Kinetics and activation of phospholipase C by P2Y purinergic agonists and guanine nucleotides

Membranes prepared from [3H]inositol-labeled turkey erythrocytes express a phospholipase C that is markedly stimulated by stable analogs of GTP (Harden, T. K., Stephens, L., Hawkins, P. T., and Downes, C. P. (1987) J. Biol. Chem. 262, 9057-9061). We now report that P2-purinergic receptor-mediated regulation of the enzyme occurs in the membrane preparation. The order of potency of a series of ATP and ADP analogs for stimulation of inositol phosphate formation, i.e. 2-methylthioadenosine 5'-triphosphate (2MeSATP) greater than adenosine 5'-O-(2-thiodiphosphate) greater than adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than 5'-adenylyl imidodiphosphate approximately ADP greater than alpha, beta-methyleneadenosine 5'-triphosphate greater than beta, gamma-methyleneadenosine 5'-triphosphate, was consistent with that for the P2Y-purinergic receptor subtype. Agonist-stimulated effects were completely dependent on the presence of guanine nucleotide. Activation of phospholipase C by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) occurred with a considerable time lag. The rate of activation followed first order kinetics and was markedly increased by increasing concentrations of a P2Y receptor agonist; in contrast, the rate of activation at a fixed agonist concentration was independent of guanine nucleotide concentration. Addition of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) prior to addition of agonist and GTP, 5'-guanylyl imidodiphosphate (Gpp(NH)p), or GTP gamma S blocked in a concentration-dependent manner the stimulatory effect of guanine nucleotide. GDP beta S, added subsequent to preactivation of membranes with 2MeSATP and GTP gamma S or Gpp(NH)p had only small inhibitory effects on the rate of inositol phosphate production observed over the subsequent 10 min. In contrast, addition of GDP beta S to GTP-preactivated membranes resulted in a rapid return of enzyme activity to the basal state within 60 s. Taken together, the data are consistent with the idea that P2Y receptor activation increases the rate of exchange of GTP and GTP analogs for GDP on the relevant guanine nucleotide regulatory protein. Once the active enzymic species is formed, hydrolysis of guanine nucleotide reverts the enzyme to the inactive state

A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. I. Purification and properties.

Eighty-three percent of polyphosphoinositide-specific phospholipase C activity was recovered in a cytosolic fraction after nitrogen cavitation of turkey erythrocytes. This activity has been purified approximately 50,000-fold when compared to the starting cytosol with a yield of 1.7-5.0%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phospholipase C preparation revealed a major polypeptide of 150 kDa. The specific activity of the purified enzyme was 6.7-14.0 mumol/min/mg of protein with phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 4-phosphate as substrate. Phospholipase C activity was markedly dependent on the presence of Ca2+. The phospholipase C showed an acidic pH optimum (pH 4.0). At neutral pH, noncyclic inositol phosphates were the major products formed by the phospholipase C, while at pH 4.0, substantial formation of inositol 1:2-cyclic phosphate derivatives occurred. Properties of the purified 150-kDa turkey erythrocyte phospholipase C were compared with the approximately 150-kDa phospholipase C-beta and -gamma isoenzymes previously purified from bovine brain (Ryu, S. H., Cho, K. S., Lee, K. Y., Suh, P. G., and Rhee, S. G. (1987) J. Biol. Chem. 262, 12511-12518). The turkey erythrocyte phospholipase C differed from the two mammalian phospholipases with respect to the effect of sodium cholate on the rate of polyphosphoinositide hydrolysis observed. Moreover, when presented with dispersions of pure inositol lipids, phospholipases C-beta and -gamma displayed comparable maximal rates of polyphosphoinositide and phosphatidylinositol hydrolysis. By contrast, the turkey erythrocyte phospholipase C displays a marked preference for polyphosphoinositide substrates

A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. II. P2Y-purinergic receptor and G-protein-mediated regulation of the purified enzyme reconstituted with turkey erythrocyte ghosts.

The preceding paper describes purification and properties of a 150-kDa polyphosphoinositide-specific phospholipase C from a cytosolic fraction of turkey erythrocytes (Morris, A. J., Waldo, G. L., Downes, C. P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507). Turkey erythrocytes express a P2Y-purinergic receptor that employs an unidentified G-protein to activate phospholipase C (Boyer, J. L., Downes, C. P., and Harden, T. K. (1989) J. Biol. Chem. 264, 884-890; Cooper, C. L., Morris, A. J., and Harden, T. K. (1989) J. Biol. Chem. 264, 6202-6206). This paper describes receptor and G-protein regulation of the purified turkey erythrocyte phospholipase C after reconstitution of the enzyme using [3H]inositol pre-labeled turkey erythrocyte ghosts as acceptor membranes. These membranes contain polyphosphoinositides labeled to high specific radioactivity and display reduced responsiveness of their endogenous phospholipase C to P2Y-purinergic receptor agonists and guanine nucleotides. Reconstitution of purified enzyme had no effect on basal inositol phosphate production, but markedly increased P2Y-purinergic receptor agonist and guanine nucleotide-dependent accumulation of inositol phosphates. Reconstitution of 5 ng of purified phospholipase C with 10 micrograms of acceptor membrane protein produced half-maximal effects, and maximal activity was observed with reconstitution of 100 ng of purified enzyme. Agonist and guanine nucleotide-regulated phospholipase C activity measured using a reconstitution assay co-purified with phospholipase C activity detected using exogenously provided phosphatidylinositol 4,5-bisphosphate during purification of the 150-kDa protein. Only the maximal rate of inositol phosphate formation attained upon activation was increased in the presence of the purified phospholipase C. K0.5 values for adenosine 5'-O-(2-thiodiphosphate), guanosine 5'-3-O-(thio)triphosphate, and A1F4- activation of the purified enzyme were the same as for the endogenous phospholipase C activity of the acceptor membranes

Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors

<p>Abstract</p> <p>Background</p> <p>Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors.</p> <p>Methods</p> <p>A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP) in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC), 13 papillary (pRCC), 10 chromophobe (chRCC), and 10 oncocytomas) and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters.</p> <p>Results</p> <p>Significant differences in methylation levels among the four subtypes of renal tumors were found for <it>CDH1 </it>(<it>p </it>= 0.0007), <it>PTGS2 </it>(<it>p </it>= 0.002), and <it>RASSF1A </it>(<it>p </it>= 0.0001). <it>CDH1 </it>hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (<it>p </it>= 0.00016 and <it>p </it>= 0.0034, respectively), whereas <it>PTGS2 </it>methylation levels were significantly higher in ccRCC compared to pRCC (<it>p </it>= 0.004). <it>RASSF1A </it>methylation levels were significantly higher in pRCC than in normal tissue (<it>p </it>= 0.035). In pRCC, <it>CDH1 </it>and <it>RASSF1A </it>methylation levels were inversely correlated with tumor stage (<it>p </it>= 0.031) and nuclear grade (<it>p </it>= 0.022), respectively.</p> <p>Conclusion</p> <p>The major subtypes of renal epithelial neoplasms display differential aberrant <it>CDH1</it>, <it>PTGS2</it>, and <it>RASSF1A </it>promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses.</p

Eight urgent, fundamental and simultaneous steps needed to restore ocean health, and the consequences for humanity and the planet of inaction or delay

The ocean crisis is urgent and central to human wellbeing and life on Earth; past and current activities are damaging the planet's main life support system for future generations. We are witnessing an increase in ocean heat, disturbance, acidification, bio‐invasions and nutrients, and reducing oxygen levels. Several of these act like ratchets: once detrimental or negative changes have occurred, they may lock in place and may not be reversible, especially at gross ecological and ocean process scales. Each change may represent a loss to humanity of resources, ecosystem function, oxygen production and species. The longer we pursue unsuitable actions, the more we close the path to recovery and better ocean health and greater benefits for humanity in the future. We stand at a critical juncture and have identified eight priority issues that need to be addressed in unison to help avert a potential ecological disaster in the global ocean. They form a purposely ambitious agenda for global governance and are aimed at informing decision‐makers at a high level. They should also be of interest to the general public. Of all the themes, the highest priority is to rigorously address global warming and limit surface temperature rise to 1.5°C by 2100, as warming is the pre‐eminent factor driving change in the ocean. The other themes are establishing a robust and comprehensive High Seas Treaty, enforcing existing standards for Marine Protected Areas and expanding their coverage, especially in terms of high levels of protection, adopting a precautionary pause on deep‐sea mining, ending overfishing and destructive fishing practices, radically reducing marine pollution, putting in place a financing mechanism for ocean management and protection, and lastly, scaling up science/data gathering and facilitating data sharing. By implementing all eight measures in unison, as a coordinated strategy, we can build resilience to climate change, help sustain fisheries productivity, particularly for low‐income countries dependent on fisheries, protect coasts (e.g. via soft‐engineering/habitat‐based approaches), promote mitigation (e.g. carbon storage) and enable improved adaptation to rapid global change.The attached document is the author(’s’) final accepted/submitted version of the journal article. You are advised to consult the publisher’s version if you wish to cite from it

Rural-to-Urban Labor Migration, Household Livelihoods, and the Rural Environment in Chongqing Municipality, Southwest China

Rural migration and its relationship to the rural environment have attracted increasing research interest in recent decades. Rural migration constitutes a key component of human population movement, while rural areas contain most of the world’s natural resources such as land and forests. This study empirically evaluates a conceptual framework incorporating rural household livelihoods as an integrative mediating factor between rural migration and the rural environment in the context of rural-to-urban labor migration in Chongqing Municipality, Southwest China. The analysis draws on data collected through household surveys and key informant interviews from four villages. Results confirm the hypothesis that labor-migrant and non-labor-migrant households differ significantly in livelihood activities including agricultural production, agricultural technology use, income and consumption, and resource use and management. Implications for the subsequent environmental outcomes of rural labor out-migration and corresponding natural resource management and policy in rural origin areas are discussed

An Interactive Internet-Based Continuing Education Course on Sexually Transmitted Diseases for Physicians and Midwives in Peru

Clinicians in developing countries have had limited access to continuing education (CE) outside major cities, and CE strategies have had limited impact on sustainable change in performance. New educational tools could improve CE accessibility and effectiveness.The objective of this study was to evaluate an interactive Internet-based CE course on Sexually Transmitted Diseases (STDs) management for clinicians in Peru. Participants included physicians and midwives in private practice drawn from a census of 10 Peruvian cities. The CE included a three-hour workshop for improving Internet skills, followed by a 22-hour online course on STD-syndrome-management, with subsequent educational support. The course used case-based clinical vignettes tailored to local STD problems. Knowledge and reported practices on STD management were assessed before, immediately after and at four months after completion of the course. Statistical analysis included parametric tests-linear regression multivariate analysis, paired t-test and repeated measures ANOVA using SPSS 14.0. Of 1,071 eligible clinicians, 510 agreed to participate, as did an additional 132 public sector clinicians. Of these 642 participants, 619 (96.4%) completed the course, and 596 (96.3%) took the four-month follow-up evaluation. Physician and midwife scores improved from 64.2% correct answers on the pre-test to 77.9% correct on the four-month follow-up test (p<0.001). Most participants (95%) found the online course useful for their work needs. Self reported STD management practices did not change.Among physicians and midwives in Peru, an Internet-based CE course was feasible, acceptable with high participation rates, and led to sustained improvement in knowledge at four months. Further studies are needed to test it as a model for improving the training of physicians, midwives, and other health care providers

Patient-reported-outcomes in subjects with painful lumbar or cervical radiculopathy treated with pregabalin: evidence from medical practice in primary care settings

The objective of this study was to evaluate the effect of pregabalin in painful cervical or lumbosacral radiculopathy treated in Primary Care settings under routine clinical practice. An observational, prospective 12-week secondary analysis was carried-out. Male and female above 18 years, naïve to PGB, with refractory chronic pain secondary to cervical/lumbosacral radiculopathy were enrolled. SF-MPQ, Sheehan Disability Inventory, MOS Sleep Scale, Hospital Anxiety and Depression Scale and the EQ-5D were administered. A total of 490 (34%) patients were prescribed PGB-monotherapy, 702 (48%) received PGB add-on, and 159 (11%) were administered non-PGB drugs. After 12 weeks, significant improvements in pain, associated symptoms of anxiety, depression and sleep disturbances, general health; and level of disability were observed in the three groups, being significantly greater in PGB groups. In routine medical practice, monotherapy or add-on pregabalin is associated with substantial pain alleviation and associated symptoms improvements in painful cervical or lumbosacral radiculopathy

The δ subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling

Cellular RNA polymerases RNAPs can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis, the RNAP amp; 948; subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP amp; 948; HelD complexes. HelD has two long arms a Gre cleavage factor like coiled coil inserts deep into the RNAP secondary channel, dismantling the active site and displacing RNA, while a unique helical protrusion inserts into the main channel, prying the amp; 946; and amp; 946; amp; 8242; subunits apart and, aided by amp; 948;, dislodging DNA. RNAP is recycled when, after releasing trapped nucleic acids, HelD dissociates from the enzyme in an ATP dependent manner. HelD abundance during slow growth and a dimeric RNAP amp; 948; HelD 2 structure that resembles hibernating eukaryotic RNAP I suggest that HelD might also modulate active enzyme pools in response to cellular cue