To examine the involvement of phospholipase D (PLD)isozymes in postnatal testis development, the expression ofPLD1 and PLD2 was examined in the mouse testis atpostnatal weeks 1, 2, 4, and 8 using Western blot analysisand immunohistochemistry. The expression of both PLD1and PLD2 increased gradually with development frompostnatal week 1 to 8. Immunohistochemically, PLDimmunoreactivity was detected in some germ cells in thetestis and interstitial Leydig cells at postnatal week 1.PLD was mainly detected in the spermatocytes andresidual bodies of spermatids in the testis after 8 weeksafter birth. The intense immunostaining of PLD in Leydigcells remained unchanged by postnatal week 8. Thesefindings suggest that PLD isozymes are involved in thespermatogenesis of the mouse testis.
Animals ; Blotting, Western ; Female ; Immunohistochemistry ; Isoenzymes ; Male ; Mice ; Mice, Inbred BALB C ; Phospholipase D/biosynthesis/*metabolism ; Spermatogenesis/physiology ; Testis/*enzymology/growth & development

Phospholipase D (PLD) hydrolyzes phosphocholine into choline and phosphatide acid, and these metabolites play an important role in regulating cell physiology and biochemistry. To study the biological function of phospholipase D3 (PLD3) during the insulin stimulation in C2C12 myoblasts, we constructed PLD3 over-expressed cell lines (C2C12/pPLD3) and investigated the phosphorylation of Akt. The results showed that the level of phosphorylated Akt (P-Akt) was significantly increased in control C2C12 cells when insulin concentration was elevated during cell treatment, whereas the level of P-Akt in C2C12/pPLD3 cells was not changed. When extending the time of insulin treatment, P-Akt level in C2C12/pPLD3 cells was increased around 2 folds, but the total level of P-Akt in C2C12/pPLD3 was still lower than that in control group. 1-Butanol, a PLD inhibitor, could completely block Akt phosphorylation in C2C12 cells that even stimulated by insulin. However, 1-Butanol did not inhibit the Akt phosphorylation in C2C12/pPLD3 cells, but increased the phosphorylation up to 6 folds higher than control cells. The level of Akt phosphorylation in control C2C12 cells was increased significantly when stimulated by phosphatidic acid (PA), while there was no change in C2C12/pPLD3 cells with the similar treatment. When cells simulated by both PA and insulin, P-Akt level in both C2C12/pPLD3 cells and C2C12 cells were down regulated. Our observations indicated that PLD3 over expression may inhibit Akt phosphorylation and further block the transduction of insulin signaling in C2C12 cells.
Cell Line ; Humans ; Insulin ; pharmacology ; Myoblasts ; cytology ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phospholipase D ; biosynthesis ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; chemistry ; drug effects ; Signal Transduction

To investigate the expression of phospholipase C-gamma1 (PLC-gamma1) in mouse embryonic tissues, serial tissue sections were prepared routinely for immunocytochemistry for PLC-gamma1. The results showed that PLC-gamma1 was expressed in the cartilage, skeletal muscles, myocardium, the collecting tubule of the kidney, connective tissues and the brain, suggesting the important role PLC-gamma1 and the related signal pathway may play in the development of mouse embryonic tissues.
Animals ; Brain ; embryology ; enzymology ; Cartilage ; embryology ; enzymology ; Embryo, Mammalian ; enzymology ; Female ; Fetal Heart ; enzymology ; Immunohistochemistry ; Kidney ; embryology ; enzymology ; Mice ; Muscle, Skeletal ; embryology ; enzymology ; Phospholipase C gamma ; biosynthesis ; Pregnancy

Phospholipase C (PLC) and related enzymes in signal transduction system are closely linked to cellular damage in ischemic encephalopathy. This study was undertaken to elucidate the time sequential changes of PLC isoenzymes (beta and gamma) in vulnerable areas of hippocampus in global ischemia and infarcted area in focal infarction. Mongolian gerbils were used because of their susceptibility to ischemic encephalopathy and divided into the following groups: the bilateral ischemia with various reperfusion periods group, unilateral progressive ischemia group, and focal ischemia group induced by infusion of iron particles through the femoral artery. The changes of PLC isoenzymes were observed immunohistochemically and matched with morphological changes. In the global ischemia with reperfusion group, the changes were most significant in hippocampus. Sequential changes of neurons such as red neurons at an early stage progressed to pknotic neurons at a later stage were noted with typical delayed neuronal damage in the corns ammonis (CA) 1 subfield of hippocampus. Red neurons and pyknotic neurons as well as intracytoplasmic inclusion in 3 to 24 hours of reperfusion showed loss of PLC isoenzymes as well as tubulin. The changes of PLC expression were corresponding to the degeneration of neurons with no discernible time sequential changes in remaining neurons. In the unilateral hemispheric progressive ischemia group, ischemic damage was far more marked and extensive with no selective injury pattern according to time and location. At 1 day, there was diffuse vacuolization and necrosis of neuropil with a loss of neuron. Admixed surviving neurons and vacuolated neuropil showed increased reaction to anti-PLC antibodies, which could be either an evidence of protein synthesis responding to ischemic insult or an artifactual change. Focal ischemia group showed time sequential changes of blood vessels and white blood cells with necrosis of surrounding tissue. Degenerating hippocampal neurons in infarction also showed a strong positive reaction to anti-PLC antibody, which was most likely due to condensation of cytoplasm rather than increased synthesis. This study showed different changes of PLC expression in global ischemic encephalopathy with reperfusion, progressive ischemia, and focal infarction, which suggested different pathophysiologic mechanism between these conditions.
Animal ; Brain Ischemia/*metabolism/pathology ; Cerebral Infarction/metabolism/pathology ; Female ; Gene Expression ; Gerbillinae ; Hippocampus/*enzymology ; Immunoenzyme Techniques ; Isoenzymes/*biosynthesis ; Male ; Neurons/enzymology/ultrastructure ; Phospholipase C/*biosynthesis ; Support, Non-U.S. Gov't ; Time Factors

OBJECTIVETo construct recombinant lentivirus that stably suppresses phospholipase C (PLC) gamma1 expression in human colorectal carcinoma LoVo cells to obtain LoVo cell lines deficient in PLC gamma1 for investigation of the role of PLC gamma1 gene.

METHODSRecombinant lentivirus producing PLC gamma1 siRNA were constructed to infect LoVo cells, and the stably transduced cells were selected with blasticidin. The protein and mRNA expression of PLC gamma1 was examined by Western blotting and RT-PCR, and the effect of the lentivirus on cell apoptosis was analyzed by flow cytometry.

RESULTS AND CONCLUSIONPLC gamma1 siRNA significantly suppressed PLC gamma1 expression in LoVo cells, suggesting high efficiency of gene silencing induced by the siRNA produced by the recombinant lentivirus. Concomitantly, cell apoptosis induced by 5-FU was significantly increased.

Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; genetics ; Blotting, Western ; Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; pathology ; DNA, Recombinant ; genetics ; Fluorouracil ; pharmacology ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Phospholipase C gamma ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

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