Protein kinase C (PKC) is localized in the equatorial segment and the principal piece of the tail of spermatozoa. Activator of PKC results in increasing flagellar motility of sperm that is blocked by PKC inhibitors such as staurosporine. A good correlation (r = 0.9, P < 0.001) is found between the content of PKC in sperm and sperm motility. Zona pellucida (ZP) stimulates the spermatozoa binding the acrosome reaction resulting in the release of hydrolytic enzymes and in the exposure of new membrane domains. ZP binding to receptors in the plasma membrane can regulate adenyl cyclase (AC) leading to elevation of cAMP and protein kinase A (PKA) activation. The PKA activates a voltage-dependent Ca2+ channel in the outer acrosomal membrane which releases Ca2+ from the interior of the acrosome to the cytosol. Activation of the PLC resulted from the rise in Ca2+ hydrolyze phosphatidyl inositol bisphosphate. The product activate PCK to open a voltage-dependent Ca2+ channel (L) in the plasma membrane, leading to the second (II) Ca2+ higher increase which result in membrane fusion and acrosome reaction. It is proposed that PKC would be involved in the regulation of motility and acrosome reaction of sperm.
Acrosome Reaction ; physiology ; Humans ; Male ; Protein Kinase C ; metabolism ; Sperm Motility ; physiology ; Spermatozoa ; enzymology ; physiology

BACKGROUNDAirway smooth muscle proliferation plays an important role in airway remodeling in asthma. But little is known about the intracellular signal pathway in the airway smooth muscle cell proliferation in asthma. The objective of this paper is to investigate the contribution of protein kinase C (PKC) and its alpha isoform to passively sensitized human airway smooth muscle cells (HASMCs) proliferation.

METHODSHASMCs in culture were passively sensitized with 10% serum from asthmatic patients, with non-asthmatic human serum treated HASMCs used as the control. The proliferation of HASMCs was examined by cell cycle analysis, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT) colorimetric assay and proliferating cell nuclear antigen (PCNA) immunofluorescence staining. The effect of PKC agonist phorbol 12-myristate 13-acetate (PMA) and PKC inhibitor Ro-31-8220 on the proliferation of HASMCs exposed to human asthmatic serum and non-asthmatic control serum was also examined by the same methods. The protein and mRNA expression of PKC-alpha in passively sensitized HASMCs were detected by immunofluorescence staining and reverse transcription-polymerase chain reaction.

RESULTSThe percentage of S phase, absorbance (value A) and the positive percentage of PCNA protein expression in HASMCs passively sensitized with asthmatic serum were (16.30 +/- 2.68)%, 0.430 +/- 0.060 and (63.4 +/- 7.4)% respectively, which were significantly increased compared with HASMCs treated with control serum [(10.01 +/- 1.38)%, 0.328 +/- 0.034 and (37.2 +/- 4.8)%, respectively] (P < 0.05). After HASMCs were passively sensitized with asthmatic serum, they were treated with PMA, the percentage of S phase, value A and the positive percentage of PCNA protein expression were (20.33 +/- 3.39)%, 0.542 +/- 0.065 and (76.0 +/- 8.7)% respectively, which were significantly increased compared with asthmatic serum sensitized HASMCs without PMA(P < 0.05). After HASMCs passively sensitized with asthmatic serum were treated with Ro-31-8220, the percentage of S phase, value A and the positive percentage of PCNA protein expression were (11.21 +/- 1.56)%, 0.331 +/- 0.047 and (38.8 +/- 6.0)% respectively, which were significantly decreased compared with asthmatic serum sensitized HASMCs without Ro-31-8220 (P < 0.05). The relative ratio of value A of PKC-alpha mRNA and the positive percentage of PKC-alpha protein expression in passively sensitized HASMCs were 1.23 +/- 0.10 and (61.1 +/- 9.4)% respectively, which were significantly increased compared with HASMCs treated with control serum [1.05 +/- 0.09 and (34.9 +/- 6.7)%, respectively] (P < 0.05).

CONCLUSIONSThe proliferation of HASMCs passively sensitized with human asthmatic serum is increased. PKC and its alpha isoform may contribute to this proliferation.

Asthma ; immunology ; pathology ; Cell Division ; physiology ; Cells, Cultured ; Humans ; Immunization, Passive ; Myocytes, Smooth Muscle ; pathology ; physiology ; Protein Kinase C ; physiology ; Protein Kinase C-alpha ; Signal Transduction ; physiology

Protein kinase C (PKC) family plays a critical role in many developmental events, including oocyte activation, completion of the second meiosis and initiation of the first mitosis, compaction, and blastocysts formation as well. But little is known of its many isozymes. Studies have shown that 10 isozymes of PKC and its anchor protein, RACK, are expressed in the course from 2 cell stage through blastocyst stage in mouse. We reviewed here the recent studies on the location pattern and expression levels of different PKC isozymes. Those studies indicated that the isozymes were very important for every stage of preimplantation embryonic development, especially at the early 4-cell stage. Some are increased temporarily in nucleus, which indicated that they might control and regulate the remolding of embryonic nucleus. We also analyzed the possible functions of PKCs in the somatic nuclear transferred embryos.
Animals ; Embryonic Development ; physiology ; Fertilization ; physiology ; Isoenzymes ; metabolism ; physiology ; Oocytes ; physiology ; Protein Kinase C ; metabolism ; physiology ; Receptors for Activated C Kinase ; Receptors, Cell Surface ; metabolism

OBJECTIVETo investigate the expressions of protein kinase C (PKC) isoforms in X-ray-exposed HepG2 cells and identify the PKC isoforms that induce radioresistance in HepG2 cells.

METHODSCultured HepG2 cells were divided into control group and 6 Gy radiation group for corresponding treatments. The fluorescence intensity (FI) and the percentage of positive cells were determined using flow cytometry.

RESULTSThe FI of PKCalpha and PKCdelta were 2.28 and 5.05 in the radiation group, respectively, significantly higher than those in the control group (P<0.05). The percentages of PKCalpha- and PKCdelta -positive cells were significantly higher in the radiation group than in the control group (P<0.05). The FI and the percentages of PKC zeta, gamma, epsilon, zeta positive cells were rather low and showed no significant differences between the two groups (P>0.05); PKCbeta expression was not detected in the two groups of cells. The apoptosis rates of the control and radiation groups were 1.73% and 20.90%, respectively.

CONCLUSIONPKCalpha and PKCdelta may be involved in protecting HepG2 cells from radiation-induced apoptosis.

Apoptosis ; physiology ; radiation effects ; Hep G2 Cells ; Humans ; Isoenzymes ; classification ; metabolism ; Protein Kinase C-alpha ; metabolism ; Protein Kinase C-delta ; metabolism ; Radiation Tolerance ; Signal Transduction ; drug effects ; physiology

Animals ; Humans ; Models, Biological ; Oxidative Stress ; genetics ; physiology ; Phosphorylation ; Protein Kinase C ; genetics ; metabolism ; physiology ; Signal Transduction ; genetics ; physiology

OBJECTIVETo examine the effect of SH2A gene in cell signal transduction and its subcellular localization.

METHODSRT-PCR method was used to amplify the coding sequence of SH2A gene. Eukaryotic recombined expression vector pcDNA 3.1-SH2A was constructed, and then Bel7402 cell and COS7 cell transfected by liposome. Multiple kinase assay was performed to examine the activity of protein kinase (PKC), mitogen activated protein kinase (MAPK), tyrosine protein kinase (TPK) in the transfected cells. Meantime, pEGFP-SH2A vector was also constructed and the cells transfected with it were examined by fluorescent microscopy.

RESULTSRecombined expression vector pcDNA3.2-SH2A and pEGFP-SH2A contained the coding sequence of SH2A cDNA. In both cell lines expressing SH2A gene, the cytoplasm PKC activity decreased by 40% or so, but no apparent alteration was found in MAPK and TPK activity. SH2A gene was found localized in the cytoplasm of transfected cells under fluorescent microscope.

CONCLUSIONSH2A gene may act as an inhibiting factor in PKC signal transduction, and it is localized in cytoplasm.

Animals ; COS Cells ; Cytoplasm ; chemistry ; Humans ; Membrane Proteins ; analysis ; genetics ; physiology ; Mitogen-Activated Protein Kinases ; metabolism ; Protein Kinase C ; physiology ; Signal Transduction ; physiology ; Transfection

Carcinoma of the uterine cervix is one of the most common malignancies among women worldwide. Human papillomaviruses (HPV) have been identified as the major etiological factor in cervical carcinogenesis. However, the time lag between HPV infection and the diagnosis of cancer indicates that multiple steps, as well as multiple factors, may be necessary for the development of cervical cancer. The development and progression of cervical carcinoma have been shown to be dependent on various genetic and epigenetic events, especially alterations in the cell cycle checkpoint machinery. In mammalian cells, control of the cell cycle is regulated by the activity of cyclin-dependent kinases (CDKs) and their essential activating coenzymes, the cyclins. Generally, CDKs, cyclins, and CDK inhibitors function within several pathways, including the p16INK4A-cyclin D1-CDK4/6-pRb-E2F, p21WAF1-p27KIP1-cyclinE-CDK2, and p14ARF-MDM2-p53 pathways. The results from several studies showed aberrant regulation of several cell cycle proteins, such as cyclin D, cyclin E, p16 INK4A, p21WAF1, and p27KIP1, as characteristic features of HPV- infected and HPV E6/E7 oncogene-expressing cervical carcinomas and their precursors. These data suggested further that interactions of viral proteins with host cellular proteins, particularly cell cycle proteins, are involved in the activation or repression of cell cycle progression in cervical carcinogenesis.
Uterine Cervical Neoplasms/*pathology ; Tumor Suppressor Protein p53/physiology ; Tumor Suppressor Protein p14ARF/physiology ; Retinoblastoma Protein/physiology ; Proto-Oncogene Proteins c-mdm2/physiology ; Humans ; Female ; E2F Transcription Factors/physiology ; Cyclin-Dependent Kinase Inhibitor p27/physiology ; Cyclin-Dependent Kinase Inhibitor p21/physiology ; Cyclin-Dependent Kinase Inhibitor p16/physiology ; Cyclin-Dependent Kinase 4/physiology ; Cyclin-Dependent Kinase 2/physiology ; Cyclin E/physiology ; Cyclin D1/physiology ; Cell Cycle/*physiology

OBJECTIVETo investigate the content and activity of M-phase promoting factor (MPF) in pleomorphic adenoma, mucoepidermoid carcinoma, buccal carcinoma and normal tissue, in order to evaluate the role of MPF in the development of tumor and the relationship between MPF and malignant degree.

METHODSThe content and activity of MPF were assessed by immunobloting and Gollicano method.

RESULTSThe cdc2 and cyclinB (two subunits of MPF) were found both in normal and tumor tissues, and their content in tumor was higher than normal tissues. Buccal carcinoma was 64% higher than normal tissues. The activity of MPF in carcinoma was higher than normal tissue and had positive relation with the malignant extent.

CONCLUSIONSThe content and activity of MPF in tumor are higher than normal tissue. PKC can activate MPF. These results show PKC may promote tumor proliferation by activating MPF and also, the activity of MPF has some relation with malignant extent.

CDC2 Protein Kinase ; analysis ; Cyclin B ; analysis ; Humans ; Immunoblotting ; Maturation-Promoting Factor ; analysis ; Mouth ; chemistry ; Mouth Neoplasms ; chemistry ; Protein Kinase C ; physiology

AIMTo explore the role of novel protein kinases C (nPKCs) in the development of cerebral hypoxic preconditioning.

METHODSBy using the mice model of hypoxic preconditioning, which was established before in our lab, the biochemistry techniques of SDS-PAGE and Western blot were applied to observe the effects of repetitive hypoxic exposure (H0-H4) on nPKCs (nPKCepsilon, delta, eta, mu and theta) membrane translocation in hippocampus and cortex.

RESULTSnPKCepsilon membrane translocation was increased in response to the hypoxic exposure times in the hippocampus (H0: 41.6% +/- 1.4% vs. H1-H4: 46.9% +/- 4.5%, 52.7% +/- 3.9%, 58.8% +/- 2.7% and 61.3% +/- 3.7%) and cortex (H0: 38.4% +/- 4.5% vs. 42.4% +/- 5.0%, 48.7% +/- 6.5%, 55.3% +/- 8.9% and 61.2% +/- 10.2%) of mice, and there were statistic significances among H2, H3 and H4 in hippocampus, and H3 and H4 in cortex respectively (P < 0.01). But for nPKCdelta, eta, mu and theta membrane translocation, there were no any significant changes in hippocampus and cortex of hypoxic preconditioned mice.

CONCLUSIONnPKCepsilon may play an important role in the development of cerebral hypoxic preconditioning, but it need more evidence to prove.

Animals ; Blotting, Western ; Brain ; metabolism ; Hippocampus ; metabolism ; Hypoxia ; metabolism ; Mice ; Mice, Inbred BALB C ; Protein Kinase C ; metabolism ; Protein Transport ; physiology

Cerebral hypoxic preconditioning (CHP), which was induced by repetitive sub-lethal hypoxic insult, is an endogenous protection of neuron against subsequent severe hypoxic injury. Although a number of possible induction pathways have been investigated, such as neuroactive cytokines, activation of glutamate receptors, the ATP-sensitive potassium channel, nitric oxide and oxidative stress, the exact mechanism underlying CHP-induced protection remains unclear. It is interesting that all the above-mentioned mechanisms are involved in the activation of protein kinases C (PKC). Recently we reported that the level of PKCs membrane translocation was significantly increased in the brain of hypoxic preconditioned mice. In order to explore the role of conventional protein kinases C (cPKC) in the development of cerebral hypoxic preconditioning, biochemical techniques of SDS-PAGE and Western bolt were applied to observe the effects of repetitive hypoxic exposure (H1-H4) on the level of cPKCalpha and gamma membrane translocation in the cortex and hippocampus of mice. Experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals. The hypoxic preconditioned mice model was adapted with minor modification from our previous report. In brief, healthy adult BALB/C mice weighing 18-20 g of either sex were randomly divided into 5 groups: control group (H0), hypoxic control group (H1, hypoxic exposure once ), hypoxic preconditioned group (H2-H4, repetitive hypoxic exposure for 2-4 times respectively). The first sign of gasping breath was taken as the end of each hypoxic exposure, and then the mice were kept in normal control condition for a 30-min interval to recover before the following hypoxic insult. We found that the level of cPKCgamma membrane translocation was increased significantly (*P<0.05, n=6) with the increase of the hypoxic exposure times in both hippocampus (H0: 100% vs H1 approximately H4: 119.2%+/-7.0% *, 139.3% +/-7.4%*, 134.2% +/-8.95%*, 184.0% +/-10.8%*) and cortex (H0: 100% vs H1-H4: 129.7% +/-13.8%, 143.3% +/-13.9%*, 204.0% +/-12.1%*, 229.5% +/-14.6%*) of mice. But there were no significant changes in cPKCalpha membrane translocation in cortex and hippocampi of hypoxic preconditioned mice. These results suggest that cPKCgamma plays an important role in the development of cerebral hypoxic preconditioning. The changes in some other forms of novel and atypical PKCs are still under investigation.
Animals ; Brain ; blood supply ; metabolism ; physiology ; Cell Membrane ; metabolism ; Female ; Hippocampus ; metabolism ; Ischemic Preconditioning ; Male ; Mice ; Mice, Inbred BALB C ; Protein Kinase C ; metabolism ; Protein Transport