Enzyme-catalyzed synthesis of radiolabeled 5-pregnen-3 beta-ol-20-one sulfate.

The enzymatic synthesis of [3H]5-pregnen-3 beta-ol-20-one sulfate using [3H]5-pregnen-3 beta-ol-20-one, 3'-phosphoadenosine-5'-phosphosulfate and hydroxysteroid sulfotransferase 1 purified from rat liver is reported. The described procedure allowed the obtainment of high specific activity [3H]5-pregnen-3 beta-ol-20-one sulfate in yields ranging from 78 to 86% with respect to [3H]5-pregnen-3 beta-ol-20-one. Two-dimensional thin-layer chromatography was used to purify [3H]5-pregnen-3 beta-ol-20-one sulfate which upon solvolysis resulted in the formation of [3H]5-pregnen-3 beta-ol-20-one. The identity both of the synthesized compound and the solvolysed one was confirmed by reversed-phase high pressure liquid chromatography, and 2-dimensional thin-layer chromatography.

Characterization of estrone sulfatase activity in human thrombocytes.

Estrone sulfatase is an important enzyme which catalyzes the production of estrone from estrone sulfate in a variety of human and animal tissues. We report, for the first time, on the presence of estrone sulfatase activity in thrombocytes from human blood. Incubation of [3H]estrone sulfate in the presence of human thrombocyte lysates resulted in the formation of [3H]estrone as assessed by two-dimensional TLC. Estrone sulfatase activity was localized in the mitochondrial-microsomal fraction in thrombocytes from human blood. The enzyme was thermostable and had an optimum pH of 5.60 in acetate buffer. The highest activity was obtained in the presence of 0.1% of either Nonidet P-40 or Triton X-100. Phosphate ions (1 mM) inhibited the enzyme activity by 64% and similar effects were observed in the presence of platelet-free plasma. Endogenous inhibitors had no effect on the observed enzyme activity under assay conditions as evidenced in this study. The apparent Km value was 3.16 +/- 0.08 microM for [3H]estrone sulfate and V was 188.5 +/- 2.6 (mean +/- SEM, n = 22) pmol.mg protein-1.h-1. Comparison between two thrombocyte preparative procedures provided evidence that thrombocyte estrone sulfatase activity should be measured in thrombocyte samples representing the whole thrombocyte population. This parameter appeared critical for accurate measurements of enzyme activity. The presence of estrone sulfatase activity in human thrombocytes provides a new non-invasive tool for the study of this activity both in physiological and pathological conditions which could be of potential clinical relevance.

Establishment of permanent human endothelial cells achieved by transfection with SV40 large T antigen that retain typical phenotypical and functional characteristics.

The plasmid pMK16 containing-SV40 replicated origin defective gene was efficiently introduced into early-passage human umbilical vein endothelial cells (HUVEC) using positively charged liposomes. The resulting cell line acquired an almost infinite lifespan, was morphologically unchanged, expressed SV40-antigen, and coexpressed von Willebrand factor (vWF), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), angiotensin conversion enzyme (ACE), and endothelin converting enzyme (ECE). In addition, these are the first immortalized human endothelial cells, to our knowledge, that biosynthesized and secreted interleukins (IL-1 beta and IL-6) in both a constitutive and regulated fashion and endothelin-1 (ET-1), the most potent vasoactive peptide, which has been suggested to be implicated in the pathogenesis of hypertension. Interestingly enough, both of the immortalized cells and the early-passage HUVEC from which the immortalized cells were obtained biosynthesized and secreted the same levels of ET-1 suggesting full maintenance of its biosynthetic pathway including the presence of active ECE, which cleaves big endothelin-1 (big-ET-1) to ET-1 and regulation factors. Moreover, the immortalized cells retained the ability to express the functional specific amino acid Na(+)-independent system Y+ transporter, which mediates L-arginine transport into endothelial cells from which endothelium-derived relaxing factor (EDRF, nitric oxide) is formed via the action of nitric oxide-synthase. Obtaining these immortalized human endothelial cells without alteration of the differentiated characteristics constitutes a useful model: (a) to study ET-1 secretion, gene regulation, and human ECE, which may be an important therapeutic target in disease conditions in which ET-1 is to be implicated; (b) to study L-arginine transport, which is a key step in the formation of EDRF; (c) to study IL-1 beta and IL-6 secretions, and gene regulations; (d) to substitute large quantities of HUVEC; and, finally, (e) to reproduce, starting with different primary endothelial cells both from human and animal origin.

[Biochemical mechanisms in the physiopathology of migraine]. / Mécanismes biochimiques dans la physiopathologie migraineuse.

Migraine is one of the few pathologies which gave rise to a tremendous number of physiopathological hypotheses. The variability of its clinical features and of the crisis initiating triggers, together with the numerous functional and/or biological abnormalities reported in migrainous patients, led to multiple 'theories' about migraine. For instance, migraine attacks may be associated with modifications of cerebral blood flow, and/or alterations at the cellular (neuronal and peripheral: platelets, mast cells, etc.) and subcellular (mainly mitochondrial) levels leading to variations of parameters such as serotonin, vasoactive neuropeptides, histamine, nitric oxide, neuroactive amino acids, etc. However, these modifications are mainly related to migraine attacks but not to migrainous patients. These emphasize how important is the distinction between the crisis mechanism(s) and the determinism of migraine illness. Despite the absence of any true animal model of migraine attack, the obtention, through the activation of the trigemino-vascular complex, of an experimental meningeal neurogenic inflammation was a clear breakthrough for the understanding of the migraine attack. Concerning the determinism of migraine, its familial characteristic has been known for a long time, but genetic studies started only recently. Despite some important contributions, the respective roles of genetic and environmental factors, as well as the transmission mode of migraine, remain largely to be determined. Practically, these genetic data, which really concern only a very peculiar form of migraine--the familial hemiplegic one--do not have presently any diagnostic or therapeutic application.

[Functional properties of a new line of immortalized human endothelial cells]. / Propriétés fonctionnelles d'une nouvelle lignée de cellules endothéliales humaines immortalisées.

An immortalized human endothelial cell line was obtained by transfecting umbilical vein endothelial cells in primary culture with plasmid pMK16 containing SV40 replicated origin defective gene. The essential functional properties demonstrated in these immortalized human endothelial cells also retaining the classical phenotypical characteristics of endothelial cells in primary culture are: (1) endothelin-1 secretion; (2) capacity to convert big endothelin-1 into endothelin-1; (3) the capacity to secrete IL1 beta and IL6 interleukins both spontaneously and after lipopolysaccharide (LPS) stimulation; (4) arginine transfer from the extracellular to the intracellular medium. Such stable cell line could facilitate studies of regulation of endothelin-1 production; (5) No-synthase activity; (6) binding and metabolisation of acetylated low-density lipoproteins.