No abstract available.
Animals ; Inositol* ; Kidney* ; Liver* ; Phospholipases* ; Rats* ; Type C Phospholipases*

No abstract available.
Phospholipases* ; Protein Kinase C* ; Protein Kinases* ; Psychotropic Drugs* ; Type C Phospholipases*

PURPOSE: The activation of phospholipase C (PLC) is one of the early events in various growth processes, including malignant transformation. Among PLC-isozymes, PLC-gamma is activated through direct interaction with growth factor receptor tyrosine kinase. In this study, we evaluated the patterns of PLC-gamma expression in benign and malignant tumors of the breast and compared the patterns with their normal counterpart. METHODS: Using immunoblot assay, we evaluated the patterns of expression in PLC-gamma in 20 breast cancer tissues, 13 fibroadenoma tissues, and normal tissues. The level of expression was analyzed by densitometry. RESULTS: All of 20 breast cancer tissues and 13 fibroadenoma tissues showed overexpression of PLC-gamma when compared with their normal counterparts. The level of the PLC-gamma expression was 3.9-fold and 17.3-fold higher in fibroadenomas and breastcancers, respectively, than in normal tissues. CONCLUSION: The result suggested that the level of PLC-gamma expression increases as the normal tissue undergoes progression to fibroadenoma, and finally to carcinoma. The pattern of expression of PLC-gamma in breast tissue implies that the PLC-gamma-mediated signal transduction may play a significant role in the progression of breast cancer from normal tissue.
Breast Neoplasms ; Breast* ; Densitometry ; Fibroadenoma ; Phospholipase C gamma ; Phospholipases* ; Protein-Tyrosine Kinases ; Signal Transduction ; Type C Phospholipases

This study was conducted to investigate the effects of extremely low frequency electromagnetic fields (EMF) on signal pathway in plasma membrane of cultured cells (RAW 264.7 cells and RBL 2H3 cells), by measuring the activity of phospholipase A2 (PLA2), phospholipase C (PLC) and phospholipase D (PLD). The cells were exposed to the EMF (60 Hz, 0.1 or 1 mT) for 4 or 16 h. The basal and 0.5 microM melittin-induced arachidonic acid release was not affected by EMF in both cells. In cell-free PLA2 assay, we failed to observe the change of cPLA2 and sPLA2 activity. Also both PLC and PLD activities did not show any change in the two cell lines exposed to EMF. This study suggests that the exposure condition of EMF (60 Hz, 0.1 or 1 mT) which is 2.4 fold higher than the limit of occupational exposure does not induce phospholipases-associated signal pathway in RAW 264.7 cells and RBL 2H3 cells.
Arachidonic Acid ; Cell Line ; Cell Membrane ; Cells, Cultured ; Electromagnetic Fields ; Magnets ; Occupational Exposure ; Phospholipase D ; Phospholipases ; Phospholipases A2 ; Pyridoxal ; Signal Transduction ; Type C Phospholipases

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.
Animals ; Calcium ; Dermatitis, Atopic ; Humans ; Ion Channels ; Ketanserin ; Mice ; Serotonin* ; Skin ; Type C Phospholipases

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.
DNA ; DNA Fragmentation* ; DNA Nucleotidylexotransferase ; Humans ; Semen ; Spermatozoa* ; Type C Phospholipases*

Phospholipase C (PLC) is a key enzyme in phosphatidyl inositol turnover during signal transduction. The 12 mammalian PLC isozymes identified to date can be divided into five subtypes, beta-type, gamma-type, delta-type, epsilon-type and zeta-type, with extensive difference in structure, regulatory mechanism and tissue distribution. PLC plays important roles not only in sperm acrosome reaction but also in egg activation. The present studies are reviewed on the structure, regulation and function of PLC, especially its function in male reproduction, including triggering Ca2+ oscillations in eggs to activate the eggs and helping embryo development. And the prospect of the clinical application of PLC is discussed.
Acrosome Reaction ; Animals ; Calcium Signaling ; Humans ; Reproduction ; physiology ; Signal Transduction ; Type C Phospholipases ; chemistry ; physiology

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.
Animals ; Atropine ; Gentamicins* ; Guanethidine ; Muscle, Smooth* ; Phosphotransferases ; Rats* ; rho-Associated Kinases ; Type C Phospholipases ; Urinary Bladder*

To clarify the effect of extracellular ATP in cultured C6 glioma cells, ATP-induced cytosolic free calcium ((Ca2+)i) mobilization and cell proliferation were investigated. ATP-induced (Ca2+)i increased in a dose-dependent manner (10-7 M apprx 10-3 M). ATP-induced (Ca2+)i increases were slightly slowed in extracellular calcium-free conditions especially in sustained phase. ATP-induced (Ca2+)i increment was also inhibited by the pretreatment of U73122, a phospholipase C (PLC) inhibitor, in a time-dependent manner. Suramin, a putative P2Y receptor antagonist, dose-dependently weakened ATP-induced (Ca2+)i mobilization. Significant increases in cell proliferation were observed at 2, 3, and 4 days after ATP was added. Stimulated cell proliferation was also observed with adenosine at days 2 and 3. This cell proliferation was significantly inhibited by the treatment with suramin. Ionomycin also stimulated cell proliferation in a concentration-dependent manner. In conclusion, we suggest that extracellular ATP stimulates C6 glioma cell proliferation via intracellular free calcium mobilization mediated by purinoceptor.
Adenosine ; Adenosine Triphosphate* ; Calcium* ; Cell Proliferation* ; Cytosol ; Glioma* ; Ionomycin ; Receptors, Purinergic ; Suramin ; Type C Phospholipases

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.
Baculoviridae ; Chimera ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Clone Cells ; Phospholipase C beta ; Phospholipases ; Polymerase Chain Reaction ; Proteins ; Retina ; Sf9 Cells ; Structure-Activity Relationship ; Type C Phospholipases