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

Recently identified second messenger molecules of signal transmission were derived from inositol phospholipid and several phospholipase C(PLC) isoenzyme were reconized. But the role of PLC in embryonal organ development, differentiation and growth is not described, yet. And, Siah gene of mouse is expressed at a higher level in a restricted number of sites during development. So, it is suggested that the Siah gene plays a significant role in development of vertebrates.Therefore, this study was aimed to investigate the role of PLC isoenzymes and Siah gene during murine fetal development by use of immunehistochemistry. The results are as follow: 1. PLC-beta1 and PLC-gammal are expressed apparently at early fetal stage, PLC-betal is observed focally, whereas PLC-gamma1 is diffusely expressed. 2. PLC-delta1 is not expressed during early and mid murine fetal age but strongly expressed in placental tissue. 3. PLC-beta1 is observed mainly in neural tissues including brain at mid fetal stage. PLC-gamma1 is expressed in entire organs, especially in liver tissue, and then their expression is reduced after mid stage. 4. Siah gene product is significantly expressed in olfactory epithelium, retina, neuron of brain and choroid plexus from mid fetal stage to late gestation. These results suggest that PLC-betal play a important role in central neural development and PLC-gamma1 in entire orgaans, especially in liver development. The strong expression of PLC-deltal in placenta suggest its involvement in early development, indirectly. Siah gene product expressed at a higher levels in olfactory epithelium, retina, neuron and choroid plexus, so Siah gene may play a important role in these organ developments.
Animals ; Brain ; Choroid Plexus ; Fetal Development* ; Gestational Age ; Immunohistochemistry* ; Inositol ; Isoenzymes ; Liver ; Mice ; Neurons ; Olfactory Mucosa ; Phospholipases* ; Placenta ; Pregnancy ; Retina ; Second Messenger Systems ; 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

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

We expressed anti-HBsAg human antibody fragment (B7 Fd) using pRSET-A vector and BL21(DE3)pLysS strain of E. coli. B7 Fd is composed only of variable domain (VH) and CH1 constant domain of IgG heavy chain molecule. This Fd molecule was solubilized using guanidine salt and then expressed in the form of inclusion body and successfully refolded into functional antibody molecule by rapid dilution in refolding buffer. B7 Fd reacted with d epitope of hepatitis B virus surface antigen (HBsAg). Its affinity was determined by competition enzyme-linked immunosorbent assay (competition ELISA). The K value of B7 Fd was 3.3 * 10.
Antigens, Surface ; Enzyme-Linked Immunosorbent Assay ; Guanidine ; Hepatitis B virus ; Humans* ; Immunoglobulin G ; Inclusion Bodies

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

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