Presence of a metallothionein-like protein in the bovine pineal gland

Summary The high concentration of zinc in the bovine pineal gland prompted us to investigate the existence of a zinc-binding protein in this organ. In this study, we report that the subcellular distribution of zinc in the bovine pineal gland is nonuniform, with the crude nuclear, mitochondrial, microsomal, and supernatant fractions having 0.264±0.038, 0.160±0.019, 0.130±0.016, and 0.287±0.010 g zinc/mg protein, respectively. Furthermore, gel filtration studies using Sephadex G-75 and a 105,000 g supernatant fraction revealed two zinc binding protein peaks that bind 1.7 and 3.7 g Zn⁺⁺/mg protein, respectively. Furthermore, purification of the protein peak with an elution volume (ve/vo) of 2.06 on anion exchange chromatography (DEAE-A 25) yielded a single protein peak which binds 10 g zinc/mg protein. The comparative high performance liquid Chromatographic (HPLC) profiles of the zinc-induced hepatic metallothionein isoform I (retention time=17.39 min) and of the bovine pineal metallothionein-like protein isoform I (retention time=17.49 min) are similar. Since zinc is a potent inhibitor of sulfhydryl-containing enzymes and receptor sites, we investigated the effects of zinc and found that it inhibited the binding of [³H]glutamate (IC 50=80 M) and of [³H]spiroperidol (IC 50=0.6 mM) to the pineal membranes. The results of these studies are interpreted to indicate that the bovine pineal gland possesses an active and dynamic zinc homeostatic mechanism, whose precise function(s) remain(s) to be delineated.     

Nitric oxide synthesis in endothelial cytosol: Evidence for a calcium-dependent and a calcium-independent mechanism

Summary Release of nitric oxide (NO) from endothelial cells critically depends on a sustained increase in intracellular free calcium maintained by a transmembrane calcium influx into the cells. Therefore, we studied whether the free cytosolic calcium concentration directly affects the activity of the NO-forming enzyme(s) present in the cytosol from freshly harvested porcine aortic endothelial cells. NO was quantified by activation of a purified soluble guanylate cyclase coincubated with the cytosol. In the presence of 1 mM L-arginine, 0.1 mM NADPH and 0.1 mM EGTA, endothelial cytosol (0.2 mg of cytosolic protein per ml) stimulated the activity of guanylate cyclase 5.0 + 0.5-fold (from 31 + 9 to 153 + 15 nmol cyclic GMP formed per min per mg guanylate cyclase). Calcium chloride increased this stimulation further in a concentration-dependent fashion by up to 136 + 15% (with 2 M free calcium; EC₅₀ 0.3 M). The calcium-dependent and -independent activation of guanylate cyclase was enhanced by superoxide dismutase (0.3 M) and was inhibited by the stereospecifically acting inhibitor of L-arginine-dependent NO formation N^G-nitro-L-arginine (1 mM) and by LY 83583 (1 M), a generator of superoxide anions. Our findings suggest a calcium-dependent and -independent synthesis of NO from L-arginine by native porcine aortic endothelial cells. Send of fprint requests to A. Mülsch, at the above address     

Synthesis and acid ionization constants of cyclic cystine peptides

Cyclic peptide disulfides of the general formula were synthesized from the corresponding peptide derivatives [Boc-Cys(Trt)(Gly)-_n-Cys(Trt)-OBu^t] by oxidation with iodine in methanol and by subsequent removal of the terminal groups with trifluoroacetic acid. Acid ionization constants of the obtained peptides were determined by potentiometric titration in aqueous KCl (0.1 mol/L) medium. All compounds have two dissociable hydrogens, corresponding to carboxyl (pK¹= 2.35–2.84) and to terminal amino group (pK₂= 5.61–6.93); pK₁, values show first an upward and then a downward trend with the increase in ring size; the opposite is true for pK₂, values. These trends could be tentatively attributed to the intramolecular salt bridge (-COO——-NH⁺₃-) formation.     

Effects of atrial natriuretic peptide on DNA synthesis and NADPH diaphorase activity in epitheliocytes and smooth muscle cells of the respiratory tract in newborn albino rats

Newborn rats received single intraperitoneal injections of atrial natriuretic peptide (1-28) in a dose of 3.2×10_-8 mol/kg on day 6 of life. Autoradiography with ³H-thymidine showed that the peptide inhibited DNA synthesis in smooth muscle cells of the respiratory tract. Pretreatment with nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester attenuated, but did not abolish the effect of atrial natriuretic peptide (1-28). Histochemical assay for NADPH diaphorase showed that nitric oxide constitutively produced in the epithelium is involved in the growth-inhibitory effect of atrial natriuretic peptide (1-28) on proliferating smooth muscle cells.     

血小板源生长因子对人血管成纤维细胞DNA及胶原蛋白合成的影响

目的:观察血小板源生长因子对培养的人血管成纤维细胞DNA及胶原蛋白合成的影响。方法:采用培养的人血管成纤维细胞,应用 [³H]-TdR和 [³H]-脯氨酸掺入的方法,观察血小板源生长因子-BB对人血管成纤维细胞DNA合成以及胶原蛋白合成的影响。结果:血小板源生长因子-BB可促进静止状态的人血管成纤维细胞DNA及胶原蛋白的合成,在 30μg/L浓度时DNA及胶原蛋白的合成达到高峰,DNA及胶原蛋白分别于 2 4h和 36h合成最为显著。结论:血小板源生长因子-BB可明显促进培养的人血管成纤维细胞DNA及胶原蛋白的合成     

Synthesis of gonadotrophins and its regulation in the pituitary gland

Regulation of the synthesis of pituitary gonadotrophins LH andFSH has been studied in the rat using either cell-free translationof pituitary mRNAs, or hybridization techniques with syntheticoligodeoxynucleotides or cloned complementary DNAs. Gonadectomygreatly increases and supplementing gonadectomized rats withgonadal steroids diminishes the rate of synthesis of the gonadotrophinsubunits. Hybridization experiments suggest that gonadal steroidsregulate the expression of the genes coding for pituitary gonadotrophinsubunit precursors. Using the incorporation of labelled methionineby pituitary cells in culture, followed by specific immunoprecipitationof LH-related subunits and SDS-poly-acrylamide gel analysisof immunoprecipitated peptides, there was evidence that gonadotrophinreleasing hormone (GnRH) significantly enhances the radioactivityincorporated into both - and LH-subunits. This effect is specific,it is not a secondary effect due to the release of LH. A cyclicAMP (cAMP) analogue, 8-Br-cAMP, as well as forskolin and choleragen,which are cAMP generators and a diacylglycerol analogue, tetradecanoylphorbolacetate (TPA), mimic the stimulatory action of GnRH on the synthesisof the polypeptide chains of LH. However, no evidence has beenobtained that either cAMP or diacylglycerols mediate this GnRHeffect. These results suggest that the synthesis of pituitarygonadotrophins is under a double control of gonadal steroidsand GnRH which exert opposite effects, inhibitory for steroidsand stimulatory for GnRH. The negative control by steroids occursat the genomic level, while the positive effect of GnRH proceedsvia different mechanisms which remain to be elucidated.     

Endothelin-1 causes a prolonged protein kinase C activation and acts as a co-mitogen in vascular smooth muscle cells

Endothelin-1 (ET-1) is known to act via G-protein coupled receptors. It has therefore been suggested that any mitogenic activity it may possess, is due to activation of phospholipase C and protein kinase C (PKC). We have therefore examined both the ability of ET-1 to act as a mitogen and its ability to activate PKC. We found that ET-1 significantly increased thymidine incorporation and enhanced platelet-derived growth factor-induced DNA synthesis, as well as causing a prolonged translocation of PKC to the cell membrane. ET-1 significantly increased PKC dependent phosphorylation of two specific substrates. The phosphorylation of MBP_4–14 (from myelin basic protein) was partially dependent on extracellular Ca²⁺, implicating activation of PKC-α, whereas phosphorylation of the so called ε-peptide was Ca²⁺-independent and prolonged. This could be due either to the δ or ζ isoform of PKC, known to be present in these cells. However, ET-1 induced little proliferation or PKC activity in a transformed smooth muscle cell line, DDT₁ MF-2, which lacks expression of the PKC-αisoform, but expresses the ζ-isoform. Thus, it would appear that ET-1-induced mitogenicity in smooth muscle cells may be related to the sustained, Ca²⁺-independent activation of PKC-δ.     

Endothelin synthesis and receptors in human endometrium throughout the normal menstrual cycle

This study was undertaken to investigate the presence of messengerRNA (mRNA) for prepro-endothelin-I (ET-1) and the known receptorsubtypes (ETA and ETB) in human endometrium at different stagesof the menstrual cycle obtained at hysterectomy. Northern blotanalysis revealed expression of ET-1 mRNA in human endometriumduring the normal menstrual cycle. The concentration of ET-1mRNA in endometrial tissue was greater during the menstrualand proliferative phases than during the ovulatory and secretoryphases. Immunoreactive ET-1 was secreted into the medium ofisolated endometrial stromal cells. Oestradiol and progesteronesignificantly attenuated ET-1 release in endometrial stromalcells cultured for 6 days. ETA and ETB mRNA were also presentin endometrial tissue of the normal cycle. The concentrationof ETA receptor mRNA was greater in the proliferative phasethan in the secretory phase, whereas expression of ETB mRNAincreased in menstrual phase. ET-1 significantly increased extracellularaccumulation of cyclic AMP (cAMP), intracellular generationof inositol phosphates and significantly enhanced DNA synthesisin cultured endometrial stromal cells from the proliferativephase. Our results showed that human endometrial cells synthesizedand released ET-1, and contained ETA and ETB receptors whichwere functionally coupled to phosphoinositide breakdown andto adenylate cyclase with the increase of cAMP by ET-1 stimulation.Our findings suggest that ET-1 may have a potential autocrineand/or paracrine function in human endometrial stromal cells.     

A new assay for leukocyte chemotaxis using cell retrieval,electronic particle counting and flow cytometry

A new micropore membrane assay for leukocyte migration has been devised. It permits the complete retrieval in monodisperse suspension of functionally intact cells that have traversed the membrane, thus allowing the application of precise, automated techniques, including flow cytometry and electronic particle counting. Hemocytometers may also be used. Direct comparison with 2 different conventional membrane methods showed that the new method performed superiorly. It was also much more economical with regard to time and labor. This technique permitted detection of functional differences between leukocytes isolated from blood in different ways. Data on the duration of concentration gradients in chemotaxis chambers are also presented.     

Permanent brain ischemia induces marked increments in hsp72 expression and local protein synthesis in synapses of the ischemic hemisphere

Transient focal ischemia induced in rat brain by occlusion of the middle cerebral artery (MCAo) elicits a generalized induction of the 72 kDa heat-shock protein (hsp72) heralding functional recovery. As this effect implies activation of protein synthesis, and local systems of protein synthesis are present in brain synapses, and may be analyzed in preparations of brain synaptosomes, we evaluated hsp72 expression and protein synthesis in synaptosomal fractions of spontaneously hypertensive rats (SHRs) subjected to permanent MCAo. SHRs were randomly divided in ischemics and sham controls, anaesthesia controls and passive controls. Focal ischemia was induced under chloral hydrate anaesthesia by unilateral permanent MCAo. Protein synthesis was determined by [³⁵S]methionine incorporation into synaptosomal proteins from ischemic and contralateral cortex/striatum, and from cerebellum. Hsp72 expression was measured in the same fractions by immunoblotting. Our data demonstrate that under these conditions synaptic hsp72 markedly increases in the ischemic hemisphere 1 and 2 days after MCAo, progressively declining in the following 2 days, while no significant change occurs in control rats. In addition, in the ischemic hemisphere the rate of synaptic protein synthesis increases more than two-fold between 1 and 4 days after MCAo, without showing signs of an impending decline. The present data provide the first demonstration that synaptic protein synthesis is massively involved in brain plastic events elicited by permanent focal ischemia.