Attachment and multiplication,morphology and protein production of human fetal primary liver cells cultured in hormonally defined media

In Vitro Cellular &; Developmental Biology - Plant -     

Evolutionary diversification of structure and function in the family of intracellular calcium-binding proteins

Summary The maximum parsimony method was used to reconstruct the genealogical history of the family of intracellular calcium-binding proteins represented by six major present-day lineages, three of which - calcium dependent modulator protein, heart and skeletal muscle troponin Cs, and alkali light chains of myosin - were found to share a closer kinship with one another than with the other lineages. Similarly, parvalbumins and regulatory light chains of myosin were depicted as more closely related, whereas the branch of intestinal calcium-binding protein proved to have the most distant separation. The computer-generated amino acid sequence for the common ancestor of these six lineages described a four domain protein in which each domain of approximately 40 amino acid residues had a mid-region, 12 residue segment that bound calcium and had properties most resembling those of the calcium dependent modulator protein. It could then be deduced that parvalbumins evolved by deletion of domain I, inactivation of calcium-binding properties in domain II, and acquisition of increased affinity for Ca⁺⁺ and Mg⁺⁺ in domains III and IV. Regulatory light chains of myosin lost the cation binding property from three domains, retaining it in I, whereas alkali light chains of myosin lost this ability from each of the four domains. In skeletal muscle troponin C all domains retained their calcium-binding activity; however, like parvalbumins, domains III and IV acquired high affinity properties. Cardiac troponin C lost its binding activity from domain I but otherwise resembled the skeletal muscle form. Finally, intestinal calcium-binding protein evolved by deletion of domains III and IV.Positive selection could be implicated in these evolutionary changes in that the rate of fixation of mutations substantially increased in the mid portions of those domains which were loosing calcium-binding activity. Likewise, when the cation binding sites were changing from low to high affinity, an accelerated rate of fixed mutations was observed. Once this new functional parameter was selected these regions showed a remarkable conservatism, as did those binding sites which were maintaining the lower affinity. Moreover even in sequence regions not directly involved in cation binding, the lineage of troponin C became very conservative over the past 300 million years, perhaps because of the necessity for maintaining specific interfaces in order for the molecule to interact with troponin I and T in a functional thin myofilament. A similar phenomenon was observed in domain II of the regulatory light chains of the myosin lineage suggesting a possible binding site with the heavy chain of myosin.This paper is dedicated to the memory of Jean-Francois Pechère, deceased     

Glutamine Supplementation Alleviates Vasculopathy and Corrects Metabolic Profile in an In Vivo Model of Endothelial Cell Dysfunction

Endothelial Cell Dysfunction (ECD) is a recognized harbinger of a host of chronic cardiovascular diseases. Using a mouse model of ECD triggered by treatment with L-Nω-methylarginine (L-NMMA), we previously demonstrated that renal microvasculature displays a perturbed protein profile, including diminished expression of two key enzymes of the Krebs cycle associated with a Warburg-type suppression of mitochondrial metabolism. We hypothesized that supplementation with L-glutamine (GLN), that can enter the Krebs cycle downstream this enzymatic bottleneck, would normalize vascular function and alleviate mitochondrial dysfunction. To test this hypothesis, mice with chronic L-NMMA-induced ECD were co-treated with GLN at different concentrations for 2 months. Results confirmed that L-NMMA led to a defect in acetylcholine-induced relaxation of aortic rings that was dose-dependently prevented by GLN. In caveolin-1 transgenic mice characterized by eNOS inactivation, L-NMMA further impaired vasorelaxation which was partially rescued by GLN co-treatment. Pro-inflammatory profile induced by L-NMMA was blunted in mice co-treated with GLN. Using an LC/MS platform for metabolite profiling, we sought to identify metabolic perturbations associated with ECD and offset by GLN supplementation. 3453 plasma molecules could be detected with 100% frequency in mice from at least one treatment group. Among these, 37 were found to be differentially expressed in a 4-way comparison of control vs. LNMMA both with and without GLN. One of such molecules, hippuric acid, an “uremic toxin” was found to be elevated in our non-uremic mice receiving L-NMMA, but normalized by treatment with GLN. Ex vivo analysis of hippuric acid effects on vasomotion demonstrated that it significantly reduced acetylcholine-induced vasorelaxation of vascular rings. In conclusion, functional and metabolic profiling of animals with early ECD revealed macrovasculopathy and that supplementation GLN is capable of improving vascular function. Metabolomic analyses reveal elevation of hippuric acid, which may further exacerbate vasculopathy even before the development of uremia.     

Disruption of the mouse mTOR gene leads to early postimplantation lethality and prohibits embryonic stem cell development

The mammalian target of rapamycin (mTOR) is a key component of a signaling pathway which integrates inputs from nutrients and growth factors to regulate cell growth. Recent studies demonstrated that mice harboring an ethylnitrosourea-induced mutation in the gene encoding mTOR die at embryonic day 12.5 (E12.5). However, others have shown that the treatment of E4.5 blastocysts with rapamycin blocks trophoblast outgrowth, suggesting that the absence of mTOR should lead to embryonic lethality at an earlier stage. To resolve this discrepancy, we set out to disrupt the mTOR gene and analyze the outcome in both heterozygous and homozygous settings. Heterozygous mTOR (mTOR(+/-)) mice do not display any overt phenotype, although mouse embryonic fibroblasts derived from these mice show a 50% reduction in mTOR protein levels and phosphorylation of S6 kinase 1 T389, a site whose phosphorylation is directly mediated by mTOR. However, S6 phosphorylation, raptor levels, cell size, and cell cycle transit times are not diminished in these cells. In contrast to the situation in mTOR(+/-) mice, embryonic development of homozygous mTOR(-/-) mice appears to be arrested at E5.5; such embryos are severely runted and display an aberrant developmental phenotype. The ability of these embryos to implant corresponds to a limited level of trophoblast outgrowth in vitro, reflecting a maternal mRNA contribution, which has been shown to persist during preimplantation development. Moreover, mTOR(-/-) embryos display a lesion in inner cell mass proliferation, consistent with the inability to establish embryonic stem cells from mTOR(-/-) embryos.     

Stereospecific synthesis of "para-hydroxymexiletine" and sodium channel blocking activity evaluation

Both enantiomers of "para-hydroxymexiletine" (PHM), one of the main metabolites of mexiletine, were synthesized and fully characterized. Properties of (R)- and (S)-PHM, in terms of blocking potency and stereoselectivity on frog skeletal muscle Na(+) channels, were evaluated. The presence of a hydroxy group on the aryloxy moiety in the 4-position, as in PHM, reduced potency with respect to mexiletine in reducing I(Na max). However, PHM showed clear use-dependent behavior similar to that of mexiletine and, in contrast with what is observed with the parent compound, maintained its stereoselectivity during the use-dependent block. Chirality 16:72-78, 2004.     

Accuracy of transrectal ultrasonography for determination of pregnancy in sheep: effect of fasting and handling of the animals

The present study was performed to investigate the effect of previous fasting and lifting of the abdomen of the ewes during transrectal ultrasonographic scanning on the results of early pregnancy diagnosis. Ewes of four flocks (A, B, C and D; all Awassi x Merino ewes, n = 1247 ) aged 0.7-10 years were used in this study. These ewes were estrus synchronized and artificially inseminated. From 2 weeks later onwards, fertile rams were kept with the ewes of flocks A, B and C ( n=949 ) for natural breeding, while ewes of flock D ( n=298 ) were re-inseminated 17 days later. Transrectal ultrasonography (5 MHz) was carried out in ewes of flocks A, B and C on four separate occasions but only once in ewes of flock D. For final analysis, animals were divided over two groups: ewes of Group 1 ( n=949 scans) were scanned in a standing position within the milking parlor. Animals of Group 2 ( n=764 scans) were scanned by the same operator and with the same scanning technique, but these ewes were fasted for 12h prior to scanning and the abdominal wall was lifted, just in front of the udder during scanning. The sensitivity of the test for diagnosing pregnancy at Days 18-24, 25-30, 31-40 and 41-50 was 21.8, 32.3, 63.3 and 50% in Group 1, and 46, 92.5, 92.3 and 96.8% in Group 2, respectively. Only within Group 1, the sensitivity of the test was higher in young ewes (0.7-2 years) than in older ones (>2-10 years). Significant differences were observed at scan periods Days 18-24 and Days 41-50 of gestation. It is concluded that, fasting prior to scanning and lifting the abdomen during scanning significantly improve the accuracy of transrectal ultrasonographic pregnancy diagnosis in Awassi x Merino ewes.     

Chronic bronchial allergic inflammation increases alveolar liquid clearance by TNF-alpha -dependent mechanism

Bronchial inflammation in allergic asthma is associated with active exudation from the bronchial tree into the interstitial space of both mucosa and submucosa. The aim of this study was to evaluate epithelial and endothelial permeability as well as alveolar fluid movement in a model of chronic allergic inflammation in Brown-Norway rats sensitized and challenged with ovalbumin (OA). Control groups were challenged with saline solution (C), and rats were immunized by OA but not challenged (Se). Lung sections showed a marked inflammatory infiltrate associated with perivascular and peribronchiolar edema in OA. To measure alveolar liquid clearance, a 5% bovine albumin solution with 1 microCi of (125)I-labeled human albumin was instilled into the air spaces. Alveolar-capillary barrier permeability was evaluated by intravascular injection of 1 microCi of (131)I-labeled albumin. Endothelial permeability was significantly increased in OA, from 0.08 +/- 0.01 in the C group to 0.19 +/- 0.03 in OA group (P < 0.05). Final-to-initial protein ratio was also statistically higher in OA (1.6 +/- 0.05) compared with C (1.38 +/- 0.03, P = 0.01) and Se groups (1.42 +/- 0.03, P = 0.04). Administration of anti-tumor necrosis factor-alpha antibodies within the instillate significantly decreased this ratio (1.32 +/- 0.08, P = 0.003 vs. OA). To conclude, we demonstrated a tumor necrosis factor-alpha-dependent increase in alveolar fluid movement in a model of severe bronchial allergic inflammation associated with endothelial and epithelial leakage.     

Effect of selective phosphodiesterase inhibitors on response of ovine pulmonary arteries to prostaglandin E2

Gao, Yuansheng, Jean-François Tolsa, Hai Shen, and J. Usha Raj. Effect of selective phosphodiesteraseinhibitors on response of ovine pulmonary arteries to prostaglandinE₂. J. Appl. Physiol. 84(1): 13-18, 1998.Several adenosine3,5-cyclic monophosphate (cAMP)-hydrolyzingphosphodiesterase isozymes are present in the pulmonary vasculature.The present study was designed to determine the effect of selectiveinhibitors of phosphodiesterase subtypes on prostaglandinE₂(PGE₂)-induced relaxation ofisolated fourth- generation pulmonary arteries of newborn lambs.PGE₂ and forskolin causedpulmonary arteries to relax and induced an increase in theintracellular cAMP content in the vessels. The relaxation and change incAMP content were augmented by milrinone and rolipram, inhibitors ofphosphodiesterase type 3 (PDE3) and type 4 (PDE4), respectively. Theaugmentation in relaxation and the increase in cAMP content caused bymilrinone plus rolipram was greater than the sum of theresponses caused by either of the inhibitors alone.8-Methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine, an inhibitor of phosphodiesterase type 1, had no effect on relaxation andchange in cAMP induced by PGE₂ andforskolin. Acetylcholine alone had no effect on cAMP content in thevessels but augmented the relaxation and the increase in cAMP inducedby PGE₂ and forskolin in arterieswith endothelium. This effect was not observed in arteries withoutendothelium or in arteries with endothelium treated withN^G-nitro-L-arginine.These results suggest that PDE3 and PDE4 are the primary enzymeshydrolyzing cAMP of pulmonary arteries of newborn lambs and that aninhibition of both PDE3 and PDE4 would result in a greater effect thanthat caused by inhibition of either one of the subtype isozymes alone.Furthermore, endothelium-derived nitric oxide may enhance cAMP-mediatedrelaxation by inhibition of PDE3.