Subregional mapping of the human gonadotropin-releasing hormone receptor (GnRH-R) gene to 4q between the markers D4S392 and D4S409

We have isolated nine yeast artificial chromosomes (YACs) containing the gene that encodes the human gonadotropin-releasing hormone receptor (GnRH-R) gene by screening the YAC library of the Centre d'Etude du Polymorphisme Humain (Hôpital Saint-Louis, Paris, France) by the use of the polymerase chain reaction. We defined the location of the GnRH-R gene relative to 4q microsatellite markers D4S392 and D4S409. The genetic positions of these markers on chromosome 4 are 76 and 77 cM, respectively. This location was further established by chromosomal in situ hybridization.     

Serum levels of soluble IL-2R, CD4 and CD8 in bronChial asthma

The aim of the present study was to compare serum levels of soluble forms of interleukin-2 receptor, CD4 and CD8, released by lymphocytes during activation of the immune system, in patients with allergic bronchial asthma, with those in healthy subjects. Significantly higher levels of soluble IL-2R and soluble CD4 were found in patients with asthma compared with the control group. In contrast, lower levels of soluble CD8 values were found in patients with asthma compared to the control group. Significant correlations were found for both sIL-2R and sCD4 and these two molecules, with lung function measured as bronchial responsiveness to inhaled methacholine. These results strengthen previous suggestions that in allergic bronchial asthma, activation of T cells plays a significant role in the disease pathogenesis.     

Transmural potential differences and short-circuit current intensity in the posterior intestine of Blennius parvicornis

Simultaneous measurements of the transmural potential difference (PD) and the short-circuit current intensity (Isc) in the posterior intestine of the fish Blennius parvicornis were made in normal Ringer and in solutions of different ionic composition. The ouabain effects on these two parameters were also tested in normal Ringer solution. The absence of K+ from the Ringer solution on both the mucosal and serosal sides has no apparent effect on the PD and Isc within the first 15 min, but it makes them null after 30 min. When Na+ is substituted in both compartments, using Tris as substitute, a serosal negativity increase is initially observed, but it gradually decreases to zero after 30 min of experimentation. Similarly the PD and Isc drop to zero in the absence of Cl- (sulfate as substitute). Ouabain diminishes the serosa negative potential difference to zero after 30 min presenting a lineal relation to the Isc. A likely transport mechanism for Cl- dependent on the Na+ - K+ pump, is discussed.     

POTASSIUM-INDUCED RELEASE OF [3H]GABA AND OF [3H]NORADRENALINE FROM NORMAL AND RESERPINIZED RAT BRAIN CORTEX SLICES. DIFFERENCES IN CALCIUMDEPENDENCY, AND IN SENSITIVITY TO POTASSIUM IONS

The release of [³H]GABA induced by elevated extracellular potassium (K)_o, from thin rat brain cortex slices, has been compared with that of [³H]noradrenaline ([³H]NA), released by the same procedures, both from normal slices, and from slices pre-treated with reserpine and nialamide, [³H]NA being predominantly a vesicular component in the former situation, and a soluble substance in the latter one. 46 mM-(K)_o released considerably more [³H]NA from normal than from drug-treated slices, while the release of GABA was about two thirds of the latter. When 4min ‘pulses’ of increasing concentrations of potassium were applied, it was observed that the release of GABA and of [³H]NA from drug-treated slices increased in proportion to (K)_o, up to 36-46 mM and then declined considerably with higher (K)_o. The dependency of potassium-induced release on the concentration of calcium in the medium, indicated that release of [³H]NA from normal slices was proportional to calcium up to 1.5-2 mM, while that of [³H]NA from drug-treated slices increased up to 0.5 mM-Calcium, and then declined with higher concentrations. GABA release also increased up to 0.5 mM-calcium, but no further changes were observed at higher concentrations. The calcium antagonist D-600 inhibited high (K)_o-induced release of [³H]NA from normal slices to a greater extent than that of [³H]GABA or of [³H]NA from drug-treated slices. These results, in which elevated (K)_o-induced release of [³H]GABA resembles considerably that of soluble NA, but differs from that of NA present in synaptic vesicles, suggest that release of [³H]GABA also occurs from the soluble cytoplasmic compartment, and that the partial calcium requirement that is found is unrelated to that of transmitter secretion. These findings are also a further indication of the lack of specificity of elevated (K)_o as a stimulus for inducing transmitter secretions.     

Mitochondrial abnormalities in some human muscular diseases and in experimental ischemic myopathy

Morphologic abnormalities have been observed in two cases of human polymyositis and in three cases of ocular myopathies. Similar findings can be observed in experimental ischemic myopathy. "Ragged red" fibres, with anomalous distribution of oxidative enzymes, mitochondrial alterations, with presence of intracristal paracrystalline inclusions and degenerative myofibrillar changes are the similar features. The similarity between some of these alterations, expecially mitochondrial changes, is remarkable, but it is difficult to correlate them to the primary etiology of described human myopathies.     

Effect of enzyme inducers on metabolism of 1-nitropyrene in human hepatoma cell line HepG2.

We measured the response of HepG2 cells to the classic cytochrome (cyt.) P-450 inducers 3-methylcholanthrene (3-MC) and phenobarbital (PB), by evaluating oxidative and/or reductive metabolism of the nitroarenes, 1-NP and 1,6-dinitropyrene (1,6-DNP), in control and induced cells. In HepG2 cells, 3-MC induces ring-hydroxylation of 1-NP, whereas PB stimulates its nitroreduction. PB induces NADPH-cyt. c reductase, but does not affect other cytosolic and microsomal enzymes which contribute to 1-NP nitroreduction in these cells. However, PB-inducible nitroreductase activity seems to be associated primarily with cyt. P-450 isoenzymatic form(s), as indicated by the requirement for NADPH and the response to specific inhibitors such as alpha-naphthoflavone and CO.     

Selective localization of receptors for urokinase amino-terminal fragment at substratum contact sites of an in vitro-established line of human epidermal cells.

We have shown the presence of surface receptors for the amino-terminal fragment (ATF) of human urokinase-type plasminogen activator (u-PA) on an in vitro-established cell line of human epidermal origin by both radio-binding assays with human 125I-u-PA-ATF and transmission electron microscopy of a gold-u-PA complex. On the basis of cross-linking experiments with 125I-u-PA-ATF and subsequent autoradiography of the gels we have observed that such receptors are not spontaneously released into the culture medium. The treatment with phosphatidylinositol-specific phospholipase C induces the release of the receptor, which behaves as a glycosyl phosphatidyl inositol(GPI)-anchored protein. Phase-partitioning experiments on cell lysates have shown that the receptor partitions into the detergent phase. By detaching cell monolayers with the chelating agent EDTA we have prepared the cell-substratum contact sites of these cells, which represent only the 3.5% of the surface membrane of monolayered cells. Such plasma membrane remnants are highly selected since they contain about 43% of total u-PA-ATF binding sites. Such binding sites show the same biochemical and morphological characteristics of u-PA-ATF receptors observed in the monolayered cells, thus indicating that u-PA is selectively concentrated at the level of cell-substratum contacts. This is likely to enable directional proteolysis for cell migration and invasion.     

XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida.

Summary The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida. This protein is activated by a variety of benzoate analogues. To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to -galactosidase were constructed but all are inactive. In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions. The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein. The intraallelic dominance of the Ile229 (Ser229 Ile) and Val274 (Asp274 Val) substitutions over the N-terminal His4l (Arg4l His) substitution, and the intraallelic dominance of Thr45 (Arg45 Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally. Combination of the Leu88 (Trp88 Leu) and Arg256 (Pro256 Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates. In contrast, the Leu88 semiconstitutive phenotype was suppressed by Va1288 (Asp288 Val), and the double mutant was susceptible to activation by benzoates. The results suggest that intramolecular interactions between the C- and N-terminal regions of XylS are critical for activation of the regulator by the effector.     

Aldosterone regulation of active sodium chloride transport in the lizard colon (Gallotia galloti)

Summary Bioelectrical parameters and unidirectional sodium and chloride fluxes were measured under voltageclamp conditions in groups of lizards submitted to single or chronic aldosterone treatment. Both acute (AT) and chronic (CT) treatment induced significant increases in the short-circuit current (I _sc), as well as in the mucosa-to-serosa (J _m-s ^Na ) and net sodium flux (J _net ^Na ). In AT tissues, aldosterone did not change net chloride flux (J _net ^Cl ) but did so in CT tissues. Amiloride reduced the aldosterone-increased I _sc in AT and CT tissues, inhibited J _net ^Na in AT tissues and abolished it in CT colons. J _net ^Cl was also reduced by the diuretic in the group of AT colons, whereas no changes were observed in the CT tissues. Addition of luminal DIDS reduced Na⁺ absorption and totally inhibited Cl^- absorption in the AT tissues, but did not change I _sc. However, in CT tissues neither Na⁺ nor Cl^- transport were affected by DIDS. A good relationship between I _sc and J _m-s ^Na was apparent after DIDS treatment in AT tissues. In this group, simultaneous addition of DIDS and amiloride totally abolished J _net ^Na and reduced I _sc to untreated control values. Addition of serosal ouabain abolished I _sc and Na⁺ absorption in AT and CT colons, but Cl^- absorption was only altered in AT tissues. These results support the hypothesis that aldosterone induces an electrogenic, amiloride-sensitive sodium absorption, and in a dose-dependent fashion suppresses electroneutral NaCl absorption in the lizard colon.Abbreviations AT acutely treated - CT chronically treated animals - DIDS 4-4-diisothiocyanatostibene-2-2-disulfonic acid - DMSO dimethylsulphoxide - G _t tissue conductance - I _sc short circuit current - PD transepithelial potential difference - SITS 4-acetamido-4-isothiocyanatostilbene-2-2-disulfonic acid - UC untreated controls Preliminary results of this paper were presented at the X ^th meeting of the European Intestinal Transport Group (EITG), Askov Hojskole, Denmark, 16–19 September 1990     

Identification of two distinct nuclear factors with DNA binding activity within the glucocorticoid regulatory region of the rat alpha-1-acid glycoprotein promoter

Efficient glucocorticoid induction of alpha-1-acid glycoprotein (AGP) mRNA in rat hepatoma cells HTC (JZ-1) requires the activity of one or more preexisting and labile proteins acting cooperatively with the glucocorticoid receptor. Inhibiting protein synthesis markedly diminishes the glucocorticoid induction of rat AGP mRNA without affecting the inducibility of other glucocorticoid inducible genes such as the mouse mammary tumor virus (MMTV) or tyrosine amino transferase (TAT). The sequences responsible for conferring glucocorticoid inducibility to the rat AGP gene have localized on the AGP promoter between nucleotides -121 and -42. A typical glucocorticoid regulatory element (GRE) is found between residues -121 and -105 and downstream of this are the sequences (-90 to -42) responsible for the cycloheximide inhibition of the hormonal induction (10). Using mobility shift assays we have characterized the binding of two proteins or complexes of proteins to this promoter region (-90 to -64). Our data show that the binding of these factors (called ANF-1 and ANF-2) to the DNA is highly specific, and is not directly affected by cycloheximide. Furthermore a second binding site for ANF-2 has been localized in the AGP regulatory region to a sequence that overlaps the GRE.