Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have isolated and mapped to the left end of chromosome III a single-copy gene (TRG1) encoding a 72-kDa glycoprotein, by screening a yeast genomic library with a DNA probe specifying the catalytic center (APWCGHCK) of thioredoxin-related proteins. the TRG1 gene sequence predicts an amino-terminal leader peptide, two thioredoxin-like domains, five N-glycosylation sites and a carboxyl-terminal HDEL retention signal. The TRG1 protein shows about equal sequence similarity to a mammalian multifunctional protein family residing in the lumen of the endoplasmic reticulum (ER), and to a putative cytosolic alpha form of phosphoinositide-specific phospholipase C. Haploid cells do not survive TRG1 gene disruptions, unless an additional wild-type copy is generated by interchromosomal transposition. Antibodies raised against synthetic amino- and carboxyl-terminal epitopes recognize a pair of lumenl ER glycoproteins (gp70/72) and a cytosolic 48-kDa protein. A 1.8-kilobase TRG1 transcript was translated by a reticulocyte lysate into a 60-kDa protein, which was translocated and processed to a 72-kDa glycoprotein in the presence of ER membrane vesicles. The TRG1 gene was placed under the control of the galactose-inducible and glucose-repressible GAL1 promoter, leading to growth arrest in glucose media. Glucose repression of the TRG1 gene caused the disappearance of gp72 and the accumulation of procarboxypeptidase. Our data indicate that the TRG1 gene encodes a growth essential lumenal ER glycoprotein involved the maturation of vacuolar carboxypeptidase.
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PMID:The Saccharomyces cerevisiae TRG1 gene is essential for growth and encodes a lumenal endoplasmic reticulum glycoprotein involved in the maturation of vacuolar carboxypeptidase. 176 54

Insulin modifies the effects of PTH on osteoblast-like cells. However, the basis for this effect is unknown. In bone and kidney cells, the effects of PTH on cellular function are mediated by second messengers generated through both the phospholipase C and adenylate cyclase systems. Therefore, we examined the effects of insulin on PTH second messenger generation in UMR-106-01 rat osteoblastic osteosarcoma cells. PTH produced a rapid, transient increase in intracellular free calcium concentration ([Ca2+]i) which was maximal at 30 sec and was only minimally reduced in the absence of extracellular calcium. Inositol-triphosphate (IP3) production was increased in parallel. PTH stimulation of [Ca2+]i was concentration-dependent from 0.5-1,000 nM, with half-maximal stimulation at approximately 50 nM PTH. A 30-sec exposure to 50 nM PTH produced 32% and 23% increases in IP1 and IP3 production, respectively (both P less than 0.05). Although insulin alone did not significantly alter basal [Ca2+]i, a 1-min exposure to 1-100 nM insulin produced a concentration-dependent suppression of the PTH-stimulated transient increase in [Ca2+]i and IP3 generation. 100 nM insulin decreased 50 nM PTH stimulation of [Ca2+]i and IP3 levels by 84% (P less than 0.02) and 80% (P less than 0.001), respectively. Preexposure to insulin also decreased PTH stimulation of intracellular cAMP levels, but to a lesser degree. A 1-min exposure to 100 nM insulin produced a 32% (P less than 0.01) decrease in PTH-stimulated cAMP generation, but lower insulin concentrations were without significant effects. These results demonstrate that in UMR-106-01 cells, insulin suppresses PTH stimulation of second messengers generated through both the phospholipase C and adenylate cyclase systems, but has a more marked effect on the former.
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PMID:Insulin acutely suppresses parathyroid hormone second messenger generation in UMR-106-01 osteoblast-like cells: differential effects on phospholipase C and adenylate cyclase activation. 185 51

Opioids elicit an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG108-15 cells, which, depending upon growth conditions, results from either Ca2+ influx in differentiated cells or Ca2+ release from internal stores in undifferentiated cells (Jin et al., 1992). In this report we describe fura-2-based digital imaging studies that demonstrate that opioid-evoked Ca2+ release in these cells results from the activation of phospholipase C (PLC) and subsequent mobilization of the inositol 1,4,5-trisphosphate (IP3)-sensitive store. D-Ala2-D-Leu5-enkephalin (DA-DLE) evoked concentration-dependent increases in [Ca2+]i (EC50 approximately equal to 4 nM). The response was blocked by naloxone (1 microM). In single cells, sequential application of selective opioid agonists (10 nM) evoked responses of the rank order DADLE = D-Pen2, D-Pen5-enkephalin (DPDPE) > trans-(+/-) 3,4-dichloro-N-methyl-N-(2-[1- pyrrolidinyl]cyclohexyl) benzeneacetamide (U50488) > D-ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO), consistent with activation of a delta-opioid receptor. Forty percent (n = 198) of the cells responded to 100 nM DADLE with a net [Ca2+]i increase of 483 +/- 40 nM. Bradykinin (100 nM) elicited a response in 91% of the cells with a mean net amplitude of 707 +/- 36 nM. The DADLE-evoked responses were not blocked by removal of extracellular Ca2+; instead, they were abolished by treatment with 10 nM thapsigargin, an agent that depletes and prevents refilling of IP3-sensitive Ca2+ stores. A 1 microM concentration of U73122, an aminosteroid inhibitor of PLC, completely blocked the DADLE-evoked [Ca2+]i increase, while an inactive analog, U73433, was without effect. To explore the possible role of G-proteins in mediating opioid-induced [Ca2+]i increases in NG108-15 cells, we pretreated cells with pertussis or cholera toxin; pertussis toxin blocked the opioid-induced response while cholera toxin was without effect, consistent with a Gi- or Go-mediated effect. Activation of the opioid inhibitory pathway previously described for these cells appears to stimulate the phosphoinositide (PI) cascade as well. Including the PI cascade among the multiple second messenger systems modulated by opioids may be key to understanding the biochemical events that underlie acute and chronic opioid action.
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PMID:Opioids mobilize calcium from inositol 1,4,5-trisphosphate-sensitive stores in NG108-15 cells. 815 47

The effects of pulsatile and steady fluid flow on the mRNA levels of proto-oncogenes c-fos, c-jun, and c-myc in cultured human umbilical vein endothelial cells (HUVEC) were investigated. c-fos mRNA levels in stationary cultures were very low. A 1 Hz pulsatile flow with an average shear stress of 16 dynes/cm2 induced a dramatic increase of c-fos mRNA levels in HUVEC 0.5 h after the onset of flow, which declined rapidly to basal levels within 1 h. Steady flow with a similar shear stress also induced a transient increase of c-fos mRNA levels, but to a lesser extent. In addition, increased c-fos mRNA levels were observed when low shear (2-6 dynes/cm2) was replaced by high shear (16-33 dynes/cm2). Pulsatile and steady flow caused a slight increase of c-jun and c-myc mRNA levels. The role of pulsatility was also investigated in platelet-derived growth factor (PDGF) expression. Pulsatile flow induced a transient increase of PDGF A- and B-chain mRNA levels with peaks at 1.5-2 h. Pulsatile flow, which was more stimulatory in mediating c-fos expression, however, was less stimulatory than steady flow in mediating PDGF expression. By using various inhibitors, protein kinase C was found to be an important mediator in flow-induced c-fos expression, with the involvement of G proteins, phospholipase C, and intracellular calcium. Protein kinase C was previously shown as a possible major mediator in flow-induced PDGF expression which, at least partly, appeared to follow the induction mechanism of c-fos, suggesting a possible connection between c-fos and PDGF induction. However, the c-fos antisense treatment, which significantly inhibited c-fos transcription, failed to block the flow-induced PDGF expression, suggesting that flow-induced c-fos expression may not play an important role in the mechanism of flow-induced PDGF expression. The difference in the induction of c-fos and PDGF expression under pulsatile as compared to steady flow indicates that a complex, flow-mediated regulatory mechanism of gene expression exists in HUVEC. The increased expression of these proto-oncogenes mediated by flow may be important in regulating long-term cellular responses.
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PMID:Pulsatile and steady flow induces c-fos expression in human endothelial cells. 841

We describe an apparatus that combines microelectrophoresis and laser trap technologies to monitor the activity of phosphoinositide-specific phospholipase C-delta1 (PLC-delta) on a single bilayer-coated silica bead with a time resolution of approximately 1 s. A 1-microm-diameter bead was coated with a phospholipid bilayer composed of electrically neutral phosphatidylcholine (PC) and negatively charged phosphatidylinositol 4,5-bisphosphate (2% PIP2) and captured in a laser trap. When an AC field was applied (160 Hz, 20 V/cm), the electrophoretic force produced a displacement of the bead, Delta(x), from its equilibrium position in the trap; Delta(x), which was measured using a fast quadrant diode detector, is proportional to the zeta potential and thus to the number of PIP2 molecules on the outer leaflet (initially, approximately 10(5)). When a solution containing PLC-delta flows past the bead, the enzyme adsorbs to the surface and hydrolyzes PIP2 to form the neutral lipid diacylglycerol. We observed a nonexponential decay of PIP2 on the bead with time that is consistent with a model based on the known structural properties of PLC-delta.
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PMID:Microelectrophoresis of a bilayer-coated silica bead in an optical trap: application to enzymology. 1132 31

Alpha-toxin gene was amplified from chromosomal DNA of Clostridium perfringens type A by polymerase chain reaction (PCR). PCR product was inserted into vector pGEM-T directly. The cloned recombinant plasmid pXCPA02 possesses positive nucleotide sequence of alpha-toxin. A 1.2 kb alpha-toxin gene fragment was cleaved with restriction endonucleases Nco I /EcoR I from plasmid pXCPA02, and then inserted into an expression vector pET-28c which cleaved with Nco I /EcoR I by blunt-end ligation. The recombinant expression plasmid pXETA02 was studied in detail by restriction endonucleases analysis and nucleotide sequencing. The results showed that the recombinant expression pXETA02 possessed a positive alpha-toxin gene sequence and reading frame. BL21 (DE3) (pXETA02) could produce alpha-toxin and the expressed products were recognized by alpha-toxin monoclonal antibodies with ELISA and Western blot. The expression optimization result indicated that the alpha toxin gene expression optimized condition with IPTG induction is culture medium pH 7.5, culture temperature 37 degrees C, joining IPTG to final concentration 0.8 mmol/L when the recombinant strain growth density OD600 achieved 0.8, and induction time 5h. The expression level of the alpha-toxin proteins were about 34.28% of total cellular protein with IPTG induction by SDS-PAGE and thin-layer gel scanning analysis. The alpha toxin gene expression optimized condition with lactose induction is culture medium pH 7.5, culture temperature 37 degrees C, joining lactose to final concentration 0.1 g/L when the recombinant strain growth density OD600 achieved 0.8, and induction time 5h. The expression level of the alpha-toxin proteins were about 23.82% of total cellular protein with lactose induction by SDS-PAGE and thin-layer gel scanning analysis. More importantly, Immunization in a mouse model with crude preparation containing the alpha-toxin protein inclusion bodies or inactivated recombinant strain induced protection against at least 1 MLD of the toxin from Clostridium perfringens type A.
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PMID:[Expression of alpha-toxin gene of Clostridium perfringens type A and its primary immunological protective function]. 1703 67