Search

We have studied the role of nuclear factor of activated T-cells (NFAT) transcription factors in the induction of vascular smooth muscle cell (VSMC) growth by platelet-derived growth factor-BB (PDGF-BB) and thrombin, the receptor tyrosine kinase (RTK) and G-protein-coupled receptor (GPCR) agonists, respectively. NFATc1 but not NFATc2 or NFATc3 was translocated from the cytoplasm to the nucleus upon treatment of VSMCs with PDGF-BB or thrombin. Translocation of NFATc1 was followed by an increase in NFAT—DNA binding activity and NFAT-dependent reporter gene expression. Cyclosporin A (CsA), a potent and specific inhibitor of calcineurin, a calcium/calmodulin-dependent serine phosphatase involved in the dephosphorylation and activation of NFATs, blocked NFAT—DNA binding activity and NFAT-dependent reporter gene expression induced by PDGF-BB and thrombin. CsA also completely inhibited PDGF-BB- and thrombin-induced VSMC growth, as measured by DNA synthesis and cell number. In addition, forced expression of the NFAT-competing peptide VIVIT for calcineurin binding significantly attenuated the DNA synthesis induced by PDGF-BB and thrombin in VSMCs. Together, these findings for the first time demonstrate a role for NFATs in RTK and GPCR agonist-induced growth in VSMCs.

The pilin glycoprotein (PilE) is the main building block of the pilus of Neisseria gonorrhoeae (gonococcus [GC]). GC pilin is known to carry a disaccharide O-glycan, which has an αGal attached to the O-linked GlcNAc by a 1–3 glycosidic bond. In this report, we describe the cloning and characterization of the GC gene, pilus glycosyl transferase A (pgtA), which encodes the galactosyl transferase that catalyzes the synthesis of this Gal–GlcNAc bond of pilin glycan. A homopolymeric tract of Gs (poly-G) is present in the pgtA gene of many GC strains, and this pgtA with poly-G can undergo phase variation (Pv). However, in many other GC, pgtA lacks the poly-G and is expressed constitutively without Pv. Furthermore, by screening a large number of clinical isolates, a significant correlation was observed between the presence of poly-G in pgtA and the dissemination of GC infection. Poly-G was found in pgtA in all (24 out of 24) of the isolates from patients with disseminated gonococcal infection (DGI). In contrast, for the vast majority (20 out of 28) of GC isolated from uncomplicated gonorrhea (UG) patients, pgtA lacked the poly-G. These results indicate that Pv of pgtA is likely to be involved in the conversion of UG to DGI.

Previously we have demonstrated that activation of p38 mitogen-activated protein kinase (MAPK) and induction of DNA synthesis in response to receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) agonists require NADH/NADPH-like oxidase activity in vascular smooth muscle cells (VSMC). Here we tested the role of p38 MAPK in RTK and GPCR agonist-induced DNA synthesis in VSMC. Platelet-derived growth factor (PDGF)-BB and thrombin (RTK and GPCR agonists, respectively) activated p38 MAPK in a time-dependent manner in VSMC. Inhibition of p38 MAPK led to a 50% decrease in the DNA synthesis induced by thrombin but not PDGF-BB. ATF-1 was found to be the predominant member of the cyclic AMP response element (CRE)-DNA complex formed in VSMC in response to PDGF-BB and thrombin, and both agonists induced its phosphorylation. Regardless of this, inhibition of p38 MAPK reduced only thrombin- but not PDGF-BB-induced ATF-1 phosphorylation. Similarly, inhibition of p38 MAPK caused a 50% decrease in thrombin- but not PDGF-BB-induced CRE promoter-dependent transcription. Ectopic expression of an inhibitory anti-ATF-1 single-chain antibody fragment, ScFv, significantly interfered with DNA synthesis induced by thrombin but not PDGF-BB. Together, these results suggest the following conclusions. 1) Both RTK and GPCR agonists activate p38 MAPK and induce CRE promoter-dependent transcription; 2) both RTK and GPCR agonists induce ATF-1 phosphorylation, and ATF-1 is a predominant member in the CRE-DNA complexes formed in response to these agents; and 3) p38 MAPK-dependent ATF-1 phosphorylation and CRE promoter-mediated transcription are associated with GPCR agonist-induced VSMC growth.

We have investigated the role of an aprotinin-sensitive protease in regulating Ca2+-ATPase activity and Ca2+ uptake (ATP-dependent and Na+-dependent) in microsomes of bovine pulmonary vascular smooth muscle during treatment with the O2-•-generating system hypoxanthine plus xanthine oxidase. Treatment of the smooth muscle microsomes with the O2-•-generating system produced a protease in a gelatin-containing zymogram with an apparent molecular mass of 16 kDa. This 16 kDa proteolytic protein was found to be inhibited by superoxide dismutase (SOD) and aprotinin but not by PMSF. Using polyclonal antiserum to aprotinin, we found that it is an ambient antiprotease of the smooth muscle microsomes. Treatment of the microsomes with the O2-•-generating system stimulated protease activity tested with a synthetic substrate N-benzoyl-dl-arginine p-nitroanilide and also enhanced Ca2+-ATPase activity. It also stimulated ATP-dependent Ca2+ uptake. In contrast, Na+-dependent Ca2+ uptake was found to be inhibited by the O2-•-generating system. Pretreatment of the microsomes with SOD and aprotinin preserved the increase in protease activity, Ca2+-ATPase activity and ATP-dependent Ca2+ uptake. In addition, O2-•-caused inhibition of the Na+-dependent Ca2+ uptake which was reversed by SOD and aprotinin. Pretreatment with PMSF did not cause any discernible alteration in the protease activity, Ca2+-ATPase activity, ATP-dependent Ca2+ uptake and Na+-dependent Ca2+ uptake in the microsomes caused by the O2-•-generating system. These results suggest that an aprotinin-sensitive protease plays a pivotal role in regulating Ca2+-ATPase and Ca2+-uptake activities in microsomes of pulmonary vascular smooth muscle under oxidant O2-•-triggered conditions.

Treatment of bovine pulmonary artery smooth muscle tissue microsomes with H2O2(1 mM) markedly stimulated protease activity tested with a synthetic substrate p‐nitroanilide (BAPNA), and also enhanced Ca2+‐ATPase activity. ATP‐dependent Ca2+uptake was found to be stimulated upon treatment of the microsomes with H2O2. Pretreatment of the microsomes with vitamin E and aprotinin prevented the H2O2‐induced stimulation of Ca2+‐ATPase activity and also ATP‐dependent Ca2+uptake. In contrast, H2O2‐induced inhibition of Na+‐dependent Ca2+uptake was reversed by vitamin E and aprotinin.

Records of 17 patients who had two or more attacks of pyogenic meningitis were collected from eight centres in the United Kingdom for retrospective analysis. Thirteen patients had intracranial abnormality; of seven with head injury five produced cerebrospinal fluid rhinorrhoea. The first of the 28 attacks seen in these occurred between a few weeks and 12 years of the head injury. Pneumococci were identified in 25 episodes in cerebrospinal fluid. Of six patients without a history of head injury, one had ‘spontaneous cerebrospinal fluid rhinorrhoea’ and five had pathological changes of the ear. Various organisms were found in the cerebrospinal fluid during the 12 attacks in these five. Four of the 17 patients had primary complement deficiency (C7, C5, C4 and C3b inhibitor); 10 (possibly 11) of 16 attacks in these cases were due to Neisseria meningitidis. Routine radiological investigations including computerized tomography did not always identify the abnormality; radioactive cisternography can help to establish cerebrospinal fluid leak. All 58 episodes of pyogenic meningitis in these 17 patients with different underlying disease responded to conventional treatment with antibiotics without mortality and without undue morbidity. Surgical procedures in intracranial disease had variable success. Correction of complement deficiency is not practical at present. In some patients prophylaxis with antimicrobial drugs is the only method of preventing future attacks.