Cellular recovery kinetics for postirradiation treatments. Postirradiation cellular recovery kinetics for delayed plating, araA, hypertonic saline (HS) treatments and their combinations, were analysed in the framework of the previously developed dsb model (Ostashevsky 1989). Two possible types of DNA double-strand break (dsb) repair kinetics were considered: cooperative, where the rate-limiting step is related to a DNA molecule, and non-cooperative, where the rate-limiting step is related to a dsb. The following estimates were made for plateau-phase V79 cells: (1) HS induces a temporary block of dsb repair. The estimates for the dsb repair block duration are about 0.6 h for a 10-min treatment and about 1.2 h for a 20-min treatment. (2) HS creates additional DNA fragments. The number of these fragments calculated for 0.37 M NaCl treatment, in a way independent of the type of dsb repair, was consistent with the number estimated for 0.5 M NaCl treatment for cooperative dsb repair, but not for noncooperative dsb repair. (3) The estimated time constant for the process restricting the formation of DNA fragments was 10-20 min. In the presence of 200-400 microM araA, the rate of this process should decrease 2-5-fold. (4) The time constant for the araA recovery kinetics coincides with that for dsb repair, in agreement with experimental data. (5) For delayed plating recovery the kinetics curve calculated for cooperative dsb repair fits well experimental data, in contrast to the curve calculated for non-cooperative dsb repair.


Induction of thermotolerance by chemical agents in enucleate erythrocytes. Erythrocytes treated with various chemical agents for 1 h at 37 degrees C showed resistance to a subsequent 1 h heat treatment at 53 degrees C. Maximal thermotolerance was observed 6 h after 3 mM DNP and 0.03 mM disulfiram treatment and 4 h after diamide exposure at 0.3 mM. Our results suggest that chemically induced thermotolerance to heat treatment in erythrocytes was similar to heat-induced thermotolerance.


Molecular cloning and sequencing of the upstream region of the major Bacillus subtilis autolysin gene: a modifier protein exhibiting sequence homology to the major autolysin and the spoIID product. The upstream region of the N-acetylmuramoyl-L-alanine amidase gene (cwlB; a major Bacillus subtilis autolysin) was cloned into Escherichia coli by chromosome walking. Sequencing of the region showed the presence of two open reading frames, one (designated as cwbA) which starts at a UUG codon and encodes a polypeptide of 705 amino acids with an M(r) of 76,725, and the other (designated as lppX), upstream of cwbA, comprising 102 amino acids and having a signal sequence characteristic of a lipoprotein. Purification of the CwbA protein and determination of its N-terminal amino acid sequence revealed that it contains a presumed signal peptide which is processed after Ala at position 25 from the N-terminal, and that the M(r) of the mature form is 75,000. The amino acid sequences of the N-terminal and C-terminal regions of CwbA were found to be highly homologous with those of the cell wall binding domain of CwlB and the spoIID gene product, respectively. CwbA stimulated the major autolysin activity approximately threefold in vitro. These data indicate that CwbA is the modifier protein of the major autolysin reported by Herbold, D. R. & Glaser, L. (1975; Journal of Biological Chemistry 250, 1676-1682). In-frame fusion between the lppX and lacZ genes demonstrated that lppX is translated in vivo and expressed during the exponential growth phase.


Differentiation of mosquito-pathogenic strains of Bacillus sphaericus from non-toxic varieties by ribosomal RNA gene restriction patterns. DNA from 17 strains of Bacillus sphaericus, including representatives of all the established DNA homology groups, was cleaved with EcoRI or HindIII and fragments were separated by agarose gel electrophoresis. Southern blots of this DNA were hybridized to a radioactively labelled DNA probe prepared from the cloned 16S rrnB ribosomal RNA operon of Escherichia coli. Banding patterns of the chromosomal DNA digests and the autoradiograms were specific to DNA homology groups I (B. sphaericus sensu stricto), IIA (mosquito-pathogenic strains), IIB (B. fusiformis) and V, but groups III and IV were not clearly distinguished. This suggests that the mosquito-pathogenic strains represent a separate subspecies.


Clonal variation of chromosome size derived from the rDNA cluster region in Candida albicans. Of the eight Candida albicans chromosomes, chromosome 2, assigned by the MGL1 probe, is more variable in size than the other chromosomes among strains. We found that the clonal variation of chromosome 2, which carries a rDNA gene, occurred at a frequency of up to 10% of the progeny clones. After total chromosomal digestion with XhoI, which has no recognition sites within the rDNA repeat unit, the fragments containing the rDNA cluster were detected by Southern hybridization. The difference in fragment sizes corresponded to the clonal size variation of chromosome 2. The intensity of hybridization with rDNA also correlated with the difference in size. In addition, there was no size change in the non-rDNA region as detected by NotI digestion of chromosome 2, and there was no observed change in the individual rDNA basic repeat unit size. From these lines of evidence, we confirmed that the clonal size variation of chromosome 2 which occurs at high frequency is derived from the size change of the rDNA cluster.


Purification and some properties of glutamate dehydrogenase and glutamine synthetase from Paracoccus denitrificans. The purification and some properties of NADP-dependent glutamate dehydrogenase (GDH) and glutamine synthetase (GS) from the facultatively anaerobic Gram-negative bacterium Paracoccus denitrificans were investigated. The enzymes were purified to homogeneity using a procedure which involved affinity chromatography on Blue Sepharose CL-6B as the major purification step. The recoveries in the purification of GDH and GS were 28% and 64%, respectively. The specific activity of purified GDH was 183 nkat (mg protein)-1 (deaminating reaction). GDH was composed of subunits of molecular mass 47 kDa and the native enzyme was either a tetramer or hexamer. The apparent Km values for L-glutamate, NADP, 2-oxoglutarate, NADPH and ammonia were 1.5 mM, 5.9 microM, 0.47 microM, 12.5 microM and 14 mM, respectively. The specific activity of purified GS was 1125 nkat (mg protein)-1 (transferase reaction). The molecular mass of native GS was 570 kDa; it was composed of 12 subunits of molecular mass 50.1 kDa. The apparent Km values for L-glutamine and hydroxylamine in the transferase reaction were 2.1 and 2.4 mM, respectively; those of ammonia, L-glutamate and ATP in the biosynthetic reaction were 0.03, 1 and 0.17 mM, respectively. After the adenylylation of GS, the Km for L-glutamine and L-glutamate increased and reached the values of 8.0 and 27 mM, respectively. The effects of the changes in GS activity on the ammonia metabolism of Paracoccus denitrificans are discussed.


NE/DA interactions in prefrontal cortex and their possible roles as neuromodulators in schizophrenia. The monoaminergic innervation of the rat prefrontal cortex arises from well-defined mesencephalic nuclei, with noradrenergic (NE) neurons located in the locus coeruleus, dopaminergic (DA) neurons located in the ventral tegmental area, and serotonergic (5-HT) neurons originating in the raphe nuclei. Specific destruction of the NE bundle was found to induce morphological (i.e., sprouting) as well as metabolic (i.e., changes in rate of DA utilization) modifications of mesocortical DA neurons, suggesting that these two catecholaminergic systems have functional interactions within the prefrontal cortex. This was substantiated by experiments showing that DA afferents modulate the sensitivity of cortical post-synaptic beta-adrenergic receptors and that, reciprocally, NE neurons control the sensitivity of cortical D1 receptors. Behavioural and pharmacological data have further indicated that the stimulation of cortical alpha-1 adrenergic receptors inhibits cortical DA transmission at D1 receptors. Secondly, we have attempted to analyze how such interactions between neuromodulatory systems may be related to the development of mental diseases such as schizophrenia. On the basis of studies in the literature describing the effects produced by the ingestion of hallucinogenic drugs or data collected regarding REM sleep, it is postulated that two modes of brain functioning exist: analogical and cognitive. Each mode is characterized by differences in the relative activities of NE, DA and 5-HT neurons. At birth, during REM sleep, and following the ingestion of hallucinogens, the mode of brain functioning is essentially analogical; in contrast, both analogic and cognitive modes are postulated to coexist in the awake state. Oscillations between these two modes are under the control of monoaminergic systems on which an increase in cortical DA release favours the cognitive processing mode, whereas intermittent activations of NE neurons would switch the brain into the analogical mode of processing. It is proposed that schizophrenic patients with "positive" symptoms suffer from an abnormal preponderance of the analogical mode while awake, whereas "negative" symptoms are due to the excessive presence of the cognitive mode. Although pure biological deficits cannot be excluded, these dysfunctions could be related to the absence of particular environmental variables early in the development of these patients. This condition is probably required to establish normal regulatory control of monoaminergic neuronal activity.


The depolarization block hypothesis of neuroleptic action: implications for the etiology and treatment of schizophrenia. Antipsychotic drugs are known to block dopamine receptors soon after their administration, resulting in an increase in dopamine neuron firing and dopamine turnover. Nonetheless, antipsychotic drugs must be administered repeatedly to schizophrenics before therapeutic benefits are produced. Recordings from dopamine neurons in rats have revealed that chronic antipsychotic drug treatment results in the time-dependent inactivation of dopamine neuron firing via over-excitation, or depolarization block. Furthermore, the clinical profile of the response to antipsychotic drugs appears to correspond to the dopamine system affected: antipsychotic drugs that exert therapeutic actions in schizophrenics inactivate dopamine neuron firing in the limbic-related ventral tegmental area, whereas drugs that precipitate extrapyramidal side effects cause depolarization block of the motor-related substantia nigra dopamine cells. One factor that remains unresolved with regard to the actions of antipsychotic drugs is the relationship between dopamine turnover and depolarization block--i.e., why does a significant level of dopamine release or turnover remain after antipsychotic drug treatment if dopamine cells are no longer firing? We addressed this question using an acute model of neuroleptic-induced depolarization block. In this model, dopamine cells recorded in rats one month after partial dopamine lesions could be driven into depolarization block by the acute administration of moderate doses of haloperidol. However, similar doses of haloperidol, which were effective at increasing dopamine levels in the striatum of intact rats, failed to change dopamine levels in lesioned rats. This is consistent with a model in which neuroleptic drugs exert their therapeutic effects in schizophrenics by causing depolarization block in DA cells, thereby preventing further activation of dopamine neuron firing in response to external stimuli. Thus, attenuating the responsivity of the dopamine system to stimuli may be more relevant to the therapeutic actions of antipsychotic drugs than receptor blockade or decreases in absolute levels of dopamine, which could presumably be circumvented by homeostatic adaptations in this highly plastic system.


Localization of adenylyl and guanylyl cyclase in rat brain by in situ hybridization: comparison with calmodulin mRNA distribution. Cyclic nucleotides are major intracellular mediators in the signal transduction events in synaptic neurotransmission of the CNS. Intracellular Ca2+ is known to regulate adenylyl cyclase (AC) in a calmodulin (CaM)-dependent manner, and guanylyl cyclase (GC), in an indirect manner through CaM-sensitive nitric oxide synthase. To ascertain the physiological significance of cyclic nucleotide second messenger systems, we have localized the mRNAs encoding AC, GC, and CaM in the rat brain by in situ hybridization using 35S-labeled RNA probes. The AC mRNA is widely distributed throughout the brain; strong hybridization signal was observed in the granular layers of the cerebellum, in the pyramidal and granule cells of the hippocampus, and in the olfactory system. These AC mRNA localizations are compatible with the distribution of Ca2+/CaM-sensitive AC activities. In contrast to AC mRNA distribution, GC mRNA has a more limited distribution. Significant signals were observed in the striatum, in the pyramidal and granule cells of the hippocampus, in the olfactory system, in the inferior and superior colliculus, in the Purkinje cells of the cerebellum, in the locus coeruleus, and in many pyramidal cells in the layers II-III and V of the cerebral cortex, and mainly, in the occipital cortex. In some discrete brain regions, a close correlation was found between enzyme activity and mRNA hybridization signal of GC. The distinct distribution of AC and GC mRNAs suggests that different cyclic nucleotide second messenger systems have specialized functions. On the other hand, CaM mRNA was colocalized with the AC and GC mRNA, but its distribution was more abundant and specific for neuronal cells, since there was little hybridization signal with CaM probe in neuronal fiber regions such as the corpus callosum and the anterior commissure. The high expression of CaM mRNA in neuronal cells is in agreement with its biochemical role in the regulation of various enzymes. Results of the present study should help in analyzing the role of cyclic nucleotides and CaM in physiological and pathological situations in the CNS.


A novel N18TG2 x mesencephalon cell hybrid expresses properties that suggest a dopaminergic cell line of substantia nigra origin. A dopaminergic neuroblastoma was derived using somatic cell fusion of rat embryonic mesencephalon cells and the murine neuroblastoma-glioma cell line N18TG2. The resulting interspecies hybrid, named MES23.5, has retained a stable phenotype and karyotype for a continuous culture period of 1 year. The hybrid exhibits several properties that suggest that the parent primary neurons originated in the substantia nigra. The cell line contains tyrosine hydroxylase, which is identifiable both by biochemical and immunological methods and synthesizes dopamine, but no other catecholamine. Additionally, the cell line expresses apparent voltage-gated CA2+ channels as measured by high-affinity omega-conotoxin binding. The MES23.5 omega-conotoxin receptors are of similar affinity class to those found in adult rat mesencephalon. No dihydropyridine receptors, as measured by PN200-100 ligand binding, are present. None of these properties are found in the N18TG2 parent. At least three neuronal features, namely, tyrosine hydroxylase, dopamine synthesis, and omega-conotoxin receptor expression, are quantitatively elevated after sustained treatment with cAMP analogs. The cell line expresses a complex range of neural properties found in the dopaminergic neurons of the substantia nigra, and may therefore be useful elucidating further details of their cell biology.


Anatomy and physiology of multipolar cells in the rat inferior collicular cortex using the in vitro brain slice technique. Coronal brain slices from 21-50-d-old hooded rats were used to characterize intracellular responses of cells in both the external and dorsal cortices of the inferior colliculus (IC). These cells could generate both sodium and calcium spikes. Depending on current amplitude, depolarizing current pulses could elicit either phasic or tonic firing patterns, with spike frequency adaptation. Spiking also occurred at the offset of a hyperpolarizing pulse. These patterns were due primarily to the activation of calcium conductances. Stimulation of the commissural pathway connecting the left and right IC produced a short-latency monosynaptic IPSP followed by an EPSP(s) and a late polysynaptic IPSP(s). Non-NMDA glutamate antagonists eliminated or reduced the amplitude of the EPSP and the late portion of the inhibition, while both IPSPs were blocked by GABAA antagonists. As described previously in guinea pig (Smith, 1986) and rat (Pierson et al., 1989), a large NMDA-mediated depolarizing event (paroxysmal depolarizing shift, or PDS) could be elicited by shocking the commissure of the IC in the presence of picrotoxin or bicuculline, NMDA, 4-aminopyridine, or in 0 Mg2+ Ringer's. The picrotoxin-induced PDS was significantly reduced or abolished in Ringer's containing aminophosphonovalerate. Cells displaying the responses described were labeled with neurobiotin. Those labeled are medium-sized multipolar cells. Their dendrites are usually spiny and can extend superficially up to the cortical surface. Their thin axons give rise to collaterals that branch profusely within the cortex. The main axons project laterally along the circumference of the IC or medially into the commissure separating the collicular hemispheres.


Hyperbaric oxygen therapy: contraindications and complications. The literature is replete with references regarding the use of hyperbaric oxygen (HBO) therapy to treat various human maladies. However, the oral and maxillofacial surgery literature is lacking in information regarding patient selection criteria and possible contraindications to HBO therapy, as well as possible risks and/or complications of such therapy. This article details patient selection criteria, discusses relative and absolute contraindications to HBO therapy, and describes the potential risks and complications of this therapy.


Cathepsin B/L-, elastase-, tryptase-, trypsin- and dipeptidyl peptidase IV-like activities in gingival crevicular fluid: a comparison of levels before and after periodontal surgery in chronic periodontitis patients. Gingival crevicular fluid (GCF) was collected from the deepest probing site of each tooth of 10 chronic periodontitis patients prior to treatment, after scaling and hygiene treatment, and after periodontal surgery. Surgery was carried out at sites which had persistent probing depths in excess of 5 mm. The patients were given a full periodontal examination, including measurements of probing depth, gingival index, bleeding index, and plaque index before each GCF collection. Cathepsin B/L-, elastase-, tryptase-, trypsin-, and dipeptidyl peptidase IV-like activities in the GCF samples were determined by fluorimetric assay with peptidyl derivatives of 7-amino-4-trifluoromethyl coumarin. There were reductions in all clinical parameters and all protease activities after scaling and hygiene treatment and further reductions after periodontal surgery. Decreases were recorded for both total enzyme activities and concentrations. The reductions were statistically significant in inter-patient comparisons using mean patient values and also in most intra-patient comparisons using site data from individual patients. GCF protease levels appear to reflect the clinical status of periodontal lesions and may prove to be of value in monitoring disease activity.


Functional subsensitivity of 5-hydroxytryptamine1C or alpha 2 adrenergic heteroreceptors mediating clonidine-induced growth hormone release in the Fawn-Hooded rat strain relative to the Wistar rat strain. Administration of various doses of clonidine increased plasma growth hormone levels. Pretreatment with the alpha 2 adrenergic antagonists, yohimbine and 1-(2-pyrimidyl)piperazine, completely blocked clonidine's effect on growth hormone levels. Pretreatment with the 5-hydroxytryptamine3 (5-HT3) receptor antagonist, MDL-72222, the 5-HT1A/5-HT2 antagonist, spiperone, and the mixed beta adrenergic/5-HT1B antagonists, l-propranolol and CGP361A, did not attenuate clonidine-induced increases in growth hormone levels. In contrast, pretreatment with the non-selective 5-HT1/2 antagonist, metergoline, and the 5-HT1C/5-HT2-selective antagonist, mesulergine, reduced clonidine-induced increases in growth hormone levels 81 to 87% without affecting clonidine-induced decreases in locomotor activity. Two other 5-HT1C/5-HT2 antagonists, ritanserin and mianserin, also attenuated (47%) clonidine-induced increases in growth hormone levels. Pretreatment with the noradrenergic neurotoxin, DSP4, did not block clonidine's effect on growth hormone levels. Clonidine administration decreased locomotor activity in both the Fawn-Hooded and the Wistar rat strains to the same extent. On the other hand, clonidine administration failed to increase growth hormone levels in the Fawn-Hooded rat strain. These findings suggest that clonidine stimulates growth hormone secretion by activation of alpha 2 adrenergic heteroreceptors present on 5-HT nerve terminals which, in turn, enhance 5-HT activity via stimulation of postsynaptic 5-HT1C receptors to promote growth hormone releasing factor. Furthermore, either 5-HT1C receptors or alpha 2 adrenergic heteroreceptors or both are functionally sub-sensitive in the Fawn-Hooded rat strain relative to the Wistar rat strain.


Ro 19-8022, a nonbenzodiazepine partial agonist at benzodiazepine receptors: neuropharmacological profile of a potential anxiolytic. The partial agonist at benzodiazepine receptors, Ro 19-8022, has been characterized as a putative anxiolytic drug with an improved side effect profile. This orally active compound is a representative of a quinolizinone structure class and shows potent anticonflict activity in mice and rats. It protects rodents from convulsions induced by pentylenetetrazol, N-methyl-D-aspartic acid and maximal electroshock, as well as against audiogenic seizures, with an efficacy comparable to that of the full agonist alprazolam. No appreciable sedative or motor-impairing effects could be detected up to a very high dose (100 mg/kg) in the horizontal wire test or the rotarod performance test in mice and rats and in spontaneous behavior in monkeys. Consistent with its characterization as a partial agonist, Ro 19-8022 antagonized the motor impairment induced by the full agonists diazepam or meclonazepam measured in horizontal wire and rotarod tests in rodents, and reduced flunitrazepam-induced effects in squirrel monkeys, with an efficacy comparable to that of the benzodiazepine receptor antagonist flumazenil. After subchronic administration of Ro 19-8022 to mice, antagonist-precipitated withdrawal syndrome was dramatically weaker than after alprazolam treatment, which is indicative of a lower physical dependence liability of Ro 19-8022. Pharmacodynamic effects recorded in convulsion and reversal of motor impairment tests after i.v. administration suggest a long duration of action of this compound. Taken together, such preclinical data suggest that benzodiazepine receptor partial agonists with a neurological and behavioral profile such as that of Ro 19-8022 may offer an innovative therapeutic approach to the treatment of anxiety disorders.


Receptor-mediated toxicity of norepinephrine on cultured catecholaminergic neurons of the rat brain stem. The mechanisms associated with the neurotoxic responses caused by prolonged exposure (48 hr) to norepinephrine (NE) were examined in cultures of brain stem of 18-day-old rat fetuses. Two separate components of NE neurotoxicity were identified and differentiated according to dose dependency, sensitivity to catalase and blockade by adrenoceptor antagonists. The first component of NE toxicity was responsible for the death of the overall cell population, affecting both neurons and astroblasts, and was mediated by NE auto-oxidation products. This toxicity was observed at high doses of NE (LD50: 100 microM), was mimicked by other catecholamines (epinephrine, isoproterenol, dopamine), was fully antagonized by catalase and could not be blocked by adrenoceptor antagonists. The second component of NE toxicity was specifically targeted at noradrenergic neurons and was mediated by alpha 1 adrenoceptors. The specific toxicity for noradrenergic neurons was seen at lower doses of NE (LD50: 20 microM) and epinephrine (LD50: 40 microM). It was mimicked by the alpha 1 agonist phenylephrine and blocked by the alpha antagonists prazosine and nicergoline. These results indicate that protracted exposure to catecholamines may be a possible cause of damage to noradrenergic neurons that can be prevented by alpha 1 adrenoceptor blockade.


Identification of the mixed disulfide of glutathione and cysteinylglycine in bile: dependence on gamma-glutamyl transferase and responsiveness to oxidative stress. Biliary excretion of glutathione disulfide (GSSG) is used as an index of oxidative stress. Analysis of endogenous thiols and disulfides in rat bile by reverse phase high performance liquid chromatography with electrochemical detection revealed an unknown disulfide which eluted immediately after GSSG. This disulfide was tentatively identified as the mixed disulfide of glutathione (GSH) and cysteinylglycine (Cys-Gly), based on its coelution on a reverse phase column with the synthetic GS-Cys-Gly. GS-Cys-Gly was also detected in bile of other species. On analyzing species differences in biliary excretion of GSH-related thiols and disulfides, it was concluded that biliary excretion of GS-Cys-Gly was related to the excretion of both GSSG and Cys-Gly, which is formed from GSH by gamma-glutamyltransferase (gamma-GT)-catalyzed hydrolysis. Species with low hepatic gamma-GT (i.e., hamsters and mice) excreted little Cys-Gly in bile. These animals excreted negligible amounts of GS-Cys-Gly even when biliary excretion of GSSG was markedly increased by paraquat-induced oxidative stress. Rats and guinea pigs, which have high hepatic gamma-GT activities, excreted large amounts of both Cys-Gly and GS-Cys-Gly. Treatment of rats with acivicin, an inhibitor of gamma-GT, decreased the biliary excretion of both Cys-Gly and GS-Cys-Gly. Paraquat treatment of rats resulted in an increase in GSSG excretion with concomitant increase of GS-Cys-Gly excretion. Rabbits, which also have high hepatic gamma-GT activity, excreted little GS-Cys-Gly into bile.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of chlorethylclonidine on arterial blood pressure and heart rate in the conscious rat. The effect of chlorethylclonidine (CEC) on arterial blood pressure and heart rate (HR) has been evaluated in the conscious rat. CEC injection (25 mg/kg i.p.) caused a statistically significant decrease in mean arterial blood pressure (MAP) that was seen 24 hr after treatment. CEC also induced a decrease in HR that was maximal at 45 min but returned to pretreatment levels after 3 hr. CEC had no effect on the ability of isoproterenol to increase HR. CEC treatment had little effect on the pressor dose-response curve of either phenylephrine or BHT 920. When injected into the brain (25 mg/kg, lateral ventricle), CEC had no effect on MAP or HR. Yohimbine injected into the lateral ventricle had no effect on the response to i.p. CEC. Prazosin, used as a standard for comparison, caused a larger fall in MAP than CEC and this hypotension was associated with tachycardia and a marked shift (greater than 300-fold) in the phenylephrine pressor dose-response curve. A reactive analog of prazosin, SZL-49 [1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(2-bicyclo[2,2,2]octa- 2,5-diene-2-carbonyl)piperazine], had effects similar to prazosin on MAP and HR.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of [3H]quinpirole binding to D2-like dopamine receptors in rat brain. The putative D2 dopamine receptor agonist quinpirole (LY 171,555) is the most widely used D2 agonist in in vivo and in vitro studies of D2 receptor-mediated effects. In addition, quinpirole may have even higher affinity for the recently described D3 dopamine receptor. The present study describes the in vitro binding properties of newly developed [3H]quinpirole in rat brain. [3H]Quinpirole binding was characterized in striatal membrane homogenate preparations using a filtration assay. Nonspecific binding was defined by 1 microM (+)-butaclamol. Specific [3H]quinpirole binding was saturable, and dependent on temperature, membrane concentration, sodium concentration and guanine nucleotides. Saturation analysis revealed high affinity binding characteristics (KD = 2.3 +/- 0.3 nM) which were confirmed by association-dissociation kinetics. The pharmacological profile of [3H]quinpirole binding in striatum was: (-)-N-n-propylnorapomorphine (+/-)-2-amino-6,7-dihydroxyl-1,2,3,4-tetrahydronaphthalene greater than or equal to quinpirole greater than apomorphine greater than bromocriptine greater than dopamine greater than SKF 38393 much greater than 5-hydroxytryptamine for putative dopamine agonists; spiperone greater than (+)-butaclamol greater than haloperidol greater than (-)-sulpiride greater than clozapine greater than SCH 23390 much greater than cinanserin for antagonists. [3H]Quinpirole binding exhibited stereoselectivity: (-)-sulpiride greater than (+)-sulpiride and (+)-butaclamol greater than (-)-butaclamol. This pharmacological profile is similar, though-not identical, to that observed for [3H] spiperone-labeled D2 receptors. The regional distribution of [3H]quinpirole binding sites roughly paralleled the distribution of [3H]spiperone binding sites, with greatest densities present in the striatum, nucleus accumbens and olfactory tubercles.(ABSTRACT TRUNCATED AT 250 WORDS)


[N-methyl-Tyr1,N-methyl-Arg7-D-Leu8]-dynorphin-A-(1-8)ethylamide, a stable dynorphin analog, produces diuresis by kappa-opiate receptor activation in the rat. The i.v. administration of E-2078 ([N-methyl-Tyr1-N-methyl-Arg7-D-Leu8]-dynorphin-A-(1-8) ethylamide) to conscious animals in doses of 15, 50 or 200 micrograms/rat caused a dose-related diuretic response associated with a significant in crease in glomerular filtration rate (GFR) and in blood pressure. The overall excretion of Na+ was not modified by the opioid, whereas it reduced K+ output and its fractional excretion. Time course studies demonstrated that the increase in GFR and in blood pressure were transient and did not parallel the changes in urine outflow. Pretreatment of the animal with 1 mg/kg of naltrexone or of naloxone reduced the pressor response but did not reduce the renal action of E-2078. Doses of naltrexone 10 times larger (10 mg/kg) were required to attenuate the diuretic effect and abolish completely the changes in K+ excretion; however, the increase in GFR was not antagonized by 10 mg/kg of naltrexone. Consonant with the studies in conscious rats, perfusion of isolated rat kidneys with 0.2 to 1.8 microM E-2078 increased urine flow in a dose-dependent manner, and this effect was prevented by the simultaneous perfusion of 2 microM naltrexone with the peptide. In pentobarbital-anesthetized animals, E-2078 elicited a diuretic response that was not parallelled by changes in GFR or electrolyte excretion. In addition, E-2078 caused a long lasting decrease in blood pressure which was blocked completely by pretreatment of the animal with 1 mg/kg of naltrexone. The diuretic effect of E-2078 was not modified by pretreatment of the animals with beta-funaltrexamine.(ABSTRACT TRUNCATED AT 250 WORDS)


Flexion characteristics of four-unit fixed partial denture frameworks using holographic interferometry. Flexion of a metal/ceramic fixed partial denture (FPD) framework under function can cause fracture of the porcelain or deterioration of the cement seal. This study evaluated the flexion under compressive load of a four-unit mandibular FPD replacing the second premolar and the first molar. Testing was accomplished with elapsed time holographic interferometry, using 39 porcelain fused-to-metal frameworks cast with a silver-palladium alloy. The results demonstrated that solder joints at the junction of the premolar and molar pontics flexed under a reduced compressive load and exhibited a higher failure rate than other connector designs.


Linear dimensional variability and tensile strengths of three solder index materials. This study compared the linear dimensional change and tensile strengths of three indexing materials used for aligning joints to be soldered. Sixty rods 6 mm in diameter and 18 mm in length were made from base metal. The rods were divided into three groups of 10 pairs each, and a simulated solder gap was created between the rods in each pair. Ten simulated gaps were indexed with Duralay resin, 10 were indexed with Relate resin, and 10 were indexed with ZAPIT cyanoacrylate material. Measurements were made of each pair, before and after indexing, and the tensile strength of each indexed pair was measured and recorded. Linear measurements showed no significant difference between any of the groups. However, significant differences in tensile strength were found among all the materials tested.


Astrocyte cultures from human embryonic brain: characterization and modulation of surface molecules by inflammatory cytokines. Astrocyte-enriched cultures were established upon passaging of primary cultures from the myelencephalon and mesencephalon of 7-9-week-old human embryos. Immunocytochemical analysis showed that third-fourth passage cultures were composed of a highly enriched population of proliferating, epithelioid cells, up to 90% of which expressed glial fibrillary acidic protein (GFAP); no macrophages and very few fibroblasts (less than 2%) were present. GFAP expression and proliferation declined upon further culturing in serum-containing medium but could be transiently reinduced by growing the cells in a serum-free chemically defined medium. Large numbers of GFAP+ astrocytes were obtained from each embryo and could be stored frozen and recultured. Using flow cytometric analysis, human astrocyte cultures were examined for basal and cytokine [interferon-gamma (IFN-gamma), interleukin-1 beta (IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha)]-induced expression of molecules that may be involved in astrocyte-T-lymphocyte interactions. Cultured human astrocytes spontaneously expressed major histocompatibility complex (MHC) class I antigens and variable levels of MHC class II; MHC class I levels were increased upon IFN-gamma and TNF-alpha treatment, whereas MHC class II antigens were induced on most of the astrocytes by IFN-gamma. Among the molecules involved in antigen-independent interactions between T lymphocytes and target cells, lymphocyte function-associated molecule-3 (LFA-3) was spontaneously expressed by most cultured human astrocytes, whereas intercellular adhesion molecule-1 (ICAM-1) was present at variable levels in non-stimulated astrocytes and was greatly induced by IFN-gamma, TNF-alpha, and IL-1 beta. In this study we also show that the above cytokines upregulate astroglial expression of adhesion molecules of the integrin family (VLA-1, VLA-2, and VLA-6) that may be involved in astrocyte-extracellular matrix interaction and play a role in the astrocyte reactive changes occurring at sites of brain injury and inflammation. The human astrocyte cultures developed here represent a useful in vitro model to further investigate mechanisms involved in bidirectional communication between central glia and cells of the immune system.


Staphylococcal exotoxin superantigens induce human immunodeficiency virus type 1 expression in naturally infected CD4+ T cells. A high proportion of Staphylococcus aureus strains of human origin produce one or more exotoxins. In vivo, these toxins may give rise to a variety of clinical syndromes. In vitro, staphylococcal exotoxins have been shown to bind both to human leukocyte antigen (HLA) class II molecules on antigen-presenting cells and to the T-cell receptors on large fractions of T cells. The result of this interaction may be proliferation of the T cells, T-cell anergy, or apoptosis, depending on several factors, including the state of the responding cells and the presence of accessory molecules. Using naturally infected peripheral blood mononuclear cells depleted of CD8+ T cells, we have shown that staphylococcal exotoxins are powerful inducers of human immunodeficiency virus type 1 expression and that they induce expression at low concentrations and with greater efficiency than other T-cell mitogens. Human immunodeficiency virus type 1 was produced entirely by CD4+ T cells in this model; monocytes were expendable both as a source of virus and as a source of HLA class II molecules as long as other cells expressing HLA class II molecules were present. The results suggest that infection by S. aureus may be a cofactor in the immunopathogenesis of AIDS.


Hantaan virus infection of human endothelial cells. The primary pathophysiologic finding of the viral disease known as Korean hemorrhagic fever, the etiological agent of which is Hantaan virus (HTV), is vascular instability. To investigate whether HTV was able to infect cells derived from human vascular tissue and alter their behavior, we infected in vitro primary adult human endothelial cells from saphenous veins (HSVEC). We were able to detect the presence of viral antigens in infected cells both by immunofluorescence and by Western blot (immunoblot) analysis as early as day 1 postinfection. HSVEC infected with HTV produce infectious virus during the first 3 days of infection but, at later times (days 4 to 8), show decreasing yields of virus. This contrasts with the HTV growth pattern observed for the permissive simian CV-7 cell line, which generates infectious virus up to day 12 after infection. Further investigation showed that the late decrease in viral production in HSVEC is the result of the induction of beta interferon and can be reversed by the addition of anti-beta interferon serum to the culture medium. At no time during the course of infection of HSVEC with HTV was any obvious cytopathic effect observed. When tests for changes in mRNA levels of other cytokines and endothelial cell gene products following HTV infection of HSVEC were done by reverse transcription and polymerase chain reaction methods, no significant changes were observed in the levels of interleukin 1, interleukin 6, or von Willebrand factor mRNA. We hypothesize that, while HTV can replicate in human vascular endothelial cells, the mechanism of microvascular damage seen with Korean hemorrhagic fever is not likely to be a direct effect of virus replication but may conceivably be the consequence of an immune-mediated endothelial injury triggered by viral infection.


Attempts to convert the cellular prion protein into the scrapie isoform in cell-free systems. The scrapie prion protein (PrPSc) is derived from a cellular isoform (PrPC) that acquires protease resistance posttranslationally. We have used several different experimental approaches in attempts to reconstitute in vitro the processes leading to protease-resistant PrPSc molecules. In the first study, we performed mixing experiments by adding mouse PrP 27-30 (MoPrP27-30), the protease-resistant core of PrPSc, to PrPC and then incubating the mixture to investigate the possibility of heterodimer formation as a first step in prion replication. We used epitopically tagged PrP molecules, synthesized in murine neuroblastoma (N2a) cells transfected with the chimeric mouse/Syrian hamster MHM2 PrP construct, which are recognized by the Syrian hamster-specific monoclonal antibody 3F4. After as long as 24 h of incubation, the reaction mixture was assayed for heterodimeric intermediates of MHM2 PrPC and MoPrPSc and for protease-resistant 3F4-reactive PrP. We were unable to identify any aggregates of MHM2 PrPC and MoPrPSc on immunoblots; furthermore, we did not observe de novo formation of protease-resistant MHM2 PrP. In a second study, MoPrPC was metabolically radiolabeled in scrapie prion-infected N2a cultured cells, and then the cell extract was homogenized and incubated under various conditions to allow for the formation of protease-resistant MoPrPSc. We observed no radiolabeled MoPrPSc by immunoprecipitation after as long as 24 h of in vitro incubation. In a third approach, Syrian hamster PrP (SHaPrP) was synthesized in a cell-free translation system supplemented with microsomal membranes derived from either normal or scrapie prion-infected cultured cells. We found that all SHaPrP species translocated across microsomal membranes from scrapie prion-infected cells were protease sensitive in the presence of detergents and displayed the same topology as those generated by microsomes from normal cells or from dog pancreas. We also studied PrP molecules that encode the codon 102 mutation that causes the rare human prion disease Gerstmann-Sträussler-Scheinker (GSS) syndrome. On the basis of our data, GSSPrP appears to yield topological forms similar to those of the wild-type PrP when processed by either normal or scrapie prion-derived microsomes.


A questionnaire study of beta-adrenergic blockade in dilated cardiomyopathy in Japan. A questionnaire study on the effect of beta-blockade in dilated cardiomyopathy was performed. In 89 cases obtained from 24 institutions, either metoprolol (72 patients, 41.4 +/- 29.3 mg/day, 14.1 +/- 13.2 months, mean +/- SD), propranolol (5 patients, 23.8 +/- 24.3 mg/day, 25.0 +/- 25.3 months), carteolol (4 patients, 7.5 +/- 2.9 mg/day, 9.0 +/- 2.8 months) or another beta-blockers (8 patients) was administered. Nine patients died during the follow-up period. Overall effectiveness as evaluated by the attending physicians showed improvement in 51 patients (57.3%), no change in 26 patients (29.2%), deterioration in 11 patients (12.4%) or was indeterminate in one patient. New York Heart Association (NYHA) functional class improved significantly from 2.6 to 2.3 (p less than 0.01). Heart rate decreased from 83.1 to 70.1 (p less than 0.01). Cardiothoracic ratio decreased from 55.5% to 53.9% (p less than 0.01). Left ventricular ejection fraction of the left ventricle measured by echocardiogram increased from 29.8% to 37.8% (p less than 0.01). Exercise tolerance during a treadmill test and ventricular arrhythmias in Holter electrocardiograms improved significantly. There was no change in blood pressure. When the patients in different functional classes were compared, the patients of NYHA class III improved more frequently than those of NYHA class II (69% vs 53% p less than 0.01). Improvement of left ventricular end-diastolic dimension and left ventricular ejection fraction was more prominent in patients of class III than in those of class II. NYHA functional class and cardiothoracic ratio were significantly improved only in class III.(ABSTRACT TRUNCATED AT 250 WORDS)


Modulation of cellular and viral promoters by mutant human p53 proteins found in tumor cells. Wild-type p53 has recently been shown to repress transcription from several cellular and viral promoters. Since p53 mutations are the most frequently reported genetic defects in human cancers, it becomes important to study the effects of mutations of p53 on promoter functions. We, therefore, have studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen (PCNA) promoter and on several viral promoters, including the herpes simplex virus type 1 UL9 promoter, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and a plasmid containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. As expected, expression of the wild-type p53 inhibited promoter function. Expression of a p53 with a mutation at any one of the four amino acid positions 175, 248, 273, or 281, however, correlated with a significant increase of the PCNA promoter activity (2- to 11-fold). The viral promoters were also activated, although to a somewhat lesser extent. We also showed that activation by a mutant p53 requires a minimal promoter containing a lone TATA box. A more significant increase (25-fold) in activation occurs when the promoter contains a binding site for the activating transcription factor or cyclic AMP response element-binding protein. Using Saos-2 cells that do not express p53, we showed that activation by a mutant p53 was a direct enhancement. The mutant forms of p53 used in this study are found in various cancer cells. The activation of PCNA by mutant p53s may indicate a way to increase cell proliferation by the mutant p53s. Thus, our data indicate a possible functional role for the mutants of p53 found in cancer cells in activating several important loci, including PCNA.


The effects of phentolamine and nitroglycerin on right-sided hemodynamics in cardiac patients can be explained by a shift of the systemic venous return curve and right-ventricular output curve. The present study investigated the effects of phentolamine (PH) and nitroglycerin (NG) on the hemodynamics of the right heart in patients with cardiac disease. The patients were divided into a well-functioning left heart group (W group, n = 15) and a poorly-functioning left heart group (P group, n = 15). Right cardiac hemodynamic parameters and plasma noradrenaline (NA) and adrenaline (A) concentrations were measured before and after administering PH (0.1 mg/kg, i.v.) or NG (0.6 mg, sublingual). In a parallel animal study we obtained a systemic venous return curve by measuring mean circulatory pressure (MCP), mean right atrial pressure (RAP) and cardiac output, before and after administering PH (0.1 mg/kg, i.v.) or NG (12.5 micrograms/kg, i.v.) to anesthetized open-chest dogs (n = 14). We used MCP data (W group: 7.5 mmHg, P group: 10 mmHg) obtained in a separate series of human studies in our laboratory. We constructed the venous return curve by connecting the MCP point on abscissa with the cardiac index (CI)-RAP plot obtained in the clinical study. We also constructed the right ventricular output curve by connecting the point of -2 mmHg on the abscissa with the CI-RAP plot. We obtained the following results: (1) PH shifted the CI-RAP plot to the left and upwards, while NG shifted the CI-RAP plot to the left almost horizontally on the CI-RAP plane, where CI was plotted on ordinate and RAP on abscissa. The length [formula: see text] C = control point, PH = point after PH) of the shift of CI-RAP plot due to PH was greater in the P group than in W group, while there was no difference in the length [formula: see text] C = control point, NG = point after NG) of the shift of CI-RAP plot due to NG between P and W groups. (2) Both PH and NG significantly elevated plasma NA and A concentrations in both the W and P groups. In the P group, PH increased the plasma NA concentration significantly more than did NG, but both drugs increased plasma A concentration to a similar extent. (3) Both PH and NG significantly decreased the mean pulmonary arterial pressure with NG doing so significantly more than PH.(ABSTRACT TRUNCATED AT 400 WORDS)


Differing actions of endothelin-1 on canine systemic resistance and capacitance vessels. In anesthetized open-chest dogs, an intravenous bolus injection of endothelin-1 (ET-1, 400 pmol/kg) caused transient hypotension (initial hypotensive phase; phase 1), followed by a continuous elevation of blood pressure (late hypertensive phase; phase 2). The constriction and dilation of the systemic capacitance and resistance vessels were evaluated from the change in mean circulatory pressure (MCP) and in total peripheral resistance (TPR) in phases 1 and 2. To examine the modification of the action of ET-1 on the blood vessels by the baroceptor reflex or by the endothelium-derived relaxing factor (EDRF) released by ET-1 in phase 1, we performed experiments in dogs under total spinal anesthesia (TSA group), methylene blue-treated dogs (MB group) as well as in the untreated dogs (control group). ET-1 decreased the TPR significantly, and increased the MCP significantly in phase 1 in the control (n = 8) and TSA (n = 8) groups; there was no difference between the groups. ET-1 had no significant effect on TPR but increased the MCP significantly in MB group (n = 8) during phase 1. The percentage increase of MCP in the MB group significantly exceeded that of the control group. ET-1 increased both the TPR and MCP significantly in phase 2 in the control group (n = 8). This study indicated that the vasoconstrictor action of ET-1 on the systemic capacitance vessels in phase 1 did not result from a baroceptor reflex, and that the vasodilator action of ET-1 on the systemic resistance vessels may be at least in part mediated via EDRF released by ET-1. We suggest that the vasoconstrictor action of ET-1 on the systemic capacitance vessels is strong, but the vasodilator action of EDRF on the systemic capacitance vessels is weak.


[Two kindreds with familial Alzheimer's disease--analysis of the APP717 mutation and the mutated genes for the prion protein]. Numerous Caucasian familial Alzheimer's disease (FAD) pedigrees have been described in the literature, while only 21 Japanese FAD families have been reported to date. Here we report the clinical findings and the result of molecular genetic analysis of 4 patients from two FAD kindreds, OS-2 and OS-3. The proband in OS-2 family has developed loss of recent memory and place disorientation age at 43. A brain CT showed severe diffuse cortical atrophy. Her younger brother had dementia at 42 years and her mother and other 3 siblings had also dementia symptoms suspected to be Alzheimer's disease. The proband in OS-3 family showed declining recent memory at 49 years and developed dysphagia, gait disturbance and emotional incontinent with cerebral atrophy at 52 years. His father and elder brother demonstrated dementia signs at 60 and 54 years old, respectively. Recently it was reported that affected members from 2 Caucasian kindreds with FAD had missense mutation in exon 17 of the gene for amyloid precursor protein (APP). Patients from three different Japanese kindreds with FAD also showed the same mutation on the APP gene. Amino acid substitution (Val-Ile) at codon 717 by this mutation is responsible for FAD in at least some kindreds. We used genomic DNA from 4 affected members of 2 families to determine whether the disease in these families is associated with a APP717 mutation and the mutated codons, 102, 117, 129, 178 and 200, on the gene for protease-resistance prion protein (PrP) which cause transmissible dementia, Creutzfelt-Jacob disease (CJD) and Gerstmann-Strausler syndrome (GSS).(ABSTRACT TRUNCATED AT 250 WORDS)


[Assistance: the handiwork and art].

[Assistance: the handiwork and art]. The problems of assistance are still not the subject of comprehensive discussion, at the same time perfection of assistance is an important link of surgery perfection. Questionnaires containing 120 basic questions were sent to 120 surgeons of the country; 73 representatives of academic and practical surgery responded. Analysis of answers and recommendations, which were not always unambiguous, allowed the problem to be considered in a wider aspect and a rich collective and individual experience to be critically comprehended. Optimization of the activity of assistants, their skilled handiwork, and the skill in teaching and learning surgery in the interest of a sick person are dwelt on.


DNA-based presymptomatic diagnosis of Wilson disease. Investigation using DNA markers in a family with Wilson disease revealed that an apparently normal child of 10 years of age with non-diagnostic copper biochemistry had the disease. The procedure used linked restriction fragment length polymorphic markers. Demonstration of increased liver copper concentration from a liver biopsy confirmed the diagnosis and the child was started on chelation therapy. Two other asymptomatic siblings were shown, using the same techniques, not to have the disease. Similar analysis was carried out on another family with just one index case.


Molecular basis of inherited medium-chain acyl-CoA dehydrogenase deficiency causing sudden child death. Deficiency of medium-chain acyl-CoA dehydrogenase (MCAD) is an important cause of sudden death in children. The majority of surviving individuals with MCAD deficiency studied to date are homozygous for a single point mutation at bp 985 of the MCAD mRNA (A985G). We have now identified a four-base-pair deletion in exon 11 of one allele of the MCAD gene in an American child who died of MCAD deficiency. The deletion mutation results in a frameshift and premature termination codon in the mutant MCAD mRNA. The second mutant allele contained the common point mutation A985G, and thus the proband was a compound heterozygote. Protein immunoblot analysis of the child's liver proteins revealed that the mutant MCAD proteins were barely detectable. Allele-specific oligonucleotide hybridization analysis performed on amplified exon 11 of the child's MCAD gene clearly identified both mutations. MCAD RFLP analysis of the patient's DNA revealed heterozygosity at the Taq I MCAD RFLP site, thus, the two mutations are associated with different haplotypes. Therefore, we have identified a new mutation in the MCAD gene and have developed a nucleic-acid-based screening approach which allows the post mortem identification of MCAD deficiency.


Gene analysis of Mennonite maple syrup urine disease kindred using primer-specified restriction map modification. Maple syrup urine disease (MSUD) is an autosomal recessive inherited disease due to a deficiency of any of the subunits, E1 alpha, E1 beta or E2, of the branched-chain alpha-ketoacid dehydrogenase complex (BCKDH). A large Mennonite kindred of MSUD has been studied in Pennsylvania, USA. In the present investigation, genomes from 70 members, including 12 patients belonging to eight different Mennonite MSUD pedigrees, were examined for possible abnormalities in the E1 alpha gene of BCKDH, by primer-specified restriction map modification. A T-to-A substitution which generates an asparagine in place of a tyrosine at amino acid 394 of the mature E1 alpha subunit was present in both alleles in all the patients and in a single allele in all obligate carriers and several siblings. We describe a new technique for rapid and easy detection of the mutant gene in this population. These family studies provide additional evidence that Mennonite MSUD is caused by a missense mutation of the E1 alpha gene of BCKDH


Hereditary tyrosinaemia type II in a consanguineous Ashkenazi Jewish family: intrafamilial variation in phenotype; absence of parental phenotype effects on the fetus. We describe an Ashkenazi Jewish family in which two adults, offspring of consanguineous parents, have persistent hypertyrosinaemia (770-1110 mumol/L; normal less than 110 mumol/L). The metabolic disorder in this family is apparently due to hepatic cytosolic tyrosine aminotransferase deficiency (hereditary tyrosinaemia, type II; McKusick, 276600), because it is associated with the oculocutaneous manifestations of Richner-Hanhart syndrome. The association of this syndrome with hereditary tyrosinaemia type II is presumed to be constant. It is not in this family. The affected female sib (age 41 years) has hypertyrosinaemia and oculocutaneous signs; the brother (age 39 years) has hypertyrosinemia but no oculocutaneous disease. Both sibs have two children; none has signs of a metabolic fetopathy. Maternal hypertyrosinaemia and maternal hyperphenylalaninaemia evidently constitute different risk factors for the fetus. Paternal hypertyrosinaemia is apparently not a risk to male infertility.


Prospective versus clinical diagnosis and therapy of acute neonatal hyperammonaemia in two sisters with carbamyl phosphate synthetase deficiency. Two female siblings were treated for acute neonatal hyperammonaemia due to complete carbamyl phosphate synthetase I deficiency. The first child was detected clinically at 65 hours of age and therapy started at 79 hours. The second child was followed from birth and therapy started at 5 hours of age. The extrapolated rate of increase of blood ammonia, in the first hours of life before therapy started, was 19 mumol L-1 h-1 in both babies. Peak blood ammonia level was 2235 mumol/L in the first (clinically detected) child and 271 mumol/L in the second (prospectively followed) child. The second child became symptomatic at 3 hours of age when blood ammonia level was as low as 90 mumol/L, whereas blood ammonia levels above 100 mumol/L caused no symptoms during recovery. The child detected clinically required haemodialysis and peritoneal dialysis to treat the hyperammonaemia. In the prospectively treated child, early therapy with intravenous sodium benzoate and sodium phenylacetate slowed the rate of increase in blood ammonia level, but this therapy did not prevent the need for peritoneal dialysis.


Mechanisms of increased hepatic glutamine uptake in the endotoxin-treated rat. The mechanisms underlying the accelerated hepatic consumption of glutamine that occurs during endotoxemia were investigated in rats 12 hr after treatment with Escherichia coli lipopolysaccharide. Hepatic glutamine delivery and consumption were calculated from measurements of hepatic blood flow and blood glutamine levels. Hepatic glutaminase activity and glutamine and glutamate content were determined. Hepatocyte plasma membrane transport activity was evaluated employing isolated hepatic plasma membrane vesicles (HPMVs). Endotoxin treatment resulted in an 11-fold increase in hepatic glutamine consumption and a 2-fold increase in the delivered load of glutamine to the liver. Hepatic glutamate content doubled while glutamine content was unaffected, not withstanding a decrease in the specific activity of glutaminase. Studies employing HPMVs demonstrated that hepatic plasma membrane transport activity was unaffected by endotoxin treatment. The enhanced hepatic consumption of glutamine secondary to endotoxemia appears to be the result of both a mass-action effect and the concurrent activation of intracellular metabolism. Responses at the level of plasma membrane transport do not appear to play an active role in mediating this enhanced hepatic uptake.


Push-pull cannula for localized application of drugs and sampling of medium, combined with electrophysiological recordings in an interface slice chamber. These experiments combined electrophysiological recordings from hippocampal slices with application of drugs to and sampling of extracellular fluid from a restricted region of the slice using a push-pull cannula placed under the slice in an interface chamber. Stable and apparently normal extracellular and intracellular recordings could be obtained directly over the tip of the cannula and solutions changed without disturbing the recording. Relatively rapid effects (1-5 min) were observed when TTX, CNQX, or medium containing 50 mM K+ were applied via the cannula and recovery from these effects was achieved. In addition, effects were restricted to the immediate vicinity of the cannula; neurones recorded several hundred micrometers away were apparently unaffected. Samples of extracellular fluid obtained as minute fractions during the application of high K+ containing medium contained higher concentrations of GABA, aspartate and glutamate than control fractions but the same levels of other amino acids, e.g., isoleucine and leucine. With appropriate design of push-pull cannula and recording chamber, therefore, stable electrophysiological recordings can be combined with localized extracellular fluid sampling and rapid and localized application of test solutions in an interface slice chamber.


Controlled trial of pulse methylprednisolone versus two regimens of pulse cyclophosphamide in severe lupus nephritis. Pulse cyclophosphamide is more effective than prednisone alone in preventing renal failure in lupus nephritis. We undertook a randomised, controlled trial to find out whether pulse methylprednisolone could equal pulse cyclophosphamide in preserving renal function in patients with lupus nephritis, and whether there was a difference between long and short courses of pulse cyclophosphamide in preventing exacerbations. 65 patients (60 female, 5 male; median [range] age 29 [10-48] years) with severe lupus nephritis were assigned randomly to monthly pulse methylprednisolone for 6 months (25 patients), monthly pulse cyclophosphamide for 6 months (20), or monthly cyclophosphamide for 6 months followed by quarterly pulse cyclophosphamide for 2 additional years (20). Patients treated with pulse methylprednisolone had a higher probability of doubling serum creatinine than those treated with long-course cyclophosphamide (p less than 0.04). Risk of doubling creatinine was not significantly different between short and long course cyclophosphamide. However, patients treated with short-course cyclophosphamide had a higher probability of exacerbations than those treated with long-course cyclophosphamide (p less than 0.01). An extended course of pulse cyclophosphamide is more effective than 6 months of pulse methylprednisolone in preserving renal function in patients with severe lupus nephritis. Addition of a quarterly maintenance regimen to monthly pulse cyclophosphamide reduces the rate of exacerbations.


Placebo-controlled trial of prednisolone in children intubated for croup. Many studies have attempted to find out whether steroid treatment is beneficial in children with croup, but the results have been inconclusive. We have done a prospective placebo-controlled study of the effect of prednisolone on two clinical endpoints--the duration of intubation and the need for reintubation. Reasons for exclusion were age under 6 months, congenital airway anomalies, and previous intubation. 70 eligible children were randomly assigned treatment with prednisolone 1 mg/kg (n = 38) or placebo (n = 32) every 12 h given by nasogastric tube until 24 h after extubation. 11 (34%) placebo-treated and only 2 (5%) prednisolone-treated patients required reintubation after accidental or elective extubation (p = 0.004, Fisher's exact test; odds ratio 8.9, 95% confidence interval 1.7-59.3). Survival analysis with log-normal regression showed that the duration of intubation was shorter with steroid therapy (p less than 0.003) and increasing age (p less than 0.02), but was not influenced by endotracheal tube size or abnormality on chest radiograph. The median duration of intubation was 138 (95% CI 118-160) h in children who received placebo and 98 (85-113) h in the prednisolone group. Steroid therapy reduces the duration of intubation and the need for reintubation in children intubated for croup.


Humanised monoclonal antibody therapy for rheumatoid arthritis. Monoclonal antibodies that target T cells have shown some benefit in rheumatoid arthritis although responses have not been long lasting. This is partly due to insufficient therapy consequent upon antibody immunogenicity. Use of humanised antibodies, which are expected to be less foreign to man than conventional rodent antibodies, might overcome this problem. We therefore assessed in a phase 1 open study the potential of a "lymphocyte depleting" regimen of the humanised monoclonal antibody CAMPATH-1H in 8 patients with refractory rheumatoid arthritis. Apart from symptoms associated with first infusions of antibody, adverse effects were negligible. Significant clinical benefit was seen in 7 patients, lasting for eight months in 1. After one course of therapy, there was no measurable antiglobulin response, although 3 out of 4 patients have become sensitised on retreatment. Humanisation reduces the immunogenicity of rodent antibodies but anti-idiotype responses may still be seen on repeated therapy, even in patients sharing immunoglobulin allotype with the humanised antibody.


Maternal relaxin concentrations in diabetic pregnancy. Maternal serum concentrations of relaxin, an insulin homologue produced both by the corpus luteum of pregnancy and by the fetoplacental unit, are highest in the first trimester and fall to their lowest level in the third trimester. Relaxin is thought to influence carbohydrate metabolism in the uterus, and it has been suggested that serum concentrations of relaxin in diabetic women are higher than those of non-diabetic women. We show that maternal serum relaxin concentrations are significantly higher at each stage of pregnancy in insulin-dependent diabetic mothers than in non-diabetic mothers. This elevation in relaxin concentrations is not related to other indices of diabetic control. The physiological importance of the higher concentrations of relaxin in the serum of diabetic women--in particular, whether they contribute to the higher incidence of major anomalies in the fetuses of diabetic mothers--is yet to be determined.


Prevention of Yersinia enterocolitica growth in red-blood-cell concentrates. In response to concern about Yersinia enterocolitica contamination of blood products, we have studied the effects on Y enterocolitica growth of holding whole blood at 22 degrees C for 20 h and then removing leucocytes. Thirty pools of three bags of blood were inoculated with Y enterocolitica (2 x 10(1)-3 x 10(4) colony-forming units/ml). One bag in each pool was processed to red-blood-cell concentrate after 6 h at 4 degrees C (RBC); the other two were held at 22 degrees C for 20 h before processing to buffy-coat-depleted RBC (BCd-RBC). One of these bags was then depleted of leucocytes by filtration (Ld-RBC). All bags were stored at 4 degrees C for 5 weeks. RBC bags showed Y enterocolitica growth after the shortest storage times, followed by BCd-RBC then Ld-RBC (p less than 0.03-0.001). We recommend that whole blood should be held at 22 degrees C to make use of inherent bactericidal activity; leucocytes should then be removed.


Stress failure of pulmonary capillaries: role in lung and heart disease. Pulmonary capillaries have extremely thin walls to allow rapid exchange of respiratory gases across them. Recently it has been shown that the wall stresses become very large when the capillary pressure is raised, and in anaesthetised rabbits, ultrastructural damage to the walls is seen at pressures of 40 mm Hg and above. The changes include breaks in the capillary endothelial layer, alveolar epithelial layer, and sometimes all layers of the wall. The strength of the thin part of the capillary wall can be attributed to the type IV collagen in the extracellular matrix. Stress failure of pulmonary capillaries results in a high-permeability form of oedema, or even frank haemorrhage, and is apparently the mechanism of neurogenic pulmonary oedema and high-altitude pulmonary oedema. It also explains the exercise-induced pulmonary haemorrhage that occurs in all racehorses. Several features of mitral stenosis are consistent with stress failure. Overinflation of the lung also leads to stress failure, a common cause of increased capillary permeability in the intensive care environment. Stress failure also occurs if the type IV collagen of the capillary wall is weakened by autoantibodies as in Goodpasture's syndrome. Neutrophil elastase degrades type IV collagen and this may be the starting point of the breakdown of alveolar walls that is characteristic of emphysema. Stress failure of pulmonary capillaries is a hitherto overlooked and potentially important factor in lung and heart disease.


Simplification of antibiotic dose adjustments in renal insufficiency: the DREM system. Many clinicians, unassisted by reference books, are unable to make the required dose adjustment of antibiotics needed when a patient has renal insufficiency. We describe the DREM (dosing in renopathy by easy-to-use multipliers) system, which simplifies the understanding and the process of dose adjustment. DREM is a two-step process: Cockcroft and Gault estimation of creatinine clearance (CLcr) from age, sex, and serum creatinine and calculation of the adjusted dose or dosing interval by multipliers. If the normal dose is multiplied by the dose multiplier (CLcr/100) and the dosing interval by the interval multiplier (100/CLcr), the adjusted dose and interval, respectively, are obtained. Theoretical trough concentrations calculated with the DREM system correlated closely (r = 0.9) with actual concentrations obtained from doses calculated by the Hull and Sarubbi method in 23 patients. With DREM, gentamicin or tobramycin trough concentrations above 2 micrograms/ml were less likely to occur. The DREM system is a simple and easily remembered method for dose adjustments of certain anti-infective agents in renal insufficiency. Dose estimates with this method are reasonably accurate and compare favourably with other standard methods of correction.


Early detection of antibodies to HIV-1 by third-generation assays. Third-generation immunoassays for detection of antibody to human immunodeficiency virus (HIV) have reduced the interval between infection and antibody detection. Might such earlier detection diminish the value of the western blot as a confirmatory assay? We compared the sensitivity of one second-generation and two third-generation anti-HIV enzyme immunoassays (EIAs) with the results from HIV-antigen testing and western blot. 1045 western-blot confirmed anti-HIV positive samples were tested with a detection rate of 100% for all three EIAs. The detection rate in 36 samples drawn from different persons in early stages of HIV infection was 89% for the second-generation EIA and 94% for both third-generation EIAs. With carefully selected seroconversion panels that included sampling intervals during seroconversion of one week or less, we found that both third-generation EIAs detected seroconversion on average 5 days earlier (range 0-13) than did the second-generation assay. Western blot is commonly used to confirm HIV infection. In 6 of 10 seroconversions, one or both third-generation EIAs were reactive before any band appeared in the western blot. Since HIV antigen was detectable in these cases, the HIV antigen test may serve as a confirmatory assay for anti-HIV EIA-positive, western-blot negative, samples.


Metabolic compartmentation of vertebrate glutamine synthetase: putative mitochondrial targeting signal in avian liver glutamine synthetase. The evolution of uricoteley as a mechanism for hepatic ammonia detoxication in vertebrates required targeting of glutamine synthetase (GS) to liver mitochondria in the sauropsid line of descent leading to the squamate reptiles and archosaurs. Previous studies have shown that in birds and crocodilians, sole survivors of the archosaurian line, hepatic GS is translated without a transient, N-terminal targeting signal common to other mitochondrial matrix proteins. To identify a putative internal targeting sequence in the avian enzyme, the amino acid sequence of chicken liver GS was derived by a combination of sequencing of cloned cDNA, direct sequencing of mRNA, and sequencing of polymerase chain reaction (PCR) products amplified from reverse-transcribed mRNA. Analysis of the first 20 or so N-terminal amino acids of the derived sequence for the chicken enzyme shows that they are devoid of acidic amino acids, contain several hydroxy amino acids, and can be predicted to form a positively charged, amphipathic helix, all of which are characteristic properties of mitochondrial targeting signals. A comparison of the N-terminus of chicken GS with the N-termini of cytosolic mammalian GSs indicates that at least three amino acid replacements may have been responsible for converting the N-terminus of the cytosolic mammalian enzyme into a mitochondrial targeting signal. Two of these, His15 and Lys19, result in additional positive charges, as well as in changes in hydrophilicity. Both could have resulted from third-base-codon substitutions. A third replacement, Ala12, may contribute to the helicity of the N-terminus of the chicken enzyme. The N-terminus of the cytosolic chicken brain GS (positions 1-36) was found to be identical to that of the liver enzyme. The complete sequence of chicken retinal GS is also identical to that of the liver enzyme. GS is coded by a single gene in birds, so these sequence data suggest that, unlike the situation in other tissue-specific compartmental isozymes, differential targeting of avian GS to the mitochondrial or cytosolic compartments is not dependent on the sequence of the primary translation product of its mRNA but may involve some other tissue-specific factor(s).


Glutamate-induced energetic stress in hippocampal slices: evidence against NMDA and glutamate uptake as mediators. The introduction of exogenous glutamate to normally respiring hippocampal slices produced substantial reductions in ATP, phosphocreatine (PCr) and intracellular pH (pHi) when the concentration exceeded 1 mM. These changes were not prevented by addition of MK-801 (an NMDA receptor antagonist), nor were they mimicked by NMDA or high potassium. In addition, the glutamate-induced metabolic alterations were not prevented by addition of aspartate-b-hydroxymate or sodium substitution by choline, both of which should inhibit high-affinity sodium-dependent glutamate uptake. These results suggest that glutamate alone can produce marked energetic stress in neural tissue, even when glucose and oxygen are maintained at control levels; and that the energetic stress does not appear to be specifically mediated by NMDA-induced depolarization, or by high-affinity uptake of glutamate.


Thy-1 modulation and cell proliferation at early steps of intrathymic bone marrow cell differentiation. Intrathymic (IT) transfer of bone marrow (BM) precursor cells in sublethally irradiated hosts has been widely used to study T cell differentiation and maturation. In this report we have used double congenic mice Ly 5.1 Thy 1.1 (host) and Ly 5.2 Thy 1.2 (donor) and detected cycling Ly 5.2+ BM cells by in vivo bromodeoxyuridine incorporation, before induction of the Thy 1.2 antigen. Until Day 9 post-transfer, some donor type cells express a high level of Thy 1.2 together with macrophage and granulocyte markers. A few days later, a Thy 1.2low population transiently B220+ was detected. Thereafter, donor type cells expressed an intermediate Thy 1.2 brightness; this population then persisted and surpassed the other subsets. Our findings permitted to establish a relationship between cell cycle and Thy 1 fluorescence intensity according to the sequence: Thy 1low resting, Thy 1low cycling, Thy 1high cycling, Thy 1high resting. Moreover, we have shown that cells from the myeloïd and B lineages can, in vivo, transiently express the Thy 1 antigen, develop and differentiate within the thymus microenvironment.


Molecular typing of Helicobacter pylori by chromosomal and plasmid DNA organization. Diverse strains of Helicobacter pylori were examined in order to initiate a molecular epidemiological typing scheme for this agent of human gastritis. Twelve differently-sized plasmids from 1.8 to 63 kbp were identified in those strains harbouring extrachromosomal DNA. Recombinant DNA probes were cloned randomly from the chromosome of the (plasmid-free) type strain (NCTC 11637), and used to probe genomic Southern blots for restriction site variation in and around homologous loci. Genus-specific probe DNAs were obtained which grouped strains on the bases of DNA base substitution or rearrangements. On the basis of the four probes examined, all strains exhibited intraspecific chromosomal divergence, indicating that H. pylori is highly diverse genetically, but nonetheless susceptible to chromosome and plasmid molecular typing.


Molecular identification of the gene responsible for congenital nephrogenic diabetes insipidus. Antidiuretic hormone (arginine vasopressin) binds to and activates V2 receptors in renal collecting tubule cells. Subsequent stimulation of the Gs/adenylyl cyclase system promotes insertion of water pores into the luminal membrane and thereby reabsorption of fluid. In congenital nephrogenic diabetes insipidus (CNDI), an X-linked recessive disorder, the kidney fails to respond to arginine vasopressin. Here we report that an affected male of a family with CNDI has a deletion in the open reading frame of the V2 receptor gene, causing a frame shift and premature termination of translation in the third intracellular loop of the receptor protein. A normal receptor gene was found in the patient's brother. Both the normal and the mutant allele were detected in his mother. A different mutation, causing a codon change in the third transmembrane domain of the V2 receptor, was found in the open reading frame of an affected male but not in the unaffected brother belonging to another family suffering from CNDI.


Centractin is an actin homologue associated with the centrosome. Actin is one of the most ubiquitous, abundant and well-conserved proteins of eukaryotes, participating in many crucial cellular processes including the maintenance of cell shape, motility and cell division. Actins from the most divergent sources still share amino-acid identities in excess of 70% (ref. 3). This may well explain why low-abundance homologues of actin have been difficult to isolate. Genes encoding distant relatives of actin in budding and fisson yeast have now been cloned. We report here the discovery of a vertebrate actin-like protein, which we name centractin. A full-length complementary DNA clone was isolated whose sequence reveals amino-acid identities with actin of over 50%, increasing to more than 70% when conservative amino-acid changes are considered. Northern analysis and western blotting indicate a ubiquitous tissue and species distribution. Morphological and biochemical criteria show that centractin is associated with centrosomes.


Effects of the local application of 3-PPP and sulpiride enantiomers into the nucleus accumbens or into the ventral tegmental area on rat locomotor activity: evidence for the functional importance of somatodendritic autoreceptors. The present series of experiments was performed in order to determine the relative role of presynaptic and somato-dendritic autoreceptors for the sedative effects produced by systemically administered dopaminergic agonists. Thus, the effects of intracerebral administration of 3-(3-hydroxyphenyl)-N-n-propylpiperidine (3-PPP), or sulpiride, enantiomers on spontaneous locomotor activity was investigated in rats. It was found that the local application of (-)3-PPP, but not (+)3-PPP, into the nucleus accumbens (1.25-80.0 micrograms, bilaterally) produced a suppression of the locomotor activity, whereas the local application of the two enantiomers into the ventral tegmental area resulted in a suppression of the locomotor activity in the same dose range. Thus, the full dopamine D2 agonist (+)3-PPP produced suppression of locomotor activity only after local application into the somato-dendritic region, suggesting that in terminal areas postsynaptic receptor stimulation effectively counterbalanced the functional consequences of presynaptic receptor stimulation. The sulpiride enantiomers both produced a suppression of locomotor activity after local application into the accumbens (0.2-5.0 micrograms, bilaterally). In the ventral tegmental area, however, (-)sulpiride administration (0.2-5.0 micrograms, bilaterally) resulted in an increased locomotion, whereas the (+)enantiomer produced no effect or, at the highest dose (5.0 micrograms), a suppression of the locomotor activity. These observations indicate that for a dopamine D2 antagonist, the postsynaptic receptor blockade in the terminal region, resulting in behavioral suppression, not only counteract compensatory effects produced via the presynaptic receptor in this region, but also to a great extent overshadow the functional consequences of somatodendritic autoreceptor blockade.(ABSTRACT TRUNCATED AT 250 WORDS)


Distinct pathways for beta-adrenoceptor-induced up-regulation of muscarinic acetylcholine receptors and inhibitory G-protein alpha-subunits in chicken cardiomyocytes. Exposure of cardiomyocytes from chicken embryos for 3 days to the beta-adrenoceptor agonist, isoproterenol, lead to a down-regulation of beta-adrenoceptors by about 70% and to a decrease in isoproterenol-stimulated adenylyl cyclase activity by about 40% (homologous desensitization). In addition, the isoproterenol treatment induced an increase in the level of muscarinic acetylcholine receptors by about 30% and an increase in pertussis toxin-catalyzed ADP-ribosylation of two about 40 kDa proteins, most probably alpha-subunits of the inhibitory G-protein (Gi), by about a factor of two (heterologous desensitization). The purpose of the present study was to characterize the role of beta-adrenoceptor-dependent and -independent mechanisms in heterologous desensitization of adenylyl cyclase. Therefore, the effect of pretreatment with the beta-adrenoceptor antagonist, propranolol, with the partial agonists, celiprolol and xamoterol, and with the beta-adrenoceptor-independent adenylyl cyclase activators, prostaglandin E1 and forskolin, on beta-adrenoceptors, muscarinic acetylcholine receptors and pertussis-toxin-catalyzed ADP-ribosylation of G-protein alpha-subunits was studied. Pretreatment of the cardiomyocytes for 3 days with xamoterol or celiprolol, but not with propranolol, induced a small decrease in beta-adrenoceptor number and in isoproterenol-stimulated adenylyl cyclase activity by about 15-20%. Exposure to prostaglandin E1 and forskolin lead to a more pronounced decrease in beta-adrenoceptor binding and in isoproterenol-mediated adenylyl cyclase stimulation by about 40-60% (heterologous desensitization). An increase in the level of muscarinic acetylcholine receptors, similar to that induced by isoproterenol exposure, was only observed after pretreatment with the partial agonists, celiprolol and xamoterol, but not after pretreatment with the beta-adrenoceptor-independent agonists, prostaglandin E1 and forskolin, nor after pretreatment with propranolol. In contrast, prostaglandin E1 and forskolin exposure lead to a similar increase in pertussis toxin-catalyzed ADP-ribosylation of about 40 kDa G-proteins as isoproterenol exposure whereas treatment with propranolol, celiprolol and xamoterol had no or only a very small effect on pertussis toxin substrates. In summary, the data suggest that, similar as shown for homologous desensitization, cyclic AMP-dependent and -independent mechanisms are also involved in heterologous desensitization of adenylyl cyclase stimulation. The beta-adrenoceptor-induced upregulation of muscarinic acetylcholine receptors and of the alpha-subunits of pertussis toxin-sensitive G-proteins, most probably of Gi, seem to be mediated via distinct pathways.


Systemic administration of baclofen and the GABAB antagonist, CGP 35348, does not affect GABA, glutamate or aspartate in microdialysates of the striatum of conscious rats. Previous in vitro experiments have shown that the GABAB agonist, baclofen, and the antagonist, CGP 35348, respectively, decrease and increase the autoreceptor-mediated release of GABA in brain slices and synaptosomes. Since it is not clear whether these autoreceptors are operative in vivo, an attempt was made to reproduce these results in brain dialysis experiments, knowing that only positive results would permit a conclusion in view of the doubts expressed in the literature with respect to the origin of extracellular GABA. Because of older reports of an inhibitory action of baclofen on the in vitro release of glutamate, which might be ascribed to the action of presynaptic GABAB heteroreceptors, extracellular glutamate and aspartate were also measured. Neither (-)-baclofen, administered systemically at a dose of 20 mg/kg i.p., nor the GABAB antagonist, CGP 35348 (300 mg/kg i.p.) had significant effects on basal overflow of GABA, glutamate, or aspartate nor on that evoked by 100 mmol/l K+ in the striatum of the conscious, freely moving rat. To ascertain this result, (-)-baclofen was also administered between two K+ stimulations, so that the first stimulation could serve as an intraindividual control of the second. The compound did not significantly affect K+ evoked overflow of any of the three transmitter amino acids under these conditions. It must be emphasized that these data do not exclude the operativity of presynaptic GABAB auto- and heteroreceptors in vivo. They only suggest that this question must, in all probability, be addressed by other techniques than brain dialysis.


Effect of alpha 2-adrenoceptor agonists on the expression of morphine-withdrawal in rats. Previous studies using clonidine indicate that alpha 2-adrenoceptors are involved in suppressing opiate-withdrawal symptoms. However, clonidine may act as a partial agonist at alpha 2-adrenoceptors and it also possesses significant alpha 1-receptor agonist activity. The aim of this study was to determine the role of alpha 2-adrenoceptors in the expression of opiate withdrawal signs using morphine-dependent rats. A range of agonists were selected for study on the basis of their differential preferences for alpha-adrenoceptors. Hooded Wistar rats were made physically dependent on morphine (s.c. injection of an emulsion releasing a total of 250 mg/kg of morphine base over 48 h). Test drugs were injected s.c. followed by naloxone (10 mg/kg i.p.) 20 min later. The incidence of 5 selected withdrawal signs was recorded during the following 20 min. The alpha 2-adrenoceptor agonists displayed different profiles of activity. Azepexole (1-10 mg/kg) reduced all signs. Clonidine (80-800 micrograms/kg) reduced all signs except paw shakes while guanfacine (25-250 micrograms/kg) reduced all except jumping and diarrhoea. Talipexole (0.1-1 mg/kg) reduced all signs except diarrhoea which was not affected and jumping which was markedly enhanced. UK 14,304 (80-800 micrograms/kg) reduced jumps, potentiated paw shakes but did not affect body shakes, teeth chattering or diarrhoea. The results suggest that there are subpopulations of alpha 2-adrenoceptors that modulate the expression of opiate withdrawal signs and/or that some of the drugs used affect receptors other than alpha 2-adrenoceptors.


Role of alpha 1A adrenoceptor subtype in production of the positive inotropic effect mediated via myocardial alpha 1 adrenoceptors in the rabbit papillary muscle: influence of selective alpha 1A subtype antagonists WB 4101 and 5-methylurapidil. In order to elucidate the contribution of alpha 1A subtype to the positive inotropic effect mediated by myocardial alpha 1 adrenoceptors, the influence of the alpha 1A selective antagonists WB 4101 and 5-methylurapidil on the alpha 1-mediated positive inotropic effect (induced by phenylephrine in the presence of a beta adrenoceptor blocking agent bupranolol) was assessed in the isolated rabbit papillary muscle. WB 4101 (10(-9)-10(-7) mol/l) shifted the concentration-response curve of the alpha 1-mediated positive inotropic effect to the right in parallel, but the slope of Schild plot did not meet the competitive antagonism: WB 4101 shifted the curve by log one unit at 10(-9) mol/l, whereas it did not cause further shift at higher concentrations of 10(-8) and 10(-7) mol/l. WB 4101 did not affect the beta adrenoceptor-mediated positive inotropic effect. 5-Methylurapidil (10(-9) to 10(-7) mol/l) shifted the curve of alpha 1-mediated positive inotropic effect to the right and downwards in a concentration-dependent manner; the slope of Schild plot calculated at the level of 20% of the maximum response to phenylephrine was close to unity. 5-Methylurapidil at 3 x 10(-7) mol/l abolished the alpha 1-mediated positive inotropic effect. In addition, 5-methylurapidil inhibited the beta adrenoceptor-mediated positive inotropic effect in the same concentration range as it antagonized the alpha 1-mediated positive inotropic effect, indicating that 5-methylurapidil is not selective for myocardial alpha 1 adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)


Pharmacokinetic interaction between carbamazepine and neuroleptics after combined prolonged treatment in rats. This study investigates how neuroleptics of phenothiazine or thioxanthene structure influence the pharmacokinetics of carbamazepine. Experiments were carried out on male Wistar rats. Carbamazepine and the neuroleptics were administered i.p., separately or together, for 2 weeks in the following daily doses (mg/kg): carbamazepine 15 during the 1st week of treatment and 20 during the 2nd week of treatment, promazine 10, chlorpromazine 2, perazine 10, chlorprothixene 2, flupenthixol 0.5. One hour after the last injection of carbamazepine and/or the neuroleptic, samples of blood plasma and brain were taken to determine the concentrations of carbamazepine and two of its metabolites: 10,11-epoxide and trans-10,11-diol. The neuroleptics increased the concentration of carbamazepine in plasma and in brain, but tended to decrease (with the exception of chlorpromazine) the concentration of the epoxide and increased the concentration of trans-10,11-diol. Metabolic in vitro studies did not show any significant differences between rats treated with carbamazepine alone and those treated with carbamazepine plus neuroleptic in the rates of the carbamazepine epoxidation, of 10,11-epoxide hydrolysis or of 1-naphthol glucuronidation.


Pertussis toxin abolishes the inhibition of Ca2+ currents and of noradrenaline release via alpha 2-adrenoceptors in chick sympathetic neurons. Effects of alpha 2-adrenoceptor agonists on whole-cell Ca2+ currents and 3H-noradrenaline release were investigated by applying the patch-clamp technique and electrical field stimulation to cultured embryonic chick sympathetic neurons. A 24-h exposure of the sympathetic neurons to pertussis toxin (100 ng/ml) abolished both the alpha 2-adrenoceptor-mediated inhibition of Ca2+ currents and the modulation of noradrenaline release caused by noradrenaline (1 mumol/l; in the presence of 10 mumol/l cocaine) or the alpha 2-adrenoceptor agonists 5-bromo-6-(2-imidazolin-2- ylamino)quinoxaline (UK 14,304, 10 mumol/l) and clonidine (10 mumol/l). These results suggest that the alpha 2-autoreceptor-mediated inhibition of noradrenaline release from chick sympathetic neurons operates through the modulation of Ca2+ channels via pertussis-toxin-sensitive GTP-binding-proteins.


Adaptation of mouse leukemia cells L1210 to vincristine. Evidence for expression of P-glycoprotein. A vincristine resistant cell line was obtained from mouse leukemia cells L1210 by long-term adaptation in a medium with stepwise increasing concentrations of vincristine. By Western blotting using monoclonal antibody C219, positive signal on the presence of P-glycoprotein was observed in the resistant cells. Moreover, hybridization of mRNA from vincristine resistant cells with radiolabeled MDR1 cDNA probe gave evidence about the expression of MDR1 gene. The observed resistance may be depressed by application of "chemosensitizers" such as (1) calcium entry blockers (verapamil and nifedipine); (2) neuroleptics (trifluorperasine) and (3) local anesthetics (lidocaine) directly to the grow medium. Any significant effect in O2 consumption as well as incorporation of [U-14C]-glucose by the sensitive or resistant cells was not detected in the absence of vincristine. Presence of vincristine induced increasing velocity of O2 consumption by resistant cells from 2.5 +/- 0.3 to 3.3 +/- 0.2 microliters/min.10(6) cells, and, on the other hand, decreasing O2 consumption by sensitive cells from 2.3 +/- 0.2 to 1.7 +/- 0.1 ml/min.10(6) cells. The presence of vincristine induced less potent decrease in glucose incorporation by resistant cells in comparison with values which were observed in sensitive cells.


Effect of chronic renal failure with and without secondary hyperparathyroidism on the activities of synaptosomal tyrosine hydroxylase and monoamine oxidase. Norepinephrine (NE) content, release and uptake by brain synaptosomes are reduced in chronic renal failure (CRF), and this has been attributed to the state of secondary hyperparathyroidism. The decrease in NE content in CRF could not be explained by changes in NE uptake or release since in normal circumstances, NE content usually remains unchanged despite fluctuation in NE uptake and release. Since NE content is determined by its production and degradation, we examined the effect of CRF with and without excess parathyroid hormone (PTH) on the Michaelis-Menton constant (Km) and Vmax of tyrosine hydroxylase (TH), the rate-limiting enzyme for NE production, and monoamine oxidase (MAO), an enzyme involved in NE degradation of brain synaptosomes. Brain synaptosomes from rats with a 21-day CRF have a significantly (p less than 0.01) lower Vmax of TH (39.5 +/- 5.3 pmol tritiated H2O/mg protein/min) than that of normal rats (61. +/- 7.5 pmol tritiated H2O/mg protein/min) and a higher Km of MAO (59 +/- 2.9 nM tyramine) than normal animals (46 +/- 1.7 nM tyramine). Parathyroidectomy (PTX) in CRF rats normalized Vmax of TH (54 +/- 4.5 pmol tritiated H2O/mg protein) and Km of MAO (48.4 +/- 2.3 nM tyramine). Cytosolic calcium, [Ca2+]i, in brain synaptosomes is significantly (p less than 0.01) higher in rats with CRF (488 +/- 8.5 nM) than in normal (355 +/- 6.0 nM) or PTX-CRF (360 +/- 8.1 nM) rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Regulation of nucleus accumbens dopamine release by the dorsal raphe nucleus in the rat. The effects of microinfusing L-glutamate, serotonin (5-HT), (+-)-8-hydroxy-2-(di-N-propylamino) tetralin (8-OH DPAT; a 5-HT1A agonist), and muscimol (a GABAA agonist) into the dorsal raphe nucleus on the extracellular levels of 5-HT, dopamine (DA) and their metabolites in the nucleus accumbens were studied in unanesthetized, freely moving, adult male Wistar rats, using the technique of microdialysis coupled with small-bore HPLC. Administration of 0.75 micrograms L-glutamate produced a 25-50% increase (P less than 0.05) in the extracellular levels of both 5-HT and DA. On the other hand, infusion of 8-OH DPAT and, to a lesser extent, 5-HT produced a significant (P less than 0.05) decrease in the extracellular levels of both 5-HT and DA. Muscimol (0.25 or 0.50 microgram) had little effect on the extracellular concentrations of 5-HT or DA following its administration. In general, the extracellular levels of the major metabolites of 5-HT and DA in the nucleus accumbens were not altered by microinfusion of any of the agents. The data indicate that (a) the 5-HT neurons projecting to the nucleus accumbens from the dorsal raphe nucleus can be activated by excitatory amino acid receptors and inhibited by stimulation of 5-HT1A autoreceptors, and (b) the dorsal raphe nucleus 5-HT neuronal system may regulate the ventral tegmental area DA projection to the nucleus accumbens.


Competitive NMDA receptor antagonists raise electrically kindled generalized seizure thresholds. A sensitive method of estimation of generalized seizure thresholds (GSTs) was used to estimate the relative anticonvulsant potencies of four competitive NMDA antagonists against fully amygdala-kindled seizures. All of the antagonists tested showed potent, dose-dependent anticonvulsant activity following focal administration at doses causing no, or only minimal, overt behavioural abnormalities. These doses were similar to those which have previously been shown to inhibit the development of the kindling process i.e. which show antiepileptogenic activity. Two novel, competitive NMDA antagonists, CGP 37849 and CGP 39551, both unsaturated analogues of the NMDA antagonist AP5, showed by far the greatest anticonvulsant potencies (211-fold and 33-fold greater activity than the parent molecule, respectively). Recent reports of oral anticonvulsant activity of these two compounds in both rodent and primate models of epilepsy (12, 13) make them leading candidates for clinical testing as novel antiepileptic agents in man. Previous reports of weak or non-existent anticonvulsant activity of competitive NMDA antagonists in the kindling model of epilepsy most likely result from the use of experimental protocols which are inherently insensitive in detecting drug-induced changes in seizure thresholds.


Comparison of alterations in tyrosine hydroxylase, dopamine levels, and dopamine uptake in the striatum of the weaver mutant mouse. Previous reports have shown that among the markers for the nigro-striatal dopamine (DA) system measured in the striatum, dopamine uptake seems to be more severely affected than the others in the weaver mutant mouse. In the present study we examined DA levels, tyrosine hydroxylase (TH) activity, and high-affinity DA uptake to determine if the DA uptake is most affected when all the measurements are made in the same striatal homogenate in the same laboratory. We found that the DA uptake activity was most altered (93% lower) compared to DA levels (68% lower) and TH activity (64% lower). The DA uptake was so low in the weaver that we could not obtain reliable kinetic parameters. For TH activity we found that the Vmax was 36% lower while the Km for L-tyrosine was 92% higher in the weaver striatum. This lower affinity for substrate suggests that the TH enzyme itself may be altered in the nigro-striatal system of the weaver mutant mouse.


The ontogeny of the uptake systems for glutamate, GABA, and glycine in synaptic vesicles isolated from rat brain. The ontogeny of the uptake of glutamate, GABA and glycine into synaptic vesicles isolated from rat brain has been investigated. The vesicular uptake of the three amino acids increased with developmental age in parallel with synaptogenesis, indicating a functional role of uptake of the amino acids by synaptic vesicles in the nerve terminals. Uptake of the amino acids by plasma membrane particles (synaptosomes) in brain homogenate showed a somewhat different developmental profile. The uptake of glutamate increased markedly with developmental time, while the uptake of GABA showed only a slight increase. Uptake of glycine by plasma membrane particles was very low and therefore not registered. The observed developmental increase in uptake of glycine by synaptic vesicles isolated from brain, supports previous reports indicating that glycine can be taken up by vesicles from non-glycine terminals.