Role of antibody and complement in opsonization of group B streptococci.
(17/3731)
A requirement for the classic complement pathway in opsonization of group B streptococci was observed by using both a chemiluminescence and a radiolabeled bacterial uptake technique. The classic pathway increased levels of opsonization for types Ia and II stock and wild strains and for some type III wild strains. In contrast, other type III wild strains and the type III stock strain had accelerated kinetics of uptake in the presence of an intact classic pathway, but the level of opsonization was unchanged from that with antibody alone. We could not demonstrate a significant role for the alternative pathway in opsonizing stock or wild strains of group B streptococci. Futhermore, electrophoretic and complement consumption analysis by hemolytic titration failed to reveal alternative pathway activation by the majority of strains of this group. Therapy aimed at supplying opsonins for these organisms will require the presence of type-specific antibody. (+info)
Peptide model of a highly conserved, N-terminal domain of apolipoprotein E is able to modulate lipoprotein binding to a member of the class A scavenger receptor family.
(18/3731)
Apolipoprotein E plays a critical role in plasma lipoprotein clearance. Peptide models of a highly conserved, N-terminal domain of this protein have been shown to increase the binding of low density lipoprotein (LDL) to fibroblast cell surfaces independently of the low density lipoprotein receptor. Here we provide data to show that these peptides not only increase the binding of LDL, but also of high density lipoprotein, though not acetylated LDL. We also have data suggesting that this novel activity is mediated, at least in part, by a member of the scavenger receptor family, SR-AI. Furthermore, we show that this activity is also prominent in macrophages, a cell relevant to atherogenesis. In addition, this current paper provides evidence suggesting that this complex binding activity is initiated by a peptide-receptor interaction, and that our peptides are able to induce activity at physiologically relevant concentrations. This study provides evidence for a possible novel receptor interaction and further anti-atherogenic properties of apolipoprotein E and raises the possibility of a therapeutic potential of our peptide models. (+info)
Rapid recovery of plasma ionized calcium after acute induction of hypocalcaemia in parathyroidectomized and nephrectomized rats.
(19/3731)
BACKGROUND: Plasma ionized calcium (Ca2+) is extremely tightly regulated in normal mammals. Even a small decline in Ca2+ is followed by a fast and steep increase of the parathyroid hormone (PTH) secretion and the current understanding of the calcium homeostasis indicates that PTH is the main factor responsible for this tight minute-to-minute regulation of the normal plasma Ca2+ concentration. However, experiments from our laboratory and some clinical experiences points towards the existence of factors, other than PTH, involved in the rapid minute-to-minute calcium homeostasis. Thus, the aim of the present study was to examine whether PTH plays an important role in the rapid upregulation of plasma Ca2+ after induction of hypocalcaemia in the rat. METHODS AND RESULTS: I. Parathyroidectomy (PTX) was performed in seven rats; 60 min later no PTH was detectable in the circulation. Then by a brief infusion of EGTA plasma Ca2+ was reduced from 1.26+/-0.02 to 0.86+/-0.02 mmol/l, P<0.001. Despite there being no PTH in the circulation plasma Ca2+ increased significantly to 0.97+/-0.02 mmol/l already 10 min after discontinuation of the EGTA infusion, P<0.04, and plasma Ca2+ was normalized within another 2 h. II. To evaluate a possible role of renal Ca2+ handling in the rapid upregulation of plasma Ca2+ a group of eight rats had acute PTX and bilateral nephrectomy (NX) performed; 60 min later plasma Ca2+ was reduced from 1.18+/-0.01 to 0.86+/-0.02 mmol/l by an EGTA infusion. Despite there being no PTH and no kidneys present plasma Ca2+ increased significantly already 10 min after discontinuation of EGTA to 0.96+/-0.02 mmol/l, P<0.02. After another 1.5 h the plasma Ca2+ reached the levels of the PTX/NX control rats. III. In order to exclude a possible action of receptor-bound PTH, which may have lasted for more than 1 h, seven rats were PTX 24 h before the induction of hypocalcaemia. Basal plasma Ca2+ was significantly reduced to 1.07+/-0.01 mmol/l, P<0.01. Then plasma Ca2+ was further reduced to 0.79+/-0.03 mmol/l by EGTA. Ten minutes after discontinuing EGTA plasma Ca2+ increased to 0.91+/-0.02 mmol/l, P<0.03 and 60 min later plasma Ca2+ reached the level of the control PTX rats. Normal rats with intact parathyroid glands had an exactly similar response of plasma Ca2+ to EGTA as that of 24 h PTX rats, but at significantly higher levels of plasma Ca2+ with a fall from 1.28+/-0.01 to 0.96+/-0.03 mmol/l and again a significant increase of plasma Ca2+ to 1.13+/-0.03 (P<0.001) 10 min after discontinuation of EGTA. After another hour basal levels were reached. CONCLUSIONS: Despite there being no PTH in the circulation a rapid increase of plasma Ca2+ occurs immediately after a brief induction of hypocalcaemia. The kidneys are not responsible for this phenomenon. The present results suggest the existence of a mechanism other than the effect of PTH, which is responsible for the rapid minute-to-minute regulation of plasma Ca2+ in the rat. (+info)
Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation.
(20/3731)
Calcineurin, a Ca2+/calmodulin dependent protein phosphatase, regulates Ca2+-dependent processes in a wide variety of cells. In the yeast, Saccharomyces cerevisiae, calcineurin effects Ca2+-dependent changes in gene expression through regulation of the Crz1p transcription factor. We show here that calcineurin dephosphorylates Crz1p and that this results in translocation of Crz1p to the nucleus. We identify a region of Crz1p that is required for calcineurin-dependent regulation of its phosphorylation, localization, and activity, and show that this region has significant sequence simlarity to a portion of NF-AT, a family of mammalian transcription factors whose localization is also regulated by calcineurin. Thus, the mechanism of Ca2+/calcineurin-dependent signaling shows remarkable conservation between yeast and mammalian cells. (+info)
Carboxy-terminal conversion of profibrillin to fibrillin at a basic site by PACE/furin-like activity required for incorporation in the matrix.
(21/3731)
Fibrillin-1, the main component of 10-12 nm microfibrils of the extracellular matrix, is synthesized as profibrillin and proteolytically processed to fibrillin. The putative cleavage site has been mapped to the carboxy-terminal domain of profibrillin-1, between amino acids arginine 2731 and serine 2732, by a spontaneous mutation in this recognition site that prevents profibrillin conversion. This site contains a basic amino acid recognition sequence (R-G-R-K-R-R) for proprotein convertases of the furin/PACE family. In this study, we use a mini-profibrillin protein to confirm the cleavage in the carboxy-terminal domain by both fibroblasts and recombinantly expressed furin/PACE, PACE4, PC1/3 and PC2. Site-directed mutagenesis of amino acids in the consensus recognition motif prevented conversion, thereby identifying the scissile bond and characterizing the basic amino acids required for cleavage. Using a PACE/furin inhibitor, we show that wild-type profibrillin is not incorporated into the extracellular matrix until it is converted to fibrillin. Therefore, profibrillin-1 is the first extracellular matrix protein to be shown to be a substrate for subtilisin-like proteases, and the conversion of profibrillin to fibrillin controls microfibrillogenesis through exclusion of uncleaved profibrillin. (+info)
Differential activation of phosphoinositide 3-kinase by endothelin and ceramide in colonic smooth muscle cells.
(22/3731)
We have investigated the hypothesis that different contractile agonists activate distinct catalytic subunits of phosphoinositide (PI) 3-kinase in smooth muscle cells. Endothelin (10(-7) M) induced a sustained increase in PI 3-kinase activity at both 30 s and 4 min of stimulation (151.5 +/- 8.5% at 30 s and 175.8 +/- 8.7% at 4 min, P < 0.005). Preincubation of smooth muscle cells with the tyrosine kinase inhibitor genistein (3 microM) resulted in a significant inhibition of both C2 ceramide-induced and endothelin-induced PI 3-kinase activation and contraction. Preincubation with herbimycin A, an Src kinase inhibitor (3 microM), inhibited only C2 ceramide-induced PI 3-kinase activation and contraction. Western blotting using Src kinase antibody showed that C2 ceramide, not endothelin, stimulated the phosphorylation of Src kinase. Western blotting and immunoprecipitation with PI 3-kinase antibodies to the regulatory subunit p85 and the catalytic subunits p110alpha and p110gamma indicated that both endothelin and C2 ceramide interacted with the regulatory subunit p85; endothelin interacted with the catalytic subunits p110alpha and p110gamma, whereas C2 ceramide interacted only with the catalytic subunit p110alpha. In summary, C2 ceramide activated PI 3-kinase p110alpha subunit by a tyrosine kinase-mediated pathway, whereas endothelin-induced contraction, unlike C2 ceramide, was not mediated by the activation of Src kinase but was mediated by G protein activation of both p110alpha and p110gamma subunits (type IA and IB) of PI 3-kinase. (+info)
Cholinergic and GABAergic regulation of nitric oxide synthesis in the guinea pig ileum.
(23/3731)
Nitric oxide (NO) synthesis was examined in intact longitudinal muscle-myenteric plexus preparations of the guinea pig ileum by determining the formation of [3H]citrulline during incubation with [3H]arginine. Spontaneous [3H]citrulline production after 30 min was 80-90 dpm/mg, which constituted approximately 1% of the tissue radioactivity. Electrical stimulation (10 Hz) led to a threefold increase in [3H]citrulline formation. Removal of calcium from the medium or addition of NG-nitro-L-arginine strongly inhibited both spontaneous and electrically induced production of [3H]citrulline. TTX reduced the electrically induced but not spontaneous [3H]citrulline formation. The electrically induced formation of [3H]citrulline was diminished by (+)-tubocurarine and mecamylamine and enhanced by scopolamine, which suggests that endogenous ACh inhibits, via muscarinic receptors, and stimulates, via nicotinic receptors, the NO synthesis in the myenteric plexus. The GABAA receptor agonist muscimol and GABA also reduced the electrically evoked formation of [3H]citrulline, whereas baclofen was without effect. Bicuculline antagonized the inhibitory effect of GABA. It is concluded that nitrergic myenteric neurons are equipped with GABAA receptors, which mediate inhibition of NO synthesis. (+info)
Hypoxia inhibits increased ETB receptor-mediated NO synthesis in hypertensive rat lungs.
(24/3731)
Although hypertensive lungs of chronically hypoxic rats express increased levels of nitric oxide (NO) synthases (NOSs) and produce increased amounts of NO-containing compounds (NOx) during normoxic ventilation, the level of NO production during hypoxic exposure is unclear. Because hypoxia inhibits NO synthesis in normotensive lungs, we investigated whether hypoxic ventilation inhibited NO synthesis in isolated hypertensive lungs and chronically hypoxic rats. Measurement of perfusate NOx concentration in hypertensive lungs from male rats exposed to 4 wk of hypobaric hypoxia showed that basal NOx production was reduced during hypoxic (0% O2) vs. normoxic (21% O2) ventilation. Similarly, plasma NOx concentration was lower in chronically hypoxic rats breathing 10% O2 than in those breathing 21% O2. Hypoxic inhibition of lung NOx production was not prevented by supplementary L-arginine or tetrahydrobiopterin and was not mimicked by inhibition of Ca2+ influx. However, it was mimicked by inhibition of constitutive NOS with NG-monomethyl-L-arginine and chelation of intracellular Ca2+. The endothelin type B-receptor antagonist BQ-788 prevented the increases in NOx production associated with normoxic ventilation in both isolated hypertensive lungs and intact chronically hypoxic rats. These results suggest that a reduced supply of the cosubstrate molecular O2 to NOS counteracts an endothelin type B receptor-mediated stimulation of NO synthesis in hypertensive rat lungs. Thus, despite increased NOS protein in the lungs and pulmonary arteries of chronically hypoxic rats, direct hypoxic inhibition of NO production may contribute to the development of pulmonary hypertension. (+info)