Protein kinase C is involved in adrenergic stimulation of pineal cGMP accumulation

The amounts of cAMP and cGMP in the rat pinealocyte are regulated by norepinephrine acting through synergistic dual receptor mechanisms involving alpha 1- and beta-adrenoceptors (Vanecek, J., Sugden, D., Weller, J.L., and Klein, D.C. (1985) Endocrinology 116, 2167-2173; Sugden, L., Sugden, D., and Klein, D.C. (1986) J. Biol. Chem. 261, 11608-11612). Based on the available evidence, it appears that Ca2+-phospholipid-dependent protein kinase is involved in the alpha 1-adrenergic potentiation of beta-adrenergic stimulation of cAMP, but not in the stimulation of cGMP (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W.B. (1985) Nature 314, 359-361). In the present study the role of protein kinase C in the adrenergic stimulation of cGMP was reinvestigated, with the purpose of determining whether protein kinase C activators would potentiate the effects of beta-adrenergic agonists on cGMP if cells were also treated with agents known to elevate intracellular free Ca2+. The protein kinase C activator 4 beta-phorbol 12-myristate 13-acetate (PMA) markedly elevated the cGMP content of beta-adrenergically stimulated pinealocytes which had also been treated with 1 microM A23187, 15 mM K+, or 1 microM ouabain. The effects of A23187 were blocked by EGTA and those of K+ were blocked by nifedipine, establishing the involvement of Ca2+. The stimulatory effects of PMA on cGMP accumulation were mimicked by other protein kinase C activators. PMA also stimulated cGMP accumulation in cells treated with cholera toxin (1 microgram/ml) and A23187 (1 microM), but not in cells treated only with cholera toxin. These results suggest that protein kinase C, which is activated in the pinealocyte by the alpha-adrenergic agonist phenylephrine, is probably involved in the adrenergic regulation of cGMP accumulation at a step distal to receptor activation.     

Comparison of ccd of F, parDE of RP4, and parD of R1 using a novel conditional replication control system of plasmid R1

A number of plasmid-encoded gene systems are thought to stabilize plasmids by killing plasmid-free cells (also termed post-segregational killing or plasmid addiction). Here we analyse the mechanisms of plasmid stabilization by ccd of F, parDE of RP4 and parD of R1, and compare them to hok/sok of R1. To induce synchronous plasmid loss we constructed a novel plasmid replication-arrest system, which possesses the advantage that plasmid replication can be completely arrested by the addition of IPTG, a non-metabolizable inducer. Using isogenic plasmid constructions we have found, for the first time, consistent correlation between the effect on steady-state loss rates and the effect on cell proliferation in the plasmid replication-arrest assay for all three systems. The parDE system had the most pronounced effect both on plasmid stabilization and on plasmid retention after replication arrest. In contrast, ccd and parD both exhibited weaker effects than anticipated from previously published results. Thus, our results indicate that the function and efficiencies of some of the systems should be reconsidered. Our results are consistent with the previously postulated hypothesis that ccd and parDE act by killing plasmid-free segregants, whereas parD seems to act by inhibiting cell division of plasmid-free segregants.     

Discovery of a novel site regulating glucokinase activity following characterization of a new mutation causing hyperinsulinemic hypoglycemia in humans

Type 2 diabetes is a global problem, and current ineffective therapeutic strategies pave the way for novel treatments like small molecular activators targeting glucokinase (GCK). GCK activity is fundamental to beta cell and hepatocyte glucose metabolism, and heterozygous activating and inactivating GCK mutations cause hyperinsulinemic hypoglycemia (HH) and maturity onset diabetes of the young (MODY) respectively. Over 600 naturally occurring inactivating mutations have been reported, whereas only 13 activating mutations are documented to date. We report two novel GCK HH mutations (V389L and T103S) at residues where MODY mutations also occur (V389D and T103I). Using recombinant proteins with in vitro assays, we demonstrated that both HH mutants had a greater relative activity index than wild type (6.0 for V389L, 8.4 for T103S, and 1.0 for wild type). This was driven by an increased affinity for glucose (S(0.5), 3.3 ± 0.1 and 3.5 ± 0.1 mm, respectively) versus wild type (7.5 ± 0.1 mm). Correspondingly, the V389D and T103I MODY mutants had markedly reduced relative activity indexes (<0.1). T103I had an altered affinity for glucose (S(0.5), 24.9 ± 0.6 mm), whereas V389D also exhibited a reduced affinity for ATP and decreased catalysis rate (S(0.5), 78.6 ± 4.5 mm; ATP(K(m)), 1.5 ± 0.1 mm; K(cat), 10.3 ± 1.1s(-1)) compared with wild type (ATP(K(m)), 0.4 ± <0.1; K(cat), 62.9 ± 1.2). Both Thr-103 mutants showed reduced inhibition by the endogenous hepatic inhibitor glucokinase regulatory protein. Molecular modeling demonstrated that Thr-103 maps to the allosteric activator site, whereas Val-389 is located remotely to this position and all other previously reported activating mutations, highlighting α-helix 11 as a novel region regulating GCK activity. Our data suggest that pharmacological manipulation of GCK activity at locations distal from the allosteric activator site is possible.     

Ethanol inhibits dual receptor stimulation of pineal cAMP and cGMP by vasoactive intestinal peptide and phenylephrine

Concurrent activation of vasoactive intestinal peptide and alpha 1-adrenergic receptor resulted in greater than 20-fold increases in pineal cAMP and cGMP accumulation. We now find that an intoxicating level of ethanol (0.2%, 34 mM) inhibits greater than 50% the large increases in pineal cAMP and cGMP produced by concurrent treatment with vasoactive intestinal peptide and phenylephrine. The potency of the various alcohols tested was directly related to their chain length. This inhibition appears to be specific since a five-fold higher concentration of ethanol does not inhibit the stimulation of cAMP and cGMP accumulation produced by concurrent treatment with isoproterenol and phenylephrine. Accordingly, it seems that one mechanism of action of ethanol on neural function may be its ability to selectively inhibit ethanol-sensitive integrative mechanisms which regulate cyclic nucleotides.     

Membrane-type MMPs enable extracellular matrix permissiveness and mesenchymal cell proliferation during embryogenesis

Peri-cellular remodeling of mesenchymal extracellular matrices is considered a prerequisite for cell proliferation, motility and development. Here we demonstrate that membrane-type 3 MMP, MT3-MMP, is expressed in mesenchymal tissues of the skeleton and in peri-skeletal soft connective tissue. Consistent with this localization, MT3-MMP-deficient mice display growth inhibition tied to a decreased viability of mesenchymal cells in skeletal tissues. We document that MT3-MMP works as a major collagenolytic enzyme, enabling cartilage and bone cells to cleave high-density fibrillar collagen and modulate their resident matrix to make it permissive for proliferation and migration. Collectively, these data uncover a novel extracellular matrix remodeling mechanism required for proper function of mesenchymal cells. The physiological significance of MT3-MMP is highlighted in mice double deficient for MT1-MMP and MT3-MMP. Double deficiency transcends the combined effects of the individual single deficiencies and leads to severe embryonic defects in palatogenesis and bone formation incompatible with life. These defects are directly tied to loss of indispensable collagenolytic activities required in collagen-rich mesenchymal tissues for extracellular matrix remodeling and cell proliferation during embryogenesis.     

Protein kinase C: subcellular redistribution by increased Ca2+ influx. Evidence that Ca2+-dependent subcellular redistribution of protein kinase C is involved in potentiation of beta-adrenergic stimulation of pineal cAMP and cGMP by K+ and A23187

Phenylephrine is known to stimulate translocation of protein kinase C in rat pinealocytes (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W. B. (1985) Nature 314, 359-361). In the present study, the receptor mediating this effect was found to belong to the alpha 1-adrenoceptor subclass. Activation of this receptor is also known to produce a sustained increase in [Ca2+]i by increasing net influx (Sugden, A. L., Sugden, D., and Klein, D. C. (1985) J. Biol. Chem. 261, 11608-11612), which points to the possible importance of Ca2+ influx in the subcellular redistribution (activation) of protein kinase C in intact cells. This possibility was investigated by reducing extracellular Ca2+ ((Ca2+]o) with EGTA or by inhibiting Ca2+ influx with inorganic Ca2+ blockers. These treatments reduced alpha 1-adrenoceptor-mediated translocation of protein kinase C. This suggested that elevation of Ca2+ influx alone triggers activation of protein kinase C. In support of this, it was found that treatments which elevate Ca2+ influx, including increased extracellular K+ and addition of the Ca2+ ionophore A23187, cause redistribution of protein kinase C. The effect of K+ was blocked by nifedipine and that of A23187 by EGTA, indicating that effects of these agents are Ca2+-dependent. The possible role of phospholipase C activation in these effects was examined by measuring the formation of [3H]diacylglycerol by cells labeled with [3H]arachidonic acid. Although [3H]diacylglycerol formation was easily detected in the presence or absence of an effective concentration of an inhibitor of diacylglycerol kinase, none of the agents which cause rapid translocation of protein kinase C were found to cause a rapid increase in the generation of [3H]diacylglycerol. These findings establish that an increase in Ca2+ influx is sufficient to trigger translocation of protein kinase C. In addition, we found that a very close correlation exists between translocation of protein kinase C by phenylephrine, K+, and A23187 and their ability to potentiate beta-adrenergic stimulation of cAMP and cGMP accumulation. This provides strong support to the proposal that translocation of protein kinase C is required for potentiation of beta-adrenergic stimulation of pinealocyte cAMP and cGMP accumulation.     

Prokaryotic toxin-antitoxin stress response loci

Although toxin-antitoxin gene cassettes were first found in plasmids, recent database mining has shown that these loci are abundant in free-living prokaryotes, including many pathogenic bacteria. For example, Mycobacterium tuberculosis has 38 chromosomal toxin-antitoxin loci, including 3 relBE and 9 mazEF loci. RelE and MazF are toxins that cleave mRNA in response to nutritional stress. RelE cleaves mRNAs that are positioned at the ribosomal A-site, between the second and third nucleotides of the A-site codon. It has been proposed that toxin-antitoxin loci function in bacterial programmed cell death, but evidence now indicates that these loci provide a control mechanism that helps free-living prokaryotes cope with nutritional stress.