Phosphorus is a major nutrient vital for plant growth and development,with a substantial amount of cellular phosphorus being used for the biosynthesis of membrane phospholipids.Here,we report that NON-SPECIFIC PHOSPHO...Phosphorus is a major nutrient vital for plant growth and development,with a substantial amount of cellular phosphorus being used for the biosynthesis of membrane phospholipids.Here,we report that NON-SPECIFIC PHOSPHOLIPASE C4(NPC4)in rapeseed(Brassica napus)releases phosphate from phospholipids to promote growth and seed yield,as plants with altered NPC4 levels showed significant changes in seed production under different phosphate conditions.Clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated nuclease 9(Cas9)-mediated knockout of Bna NPC4 led to elevated accumulation of phospholipids and decreased growth,whereas overexpression(OE)of Bna NPC4resulted in lower phospholipid contents and increased plant growth and seed production.We demonstrate that Bna NPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in vitro,and plants with altered Bna NPC4 function displayed changes in their sphingolipid and glycerolipid contents in roots,with a greater change in glycerolipids than sphingolipids in leaves,particularly under phosphate deficiency conditions.In addition,Bna NPC4-OE plants led to the upregulation of genes involved in lipid metabolism,phosphate release,and phosphate transport and an increase in free inorganic phosphate in leaves.These results indicate that Bna NPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in rapeseed to enhance phosphate release from membrane phospholipids and promote growth and seed production.展开更多
Background In most colorectal carcinomas, the level of phospholipase C (PLC)-gamma 1 expression is greatly elevated. Increased expression of PLC-gamma 1 may play an important role in colon carcinogenesis, but the me...Background In most colorectal carcinomas, the level of phospholipase C (PLC)-gamma 1 expression is greatly elevated. Increased expression of PLC-gamma 1 may play an important role in colon carcinogenesis, but the mechanism is not well known. The aim of this study was to evaluate the role of PLC-gamma 1 in colon carcinogenesis by using recombinant lentivirus that stably suppressed the PLC-gamma 1 expression in human colorectal carcinoma LoVo cells. Methods Recombinant lentivirus producing PLC-gamma 1 siRNA were prepared. After LoVo cells were transduced by each lentivirus, stably transduced cells were selected by Blasticidin. The protein and mRNA expression of PLC-gamma 1 were examined by Western-blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis, and the effects of the lentivirus on the cell adhesion, migration and apoptosis were analyzed. Results Stable LoVo cell line deficient in PLC-gamma 1, was established. Notably, PLC-gamma 1 was silenced without affecting the levels of other subtypes of PLC so that the role of PLC-gamma 1 in colon carcinogenesis could be examined Silencing of endogenous PLC-gamma 1 resulted in efficient inhibition of the adhesion and migration of LoVo cells in vitro and a great increase of 5-fluorouracil induced apoptosis (30%-40%) of LoVo cells.Conclusions PLC-gamma 1 may play an important role in metastasis and anti-apoptosis in human colorectal carcinomas.展开更多
Background The pathogenesis of gastric cancer (GC) involves environmental and genetic factors.Recently,two genome-wide association studies found that phospholipase C epsilon 1 (PLCE1) polymorphisms might be relate...Background The pathogenesis of gastric cancer (GC) involves environmental and genetic factors.Recently,two genome-wide association studies found that phospholipase C epsilon 1 (PLCE1) polymorphisms might be related to GC risk,and several studies further validated this finding.However,these studies yielded inconsistent results.Methods A comprehensive database search was performed to identify eligible studies.Odds ratios with 95% confidence intervals were calculated to assess the strength of the association between PLCE1 rs2274223,rs753724,and rs11187842 and risk of GC.Subgroup analyses,publication bias,and sensitivity analyses were also conducted.Results Eleven studies (12 cohorts) were included in the meta-analysis.Based on 13 676 cases and 23 569 controls,a significant association between PLCE1 rs2274223 and GC risk was detected under various genotypic models.In the subgroup analyses,the association was significant for cardia GC,but weak for non-cardia GC.The association under the heterozygote model was detected for PLCE1 rs753724 and rs11187842 based on three studies involving 2768 cases and 3890 controls.Conclusions Our findings demonstrate that the presence of the G allele at rs2274223 of the PLCE1 gene may contribute to susceptibility to GC,especially cardia GC.PLCE1 rs753724 and rs11187842 are associated with GC risk under the heterozygote model.Further well-designed large studies are warranted to validate these findings.展开更多
Apolygus lucorum is the dominant pathogenic insect attacking Bacillus thuringiensis(Bt)cotton in China.Additionally,20-hydroxyecdysone(20E)has important functions in many biological processes,including insect reproduc...Apolygus lucorum is the dominant pathogenic insect attacking Bacillus thuringiensis(Bt)cotton in China.Additionally,20-hydroxyecdysone(20E)has important functions in many biological processes,including insect reproduction.Phospholipase C(PLC),which is an essential enzyme for phosphoinositide metabolism,is involved in 20E signal transduction,but its function in 20E-mediated reproduction in A.lucorum remains unclear.In this study,20E increased A/PLCγ transcription as well as the abundance and activity of the encoded protein during molting and metamorphosis.The 20E treatment also induced the considerable accumulation of two second messengers,inositol triphosphate and diacylglycerol.The expression levels of genes encoding vitellogenin(AlVg)and soluble trehalase(AlTre-1)were similar to those of AlPLCy,and were upregulated in response to 20C.The silencing of AlPLCγ resulted in downregulated expression o f AITre-γ smdAlVg.However,the silencing of AlTre-1 and AlVg did not affect AlPLCγ expression.Moreover,the silencing of AlVg did not alter AlTre-1 expression.Furthermore,an examination of the insect specimens indicated that AlPLCy is required for female adult reproduction,and that downregulated expression of this gene is associated with decreases in fecundity,adult longevity,and egg hatching rate as well as delayed oocyte maturation.We propose that 20E regulates AlTre-1 expression via AlPLCy and affects Vg expression as well as ovary development to facilitate the reproductive activities of A.lucorum females.展开更多
Phospholipase C zeta(PLCζ)is a sperm-specific protein that triggers oocyte activation.The analysis of PLCζexpression in human spermatozoa can be used as a diagnostic marker for oocyte activation deficiency.Our labor...Phospholipase C zeta(PLCζ)is a sperm-specific protein that triggers oocyte activation.The analysis of PLCζexpression in human spermatozoa can be used as a diagnostic marker for oocyte activation deficiency.Our laboratory has previously optimized a standard"in-house"assay to determine PLCζexpression in human spermatozoa.However,one study has suggested that an antigen unmasking method(AUM)would be more efficient in visualizing PLCζin human sperm.This study aimed to compare our established assay and AUM(involving HCl,acidic Tyrode's solution[AT],and heat).The mean relative fluorescence(RF)intensity of PLCζin frozen-thawed spermatozoa from fourteen fertile donors stained with the in-house method was significantly higher than three other AUM groups(in-house[mean±standard error of mean]:18.87±2.39 arbitrary units[a.u.]vs non-AUM:11.44±1.61 a.u.,AT-AUM:12.38±1.89 a.u.,and HCl-AUM:12.51±2.16 a.u.,P<0.05,one-way analysis of variance).The mean RF intensity of PLCζin AT-and HCl-treated spermatozoa from 12 infertile males was not significantly different from that of the non-AUM group.However,the in-house method resulted in the highest RF intensity(12.11±1.36 a.u.,P<0.01).Furthermore,specificity testing of antibody-antigen binding indicated that the in-house method showed more specific binding than spermatozoa treated by the AUM.In conclusion,our in-house method showed superior visualization and reliability than the AUM,thus supporting the continued use of our in-house assay for clinical research screening.展开更多
Prior to fertilization sperm has to undergo an activation process known as capaciation,leading to the acrosome reaction.Till now,little is known about the mechanism for preventing premature capacitation in sperm altho...Prior to fertilization sperm has to undergo an activation process known as capaciation,leading to the acrosome reaction.Till now,little is known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources have been thought to be involved.In this study,we report that NYD-SP27,an isoform of phospholipase C Zeta 1(PLCZ1),is localized to the sperm acrosome in mouse and human spermatozoa by immunofluorescence using a specific antibody.Western blot and double staining analyses show NYD-SP27 becomes detached from sperm,as they undergo capacitation and acrosome reaction.The absence of HCO_(3)^(-),a key factor in activating capacitation,from the capacitation-inducing medium prevents the loss of NYD-SP27 from sperm.The anti-NYD-SP27 antibody also prevents the loss of NYD-SP27 from sperm,reduced the number of capacitated sperm,inhibited the acrosome reaction induced by ATP and progesterone,and inhibited agonist-induced PLC-coupled Ca^(2+)mobilization in sperm,which can be mimicked by the PLC inhibitor,U73122.These data strongly suggest that NYD-SP27 is a physiological inhibitor of PLC that acts as an intrinsic decapacitation factor in sperm to prevent premature capacitation and acrosome reaction.展开更多
BACKGROUND:This study aimed to explore the effects of TNF-a on the expression of IP_3R1mRNA and protein in human mesangial cells(HMCs),and to elucidate the mechanism of TNF-a relating to IP_3R1 expression in the occur...BACKGROUND:This study aimed to explore the effects of TNF-a on the expression of IP_3R1mRNA and protein in human mesangial cells(HMCs),and to elucidate the mechanism of TNF-a relating to IP_3R1 expression in the occurrence of hepatorenal syndrome(HRS).METHODS:HMCs were stimulated by tumor(TNF-a) with 100 ng/mL for different hours(2,4,8,and 24 hours).The expression changes of IP_3R1 mRNA and protein were detected by quantitative real-time polymerase chain reaction and immunoblotting.Several inhibitors including D609,U73122,PP1,safingol,rottlerin and non-radioactive protein kinase C(PKC) were used to examine the mechanism of signal transduction ofTNF-a-regulated IP_3R1 in HMCs.RESULTS:The levels of IP_3R1 mRNA at 2 hours after TNF-a exposure were significantly enhanced and peaked at 8 hours in HMCs(P<0.01),then descended at 24 hours(P<0.01).The levels of IP_3R1 protein at 4 hours after TNF-a exposure were obviously increased and peaked at24 hours after TNF-a exposure(P<0.01).Compared to the control group,safingol(PKCa inhibitor)and D609(phosphatidylcholine-specific phospholipase C inhibitor) significantly blocked the TNF-ainduced expression of IP_3R1 mRNA(3.30±0.81 vs.1.95±0.13,P<0.05;2.10±0.49,P<0.01) and IP_3R1protein(3.09±0.13 vs.1.86+0.39,P<0.01;1.98±0.02,P<0.01).TNF-a promoted PKCa activation with maximal PKCa phosphorylation that occurred 8 hours after stimulation measured by non-radioactive PKC assay,and the effect was markedly attenuated by pretreatment with D609 or safingol.CONCLUSION:TNF-a increased the expression of IP_3R1 and this was mediated,at least in part,through the PC-PLC/PKCa signaling pathways in HMCs.展开更多
Understanding the growth regulatory mechanisms in filamentous fungi is very important for the production of medicines for antifungal therapies. It is well established that Ca2+ gradient is essential for hyphal growth ...Understanding the growth regulatory mechanisms in filamentous fungi is very important for the production of medicines for antifungal therapies. It is well established that Ca2+ gradient is essential for hyphal growth and that one mechanism responsible for the Ca2+ cellular concentration starts with the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by receptor-regulated forms of phosphoinositide-specific phospholipase C (PI-PLC). In the present study the levels of calcium in Aspergillus nidulans wild type (A26) and plcA-deficient mutant (AP27) growing in a carbon source readily assimilated, as glucose or pectin a non-readily assimilated carbon source was investigated. Intracellular calcium levels in A26 were higher in the presence of glucose than in pectin, but lower in AP27 independently of the carbon source and in AP27 the vesicular calcium distribution occurred mainly at the apex of the hyphae. Delay in nuclear division was also observed if A26 and AP27 were grown in pectin presence when compared with growth in glucose. For the first time, it is demonstrated that the levels of intracellular Ca2+ were higher when A. nidulans was growing in glucose than in a non readily assimilated carbon source as pectin. Further, it also showed that the plcA gene, although not essential, may be responsible for high-molecular weight carbon source recongnation, for the intracellular Ca2+ levels maintenance and consequently by the nuclear division in A. nidulans.展开更多
An oligopeptide permease A(OppA)was purified from the extracellular product of Vibrio harveyi SF-1.The molecular weight of the purified protein was estimated to be 58 kDa on SDS-PAGE.The purified protein showed phosph...An oligopeptide permease A(OppA)was purified from the extracellular product of Vibrio harveyi SF-1.The molecular weight of the purified protein was estimated to be 58 kDa on SDS-PAGE.The purified protein showed phospholipase C activity at the optimal values of temperature 50℃ and pH 8.0.The enzymatic activity decreased when the temperature increased to 40℃.The N-terminal sequence of the purified protein was determined as ADVPAGTKLA,which is similar to that of OppA.The OppA pre-cursor gene was cloned from the genome of V.harveyi SF-1.The gene consisted of 1665 base pairs and encoded a 554 amino acid polypeptide,which showed a high similarity to those of the OppAs of V.harveyi and other Vibrio species.The gene was subcloned into pET-28a(+)and expressed in Escherichia coli.The expressed recombinant protein was purified by Ni-NTA metal affinity chro-matography.The expressed recombinant protein showed a 58 kDa band on SDS-PAGE and exhibited phospholipase C activity with the optima of temperature 50℃ and pH 8.0.The purified protein was toxic to the flounder gill cells.An OppA mutant of V.harveyi SF-1 was constructed by homologous recombination.The mutant strain was less virulent when it was intraperitoneally inoculated to zebra fish,with the LD50 of 5.46×105 CFU fish-1,compared to 3.11×104 CFU fish-1 of the wild-type strain,which indicated that the OppA might play an important role in the pathogenicity of V.harveyi.展开更多
Clostridium perfringens phospholipase C(plc),also calledα-toxin,is encoded by the plc gene Clostridium perfringers.The production ofα-toxin can lead to the occurrence of gas gangrene.Vaccination is considered as one...Clostridium perfringens phospholipase C(plc),also calledα-toxin,is encoded by the plc gene Clostridium perfringers.The production ofα-toxin can lead to the occurrence of gas gangrene.Vaccination is considered as one of the best solutions against Clostridium infections.In this study,an anti-Cpα-toxin monoclonal antibody(mAbs)A10E5 was successfully prepared,which had better biological reactivity.Then,the phage random 12-peptide library was used to screen mAb A10E5 protein.After four rounds of screening,three peptides with high affinity to the anti-α-toxin mAbs were screened.Two 12-peptide peptide Q and peptide E with higher inhibition rate were obtained by indirect ELISA.Two polypeptides of 500μg·mL^(-1)synthesized in vitro were mixed with30μg·mL^(-1)α-toxin at a concentration to treat Hela cells.Cell viability was determined by MTT assay.The results showed that both of the peptides significantly increased the survival rate of Hela cells compared with theα-toxin group,and the effect of peptide Q was more obvious.The chickens were immunized with phages expressing two different affinity polypeptides and then challenged.The results of chicken weight change,intestinal lesion score,bacterial count,and antibody titer in peripheral blood showed that the two phages expressing the polypeptides had a certain protective effect on the chickens compared with the PBS group,and peptide Q had better protection effect.In conclusion,the high affinity peptide with mAb A10E5 was screened in this study,and the protective effect of the plc polypeptide vaccine was verified by in vivo and in vitro experiments,which was of great significance for the comprehensive prevention and treatment of the disease.展开更多
The effect of U50488, a selective K-opioid receptor agonist, on cardiac rhythm in the isolated perfused rat heart and intracellular calcium ([Ca2+] i) in the single ventricular myocyte were studied. The results showed...The effect of U50488, a selective K-opioid receptor agonist, on cardiac rhythm in the isolated perfused rat heart and intracellular calcium ([Ca2+] i) in the single ventricular myocyte were studied. The results showed that U50488 can induce arrhythmias dose-dependently in the isolated perfused rat heart and increase [ Ca2 + ] i in the single ventricular myocyte. The effect of U50488 can be blocked by a selectivek-receptor antagonist, nor-binaltorphimine. The arrhythmogenic effects and the increase of [ Ca2+]i induced by U50488 were blocked by U73122, neomycin and streptomycin, which are selective phospolipase C inhibitors, but not by U73433, the inactive structural analog of U73122. These results demonstrated that the arrhythmogenic effect of cardiac K-receptor is due to activation of phosphoinositol/Ca2+ pathway.展开更多
Phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphocholine and diacylglycerol (DAG). PC-PLC has a long tradition in animal signal tra...Phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphocholine and diacylglycerol (DAG). PC-PLC has a long tradition in animal signal transduction to generate DAG as a second messenger besides the classical phosphatidylinositol splitting phospholipase C (PI-PLC). Based on amino acid sequence similarity to bacterial PC-PLC, six putative PC-PLC genes (NPC1 to NPC6) were identified in the Arabidopsis genome. RT-PCR analysis revealed overlapping expression pattern of NPC genes in root, stem, leaf, flower, and silique. In auxin-treated PNPc3:GUS and PNPc4:GUS seedlings, strong increase of GUS activity was visible in roots, leaves, and shoots and, to a weaker extent, in brassinolide-treated (BL) seedlings. PNPc4:GUS seedlings also responded to cytokinin with increased GUS activity in young leaves. Compared to wild-type, T-DNA insertional knockouts npc3 and npc4 showed shorter primary roots and lower lateral root density at low BL concentrations but increased lateral root densities in response to exogenous 0.05-1.0 I^M BL BL-induced expression of TCH4 and LRX2, which are involved in cell expansion, was impaired but not impaired in repression of CPD, a BL biosynthesis gene, in BL-treated npc3 and npc4. These observations suggest NPC3 and NPC4 are important in BL-mediated signaling in root growth. When treated with 0.1 I^M BL, DAG accumulation was observed in tobacco BY-2 cell cultures labeled with fluorescent PC as early as 15 min after application. We hypothesize that at least one PC-PLC is a plant signaling enzyme in BL signal transduction and, as shown earlier, in elicitor signal transduction.展开更多
The development of the nervous system is under the strict control of a number of signal transduction pathways, often interconnected. Among them, the phosphoinositide (PI) pathway and the related phospholipase C (PI...The development of the nervous system is under the strict control of a number of signal transduction pathways, often interconnected. Among them, the phosphoinositide (PI) pathway and the related phospholipase C (PI-PLC) family of enzymes have been attracting much attention. Besides their well-known role in the regulation of intracellular calcium levels, PI-PLC enzymes interact with a number of molecules belonging to further signal transduction pathways, contributing to a specific and complex network in the developing nervous system. In this review, the connections of PI signalling with further transduction pathways acting during neural development are discussed, with special regard to the role of the PI-PLC family of enzymes.展开更多
Sperm-specific phospholipase C zeta(PLCζ)initiates intracellular calcium(Ca2+)transients which drive a series of concurrent events collectively termed oocyte activation.Numerous investigations have linked abrogation ...Sperm-specific phospholipase C zeta(PLCζ)initiates intracellular calcium(Ca2+)transients which drive a series of concurrent events collectively termed oocyte activation.Numerous investigations have linked abrogation and absence/reduction of PLCζwith forms of male infertility in humans where oocyte activation fails.However,very few studies have examined potential relationships between PLCζand advancing male age,both of which are increasingly considered to be major effectors of male fertility.Initial efforts in humans may be hindered by inherent PLCζvariability within the human population,alongside a lack of sufficient controllable repeats.Herein,utilizing immunoblotting,immunofluorescence,and quantitative reverse transcription PCR(qRT-PCR)we examined for the first time PLCζprotein levels and localization patterns in sperm,and PLCζmRNA levels within testes,from mice at 8 weeks,12 weeks,24 weeks,and 36 weeks of age,from two separate strains of mice,C57BL/6(B6;inbred)and CD1(outbred).Collectively,advancing male age generally diminished levels and variability of PLCζprotein and mRNA in sperm and testes,respectively,when both strains were examined.Furthermore,advancing male age altered the predominant pattern of PLCζlocalization in mouse sperm,with younger mice exhibiting predominantly post-acrosomal,and older mice exhibiting both post-acrosomal and acrosomal populations of PLCζ.However,the specific pattern of such decline in levels of protein and mRNA was strain-specific.Collectively,our results demonstrate a negative relationship between advancing male age and PLCζlevels and localization patterns,indicating that aging male mice from different strains may serve as useful models to investigate PLCζin cases of male infertility and subfertility in humans.展开更多
基金supported by grants from the National Key Research and Development Program of China (2022YFD1200400)the Key Research and Development Plan of Hubei Province (2021ABA011)+3 种基金Fundamental Research Funds for the Central Universities (2662022ZKPY001)a Higher Education Discipline Innovation Project (B20051)an Agriculture and Food Research Initiative (AFRI)award[2020-67013-30908/project accession number 1022148]of the US Department of Agriculture National Institute of Food and Agriculturethe China Postdoctoral Science Foundation (2023M731230)。
文摘Phosphorus is a major nutrient vital for plant growth and development,with a substantial amount of cellular phosphorus being used for the biosynthesis of membrane phospholipids.Here,we report that NON-SPECIFIC PHOSPHOLIPASE C4(NPC4)in rapeseed(Brassica napus)releases phosphate from phospholipids to promote growth and seed yield,as plants with altered NPC4 levels showed significant changes in seed production under different phosphate conditions.Clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated nuclease 9(Cas9)-mediated knockout of Bna NPC4 led to elevated accumulation of phospholipids and decreased growth,whereas overexpression(OE)of Bna NPC4resulted in lower phospholipid contents and increased plant growth and seed production.We demonstrate that Bna NPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in vitro,and plants with altered Bna NPC4 function displayed changes in their sphingolipid and glycerolipid contents in roots,with a greater change in glycerolipids than sphingolipids in leaves,particularly under phosphate deficiency conditions.In addition,Bna NPC4-OE plants led to the upregulation of genes involved in lipid metabolism,phosphate release,and phosphate transport and an increase in free inorganic phosphate in leaves.These results indicate that Bna NPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in rapeseed to enhance phosphate release from membrane phospholipids and promote growth and seed production.
基金This study was supported by the grants from National Natural Science Foundation of China(No.39870381)Natural Science Foundation of Guangdong Province(No.020015)Emphasis Natural Science Foundation of Guangdong Province(No.05102580).
文摘Background In most colorectal carcinomas, the level of phospholipase C (PLC)-gamma 1 expression is greatly elevated. Increased expression of PLC-gamma 1 may play an important role in colon carcinogenesis, but the mechanism is not well known. The aim of this study was to evaluate the role of PLC-gamma 1 in colon carcinogenesis by using recombinant lentivirus that stably suppressed the PLC-gamma 1 expression in human colorectal carcinoma LoVo cells. Methods Recombinant lentivirus producing PLC-gamma 1 siRNA were prepared. After LoVo cells were transduced by each lentivirus, stably transduced cells were selected by Blasticidin. The protein and mRNA expression of PLC-gamma 1 were examined by Western-blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis, and the effects of the lentivirus on the cell adhesion, migration and apoptosis were analyzed. Results Stable LoVo cell line deficient in PLC-gamma 1, was established. Notably, PLC-gamma 1 was silenced without affecting the levels of other subtypes of PLC so that the role of PLC-gamma 1 in colon carcinogenesis could be examined Silencing of endogenous PLC-gamma 1 resulted in efficient inhibition of the adhesion and migration of LoVo cells in vitro and a great increase of 5-fluorouracil induced apoptosis (30%-40%) of LoVo cells.Conclusions PLC-gamma 1 may play an important role in metastasis and anti-apoptosis in human colorectal carcinomas.
文摘Background The pathogenesis of gastric cancer (GC) involves environmental and genetic factors.Recently,two genome-wide association studies found that phospholipase C epsilon 1 (PLCE1) polymorphisms might be related to GC risk,and several studies further validated this finding.However,these studies yielded inconsistent results.Methods A comprehensive database search was performed to identify eligible studies.Odds ratios with 95% confidence intervals were calculated to assess the strength of the association between PLCE1 rs2274223,rs753724,and rs11187842 and risk of GC.Subgroup analyses,publication bias,and sensitivity analyses were also conducted.Results Eleven studies (12 cohorts) were included in the meta-analysis.Based on 13 676 cases and 23 569 controls,a significant association between PLCE1 rs2274223 and GC risk was detected under various genotypic models.In the subgroup analyses,the association was significant for cardia GC,but weak for non-cardia GC.The association under the heterozygote model was detected for PLCE1 rs753724 and rs11187842 based on three studies involving 2768 cases and 3890 controls.Conclusions Our findings demonstrate that the presence of the G allele at rs2274223 of the PLCE1 gene may contribute to susceptibility to GC,especially cardia GC.PLCE1 rs753724 and rs11187842 are associated with GC risk under the heterozygote model.Further well-designed large studies are warranted to validate these findings.
基金This study was supported by Chinese Agricultural Research System(CARS-15-18),the Integration Research and Demonstration of the Technology of Cotton Fertilizer and Pesticide Reduction of China(2017YFD0201900)Jiangsu Agricultural Science and Technology Innovation Fund(CX(19)3098)+1 种基金grants from the National Natural Science Fund of China(31301668)JAAS Research Foundation(6111613).
文摘Apolygus lucorum is the dominant pathogenic insect attacking Bacillus thuringiensis(Bt)cotton in China.Additionally,20-hydroxyecdysone(20E)has important functions in many biological processes,including insect reproduction.Phospholipase C(PLC),which is an essential enzyme for phosphoinositide metabolism,is involved in 20E signal transduction,but its function in 20E-mediated reproduction in A.lucorum remains unclear.In this study,20E increased A/PLCγ transcription as well as the abundance and activity of the encoded protein during molting and metamorphosis.The 20E treatment also induced the considerable accumulation of two second messengers,inositol triphosphate and diacylglycerol.The expression levels of genes encoding vitellogenin(AlVg)and soluble trehalase(AlTre-1)were similar to those of AlPLCy,and were upregulated in response to 20C.The silencing of AlPLCγ resulted in downregulated expression o f AITre-γ smdAlVg.However,the silencing of AlTre-1 and AlVg did not affect AlPLCγ expression.Moreover,the silencing of AlVg did not alter AlTre-1 expression.Furthermore,an examination of the insect specimens indicated that AlPLCy is required for female adult reproduction,and that downregulated expression of this gene is associated with decreases in fecundity,adult longevity,and egg hatching rate as well as delayed oocyte maturation.We propose that 20E regulates AlTre-1 expression via AlPLCy and affects Vg expression as well as ovary development to facilitate the reproductive activities of A.lucorum females.
文摘Phospholipase C zeta(PLCζ)is a sperm-specific protein that triggers oocyte activation.The analysis of PLCζexpression in human spermatozoa can be used as a diagnostic marker for oocyte activation deficiency.Our laboratory has previously optimized a standard"in-house"assay to determine PLCζexpression in human spermatozoa.However,one study has suggested that an antigen unmasking method(AUM)would be more efficient in visualizing PLCζin human sperm.This study aimed to compare our established assay and AUM(involving HCl,acidic Tyrode's solution[AT],and heat).The mean relative fluorescence(RF)intensity of PLCζin frozen-thawed spermatozoa from fourteen fertile donors stained with the in-house method was significantly higher than three other AUM groups(in-house[mean±standard error of mean]:18.87±2.39 arbitrary units[a.u.]vs non-AUM:11.44±1.61 a.u.,AT-AUM:12.38±1.89 a.u.,and HCl-AUM:12.51±2.16 a.u.,P<0.05,one-way analysis of variance).The mean RF intensity of PLCζin AT-and HCl-treated spermatozoa from 12 infertile males was not significantly different from that of the non-AUM group.However,the in-house method resulted in the highest RF intensity(12.11±1.36 a.u.,P<0.01).Furthermore,specificity testing of antibody-antigen binding indicated that the in-house method showed more specific binding than spermatozoa treated by the AUM.In conclusion,our in-house method showed superior visualization and reliability than the AUM,thus supporting the continued use of our in-house assay for clinical research screening.
基金National 973 Project of China(No.2006CB504002),Chinese National Prominent Youth Foundation(No.30425006)Program for Changjiang Scholars and Innovative Research Team in University(PCSIRT)(No.IRT0631)+1 种基金Research Grants Council(RGC)of Hong Kong(No.CUHK4524/05M)Li Ka Shing Institute of Health Sciences and Focused Investment of the Chinese University of Hong Kong,China.
文摘Prior to fertilization sperm has to undergo an activation process known as capaciation,leading to the acrosome reaction.Till now,little is known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources have been thought to be involved.In this study,we report that NYD-SP27,an isoform of phospholipase C Zeta 1(PLCZ1),is localized to the sperm acrosome in mouse and human spermatozoa by immunofluorescence using a specific antibody.Western blot and double staining analyses show NYD-SP27 becomes detached from sperm,as they undergo capacitation and acrosome reaction.The absence of HCO_(3)^(-),a key factor in activating capacitation,from the capacitation-inducing medium prevents the loss of NYD-SP27 from sperm.The anti-NYD-SP27 antibody also prevents the loss of NYD-SP27 from sperm,reduced the number of capacitated sperm,inhibited the acrosome reaction induced by ATP and progesterone,and inhibited agonist-induced PLC-coupled Ca^(2+)mobilization in sperm,which can be mimicked by the PLC inhibitor,U73122.These data strongly suggest that NYD-SP27 is a physiological inhibitor of PLC that acts as an intrinsic decapacitation factor in sperm to prevent premature capacitation and acrosome reaction.
基金supported by a grant from Health Bureauof Jiangxi Province
文摘BACKGROUND:This study aimed to explore the effects of TNF-a on the expression of IP_3R1mRNA and protein in human mesangial cells(HMCs),and to elucidate the mechanism of TNF-a relating to IP_3R1 expression in the occurrence of hepatorenal syndrome(HRS).METHODS:HMCs were stimulated by tumor(TNF-a) with 100 ng/mL for different hours(2,4,8,and 24 hours).The expression changes of IP_3R1 mRNA and protein were detected by quantitative real-time polymerase chain reaction and immunoblotting.Several inhibitors including D609,U73122,PP1,safingol,rottlerin and non-radioactive protein kinase C(PKC) were used to examine the mechanism of signal transduction ofTNF-a-regulated IP_3R1 in HMCs.RESULTS:The levels of IP_3R1 mRNA at 2 hours after TNF-a exposure were significantly enhanced and peaked at 8 hours in HMCs(P<0.01),then descended at 24 hours(P<0.01).The levels of IP_3R1 protein at 4 hours after TNF-a exposure were obviously increased and peaked at24 hours after TNF-a exposure(P<0.01).Compared to the control group,safingol(PKCa inhibitor)and D609(phosphatidylcholine-specific phospholipase C inhibitor) significantly blocked the TNF-ainduced expression of IP_3R1 mRNA(3.30±0.81 vs.1.95±0.13,P<0.05;2.10±0.49,P<0.01) and IP_3R1protein(3.09±0.13 vs.1.86+0.39,P<0.01;1.98±0.02,P<0.01).TNF-a promoted PKCa activation with maximal PKCa phosphorylation that occurred 8 hours after stimulation measured by non-radioactive PKC assay,and the effect was markedly attenuated by pretreatment with D609 or safingol.CONCLUSION:TNF-a increased the expression of IP_3R1 and this was mediated,at least in part,through the PC-PLC/PKCa signaling pathways in HMCs.
基金J.A.R.received a Doctoral Fellowship from Coordenacao de Apoio de Pessoal de Nível Superior(CAPES).
文摘Understanding the growth regulatory mechanisms in filamentous fungi is very important for the production of medicines for antifungal therapies. It is well established that Ca2+ gradient is essential for hyphal growth and that one mechanism responsible for the Ca2+ cellular concentration starts with the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by receptor-regulated forms of phosphoinositide-specific phospholipase C (PI-PLC). In the present study the levels of calcium in Aspergillus nidulans wild type (A26) and plcA-deficient mutant (AP27) growing in a carbon source readily assimilated, as glucose or pectin a non-readily assimilated carbon source was investigated. Intracellular calcium levels in A26 were higher in the presence of glucose than in pectin, but lower in AP27 independently of the carbon source and in AP27 the vesicular calcium distribution occurred mainly at the apex of the hyphae. Delay in nuclear division was also observed if A26 and AP27 were grown in pectin presence when compared with growth in glucose. For the first time, it is demonstrated that the levels of intracellular Ca2+ were higher when A. nidulans was growing in glucose than in a non readily assimilated carbon source as pectin. Further, it also showed that the plcA gene, although not essential, may be responsible for high-molecular weight carbon source recongnation, for the intracellular Ca2+ levels maintenance and consequently by the nuclear division in A. nidulans.
基金supported by grants of the National High-Tech R and D Program (2007AA09Z416)the Natural Science Foundation of China (30972275)
文摘An oligopeptide permease A(OppA)was purified from the extracellular product of Vibrio harveyi SF-1.The molecular weight of the purified protein was estimated to be 58 kDa on SDS-PAGE.The purified protein showed phospholipase C activity at the optimal values of temperature 50℃ and pH 8.0.The enzymatic activity decreased when the temperature increased to 40℃.The N-terminal sequence of the purified protein was determined as ADVPAGTKLA,which is similar to that of OppA.The OppA pre-cursor gene was cloned from the genome of V.harveyi SF-1.The gene consisted of 1665 base pairs and encoded a 554 amino acid polypeptide,which showed a high similarity to those of the OppAs of V.harveyi and other Vibrio species.The gene was subcloned into pET-28a(+)and expressed in Escherichia coli.The expressed recombinant protein was purified by Ni-NTA metal affinity chro-matography.The expressed recombinant protein showed a 58 kDa band on SDS-PAGE and exhibited phospholipase C activity with the optima of temperature 50℃ and pH 8.0.The purified protein was toxic to the flounder gill cells.An OppA mutant of V.harveyi SF-1 was constructed by homologous recombination.The mutant strain was less virulent when it was intraperitoneally inoculated to zebra fish,with the LD50 of 5.46×105 CFU fish-1,compared to 3.11×104 CFU fish-1 of the wild-type strain,which indicated that the OppA might play an important role in the pathogenicity of V.harveyi.
基金Supported by the National Natural Science Foundation of China(31370140,31372438)。
文摘Clostridium perfringens phospholipase C(plc),also calledα-toxin,is encoded by the plc gene Clostridium perfringers.The production ofα-toxin can lead to the occurrence of gas gangrene.Vaccination is considered as one of the best solutions against Clostridium infections.In this study,an anti-Cpα-toxin monoclonal antibody(mAbs)A10E5 was successfully prepared,which had better biological reactivity.Then,the phage random 12-peptide library was used to screen mAb A10E5 protein.After four rounds of screening,three peptides with high affinity to the anti-α-toxin mAbs were screened.Two 12-peptide peptide Q and peptide E with higher inhibition rate were obtained by indirect ELISA.Two polypeptides of 500μg·mL^(-1)synthesized in vitro were mixed with30μg·mL^(-1)α-toxin at a concentration to treat Hela cells.Cell viability was determined by MTT assay.The results showed that both of the peptides significantly increased the survival rate of Hela cells compared with theα-toxin group,and the effect of peptide Q was more obvious.The chickens were immunized with phages expressing two different affinity polypeptides and then challenged.The results of chicken weight change,intestinal lesion score,bacterial count,and antibody titer in peripheral blood showed that the two phages expressing the polypeptides had a certain protective effect on the chickens compared with the PBS group,and peptide Q had better protection effect.In conclusion,the high affinity peptide with mAb A10E5 was screened in this study,and the protective effect of the plc polypeptide vaccine was verified by in vivo and in vitro experiments,which was of great significance for the comprehensive prevention and treatment of the disease.
文摘The effect of U50488, a selective K-opioid receptor agonist, on cardiac rhythm in the isolated perfused rat heart and intracellular calcium ([Ca2+] i) in the single ventricular myocyte were studied. The results showed that U50488 can induce arrhythmias dose-dependently in the isolated perfused rat heart and increase [ Ca2 + ] i in the single ventricular myocyte. The effect of U50488 can be blocked by a selectivek-receptor antagonist, nor-binaltorphimine. The arrhythmogenic effects and the increase of [ Ca2+]i induced by U50488 were blocked by U73122, neomycin and streptomycin, which are selective phospolipase C inhibitors, but not by U73433, the inactive structural analog of U73122. These results demonstrated that the arrhythmogenic effect of cardiac K-receptor is due to activation of phosphoinositol/Ca2+ pathway.
文摘Phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphocholine and diacylglycerol (DAG). PC-PLC has a long tradition in animal signal transduction to generate DAG as a second messenger besides the classical phosphatidylinositol splitting phospholipase C (PI-PLC). Based on amino acid sequence similarity to bacterial PC-PLC, six putative PC-PLC genes (NPC1 to NPC6) were identified in the Arabidopsis genome. RT-PCR analysis revealed overlapping expression pattern of NPC genes in root, stem, leaf, flower, and silique. In auxin-treated PNPc3:GUS and PNPc4:GUS seedlings, strong increase of GUS activity was visible in roots, leaves, and shoots and, to a weaker extent, in brassinolide-treated (BL) seedlings. PNPc4:GUS seedlings also responded to cytokinin with increased GUS activity in young leaves. Compared to wild-type, T-DNA insertional knockouts npc3 and npc4 showed shorter primary roots and lower lateral root density at low BL concentrations but increased lateral root densities in response to exogenous 0.05-1.0 I^M BL BL-induced expression of TCH4 and LRX2, which are involved in cell expansion, was impaired but not impaired in repression of CPD, a BL biosynthesis gene, in BL-treated npc3 and npc4. These observations suggest NPC3 and NPC4 are important in BL-mediated signaling in root growth. When treated with 0.1 I^M BL, DAG accumulation was observed in tobacco BY-2 cell cultures labeled with fluorescent PC as early as 15 min after application. We hypothesize that at least one PC-PLC is a plant signaling enzyme in BL signal transduction and, as shown earlier, in elicitor signal transduction.
文摘The development of the nervous system is under the strict control of a number of signal transduction pathways, often interconnected. Among them, the phosphoinositide (PI) pathway and the related phospholipase C (PI-PLC) family of enzymes have been attracting much attention. Besides their well-known role in the regulation of intracellular calcium levels, PI-PLC enzymes interact with a number of molecules belonging to further signal transduction pathways, contributing to a specific and complex network in the developing nervous system. In this review, the connections of PI signalling with further transduction pathways acting during neural development are discussed, with special regard to the role of the PI-PLC family of enzymes.
基金This study was supported by a National Science,Technology,and Innovation plan(NSTIP)project grant(15-MED4186-20)awarded by the King Abdulaziz City for Science and Technology(KACST)to JK,AMA,and FAL.
文摘Sperm-specific phospholipase C zeta(PLCζ)initiates intracellular calcium(Ca2+)transients which drive a series of concurrent events collectively termed oocyte activation.Numerous investigations have linked abrogation and absence/reduction of PLCζwith forms of male infertility in humans where oocyte activation fails.However,very few studies have examined potential relationships between PLCζand advancing male age,both of which are increasingly considered to be major effectors of male fertility.Initial efforts in humans may be hindered by inherent PLCζvariability within the human population,alongside a lack of sufficient controllable repeats.Herein,utilizing immunoblotting,immunofluorescence,and quantitative reverse transcription PCR(qRT-PCR)we examined for the first time PLCζprotein levels and localization patterns in sperm,and PLCζmRNA levels within testes,from mice at 8 weeks,12 weeks,24 weeks,and 36 weeks of age,from two separate strains of mice,C57BL/6(B6;inbred)and CD1(outbred).Collectively,advancing male age generally diminished levels and variability of PLCζprotein and mRNA in sperm and testes,respectively,when both strains were examined.Furthermore,advancing male age altered the predominant pattern of PLCζlocalization in mouse sperm,with younger mice exhibiting predominantly post-acrosomal,and older mice exhibiting both post-acrosomal and acrosomal populations of PLCζ.However,the specific pattern of such decline in levels of protein and mRNA was strain-specific.Collectively,our results demonstrate a negative relationship between advancing male age and PLCζlevels and localization patterns,indicating that aging male mice from different strains may serve as useful models to investigate PLCζin cases of male infertility and subfertility in humans.