Occurrence of mitochondrial monoamine oxidase in human semen.

Mitochondrial monoamine oxidase (MAO) was found in human semen, showing its Km and Vmax values of 91.7 microM and 290 pmoles/mg of protein/60 min, respectively, with kynuramine as substrate. A major part of the activity was due to type A MAO.     

Occurrence of mitochondrial monoamine oxidase in human semen

Summary Mitochondrial monoamine oxidase (MAO) was found in human semen, showing its K_m and V_max values of 91.7 M and 290 pmoles/mg of protein/60 min, respectively, with kynuramine as substrate. A major part of the activity was due to type A MAO.     

Alpha 1-adrenergic stimulation of ketogenesis and fatty acid oxidation is associated with inhibition of lipogenesis in rat hepatocytes

The effect of norepinephrine on fatty acid synthesis (3H2O incorporation into fatty acids), on fatty acid oxidation to CO2 and on ketogenesis was studied in isolated hepatocytes of fed rats. After incubation with norepinephrine (50 microM), lipogenesis was lower (5.7 +/- 1.1 nmoles 3H2O incorporated into fatty acids/mg dry weight/30 min) than in controls (7.5 +/- 1.7; n = 6, p less than 0.02). In contrast, (1-14C) palmitate conversion into total ketone bodies was increased to 10.9 +/- 1.8 nmoles/mg/30 min with norepinephrine, vs 8.5 +/- 1.6 in controls (p less than 0.05), and more (1-14C) palmitate was converted to 14CO2 with norepinephrine than in controls (1.48 +/- 0.10 nmoles/mg/30 min vs 1.06 +/- 0.11, p less than 0.05). The inhibitory effect of norepinephrine on lipogenesis was abolished by addition of the alpha 1-receptor blocker prazosin, but not by alpha 2 or beta-blockers. The results demonstrate that the ketogenic effect of norepinephrine is coupled with an inhibitory effect on lipogenesis which may be explained by diminished activity of acetyl-CoA carboxylase, diminished formation of malonyl-CoA and decreased activity of carnitine palmitoyl transferase I.     

Bombesin-like immunoreactivity in the pituitary gland

Bombesin has been shown to stimulate release of anterior pituitary hormones both in vivo and in vitro. The aim of this study was to determine whether bombesin-like immunoreactivity could be detected in the human pituitary. Significant concentrations were found in the human anterior gland (4.6 +/- 1.5 pmoles/g), posterior gland (1.5 +/- 0.4 pmoles/g) and stalk (8.1 +/- 0.8 pmoles/g). Significant amounts were also observed in the guinea-pig pituitary. Gel permeation chromatography revealed the presence of 2 major molecular forms of bombesin-like immunoreactivity, one co-eluting with porcine gastrin-releasing peptide and the other with amphibian bombesin.     

Synergistic effect of acute hypoxia on flow-induced release of ATP from cultured endothelial cells

Human umbilical vein endothelial cells (HUVECs) in primary cultures were perfused under normoxic or hypoxic conditions. These cells were stimulated twice for 3 min by increased flow (from 0.5 to 3.0 ml/min). Under hypoxic conditions the basal release of ATP was the same as under normoxic conditions, but during increased flow the release was greater (0.58±0.07>0.32±0.04 pmoles/ml/10⁶ cells (+78%), for the first period of stimulation; 0.39±0.05>0.22±0.03 pmoles/ml/10⁶ cells (+79%) for the second period). Further experiments with sequential increments in flow rate showed that under both normoxic and hypoxic conditions, a positive correlation existed between ATP release and the rate of flow but there was always more ATP released under hypoxic conditions regardless of the flow rate.HUVECs in secondary culture (second passage) were similarly stimulated. No differences were observed between normoxic and hypoxic conditions. In both cases, the quantity of ATP released during high flow (0.050±0.004 pmoles/ml/10⁶ cells) was significantly smaller than the quantity of ATP released during low flow (0.09±0.01 pmoles/ml/10⁶ cells).To conclude, since hypoxia alone did not affect ATP release, there appears to be a synergistic relationship between increased shear stress and hypoxia in the stimulation of ATP release from HUVECs. Moreover, the release of ATP under these conditions seems to be a property of highly differentiated endothelial cells.     

Alterations of beta-adrenoceptor-density and cAMP-synthesis in rat-erythrocytes after stress erythropoiesis.

During the maturation of red blood cells from rats after stress erythropoiesis, adenyl cyclase activity and beta-adrenoceptor density (pmoles/mg protein) decrease at distinctly different rates suggesting a different turnover of these membrane units.     

Net ATP synthesis by running the red cell calcium pump backwards.

Ca2+ loaded inside-out vesicles from human red blood cells, yielding C2+ into a Ca2+ free medium with 4 mM EGTA, 2 mM ADP and 10 mM phosphate, produced an excess of 14.9 pmoles . min-1 . (mg protein)-1 of ATP compared to controls in which the transmembrane Ca2+ gradient was abolished by the ionophore A 23 187.     

Demonstration of high affinity binding of estradiol in adrenal cortex tissue]

Evidence is presented demonstrating the presence of a high affinity (Ka10(8)M-1), limited capacity (3-4 pmoles/mg protein) estradiol binder in the soluble fraction of the Bovine, Rat and Human adrenal cortex. The binding appears specific to the estrogen structure whereas C19 and C21 steroids do not bind. Upon sucrose density gradient centrifugation, the estradiol binder sedimented at 9 S at low ionic strength and was shifted to 4.5 S in the presence of 0.5 M KCl. This demonstration of a receptor-like moiety for estradiol in the adrenal cortex lends biochemical support to previous observations suggesting that adrenal cortex functions may be modulated by a direct effect of gonadal steroid hormones.     

Alterations ofβ-adrenoceptor-density and cAMP-synthesis in rat-erythrocytes after stress erythropoiesis

Summary During the maturation of red blood cells from rats after stress erythropoiesis, adenyl cyclase activity and -adrenoceptor density (pmoles/mg protein) decrease at distinctly different rates suggesting a different turnover of these membrane units.Acknowledgments. This work was supported by the Deutsche Forschungsgemeinschaft. The skillful technical assistance of Mrs I. Groth and Mrs C. Kiele is gratefully acknowledged. The liquid scintillation spectrometer was a gift from the Dr Robert-Pfleger-Stiftung.     

Specific in vivo binding of d-LSD in rat brain

A reproducible in vivo d-LSD binding method in rat brain is described, with high affinity (Kd of 5 pmoles/g wet wt), stereospecificity (d- vs. 1-LSD) and regional selectivity. It may be a useful adjunct to in vitro methods for measuring changes in turnover at the synaptic level related to the intact receptor.     

Bombesin-like immunoreactivity in the pituitary gland

Summary Bombesin has been shown to stimulate release of anterior pituitary hormones both in vivo and in vitro. The aim of this study was to determine whether bombesin-like immunoreactivity could be detected in the human pituitary. Significant concentrations were found in the human anterior gland (4.6±1.5 pmoles/g), posterior gland (1.5±0.4 pmoles/g) and stalk (8.1±0.8 pmoles/g). Significant amounts were also observed in the guinea-pig pituitary. Gel permeation chromatography revealed the presence of 2 major molecular forms of bombesin-like immunoreactivity, one co-eluting with porcine gastrin-releasing peptide and the other with amphibian bombesin.     

Hydrogen peroxide generation in Trypanosoma cruzi.

Homogenates from T. cruzi epimastigotes produced 3.4 pmoles H2O2/min 10(6) cells, as detected by the cytochrome c peroxidase assay. Addition of NADH or NADPH increased H2O2 production by a factor of 3 and 5, respectively. When supplemented with NADH and NADPH, the mitochondrial, microsomal and supernatant fractions produced H2O2, the soluble fraction and the mitochondrial membranes being apparently the main generators of H2O2. The epimastigote homogenates showed cyanide-sensitive superoxide dismutase activity, equivalent to 0.28 microgram bovine superoxide dismutase per mg homogenate protein.     

Binding of indole-acetic acid to cytosol proteins of embryonic chicken liver

3H-Indole acetic acid bound to cytosol proteins of embryonic chicken liver in vitro to an average capacity of at least 20 pmoles/mg binding protein. The auxin-binding protein complexes could be resolved into 4 major zones by anion exchange chromatography; they sedimented at 3-7 S in sucrose density gradients, and were also heterogenous in agarose gel electrophoresis.     

Specific in vivo binding of d-LSD in rat brain

Summary A reproducible in vivo d-LSD binding method in rat brain is described, with high affinity (K_d of 5 pmoles/g wet wt), stereospecificity (d-vs, vs. l-LSD) and regional selectivity. It may be a useful adjunct to in vitro methods for measuring changes in turnover at the synaptic level related to the intact receptor.     

A gonadotropin releasing hormone analog induces spermiation in intact and hypophysectomized frogs, Rana esculenta

The sperm-releasing activity of a gonadotropin releasing hormone (GnRH) agonist, Buserelin (GnRH) and hypophysis homogenate (PD) preparations was studied in intact and hypophysectomized (PDX) frogs, Rana esculenta. In addition, human chorion gonadotropin (hCG) was tested in PDX animals, and GnRH antagonist (GnRHA) treatments were carried out in intact and PDX animals, in combination with the hormonal injections. GnRH or PD treatments were able to elicit spermiation in intact and PDX animals. While GnRH, injected 24 h later, was again effective in inducing spermiation in intact animals, this was not the case in PDX frogs. GnRHA counteracted GnRH effects in intact frogs. Moreover, in PDX animals GnRHA injections counteracted the sperm-releasing activity induced by hCG or GnRH, but failed to inhibit sperm-releasing activity induced by PD homogenate.     

Ceramides induce apoptosis in HeLa cells and enhance cytochrome c-induced apoptosis in Xenopus egg extracts

Ceramide has been reported to induce typical apoptotic changes in nuclei incubated in a cell-free system, and that the addition of ceramide bypasses the requirement for mitochondria. Here, we explore the possible pathways by which ceramide induces apoptosis either in intact cells or in a cell-free system which we have developed. We found that in the cell-free system, C2-ceramide is not able to induce apoptosis in nuclei whereas cytochrome c does, but it is able to induce HeLa cells to undergo apoptosis. Ceramide is also not able to induce apoptosis when added into the cell-free system together with purified mitochondria. Further investigation showed that C2-ceramide at certain concentrations greatly increases nuclear apoptosis caused by cytochrome c in the cell-free system. From these results we conclude that the induction of apoptosis by ceramide may require intact cells in which some unknown signal transduction pathways are involved.     

Evidence for the extranuclear localization of thymosins in thymus.

A new radioimmunoassay has been developed for thymosin beta 4 by generating rabbit polyclonal antibodies against the synthetic N-terminal peptide fragment 1-15 coupled to KLH. The synthetic analogue [Tyr12]-thymosin beta 4 (1-15) was used as tracer. This radioimmunoassay, with a useful range of 10-1000 pmoles, showed cross-reactivity with the second homologous beta-thymosin of man and rat (thymosin beta 10) but not of calf (thymosin beta 9). This radioimmunoassay, together with an improved radioimmunoassay for the N-terminus of parathymosin alpha, was employed for the measurement of the levels of thymosin beta 4 and parathymosin alpha in nuclear and extranuclear extracts of calf thymus. The bulk of these polypeptides was found in the extranuclear material whereas only traces were observed in the nuclear environment, which indicates the extranuclear localisation of alpha- and beta-thymosins.     

Aryl-hydrocarbon receptor-dependent alteration of FAK/RhoA in the inhibition of HUVEC motility by 3-methylcholanthrene

We previously demonstrated the antiproliferative and antiangiogenic effects of 3-methylcholanthrene (3MC), an aryl-hydrocarbon receptor (AhR) agonist, in human umbilical vascular endothelial cells (HUVECs). Herein, we unraveled its molecular mechanisms in inhibiting HUVEC motility. 3MC down-regulated FAK, but up-regulated RhoA, which was rescued by AhR knockdown. It led us to identify novel AhR binding sites in the FAK/RhoA promoters. Additionally, 3MC increased RhoA activity via suppression of a negative feedback pathway of FAK/p190RhoGAP. With an increase in membrane-bound RhoA, subsequent stress fiber and focal adhesion complex formation was observed in 3MC-treated cells, and this was reversed by a RhoA inhibitor and AhR antagonists. Notably, these compounds significantly reversed 3MC-mediated anti-migration in a transwell assay. The in vitro findings were further confirmed using an animal model of Matrigel formation in Balb/c mice. Collectively, AhR’s genomic regulation of FAK/RhoA, together with RhoA activation, is ascribable to the anti-migration effect of 3MC in HUVECs.     

A tonic component in the motility of the upper urinary tract (renal pelvis-ureter)

In isolated muscle strips of porcine renal pelvis and ureter, the calcium antagonist nifedipine (3.10(-7) moles/l) completely suppressed spontaneous phasic mechanical activity and the phasic components of an adrenaline-induced activation (P-component). In the presence of nifedipine, adrenaline induced in pelvis preparations (but not in the ureter) a tonic contraction (T-component) which was on average 61% of the control reaction (SD +/- 26%; n = 35).     

Hydrogen peroxide generation inTrypanosoma cruzi

Summary Homogenates from T. cruzi epimastigotes produced 3.4 pmoles H₂O₂/min 10⁶ cells, as detected by the cytochrome c peroxidase assay. Addition of NADH or NADPH increased H₂O₂ production by a factor of 3 and 5, respectively. When supplemented with NADH and NADPH, the mitochondrial, microsomal, and supernatant fractions produced H₂O₂, the soluble fraction and the mitochondrial membranes being apparently the main generators of H₂O₂. The epimastigote homogenates showed cyanide-sensitive superoxide dismutase activity, equivalent to 0.28 g bovine superoxide dismutase per mg homogenate protein.This investigation was supported by grants from Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET) Argentina and the Scientific Office, American States Organization.Career Investigator of CONICET.