Women among a population at a psychiatric service in Senegal (psychosocial aspects)

Exposure of porin from Escherichia coli to glutardialdehyde followed by SDS-gel electrophoresis in 3% polyacrylamide yielded three bands that were identified in order of decreasing mobility as monomers and two and three cross-linked polypeptide chains. The distribution of protein among the three species for different extents of reaction showed a remarkably good agreement with corresponding values predicted from cross linking theory for an oligomer composed of three identical subunits arranged according to a 3-fold rotation axis. Electrophoresis performed in 7.5% polyacrylamide yielded four bands that were assigned to polypeptide chain monomers, dimers, and two types of trimers carrying two and three intersubunit cross-links. Our findings provide evidence that the central premise of cross-linking theory, viz. that the intersubunit cross-links formed upon exposure of a protein to a bifunctional reagent be governed by the symmetry of the molecule, is valid. Careful interpretation of cross-linking experiments thus proves an effective method to assess the oligomeric structure of a protein and reveal the symmetry underlying the spatial arrangement of the subunits within the molecule.     

The structure and organization of the human erythroid anion exchanger (AE1) gene

The AE1 (anion exchanger, band 3) protein is expressed in erythrocytes and in the A-type intercalated cells of the kidney distal collecting tubule. In both cell types it mediates the electroneutral transport of chloride and bicarbonate ions across the lipid bilayer, and, in erythrocytes, it also serves as the critical attachment site of the peripheral membrane skeleton. We have characterized the human AE1 gene using overlapping clones isolated from a phage library of human genomic DNA. The gene spans approximately 20 kb and consists of 20 exons separated by 19 introns. The structure of the human AE1 gene corresponds closely with that of the previously characterized mouse AE1 gene, with a high degree of conservation of exon/intron junctions, as well as exon and intron nucleotide sequences. The putative upstream and internal promoter sequences of the human AE1 gene used in erythroid and kidney cells, respectively, are described. We also report the nucleotide sequence of the entire 3' noncoding region of exon 20, which was lacking in the published cDNA sequences. In addition, we have characterized 9 Alu repeat elements found within the body of the human AE1 gene that are members of 4 related subfamilies that appear to have entered the genome at different times during primate evolution.     

Defensin stimulates the binding of lipoprotein (a) to human vascular endothelial and smooth muscle cells

There is evidence to suggest that elevated plasma levels of lipoprotein (a) [Lp(a)] represent a risk factor for the development of atherosclerotic vascular disease, but the mechanism by which this lipoprotein localizes to involved vessels is only partially understood. In view of studies suggesting a link between inflammation and atherosclerosis and our previous finding that leukocyte defensin modulates the interaction of plasminogen and tissue-type plasminogen activator with cultured human endothelial cells, we examined the effect of this peptide on the binding of Lp(a) to cultured vascular endothelium and vascular smooth muscle cells. Defensin increased the binding of Lp(a) to endothelial cells approximately fourfold and to smooth muscle cells approximately sixfold. Defensin caused a comparable increase in the amount of Lp(a) internalized by each cell type, but Lp(a) internalized as a consequence of defensin being present was not degraded, resulting in a marked increase in the total amount of cell-associated lipoprotein. Abundant defensin was found in endothelium and in intimal smooth muscle cells of atherosclerotic human cerebral arteries, regions also invested with Lp(a). These studies suggest that defensin released from activated or senescent neutrophils may contribute to the localization and persistence of Lp(a) in human vessels and thereby predispose to the development of atherosclerosis.     

Electron-microscopic radioautography of DNA-synthesizing cells of growing rat smooth muscle tissue

DNA-synthesizing cells of rat stomach muscle tissue following 50% resection of the fundal part, identical cells of the vena cava after disturbance of the blood out flow, and of the appendix on contraction of the ascending part of the large intestine were studied by electron microscopic autoradiography. DNA synthesis in the differentiated myocyte nuclei of the stomach muscle tissue, and of the "activated" myocytes of the stomach and the vein was observed. There were signs of asynchronous DNA synthesis in the nuclei of some smooth muscle cells and fibroblasts.     

The cytoskeleton of the vertebrate smooth muscle cell

Four experiments were conducted to determine the effects of dopamine (DA) antagonists and DA depletions on progressive-ratio responding for food reinforcement. On this schedule, ratio requirement increased by one response after each reinforcer was obtained, and rats were tested in 30-min sessions. Response rates and highest ratio completed were reduced in a dose-related manner by systemic injections of the D1 antagonist SCH 23390, and also by the D2 antagonists haloperidol and raclopride. Drug-treated rats also showed reductions in time to complete the last ratio, demonstrating that they had stopped responding before the end of the session. DA depletions produced by injections of 6-OHDA directly into the nucleus accumbens substantially decreased both the number of responses and the highest ratio completed. The deficits in response number and highest ratio completed induced by DA depletions persisted through the first 3 weeks of postsurgical testing, with some recovery by the fourth week. However, the deficits resulting from dopamine depletions were largely a manifestation of a decrease in response rate; although time to complete the last ratio was significantly reduced by dopamine depletions in the first few days of testing, rats recovered on this measure by the fifth day after surgery. Although previous work has shown that performance on several schedules (e.g., continuous, low value ratios, variable interval) is relatively unaffected by accumbens DA depletions, the present data demonstrate that such depletions do produce a substantial and persistent impairment of progressive ratio response output. Rats with accumbens DA depletions appear to have deficits in maintaining the high work output necessary for responding at large ratio values. The relative sparing of responding on some simple schedules, together with the present progressive ratio results, suggest that rats with accumbens DA depletions remain directed toward the acquisition and consumption of food, but they show deficits in work output for food.     

The anatomical organization of the rat fascia dentata: new aspects of laminar organization as revealed by anterograde tracing with Phaseolus vulgaris-Luecoagglutinin (PHAL)

The rat fascia dentata is characterized by a simple cytoarchitecture and characteristic lamination of afferents. Entorhinal afferents are believed to terminate exclusively in the outer two thirds of the molecular layer, whereas commissural fibers are believed to terminate exclusively in the inner molecular layer of the fascia dentata. A sharp border divides these two major afferent fiber systems and is regarded as the main boundary of the fascia dentata. This concept of a highly laminated brain structure has made the fascia dentata attractive for studies analyzing normal or pathological processes of the brain. Recently, entorhinal as well as commissural fibers have been identified which do not follow the classical lamination of the fascia dentata. Using anterograde tracing with Phaseolus vulgaris-Leucoagglutinin, an entorhino-dentate projection to the molecular layer, granule cell layer, and hilus of the fascia dentata was described. With the same technique, GABAergic commissural fibers to the outer molecular layer of the fascia dentata were revealed and a previously unknown heterogeneity of the commissural projection was demonstrated. These previously unknown fiber systems complicate the interpretation of lesion effects in this brain region and have to be taken into account as possible sources of sprouting fibers following the partial denervation of the fascia dentata.     

The structure and organization of the human carnitine/acylcarnitine translocase (CACT1) gene2

One hundred and eighty-one families with multiple endocrine neoplasia type 2A (MEN-2A) or familial medullary thyroid carcinoma (FMTC) have been investigated for mutations in the ret protooncogene in Germany. In 8 families with FMTC or MEN-2A, no mutation could be detected in the cysteine-rich domain encoded in exons 10 and 11 of the ret protooncogene. DNA sequencing of additional exons (no. 13-15) revealed rare noncysteine mutations in 3 families (codons 631, 768, and 844). In contrast to these rare events, heterozygous missense mutations in exon 13, codons 790 and 791, were found in 5 families (4 with MTC only; 1 family with MTC and pheochromocytoma) and 11 patients with apparently sporadic tumors. Two different mutations in codon 790 (TTG-->TTT, TTG-->TTC; Leu790Phe) and one mutation in codon 791 (TAT-->TTT; Tyr791Phe) created a phenylalanine residue. We conclude that codons 790 and 791 of the ret protooncogene represent a new hot spot for FMTC/MEN-2A causing mutations. With the discovery of these considerably common mutations in codons 790 and 791 and the identification of some rare mutations, 100% of the German FMTC/MEN-2A families could be characterized by a mutation in the ret protooncogene.     

Endothelin converting enzyme (ECE) activity in human vascular smooth muscle

1. We have characterized the human smooth muscle endothelin converting enzyme (ECE) present in the media of the endothelium-denuded human umbilical vein preparation. 2. Endothelin-1 (ET-1) and ET-2 were potent constrictors of umbilical vein with EC50 values of 9.2 nM and 29.6 nM, respectively. ET-1 was at least 30 times more potent than ET-3 suggesting the presence of constrictor ETA receptors. Little or no response was obtained to the ETB-selective agonist sarafotoxin 6c. These data suggest that endothelin-mediated vasoconstriction is via ETA receptors in this preparation. 3. Autoradiographical visualization of endothelin receptors with subtype selective ligands confirmed the predominance of the ETA receptor in the media of umbilical vein. High density of binding was obtained with the ETA selective [125I]-PD151242, with much lower levels detected with the ETB selective [125I]-BQ3020. 4. Big ET-1 (EC50 = 42.7 nM) and big ET-2(1-38) (EC50 = 99.0 nM) were less potent than ET-1 and ET-2, respectively. Big ET-2(1-38) was more potent than its isoform big ET-2(1-37) with concentration-response curves to big ET-2(1-37) incomplete at 300 nM. No response was obtained to big ET-3 at concentrations up to 700 nM. The C-terminal fragments, big ET-1(22-38) and big ET-2(22-38) were inactive. 5. Responses to ET-1 were unaffected by either the neutral endopeptidase (NEP) inhibitor thiorphan (10(-5) M) or by the dual NEP/ECE inhibitor phosphoramidon (10(-5) M). Big ET-1 was also unaffected by thiorphan but antagonized in a concentration-dependent manner by phosphoramidon (10(-5) M and 10(-4) M). 6. Addition of all four big endothelin peptides to human umbilical vein preparations resulted in detectable amounts of ET-IR in the bathing medium. Therefore, although big ET-3 was functionally inactive this reflects the low potency of ET-3 at the ETA receptor rather than the lack of ability of this smooth muscle ECE to convert big ET-3 to ET-3. 7. To conclude we have demonstrated the presence of a phosphoramidon-sensitive ECE on the smooth muscle layer of the human umbilical vein which can convert big ET-1, big ET-2(1-37), big ET-2(1-38) and big ET-3 to their mature biologically active forms. The precise subcellular localization of this enzyme and its physiological relevance remains to be determined.     

Beta(3)-adrenoceptors mediate smooth muscle relaxation in the rat lower oesophageal sphincter

BACKGROUND: The neurological literature concerning disinhibition syndromes and secondary mania has run in parallel to clinical reports of bipolar disorder in old age. METHODS: A critical review was conducted of both the neurological and geriatric psychiatry literature in an attempt to integrate the two streams. RESULTS: Disinhibition syndromes include lateralization to the right hemisphere and localization of lesions to the orbito-frontal and basotemporal cortex involving limbic and frontal connections (orbito-frontal circuit). Mania in old age is associated with late onset, heterogeneous neurological disorders and poor outcome. CONCLUSION: Bipolar disorders in old age may be understood in the context of affective vulnerability influenced by a specific neurobiologic substrate. LIMITATIONS: The clinical literature consists predominantly of small case series and anecdotal reports. CLINICAL RELEVANCE: Improved understanding of these syndromes may elucidate the pathogenesis and etiology of bipolar disorders and the neuropsychiatric syndromes affecting mood, motivation and behavioural disinhibition.     

Myosin light chains (MLCs) heterogeneity in mammalian smooth and cardiac muscle

Tuberous sclerosis (TS) is well known to be occasionally associated with subependymal giant cell astrocytoma (SGCA). SGCA is considered to be a benign tumor in its clinical course and morphology. However, this tumor is grown sometimes so rapid and caused hydrocephalus. To our knowledge, little is known about hemocirculation and metabolism, particularly in relation with proliferating activity of TS and SGCA. We measured hemocirculation and metabolism of SGCA developed in a case of TS using positron emission tomography (PET). A 13-year-old-boy who had frequently developed convulsions four months after birth. He was diagnosed as TS and had been medically treated with anticonvulsants, since multiple intraventricular calcifications were detected by CT, at the age of five months. The convulsions had been well controlled. In March 1993, he presented with syncopal attack and admitted to our hospital. CT showed multiple subependymal nodules. Among the nodules, one of the left anterior horn exceeded 2cm in size obliterated Monro's foramen. The tumor was homogeneously enhanced with contrast medium. The lesion detected by postcontrast T1-weighted MR imaging had almost the same status as that by CT. T2-weighted image revealed cortical tubers as high intensity area at the left frontal and parietooccipital regions. PET was performed with the Headtome IV. Hemocirculation of the tumor was lower than that of contralateral gray matter, which suggested poor blood supply. The oxygen and glucose metabolism of the tumor were decreased compared with contralateral gray matter, indicative of a low activity of proliferation and a clinically benign tumor in the present case.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenovirus-mediated overexpression of human transforming growth factor-beta 1 in rat cardiac fibroblasts, myocytes and smooth muscle cells

Transforming growth factor-beta 1 (TGF-beta 1) is known to regulate cardiac cell function and its overexpression in the heart is thought to contribute to the development of cardiac hypertrophy and fibrosis. We wished to develop a high efficiency gene transfer method that could be used both in vitro and in vivo and result in the overexpression of TGF-beta 1. For this purpose, we constructed a replication-deficient human adenovirus 5 vector encoding for human TGF-beta 1 and used for control purposes an adenovirus lacZ vector. The adenovirus 5 construct was capable of infecting neonatal rat cardiac myocytes, fibroblasts and VSMCs. Of the three cell types, cardiac myocytes appear more susceptible to infection by the adenovirus 5 construct as assessed through beta-galactosidase staining. Infection of cardiac fibroblasts, myocytes and VSMCs with the hTGF-beta 1 adenovirus leads to the expression of hTGF-beta 1 mRNA and enhanced levels of bioactive and total TGF-beta 1 protein. Infection with hTGF-beta 1 adenovirus also results in enhanced levels of collagen type III gene expression in VSMCs and fibroblasts whereas in cardiac myocytes it leads to increased levels for sarcomeric and beta-actin. Thus, this adenoviral vector might be used for the exploration of in vivo effects of altered levels of cardiac TGF-beta 1.     

The structure and organization of the human NPAT gene

Ataxia telangiectasia (AT) is an autosomal recessive gene disorder, and ATM, a housekeeping gene, has been identified as the gene responsible for AT. Recently we found that another housekeeping gene, NPAT, is located upstream of ATM on human chromosome 11. The two housekeeping genes are transcribed in opposite directions and share a 0.5-kb 5' flanking sequence. The structure and organization of NPAT were determined by direct sequencing of cosmid clones carrying the gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon/intron boundaries and all of the exons. The gene spans at least 44 kb and consists of 18 exons and 17 introns. It has been suggested that AT heterozygotes have an increased risk of developing cancer, especially breast cancer in women. Frequently, loss of heterozygosity at loci on 11q22-q24 has been observed in DNA isolated from tumors of the breast, uterine cervix, and colon, perhaps suggesting the location of a tumor suppressor gene in 11q22-q24. For investigation of the role of NPAT in AT and these tumors with allelic loss of 11q22-q24, appropriate primer sequences and PCR conditions for amplification of all the NPAT exons from genomic DNA were determined. We previously reported that no recombinations are found among Atm, Npat, and Acat1 (acetoacetyl-CoA thiolase) loci as determined by fine genetic linkage mapping of the mouse AT region. The results of the LA-PCR analysis using NPAT- and ACAT-specific primers and human genomic DNA allowed us to map ACAT 12 kb centromeric to NPAT.     

Neocuproine, a selective Cu(I) chelator, and the relaxation of rat vascular smooth muscle by S-nitrosothiols

1. A study has been made of the effect of neocuproine, a specific Cu(I) chelator, on vasodilator responses of rat isolated perfused tail artery to two nitrosothiols: S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and S-nitroso-glutathione (GSNO). 2. Bolus injections (10 microl) of SNAP or GSNO (10(-7)-10(-3) M) were delivered into the lumen of perfused vessels pre-contracted with sufficient phenylephrine (1-7 microM) to develop pressures of 100-120 mmHg. Two kinds of experiment were made: SNAP and GSNO were either (a) pre-mixed with neocuproine (10(-4) M) and then injected into arteries; or (b) vessels were continuously perfused with neocuproine (10(-5) M) and then injected with either pure SNAP or GSNO. 3. In each case, neocuproine significantly attenuated vasodilator responses to both nitrosothiols, although the nature of the inhibitory effect differed in the two types of experiment. We conclude that the ability of exogenous nitrosothiols to relax vascular smooth muscle in our ex vivo model is dependent upon a Cu(I) catalyzed process. Evidence is presented which suggests that a similar Cu(I)-dependent mechanism is responsible for the release of NO from endogenous nitrosothiols and that this process may assist in maintaining vasodilator tone in vivo.     

The actions of ketamine on vascular smooth muscle

The responses of rabbit aortic strips superfused with noradrenaline, adrenaline and 5-HT were studied alone and in combination with ketamine (50 mug/ml). Ketamine caused a slight depression of the isolated aorta but potentiated responses to adrenaline but not to noradrenaline or 5-hydroxytryptamine. Ketamine did not potentiate aortic strips contracted to a stable level by pyrogallol and adrenaline. Experiments carried out with COMT from homogenates of rat liver showed that, in contrast to pyrogallol (10(-5) M), ketamine (10(-3) M) did not inhibit the enzyme. Other experiments with rabbits given 6-hydroxydopamine showed that aortas of these rabbits responded in a similar manner to controls when treated with ketamine and catecholamines. Results obtained with aortas contracted by adrenaline and noradrenaline with ketamine present, followed by oil immersion, showed that ketamine prolonged greatly the relaxation induced by adrenaline and to a lesser extent the relaxation induced by noradrenaline. The results of these studies indicate that ketamine prevented catecholamines from reaching the intracellular site of COMT. In this respect, ketamine can be termed an inhibitor of uptake site 2. If this hypothesis is valid then the action of ketamine on vascular tissue might explain the cardiovascular effects of the drug in man and experimental animals.     

Calponin phosphatase from smooth muscle: a possible role of type 1 protein phosphatase in smooth muscle relaxation

Smooth muscle myosin bound phosphatase (MBP) purified from chicken gizzard, which is a holoenzyme of type 1 delta protein phosphatase and dephosphorylated intact myosin, catalyzed the dephosphorylation of calponin phosphorylated by protein kinase C (PK-C). The Km of MBP for calponin was 0.6 microM and the Vmax was 350 nmol/min/mg. All of the multiple sites of phosphorylation by PK-C of calponin were completely dephosphorylated by MBP. Functionally, calponin dephosphorylated by MBP recovered its inhibitory effect on the actin-activated Mg(2+)-ATPase activity of myosin. Therefore, these results suggest that a type 1 delta protein phosphatase causes relaxation of smooth muscle by the dephosphorylation not only of myosin but also of calponin.