholesterol and its sulfate derivative serve as precursors of different neurosteroids, which bidirectionally modulate neuronal excitability, by potentiating or inhibiting function of the GABAA receptors. The regulation of GABAA receptors in the CNS by the steroids of central or peripheral origin may constitute a vital means of brain-body communication, essential for integrated whole organism responses to external stimuli or internal signals. Modulation of the brain GABA receptors by neurosteroids may form the basis of a myriad of psychophysiological phenomena, such as memory, stress, anxiety, sleep, depression, seizures and others. Therefore, the aberrant synthesis of centrally-active steroids may contribute to defects in neurotransmission, resulting in a variety of neural and affective disorders. The biosynthesis of neurosteroids may also be altered by diet and certain psychotropic drugs, thereby affecting excitation of neurons. Hereditary differences in the level of synthesis and catabolism of different neurosteroids may underlie individual variations in CNS excitability, contributing to differences in personality traits, including the inherited susceptibility to drug addition.


Glucose- and concentration-dependence of vasopressin-induced hormone release by mouse pancreatic islets. The effects of arginine-vasopressin (AVP) on hormone release by the endocrine pancreas have been studied with incubated islets from normal mice. A wide range of AVP concentrations (1 pM-100 nM) were tested in the presence of various glucose concentrations. AVP did not affect somatostatin release in a glucose-free medium but increased it in the presence of all tested glucose concentrations (3-30 mM). The lowest effective concentration was 1 mM and the effect was not yet maximal at 100 nM AVP. AVP markedly increased glucagon release in the absence of glucose. Its effect was attenuated but not abolished when glucagon release was inhibited by glucose. Surprisingly, the attenuation of the effect of AVP was stronger in 3-10 mM than in 15-30 mM glucose. The lowest effective concentration was 1 nM and the effect was not yet maximal at 100 nM AVP. AVP was ineffective on basal insulin release (0, 3 and 7 mM glucose), but potentiated the effect of 10, 15 and 30 mM glucose. The lowest effective concentration was 0.1-1 nM AVP and the maximal effect was produced by 10-100 nM AVP. The results suggest a direct action of AVP on each of the three islet cell types which display a roughly similar sensitivity to the peptide. This sensitivity is too low to make islet cells a possible target for circulating AVP under physiological conditions. On the other hand, the presence of AVP in the pancreas suggests that it might be involved in the peptidergic control of islet function.


Making sense of NSAID gastropathy and considering the therapeutic options. There is increasing recognition that nonsteroidal anti-inflammatory drugs (NSAIDs), while quite effective in treating arthritic symptoms, are associated with gastrointestinal mucosal damage. Although therapy for patients with NSAID-induced ulcers or those at high risk of developing them is still a matter of debate, the current literature supports the use of misoprostol as the only drug effective for the prevention of both NSAID-induced gastric and duodenal ulceration. It has also been shown to treat NSAID-induced gastropathy in patients continuing their NSAID therapy. In a study of misoprostol (100 or 200 micrograms QID) plus NSAID, after three months of continuous therapy, a significantly lower frequency of gastric ulcers in the misoprostol-treated group was revealed: 100 micrograms misoprostol, 5.6%; 200 micrograms misoprostol, 1.4%; placebo, 21.7%. A recent three-month, placebo-controlled study established the efficacy of misoprostol 200 micrograms QID in the prevention of NSAID-induced duodenal ulcers in 429 patients with osteoarthritis (OA), rheumatoid arthritis (RA), or other rheumatic disease, who were receiving daily treatment with various NSAIDs. The incidence of duodenal ulcer development over three months was 1.0% in patients treated with misoprostol versus 6.3% in placebo-treated patients (P = 0.004). The same trial also evaluated the efficacy of misoprostol 200 micrograms QID versus placebo in preventing NSAID-induced gastric ulcers. The incidence of gastric ulcer over the three-month treatment period was 1.5% in patients treated with misoprostol versus 9.0% in placebo-treated patients (P = 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Ablation of stimulation of a cAMP-responsive promoter in CHO cell lines defective in their cAMP-dependent protein kinase system. We have studied the requirement for an intact cAMP-dependent protein kinase (PKA) system to regulate cAMP-mediated gene transcription in Chinese hamster ovary (CHO) cells. Wild-type CHO cells and mutant CHO cell lines selected for their resistance to the growth inhibitory effect of 8-Br-cAMP and defective in their PKA system were transiently transfected with reporter plasmids containing 2.5 and 3.0 kb of the 5'-flanking sequence of the rat tyrosine aminotransferase (TAT) gene promoter. This segment of DNA contains no CRE-like sequences, yet wild-type transfectants exhibited a specific increase in TAT promoter activity following growth in medium containing 8-Br-cAMP. In CHO cell lines defective in their PKA, the transfected TAT promoter failed to respond to cAMP treatment. We conclude that an intact PKA system is necessary for the cAMP-mediated increase in TAT promoter activity in CHO cells and that there is no requirement for a CRE to see this effect.


A high frequency of the A30, B18, DR3, DRw52, DQw2 extended haplotype in Sardinian celiac disease patients: further evidence that disease susceptibility is conferred by DQ A1*0501, B1*0201. This study characterizes by serological and molecular methods the HLA class I and class II alleles in a group of celiac disease children, their parents and a control group of Sardinian descent. We found the DR3-DQw2 haplotype in all patients which was, in almost all cases (84%), associated with the HLA-A30, B18, DR3, DRw52, DQw2 extended haplotype named "Sardinian haplotype" because of its frequency (12-15%) in this Caucasian population. This is the first time that this DQw2-linked haplotype has been reported with such a high frequency in CD. However, no different distribution of "Sardinian haplotype" was found comparing CD patients with 91 haplotyped DQw2-positive controls. This finding indicates that the DQw2 antigen in Sardinians is almost always associated with the A30, B18, DR3, DRw52, DQw2 extended haplotype. The DQA1 and DQB1 second exon sequence analysis of the B18,DR3 and B8,DR3 haplotypes showed the DQA1*0501 and DQB1*0201 alleles which shared the already published sequences. DPB1 subtyping showed the DPB1*0301 allele more frequently (p less than 0.005) in CD patients but this difference was no longer significant when patients and controls, both heterozygous for the DR3-DQw2 haplotype, were compared. We suggest that the divergent HLA extended haplotypes and DP allele associated with CD, described in different Caucasian populations, can be explained by the particular DQw2 linkage disequilibrium in each population.


Developments in the drug treatment of schizophrenia. Despite its efficacy in many cases, the drug treatment of schizophrenia remains problematic. A substantial proportion of patients do not improve, and many others suffer from unpleasant side-effects. In this review, Gavin Reynolds describes the new approaches to antipsychotic drug development that attempt to address these problems, and relates some of these approaches to growing evidence for neuronal pathology in the brain in schizophrenia.


Induction of protective class I MHC-restricted CTL in mice by a recombinant influenza vaccine in aluminium hydroxide adjuvant. Induction of class I MHC-restricted cytotoxic T lymphocyte (CTL) responses by soluble proteins or peptides requires complex adjuvants or carrier systems which are not licensed for use with human vaccines. The data presented in this report show that vaccination with a highly purified recombinant influenza protein antigen in aluminium hydroxide adjuvant, the only adjuvant currently licensed for clinical use, elicited class I restricted CTL and protection from lethal challenge with H1N1 and H2N2 viruses. The antigen (D protein, SK&F 106160) is produced by expression of H1N1 influenza virus-derived cDNA (strain A/PR/8/34) in Escherichia coli, and is composed of the first 81 N-terminal amino acids (aa) of the non-structural protein 1 (NS1) fused via a nine nucleotide non-viral linker sequence to the 157 C-terminal aa of the haemagglutinin 2 subunit (HA2). Previous work by Kuwano et al demonstrated that in vitro stimulation of spleen cells from influenza virus-primed mice, with a partially purified preparation of the D protein, selected for CD8+ CTL clones which facilitated lung clearance of H1N1 and H2N2 viruses. In the current study, these results were extended by studying the responses of mice actively immunized with highly purified D protein in the presence or absence of adjuvants. Vaccination of CB6F1 (H-2dxb) mice with D protein in aluminum hydroxide or Freund's complete adjuvant generated H1N1 cross-reactive, H-2d-restricted, CD8+ CTL directed against an immunodominant HA2 epitope (aa 189-199). D protein without adjuvant did not elicit CTL, regardless of the route of injection. However, long-lived (greater than 6 months) splenic memory CTL were elicited by boosting mice intraperitoneally (i.p.) with the D protein in the absence of adjuvant. In mice injected subcutaneously with D protein in aluminium hydroxide at weeks 0 and 3, survival was increased relative to controls up to 16 weeks beyond the second vaccination, after which time additional boosting was required for protection. Studies in H-2b and H-2k mice vaccinated with the D protein showed that induction of CD4+ T-cell or antibody responses, in the absence of CD8+ CTL, did not correlate with protection. Passive transfer of immune sera from CB6F1 mice was also not protective. This prototype H1N1 recombinant subunit vaccine in aluminium adjuvant should directly address the feasibility of achieving a protective cell-mediated immune response in human influenza.


Characterization of mutants of the yeast Yarrowia lipolytica defective in acetyl-coenzyme A synthetase. The expression of the glyoxylate cycle enzymes is required for growth of the yeast Yarrowia lipolytica on acetate or fatty acids as sole carbon source. Acetyl-coenzyme A, which is produced by acetyl-coenzyme A synthetase (ACS) from acetate, is needed for induction of this expression. Acetate-non-utilizing mutants of this yeast were investigated in order to identify mutants which express no or strongly reduced activity of this enzyme. Mutations in gene ICL2 exhibited the strongest effects on the activity. In icl2 mutants, lack of ACS activity resulted in a non-induced glyoxylate cycle on acetate; however, induction on fatty acids was not affected. Gene ICL2 was identified as the structural gene encoding the monomer of ACS. It is shown that a high level of ACS activity is necessary for full expression of the glyoxylate cycle enzymes. Mutations in gene ICL1, which encodes isocitrate lyase, resulted in overproduction of ACS without any growth on acetate. A new gene (GPR1 = glyoxylate pathway regulation) was detected in which trans-dominant mutations inhibit expression of ACS and the glyoxylate cycle on acetate as carbon source.


The effect of selective beta 1-blockade on glucose thresholds for release of counterregulatory hormones and symptoms in insulin-dependent diabetes mellitus. To evaluate the effect of beta 1-blockade (metoprolol) on the plasma glucose thresholds initiating counterregulatory hormone responses and symptoms of hypoglycemia, we used a modified glucose clamp technique to produce a standardized gradual glucose decline from 5.0 to 2.0 mmol/l in nine patients with insulin-dependent diabetes mellitus (IDDM) (HbAlc range 6.7-10.3%, duration of diabetes 5-18 years, autonomous neuropathy present in three of the patients). The responses were studied once with metoprolol and once with placebo, in random order. With the beta 1-selective blockade, epinephrine release was triggered at a significantly higher (p less than 0.02) plasma glucose level (3.5 mmol/l) than it was with placebo (3.0 mmol/l). Metoprolol did not change thresholds for growth hormone (3.7/3.5 mmol/l), cortisol (2.9/2.9 mmol/l), glucagon (2.8/2.8 mmol/l) or for pancreatic polypeptide (2.8/2.7 mmol/l). The peak responses of epinephrine and growth hormone were significantly higher (p less than 0.01) with the beta 1-blockade. Metoprolol did not change the thresholds for neuroglycopenic and autonomic symptoms. Six out of the seven patients who answered yes to having hypoglycemia did so at a higher blood glucose with metoprolol than without. In our study, the beta 1-selective blockade altered the responses of counterregulatory hormones, but it did not change the thresholds for hypoglycemic symptoms.


Colocalization of prion protein and beta protein in the same amyloid plaques in patients with Gerstmann-Sträussler syndrome. We examined paraffin-embedded brain sections from three patients with Creutzfeldt-Jakob disease (CJD) and four patients with Gerstmann-Sträussler syndrome (GSS) who also had beta protein deposits in the brains. Immunostaining using anti-prion protein (PrP) and anti-beta protein coupled with formic acid pretreatment, revealed PrP deposits and beta protein deposits, respectively. In all four GSS patients examined, sequential double immunostaining and single immunostaining in serial sections or simultaneous double immunofluorescence revealed the colocalization of PrP and beta protein in the same amyloid plaques. The plaques labeled with both antibodies were designated as beta-PrP plaques. Small kuru plaques of less than 15 microns in diameter were rarely found to coexist with beta deposits. The percentages of beta-PrP plaques in larger kuru plaques were not constant among the four GSS patients. The colocalization patterns of both deposits were observed as being roughly of two types as follows: (1) diffuse beta protein deposits located around the PrP core; and (2) a beta protein core and PrP core simultaneously existing in one amyloid plaque. Under an electron microscope, we were able to confirm the presence of both beta protein and PrP in a single plaque in four GSS patients older than 60 years old. In contrast, no colocalization of either deposits was seen in the amyloid plaque core fractions of a young GSS patient who had no beta protein deposits, even at the electron microscopic level. Therefore, the colocalization of both proteins in a single plaque is believed to be age-related and incidental in GSS patients but suggests a similar morphogenesis of both amyloid deposits.


Proliferating cell nuclear antigen counts in formalin-fixed paraffin-embedded tissue correlate with Ki-67 in fresh tissue. Cell proliferation can be studied by a variety of techniques. However, most require fresh tissue. To evaluate cell proliferation in formalin-fixed paraffin-embedded sections, the authors immunohistochemically studied 35 tumors and 11 samples of normal/hyperplastic tissue with PC10, a monoclonal antibody directed against proliferating cell nuclear antigen. Results were compared with those obtained with Ki-67 on fresh tissues. There was no significant difference between proliferating cell nuclear antigen and Ki-67 counts, which were strongly correlated (r = 0.8). Proliferating cell nuclear antigen positivity was easier to evaluate because morphology was better preserved in formalin-fixed tissue. The authors conclude that PC10 is an alternative to Ki-67 in evaluating cell proliferation and has the advantage of reacting with formalin-fixed paraffin-embedded tissue.


Frosted branch angiitis associated with cytomegalovirus retinitis. We examined three patients with acquired immunodeficiency syndrome who had frosted branch angiitis associated with small patches of cytomegalovirus retinitis. Each patient had a low CD4-helper T-lymphocyte count and a T-lymphocyte helper-suppressor ratio of less than 0.1. Treatment with intravenous anticytomegalovirus antibiotics caused the vascular sheathing to resolve within two weeks in all three patients, but each patient continued to have a smoldering retinitis. Retinal biopsy in one of the patients demonstrated virions whose morphologic characteristics were consistent with cytomegalovirus on electron microscopy and the identity of which was confirmed by immunohistochemistry. Although frosted branch angiitis in otherwise healthy patients responds to corticosteroids, similar treatment with corticosteroids for frosted branch angiitis associated with cytomegalovirus retinitis in patients with AIDS does not seem to be indicated. Before corticosteroid treatment is started for a patient with the clinical signs and symptoms of frosted branch angiitis, careful medical examination of the patient is necessary.


The debate on treating individuals incompetent for execution. The question of whether to provide mental health treatment to prisoners under death sentence who have been judged incompetent for execution presents a powerful ethical dilemma for mental health professionals. Arguments that favor or oppose the provision of treatment are discussed in the context of the nature of the disorder to be treated, the type of treatment to be provided, the goals of treatment, and the relevant legal standard for determining competency for execution. Arguments against treating the incompetent include 1) the need to avoid harming those who are treated, 2) the risk that disclosures in therapy will be used for assessment purposes, 3) the need for paternalism when sufficient harm is necessary, 4) the adverse impact on the clinician, 5) the potential undermining of patient and public perceptions of mental health professionals, and 6) the poor allocation of limited resources. Arguments for treating the incompetent include 1) respect for the wishes of the prisoner, 2) the need to clarify the values underlying the refusal to treat, 3) the low risk of harm from some forms of treatment, and 4) the adverse impact on the milieu stemming from failure to treat. The authors conclude that treating incompetent prisoners may not violate ethical standards under some circumstances, and that some forms of treatment will require the informed consent of the prisoner.


Randomized, double-blind, crossover, placebo-controlled comparison of propranolol and betaxolol in the treatment of neuroleptic-induced akathisia. Beta-blocking agents, particularly propranolol, are considered effective in the treatment of neuroleptic-induced akathisia, but considerable controversy exists about the involved receptor subtype(s). The authors conducted a randomized, controlled trial comparing the effects of propranolol and betaxolol to determine whether central beta 1-adrenoceptor blockade is sufficient to correct neuroleptic-induced akathisia. The subjects were 19 patients whose neuroleptic-induced akathisia responded to 20 mg/day of propranolol and subsequently reemerged during a placebo washout period. They were randomly assigned to propranolol (20 or 40 mg/day) or betaxolol (10 or 20 mg/day) and, after another placebo period, were switched to the second beta blocker. There was no significant difference in the antiakathisia effects of propranolol and betaxolol. The lack of difference between propranolol and betaxolol suggests that beta 1-adrenoceptor blockade is sufficient to improve neuroleptic-induced akathisia. The results of this explanatory study need therapeutic confirmation.


Changes in chronic nightmares after one session of desensitization or rehearsal instructions. The purpose of this study was to examine the effects of one session of instructions on the frequency of chronic nightmares and on self-rated distress. Twenty-eight volunteers with chronic nightmares (mean duration = 19 years) were treated with either one session of desensitization with instructions on how to practice this treatment or with one session of instructions to change the nightmare and how to rehearse the new version. The authors administered four scales of the SCL-90 and the corresponding scales of the Symptom Questionnaire. At 7-month follow-up of 23 patients, there was a significant reduction in the frequency of nightmares and significant decreases in self-rated depression, anxiety, and hostility. There were no significant differences between the effects of the two types of treatment. In four patients, whose mean duration of nightmares was 23 years, the nightmares ceased. The results of this preliminary study suggests that the instructions given to the patients reduced the frequency of their chronic nightmares and decreased their self-rated distress.


Weight gain among schizophrenic patients treated with clozapine. A retrospective chart review was used to assess weight changes in 36 chronic schizophrenic inpatients who were treated with clozapine after being treated with standard neuroleptics. The average weight gain during 6 months of clozapine treatment was 16.9 lb; 75.0% of the patients gained at least 10 lb. The results confirm previous findings of clozapine-associated weight gain.


Knowledge and beliefs about malaria on the Pacific coastal plain of Guatemala. Surveys of residents of the Pacific coast of Guatemala revealed a lack of knowledge and many misconceptions about the transmission and treatment of malaria, which could adversely affect malaria control measures and antimalarial therapy. Although mosquitoes are known to play an important role in malaria transmission and are thought to become infected by biting individuals with malaria, 75% of people interviewed believe that the mosquitoes can also acquire infections from contaminated water or by biting snakes and frogs. Furthermore, most residents believe that malaria can be acquired in other ways, such as by bathing too frequently or by drinking unboiled water. Although self-treatment of malaria with oral and injectable drugs purchased at stores and pharmacies is very common, less than 10% of the respondents were aware of the correct curative dose of chloroquine. Chloroquine injections are preferred to tablets and believed to be approximately three times as potent as tablets of the same concentration. Nearly two-thirds of the interviewees believed that pregnant and lactating women with malaria should avoid the use of chloroquine because it may cause a spontaneous abortion or dry up breast milk. Similar surveys of National Malaria Service workers and village malaria workers revealed higher levels of knowledge, although the village workers had many misconceptions about malaria transmission. An educational campaign directed at correcting some of these misconceptions should result in more appropriate self-treatment of malaria and greater acceptance by residents of personal protection methods and vector control and drug treatment programs.


Chromogranin A immunoreactivity and Grimelius' argyrophilia. A correlative study in mammalian endocrine cells. Various endocrine cells can be stained by the argyrophil reaction of Grimelius. This silver stain has recently been attributed to chromogranin A, an acidic glycoprotein, that is present in many endocrine cells. Using serial sections of plastic-embedded tissues (adrenal medulla, pancreas, gastric mucosa) various endocrine cells were investigated for their content of chromogranin A immunoreactivity and for their argyrophilia. The findings in four species (man, cattle, pig, guinea pig) showed that chromogranin A immunoreactivity and argyrophil stain partly overlap in identical endocrine cells, but do not necessarily coincide in the majority of endocrine cells. We found that endocrine cells could be positive for chromogranin A and argyrophilia (e.g., aminergic endocrine cells); or positive for chromogranin A but negative for argyrophilia (e.g., insulin cells of all species; somatostatin cells of cattle and pig); or negative for chromogranin A but positive for argyrophilia (e.g., glucagon cells of pig and guinea pig); or negative for chromogranin A and argyrophilia (e.g., somatostatin cells of man and guinea pig). Such heterogeneities of the staining pattern for chromogranin A and argyrophil silver reaction were also observed in individual endocrine cells of a given population (e.g., gastrin cells). Hence, although recent dot-blot tests have shown that chromogranin A is an argyrophilic substance, in tissue sections chromogranin A immunostaining and Grimelius' silver staining did not coincide in various endocrine cells, for unknown reasons. Therefore, it is recommended to use both chromogranin A immunohistochemistry and the classical Grimelius' silver stain to "mark" that vast majority of endocrine cells in tissue sections.


Anesthetic properties of riluzole (54274 RP), a new inhibitor of glutamate neurotransmission. It has been suggested that some anesthetic agents could exert their hypnotic/anesthetic effects by selectively blocking receptors involved in the central excitatory neurotransmission mediated by glutamate. In the present study, we analyzed whether riluzole (54274 RP), a novel compound that inhibits both the release and some postsynaptic effects of glutamate in some brain structures, has anesthetic properties in rats. For this purpose, we investigated whether 1) riluzole administered intraperitoneally (ip) at doses ranging from 2.5 to 45 mg/kg induces loss of righting reflex (LRR); 2) riluzole (2.5 and 5 mg/kg) prolongs sleep-times induced by either ketamine (30 or 80 mg/kg ip) or thiopental (25 or 35 mg/kg ip); 3) a 5-mg/kg subanesthetic riluzole dose affects the minimum alveolar concentration of halothane (MACh). Onset of drug action was defined as the period of time from the ip injection to LRR. Sleep-time was considered the period of time from LRR to restoration of righting reflex. Riluzole at doses greater than 15 mg/kg was able to induce LRR (riluzole dose for which LRR was achieved in 50% of the rats [ED50 = 25.6 mg/kg]). A positive correlation was found between the dose of riluzole and sleep-time (r = 0.92, P less than 0.001). A 5-mg/kg (but not 2.5-mg/kg) riluzole dose significantly prolonged sleep-times induced by both ketamine (30 and 80 mg/kg) and thiopental (25 but not 35 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)


Does impairment of energy metabolism result in excitotoxic neuronal death in neurodegenerative illnesses? The etiology of nerve cell death in neuronal degenerative disease is unknown, but it has been hypothesized that excitotoxic mechanisms may play a role. Such mechanisms may play a role in diseases such as Huntington's disease, Parkinson's disease, amyotropic lateral sclerosis, and Alzheimer's disease. In these illnesses, the slowly evolving neuronal death is unlikely to be due to a sudden release of glutamate, such as occurs in ischemia. One possibility, however, is that a defect in mitochondrial energy metabolism could secondarily lead to slow excitotoxic neuronal death, by making neurons more vulnerable to endogenous glutamate. With reduced oxidative metabolism and partial cell membrane depolarization, voltage-dependent N-methyl-D-aspartate (NMDA) receptor ion channels would be more easily activated. In addition, several other processes involved in buffering intracellular calcium may be impaired. Recent studies in experimental animals showed that mitochondrial toxins can result in a pattern of neuronal degeneration closely resembling that seen in Huntington's disease, which can be blocked with NMDA antagonists. NMDA antagonists also block neuronal degeneration induced by 1-methyl-4-phenylpyridium, which has been implicated in experimental models of Parkinson's disease. The delayed onset of neurodegenerative illnesses could be related to the progressive impairment of mitochondrial oxidative phosphorylation, which accompanies normal aging. If defective mitochondrial energy metabolism plays a role in cell death in neurodegenerative disorders, potential therapeutic strategies would be to use excitatory amino acid antagonists or agents to bypass bioenergetic defects.


Genetic association and linkage analysis of the apolipoprotein CII locus and familial Alzheimer's disease. We previously reported a genetic association between the 3.5 kb (F) Taq I restriction fragment length polymorphism allele of the apolipoprotein CII gene on chromosome 19 and familial Alzheimer's disease. Here, we report an additional analysis of this association performed on an expanded and better defined data set of 23 families with familial Alzheimer's disease. The F allele frequency in affected family members in the expanded set was 0.62 +/- 0.06 (mean +/- standard error, n = 51 subjects), which differed significantly from a frequency of 0.39 +/- 0.02 (n = 226) for unrelated control subjects (Z = 3.75, p less than 0.0002). These results are consistent with our previous findings and suggest an association between the F allele of apolipoprotein CII and familial Alzheimer's disease. When the apolipoprotein CII locus was tested for linkage to familial Alzheimer's disease, LOD scores summed for the complete group of families were negative and close linkage was excluded. Close linkage was also excluded for early-onset families (mean onset age less than or equal to 60 years), but small positive LOD scores were obtained for late-onset kindreds.


Soluble methane monooxygenase component B gene probe for identification of methanotrophs that rapidly degrade trichloroethylene. Restriction fragment length polymorphisms, Western blot (immunoblot) analysis, and fluorescence-labelled signature probes were used for the characterization of methanotrophic bacteria as well as for the identification of methanotrophs which contained the soluble methane monooxygenase (MMO) gene and were able to degrade trichloroethylene (TCE). The gene encoding a soluble MMO component B protein from Methylosinus trichosporium OB3b was cloned. It contained a 2.2-kb EcoRI fragment. With this cloned component B gene as probe, methanotroph types I, II, and X and environmental and bioreactor samples were screened for the presence of the gene encoding soluble MMO. Fragments produced by digestion of DNA with rare cutting restriction endonucleases were separated by pulsed-field gel electrophoresis and transferred to Zeta-Probe membrane (Bio-Rad) for Southern blot analysis. Samples were also analyzed for the presence of soluble MMO by Western blot analysis and the ability to degrade TCE. The physiological groups of methanotrophs in each sample were determined by hybridizing cells with fluorescence-labelled signature probes. Among twelve pure or mixed cultures, DNA fragments of seven methanotrophs hybridized with the soluble MMO B gene probe. When grown in media with limited copper, all of these bacteria degraded TCE. All of them are type II methanotrophs. The soluble MMO component B gene of the type X methanotroph, Methylococcus capsulatus Bath, did not hybridize to the M. trichosporium OB3b soluble MMO component B gene probe, although M. capsulatus Bath also produces a soluble MMO.


Immunochemical characterization of rat testicular asparagine synthetase. We studied immunochemical properties of rat testicular asparagine synthetase. Western blot analysis of testis extract with polyclonal antibody raised against purified asparagine synthetase revealed an immunoreactive band at 62 kDa. The pancreas, brain, thymus, and spleen also showed 62-kDa bands. The intensities of these bands were roughly proportional to the specific activities of the enzyme in these tissues. The antibody showed some degree of cross-reactivity to asparagine synthetases from human, beef, pig, mouse, guinea pig, chicken, and frog, but not carp. But the enzyme from human HL-60 cells and lower vertebrates reacted with the antibody less strongly than enzyme from rats. The N-terminal amino acid sequence of the enzyme, determined by the Edman degradation method, in 10 recovered residues was identical to that of human asparagine synthetase deduced from corresponding cDNA (I.L. Andrulis et al., 1987, Mol. Cell. Biol. 7, 2435-2443). Immunohistochemical staining of the testis showed the presence of asparagine synthetase mainly in Sertoli cells in the seminiferous tubules.


Purification and characterization of chaperonin 10 from Chromatium vinosum. Chromatium vinosum contains a polypeptide that is functionally and structurally similar to the Escherichia coli chaperonin 10. The protein has been purified to homogeneity by sucrose density gradient centrifugation followed by gel filtration using a Bio-Gel A-1.5 m column. The molecular mass of chaperonin 10, as determined by gel filtration or nondenaturing polyacrylamide gel electrophoresis, is 95 kDa. The oligomer is composed of seven or eight subunits. Comparisons of the overall amino acid composition and N-terminal sequences among chaperonin 10 species from C. vinosum and E. coli reflect a high degree of similarity. A physical association between chaperonins 60 and 10 from C. vinosum, in vitro, is supported by three experimental approaches. First, the proteins form a stable binary complex in sucrose density gradients, gel filtration chromatography, and nondenaturing polyacrylamide gel electrophoresis, solely in the presence of ATP and Mg2+. Second, chaperonin 10 from C. vinosum binds, selectively, to a chaperonin 60-coupled Affi-Gel 10 matrix column. Third, a slight molar excess of chaperonin 10 is able to abolish, almost completely, the ATPase in chaperonin 60. The rate for ATPase activity of chaperonin 60 from C. vinosum is enhanced when supplemented with monovalent cations.


Primary adenocarcinoma of third and fourth portions of duodenum. Favorable prognosis after resection. Duodenal adenocarcinoma, a rare malignant lesion, is associated with a poor 5-year survival. Few series have addressed differences between resectable tumors of the proximal and distal duodenum. We reviewed records of 17 consecutive patients with adenocarcinoma of the duodenum who underwent resection: 10 had adenocarcinoma of the proximal duodenum, and seven had tumors of the distal duodenum. Most patients underwent pancreatoduodenectomy. Five patients with adenocarcinoma of the distal duodenum underwent segmental resection. No perioperative deaths occurred. Six of 10 patients with proximal tumors died of metastatic disease. Of the seven patients with tumors of the distal duodenum, five are alive without evidence of disease, and two died of unrelated causes. The survival of patients with adenocarcinoma of the distal duodenum is surprisingly good, and segmental resection is the procedure of choice.


Serum lipids, hepatic glycerolipid metabolism and peroxisomal fatty acid oxidation in rats fed omega-3 and omega-6 fatty acids. Rats were fed, for 3 weeks, high-fat (20% w/w) diets containing sunflower-seed oil, linseed oil or fish oil. Chow-fed rats were used as a low-fat reference. The high-fat diets markedly reduced non-fasting-rat serum triacylglycerol as compared with the low-fat reference, and the highest reduction (85%) was observed with the fish-oil group, which was significantly lower than that of the other high-fat diets. The serum concentration of phospholipids was significantly reduced (30%) only in the fish-oil-fed animals, whereas serum non-esterified fatty acids were reduced 40-50% by both the fish-oil- and linseed-oil-fed groups. The liver content of triacylglycerol showed a 1.7-fold increase with the fish-oil diet and 2-2.5-fold with the other dietary groups when compared with rats fed a low-fat diet, whereas the hepatic content of phospholipids was unchanged. Peroxisomal fatty acid oxidation (acyl-CoA oxidase) was 2-fold increased for the rats fed fish oil; however this was not significantly higher when comparison was made with rats fed the linseed-oil diet. There was no difference in phosphatidate hydrolysis (microsomal and cytosolic fractions) among animals fed the various diets. Acyl-CoA:diacylglycerol acyltransferase activity was increased by all high-fat diets, but the fish-oil-diet-fed group showed a significantly lower enzyme activity than did rats fed the other high-fat diets. A linear correlation between acyl-CoA:diacylglycerol acyltransferase activity and liver triacylglycerol was observed, and the microsomal enzyme activity was decreased 40-50% by incubation in the presence of eicosapentaenoyl-CoA. CoA derivatives of arachidonic, linolenic and linoleic acid had no inhibitory effect when compared with the control. These results indicate that dietary fish oil may have greater triacylglycerol-lowering effect than other polyunsaturated diets, owing to decreased triacylglycerol synthesis caused by inhibition of acyl-CoA:diacylglycerol acyltransferase. In addition, increased peroxisomal fatty acid oxidation and decreased availability of non-esterified fatty acids could also contribute by decreasing the amounts of fatty acids as substrates for triacylglycerol synthesis and secretion.


Synergistic action of tiazofurin and difluorodeoxycytidine on differentiation and cytotoxicity. Tiazofurin (TR), an inhibitor of IMP dehydrogenase, causes remissions and induced differentiation in human leukemia through lowering the concentrations of GTP and dGTP. A deoxycytidine analog, difluorodeoxycytidine (DFDC), is an anti-tumor agent phosphorylated by deoxycytidine kinase, resulting in decreased concentration of dCTP, leading to inhibition of DNA synthesis. In HL-60 cells DFDC induced differentiation and inhibited proliferation in a dose-dependent manner (IC50 = 4 nM); TR provided synergism with DFDC. DFDC inhibited proliferation in OVCAR-5 human ovarian carcinoma cells (IC50 = 25 nM) and colony formation in PANC-1 human pancreatic carcinoma cells (IC50 = 2 nM) and rat hepatoma 3924A cells (IC50 = 22 nM). TR and DFDC are synergistically cytotoxic in hepatoma cells and additive in PANC-1 cells. The two drugs together should be helpful in treating leukemias and solid tumors in humans.


Analysis of carrot genes for proliferating cell nuclear antigen homologs with the aid of the polymerase chain reaction. We designed a polymerase chain reaction-based strategy to obtain information about the origin and distribution of a newly discovered proliferating cell nuclear antigen (PCNA) homolog. Carrot genomic segments were amplified using degenerate primers for two conserved regions of known PCNA homologs. The genes encoding the larger PCNA as well as typical PCNA contained introns. Thus, unlike processed PCNA pseudogenes in mammals, the larger homolog is not generated through reverse transcription of a typical PCNA mRNA. Moreover, introns of the larger PCNA homolog were positioned at the characteristic sites in plant PCNA genes. Attempts to amplify cDNA for an additional PCNA homolog from mammalian cells have been unsuccessful. These results suggest that the larger PCNA homolog was generated, presumably through gene duplication, after the divergence of the Planta and Animalia.