Human platelet membrane proteins were phosphorylated by exogenous, partially purified Ca2 +-activated phospholipid-dependent protein kinase (protein kinase C). The phosphorylation of one of the major substrates for protein kinase C (Mr = 41000) was specifically suppressed by the fl subunit of the inhibitory guaninenucleotide-binding regulatory component (Gi, Ni) of adenylate cyclase. The free a subunit of Gi ( M , = 41 000) also served as an excellent substrate for the kinase (> 0.5 mol phosphate incorporated per mol of subunit), but the Gi oligomer (a . fl. y) did not. Treatment of cyc-S49 lymphoma cells, which are deficient in GJNS (the stimulatory component) but contain functional Gi/Ni, with the phorbol ester, 12-0-tetradecanoylphorbol 13-acetate, a potent activator of protein kinase C, did not alter stimulation of adenylate cyclase catalytic activity by forskolin, whereas the Gi/Ni-mediated inhibition of the cyclase by the hormone, somatostatin, was impaired in these membranes. The results suggest that the a subunit of the inhibitory guanine-nucleotide-binding regulatory component of adenylate cyclase may be a physiological substrate for protein kinase C and that the function of the component in transducing inhibitory hormonal signals to adenylate cyclase is altered by its phosphorylation.
Polysaccharides are a promising material for nonfouling surfaces because their chemical composition makes them highly hydrophilic and able to form water-storing hydrogels. Here we investigated the nonfouling properties of hyaluronic acid (HA) and chondroitin sulfate (CS) against marine fouling organisms. Additionally, the free carboxyl groups of HA and CS were postmodified with the hydrophobic trifluoroethylamine (TFEA) to block free carboxyl groups and render the surfaces amphiphilic. All coatings were tested with respect to their protein resistance and against settlement and adhesion of different marine fouling species. Both the settlement and adhesion strength of a marine bacterium (Cobetia marina), zoospores of the seaweed Ulva linza, and cells of a diatom (Navicula incerta) were reduced compared to glass control surfaces. In most cases, TFEA capping increased or maintained the performance of the HA coatings, whereas for the very well performing CS coatings the antifouling performance was reduced after capping.
The influence of the phorbol ester, 12-0-tetradecanoylphorbol 13-acetate (TPA), a direct activator of the Ca2 +-activated, phospholipid-dependent protein kinase (protein kinase C), was studied on regulation of human platelet adenylate cyclase. Intact platelets were pretreated with the phorbol ester and, thereafter, membranes were prepared and the regulation of the hormone-sensitive adenylate cyclase in these membranes was studied. The following data were obtained: The TPA treatment applied had apparently no effect on the activity of the catalytic moiety of the platelet adenylate cyclase nor on the stimulatory N, protein nor on stimulatory hormone receptors (prostaglandin El) and the mutual interactions of these components of the stimulatory hormone-sensitive pathway. However, the TPA treatment of intact platelets largely impaired the GTP-dependent, hormone-sensitive inhibitory pathway to the adenylate cyclase, involving the inhibitory Ni protein. The pretreatment led to a large reduction or loss of adenylate cyclase inhibition by GTP itself and by the inhibitory agonists, epinephrine and thrombin, inhibiting the untreated enzyme via separate receptors by an Ni-mediated process. In contrast, platelet adenylate cyclase inhibition not involving the Ni protein was not affected by the TPA treatment. The observed effects of TPA were very rapid in onset and were not shared by a derivative of TPA which did not activate protein kinase C. The data obtained suggest than protein kinase C activated by the phorbol ester interferes with the platelet adenylate cyclase system, leading to a specific alteration of the Ni-protein-mediated signal transduction to the adenylate cyclase.The adenylate cyclase of human platelet membranes is regulated by several stimulatory and inhibitory hormone-like factors affecting the catalytic activity upon receptor interaction through an action at the stimulatory (Ns, G,) and inhibitory (Ni, Gi) guanine-nucleotide-binding coupling component, respectively. For example, various prostaglandins such as prostaglandin El (PGE1), I2 and D2 cause an N,-mediated stimulation of adenylate cyclase following receptor interaction. This enzyme stimulation observed in membrane preparations is also reflected in an increase in total platelet cyclic AMP levels [l]. On the other hand, the platelet adenylate cyclase is also influenced by various inhibitory hormonal factors causing enzyme inhibition mediated by the inhibitory Ni protein. In membrane preparations, such a receptor-induced, Ni-mediated adenylate cyclase inhibition has been described for epinephrine and norepinephrine (acting via a2 adrenoceptors), for ADP, vasopressin (via VI receptors), platelet-activating factor and, very recently, also for thrombin [2-61. However, this enzyme inhibition observed in membrane preparations is not reflected in each case in a respective fall in total platelet cyclic AMP levels. For example,
The influence of ammonia on the anaerobic degradation of peptone by mesophilic and thermophilic populations of biowaste was investigated. For peptone concentrations from 5 g l-1 to 20 g l-1 the mesophilic population revealed a higher rate of deamination than the thermophilic population, e.g. 552 mg l-1 day-1 compared to 320 mg l-1 day-1 at 10 g l-1 peptone. The final degree of deamination of the thermophilic population was, however, higher: 102 compared to 87 mg NH3/g peptone in the mesophilic cultures. If 0.5-6.5 g l-1 ammonia was added to the mesophilic biowaste cultures, deamination of peptone, degradation of its chemical oxygen demand (COD) and formation of biogas were increasingly inhibited, but no hydrogen was formed. The thermophilic biowaste cultures were most active if around 1 g ammonia l-1 was present. Deamination, COD degradation and biogas production decreased at lower and higher ammonia concentrations and hydrogen was formed in addition to methane. Studies of the inhibition by ammonia of peptone deamination, COD degradation and methane formation revealed a Ki (50%) for NH3 of 92, 95 and 88 mg l-1 at 37 degrees C and 251, 274 and 297 mg l-1 at 55 degrees C respectively. This indicated that the thermophilic flora tolerated significantly more NH3 than the mesophilic flora. In the mesophilic reactor effluent 4.6 x 10(8) peptone-degrading colony-forming units (cfu)/ml were culturable, whereas in the thermophilic reactor effluent growth of only 5.6 x 10(7) cfu/ml was observed.
Amphiphilic coatings are promising candidates for fouling-release applications. As hydrophilic components, polysaccharides are interesting and environmentally benign building blocks. We used covalently coupled alginic acid (AA) and hyaluronic acid (HA) and postmodified them with a hydrophobic fluorinated amine. The surfaces showed good stability under marine conditions and fluorination led to a decreased uptake of Ca(2+) ions after modification. In single species settlement assays (bacteria, diatoms, barnacle cypris larvae), the modification decreased the settlement density and/or the adhesion strength of many of the tested species. Field studies supported findings of the laboratory experiments, as hydrophobic modification of AA and HA decreased diatom colonization.
The influence of zwitterionic self-assembled monolayers on settlement and removal of algae was studied. The monolayers were constructed either from zwitterionic thiols or from solutions of positively and negatively charged thiols. The cationic component was composed of quaternary ammonium terminated thiols and the anionic component contained sulfate or carboxylate termination. During assembly, all surfaces showed a strong tendency for equilibration of the surface charge. Settlement and adhesion assays with zoospores of Ulva linza and the diatom Navicula incerta, and field tests of the initial surface colonization revealed the relevance of charge equilibration for the biological inertness of the prepared surfaces.
Homogenates, membranes and cytosol of rat and human platelets were found to contain cGMP-dependent protein kinase immunoreactivity. Specific cGMP-dependent protein kinase immunoreactivity was about I .7 pmol protein kinase/mg protein for homogenates of human platelets and 0.7 pmol/mg for homogenates of rat platelets; the majority appeared to be associated with the membrane fraction.In membranes of platelets low concentrations of cAMP (0.5 -2 pM) stimulated the phosphorylation of five major proteins with apparent relative molecular masses, M,, of 240000,130000,50000,42000 and 22000 while low concentrations of cGMP (0.5 -2 pM) stimulated the phosphorylation of three major proteins with apparent M , of 130000, 50000 and 46000. An affinity-purified antibody against the cGMP-dependent protein kinase was prepared which specifically inhibited the activity of cGMP-dependent protein kinase. In membranes of human platelets this affinity-purified antibody inhibited the cGMP-stimulated phosphorylation of the three proteins with M, of 130000,50000 and 46000 while it had no effect on the CAMP-dependent and cyclic-nucleotide-independent protein phosphorylation.The results demonstrate that platelets contain a cGMP-dependent protein kinase and at least three specific substrates for this enzyme. Two of these substrates, the proteins with apparent molecular M , of 130000 and 50000, are substrates for both CAMP-and cGMP-dependent protein kinase. The protein with apparent M , of 130000 appears to be closely related to an intrinsic plasma membrane protein of vascular smooth muscle cells which is a substrate for a membrane-associated cGMP-dependent protein kinase. Therefore, cGMP-dependent protein kinase and cGMP-regulated phosphoproteins may mediate in platelets the intracellular effects of those hormones, vasodilators and drugs which elevate the level of cGMP and inhibit platelet aggregation.Platelets are an excellent model for studying the molecular mechanisms of receptor-linked signal transduction since several second-messenger systems appear to interact in the regulation of platelet activation. Thrombin, collagen, ADP and platelet-activating factor cause the aggregation of platelets with a simultaneous release of serotonin [l], and this platelet response can be inhibited by CAMP-and cGMP-elevating
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