Advancing male age differentially alters levels and localization patterns of PLCzeta in sperm and testes from different mouse strains / 亚洲男科学杂志(英文版)

Sperm-specific phospholipase C zeta (PLCζ) initiates intracellular calcium (Ca

Significance of phosphoinositol metabolism by DNA methylation may contribute in oral leukoplakia carcinogenesis / 口腔疾病防治

Objective @# To study the role of DNA methylation in oral leukoplakia carcinogenesis.@*Methods@# DNA methylation was detected in forty cases of oral squamous cell carcinoma (OSCC), twenty-eight cases of oral leukoplakia (OLK) and forty cases of healthy oral mucosa. Download the expression profile data of OSCC, OLK and healthy oral mucosa from Gene Expression Omnibus (GEO) database. DNA methylation data and expression profile data were compared for repeatability, DNA methylation data for difference analysis and corresponding expression profile data for IPA pathway analysis.@*Results @#The data analysis showed that DNA methylation had greater flexibility and instability. Ingenuity Pathway Analysis (IPA) analysis showed that genes related to OLK differential methylation sites were mainly concentrated in the process of cell movement and differentiation. Genes related to differential methylation sites of OSCC are mainly enriched in cell proliferation, migration, oxidation regulation, and anti-apoptosis processes. The genes associated with OLK and OSCC differential methylation sites are co-enriched in phosphoinositol metabolism and phospholipase C signaling pathway.@* Conclusion@#DNA methylation is involved in the formation of oral squamous cell carcinoma, and the activation of phosphoinositol metabolism may promote oral leukinoma.

Banxia Xiexin Decoction () Treats Diabetic Gastroparesis through PLC-IP / 中国结合医学杂志

OBJECTIVE@#To test the effect of Banxia Xiexin Decoction (, BXD) on the contraction and relaxation of gastric smooth muscle (SM) in diabetic gastroparesis (DGP) model rats, and to explore the mechanism of BXD in the prevention and treatment of DGP through experiments of signal pathway both in vivo and in vitro.@*METHODS@#Sixty Sprague-Dawley rats were divided into 6 groups according to a random number table: control group, model group, high-, medium- and low-dose BXD groups (9.2, 4.6 and 1.8 g/(kg·d), respectively), and domperidone group (10 mg/(kg·d)), 10 rats per group. DGP model was established initially by a single intraperitoneal injection of streptozotocin (STZ), and was confirmed by recording gastric emptying, intestinal transport velocity and gastric myoelectric activity of rats after 2 months. Each group was treated with a corresponding drug for 4 weeks. The mRNA and protein expressions of phospholipase C (PLC), inositol triphosphate (IP@*RESULTS@#Compared with the model group, high- and medium-dose BXD and domperidone significantly increased the expressions of PLC, IP@*CONCLUSIONS@#Treatment with high- and medium-dose BXD significantly attenuated STZ-induced experimental DGP in rats. The therapeutic effect of BXD on DGP rats might be associated with the PLC-IP

Effect of Zhizhuwan on Intestinal Conduction and PLC-γ1/PLC-γ2 Signaling Pathway of Slow Transit Constipation Model Mouse with Spleen Deficiency Syndrome / 中国实验方剂学杂志

Objective:To explore the effect of Zhizhuwan on intestinal conduction and expressions of Phospholipase C-γ1 (PLC-γ1)/Phospholipase C-γ2 (PLC-γ2) signaling pathway of slow transit constipation (STC) with spleen deficiency syndrome. Method:Special pathogen free (SPF) healthy mice were randomly divided into normal group and model making group. Folium Sennae gavage was used to induce the spleen deficiency status, and then diet and drinking water were controlled to establish the mice model of spleen deficiency constipation. After the modeling, the mice in modelling group were randomly divided into model group, Zhizhuwan group and mosapride group. Zhizhuwan group was given drug at the dose of 9.0 g·kg-1·d-1, mosapride group was given 2.5 mg·kg-1·d-1, model group and normal group were given the equal dose of distilled water for 7 consecutive days. At the end of the treatment, the length of Indian ink in the colon was used to calculate the intestinal propulsion rate of the mice. The D-xylose kit was used to determine the content of D-xylose in serum of mice. The hematoxylin eosin (HE) staining was used to observe the pathological changes of colon tissues in mice. The immunohistochemistry and Western blot were used to detect the expression levels of PLC-γ1 and PLC-γ2 proteins in colon tissues of mice. Real-time PCR was used to detect the mRNA expression levels of PLC-γ1 and PLC-γ2. Result:Compared with normal group, the intestinal propulsive rate and the serum D-xylose value in model group were significantly decreased (Pγ1 and PLC-γ2 in colon tissues were significantly decreased (PD-xylose in Zhizhuwan group and mosapride group were significantly increased (Pγ1 and PLC-γ2 were significantly increased (PConclusion:Zhizhuwan can promote the intestinal movement in slow transit constipation model mouse with spleen deficiency syndrome, and alleviate the symptoms of constipation in mice. The related mechanism may be related to the increase of the expressions of PLC-γ1 and PLC-γ2 in colon tissues of mice with spleen deficiency and constipation.

The change of signaling pathway on the electrical stimulated contraction in streptozotocin-induced bladder dysfunction of rats

Bladder dysfunction is a common complication of diabetes mellitus (DM). However, there have been a few studies evaluating bladder smooth muscle contraction in DM in the presence of pharmacological inhibitors. In the present study, we compared the contractility of bladder smooth muscle from normal rats and DM rats. Furthermore, we utilized pharmacological inhibitors to delineate the mechanisms underlying bladder muscle differences between normal and DM rats. DM was established in 14 days after using a single injection of streptozotocin (65 mg/kg, intraperitoneal) in Sprague-Dawley rats. Bladder smooth muscle contraction was induced electrically using electrical field stimulation consisting of pulse trains at an amplitude of 40 V and pulse duration of 1 ms at frequencies of 2–10 Hz. In this study, the pharmacological inhibitors atropine (muscarinic receptor antagonist), U73122 (phospholipase C inhibitor), DPCPX (adenosine A₁ receptor antagonist), udenafil (PDE5 inhibitor), prazosin (α₁-receptor antagonist), verapamil (calcium channel blocker), and chelerythrine (protein kinase C inhibitor) were used to pretreat bladder smooth muscles. It was found that the contractility of bladder smooth muscles from DM rats was lower than that of normal rats. In addition, there were significant differences in percent change of contractility between normal and DM rats following pretreatment with prazosin, udenafil, verapamil, and U73122. In conclusion, we suggest that the decreased bladder muscle contractility in DM rats was a result of perturbations in PLC/IP₃-mediated intracellular Ca²⁺ release and PDE5 activity.

Fluorescence Assay for Phospholipase C Activity Using Liposome Probes / 分析化学

A simple assay for detection of phospholipase C (PLC) activity was developed based on a fluorescence liposome probe using the Liss Rhod PE-loaded phospholipid liposomes.The liposome probe was prepared by the coassembly of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and fluorescent lipid (Liss Rhod PE).The probe showed very low background fluorescence due to fluorescence self-quenching effect of Liss Rhod PE.As the PLC enzyme selectively digested lipid, the Rhod fluorescence was recovered from its quenched state, leading to the sensitive detection of PLC.This assay provided a limit of detection (at a signal-to-noise ratio of 3) of 2 U/L for PLC.In the presence of PLC inhibitor, the fluorescent response of the sensor for PLC decreased, indicating that the assay could also be used for screening PLC inhibitors.

Glucosylsphingosine Activates Serotonin Receptor 2a and 2b: Implication of a Novel Itch Signaling Pathway

Recent reports claimed that glucosylsphingosine (GS) is highly accumulated and specifically evoking itch-scratch responses in the skins of atopic dermatitis (AD) patients. However, it was unclear how GS can trigger itch-scratch responses, since there were no known molecular singling pathways revealed yet. In the present study, it was verified for the first time that GS can activate mouse serotonin receptor 2a (mHtr2a) and 2b (mHtr2b), but not 2c (mHtr2c) that are expressed in HEK293T cells. Specifically, effects of GS on all mouse serotonin receptor 2 subfamily were evaluated by calcium imaging techniques. The GS-induced intracellular calcium increase was dose-dependent, and antagonists such as ketanserin (Htr2a antagonist) and RS-127445 (Htr2b antagonist) significantly blocked the GS-induced responses. Moreover, the proposed GS-induced responses appear to be mediated by phospholipase C (PLC), since pretreatment of a PLC inhibitor U-73122 abolished the GS-induced responses. Additionally, the GS-induced calcium influx is probably mediated by endogenous TRPC ion channels in HEK293T cells, since pretreatment of SKF-96365, an inhibitor for TRPC, significantly suppressed GS-induced response. In conclusion, the present study revealed for the first time that GS can stimulate mHtr2a and mHtr2b to induce calcium influx, by utilizing PLC-dependent pathway afterwards. Considering that GS is regarded as a pruritogen in AD, the present study implicates a novel GS-induced itch signaling pathway.

Interactions of aminoglycosides with RNAs and proteins via carbohydrate microarray / 生物工程学报

Aminoglycosides are broad-spectrum antibacterials to treat bacterial infections, especially gram-negative bacteria infections. However, aminoglycosides are losing efficacy because of the increase in antibiotic resistance and their inherent toxicity, attracting more interests in developing new aminoglycosides. Several clinically used aminoglycosides are mainly exerted by inhibition of protein synthesis through binding to bacterial rRNA. The bacterial ribosome RNA is the most currently exploited RNA drug target. Identification of new compounds that target RNAs is indispensable to fight with the growing threat that bacteria pose to human safety. In this work, we used carbohydrate microarrays to probe interactions of low molecular weight ligands with RNAs and proteins. Carbohydrate microarrays, comprising hundreds to thousands of different glycan structures on surfaces in a spatially discrete pattern, are sensitive and versatile tools to study the interactions between biological macromolecules. Herein, aminoglycosides have been immobilized onto the modified glass microscope slides and their interactions with RNAs and proteins are then measured through the labeled fluorescence. The results displayed that microarray can be used to detect the binding of aminoglycosides with three types of target molecules, including the small RNA oligonucleotide mimics of aminoglycoside binding sites in the ribosome (rRNA A-site mimics), the large group I ribozyme RNA (approximately 400 nucleotide) and certain proteins (toxicity-causing enzymes, such as DNA polymerase and phospholipase C). For rRNA A-site mimics, the fluorescence intensities of 16S rRNA is stronger than that of 18S rRNA, illustrating that as a screen technique, the microarray method can not only determine the binding affinity to RNA but also detect the specific binding to bacterial rRNA mimic. The ability to screen group I ribozyme RNA can be helpful to the discovery of new RNA therapeutic targets. Binding of immobilized aminoglycosides to toxicity-causing proteins (DNA polymerase and phospholipase C) is a new method to study of aminoglycoside toxicity. These studies lay the foundation for rapid identification of new RNA-binding ligands with strong and specific binding affinity for their desired targets.

The Inhibitory Mechanism of Gentamicin on Electrical Field Stimulation Response in Rat Bladder Smooth Muscle

To see the inhibitory mechanism of gentamicin in response to electrical field stimulation (EFS) using the rat bladder smooth muscle, atropine or guanethidine was treated but had no effect. Methylsergide, a non-selective 5-HT1, 5-HT2 receptor antagonist was also treated but had on effect. Kinase inhibitors, such as chelerythrine (PKC inhibitor), ML-9 (MLCK inhibitor), or Y27632 (rho kinase inhibitor) were pretreated before gentamicin treatment, but did not have effect. For U73122, a phospholipase C (PLC) inhibitor however, the inhibitory effect to gentamicin was significantly attenuated in all frequencies given by the EFS. Therefore gentamicin induced inhibitory effect on EFS response in rat bladder smooth muscle was not mediated by the activation of adrenergic, cholinergic, or serotonergic receptor. The inhibition of gentamicin might be mediated through the PLC dependent pathway, but not through the PKC, MLCK or rho kinase dependent pathway.

PLCE1 suppresses apoptosis of lung adenocarcinoma cells via modulating p53 expression / 中国病理生理杂志

AIM:To investigate the role of phospholipase C epsilon 1 ( PLCE1 ) in modulating the apoptotic mechanism in lung adenocarcinoma A 549 cells.METHODS:PLCE1 inhibitor U-73122 was used to suppress the expres-sion of PLCE1.The expression of PLCE1 and p53 in A549 cells was evaluated by quantitative real-time PCR and Western blotting.Apoptosis was assessed by flow cytometry .RESULTS:A549 cells expressed high level of PLCE1 and low level of p53.Inhibition of PLCE1 markedly increased the expression of p 53, and increased the apoptosis of A 549 cells.CON-CLUSION:PLCE1 suppresses apoptosis of A549 cells via inhibiting the expression of p53.

Relationship between phospholipase C zeta immunoreactivity and DNA fragmentation and oxidation in human sperm

OBJECTIVE: The study aimed to evaluate the feasibility and reproducibility of measuring phospholipase C zeta (PLCzeta) using immunostaining in human sperm and to investigate the relationship between PLCzeta immunoreactivity and DNA fragmentation and oxidation in human sperm. METHODS: Semen samples were obtained from participants (n=44) and processed by the conventional swim-up method. Sperm concentration, motility, normal form by strict morphology, DNA fragmentation index assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling method and immunofluorescent expression for 8-hydroxy-2'-deoxyguanosine (8-OHdG) and PLCzeta were assessed. RESULTS: When duplicate PLCzeta tests were performed on two sperm samples from each of the 44 participants, similar results were obtained (74.1+/-9.4% vs. 75.4+/-9.7%). Two measurements of PLCzeta were found to be highly correlated with each other (r=0.759, P<0.001). Immunoreactivity of PLCzeta was not associated with donor's age, sperm concentration, motility, and the percentage of normal form as well as DNA fragmentation index. However, immunoreactivity of PLCzeta showed a significant negative relationship with 8-OHdG immunoreactivity (r=-0.404, P=0.009). CONCLUSION: Measurement of PLCzeta by immunostaining is feasible and reproducible. Lower expression of PLCzeta in human sperm may be associated with higher sperm DNA oxidation status.

Cardiovascular and biochemical studies on the effects of thrombin and dabigatran and the interaction with vasopressor molecules.

Background: The effect of serine protease thrombin and its directly acting inhibitor dabigatran were evaluated on the heart rate, blood pressure, and phospholipase C (PLC) enzyme activity and the intracellular calcium levels in the platelets. Methods: Heart rate and blood pressure were estimated using electrophysiology equipment. Results: While thrombin was unable to significantly affect the heart rate and blood pressure, the inhibitor dabigatran was able to reduce the heart rate appreciably but its effects on the blood pressure were minimal. The thrombin induced increase in PLC enzyme activity, and intracellular calcium levels were attenuated by dabigatran in the platelets. The posterior pituitary hormone, vasopressin, and the adrenergic agonist noradrenaline were used to stimulate the PLC and calcium levels in platelets. Conclusion: The thrombin inhibitor, dabigatran reduces vascular oxidative stress and inflammation, improves endothelial function, and decreases atherosclerosis in rodents.

Increased activity of goat liver plasma membrane alkaline phosphatase upon release by phosphatidylinositol-specific phospholipase C.

Mammalian alkaline phosphatase (ALP) is attached to the plasma membrane by a unique glycosylphosphatidylinositol (GPI) anchor. The influence of such a complex anchoring device on the enzyme function is not fully understood. Here, we report the effect of cleavage of the GPI anchor on the activity of goat liver plasma membrane ALP (GLPM-ALP). Phosphatidylinositol-specific phospholipase C (PI-PLC) purified from Bacillus cereus was used for the cleavage of the GPI anchor (delipidation) and hence for release of ALP from the membrane. Detergents — octyl-β-D-glucopyranoside (OG) and triton X100 (TX100) were also used for solubilization of ALP from the membrane. Resistance to solubilization by TX100 suggested the association of GPI-ALP with lipid rafts. Solubilization of GLPM-ALP with OG had no effect on the enzyme activity; however, delipidation with PI-PLC resulted in enhanced ALP activity. Kinetic analysis showed catalytic activation of PI-PLC-treated GLPM-ALP with an increase in Vmax (35%) without a significant change in Km. Moreover, this change in Vmax was observed to be independent of pH and buffer. The results suggested the implication of GPI anchor in modulating the catalytic property of GLPM-ALP, thus indicating the role of this special anchoring structure in the enzyme regulation.

Phospholipase C from Pseudomonas aeruginosa and Bacillus cereus; characterization of catalytic activity

Objective: To study characteristics of phospholipases C (PLCs), their importance for producing microorganisms as well as the potential of their use for industrial purposes. Method: PLC from Bacillus cereus (B. cereus) D101 was selected as an example of Gram-positive PLCs and PLC from Pseudomonas aeruginosa (P. aeruginosa) D183 of Gram-negative ones. Enzymes were partially purified by ammonium sulfate precipitation followed by membrane dialysis. Partially purified preparations were used to study effect of different factors on activities as well as in substrate specificity tests which were conducted using a turbidimetric assay method. Results: Maximum activity was at pH 7 and 8 and 40 °C for P. aeruginosa PLC, and pH 8-10 and 37 °C for B. cereus PLC. Both PLCs were inhibited by Pi at 5 mM or higher, whereas, PLC from B. cereus only was inhibited by EDTA. Activity of P. aeruginosa PLC was not affected by removing Zn

Pirarubicin inhibits proliferation of bladder cancer cells through PLCε-Bcl-2 pathway / 第二军医大学学报

Objective: To investigate the effects of pirarubicin on proliferation of bladder cancer cells and the related mechanism. Methods: After bladder cancer cell lines T24 and BIU-87 were treated with 0.4, 0.8, 1.6, and 3.2 mg/L pirarubicin for 24, 48, and 72 h, the cell proliferation was detected by MTT. Flow cytometry was used to examine the apoptosis of T24 and BIU-87 cells. qRT-PCR and RT-PCR were used to examine the mRNA expression of phospholipase C ε (PLCε),Bcl-2 in T24 and BIU-87 cell lines; the protein expression of PLCε in pirarubicin-treated cells was determined by Western blotting analysis. Bladder cancer cells were designed as blank group, pirarubicin treatment group, Ad-shPLCε treatment group, and Ad-shPLCε plus pirarubicin treatment group; cell proliferation was observed and protein expression of Bcl-2 was examined and compared between different groups. Results: Pirarubicin showed a dose-and time-dependent inhibitory effect against proliferation of T24 and BIU-87 cell lines. Moreover, pirarubicin promoted cell apoptosis in T24 and BIU-87 cells and suppressed the expression of PLCε and Bcl-2. Pirarubicin treatment group, Ad-shPLCε treatment group, and Ad-shPLCε plus pirarubicin treatment group all had suppressed cell proliferation and Bcl-2 expression, and pirarubicin plus Ad-shPLCε group exhibited significantly stronger inhibitory effects compared with pirarubicin treatment group (P<0.05). Conclusion: Pirarubicin can effectively inhibit cell proliferation of bladder cancer cells, which may be through suppressing the expression of PLCε and Bcl-2.

Phospholipase C from Pseudomonas aeruginosa and Bacillus cereus; characterization of catalytic activity

OBJECTIVE@#To study characteristics of phospholipases C (PLCs), their importance for producing microorganisms as well as the potential of their use for industrial purposes.@*METHOD@#PLC from Bacillus cereus (B. cereus) D101 was selected as an example of Gram-positive PLCs and PLC from Pseudomonas aeruginosa (P. aeruginosa) D183 of Gram-negative ones. Enzymes were partially purified by ammonium sulfate precipitation followed by membrane dialysis. Partially purified preparations were used to study effect of different factors on activities as well as in substrate specificity tests which were conducted using a turbidimetric assay method.@*RESULTS@#Maximum activity was at pH 7 and 8 and 40 °C for P. aeruginosa PLC, and pH 8-10 and 37 °C for B. cereus PLC. Both PLCs were inhibited by Pi at 5 mM or higher, whereas, PLC from B. cereus only was inhibited by EDTA. Activity of P. aeruginosa PLC was not affected by removing Zn(2+) ions from reaction mixture or their replacement with Ca(2+), Ba(2+), Mg(2+) or Mn(2+) ions. Vis-à-vis, activity of B. cereus PLC was found to be metal ion dependent. PLCs from both isolates were relatively thermostable and showed maximum affinity toward phosphatidylcholine. Sphingomyelin and phosphatidylethanolamine were not good substrates and phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and cardiolipin could be considered non-substrates.@*CONCLUSION@#Human body physiological conditions could favor activity of P. aeruginosa and B. cereus PLCs. These enzymes may participate in phosphate scavenging and virulence of producing isolates but not in autolysis. PLCs from both isolates are potential candidates for industrial use.

Identification of phospholipase C in Leptospira interrogans and the mechanism of macrophage apopto-sis induced by phospholipase C / 中华微生物学和免疫学杂志

Objective To investigate the function of phosphatidylinositol phospholipase C encoded by LB361 gene of L.interrogans ( L-PI-PLC) and its mechanism in inducing macrophage apoptosis .Meth-ods The PI-PLC domains in the sequence of LB 361 gene of L.interrogans serovar Lai strain were analyzed by bioinformatics method .Prokaryotic expression system was established to express the recombinant L -PI-PLC ( rL-PI -PLC).The enzymatic activity of rL-PI-PLC in hydrolyzing phosphatidylinositol -4,5-bisphos -phate (PIP2) substrate into inositol-1,4,5-trisphosphate (IP3) was determined by IP3 fluorescence polariza-tion assay.LB361gene expressions at mRNA and protein levels as well as the secretion of LB 361gene prod -ucts were detected by real-time fluorescent quantitative RT -PCR and Western blot assay after infection of hu-man THP-1 macrophages with L.interrogans serovar Lai strain.A LB361 gene-transfected THP -1cell line was generated for evaluation of the mechanism of LB 361 gene products in elevating intracellular free Ca 2+( [Ca 2+] i) concentration and inducing the apoptosis of transfected THP -1 cells with the use of laser confocal microscopy and flow cytometry.Re sul ts The rL-PI-PLC hydrolyzed PIP2 into IP3 with a Km of 199 μmol/L and a Kcat of 8.566×10-5 S-1 .The expressions of LB361gene at mRNA and protein levels were both signifi -cantly up-regulated after infection of THP-1 cells with L.interrogans serovar Lai strain .Moreover , the exter-nal secretion of L-PI-PLC was also found during infection .The concentrations of IP 3 and [ Ca2+] i in the LB361 gene-transfected THP-1 cells were significantly increased compared to those in the non-transfected THP-1 cells, resulting in a high [Ca2+]i-dependent apoptosis of partial THP-1 cells.Conclusion PI-PLC is encoded by the LB361 gene of L.interrogans, which could induce the apoptosis of macrophages through el-evating [ Ca2+] i concentration during infection of microphages with L.interrogans.

PKCα signaling pathway involves in TNF-α-induced IP3R1 expression in human mesangial cells / 世界急诊医学杂志（英文）

BACKGROUND: This study aimed to explore the effects of TNF-α on the expression of IP3R1 mRNA and protein in human mesangial cells (HMCs), and to elucidate the mechanism of TNF-α relating to IP3R1 expression in the occurrence of hepatorenal syndrome (HRS). METHODS: HMCs were stimulated by tumor (TNF-α) with 100 ng/mL for different hours (2, 4, 8, and 24 hours). The expression changes of IP3R1 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 of TNF-α-regulated IP3R1 in HMCs. RESULTS: The levels of IP3R1 mRNA at 2 hours after TNF-α 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 IP3R1 protein at 4 hours after TNF-α exposure were obviously increased and peaked at 24 hours after TNF-α exposure (P<0.01). Compared to the control group, safingol (PKCα inhibitor) and D609 (phosphatidylcholine-specific phospholipase C inhibitor) significantly blocked the TNF-α-induced expression of IP3R1 mRNA (3.30±0.81 vs. 1.95±0.13,P<0.05; 2.10±0.49,P<0.01) and IP3R1 protein (3.09±0.13 vs. 1.86+0.39,P<0.01; 1.98±0.02,P<0.01). TNF-α promoted PKCα activation with maximal PKCα 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-α increased the expression of IP3R1 and this was mediated, at least in part, through the PC-PLC/PKCα signaling pathways in HMCs.

Expression and Purification of Phospholipase C-beta4, and Chimeric Phospholipase C and Characterization of Them

BACKGROUND: Phospholipase C-beta4 (PLC-beta4) is known to be one of the most important signal transducing molecules; however, its biophysical and chemical characteristics are not well known due to the difficulty in purifying PLC-beta4 from bovine retina. In the present study, we used the baculovirus expression system in order to express and purify large amounts of PLC-beta4. With this system, we also tried to produce chimeric PLC-beta3/beta4 and PLC-beta4/beta3 protein in order to study the structure-activity relationship between N terminal and C terminal portion of PLC-betas. METHODS: I cloned PLC-beta4 to the baculovirus expression system by the polymerase chain reaction method and infected the PLC-beta4 to Sf9 cells. I purified recombinant PLC-beta4 proteins using sequential high performnance liquid chromatography (HPLC) by using the TSK phenyl-5PW column and the TSK heparin-5PW column. With this similar method, I was able to express chimeric PLC-beta3/beta4 and PLC-beta4/beta3 proteins. RESULTS: With the two step HPLC, I was able to purify PLC-beta4 by 30-fold; this purified PLC-beta4 contained PLC activity. I also expressed chimeric PLC-beta3/beta4 and PLC-beta4/beta3 using the baculovirus system, and their expression was confirmed by the immunoblot method. However, chimeric PLC-beta4/beta3 did not show PLC activity, while chimeric PLC-beta3/beta4 retained its PLC-activity. CONCLUSION: Expression of chimeric PLC-beta4 using the baculovirus system was an efficient method to obtain a large amount of protein. Moreover, this expression and purification method would be useful in studying the physical and chemical characteristics of this protein. In my study using chimeric PLC-beta protein by swapping the N terminal and C terminal portions of PLC-beta3 and beta4, chimeric protein lost its activity completely in PLC-beta4/beta3 chimera. This result suggested a minute change in the tertiary structure of the protein, which may significantly affect its function.

A simple technique for estimation of phospholipase d and phospholipase c enzymes in smooth muscle and its cells.

This research article describes two novel and simple techniques for the estimation of phospholipase D and phospholipase C enzymes in aortic smooth muscle and cells cultured from the bovine aorta. The techniques encompass the use of ion exchange chromatography and liquid scintillation spectrophotometry.