BACKGROUND: Cardiac hypertrophy is an adaptive mechanisms in response to an increased cardiac work load. Alterations in gene expression play an important role in this adaptive process. Recent investigations have indicated that the alpha-1 adrenergic stimulation in vitro induces hypertrophic change of neonatal cardiomyocytes. The signalling mechanisms of this alpha-1 agonist induced cardiomyocyte hypertrophy are largely unknown. however, recent evidence favors an effector pathway that involves phospholipase C(PLC) mediated hydrolysis of phosphatidylinositol 4,50 bisphosphate. It should be recognized that the demonstration of enhanced phosphoinositol turnover in the presence of alpha-1 adrenergic agonist in vitro does not necessarily imply that a similar response is operative in vivo. Furthermore, the role of subtypes of phospholipase C in this system should be determined. In this context, we produced in vivo cardiac hypertrophy by repeated injection of alpha-1 adrenergic agonist, phenylephrine, and tried to evaluate any change of phospholipase C subtypes by immunohistochemistry and immunoblotting technique and also measured the phosphatidylinositol hydrolyzing activity of the enzyme. METHOD: To produce cardiac hypertrophy, we injected phenylephrine 12mg/kg i.p. to the 28 female S-D rats weighing 150-250g daily for 5 days. This measures produced 22% increase of heart weight/body weight ratio. After 5 days. rats were sacrificed and hearts were rapid excised and freezed for next procedure. The immunohistochemical stainings of myocardium were carried out using monoclonal antibodies against PLC-beta1,-gamma1,-delta1 with Avidine-Biotin Complex method. Immunoblotting was done with monoclonal anti-PLC-gamma1 antibody after immnoprecipitation. The activity of PLC-gamma1 was determined in the assay mixture containing [3H] phosphatidylinositol of 20,000 cpm. The reaction was performed by incubating with resuspended immunoprecipttol of 20,000 cpm. The reaction was performed by incubating with resuspended immunoprecipitate for 10 min and supernatant was collected for -scintillation counting. RESULTS: Immunohistochemical staining demonstrated increased staining of PLC-gamma1 in the phenylephrine induced hypertrophied heart as compared with normal control heart. PLC-beta1 and-o1 did not showed any change. Elghteen out of 20 hypertrophied cardiac tissue(90%) demonstrated increased expression of the PLC-gamma1 compared with control heart tissue in immunoblotting. [3H] PI hydrolyzing activity of PLC-gamma1 in the immunoprecipitates of the hypertrophied hearts(4650+/-614 cpm) were increased consistently in 6 samples as compared with control normal hearts (2387+/-651 cpm). CONCLUSION: In the present experiments we demonstrated that Phospholipase C-gamma1 was overexpressed compared with control normal heart of rat by immunohistochemistry and immunoblotting technique and showed that the activity of this isoenzyme was elevated. Our findings of increased PLC-gamma1 expression in the alpha1-adrenergic agonist induced cardiac hypertrophy tissue suggest that the phosphatidylinositol signalling pathway is important in the genesis of cardiac hypertrophy and the isoenzyme of PLC-gamma1 may play a central role in this mechanism.
Adrenergic Agonists ; Animals ; Antibodies, Monoclonal ; Cardiomegaly* ; Female ; Gene Expression ; Heart ; Humans ; Hydrolysis ; Hypertrophy ; Immunoblotting ; Immunohistochemistry ; Myocardium ; Myocytes, Cardiac ; Phenylephrine* ; Phosphatidylinositols ; Phospholipases* ; Rats* ; Signal Transduction ; Type C Phospholipases

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.
Adult ; Animals ; Brain* ; Cerebellum ; Cerebral Cortex ; Dentate Gyrus ; Embryonic Structures ; Hippocampus ; Humans ; Inositol ; Isoenzymes* ; Nervous System ; Olfactory Bulb ; Parturition ; Phospholipase C beta* ; Phospholipases* ; Purkinje Cells ; Putamen ; Rats* ; RNA, Messenger* ; Second Messenger Systems ; Signal Transduction ; Thalamus

Phospholipase C (PLC) plays a pivotal role in transmembrane signal transduction pathway for cellular proliferation differentiation and growth. Thus far, there have been few reports in which PLC activity was investigated in human malignant neoplastic tissues. In the present study, we evaluated PLC activity in 23 human gastric cancer tissues and normal mucosal tissues to investigate whether alteration of PLC activity is associated with gastric cancer. The amount of [14C] diacylglycerol, one of the earliest products of inositol phospholipid hydrolysis by PLC, was measured by thin layer chromatography. Also, expression of PLC-gamma1, which is one of the most important PLC isozymes,was examined by immunohistochemistry using specific monoclonal antibody directed against PLC-gamma1. The results are summarized as follows. PLC activity in all 23 gastric cancer tissues (1.35+/-1.04 units/mg of protein) was significantly higher than normal mucosal tissues (0.28+/-0.21 units/mg of protein) (P<0.001). PLC activity in gastric cancer tissues was not correlated with histologic grade (P>0.05). PLC-gamma1 immunoreactivity was detected in all of 23 cases studied. The intensity and extent of PLC-gamma1 immunoreactivity was not correlated with PLC enzyme activity, although stronger intensity was demonstrated in malignant cells in comparison to normal gland epithelial cells. The present study provides the first evidence of significant elevation of PLC activity in human stomach cancer tissues. Our results strongly suggest that PLC might be involved in tumorigenesis and/or progression(uncontrolled continuous cycling of cells) of human gastric cancer. Further studies are needed to elucidate the role of elevated PLC activity in cancer tissues.
Humans ; Cell Transformation, Neoplastic ; Stomach Neoplasms

Phospholipase C (PLC) plays a role in ligand-mediated signal transduction for cellular activity such as proliferation and differentiation. A recent observation that PLC- gamma1 is highly expressed in some kinds of human cancer tissue supports the view that PLC-gamma1 may be involved in proliferation and carcinogenesis. PLC-gamma2 is known to be involved in B cell differentiation and maturation. However, there have been few studies about the expressions of PLC-gamma1 and gamma2 in human lymphoid malignancy. In the present study, we examined the contents of PLC-gamma1 and gamma2 in 10 cases of B cell, 10 cases of T cell non-Hodgkin's lymphoma and 5 cases of Hodgkin's lymphoma to find out whether these enzymes play any role in the carcinogenesis by immunohistochemistry and immunoprecipitation. Immunoprecipitation analysis revealed that in contrast to increased expression of PLC-gamma2 only in B cell lymphoma, a considerably higher level of PLC-gamma1 was detected in both B and T cell lymphoma. Immunohistochemical finding confirmed this observation. PLC-gamma1 and PLC-gamma2 were expressed in the cytoplasm of most tumor cells. PLC-gamma2 was also expressed in mature B cells, while PLC-gamma1 was not expressed in reactive non-tumor cells. These results suggest that PLC-gamma1 mediated signal transduction implicates a significant role in the carcinogenesis of all types of lymphoid tissue, and PLC-gamma2 may play a role in the carcinogenesis of B cell lymphoma as well as B cell differentiation.
B-Lymphocytes ; Carcinogenesis ; Cell Differentiation ; Cytoplasm ; Hodgkin Disease* ; Humans ; Immunohistochemistry ; Immunoprecipitation ; Lymphoid Tissue ; Lymphoma, B-Cell ; Lymphoma, Non-Hodgkin ; Lymphoma, T-Cell ; Phospholipases* ; Signal Transduction ; Type C Phospholipases

5'-upstream region of the phospholipase C-beta2 gene, 810 bp, was cloned and characterized. S1 nuclease mapping and primer extension analyses revealed that a single transcriptional start site locates at 284 nucleotides upstream from the beginning of translation. The 5-upstream region lacks both TATA motif and typical initiator sequence, but retains GC-rich segment. Two putative regulatory regions, a negative region (-636/-588) and a positive region (-98/ -13) were identified in the upstream region of PLC-beta2 gene. We suggest that the transcription of PLC-beta2 may be regulated by binding of regulatory proteins to the negative and/or positive regulatory regions located in the upstream of the gene.
Aspergillus Nuclease S1/metabolism ; Base Sequence ; Cells, Cultured ; Chloramphenicol O-Acetyltransferase/metabolism ; Cloning, Molecular ; Conserved Sequence ; Gene Deletion ; Isoenzymes/*chemistry/*genetics ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Phospholipase C/*chemistry/*genetics ; Promoter Regions (Genetics) ; Protein Binding ; Support, Non-U.S. Gov't ; Transcription, Genetic ; Transfection

FTY720, a synthetic sphingoid base analog, was examined as a new sphingosine kinase inhibitor, which converts endogenous sphingosine into its phosphate form. With 20 micrometer of FTY720, sphingosine accumulated in the LLC-PK1 cells in a time- and dose-dependent manner. The FTY720 treated cells showed a high concentration of fragmented DNA, a high caspase-3 like activity and TUNEL staining cells. It was also found that the sphingosine and sphinganine level increased in a time- and dose-dependent manner within 12 h after the FTY720 treatment. The sphingosine kinase activity was reduced by FTY720 as much as other sphingosine kinase inhibitors, N, N-dimethylsphingosine (DMS), dl-threo-dihydrosphingosine (DHS). The fragmented DNA content as a result of the 20 micrometer of FTY720 treatment and by 5 micrometer of the exogenously added BSA-sphingosine complex indicated typical apoptosis. Under similar conditions, the accumulated sphingosine concentration in all the cells was almost identical even though the sphingosine distribution inside the cells was somewhat different. These results indicate that the FTY720 induced apoptosis is associated with the inhibition of the sphingosine kinase activity and is strongly associated with the successive accumulation of sphingosine.
Animals ; Apoptosis/*physiology ; Caspases/biosynthesis ; Cell Line ; DNA Fragmentation ; Endothelial Cells/drug effects ; Enzyme Inhibitors/*pharmacology ; Kidney/cytology ; Phosphotransferases (Alcohol Group Acceptor)/*antagonists & inhibitors/physiology ; Propylene Glycols/*pharmacology ; Research Support, Non-U.S. Gov't ; Sphingosine/pharmacology/*physiology ; Swine ; Up-Regulation

PURPOSE: Phospholipase C (PLC) isozymes ply significant roles in transmembrane signal transduction. PLC-i 1 acts as the intracellular effector in signal transduction for cellular proliferation and differentiation. Ras oncoprotein is also involved in cell growth. We determined the biological significance of PLC and ras oncoprotein in regeneration following radiation and the effect of different modes of administration of 5-FU. MATERIALS AND METHODS: To determine the effect of the administration mode of 5-FU on the regeneration of intestinal mucosa of rats following radiation, we compared the expression of PLC and ras oncoprotein in six groups. Group I had no treatment. GroupII received radiation( 8 Gy) only. Group III received radiation(8 Gy) and 5-FU(150mg/kg) continuous intravenous (iv) infusion for 12 hours. Group iv bolus injection. Group V received only 5-FU(150mg/kg) iv bolus injection. Through immunoblotting and immunohistochemistry, we examined the expression of PLC and ras oncoprotein in rat jejunum at 96 hours after radiation or 5-FU administration and at 120 hours after radiation and 5-FU administration. We also investigated the histological findings using hematoxylin and eosin stain. RESULTS: In the immunohistochemistry study, PLC-i 1 expression was the highest in group III followed by groups II and VI in that order and was weakly positive in groups V and VI. PLC-i 1 was hardly detected in the control group. The expression of ras oncoprotein wss the same as the PLC-i 1 expression for all groups. These results were confirmed by the histological findings regarding the mucosal regeneration. In the immunoblotting analysis, PLC-i 1 expression was the highest in group III followed by group IV and II in that order. This difference between the immunoblotting and immunohistochemistry study was due to the high expression of PLC-i 1 on the damaged surface epithelium rather than to its expression in the regeneration region as observed in the immunohistochemistry study for group IV. The expression of PLC- 1 was positive only in group V and VI, which received both radiation and 5-FU, and the expression of PLC-b 1was negligible for all groups. CONCLUSION: These results suggest that PLC-i 1 mediated signal transduction and ras oncoprotein may have a significant role in mucosal regeneration after radiation, and that continuous iv infusion of 5-FU may induce active regeneration in intestinal mucosa following radiation. In addition, the expression of PLC- 1 in combined group of radiation and 5-FU implies that PLC- 1 may be involved in signal transduction mediated by concerted action between radiation and 5-FU.
Animals ; Cell Proliferation ; Eosine Yellowish-(YS) ; Epithelium ; Fluorouracil* ; Hematoxylin ; Immunoblotting ; Immunohistochemistry ; Intestinal Mucosa* ; Isoenzymes ; Jejunum ; Phospholipases* ; Rats* ; Regeneration* ; Signal Transduction ; Type C Phospholipases*

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-gamma1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Rats ; Protein Interaction Mapping ; Protein Biosynthesis/drug effects ; Protein Binding/drug effects ; Phosphoproteins/chemistry/*metabolism ; Phospholipase C gamma/antagonists & inhibitors/chemistry/*metabolism ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Peptides/chemistry/metabolism ; PC12 Cells ; Neurofilament Proteins/chemistry/*metabolism ; Nerve Growth Factor/pharmacology ; Molecular Weight ; Molecular Sequence Data ; Microtubules/metabolism ; Microscopy, Fluorescence ; Isoenzymes/metabolism/pharmacology/physiology ; Glutathione Transferase/metabolism ; Blotting, Far-Western ; Blood Proteins/chemistry/*metabolism ; Binding Sites ; Animals ; Amino Acid Sequence

In our previous report an anti-HBsAg human monoclonal antibody was generated using antibody phage display library technique. Using pComb3 filamentous phagemid vector, Fab molecule was expressed in fusion form to a phage coat protein in the periplasm of E. coli. A clone of HBsAg binder was selected after panning and designated as B7. In order to select the clones with higher affinity and to examine which chain contributes most to the affinity of B7, the light and heavy chain of B7 was sequentially deleted and replaced with new library. HBsAg-binders were selected and tested by EIA (enzyme immunoassay). It was revealed that the affinity of B7 depends only on the heavy chain of Fd. B7 Fd was constructed without light chain and specificity and affinity was further confirmed by western blot analysis. This human monoclonal Fd antibody was found to react with d antigenic determinant of HBsAg as the original clone did. The nucleotide sequence analysis revealed that VH of B7 could be classified into Kabat's subgroup II and human IgG heavy chain family IV. The CH1 of B7 was IgG1.
Bacteriophages* ; Base Sequence ; Blotting, Western ; Clone Cells ; Hepatitis B Surface Antigens ; Humans ; Immunoglobulin G ; Periplasm ; Sensitivity and Specificity

Phospholipase C (PLC) isozymes play significant roles in transmembrane signal transduction. PLC- 1 is one of the key regulatory enzymes in signal transduction for cellular proliferation and differentiation. The exact mechanisms of this signal transduction of tissue damage and subsequent regeneration, however, were not clearly documented. This study was planned to determine the biological significance of PLC isozymes following irradiation in rat small intestine. Sprague-Dawley rats were irradiated to the entire body by a single dose of 8 Gy. The rats were divided into 5 groups according to the sacrifice days after irradiation. The expression of PLCs in each group was examined by the immunohistochemistry and immunoblotting. The histologic findings were observed using hematoxylin and eosin staining. The regenerative activity, which was estimated by mitotic count and proliferatin cell nuclear antigen (PCNA) immunostaining, was highest in Group III (5th day after irradiation). By the immunohistochemistry, the expression of PLC- 1 was higher in Group III and Group II (3rd day after irradiation), and was found in the regenerative zone of the mucosa. The expression of PLC- 1 was highest in Group I (1st day after irradiation) and was dominantly in the damaged surface epithelium. The immunostaining of PLC- 1 was negative in all groups. The results of the immunoblotting study was compatible to that of the immunohistochemical study. Group II and III showed positive bands for PLC- 1, and group I and II for PLC- 1. These results suggest that PLC- 1 plays a significant role in mucosal regeneration following irradiation. PLC- 1 may play a role in radiation - induced mucosal damage.
Animals ; Cell Proliferation ; Eosine Yellowish-(YS) ; Epithelium ; Hematoxylin ; Immunoblotting ; Immunohistochemistry ; Intestine, Small* ; Isoenzymes* ; Mucous Membrane ; Phospholipases* ; Rats ; Rats, Sprague-Dawley ; Regeneration* ; Signal Transduction ; Type C Phospholipases*