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.

Rapid inhibitory effect of glucocorticoids on peak of Ca2+i and PLC in airway smooth muscle / 中国应用生理学杂志

<p><b>OBJECTIVE</b>In this study, we pretreated the mice ASMCs by dexamethasone (Dex) within 10 min, to test the peak of [Ca2+]i and phospho-PLCbeta (ser1105) in the cells by treated with Ach.</p><p><b>METHODS</b>The peak of [Ca2+]i was measured by Fura-2/AM methods and the phospho-PLCbeta-ser1105 was by Western blot, and compared with dexamethasone pretreated groups. Glucocorticoid receptor antagonist RU486 and the protein synthesis inhibitor cycloheximide groups were settled in our study.</p><p><b>RESULTS</b>Glucocorticoids (GCs) significantly decreased the resting values and peak of [Ca2+]i elevation and elevated the intracellular levels of phospho-PLCbeta (ser1105) in 10 min. Neither the RU486 nor cycloheximide could alter the inhibitory effects of glucocorticoids stated above.</p><p><b>CONCLUSION</b>Our results demonstrate that glucocorticoids exert rapid inhibitory effects. The series of signal changes in this process that restrain the peak of [Ca2+]i may be responsible for the rapid nongenomic inhibitory effects of GCs by reducing the activity of PLC.</p>

Suppressive effect in vitro of resveratrol on ADP induced human platelet aggregation and its active mechanism / 药学学报

Resveratrol (RESV) is a polyphenolic compound existed in native plants such as grape, fleeceflower root, and peanut, etc. The aim of this study was to investigate the effects in vitro of RESV on adenosine diphosphate (ADP)-induced platelet aggregation, platelet membrane-bound fibrinogen (PFig) its mechanism of action. The effects of RESV and phospholipase Cbeta inhibitor (U73122) on ADP-induced healthy human volunteers platelet aggregation, PFig, and the expression of phospho-phospholipase Cbeta3 (P-PLCbeta3) and total-phospholipase Cbeta3 (T-PLCbeta3) were studied with platelet aggregometer, flow cytometry and Western blotting, respectively. Compared with control group, RESV at 25, 50 and 100 micromol x L(-1) inhibited ADP-induced platelet aggregation and PFig in a dose dependent manner, and RESV at 25 micromol x L(-1) obviously reduced expression of P-PLCbeta3 and ratio of P-PLCbeta3 to T-PLCbeta3 in platelet of healthy human volunteers. Furthermore, RESV and U73122 had additive effect in inhibiting platelet aggregation and PFig. All these suggested that RESV inhibited platelet aggregation and PFig induced by ADP partly through decreasing the activity of PLCbeta of platelets, and that RESV had definite effect of antiplatelet and might be developed as a novel antithrombotic agent.

Angiotensin II induced upregulation of G alpha q/11, phospholipase C beta 3 and extracellular signal-regulated kinase 1/2 via angiotensin II type 1 receptor / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The role of the G alpha q/11-mediated signal transduction pathway in angiotensin II (AngII) induced cardiac hypertrophy remains unclear. This study was to investigate the role of the G alpha q/11 signal transduction pathway in the development of cardiac hypertrophy in 2K1C hypertensive rats and in cultured neonatal rat ventricular myocytes (NRVMs) and to elucidate the effects of the pathway on AngII induced cardiac hypertrophy.</p><p><b>METHODS</b>Renal hypertension was induced in 2K1C hypertensive rats by placing a silver clip around the left renal artery. At 8 weeks after operation, the systolic blood pressure, the ratio of left ventricular weight to body weight (LV/BW), and the concentration of AngII in the heart were measured. The protein levels of G alpha q/11 and extracellular signal-regulated kinase 1/2 (ERK1/2) were assayed by Western blot analysis, and the activity of phospholipase C (PLC) in the myocardium was detected using [(3)H]-PIP2 as a substrate. Changes in [(3)H]-leucine incorporation and in the protein levels of the signal molecules G alpha q/11, PLC beta 3, and ERK1/2 were measured after NRVMs were stimulated with 10(-7) mol/L AngII.</p><p><b>RESULTS</b>The protein levels of G alpha q/11 and ERK1/2 in the hearts of 2K1C rats increased by 35.8% and 31.9%, respectively, compared with the sham group. The PLC activity in the 2K1C group was also significantly increased (P < 0.05). The levels of G alpha q/11, PLC beta 3, and ERK1/2 increased significantly after NRVMs were stimulated by AngII. The upregulation of G alpha q/11, PLC beta 3 and ERK1/2 in NRVMs occurred prior to [(3)H]-leucine incorporation increases, and could be inhibited with losartan.</p><p><b>CONCLUSION</b>AngII can initiate cardiac hypertrophy and upregulate signal molecules in the G alpha q/11-mediated signal transduction pathway, such as G alpha q/11, PLC beta 3 and ERK1/2, at both tissue and cellular levels.</p>

Cooperation of Gbeta and Galphaq Protein in Contractile Response of Cat Lower Esophageal Sphincter (LES)

We previously shown that LES contraction depends on M3 receptors linked to PTX insensitive Gq protein and activation of PLC. This results in production of IP3, which mediates calcium release, and contraction through a CaM dependent pathway. In the esophagus ACh activates M2 receptors linked to PTX sensitive Gi3 protein, resulting in activation of PLD, presumably, production of DAG. We investigated the role of PLC isozymes which can be activated by Gq or Gbeta protein on ACh-induced contraction in LES and esophagus. Immunoblot analysis showed the presence of 3 types of PLC isozymes, PLC-beta1, PLC-beta3, and PLC-gamma1, but not PLC-beta2, PLC-beta4, PLC-gamma2, PLC-delta1, and PLC-delta2 from both LES and esophageal muscle. ACh produced contraction in a dose dependent manner in LES and esophageal muscle cells obtained by enzymatic digestion with collagenase. PLC-beta1 or PLC-beta3 antibody incubation reduced contraction in response to ACh in LES but not in esophageal permeabilized cells, but PLC-gamma1 antibody incubation did not have an inhibitory effect. The inhibition by PLC-beta1 or PLC-beta3 antibody on Ach-induced contraction was antibody concentration dependent. The combination with PLC-beta1 and PLC-beta3 antibody completely abolished the contraction, suggesting that PLC-beta1 and PLC-beta3 have a synergism to inhibit the contraction in LES. PLC-beta1, -beta3 or -gamma1 antibody did not reduce the contraction of LES cells in response to DAG (10 (-6) M), suggesting that this isozyme of PLC may not activate PKC. When Gq/11 antibody was incubated, the inhibitory effect of the incubation of PLC beta3, but not of PLC beta1 was additive (Fig. 6). In contrast, when Gbeta antibody was incubated, the inhibitory effect of the incubation of PLC beta1, but not of PLC beta3 was additive. This data suggest that Gq/11 or Gbeta may activate cooperatively different PLC isozyme, PLCbeta1 or PLCbeta3 respectively.

Localization of Phosphoinositide Specific Phospholipase Cbeta Isozymes in Rat Cochlea / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Phosphoinositide specific phospholipase C (PLC) plays a pivotal role in the transmembrane signal transduction pathways by catalyzing the hydrolysis of phosphoinositide 4,5-bisphosphate (PIP2) to yield the intracellular second messengers, diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3), in response to the interaction of various ligands with the cell surface receptors. The question arises as to the physiological roles of the phosphoinositide second messenger system in the inner ear. The purpose of this study was to determine whether PLCbeta isozymes are present at the cochlea and what portion of cochlea each PLCbeta isozymes are distributed in. MAERIALS AND METHODS: Two methods, an immunohistochemical staining and western blot for PLCbeta isozymes were used in the rat cochlea. Frozen section and surface preparation were prepared for immunohistochemical staining. The PLCbeta isozymes or proteolytic digests were separated by SDS-polyacrylamide gels and then electrophoretically transferred to nitrocellulose membranes. Rabbit polyclonal antibodies raised against four PLCbeta isozymes were used. RESULTS: Each PLCbeta isozymes showed differential expressions in the cochlea. PLCbeta1 immunoreactivity was observed in the inner and outer hair cells and the spiral ganglion cells; PLCbeta2 in the stria vascularis and PLCbeta3 mainly in the inner hair cells. PLCbeta4 was not observed in cochlea. In western blots of rat cochlea extracts, the PLCbeta isozymes stained several bands corresponding to the known molecular weight of PLCbeta monomers, which are probably proteolytic digests. CONCLUSION: These results suggest that differentially localized each PLCbeta isozymes in the cochlea may have specific roles in signal transduction pathway of auditory system.

The mRNA Expression of Phosphoinositide-specific Phospholipase C-beta Isozymes in Postnatal Rat Brains / 대한해부학회지

Phosphoinositide-specific phospholipase C(PLC) is known as a key enzyme which produces two major second messengers: diacylglycerol and inositol 1,4,5 trisphosphate. Although it has been suggested that PLC beta isozymes have important roles in nervous system, less is known about the function of PLC beta in development of nervous system. We have localized the mRNA expressions of PLC beta isozymes in the postnatal rat brains by id firm hybridization histochemistry. In the postnatal rat brains, each isozyme of PLC beta showed differential expression pattern. The expression of PLC beta1 mRNA was found in various areas including olfactory bulb, cerebral cortex, caudate putamen, hippocampus, dentate gyrus, and cerebellum. In general, the expression in these areas was gradually increased after birth (PO) until postnatal day 21 (P2l) and slightly decreased to adult level. The expression of PLC beta2 mRNA was not found in postnatal rat brains. The expression of PLC beta3 mRNA was found from P0, peaked at Pl4, and decreased to adult level in the purkinje cells of cerebellum. PLC beta4 mRNA was strongly expressed in the thalamus, cerebellum, cerebral cortex, and olfactory bulb. In these areas, the expression was gradually increased after birth, peaked at P2l, and decreased to adult level. In whole body parasagittal sections of 18 day old rat embryo, PLC betal mRNA was exclusively expressed in nervous tissue, PLC beta3 and PLC beta4 were expressed in various tissues, and the expression of PLC beta2 was not found in any kind of rat tissues. From the different spatiotemporal mRNA expression patterns of PLC beta isozymes in the postnatal rat brains, it is suspected that each PLC beta isozyme may have specific role in signal transduction for postnatal development of rat brain.

Expression of Phospholipase C-beta1 and Phospholipase C-gamma1 on Cholesteatoma / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: A histological finding that is the most characteristic of cholesteatoma is the proliferation of the squamous cell. Signal transduction through phospholipase C(PLC) participates in the regulation of epidermal cell growth and differentiation. EGF, PDGF, and TGF-alpha bind to their receptors and thereby induce tyrosine phosphorylation of the phospholipase C-gamma1 (PLC-gamma1). PLC-gamma1 is a substrate for several receptor tyrosine kinases and its catalytic activity is increased by tyrosine phosphorylation. Tyrosine kinase phosphorylation of PLC-gamma1 stimulates PLC activation and cell proliferation. The G-protein has been shown to specifically activate PLC-beta1. However, the signal transduction pathway and the significance of PLC in cholesteatoma is unknown. This study attempted to provide some evidence that PLC plays a role in cholesteatoma by investigating the distribution and quantity of PLC-beta1 and PLC-gamma1 in the posterior auricular skin and cholestsatoma. MATERIALS AND METHODS: Western blotting and immunohistochemical study were performed for 20 cholesteatoma specimens obtained from patients who underwent operation. RESULTS: Western blot analyses revealed that PLC-beta1 protein and PLC-gamma1 protein were detectable in cholesteatoma and that these proteins were in higher levels compared with the control. In the imm-unohistochemical study, PLC-gamma1 was detected in the horny cell layer of posterior auricular skin but not in the suprabasal layer and the horny cell layer of cholesteatoma. PLC-beta1 was detected in the primary basal layer and a minor reaction was also noted in the spinous layer of posterior auricular skin. However, there were detactable reactions in both the basal and the suprabasal layers of cholesteatoma. CONCLUSION: The results of this study suggest that there are signal transduction pathways through PLC, over-expression of PLC, the different signaling mechanism by PLC in the basal and the suprabasal layer of cholesteatoma.

Distribution of Phospholipase C Isozymes in Rat Retina: Immunohistochemical Study

In order to explore the existence and distribution of phospholipase (PLC) isozymes in the rat retina, immunohistochemical staining was applied using monoclonal antibodies against PLC isozymes (PLC beta; K92, PLC gamma; D7, F7, PLC delta; R32, S11). For immunohistochemical detection, avidin-biotin peroxidase complex (ABC) method was performed on frozed tissue sections of rat retina. Our study showed that PLC isozymes have particular distributional patterns in the retina. Namely, PLC beta is broadly distributed in the outer and inner segments of photoreceptor cell layer, nuclear layer and ganglion cell layer. PLC gamma is mainly appeared in the nerve fiber layer, ganglion cell layer and inner nuclear layer. PLC delta is confined only in the ganglion cell layer. These results clearly demonstrate the PLC isozymes may have their own role in the transduction of light pathway in the retina. However, further studies will be required to verify theirs precise role in the photoreception.

Distribution of Phospholipase C Isozymes in Rat Retina: Immunohistochemical Study

In order to explore the existence and distribution of phospholipase (PLC) isozymes in the rat retina, immunohistochemical staining was applied using monoclonal antibodies against PLC isozymes (PLC beta; K92, PLC gamma; D7, F7, PLC delta; R32, S11). For immunohistochemical detection, avidin-biotin peroxidase complex (ABC) method was performed on frozed tissue sections of rat retina. Our study showed that PLC isozymes have particular distributional patterns in the retina. Namely, PLC beta is broadly distributed in the outer and inner segments of photoreceptor cell layer, nuclear layer and ganglion cell layer. PLC gamma is mainly appeared in the nerve fiber layer, ganglion cell layer and inner nuclear layer. PLC delta is confined only in the ganglion cell layer. These results clearly demonstrate the PLC isozymes may have their own role in the transduction of light pathway in the retina. However, further studies will be required to verify theirs precise role in the photoreception.