Alpha-methyl-para-tyrosine pretreatment protects from striatal neuronal death induced by four-vessel occlusion in the rat.

Rats were treated with alpha-methyl-para-tyrosine (AMT, 250 mg/kg, i.p), an hydroxylase inhibitor, in order to decrease brain levels of catecholamines. Six hours later, when cerebral dopamine (DA) and norepinephrine were reduced by about 80%, a transient forebrain ischemia of 30 min duration was induced by four-vessel occlusion technique. Evaluation of brain damage 72 hours after ischemia showed that AMT treatment significantly decreased neuronal necrosis in the striatum but had no cytoprotective effect in the CA1 sector of the hippocampus and in the neocortex. AMT treatment reduced mortality within the ischemic period but did not affect either the mortality within the recirculation period or the postischemic neurologic deficit. These results suggest that the striatal cytoprotective effect of AMT is linked to cerebral DA depletion and that excessive release of DA during ischemia or dopaminergic hyperactivity during recirculation play a detrimental role in the development of ischemic cell damage in the striatum.


[The usefulness of dietetic supplementation in feeding after gastric and pancreatic surgeries].

We evaluated the efficacy of an oral artificial supplementation in 22 patients who underwent surgery for gastric or pancreatic cancer. From 8th to 14th postoperative day, 11 patients (cases) received a diet consistent in their REE, and an oral integrator (40% of REE); controls received only the diet. On 7th and 15th day, nutritional and anthropometric parameters were evaluated, and bioelectrical impedance analysis (BIA) was performed to assess body composition. The dietary caloric input was similar in cases (1154 kcal, 86.0% of REE) and controls (1393 kcal, 92.3% of REE). Due to the integrator, cases reached 121.4% of REE (p less than 0.001). The nutritional and anthropometric parameters studied did not show significant variations in the two groups, but BIA showed a decrease of fat mass in controls with respect to cases (p less than 0.02). Our results demonstrate that the oral artificial supplementation was well tolerated, and did not reduce food intake, but induced a significant increase of total caloric input.


Measuring the neuromodulatory effects of drugs in man with positron emission tomography.

Cognitive activation in conjunction with pharmacological challenge was used to demonstrate neuromodulation in man. Using positron emission tomography (PET), measurements of regional cerebral blood flow were made during the performance of memory tasks, before and after the administration of apomorphine (dopamine agonist), buspirone (5-HT1A partial agonist) or placebo. Drug effects on memory-induced increases in regional cerebral blood flow were assessed, on a voxel-by-voxel basis, using statistical parametric mapping. Increases of regional cerebral blood flow in response to the memory challenge were attenuated by apomorphine in the dorsolateral prefrontal cortex and augmented in the retrosplenial region of the posterior cingulate. Conversely, buspirone attenuated blood flow increases in the retrosplenial region. These interactions between drugs and a cognitive challenge can best be interpreted as neuromodulatory effects.


Differential effects of in vivo estrogen administration on hypothalamic growth hormone releasing hormone and somatostatin gene expression.

The aim of this study was to investigate the effect of in vivo estrogen administration on hypothalamic growth hormone releasing hormone (GHRH) and somatostatin (SS) gene expression. We found that estrogen administration (estradiol valerate 250 micrograms/every 3 days, subcutaneously) to male rats induced a decrease in both hypothalamic GHRH mRNA levels and GHRH content, that was significant after 3 and 8 days of treatment. In contrast SS mRNA levels were transiently elevated after 1 and 3 days of estrogen administration, returning to normal values after 8 days of treatment. These data suggest that the existence of sexual dimorphism in GH secretion in the rat could be mediated to some extent by gonadal hormones regulating somatostatin and GHRH gene expression in the hypothalamus.


Developmental changes in enzymatic systems involved in protection against peroxidation in isolated rat brain microvessels.

Activities of 3 enzymes involved in the major detoxification pathway for peroxides were assessed in rat brain microvessels. Between the 7th and 60th day after birth, glutathione peroxidase specific activity remained constant in microvessels, while glutathione reductase specific activity increased from day 14 to day 60. On the other hand, the specific activity of these two enzymes evolved similarly in total brain homogenate: they increased between day 7 and day 30, and then reached a plateau. In contrast, catalase specific activity in microvessels was markedly decreased from day 7 to day 60. A significant decrease in this enzyme specific activity was also observed in brain homogenate during development. However, in microvessels, catalase specific activity remained higher than that of brain homogenate throughout the time period studied. Our results support the idea that enzymatic mechanisms against peroxidative damage are required in early age, and could be potent at the level of the blood-brain barrier.


Regional variation of excitatory and inhibitory amino acid-induced responses in rat dissociated CNS neurons.

Regional differences in glutamate (Glu), aspartate (Asp), gamma-aminobutyric acid (GABA) and glycine (Gly) responses in CNS neurons were investigated by means of the whole-cell mode of the patch-clamp technique. The neurons were freshly dissociated from rat cortex, limbic system (hippocampal CA1 region), diencephalon (ventromedial hypothalamus), medulla (nucleus paragigantocellularis lateralis) and spinal cord (spinal dorsal horn). The current amplitudes induced by Glu and GABA did not show any regional differences whereas those of Asp- and Gly-induced responses were significantly different among CNS regions. The enhancement of Asp response by Gly was observed in all regions, and the facilitatory ratio did not differ among these regions. Even though the NMDA response in cortical neurons was significantly greater than that in spinal neurons, the ratios of NMDA response facilitation by Gly were also the same in both regions. When the current amplitudes induced by individual amino acids were estimated for the unit surface area of respective neurons (current density), the Glu, Asp and Gly responses showed regional heterogeneity whereas the GABA response did not.


Congenital ectopic vas deferens with hypospadias.

A case of an ectopic vas deferens of an undescended testis is presented which was associated with hypospadias. These abnormalities came to light because of the systematic approach we have adopted for the investigation of hypospadias in our hospital.


Efficient isolation and mapping of rad genes of the fungus Coprinus cinereus using chromosome-specific libraries.

We have constructed cosmid libraries from electrophoretically separated chromosomes of the basidiomycete Coprinus cinereus. These libraries greatly facilitate the isolation of genes by complementation of mutant phenotypes and are particularly useful for map-based cloning strategies. From a library constructed from two co-migrating C.cinereus chromosomes, we isolated a clone that complements the C.cinereus rad9-1 mutation. Examination of this clone showed that it complements both the repair and meiotic defects of this mutant. Restriction fragment length polymorphism mapping using a portion of this clone showed that it maps to the rad9 locus. In addition, a single copy of transforming DNA is sufficient to complement the rad9-1 defects. Thus, we believe we have cloned the rad9 gene itself. We also used a chromosome-specific library and backcrossed isolates to rapidly identify a cosmid clone which is tightly linked to the rad11 locus and is therefore a suitable starting point for a chromosome walk. These rapid methods of gene mapping and isolation should be applicable to any organism with separable chromosomes.


cDNA cloning of a novel heterogeneous nuclear ribonucleoprotein gene homologue in Caenorhabditis elegans using hamster prion protein cDNA as a hybridization probe.

The mammalian prion protein (PrPc) is a cellular protein of unknown function, an altered isoform of which (PrPsc) is a component of the infectious particle (prion) thought to be responsible for spongiform encephalopathies in humans and animals. The evolutionary conservation of the PrP gene has been reported in the genomes of many vertebrates as well as certain invertebrates. In the genome of nematode Caenorhabditis elegans, the sequence capable of hybridizing with the mammalian PrP cDNA probe has been demonstrated, predicting the presence of the PrP gene homologue in C.elegans. In this study, Southern analysis with the hamster PrP cDNA (HaPrP) probe confirmed the previous observation. Moreover, Northern analysis revealed that the sequence is actively transcribed in adult worms. Thus, we screened C.elegans cDNA libraries with the HaPrP probe and isolated a cDNA that hybridizes to the same sequence in C.elegans that hybridized with the HaPrP probe in the Southern and Northern analyses. The deduced amino acid sequence of this cDNA, however, is substantially homologous with heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins rather than mammalian PrPc. The hnRNPs contain the glycine-rich domain in the C-terminal half of the molecule, which also seemed to be in PrPc at the N-terminal half of the molecule. Both of the glycine-rich domains are composed of tracts with high G + C content, indicating that these tracts may due to the hybridizing signals. These results suggest that this cDNA clone is derived from a novel hnRNP gene homologue in C.elegans but not from a predicted PrP gene homologue.


Calf thymus RF-C as an essential component for DNA polymerase delta and epsilon holoenzymes function.

By using a complementation assay that enabled DNA polymerase delta and DNA polymerase epsilon to replicate a singly-DNA primed M13 DNA in the presence of proliferating cell nuclear antigen (PCNA) and Escherichia coli single-stranded DNA binding protein (SSB), we have purified from calf thymus in a five step procedure a multipolypeptide complex with molecular masses of polypeptides of 155, 70, 60, 58, 39 (doublet), 38 (doublet) and 36 kDa. The protein is very likely replication factor C (Tsurimoto, T. and Stillman, B. (1989) Mol. Cell. Biol. 9, 609-619). This conclusion is based on biochemical and physicochemical data and the finding that it contains a DNA stimulated ATPase which is under certain conditions stimulated by PCNA. Together RF-C, PCNA and ATP convert DNA polymerases delta and epsilon to holoenzyme forms, which were able to replicate efficiently SSB-covered singly-DNA primed M13 DNA. Calf thymus RF-C could form a primer recognition complex on a 3'-OH primer terminus in the presence of calf thymus PCNA and ATP. Holoenzyme complexes of DNA polymerase delta and epsilon could be isolated suggesting that these enzymes directly interact with the auxiliary proteins in a similar way. Under optimal replication conditions on singly-DNA primed M13 DNA the DNA synthesis rate of DNA polymerase delta was higher than of DNA polymerase epsilon. Based on these functional date possible roles of these two DNA polymerases in eukaryotic DNA replication are discussed.


Structure of the gene encoding hepatocyte nuclear factor 1 (HNF1).

Genomic clones have been isolated that cover the entire gene for the transcription factor HNF1 (hepatocyte nuclear factor 1). This protein governs the expression of many genes, synthesized in the liver in a tissue-specific manner. We have determined the intron/exon structure of the HNF1 gene, which is strictly conserved between rat and mouse and estimate that it spans not more than 40kb in the rat genome. Whereas most homeoprotein genes do not contain introns within the homeodomain, HNF1 displays an intron between the regions encoding the second and the third helices. We discuss possible evolutionary mechanisms leading to this homeobox intron/exon pattern.


Variable inhibition of cell-free translation by HIV-1 transcript leader sequences.

The 5' ends of all human immunodeficiency virus type I (HIV-1) transcripts have the potential to coordinately regulate translation of HIV-1 mRNAs. Conflicting observations of the translational impact of these sequences in various systems stimulated these analyses of translation in reticulocyte lysates. We report a sensitive, rapid, quantitative, and inexpensive cell-free translation assay in which translational efficiency is monitored by enzymatic assay of the translation products. Using this assay and conventional radiolabeling assays, we demonstrate that the HIV-1 transcript leader inhibits downstream translation and that the stem-loop structure is required. Under our assay conditions, this inhibition occurs predominantly in cis and is not mediated by the 68 kD, interferon-induced, double-stranded RNA-activated kinase (p68). However, under other assay conditions the HIV-1 leader may activate p68 and inhibit translation in trans. We show that variation between individual preparations of cell-free extracts can dramatically alter the magnitude of the translational inhibition by the HIV-1 leader. Further, we provide evidence that a heat-labile factor is required for efficient translation of transcripts containing the HIV-1 leader. These observations provide a foundation for identifying factors required for translation of HIV-1 transcripts.


Alpha- and beta-adrenoceptors in hypertension: molecular biology and pharmacological studies.

Recent years have witnessed astonishing progress in our understanding of the molecular basis of adrenoceptor structure, function and regulation and revealed an unexpected heterogeneity of adrenoceptors demonstrating the existence of at least 11 subtypes. This paper discusses the implications of these advances on studies regarding a specific role of adrenoceptors in the development of genetic hypertension. The available data indicate that among the alpha-adrenoceptor subtypes the alpha 2A-adrenoceptor is the most likely candidate for an alteration specifically linked to genetic hypertension in the animal model of the spontaneously hypertensive rat and possibly in some patients. Alterations of other alpha-adrenoceptor subtypes may be specific for some forms of genetic hypertension but are unlikely to play an important role for blood pressure regulation. Most beta-adrenoceptor alterations appear to occur secondary to blood pressure elevation independently of whether hypertension has occurred on a genetic basis or not. Moreover, the mechanisms regulating alpha- and beta-adrenoceptor responsiveness upon prolonged agonist exposure may be altered in hypertension and thereby contribute to the pathophysiology of this disease.


Serotonergic receptors and drugs in hypertension.

The possible role of 5-hydroxytryptamine (5HT) and 5HT-receptors in hypertension, already suggested by Page in 1954, has been subject to a renaissance of interest owing to the development of antihypertensive drugs which interact with 5HT-receptors. These drugs, like ketanserin, urapidil and flesinoxan are used as tools to study the role of 5HT and its receptors in hypertension. Some arguments would plead in favour of a certain role of 5HT and 5HT-receptors in the pathogenesis and maintenance of hypertension: hyperresponsiveness of blood vessels from hypertensive patients and animals to 5HT-induced constriction; the antihypertensive/vasodilator activity of the 5HT2-receptor antagonist ketanserin; enhanced sensitivity of platelets from hypertensives to 5HT. However, there are also several arguments which do not support a causal role of 5HT in hypertensive disease: 5HT is not a generally accepted pressor agent, whereas its concentration in the circulating blood is subthreshold; the 5HT2-receptor antagonist ketanserin is the only agent of this type which lowers blood pressure, other 5HT2-receptor blockers (ritanserin; LY 53587) being inactive. The various data and arguments available do not unequivocally support a relevant role of peripheral 5HT and its receptors in hypertensive disease. 5HT2-receptor blockade may, however, have a favourable effect on the microcirculation under pathological conditions. The stimulation of central 5HT1A-receptors by drugs like urapidil, 8-OH-DPAT or flesinoxan, has been demonstrated to induce peripheral sympathoinhibition and a fall in blood pressure. This mechanism appears to be a novel target for centrally acting antihypertensives, clearly different from that of clonidine and related drugs, which are centrally acting alpha 2-adrenoceptor agonists.


Five years' experience of prenatal diagnosis of cystic fibrosis in the former U.S.S.R.

From a total of 490 cystic fibrosis (CF) high-risk families under supervision (mostly Russian Slavs from the European part of the country), DNA data including both direct screening for some CF gene (CFTR) mutations (delF508, G551D and 1677delTA) and allelic polymorphism studies with tightly CF linked DNA markers were collected from 261 families. All full families (129) and 86 CF families with a decreased index child were found to be either fully (42 per cent) or partially (40 per cent) informative for DNA analysis. Prenatal diagnosis (PD) was carried out in 161 CF families. Microvillar enzyme (MVE) assay was applied to all 140 PD at the second trimester either as a single test (88) or in conjunction with DNA analysis (52). The frequency of false-negative results of the MVE assay was 1.3 per cent and that of false-positive results, as judged by the albumin meconium test, was 5.0 per cent. Ambiguous results of MVE analysis were found in 30 cases, 12 of which were verified by DNA analysis. Molecular diagnosis of CF at the first trimester was carried out in 21 cases and four pregnancies were terminated. Altogether, 39 pregnancies with a predicted high risk of CF fetuses were terminated. The low average frequency of delF508 in CF chromosomes of Russian Slavs (50 per cent), its remarkable inter-population variation, and the significant proportion of at-risk families without an affected child determine the necessity of combined molecular and biochemical (MVE assay) approaches for efficient prenatal diagnosis of CF in the former U.S.S.R.


[Drug resistance genes].

Among the different mechanisms of multidrug resistance, the overexpression of the mdr1 gene has been actively investigated during the last 5 years. This gene encodes a 170 kDa protein, named P-gp, a member of a transporter superfamily, the ABC (ATP Binding Cassette) proteins. P-gp actively expels out of the tumoral cell different drugs like anthracyclins, vinca alkaloids, epipodophyllotoxins. The involvement of mdr1 gene in clinical drug resistance is now demonstrated, and several trials using P-gp modulators and chemotherapy are going on in resisting tumors. Other intrinsic drug resistance mechanisms, such as increase of intracellular glutathione content or decrease of topoisomerase activity, could be involved in clinical drug resistance.


Root resorption following traumatic dental injuries.

Permanent teeth are usually not attacked by osteoclasts despite their situation in a site where active bone resorption constantly takes place as a result of local and systemic osteoclast activating factors. This fact points to antiresorption factors residing in both the periodontal ligament (PDL) and the pulp. Concerning the PDL homeostasis factor (i.e. permanency of a separation between the alveolar socket and the root surface and protection of the root surface against osteoclastic activity), recent studies have shown that this factor, at least with respect to trauma and wound healing, is linked to, and probably resides in, the cementoblast layer and/or the cells next to this layer. If there is loss of this tissue integrity, root resorption may occur; especially if non-PDL derived cells gain access to the site. With respect to the pulp, no systematic research has been performed regarding the homeostasis of this structure (i.e. permanency of the pulpal organ with its specific anatomy and functional stability). In evaluating the events where resorption does occur, it appears that the loss of tissue components within the pulp (including odontoblasts) implies a risk of root canal resorption if nonpulpally derived cells gain access to the site. Root resorption following traumatic dental injuries, whether located along the root surface or within the root canal appears to be a sequel to wound healing events, where a significant amount of the PDL or pulp has been lost due to the effect of acute trauma. The goal of these processes is removal of injured tissue from zones of trauma, space creation for neovascularization or control of infection. Irrespective of the goal, these processes have a potential for root resorption. The type of tissue repair, i.e. repair originating from the dental pulp, the PDL or bone or a combination, seems to be of importance in determining the risk of root resorption during the healing process.


Response of blood serum constituents to production of and recovery from a kwashiorkor-like syndrome in the young pig.

Twenty-six 3-week-old genetically obese pigs were fed in two experiments to determine the serum chemistry profile during severe protein malnutrition and repletion. Severe protein deficiency was produced in pigs fed the high-fat, low-protein diet (growth failure, rough hair, low serum total protein and albumin). In Experiment 1, blood was sampled from the anterior vena cava of each pig five times during depletion and three times during repletion to determine serum total cholesterol, high density lipoprotein (HDL)-cholesterol, triglycerides, total protein, albumin, glucose, Ca, inorganic P, Mg, Na, K, Cl, total bilirubin, urea N, creatinine, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyltransferase. In Experiment 2, blood was sampled weekly for 8 weeks for serum total cholesterol, HDL-cholesterol, triglycerides, albumin, glucose, Ca, P, Mg and alkaline phosphatase. HDL-cholesterol was increased (P less than 0.01) and albumin was decreased (P less than 0.01) in protein-deficient pigs in both experiments. Creatinine, total bilirubin, gamma-glutamyltransferase, alanine aminotransferase, and aspartate aminotransferase were elevated in protein-deficient pigs compared with controls after 7 weeks of depletion. Inorganic P (P less than 0.01), Ca (P less than 0.01), and Mg (P less than 0.05) concentrations were depressed in protein-depleted pigs compared with controls in both experiments. After 8 weeks of repletion in Experiment 1, all elements except inorganic P were similar in the two groups. Short-term, severe, protein malnutrition affected lipid, electrolyte, and structural mineral metabolism and indices of liver function in the absence of parasites, diarrhea, and infection. The effects were reversed after 8 weeks of repletion. We conclude that the elevated serum cholesterol in protein deficiency is related primarily to an increase in the HDL fraction.


Cardioprotective effects of the vasodilator/beta-adrenoceptor blocker, carvedilol, in two models of myocardial infarction in the rat.

The purpose of this study was to evaluate the cardioprotective effects of carvedilol, a beta-adrenergic blocker and vasodilator, in two models of ischemic myocardial damage in the rat. Following coronary artery occlusion for 0.5 h and reperfusion for 24 h (MI/R group), left ventricular (LV) injury was determined by planimetric analysis of triphenyltetrazolium chloride-stained tissue, and polymorphonuclear leukocyte infiltration was assessed by measuring myeloperoxidase (MPO) activity. In the vehicle-treated MI/R group, infarct size was 14.2 +/- 1.3% of the LV (n = 16), and MPO activity was increased to 2.8 +/- 0.7 from 0.14 +/- 0.03 U/g tissue in the vehicle-treated sham-occluded group (p less than 0.01). Carvedilol (1 mg/kg i.v., 15 min prior to coronary artery occlusion and at 3.5 h following reperfusion) reduced myocardial infarct size to 7.5 +/- 1.2% of the LV (n = 14; p less than 0.01) and attenuated the increase in MPO activity to 1.4 +/- 0.4 U/g tissue (p less than 0.05). A lower dose of carvedilol (i.e. 0.3 mg/kg i.v.) did not limit myocardial infarct size or the increase in MPO activity. In a model of permanent coronary artery occlusion, 24-hour survival was reduced from 85% in sham-occluded animals (n = 38) to 44% in the vehicle-treated MI group (n = 84; p less than 0.01). In comparison to the vehicle-treated MI group, carvedilol (0.3 mg/kg i.v., 15 min prior to coronary artery occlusion and 1 mg/kg 4 h after occlusion) improved survival by 55% (n = 64; p less than 0.05, compared to the vehicle-treated MI group), whereas the same dose of propranolol (n = 42) had no significant effect on survival. These results indicate that carvedilol reduces myocardial ischemia/reperfusion injury, and significantly improves survival in a permanent coronary artery occlusion model of myocardial infarction.


Effect of pinacidil on spontaneous and evoked contractile activity.

Uninhibited bladder contractions have been associated with a variety of bladder dysfunctions including outlet obstruction, neurogenic bladder, incontinence, and other neurologic and nonneurogenic bladder disorders. One class of compounds that is gaining popularity and support for the treatment of hyperreflexia is potassium channel openers, such as pinacidil and cromakalim. In general, these agents act by hyperpolarizing the smooth muscle membrane, resulting in an increase in membrane stability which in turn would be expected to inhibit spontaneous and evoked contraction. It is the purpose of this study to compare the potency and selectivity of pinacidil at inhibiting both hyperreflexia in vivo, and several forms of in vitro contractile stimulation in the rabbit. The following is a summary of the results. (1) Pinacidil is an effective inhibitor of hyperreflexia in the in vivo rabbit model. (2) Pinacidil is a substantially more potent inhibitor of the amplitude of the hyperreflexia than the frequency. (3) Pinacidil was substantially more potent at inhibiting the contractile response to 2-Hz stimulation than to 32-Hz stimulation, but was equally effective at inhibiting field stimulation of the bladder base and body. (4) Pinacidil was significantly more potent at inhibiting the peak response to field stimulation than the rate of tension generation. (5) Pinacidil was equally potent and effective at inhibiting the phasic and tonic components of the response to field stimulation. (6) Pinacidil was a more potent inhibitor of methoxamine stimulation of the bladder base than bethanechol stimulation of the bladder body. (7) Pinacidil was a noncompetitive or mixed inhibitor of both methoxamine and bethanechol stimulation, whereas it was a competitive inhibitor of KCl stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)


Receptor protection studies with phenoxybenzamine indicate that a single alpha 1-adrenoceptor may be coupled to two signal transduction processes in vascular smooth muscle.

Full alpha 1-adrenoceptor agonists, such as (-)-norepinephrine, produce vasoconstriction in the rat aorta primarily through the mobilization of intracellular stores of calcium, whereas partial alpha 1-adrenoceptor agonists, such as (-)-dobutamine, produce vasoconstriction primarily through the translocation of extracellular calcium. The different pools of calcium utilized by full and partial alpha 1-adrenoceptor agonists have been proposed to result from the activation of different alpha 1-adrenoceptor subtypes. The irreversible alpha 1-adrenoceptor antagonist, phenoxybenzamine, selectively eliminates only that component of an alpha 1-adrenoceptor-mediated response in the rat aorta that is dependent upon the mobilization of intracellular stores of calcium. In order to determine whether in the rat aorta there exist two distinct alpha 1-adrenoceptor subtypes linked separately to the mobilization of intracellular and extracellular calcium, we utilized the full and partial alpha 1-adrenoceptor agonists, (-)-norepinephrine and (-)-dobutamine, respectively, and the irreversible antagonist, phenoxy-benzamine, as pharmacologic tools in a classical receptor-protection study to probe these alpha 1-adrenoceptor-mediated vasoconstrictor process(es). Our logic was that if the intracellular and extracellular pools of calcium were coupled to different alpha 1-adrenoceptor subtypes, then only (-)-norepinephrine, and not (-)-dobutamine, would protect against alpha 1-adrenoceptor alkylation by phenoxybenzamine, since phenoxybenzamine only eliminates the process that depends on intracellular calcium. Alternatively, if both (-)-norepinephrine and (-)-dobutamine produce a similar degree of alpha 1-adrenoceptor protection against phenoxybenzamine, our results would suggest that a single alpha 1-adrenoceptor subtype exists which activates both the translocation of extracellular calcium and the mobilization of intracellular calcium. Phenoxybenzamine (30 nM) abolished contractions of the rat aorta produced by (-)-norepinephrine, as expected. Pretreatment of the tissues with either (-)-norepinephrine or (-)-dobutamine, at concentrations that produced equivalent degrees of alpha 1-adrenoceptor occupancy, resulted in equal protection against alkylation of alpha 1-adrenoceptors by phenoxybenzamine, arguing against the existence of two distinct alpha 1-adrenoceptor subtypes in the rat aorta. These results are consistent with our previous hypothesis that two different signal-transduction processes may be activated in the rat aorta by a single alpha 1-adrenoceptor population, with the intrinsic efficacy of the agonist determining which signal-transduction process is activated.


Dynorphin receptor changes in hippocampus of the spontaneously hypertensive rat.

Dynorphin receptor binding sites in hippocampal membrane preparations were assessed in spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats at 4, 8, 12 and 16 weeks of age. At 4 weeks of age, before hypertension is manifested, SHRs had significantly more hippocampal dynorphin receptor binding sites than WKY controls. At 8, 12 and 16 weeks of age, however, when hypertension is seen, SHRs showed significantly fewer hippocampal binding sites than WKY rats. No receptor affinity changes for dynorphin were seen between the two strains of rats at any age. These results suggest that hippocampal receptor changes involving the opioid system may play a role in the central component of blood pressure control.


[Drug management and therapy resistance. A cross-sectional study of schizophrenic patients in a large psychiatric hospital].

In a cross-sectional study 473 schizophrenic patients of a great psychiatric hospital were served. It was looked for differences in patients' characteristics and pharmacotherapy according to the therapeutic-functional structure of the hospital (admission-Unit, sociotherapeutic and long-time wards). Differences in drug-management became evident on basis of a regimen, which applies high doses (number of psychotropic drugs, chlorpromacin-units) in general, despite recommendations in the literature. The treatment refractory patients in the long-time wards are found to be heterogeneous and the authors suggest alternative pharmacological and psychosocial interventions.


[Neuroleptic malignant syndrome--a case report on diagnosis, differential diagnosis and therapy of a life threatening complication of treatment with neuroleptics].

The neuroleptic malignant syndrome is a potentially lethal consequence of treatment with neuroleptics. It is possible that a patient, suffering from neuroleptic malignant syndrome now, had no such illness in the past, while being treated with neuroleptics; furthermore he may respond well to rechallenge with the original agent in the future. The malignant neuroleptic syndrome of a 29 year old man, whom we treated with benperidole, is described. The course of the disease and the differential-diagnosis is stated. The ways of treatment with bromocriptine, dantrolene sodium and amantadine, as described in the literature, are presented. As the neuroleptic malignant syndrome ist rare many cases should be collected in order to enhance the knowledge of the etiology and the pathogenesis of that life--threatening complication of treatment with neuroleptic drugs.


[Acute dyskinesia as the cause of jaw dislocation].

A case of an acute dyskinesia of a young, schizophrenic woman caused by neuroleptic therapy is reported. The acute dyskinesia was the reason for a complete luxation in the jaw-joint. The patient showed phenomenons (brain-atrophy, cognitive dysfunction, negative symptoms) which are discussed to be connected with acute dyskinesia. They also are a danger-signal regarding the beginning of a tardive dyskinesia.


[Effect of combined pharmaco- and psychotherapy in schizophrenia ambulatory care on rehospitalization incidents and treatment costs].

For a total of 89 outpatients of a schizophrenia outpatient clinic, the times of hospitalization up to 5 years before treatment in this institution, have been compared to the frequency of rehospitalization within up to 5 years of posthospital care. This treatment contained between 1 1/2 and 2 years neuroleptic medication (one half as long-term prophylaxis, the other half as interval medication) and psychotherapy in form of single talk of partly in groups for patients and their relatives. Thru this, the duration of hospitalization, before 10 weeks by average per anno, could be reduced to 2.1 weeks yearly. Time-intensive and multi-dimensional posthospital care able to reduce the duration of hospitalizations essentially. This has also a positive influence on the total costs of treatment. The positive effect lasts only in part, by the end of posthospital care the times of rehospitalization increase slightly, especially by repeated sick patients.


[Quality assurance in the psychiatric hospital--joint medical-pharmaceutical visits].

The authors report on two years of experience collected during jointly performed medico-pharmaceutical visits in a psychiatric hospital. From the viewpoint of quality assurance and quality control such a procedure has now gained topical importance. Attention is drawn to problematic drug regimens and interactions between drugs.


Attentional fatigue following breast cancer surgery.

Attentional fatigue usually follows intense use of mental effort and is manifested as a decreased capacity to concentrate, that is, to direct attention. The purpose of this study was to examine the capacity to direct attention in persons with cancer during the initial phase of illness. The sample consisted of 32 women without cognitive or affective disorders who underwent surgery for localized (Stage I or II) breast cancer. Subjects manifested attentional deficits of varying intensity on a battery of tests of directed attention on the day before discharge from the hospital, which was a mean of 3 days following mastectomy or breast conservation surgery. Unexpectedly, the two surgical groups did not differ significantly in attentional capacity and functioning. Attentional test scores were not significantly correlated with narcotic pain medication interval, mood state, or self-ratings of attentional functioning. However, as number of days postsurgery increased, attentional performance decreased. The theoretical basis for further examination of attentional fatigue in people with cancer or other life-threatening illnesses is discussed.


A seroepidemiological survey of antibodies to HTLV-I/HTLV-II in selected population groups in Paraguay.

Between March 1987 and November 1989 a cross-sectional serological survey was conducted on 884 residents of Paraguay to obtain data on the prevalence of antibodies to human T-cell leukemia virus type I/II (HTLV-I/II). Sera from 8/884 individuals (0.9%) were positive, confirmed by Western blotting and radioimmunoprecipitation (RIPA). This study shows that HTLV-I/II is very rare (or absent) among the general (healthy) population (0/338) and ethnic Japanese (0/227) in Paraguay. However, it can be detected at a rate of 2-3% in prostitutes (4/178) and homosexuals (4/117), suggesting sexual transmission as an important route for spread of HTLV-I/II in Paraguay.


A seroepidemiological study of HTLV-1 infection in Nepal.

A seroepidemiological survey of antibodies to human T-lymphotropic retrovirus type-1 (HTLV-1) was carried out among 413 residents of Chitwan, Dhapakhel and Katmandu in Nepal. Donor screening was first carried out by the gelatin particle agglutination (PA) tests and positive sera were retested by an improved PA test, indirect immunofluorescence (IF) and Western blotting (WB). Nine sera showed positive reaction in the first PA screening. Among these positive sera, 1 serum was positive in the improved PA test and the IF test but negative in the WB test. This study suggests that the prevalence of HTLV-1 in Nepalese people is negligible.


Maintenance of in vivo tolerance by persistence of antigen.

T cells of the immune system respond only to foreign antigens because those cells with reactivity for self proteins are either deleted during their development or rendered nonresponsive (anergic). The maintenance of the nonresponsive state was found to require the continual exposure of the anergic T cells to antigen. When anergic T cells were removed from the self antigen by adoptive transfer to a mouse strain lacking the antigen or by in vitro culture, nonresponsiveness was reversed and the anergic cells returned to normal functional status.


[Testicular autotransplant and laparoscopic orchiectomy in a case of bilateral adult cryptorchism].

B.L. a 27 year old bilaterally cryptorchid patient underwent right testicular autotransplantation in the presence of a quite normal testis. After one year the patency of microsurgical anastomosis was confirmed by means Doppler flowmetry and scrotal echography demonstrated the presence into the scrotum of a testis provided of a normal echogenicity. Left laparoscopic orchiectomy was planned. A small semilunar skin incision was made just below the rime of the umbilicus. Veress needle was introduced: as soon as the needle pierced the parietal peritoneum, its spring mechanism was released allowing the sharp needle point to retract leaving only the blunt tip protruding. Carbon dioxide gas was insufflated through the side part of the Veress needle until adequate abdominal distension was achieved. After having removed the Veress needle, the laparoscope on its sharp-pointed trocar was introduced into the peritoneal cavity and left testis was easily localized. Four trocars were introduced up to proceed to laparoscopic orchiectomy. The patient was discharged two days after. In our opinion in the presence of a bilateral cryptorchism in the adult, is better to plan a monolateral autotransplantation. After having verified the long-term result of microsurgery we can decide if a contralateral orchiectomy has to be planned.


[The content of gastrin, bombesin and somatostatin in the blood and gastric juice of patients with duodenal and gastric peptic ulcer].

Ninety patients suffering from peptic ulcer and 25 healthy subjects were examined for the content of gastrin, bombesin and somatostatin in blood and gastric juice. Among patients with duodenal ulcer, 2 groups were distinguished: group I included patients in whom peptic ulcer occurred before 30 years; the majority of the patients manifested blood hypergastrinemia, a decrease of bombesin concentration and normal somatostatin concentration; gastric juice was characterized by a lowering of somatostatin concentration and unchanged gastrin concentration; group II was made up of patients who developed peptic ulcer after 30: in the majority of the patients, gastrin concentration was reduced under basal conditions, after loading it was unchanged; in part of the patients, blood somatostatin concentration was elevated, in 16 in exacerbation and in 19 in remission; in the remainder, it was unchanged. The concentration of bombesin in blood remained unchanged. In gastric juice, gastrin concentration was increased only after histamine administration, somatostatin concentration was unchanged whatever the disease stage. In patients with gastric ulcer, gastrin concentration in blood was elevated only under basal conditions, being unchanged in gastric juice irrespective of the disease stage. Meanwhile, the concentration of bombesin was lowered both under basal conditions and after insulin administration, the concentration of somatostatin was decreased both in blood and gastric juice whatever the disease stage.


Neural tube and other developmental anomalies in the guinea pig following maternal hyperthermia during early neural tube development.

Guinea pigs were exposed to hyperthermia for 1 hr once or twice on day 11, 12, 13, or 14 (E11-E14) of pregnancy. The mean rectal temperatures were elevated by 3.4 degrees C-4.0 degrees C. This treatment resulted in a marked elevation of rates of resorption and developmental defects in embryos examined at day E23. The defects observed were those affecting the neural tube (NTD) (exencephaly, encephaloceles, and microphthalmia), kyphosis/scoliosis, branchial arch defects, and pericardial edema. Embryos with NTD and kyphosis/scoliosis have not been found among newborn guinea pigs to date following maternal heat exposure on days E12-E14. It appears that embryos with these defects are filtered out by resorption or abortion by days E30-E35.


Modulation of cyclophosphamide mutagenicity by vitamin C in the in vivo rodent micronucleus assay.

The modulatory effect of vitamin C (Vit C) on the mutagenic effect of the antineoplastic drug cyclophosphamide (CP) was assessed in the in vivo micronucleus test in Swiss mice. Simultaneous oral administration of Vit C with i.p. administration of CP was found to decrease the frequency of micronucleated polychromatic erythrocytes elevated by CP. Vit C exhibited a significant antimutagenic effect over a wide dose range (1.56-200 mg/kg). The dose-response relationship was highly significant. These results demonstrated the ability of the in vivo micronucleus test to detect in vivo modulation of CP mutagenicity by Vit C. Our earlier results and those from other laboratories also indicate that this model system is suitable for primary in vivo screening of modulation of mutagenesis.


Tobramycin-induced changes in renal histology of fetal and newborn Sprague-Dawley rats.

Effects on renal development were studied using tobramycin (TBM) as a model compound. Pregnant Sprague-Dawley rats were injected i.p. with TBM at 30 or 60 mg/kg body weight/day on gestational days (GD) 10-19. Kidneys from dams and conceptuses were examined on GD 20 and on postnatal day (PD) 9. The dosing regimen caused in dams moderate proximal tubular alterations and increased concentrations in serum creatinine. Fetal kidneys showed granularity and swelling of proximal tubule cells at the 30 mg/kg dose, poor glomerular differentiation at the 60 mg/kg dose, increased glomerular density at both doses, and no changes on macroscopic examination at either dose. In newborns were observed a moderate developmental delay and tubular lesions at the higher dose, and dose-related increases of glomerular density and relative medullary area at both doses. All findings were more pronounced in males. A maturational disruption of the tubular structures possibly leading to increased glomerular density was attributed to TBM exposure during renal organogenesis in the rat.


Evaluation in a battery of in vivo assays of four in vitro genotoxins proved to be noncarcinogens in rodents.

2-Chlorethanol, 8-hydroxyquinoline, 2,6-toluenediamine, and eugenol, previously found to behave as genotoxins in in vitro systems and as noncarcinogens in rodents, were evaluated for their ability to induce genotoxic effects in vivo. Rats were given by gavage a single or two successive doses equal to one-half the corresponding LD50, killed at different times after treatment, and examined for the following end points: the frequency of both micronucleated polychromatic erythrocytes in the bone marrow and micronucleated hepatocytes (after partial hepatectomy); the in vivo-in vitro induction of DNA fragmentation, as measured by the alkaline elution technique, and of unscheduled DNA synthesis, as measured by autoradiography, in hepatocyte primary cultures. The two latter end points were also evaluated after in vitro exposure of hepatocytes to log-spaced subtoxic concentrations. 2-Chloroethanol, 8-hydroxyquinoline, and eugenol never produced effects indicative of genotoxic activity. The same happened with 2,6-toluenediamine, with the exception of a significant increase over controls in the amounts of DNA damage and repair displayed by hepatocyte cultures obtained from rats given two 1/2 LD50 separated by a 24 h interval. Our results, which, apart the above mentioned exception, are in concordance with the rodent carcinogenicity results, contribute to underline the role of in vivo short-term tests for the detection of potential genotoxic carcinogens.


Diphenylhydantoin is not genotoxic in a battery of short-term cytogenetic assays.

5,5-Diphenylhydantoin (DPH) is an antiepileptic drug associated with an increase in malformations in infants born to women taking DPH during pregnancy. Positive and negative results have been reported by various investigators for in vivo and in vitro chromosome aberration (CAB) assays, in vivo and in vitro sister chromatid exchange (SCE) assays, and in vivo micronucleus tests (MNT). In this laboratory, DPH was tested in an in vitro CAB assay using Chinese hamster ovary cells with and without an S-9 activation system, an in vivo SCE assay in female CD-1 mice, an in vivo MNT, using both male and female CD-1 mice, and a transplacental micronucleus test. The results from this comprehensive battery of cytogenetic tests were uniformly negative and support a conclusion that the known teratogen, DPH, is not clastogenic.


Monoclonal antibody-based enzyme-linked immunoassays for the measurement of palytoxin in biological samples.

Mouse monoclonal and rabbit polyclonal antibodies were produced against conjugates of keyhole limpet hemocyanin and chemically defined palytoxin haptens. Palytoxin haptens were produced by derivatization of the primary amino group with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate or succinimidyl 3-(2-pyridyldithio)propionate. Selected antibodies were used to develop five palytoxin-specific enzyme-linked immunoassay formats for the quantitation of palytoxin in biological matrices, including crude extracts of Palythoa tuberculosa. The formats developed include an indirect competitive inhibition enzyme-linked immunoassay, two types of direct competitive inhibition enzyme-linked immunoassays, and both indirect and direct sandwich enzyme-linked immunosorbent assays. The sandwich enzyme-linked immunosorbent assays are capable of detecting as little as 10 pg palytoxin per test, but may be subject to matrix interference. The direct competitive inhibition enzyme-linked immunoassays detect as little as 30 pg palytoxin per test with a total assay time of only 4 hr. The enzyme-linked immunoassays do not cross-react with the other marine toxins tested, but do cross-react with certain non-toxic, treated preparations of palytoxin. The enzyme-linked immunoassays were used to quantitate palytoxin in P. tuberculosa extracts and to monitor toxin isolation. These enzyme-linked immunoassay systems can substitute for the mouse bioassay of palytoxin, providing a rapid, sensitive, and accurate means of toxin detection.


Beachside preparation of jellyfish nematocyst tentacles.

A comparison of methods for preparing a jellyfish nematocyst suspension from sea nettle (Chrysaora quinquecirrha) fishing tentacles at the beachside was conducted. Autolysis of the tentacle followed by straining and sedimentation on ice was found to be a satisfactory technique. This procedure utilized a tea strainer, plastic cup and conical centrifuge tube, all of which could be made available at a minimally equipped laboratory.


Pharmacological prospects for alpha 2-adrenoceptor antagonist therapy.

The discovery of various alpha 2-adrenoceptor subtypes in numerous tissues and studies of alpha 2-adrenoceptor-mediated mechanisms has generated considerable interest in their physiological functions. It has also increased possibilities for the design of new pharmacological tools and for the study of the pharmacological impact of new drugs. Alpha 2-adrenoceptors are located pre- and postsynaptically both in the central noradrenergic pathways and on the autonomic nerve endings. It is difficult to dissociate alpha 2-adrenoceptor-mediated autoregulation, involving presynaptic receptors, from actions dependent on post- and extrajunctional alpha 2-adrenoceptor activation. A lot of alpha 2-adrenoceptors are subject to permanent tonic activation by the sympathetic nervous system. Max Lafontan and colleagues review the major actions of alpha 2-adrenoceptors and consider the sites of impact of alpha 2-antagonists that could initiate further research for putative applications of these drugs. Many of the possible targets for alpha 2-adrenoceptor antagonists have not yet been explored clinically.


[Pattern of choice in preparation of attempted suicide by poisoning--with particular reference to changes in the pattern of prescriptions].

This investigation was initiated in 1990 to investigate the choice of preparation in cases of attempted suicide treated in the Emergency Unit in Odense University Hospital during the past decade. The results show that 1) an increase has occurred particularly among young women in the use of analgesics obtainable without prescription (e.g. 13% of young women employed paracetamol as the toxic agent), 2) the number of persons employed dextropropoxyphene did not decrease and 3) a great increase was observed in the number of cases of poisoning with ketobemodoni chloridum. In 1985, several barbiturates were withdrawn from the Danish Medical Codex and this was followed by a decrease in the number of cases of poisoning among women but not among men. At present, approximately 10% of men who poison themselves still employ barbiturates. The number of cases of poisoning with barbiturates in men is thus not reduced but phenobarbital is the preparation of choice. The authors conclude that more attention and care should be paid when prescribing dextropropoxyphene, ketobemodoni chloridum and phenobarbital and the availability of paracetamol without prescription must be debated critically.


Nitric oxide mediates relaxation in rabbit and human corpus cavernosum smooth muscle.

We investigated in vitro the relaxant effect of exogenous acetylcholine (ACh) and electric-field stimulation (EFS) on rabbit and human corpus cavernosum smooth muscle strips (CC) precontracted with phenylephrine. The effects of EFS and ACh were monitored alone, after muscarinic receptor blockade and after inhibition of nitric oxide (NO) formation with L-N-nitro-arginine (L-NOARG). In rabbit and human CC, both atropine and L-NOARG abolished the relaxant effects of ACh. The relaxant effects of EFS, however, were only slightly reduced by atropine to 97.5 +/- 17.5% in human CC and to 89.0 +/- 6.1% in rabbit CC. L-NOARG further reduced the EFS effects to 0.8 +/- 1.7% in human CC and to 16.2 +/- 8.7% in rabbit CC. In strips obtained from impotent patients with diabetes mellitus, the relaxant effects appeared to be significantly less than in strips from nondiabetic impotent men. Tetrodotoxin blocked the relaxant EFS effects in human and rabbit strips completely. The data indicate the important role of NO in cholinergically induced relaxation of cavernous smooth muscle in rabbits and humans. Our findings support the idea of NO as the nonadrenergic noncholinergic neurotransmitter in penile erection in both species. Rabbit erectile tissue might serve as an in vitro animal model for further investigation.


Alpha-adrenoceptor function before and after chemical sympathectomy in human and feline detrusor muscles.

Isolated bladder segments from man and cat were treated with 6-hydroxydopamine (6-OHDA) in vitro. Chemical sympathectomy was evaluated with fluorescence microscopy and found to be very similar to the effect of 6-OHDA administered in vivo to cats. Isometric smooth muscle contractile responses were induced by field stimulation (FS). The amplitude of the responses increased after denervation. The effects of alpha-adrenoceptor agonists and antagonists on the FS-induced contractile responses were compared before and after treatment with 6-OHDA. The reduction in the contractile responses after the addition of noradrenaline to the feline bladder strips was more pronounced after treatment. Phentolamine induced an increase in contractile responses before treatment, an effect not seen afterwards in human bladder strips but which persisted in feline bladder strips. Selective alpha-adrenoceptor agonists did not alter the contractile responses in denervated strips. It is suggested that the function of the alpha-adrenoceptors in the detrusor is to inhibit neuronally mediated contractile responses of smooth muscle.


Acute poisonings with ethyle loflazepate, flunitrazepam, prazepam and triazolam in children.

Even though acute poisonings with benzodiazepines are extremely common, less is known of the clinical toxicity of recent derivatives, particularly in children. 1,989 cases involving ethyle loflazepate, flunitrazepam, prazepam or triazolam recorded at the Lyons Poison Center and due to 1 compound and associated with clinical symptoms were selected for study. Children less than 16-y of age accounted for 482 cases. Sleepiness, agitation and ataxia were significantly more frequent in the children. Hypotonia was seldom observed but was indicative of severe poisoning. The dangerous toxic dose of these compounds in children is suggested to be 0.78-0.90 mg ethyle loflazepate/kg, 0.26-0.29 mg flunitrazepam/kg, 7.80-9.00 mg prazepam/kg and 0.06-0.07 mg triazolam/kg. These results are in keeping with the relatively low acute toxicity of the older benzodiazepines.


[Third generation beta blockers in the treatment of hypertension].

Beta-blockers of the third generation are hybrid antihypertensive drugs which reduce the blood pressure by two mechanisms--by beta-blocking and by a dilatating action. The author presents experience assembled in Czechoslovakia with bopindolol and nebivolol which belong into the class of these substances. Treatment with beta-blockers is effective also in hypertension in elderly subjects. The author mentions a group of extensive investigations conducted abroad, comparing beta-blockers and diuretics in the treatment of hypertension. From the analysis it is apparent that beta-blockers exert a favourable effect also in primary prevention of ischaemic heart disease. In the conclusion the author deals with the strategy of treatment of hypertension associated with other diseases, using different types of beta-blockers.


[Drug prevention and therapy of acute gastroduodenal lesions (stress ulcers)].

The incidence of acute lesions of the gastroduodenal mucosa is high in intensive care patients. A disturbed balance between aggressive and defensive factors is the cause of the mucosal damage. H2-receptor antagonists reduce gastric acidity, the most important aggressive agent, and are, therefore, still the drugs of choice for the prevention of stress ulcerations. Bleeding from mucosal lesions is the most common complication of stress ulcers. In addition to endoscopic and surgical techniques to stop bleeding, and prevent rebleeding, medical treatment with drugs like omeprazole have gained importance in recent years.


Carrier detection in agammaglobulinemia by X chromosome inactivation analysis.

Using a recently developed strategy to analyze patterns of X chromosome inactivation in cell populations, we found that two mothers and a sister were carriers in three atypical or sporadic cases of patients with agammaglobulinemia, two of whom were brothers. In this study, a phosphoglycerate kinase 1 (PGK1) gene probe was used to detect patterns of methylation of X-chromosome genes. A random pattern of X inactivation was observed in isolated peripheral blood granulocytes. In contrast, one of the two X chromosomes was preferentially active in the Epstein-Barr virus (EBV)-transformed peripheral B cells of the family members of these patients. The volume of the blood specimen could be significantly reduced using EBV-transformed B cell lines which contained multiple clones. The analysis described here can be used to distinguish between X-linked agammaglobulinemia (XLA) and other forms of a- or hypo-gammaglobulinemia as well as to detect the carrier state.


GABA- and glutamate-gated ion channels as molecular sites of alcohol and anesthetic action.

The evidence presented above indicates that GABA- and glutamate-activated ion channels are molecular sites of alcohol and anesthetic action. In view of the important role that these channels play in CNS excitability, it seems likely that the actions of alcohol and anesthetics on these channels contribute significantly to the behavioral effects of these agents. Although the behavioral effects of alcohol and anesthetics may well result from a combination of actions on different ion channels and other molecular sites in the CNS, it is of interest to consider whether the actions of these agents on particular types of ion channels may contribute to particular behavioral effects. In this regard, it should be noted that benzodiazepines potentiate GABAA responses, but do not produce intoxication or general anesthesia in their clinical dose range. Benzodiazepines are widely used clinically, primarily for their anxiolytic actions (26), suggesting that the potentiation of GABAA responses by ethanol and barbiturates may contribute to the anxiolytic effects of these agents. Since kainate and quisqualate channels mediate fast excitatory transmission in the CNS, inhibition of kainate and quisqualate receptor-activated responses would be expected to result in general CNS depression. This suggests that inhibition of kainate and quisqualate receptor-mediated responses may contribute to the general anesthetic effects of ethanol, trichloroethanol and barbiturates. NMDA channels are thought to mediate complex excitatory neural phenomena and cognitive function. In view of this, the observation that ethanol inhibits NMDA receptor-mediated responses over the concentration range that produces intoxication and the correlation between the potency of different alcohols for inhibiting NMDA-activated current and their potency for producing intoxication suggest that ethanol-induced inhibition of NMDA receptor-mediated responses may contribute to the intoxicating effects of ethanol. Although these speculations are no doubt oversimplifications, the recognition that GABA- and glutamate-gated ion channels are molecular sites of alcohol and anesthetic action provides a basis for investigating the molecular mechanisms involved in the action of these agents and the behavioral significance of those actions.


Levocabastine: pharmacological profile of a highly effective inhibitor of allergic reactions.

Levocabastine, selected from a series of cyclohexylpiperidine derivatives protects rats from compound 48/80-induced anaphylactic shock for at least 16 h at the oral dose of 0.0015 mg/kg. At the same dose histamine skin reactions and at slightly higher doses passive cutaneous anaphylactic reactions are inhibited. Blockade of passive cutaneous anaphylactic reactions is obtained with levocabastine, despite absence of peripheral serotonin antagonism and any other known non-specific action that may facilitate inhibition of passive anaphylaxis. In dogs allergic reactions are inhibited at oral doses 40 times lower than ketotifen. In guinea-pigs orally and topically administered levocabastine are remarkably effective against allergic conjunctivitis.


Epidemiology of Plasmodium falciparum in a rice field and a savanna area in Burkina Faso. Comparative study on the acquired immunoprotection in native populations.

A longitudinal study, including entomological, parasitological, immunological and clinical data, was carried out in a rice field and a savanna village in Burkina Faso. In this study, the authors followed the evolution of several parasitological parameters in order to compare the level of immunoprotection in children of these two areas. In particular, the percentages of recently 'infected' or 'recovered' children were calculated, during the interval separating two consecutive surveys. In both areas, parasite densities quickly increased in children from 0 to 14 years old, immediately after the beginning of the transmission period. In savanna, during the rainy season (May-October), parasite densities decreased and the proportion of recently 'recovered' children from 0 to 4 years old (becoming parasitologically negative between two consecutive surveys) was very low. On the other hand, parasite densities decreased and the recovery rate was higher in children from 10 to 14 years old before the end of the rainy season, while the transmission was going on. In the rice field area, Plasmodium falciparum densities decreased only at the end of the transmission period (December) and had the same levels as those found in savanna, in spite of a lower inoculation rate. The second peak of transmission seemed neither to increase the proportion of recovered children, nor to boost the immunoprotection of these children.


Failure of chemoprophylaxis against bovine trypanosomiasis on Galana Ranch in Kenya.

The duration of prophylaxis provided by 1 mg kg-1 bodyweight of homidium bromide was compared with that provided by 1 mg kg-1 bodyweight of isometamidium chloride in a 12 month field trial involving 90 Boran cattle exposed to trypanosome challenge on Galana Ranch in Kenya. Weekly trypanosome prevalences of over 30% were observed during 4 of the 12 months. During these periods of heavy challenge, parasites were detected 2-3 weeks after administration of both homidium bromide and isometamidium chloride. Both prophylactic drugs were administered, on a group basis, eight times over the 12 month trial and in addition individual infections were also treated with diminazene aceturate. Isometamidium chloride provided slightly longer periods of prophylaxis than homidium bromide, 28.4 days compared with 25.4 days. There was a highly significant difference in the productivity of the two groups during a period of poor grazing. 27% of the isometamidium chloride herd died from a severe wasting condition with substantial liver damage evident on post mortem. The condition was not observed in the homidium bromide herd. The surviving animals in the isometamidium chloride herd had a mean annual weight gain of 24 kg less than that recorded in the homidium bromide herd.


Absence of the LiTat 1.3 (CATT antigen) gene in Trypanosoma brucei gambiense stocks from Cameroon.

Antibodies to the variable antigen type (VAT) designated LiTat 1.3 are common in sera from parasitologically confirmed patients with gambian sleeping sickness. For this reason, LiTat 1.3 has been considered a suitable antigen for detecting Trypanosoma brucei gambiense in the Card Agglutination Test for Trypanosomiasis (CATT; Testryp-CATT, Smith Kline-RIT). However, surveys in the T.b. gambiense endemic focus of Fontem in Cameroon have suggested that expression of LiTat 1.3 might be rare or absent. We show here that the gene for LiTat 1.3 was indeed absent from some T.b. gambiense stocks isolated from this focus, and a LiTat 1.3-like gene was present in others. The divergent gene differed from the cloned version of LiTat 1.3. In addition, antibodies to LiTat 1.3 could not be detected in rabbits infected with either of the two kinds of T.b. gambiense from the Fontem area. We suggest that the absence of LiTat 1.3 expression in this focus may have important implications for the epidemiology and control of sleeping sickness, especially if heavy reliance is placed on the CATT.


The antibody response to well-defined malaria antigens after acute malaria in individuals living under continuous malaria transmission.

The IgG and IgM antibody responses to the C-terminal 783 amino acids of the P. falciparum glutamate-rich protein, GLURP489-1271, expressed as an E. coli fusion protein, the IgG response to a 18-mer synthetic peptide EDKNEKGQHEIVEVEEIL (GLURP899-916) representing the C-terminal repeats of GLURP, and a synthetic peptide (EENV)6 representing the C-terminal repeats from Pf155/RESA, were investigated longitudinally in 13 children and 7 adults living under conditions of continuous, intense malaria transmission. Some subjects did not recognize the antigens after malaria infection, and in subjects recognizing the antigens, the responses were often short-lived. In adults, the antibody responses to the GLURP489-1271 fusion protein and the (EENV)6 peptide peaked after 2 weeks, and not all individuals responded to all antigens. The antibody response, even against large fragments of conserved antigens, is not uniformly elicited by natural malaria infection in previously primed donors.


Eye lesions, blindness and visual impairment in the Taraba river valley, Nigeria and their relation to onchocercal microfilariae in skin.

2876 persons in fourteen communities in the Taraba River Valley, Nigeria were examined for eye lesions and tested for visual acuity using the 'tumbling E'. The individuals were also examined for microfilaria of Onchocerca volvulus. More than one-tenth of the population were blind, while another 16.1% had visual impairment. The prevalence of blindness was in excess of 20% in six communities, with one community recording 71.9% blindness rate. All forms of visual involvement increased with age but were similar between sexes. Eye lesions were related to the level of vision. Both eye lesions and vision deteriorate with increase in age. Vision seems to worsen with increase in prevalence and intensity of O. volvulus. Large microfilarial loads were associated with severe eye damage and blindness. These findings indicate that the Taraba river valley could be one of West Africa's worst foci of onchocercal blindness.


Evaluation of haematuria as an indirect screening test for schistosomiasis haematobium: a population-based study in the White Nile province, Sudan.

Haematuria elicited in the history, seen macroscopically or detected by reagent strips, was used as an indirect screening test for Schistosoma haematobium infection in Um-Hani Irrigation Scheme in the White Nile province, Sudan. These approaches were used separately or combined in different sequences. Reagent strips alone detected 81% of cases and 88% of those who excreted 50 egg/10 ml of urine or more. The sequence of observation of gross haematuria followed by screening with reagent strips and then taking history of haematuria had the highest sensitivity of all the orders, 0.87, and it saved 18% of reagent strips. If history and inspection were done first, followed by reagent strips, the sensitivity would be 0.86 and 47% of strips would be saved. The specificity of haematuria as a diagnostic index for schistosomiasis, however, was low.


Effect of maximal medical therapy on refractoriness of unstable angina pectoris.

A group of 125 patients with unstable angina were studied over a 5-year period to define the incidence of refractory unstable angina in the current era of 5-drug medical therapy with intravenous heparin, aspirin, nitrates, calcium antagonists and beta blockers. All patients had greater than 20 minutes of chest pain at rest with reversible electrocardiographic changes occurring in the absence of myocardial infarction. Patients were considered refractory only if chest pain continued despite treatment with maximal 5-drug therapy. At the time of transfer to the center, 65 patients continued to have ischemic chest pain at rest and were considered "medically refractory" by their referring physicians. A more aggressive medical regimen was used, and 54 patients (83%) were rendered chest pain-free. Of the 11 truly refractory patients (8.8%), coronary arteriography revealed an increased likelihood of left main or 3-vessel disease (7 of 11 vs 26 of 114; p = 0.01). In-hospital treatment strategies for the 114 patients stabilized with medical therapy included continued medical therapy (n = 37), coronary angioplasty (n = 46) and bypass grafting (n = 31). The rate of myocardial infarction or death in patients managed medically was 3%. Coronary angioplasty in medically stabilized patients was complicated by an abrupt closure rate of 26%, and a 17% rate of myocardial infarction, death or need for emergency bypass grafting. Medically stabilized patients undergoing bypass grafting had a 9% rate of myocardial infarction or death. Unstable angina truly refractory to current, maximal medical therapy is infrequent (8.8%).(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of acute and chronic ibopamine administration on resting and exercise hemodynamics, plasma catecholamines and functional capacity of patients with chronic congestive heart failure.

The effects of acute and chronic ibopamine treatment on resting and exercise hemodynamics, exercise capacity and plasma catecholamines were evaluated in 25 patients with chronic heart failure, using a double-blind, parallel, placebo-controlled design. During 2 months of therapy with either placebo or ibopamine (100 mg, 3 times daily), 1 patient was withdrawn from each group for worsening heart failure, New York Heart Association functional class improved in 4 patients on ibopamine and in 1 on placebo, and furosemide dose could be decreased in 4 on ibopamine and in no patient on placebo. Acute ibopamine administration induced, in comparison with placebo, a significant increase of cardiac and stroke volume indexes both at rest and peak exercise, with a reduction of systemic vascular resistance. These hemodynamic changes were maintained also after chronic therapy, with no evidence of tolerance development. Exercise capacity (evaluated as peak exercise duration and oxygen consumption, and ventilatory threshold) did not significantly change. Resting and peak exercise norepinephrine plasma levels were significantly reduced after both acute and chronic ibopamine administration. Thus, the hemodynamic and neurohumoral effects of ibopamine make this drug potentially useful for the chronic treatment of congestive heart failure.


The proliferative cell fraction in cytology specimens. A study of human esophageal carcinoma.

Immunostaining of cell cycle-related antigens, especially Ki-67, DNA polymerase alpha, and proliferating cell nuclear antigen, has become an important method to assess the proliferative activity of tumors. These three nuclear antigens were studied by immunohistochemical analysis of cytologic smears. These smears were obtained by scraping the cut surface of 10 cases of esophageal squamous cell carcinoma and were fixed and prepared by different methods. The results were compared with those of tissue sections to apply the immunocytochemical findings of these antigens to cytology specimens. Smears that were placed on Denhardt- or Neoprene-coated slides and subsequently fixed in 4% paraformaldehyde and methanol exhibited the best cell adherence to the slides, had minimal loss of antigenicity, and had good preservation of cell morphologic features for all three antigens examined. The percentage of positive tumor cells in the cytology smear was generally in good agreement with that in the tissue section. For these three antigens, proliferating cell nuclear antigen demonstrated a much higher percentage of positive cells than either Ki-67 or DNA polymerase alpha, in both the smears and the tissue sections. In summary, Ki-67, DNA polymerase alpha, and proliferating cell nuclear antigen can be immunolocalized successfully in cytology smears and may become another parameter to assess the proliferative activity of tumors in the field of diagnostic pathology.


Hormone and autacoid regulation of cAMP production in rat IMCD subsegments.

The inner medullary collecting duct (IMCD) of the rat consists of two structurally and functionally distinct segments, i.e., the initial and the terminal IMCD. To identify factors that may regulate the transport function in the IMCD segments, we assessed whether catecholamines, carbachol, prostaglandin E2 (PGE2), bradykinin, glucagon, calcitonin, parathyroid hormone, or epidermal growth factor affects adenosine 3',5'-cyclic monophosphate (cAMP) production in microdissected tubules in the presence and absence of arginine vasopressin (AVP, 0.1 nM). All experiments were performed in the presence of 3-isobutyl-1-methylxanthine, and cAMP was measured by radioimmunoassay. Epinephrine (greater than or equal to 50 nM) and clonidine (greater than or equal to 1 microM) markedly decreased AVP-induced cAMP levels in both IMCD segments. However, phenylephrine did not show an effect. The inhibitory effect of epinephrine was blocked by yohimbine (50 nM) but not by prazosin (50 nM). In isolated perfused terminal IMCDs, epinephrine inhibited AVP-stimulated urea permeability. Isoproterenol (1 microM), in the absence of AVP, caused a significant increase in cAMP level only in the initial IMCD. Propranolol (1 microM) inhibited this isoproterenol effect, but atenolol did not. Dopamine (less than or equal to 1 microM) had no effect on cAMP levels in either IMCD segment. Carbachol, PGE2, and the various peptide hormones had no effect on cAMP levels (+/- AVP) in either IMCD segment. We conclude that an adrenergic beta 2-receptor is present only in the initial IMCD, where its occupation increases cAMP production. We conclude also that an adrenergic alpha 2-receptor is present in both IMCD segments, where its occupation inhibits AVP-induced cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)


Characteristics and development of myocardial stunning in the pig.

Regional left ventricular function associated with consecutive ischemic periods of 2, 2, 5, 10, and 2 min was recorded by ultrasonic technique in pentobarbital-anesthetized pigs. All systolic and diastolic derangements first appeared after 5 min of ischemia, and all worsened after 10 min of ischemia. Percent systolic segment length shortening reached nadirs 23 (17-29)% (P less than 0.001) and 54 (45-65)% (P less than 0.001) below baseline 30 min after 5 and 10 min of ischemia. During reperfusion all recorded systolic and diastolic variables transiently recovered and then deteriorated with a closely similar time course. This covariance indicates that systolic and diastolic derangements are causally related, and because diastolic compliance was preserved in stunned myocardium we conclude that all derangements largely result from reduced systolic tension development. Transient postischemic hypercontractility followed all occlusions and was not attenuated by beta-blockade and not mimicked by hyperemia alone. Postischemic hypercontractility was greatly enhanced in stunned myocardium, and we hypothesize that more pronounced and sustained postischemic elevation of intracellular Ca2+ concentration explains this observation.


Surgical and pharmacological dissociation of cardiovascular and emetic responses to intragastric CuSO4.

Under control (intact and laparotomized) conditions, arterial pressure of urethan-anesthetized ferrets rose significantly in response to intragastric copper sulfate (CuSO4; 10 ml of 5 mg/ml). CuSO4 was emetic 75-90% of the time, and a cardiovascular response always immediately preceded and accompanied emetic responses. The cardiovascular response was significantly reduced or abolished by hexamethonium (0.35 mg/kg) pretreatment combined with atropine methyl bromide (1 mg/kg). Addition of the alpha 2-receptor antagonist 2,3-dichloro-alpha-methylbenzylamine HCl (DCMB, 10 mg/kg) and the beta-receptor antagonist propranolol (3 mg/kg) to the hexamethonium and atropine combination prevented its reduction of the cardiovascular response. Given individually, these agents did not alter cardiovascular response, nor did yohimbine (0.30 mg/kg), RS(+/-)-zacopride (0.30 mg/kg), or granisetron (0.50 mg/kg). Only RS(+/-)-zacopride significantly reduced incidence of emesis. Atropine methyl bromide alone, with hexamethonium, or with hexamethonium, propranolol, and DCMB significantly delayed the first emetic episode. Conversely, bilateral abdominal vagotomy significantly reduced the number of vomiting animals without affecting cardiovascular response. These results suggest that the neural pathways mediating the two events are not identical. In another series of experiments, a significantly greater proportion of animals vomited in response to intragastric CuSO4 than to intraduodenal CuSO4, suggesting that the primary site of CuSO4 action in the ferret is the stomach.


Excitatory amino acids may mediate nucleus tractus solitarius input to rat parabrachial neurons.

The pontine parabrachial nucleus (PBN) is a recipient of predominantly excitatory input from the nucleus of the solitary tract (NTS). The presence of glutamate-like immunoreactivity at these brain stem sites suggests a role for excitatory amino acids (EAAs) in neurotransmission within the projection. We utilized electrophysiological studies in vivo to examine the ability of specific EAA antagonists, applied locally, to alter glutamate (GLU)-induced and NTS-evoked excitation of PBN neurons. Nonselective EAA antagonist kynurenic acid (KYN), the selective N-methyl-D-aspartate (NMDA) antagonist DL-2-amino-5-phosphonovalerate (APV), and non-NMDA quinoxalinedione group of blockers 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulfamobenzoquinoxaline-2,3-dione (NBQX) were applied by iontophoresis or micropressure ejection from multibarreled pipettes attached to the recording electrode. Extracellular recordings in urethan-anesthetized rats were obtained from 58 PBN neurons that displayed an excitatory response following electrical stimulation within the NTS. Poststimulus histogram data revealed that NTS-evoked excitation could be reversibly blocked by KYN, APV, and CNQX in 21/37 (57%), 11/21 (52%), and 10/19 cells (53%), respectively. Both NMDA and non-NMDA antagonists reversibly attenuated or blocked GLU-evoked excitation in 21 of 29 PBN neurons. These observations suggest a role for both NMDA and non-NMDA receptors in mediating the excitatory input from NTS to the PBN.


Influence of levocabastine suspension on ciliary beat frequency and mucociliary clearance.

The effects of levocabastine, a new fast-acting, highly potent H1-antagonist, on nasal ciliary epithelial function were investigated in an in vitro and in vivo study. In the in vitro study, a suspension of levocabastine in Locke-Ringer solution was applied to 10 bioptic specimens of ciliated human adenoid tissue. Each specimen was exposed to the test solution for 60 min. Ciliary beat frequency (CBF) was recorded with a photoelectric recording device at 10-min intervals. There were small, insignificant decreases in CBF, which were minimal compared to that observed with ciliotoxic agents. In the in vivo study, 8 healthy volunteers were given, intranasally, one droplet of the levocabastine suspension. Mucociliary transit time (MTT) was measured by placing a saccharin particle drenched in indigo carmine in the nose just below the top of the concha and measuring the time until appearance of the dye in the pharyngeal cavity. No statistically significant differences were found in the MTT before and after application of the levocabastine suspension. The studies thus indicate that nasally administered levocabastine does not interfere with ciliary beat frequency and mucociliary function.


Effects of atipamezole, an alpha 2-adrenoceptor antagonist, on the anesthesia induced by barbiturates and medetomidine.

We studied the effects of atipamezole, an alpha 2-adrenoceptor antagonist, on hypnosis induced by medetomidine, an alpha 2-adrenoceptor agonist (1 mg/kg IP), and pentobarbital (40 mg/kg IP) by testing the righting reflex in the rat. The duration of antinociception was assessed with repeated pinch tests. Medetomidine-induced hypnosis and antinociception were inhibited by atipamezole at doses greater than 0.1 mg/kg. Atipamezole restored the righting reflex at a dose ratio that was 1:10 or more to that of medetomidine used to induce hypnosis. Subcutaneous atipamezole (1.5 mg/kg) increased the duration of hypnosis induced by pentobarbital (40 mg/kg) and pentobarbital + medetomidine (0.3 mg/kg). Hypnosis induced by methohexital (60 mg/kg IP) was also prolonged by atipamezole. The capacity of atipamezole to reverse the effects of medetomidine is also reduced in the presence of barbiturates. Thus, atipamezole should be used only at low doses to reverse a combination anesthesia induced by barbiturates and medetomidine.


Which patients benefit from adding theophylline to beta 2-agonist treatment in severe acute asthma?

The aim of this investigation was to study whether certain patients benefit from adding theophylline to the beta 2-agonist treatment of acute asthma. The study group comprised 101 patients who were taking oral theophylline. The patients received inhaled salbutamol (albuterol) (0.15 mg/kg x 2) (n = 53) or IV salbutamol (5 micrograms/kg) (n = 48). Aminophylline (3 mg/kg) was infused intravenously 60 minutes after the start of the salbutamol treatment. The mean increase in peak flow (PEF) after the aminophylline infusion was 22 +/- 33 L/min or 4% +/- 6% of the predicted value (mean +/- SD). A change in PEF correlated negatively to plasma theophylline before treatment (P less than .01) and positively to a change in plasma-theophylline after treatment (P less than .05). All patients with an increase in PEF of more than 10% of the predicted value (n = 14) after the theophylline infusion had plasma-theophylline levels before treatment of below 7.5 mg/L (41 mumol/L). No significant difference in the change in PEF after the theophylline infusion was found between patients who had received inhaled or intravenous salbutamol. This investigation could indicate that IV theophylline as an additive to beta 2-agonist treatment should be reserved for patients who are either not taking theophylline or who have only taken a low dose before arriving for the emergency treatment of acute asthma.


Pharmacodynamics and pharmacokinetics of mizolastine (SL 85.0324), a new nonsedative H1 antihistamine.

The antihistaminic activity, clinical safety, and pharmacokinetics of mizolastine (SL 85.0324) were studied in a 5-way, double-blind crossover study of ten healthy volunteers with doses of 1 to 75 mg. Inhibition of the histamine-induced wheal and flare showed clear dose-dependent antihistaminic activity beginning from the 2-mg dose with a maximum attained between 10 and 20 mg. The onset of action was rapid (one hour) and the effect persisted for more than 24 hours after a 10-mg dose or more. Mizolastine was well tolerated at doses up to 75 mg; subjective and objective signs of transient sedative activity were not observed at doses below 30 mg. The pharmacokinetic profile (rapid absorption with Tmax congruent to 1 h and elimination T1/2 of about eight hours) parallels the pharmacodynamic activity. Within the considered dose range, the pharmacokinetics was linear with no saturation phenomena.


Inotropic mechanisms of dopexamine hydrochloride in horses.

Mechanisms responsible for the positive inotropic effects of dopexamine were investigated in 8 halothane-anesthetized horses. The hemodynamic effects of increasing infusions of dopexamine (5, 10, 15 micrograms/kg of body weight/min) were determined before and after sequential administration of specific antagonists. Using glycopyrrolate and chlorisondamine, and atenolol and ICI 118,551, muscarinic and nicotinic ganglionic, and beta 1, and beta 2-adrenergic receptor blockade, respectively, was induced. Dopexamine infusions induced increase in heart rate, cardiac output, systolic and mean arterial blood pressure, and maximal rate of left ventricular pressure development (+dP/dtmax). Right atrial pressure and systemic vascular resistance decreased. Parasympathetic and ganglionic blockade attenuated cardiac output, systolic and mean aortic blood pressures, and +dP/dtmax responses to dopexamine infusion. Dopexamine-induced increase in heart rate was potentiated by parasympathetic and ganglionic blockade. beta 1-Adrenergic receptor blockade decreased heart rate, cardiac output, arterial blood pressure, and +dP/dtmax from baseline values and markedly reduced the response to dopexamine infusion. beta 2-Adrenergic receptor blockade induced further decrease in hemodynamic variables from baseline values and completely abolished the cardiostimulatory effects of dopexamine on +dP/dtmax. These data indicate that baroreflex activity, beta 1- and beta 2-adrenergic receptor stimulation may be an important cause of dopexamine's positive inotropic effects in horses.


[Effect of various unsaturated fatty acids on the stability of the erythrocyte membrane in the rat].

In rats restrictive feeding of a half-synthetic diet, with coconut fat as the dietary fat, caused an essential fatty acid deficiency with increased osmotic fragility of erythrocytes against hypotonic saline solutions. A 60% replacement of the coconut fat in the basal diet by pure oleic acid (18:1 n-9) or linoleic acid (18:2 n-6) by 0.6% alpha-linolenic acid (18:3 n-3), eicosatrienoic acid (20:3 n-3) or eicosapentaenoic acid (20:5 n-3) led, in the case of linoleic acid and eicosapentaenoic acid, to a significant decrease in the fragility of rat erythrocytes in comparison with the basal diet. The inefficacy of the alpha-linolenic acid treatment is possibly the consequence of a too low dietary supplementation.


Diagnosis of familial hypercholesterolemia using DNA haplotype analysis in three large families with two hyperlipidemic parents.

Since the cloning of the human LDL receptor (LDLR) gene, familial hypercholesterolemia (FH) can be diagnosed by recombinant DNA technology either using restriction enzyme mapping to detect major rearrangements of the gene or using restriction fragment length polymorphisms (RFLPs) and linkage analysis in family studies. Genotypes and haplotypes of four RFLPs (StuI, ApaII 5', PvuII, NcoI) were used to study the inheritance of the detective LDLR gene in three families. Diagnosis of FH based on the lipid levels alone was not possible because in these kindreds both parents exhibit elevated lipid levels. However, in two families using haplotype analysis, elevated cholesterol levels in certain relatives could be attributed to the inheritance of a defective LDRL gene and thereby distinguished from hypercholesterolemia due to familial combined hyperlipidemia. In the third family where both hypercholesterolemic parents carried a defective LDLR gene, a case of homozygous FH could be excluded in a child by demonstrating the inheritance of a normal LDLR gene.


Characterization of the chromosomal aac(6')-Ic gene from Serratia marcescens.

The DNA sequence of the chromosomal aac(6')-Ic gene from Serratia marcescens, which had been previously cloned (H. M. Champion, P. M. Bennett, D. A. Lewis, and D. S. Reeves, J. Antimicrob. Chemother. 22:587-596, 1988) was determined. High-pressure liquid chromatographic analysis of extracts prepared from Escherichia coli carrying the chromosomal aac(6')-Ic gene on a plasmid confirmed the presence of 6'-N-acetyltransferase activity in this strain, which was suggested by the aminoglycoside resistance profile. DNA sequence analysis of the cloned 2,057-bp PstI fragment revealed several regions of homology to previously characterized sequences from GenBank, including the rpoD and tRNA-2 genes of E. coli. Subcloning experiments confirmed the coding sequence of the aac(6')-Ic gene to be at positions 1554 to 1992. The predicted amino acid sequence of the AAC(6')-Ic protein suggested that it was the third member of a family of AAC(6') proteins which included a coding region identified between the aadB and aadA genes of Tn4000 and an AAC(6') protein encoded by pUO490, which was isolated from Enterobacter cloacae. Primer extension analysis suggested that the -35 region of the aac(6')-Ic promoter overlapped a large palindromic sequence which may be involved in the regulation of the aac(6')-Ic gene. Hybridization experiments utilizing a restriction fragment from the aac(6')-Ic gene showed that all S. marcescens organisms carried this gene whether or not the AAC(6')-I resistance profile was expressed. Organisms other than Serratia spp. did not hybridize to this probe.


Muscarinic receptors in the cerebellar vermis modulate the gain of the vestibulospinal reflexes in decerebrate cats.

1. The Purkinje (P)-cells of the cerebellar vermis, which exert a prominent influence on posture as well as on the gain of vestibulospinal (VS) reflexes, are under the control not only of the classic mossy fibers and climbing fibers which liberate excitatory amino acids as neurotransmitter, but also of cholinergic afferents. The role of these afferents was investigated in precollicular decerebrate cats by using the method of local microinjection of cholinergic agents into appropriate areas of the cerebellar cortex. 2. Unilateral injection into the vermal cortex of the culmen of the non-selective cholinergic agonist carbachol (0.25 microliters at 0.5 micrograms/microliters saline) produced a postural asymmetry, characterized by a slight decrease of the extensor tonus in the ipsilateral forelimb and an increased tonus in the contralateral forelimb. Moreover, the gain of the EMG responses of the ipsilateral and the contralateral triceps brachii to animal tilt increased significantly, while no significant changes in the phase angle of the responses were observed. These effects started 5-10 min after the injection and persisted for at least 2 hours before disappearing. Similar but smaller effects were obtained after injection of eserine, an inhibitor of acetylcholinesterase. Thus, the effects could be produced by increasing the naturally present amount of acetylcholine (ACh). 3. The changes in posture and gain of the VS reflexes described above utilized in part at least muscarinic receptors, since effects similar to those induced by carbachol injection were also obtained after unilateral microinjection into the vermal cortex of the culmen of the muscarinic agonist bethanechol (0.25 microliters at 0.1 micrograms/microliters). On the other hand opposite effects, characterized by an increased postural activity in the ipsilateral forelimb associated with a decreased activity in the contralateral forelimb, as well as by a reduced gain of the EMG responses of the triceps brachii of both sides to animal tilt were observed in other experiments after local microinjection of the muscarinic antagonist scopolamine (0.25 microliter at 4-8 micrograms/microliters saline). Evidence for muscarinic supersensitivity was obtained following repetitive injections of scopolamine into the cerebellar vermis. 4. The area which upon injection of the cholinergic agents modified the postural activity as well as the gain of the VS reflexes was located within the third and/or the fourth folium rostral to the fissura prima (culmen), at the laterality of 1.4-1.8 mm with respect to the midline.(ABSTRACT TRUNCATED AT 400 WORDS)


Cloning and expression of the mouse homolog of the human alpha 2-C2 adrenergic receptor.

Three subtypes of alpha 2 adrenergic receptors have been identified in the human and rat. The subtype located on human chromosome 2 (alpha 2-C2) is unique in that it is expressed mainly in the peripheral tissues and lacks sites for N-linked glycosylation. We isolated the gene encoding the mouse homolog of the human alpha 2-C2 adrenergic receptor (M alpha 2-2H). The deduced amino acid sequence of the M alpha 2-2H shows 82% and 96% identity to the human alpha 2-C2 and the rat RNG alpha 2 adrenergic receptors, respectively. Southern blot analysis demonstrated that the M alpha 2-2H was encoded by a single copy gene and was distinct from the mouse homologs of the alpha 2-C4 and alpha 2-C10 adrenergic receptors. When expressed in COS-7 cells, the M alpha 2-2H exhibited a pharmacological profile similar to the human alpha 2-C2 and rat RNG alpha 2 receptors.


Heterogeneous responses of recombinant human thyrotropin receptor to immunoglobulins from patients with Graves' disease.

Non-thyroid mammalian cells, CHO-K1 cells, stably expressing human thyrotropin receptor (CHO-TSH-R cells) were used for the assay of thyroid stimulating antibody (TSAb) activities of IgGs from 24 patients with Graves' disease and we compared them with the values obtained in porcine thyroid cells. A significant positive correlation was observed between the results given by CHO-TSH-R cells (hTSAb) and porcine thyrocytes (pTSAb) (r = 0.94, p less than 0.001). However, we found that hTSAb values of IgGs from 5 patients were extremely different from their hTSAb values. Four out of these 5 IgGs showed strong pTSAb activity but exhibited a weak or negative hTSAb activity. Conversely, one out of 5 autoantibodies was very strong for hTSAb but its pTSAb was low. These heterogeneous responses of recombinant hTSH-R to Graves' IgGs suggest that there exist different types of TSAb and also that the epitope(s) for TSAb may be different from case to case.


Conjugated bile acid uptake by Xenopus laevis oocytes induced by microinjection with ileal Poly A+ mRNA.

Apical membranes of ileal enterocytes contain the major Na+/bile acid cotransporter activity in mammals. Microinjection of guinea pig ileal mucosal Poly A+ mRNA (25 ng) into Xenopus oocytes resulted in 22,23-3H-cholyltaurine uptake at day 3 after injection (453 fmol/oocyte-hr), while control viral mRNA (25 ng) gave an uptake rate of 133 fmol/oocyte-hr. The transport rate increased in direct relationship to the concentration of injected mRNA, cholyltaurine, or Na+ in the incubation media. Uptake of cholyltaurine using rabbit ileal mucosal Poly A+ mRNA was 3891 fmole/oocyte-hr compared to rabbit jejunal-mucosa Poly A+ mRNA (control) injections inducing 728 fmol/oocyte-hr. Such expression of the ileal Na+/bile acid cotransporter may facilitate cloning of this key mammalian gene.


Inhibition of phosphatidylserine synthesis by glutamate, acetylcholine, thapsigargin and ionophore A23187 in glioma C6 cells.

Phosphatidylserine synthesis was studied in glioma C6 cells with [14C]serine and in the presence or absence of agents which increase the level of [Ca2+]i. It was found that glutamate and acetylcholine inhibited this synthesis by up to 40%, whereas thapsigargin and the ionophore A23187 inhibited by up to 70%. The inhibitory effect of thapsigargin and the A23187 was observed in Ca(2+)-free medium. The data show that the inhibition of this synthesis is caused by the Ca(2+)-depletion from endoplasmic reticulum, suggesting that the synthesis of phosphatidylserine occurs on the luminal side of these structures and can be regulated by transmembrane signaling systems.


Cytotoxicity of a new IMP dehydrogenase inhibitor, benzamide riboside, to human myelogenous leukemia K562 cells.

COMPARE computer program suggested that benzamide riboside, BR, 3-(1-deoxy-beta-D-ribofuranosyl)benzamide, should have a similar mechanism of action as that of tiazofurin, an inhibitor of IMP dehydrogenase (IMPDH). This hypothesis was tested in K562 cells in culture. BR was cytotoxic to K562 cells with an IC50 of 2 microM. Incubation of K562 cells with BR resulted in a significant decrease in GMP and GTP levels with a concurrent increase in IMP pools, and with a significant inhibition of IMPDH activity. However, 290-fold higher BR concentration was needed to demonstrate in vitro inhibition of IMPDH activity, suggesting that the agent may require metabolism to exert its action. These results provide evidence that BR is a new inhibitor of IMPDH. This investigation should be helpful to design new analogues having activity against IMPDH.


The primary structure of rat ribosomal protein S25.

The amino acid sequence of the rat 40S ribosomal subunit protein S25 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein S25 has 125 amino acids and has a molecular weight of 13,733. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 19 to 22 copies of the S25 gene. The mRNA for the protein is about 550 nucleotides in length. Rat S25 is homologous to ribosomal proteins from other eukaryotes (human and yeast).


Hypothalamic hormones modulate G protein levels and second messenger responsiveness in GH3 rat pituitary tumour cells.

Thyroliberin (TRH), vasoactive intestinal peptide (VIP) and somatostatin (SRIF) act through receptors that are coupled to guanine nucleotide-binding regulatory proteins (G proteins). Regulation of hormone action may occur at the level of G protein coupling to the receptor or effector systems. In this study we demonstrate that prolonged exposure (for up to 48 hr) of cultured rat pituitary adenoma GH3 cells to these hormones caused homologous and to some extent heterologous attenuation of the adenylyl cyclase (AC) (EC 4.6.1.1) responsiveness. In addition, TRH and SRIF diminished both TRH- and guanosine 5'-[beta gamma-imido]-triphosphate-enhanced phospholipase C (PLC) (EC 3.1.4.3) activity within the same time-course. Measurements of cells membrane levels of Gs protein alpha-subunit (Gs alpha), G(i)-1 alpha/G(i)-2 alpha, G(i)-3 alpha, G(o) alpha and G beta by immunoblotting were performed. TRH and VIP upregulated levels of all G proteins except G(o) alpha and G beta. In contrast, SRIF caused a marked reduction of G beta levels. Thus, TRH and VIP, both acting through Gs, both modulated the alpha-subunit levels of this signal transducer, whereas SRIF, which possibly acts through G(i)-2, did not change the steady state level of G(i)-2 alpha. The actions of TRH, VIP and SRIF are multifaceted at the G protein level, where modulations of subtypes not directly involved in their actions may occur. These findings emphasize the complexity expected to be found in the in vivo situation.