Chronic treatment with a stable obestatin analog significantly alters plasma triglyceride levels but fails to influence food intake; fluid intake; body weight; or body composition in rats

Obestatin (OB(1-23) is a 23 amino acid peptide encoded on the preproghrelin gene, originally reported to have metabolic actions related to food intake, gastric emptying and body weight. The biological instability of OB(1-23) has recently been highlighted by studies demonstrating its rapid enzymatic cleavage in a number of biological matrices. We assessed the stability of both OB(1-23) and an N-terminally PEGylated analog (PEG-OB(1-23)) before conducting chronic in vivo studies. Peptides were incubated in rat liver homogenate and degradation monitored by LC-MS. PEG-OB(1-23) was approximately 3-times more stable than OB(1-23). Following a 14 day infusion of Sprague-Dawley rats with 50 nmol/kg/day of OB(1-23) or a N-terminally PEGylated analog (PEG-OB(1-23)), we found no changes in food/fluid intake, body weight and plasma glucose or cholesterol between groups. Furthermore, morphometric liver, muscle and white adipose tissue (WAT) weights and tissue triglyceride concentrations remained unaltered between groups. However, with stabilized PEG-OB(1-23) we observed a 40% reduction in plasma triglycerides. These findings indicate that PEG-OB(1-23) is an OB(1-23) analog with significantly enhanced stability and suggest that obestatin could play a role in modulating physiological lipid metabolism, although it does not appear to be involved in regulation of food/fluid intake, body weight or fat deposition.     

GPR30, the non-classical membrane G protein related estrogen receptor, is overexpressed in human seminoma and promotes seminoma cell proliferation

Background

Testicular germ cell tumours are the most frequent cancer of young men with an increasing incidence all over the world. Pathogenesis and reasons of this increase remain unknown but epidemiological and clinical data have suggested that fetal exposure to environmental endocrine disruptors (EEDs) with estrogenic effects, could participate to testicular germ cell carcinogenesis. However, these EEDs (like bisphenol A) are often weak ligands for classical nuclear estrogen receptors. Several research groups recently showed that the non classical membrane G-protein coupled estrogen receptor (GPER/GPR30) mediates the effects of estrogens and several xenoestrogens through rapid non genomic activation of signal transduction pathways in various human estrogen dependent cancer cells (breast, ovary, endometrium). The aim of this study was to demonstrate that GPER was overexpressed in testicular tumours and was able to trigger JKT-1 seminoma cell proliferation.

Results

We report here for the first time a complete morphological and functional characterization of GPER in normal and malignant human testicular germ cells. In normal adult human testes, GPER was expressed by somatic (Sertoli cells) and germ cells (spermatogonia and spermatocytes). GPER was exclusively overexpressed in seminomas, the most frequent testicular germ cell cancer, localized at the cell membrane and triggered a proliferative effect on JKT-1 cells in vitro, which was completely abolished by G15 (a GPER selective antagonist) and by siRNA invalidation.

Conclusion

These results demonstrate that GPER is expressed by human normal adult testicular germ cells, specifically overexpressed in seminoma tumours and able to trigger seminoma cell proliferation in vitro. It should therefore be considered rather than classical ERs when xeno-estrogens or other endocrine disruptors are assessed in testicular germ cell cancers. It may also represent a prognosis marker and/or a therapeutic target for seminomas.     

Molecular connexin partner remodeling orchestrates connexin traffic: From physiology to pathophysiology

Connexins, through gap junctional intercellular communication, are known to regulate many physiological functions involved in developmental processes such as cell proliferation, differentiation, migration and apoptosis. Strikingly, alterations of connexin expression and trafficking are often, if not always, associated with human developmental diseases and carcinogenesis. In this respect, disrupted trafficking dynamics and aberrant intracytoplasmic localization of connexins are considered as typical features of functionality failure leading to the pathological state. Recent findings demonstrate that interactions of connexins with numerous protein partners, which take place throughout connexin trafficking, are essential for gap junction formation, membranous stabilization and degradation. In the present study, we give an overview of the physiological molecular machinery and of the specific interactions between connexins and their partners, which are involved in connexin trafficking, and we highlight their changes in pathological situations.     

Quantitative determination of the calcium involved in the regulation of the Ca2+-ATPase activity in sarcoplasmic reticulum vesicles

The dependence of the Ca²⁺-ATPase activity of sarcoplasmic reticulum vesicles upon the intravesicular concentration of calcium accumulated after active uptake was studied. The internal calcium concentration was modified by addition of the ionophore A23187 at the steady state of accumulation. About half of the calcium accumulated could be released at low ionophore concentration without any concomitant activation of the Ca²⁺-ATPase. This population of calcium might consist of calcium free in the lumen of the vesicles or bound to the bilayer at sites which do not interact with the ATPase activity. At higher concentrations of ionophore (above 1.75 nmol A23187/mg protein) the release of calcium activated this enzyme. This phenomenon was independent of the extravesicular calcium concentration and might be explained by assuming second species of calcium ions bound to the inner side of the membrane and in close functional interaction with the Ca²⁺-ATPase.     

Movement of cholesterol in vitro in rat blood and quantitation of the exchange of free cholesterol between plasma and erythrocytes

After administration of [4-(14)C]cholesterol to rats, blood was obtained and incubated for 6 hr or less. Incubation resulted in a net loss of erythrocyte cholesterol and, simultaneously, in an increase of esterified cholesterol in plasma and alpha-lipoproteins. Erythrocyte labile cholesterol was shown to be the sole precursor of esterified cholesterol. However, the relation between loss and esterification was not absolute. Loss of erythrocyte cholesterol could be inhibited without affecting esterification and vice versa. A catenary turnover model is proposed, which links in vivo erythrocyte labile cholesterol and plasma esterified cholesterol. Free cholesterol also exchanged between erythrocytes and lipoproteins. The topological model, as tested by analog computer, appears to be a bicompartmental system governed by nonconstant exchange fluxes. They are exponential functions of time and vary from 0.065 to 0.020 mg/hr/g of blood. The fitting of the curves obtained by analog computer analysis to the experimental curves requires esterification as described above. Variation of the exchange fluxes would be the consequence of lipoprotein structural alterations. If this is true, the initial value of the measured flux in vitro is identical with the in vivo value, and the turnover time of erythrocyte cholesterol is 9.2 hr. Initial exchange flux is not dependent on plasma cholesterol level or on the age of the rats, but it is temperature dependent. Addition of amphotericin B to the plasma does not modify exchange fluxes, but erythrocyte cholesterol loss is increased.     

The chemical synthesis of glucosaminylphosphatidylglycerol. Comparison with a new phospholipid isolated from Bacillus megaterium

1. We describe the synthesis of a glucosamine derivative of phosphatidylglycerol having the same structure as that of the natural compound isolated from Bacillus megaterium. 2. 2-O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-d-glucopyranosyl)-3-O-benzyl-1-iodo-sn-glycerol was prepared by a Königs–Knorr condensation between 3-O-benzyl-1-toluene-p-sulphonyl-sn-glycerol and 3,4,6-tri-O-acetyl-1-bromo-2-deoxy-2-phthalimido-d-glucopyranose followed by replacement of the toluene-p-sulphonyl group with iodine. The iodide was treated with the silver salt of 2-isolauroyl-1-oleoyl-sn-glycerol 3-(monobenzyl hydrogen phosphate) to form the fully protected phosphoglycolipid. 3. Removal of benzyl protecting groups by catalytic hydrogenolysis, phthaloyl group with hydrazine and acetyl groups with pH10 buffer furnished 2-O-(2-amino-2-deoxy-d-glucopyranosyl)-1-(2-isolauroyl-1-stearoyl-sn-glycero-3-phosphoryl)-sn-glycerol. 4. The synthetic and natural compounds appeared identical when compared by chromatography and by identification of hydrolysis products from chemical and enzymic degradations.     

A METHOD FOR THE STUDY OF CHOLESTEROL BIOSYNTHESIS IN THE CENTRAL NERVOUS SYSTEM

Abstract—

• 1 After intraperitoneal injection, there is negligible incorporation of [2-¹⁴C]-mevalonic lactone into the CNS of the adult rat.
• 2 Mevalonic lactone injected into the CSF is quickly transferred to blood.
• 3 Mevalonic lactone injected in the cistema magna or the lateral ventricle of the brain does not diffuse readily into the whole CSF. Spinal cord cholesterol is most heavily labelled after intracisternal injection, as is brain cholesterol after intraventricular administration.
• 4 After intraventricular perfusion, the diffusion of mevalonic lactone into the ventricle opposite the side of the injection is increased when the rate of perfusion is doubled from 5 to 10 μ1/hr. After injection, optimal homogeneity is obtained if a large volume (70μl) is administered.
• 5 An increase in the volume of injection from 70 μl to 130μl does not alter the distribution of activity between the left and right ventricles, nor does it increase the diffusion of mevalonic lactone from ventricle to spinal cord CSF.
• 6 The mean yield of mevalonic lactone incorporation into brain cholesterol is much higher after injection than after perfusion of precursor into the lateral cerebral ventricle.

     

Uv Sensitivity of Several Genetic Markers of HAEMOPHILUS INFLUENZAE DNA

Studies on inactivation by UV of several genetic markers of the streptomycin and novobiocin resistance regions of H. influenzae DNA have shown that two kinds of markers may be found with respect to frequency of integration, UV sensitivity and reactivability. One class of markers has high integration frequency and low UV sensitivity, which can be partly explained by the reactivability of the irradiated markers. The second class of markers has low integration frequency and high UV sensitivity. Markers of each class have been found in the novobiocin region.-In one recipient strain with decreased transformability the difference in integration frequency between the two classes is enhanced.