Inhibition by sulfatide of 21-kDa protein phosphorylation by protein kinase C in cow mammary gland and its reversal by phosphatidylserine

The effect of sulfatide, a sulfated sphingolipid, on phosphorylation of endogenous proteins by protein kinase C (PKC) was examined in cow mammary gland. Several proteins, including 21-kDa, 43-kDa and 56-kDa proteins in the cytosolic fraction, were found to be substrates for PKC by phosphorylation in the absence or presence of the cofactors 1-oleoyl-2-acetyl-sn-glycerol (OAG), phosphatidylserine (PS) and Ca2+. Sulfatide inhibited the 21-kDa phosphorylation, whereas it enhanced the 56-kDa and 43-kDa phosphorylation. Experiments were then conducted to examine whether other sphingolipids, including sphingosine, dihydrosphingosine, ceramides, galactocerebrosides, psychosine and sphingomyelin, modulated phosphorylation of the PKC substrates. Sphingosine, dihydrosphingosine and psychosine did not inhibit the 21-kDa phosphorylation; however, they enhanced the 56-kDa and 43-kDa phosphorylation. Ceramides, galactocerebrosides and sphingomyelin did not inhibit the 21-kDa or enhance the 56-kDa and 43-kDa phosphorylation. The inhibition by sulfatide of the 21-kDa phosphorylation was reversed by excess addition of PS, but not by OAG or Ca2+; whereas the enhancement by sulfatide, as well as sphingosine, dihydrosphingosine and psychosine, of 56-kDa and 43-kDa phosphorylation was not affected by PS, OAG or Ca2+. It is suggested that sulfatide is involved in the regulation of PKC-dependent phosphorylation by modulating the association of PKC substrates, in particular the 21-kDa protein, with membrane phospholipids in cow mammary gland.     

Similarities of cow mammary gland cytosolic 21-kDa protein, the substrate for protein kinase C, to the 20-kDa myosin light chain from smooth muscle

In the cytosol of cow mammary gland, several proteins are phosphorylated in the presence of the protein kinase C (PKC) cofactors 1-oleoyl-2-acetyl-sn-glycerol (OAG), phosphatidylserine (PS) and Ca2+. Of the substrates, the 21-kDa protein is inferred to be a 20-kDa regulatory myosin light chain (MLC20) from smooth muscle because of its molecular mass, its distribution in the cytosol, its association with melittin and sphingosine (the PKC modulators), and phosphorylation by PKC as well as by Ca2+/calmodulin-dependent myosin light chain kinase (MLCK). The present study was undertaken to examine whether the 21-kDa protein could be identified as MLC20, by adding cow uterine MLC20 to the reaction mixture containing cytosol with or without the PKC cofactors and/or calmodulin. In the absence of MLC20, the 21-kDa protein was phosphorylated when the PKC cofactors and calmodulin were added to the reaction mixture. Phosphorylation of the 21-kDa protein was inhibited by melittin or sphingosine, and the inhibition was reversed by PS, but not by calmodulin. When MLC20 was included in the reaction mixture, it was phosphorylated in the presence of the PKC cofactors, and the phosphorylated MLC20 band overlapped that of the 21-kDa protein. The indistinguishably overlapped band of the two proteins was inhibited by melittin and by sphingosine, and their inhibition was reversed by PS, not by calmodulin. It is suggested that the 21-kDa protein is the smooth muscle MLC20 and also that the 21-kDa MLC20 is phosphorylated by PKC, but not by MLCK.     

Inhibition by melittin of phosphorylation by protein kinase C of annexin I from cow mammary gland

Protein kinase C (PKC) is a phosphotransferase activated by diacylglycerols, phospholipids and Ca(2+), that regulates a wide variety of biological functions by phosphorylating multiple protein substrates such as annexin I. Annexin I is a phospholipid/Ca (2+)-binding protein distributed in various tissues, including the mammary gland, and is thought to mediate the anti-inflammatory actions of glucocorticoids by inhibiting phospholipase A(2). Melittin, a phospholipase A(2) activator in bee venom, is known to inhibit PKC activity when lysine-rich histone is used as the substrate. The purpose of the present study was to examine whether phosphorylation by PKC of annexin I from cow mammary gland was inhibited by melittin. Melittin inhibited annexin I phosphorylation by PKC in a dose-dependent manner, and its IC(50) value (concentration causing 50% inhibition) was 0.8 microM. The phosphorylation of annexin I was also inhibited by the amphiphilic polypeptides mastoparan and polymyxin B, and their inhibitory effects were comparable to that of melittin. The surface-inactive polypeptide bacitracin was less effective. The inhibition by melittin was effectively reversed by the excess addition of phosphatidylserine, but not distinctly by 1-oleoyl-2-acetyl-sn-glycerol or Ca(2+), suggesting that melittin inhibited the phosphorylation of annexin I by interacting with phosphatidylserine. The inhibition by melittin of PKC phosphorylation of annexin I seems to be pathophysiologically important, because a melittin-like phospholipase A(2)-stimulatory protein is present in bovine endothelial cells.     

PKA implicated in the phosphorylation of Cx43 induced by stimulation with FSH in rat granulosa cells

Connexin 43 (Cx43)-mediated gap junctional communication in granulosa cells is crucial for germ line development and postnatal folliculogenesis. We previously showed that follicle-stimulating hormone (FSH) promoted phosphorylation of Cx43 in rat primary granulosa cells. We further identified Ser365, Ser368, Ser369, and Ser373 in the carboxy-terminal tail as the major sites of phosphorylation by FSH, and found that the phosphorylation of these residues was essential for channel activity. In this study, we investigated the protein kinase(s) responsible for FSH-induced phosphorylation. H89, a cyclic AMP-dependent protein kinase (PKA) inhibitor, inhibited FSH-induced phosphorylation both in vivo and in vitro, whereas PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor, had little effect on the phosphorylation level. Ca2+-dependent protein kinase (PKC) appeared to negatively regulate phosphorylation. Phosphopeptide mapping with or without H89 treatment indicated that PKA could be responsible for phosphorylation of the four serine residues. In addition, the purified catalytic subunit of PKA could phosphorylate the recombinant C-terminal region of Cx43, but not the variant in which all four serine residues were substituted with alanine. These results suggest that FSH positively regulates Cx43-mediated channel formation and activity through phosphorylation of specific sites by PKA.     

Reduced protein kinase C activity and endogenous protein phosphorylation in ethionine-induced fatty liver in cows

Protein kinase C (PKC) activity was evaluated and the phosphorylation of its endogenous substrates was explored in fatty liver induced by administration of ethionine (an analogue of methionine) to cows in order to assess the relevance of PKC-dependent phosphorylation in the development of fatty liver. PKC activity was decreased in both the cytosolic and the total particulate fractions from fatty livers, compared to the corresponding fractions from control liver. The mode of activation by the PKC cofactors (1-oleoyl-2-acetyl-sn-glycerol, 12-O-tetradecanoylphorbol-13-acetate, phosphatidylserine and Ca²⁺) was similar in both control and fatty livers, suggesting a quantitative but not a qualitative change in PKC in fatty liver. At least three substrate proteins (34 kDa, 26 kDa and 19 kDa) were found in the cytosolic fraction and their phosphorylation was reduced in fatty liver. These results suggest that impairment of the signal transduction pathway mediated by PKC is involved in the pathogenesis of fatty liver in cows.Abbreviations ATP adenosine triphosphate - EGTA ethylene glycol bis(-aminoethylether)-N,N,N,N-tetraacetic acid - NEFA non-esterified fatty acid - OAG 1-oleoyl-2-acetyl-sn-glycerol - PKC protein kinase C - PS phosphatidylserine - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TG triglyceride - TPA 12-O-tetradecanoylphorbol-13-acetate     

表皮生长因子调控猪肠上皮细胞中钠依赖Ⅱb型磷转运蛋白表达的细胞信号通路研究

本研究旨在探讨表皮生长因子(EGF)调控猪小肠上皮细胞IPEC-J2中钠依赖Ⅱb型磷转运蛋白(NaPi-Ⅱb)表达的分子机制。试验分别用EGF受体酪氨酸激酶抑制剂(tyrphostin AG1478)、蛋白激酶A(PKA)抑制剂(H89)、蛋白激酶C(PKC)抑制剂(k4393)、p38抑制剂(SB203580)、细胞外信号调节激酶(ERK)抑制剂(PD98059)、c-Jun氨基末端激酶(JNK)抑制剂(anisomycin)与EGF共同处理IPEC-J2细胞,利用Western blot检测相关通路蛋白及目的蛋白(NaPi-Ⅱb)的表达水平。结果显示:相较于对照组,EGF处理后NaPi-Ⅱb表达水平显著降低(P0.05);相较于无抑制剂组,EGF受体、PKA、PKC、丝裂原活化蛋白激酶(MAPK)/p38、MAPK/ERK1/2、MAPK/JNK的特异性抑制剂处理IPEC-J2后,NaPi-Ⅱb表达水平显著提高(P0.05),其中添加MAPK/ERK1/2特异性抑制剂显著降低了MAPK/ERK1/2在Tyr204位点的磷酸化水平(P0.05),添加MAPK/JNK的特异性抑制剂显著降低了MAPK/JNK1/2/3在Thr183和Tyr185位点的磷酸化水平(P0.05),说明该2组抑制剂对该通路的抑制作用是通过降低上述位点的磷酸化水平实现的。本研究结果表明EGF受体、PKA、PKC、p38、ERK和JNK均介导了EGF调控IPEC-J2细胞中NaPi-Ⅱb的表达。     

Growth factor and cytokine interactions in myogenesis. Part II. Expression of IGF binding proteins and protein kinases essential for myogenesis in mouse C2C12 myogenic cells exposed to TNF-alpha and IFN-gamma

The aim of the study was to examine potential interactions among IGF-I and proinflammatory cytokines, TNF-alpha and IFN-gamma, in the regulation of local IGF-I bioavailability and cellular proteins mediating myogenic signals. We investigated levels of IGFBP-4, -5, -6, protein kinase Czeta (PKC zeta), p38 and extracellular signal-regulated kinase (ERK1/2) in differentiating mouse C2C12 myoblasts. IGF-I significantly stimulated expression of IGFBP-5. TNF-alpha and IFN-gamma attenuated the expression of IGFBP-4 and -6 under basal conditions and in the presence of IGF-I, and inhibited IGF-I-induced IGFBP-5 expression during 5-day myogenesis. TNF-alpha and IFN-gamma markedly attenuated p38 expression in the presence of IGF-I on the 5th day of myogenesis. When combined with IGF-I the cytokines exerted opposite effects on the PKC zeta level, i.e. TNF-alpha caused an increase, whereas IFN-gamma reduced the cellular content of this kinase. Exposition of C2C12 myoblasts to IGF-I or cytokines led to the stimulation of ERK1/2 phosphorylation; however, both TNF-alpha and IFN-gamma exerted an inhibitory effect on the activation of ERK1/2 in myoblasts cultured in the presence of IGF-I. We concluded as follows: i) TNF-alpha and IFN-gamma present in the extracellular environment of differentiating C2C12 myoblasts can alter the local bioavailability of IGF-I by inhibiting the expression of IGFBP-4, -5, and -6, ii) the decrease in p38 expression and ERK1/2 phosphorylation in C2C12 myoblasts exposed to cytokines can lead to disturbances in IGF-I-regulated myogenesis.     

Bovine metalloprotease characterization and in vitro connective tissue degradation

Metalloproteases that selectively hydrolyze connective tissue proteins may tenderize meat without creating texture problems associated with myofibrillar protein degradation. Our objective was to characterize the activity of bovine placental proteases to determine whether they can improve meat tenderness through disruption of the connective tissue matrix. Enzymes were extracted, crudely purified, and proteolytic activity was assessed against gelatin and collagen under varying pH and temperature conditions using both SDS-PAGE and zymography. Gelatin zymography revealed proteolysis between 57 and 63 kDa, with decreased activity as buffer pH decreased from pH 7.4 to 5.4 (37 degrees C). Proteolytic activity was pronounced at 37 degrees C, moderate at 25 degrees C, and absent at 4 degrees C following 48-h incubation (pH 7.4). Placental enzymes were metalloproteases inhibited by excess EDTA. Maximum proteolysis was achieved in the presence of Ca2+, with or without Mg2+ and Zn2+. Absence of Ca2+ decreased proteolytic activity. Complete degradation of both the 125- and 120-kDa proteins of the alpha-chains of gelatin was achieved following enzyme incubation for 6 h at 37 degrees C or 24 h at 25 degrees C. No degradation was observed following enzyme incubation with native Type I collagen. Given the marked decrease in enzyme activity at pH 5.4 and 4 degrees C (standard industry conditions), bovine placental metalloproteases would not be expected to contribute to connective tissue degradation or improve meat tenderness.     

黄芪多糖对小鼠免疫细胞信号转导相关分子的影响

为探讨黄芪多糖(APS)免疫调节的作用机理，观察了黄芪多糖对小鼠腹腔巨噬细胞一氧化氮(NO)生成、对体外培养的小鼠脾脏淋巴细胞内游离钙离子水平和蛋白激酶C(PKC)活性的影响。结果显示黄芪多糖能明显促进小白鼠腹腔巨噬细胞NO生成，显著升高小鼠脾淋巴细胞内游离钙离子的水平，引起细胞PKC活性明显升高。提示黄芪多糖通过NO介导信号传递通路，调节脾淋巴细胞内游离钙离子的浓度，升高细胞蛋白激酶活性而影响机体免疫细胞的信号转导，发挥其免疫调节作用。     

Prolactin signaling in porcine adrenocortical cells: involvement of protein kinases

Prolactin (PRL) was found to have a stimulatory effect on adrenal steroidogenesis in vivo and in vitro in several species including pigs. PRL signal transduction pathways, however, in adrenocortical cells are poorly recognized. Therefore, the goal of this paper is to ascertain the involvement of protein kinase C (PKC) and tyrosine kinases in PRL signaling in porcine adrenal cortex. Adrenals were harvested from locally slaughtered mature gilts. Cortical cells were dispersed by sequential treatment with collagenase. The cells were seeded into 24-well culture plates at a density of 3×10⁵/mL. Cells were incubated with or without PRL (500 ng/mL), ACTH (5 nM—a positive control), tyrosine kinase inhibitor—genistein (1; 2.5 or 5 μM), PKC inhibitor—sphingosine (20–1000 nM) and PKC activators—diacylglycerol (DiC8; 10–100 μM) and phorbol ester (PMA; 1–1000 nM). All incubations were performed for 8 h (95% air and 5% CO₂, 37°C). PRL and ACTH (P<0.05) increased cortisol and androstenedione (A₄) secretion. DiC8 and PMA mimicked the stimulatory effect of PRL. Sphingosine (P<0.05) suppressed basal and PRL-stimulated steroid secretion. Genistein inhibited (P<0.05) PRL-stimulated cortisol secretion and enhanced (P<0.05) basal and PRL-stimulated A₄ secretion. Moreover, PKC activation was assessed by measuring the specific association of [³H]phorbol dibutyrate ([³H]PDBu) with adrenocortical cells after treatment with PRL or ionomycin (a positive control). PRL (within 2–3 min) and ionomycin (within 2–5 min) increased (P<0.05) specific binding of [³H]PDBu to the porcine adrenocortical cells. In addition, PRL did not augment the cortisol and A₄ secretion by PKC-deficient adrenocortical cells. In conclusion, presented results support the hypothesis that PKC and tyrosine kinases are involved in PRL signaling in adrenocortical cells in pigs. Moreover, activation of PKC is associated with the increased secretion of cortisol and A₄.     

Antibodies to Borna disease virus in infected adult rats: an early appearance of anti-p10 antibody and recognition of novel virus-specific proteins in infected animal brain cells

The time course for appearance of antibodies to Borna disease virus (BDV) major antigens, p40, p24, p18 and p10 were investigated in BDV-inoculated adult rats by Western blotting. Anti-p10 antibodies were detected in sera as early as anti-p40 and -p24 antibodies at four or five weeks after inoculation. Furthermore, in addition to these major antigens of BDV, the rat serum could detect additional 80-, 58-, 43-, 20-, and 16-kDa proteins in BDV-infected cultured cells and/or animal brain cells by Western blot analysis. Of these proteins, the 20- and 16-kDa proteins were shown to be related to p24 protein by their reactivity with anti-p24 monoclonal antibody. Interestingly, the 58- and 24-kDa were found only in BDV-infected animal brain cells but not in cultured cells. The results in this study could provide a useful information on the mechanism for the viral replication and pathogenesis.     

Lack of immunological cross-reactivity of 36-kDa secretory salivary gland antigen of Hyalomma anatolicum anatolicum with Hyalomma dromedarii and Boophilus microplus ticks.

During feeding of ticks of the species Hyalomma anatolicum anatolicum, most of the proteins in salivary gland extracts (SGEs) remained unchanged from the unfed to the fully fed state (from day 1 to day 7 of the experiment), as revealed by SDS-PAGE. However, a 45-kDa protein band disappeared and 26-, 32- and 33-kDa bands appeared when feeding commenced. Some of the protein bands changed their intensity. When probed with anti-H. anatolicum anatolicum hyperimmune sera, transblotted SGE proteins of unfed H. anatolicum anatolicum and Hyalomma dromedarii revealed two common bands of 105 and 80 kDa. A 36-kDa protein band present in H. anatolicum anatolicum SGE could not be detected in H. dromedarii. None of these proteins were detected in partly fed Boophilus microplus when probed with anti-H. anatolicum anatolicum hyperimmune serum. This H. anatolicum anatolicum specific 36-kDa protein was strongly recognized throughout feeding, and thus may be an immunogen of importance for the development of an H. anatolicum anatolicum specific serodiagnostic assay.     

猪缝隙连接蛋白43(GJA1)结构和功能的预测与分析

为了探究猪缝隙连接蛋白43（GJA1）的结构和相关功能，本研究对16个哺乳动物的GJA1蛋白进行多重序列比对及系统发育分析，并通过相关的生物信息学数据库和软件从理化性质、蛋白修饰位点、跨膜区、亚细胞定位、二级结构、三级结构、多态位点、功能区域和互作蛋白等方面对猪GJA1蛋白的结构和功能进行系统分析。系统发育分析结果显示，GJA1蛋白在16种哺乳动物中变异未达到饱和，物种间遗传距离小且同源性高，其中猪与马的GJA1蛋白序列最为接近，同源性达93.3%，在进化树上的分类也证实了这一结果。蛋白结构分析结果显示，猪GJA1蛋白分子质量为43.08 ku，理论等电点为8.88，由20种氨基酸组成，不稳定指数为44.06，平均疏水系数为-0.240，脂溶指数为82.67，不分泌信号肽，有4个跨膜区、6个O-糖基化位点和38个磷酸化位点，最有可能位于细胞质膜和高尔基体内，其二级结构和三级结构主要由α-螺旋和无规则卷曲组成。蛋白功能分析结果显示，在猪GJA1蛋白的CDS序列中有2个超级家族结构功能区域和6个错义突变位点，且猪GJA1蛋白还与ZO-1和Cx36等蛋白及多种蛋白激酶存在相互作用。本研究通过生物信息学分析系统地揭示了猪GJA1蛋白的结构和功能，为后续进一步研究GJA1蛋白在猪体内发挥生物学功能的遗传调控机制提供一定的理论支持。     

The regulation of calcium/calmodulin-dependent protein kinase II during oocyte activation in the rat

Increases in intracellular Ca2+ are required for oocyte activation and subsequent development. Calmodulin-dependent protein kinase II (CaMKII) plays a crucial role in oocyte activation. However, how CaMKII is regulated during this process is not well characterized. We show here for the first time in rat oocytes that CaMKII is phosphorylated during oocyte activation. CaMKII phosphorylation was suppressed by KN93, a CaMKII inhibitor, but not KN92, which is the inactive analogue of KN93. Electrical stimulation of rat oocytes resulted in degradation of both cyclin B and Mos, presumably due a rise in Ca2+ induced by the electrical pulse. KN93 blocked the degradation of both proteins induced by the electrical pulse. Addition of a protein phosphatase inhibitor, okadaic acid (OA), further increased the amount of CaMKII and also increased the amount of phosphorylated enzyme. Importantly, in oocytes undergoing spontaneous activation, accumulation and phosphorylation of CaMKII also occurs in a time-dependent manner. Consistent with this, addition of KN93 inhibited spontaneous activation. Collectively, our results show that CaMKII is phosphorylated during oocyte activation and that this phosphorylation is involved in inactivation of p34cdc2 kinase and somewhat involved in degradation of Mos. Furthermore, CaMKII phosphorylation is negatively regulated by a protein phosphatase.     

Characterization of growth hormone-binding protein in cattle plasma: Prolactin-binding activity and 24-hour profile

The purpose of this study was to characterize circulating growth hormone-binding proteins (GHBP) and prolactin-binding proteins (PRLBP) in cattle blood plasma. In particular, the 24-hr profile of these molecules was investigated. The preincubation of bull plasma with iodinated bovine growth hormone (bGH) or bovine prolactin (bPRL), followed by gel filtration chromatography (Superdex 200; 1.6 × 60 cm column), resulted in the formation of essentially two complexes. The majority of [¹²⁵I]bPRL eluted with the first one (M_r∼600 kDa), whereas [¹²⁵I]bGH mainly appeared in the second one (M_r ∼70 kDa). The fractions corresponding to these two peaks were analyzed by western ligand blotting (WLB), under reducing conditions. WLB revealed, respectively, 190-, 56-, 52-, and 28-kDa bands for the first peak and only 52- and 28-kDa bands for the second one. The nature of the 600-kDa peak is at present undetermined, but the 70-kDa one was previously identified as high-affinity GHBP. Displacement studies demonstrated that bGH and bPRL were both able to bind to this GHBP, because the bGH- and bPRL-binding activities of this protein could be saturated by an excess of either of these two hormones. This was indirectly confirmed by the close correlation (r = 0.615; P = 0.0001; n = 155) observed between plasma bGH- and bPRL-binding activities, because this correlation could suggest that both ligands are bound to the same proteins. The temporal concentrations of plasma GHBP were measured in samples collected at 20-min intervals for 24 hr from 8 young bulls. The evaluation of GHBP was realized by WLB, followed by densitometric analysis. Some fluctuations were observed, but these were not correlated with bGH release, even with a ±2-hr lag period. In summary, we found that bovine high-affinity GBHP binds not only bGH, but also bPRL. A second type of protein, of higher molecular weight, also binds these two hormones, but further investigations are needed to determine its nature. Finally, GHBP concentrations in cattle blood plasma apparently show fluctuations over a 24-hr period, but no correlation was found between these fluctuations and plasma growth hormone concentrations.     

Detection and antigenicity of chlamydial proteins that bind eukaryotic cell membrane proteins.

Chlamydia psittaci proteins capable of binding eukaryotic cell membranes were identified and antigenically characterized. Cell membrane proteins (CMP) of noninfected cells were labeled with biotin (B-CMP), then were extracted with 1% Triton X-100. Nitrocellulose membrane strips containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of chlamydial elementary bodies (EB) were reacted with the B-CMP extract, followed by addition of streptavidin-conjugated horse radish peroxidase. Among the various strains of chlamydiae examined, a protein of approximately 16 to 18 kDa consistently bound B-CMP. A second larger protein, ranging in molecular mass from 24 to 32 kDa, also bound B-CMP. Immunoblotting techniques were used to analyze the reactions of antisera from immunized and experimentally infected animals to these proteins. A rabbit polyclonal antiserum produced against the 18-kDa adhesin of a serovar-1 strain of C psittaci (B577) reacted strongly with 18-kDa proteins of all C psittaci strains, but weakly with that of C trachomatis. Mouse antisera raised against the serovar-2 (FC-Stra) 28-kDa protein reacted only with proteins of the homologous serovar. Sera from experimentally infected animals did not react with the C trachomatis 18-kDa adhesion protein, but did react in 2 patterns with related and nonrelated C psittaci isolates. Two rabbits inoculated with infective serovar-1 EB and 1 rabbit inoculated with a serovar-2 strain reacted specifically with the 18-kDa proteins of their homologous serovars. In contrast, 2 other rabbits inoculated with the same serovar-2 strain produced antisera that reacted with all C psittaci 18-kDa proteins, as did serum from a similarly inoculated bull.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dependence of superoxide anion production on extracellular and intracellular calcium ions and protein kinase C in PMA-stimulated bovine neutrophils.

The involvement of both intracellular and extracellular calcium, as well as the activation of protein kinase C (PKC), in phorbol myristate acetate (PMA)-stimulated respiratory burst in bovine neutrophils has been studied. PMA significantly stimulated the superoxide anion production by these cells. The increased production of superoxide anion was inhibited by BAPTA/AM, an intracellular calcium ([Ca2+]i) chelator, but not affected by EGTA, an extracellular calcium ([Ca2+]0) chelator. PMA also induced PKC activation, and a PKC inhibitor, calphostin C, blocked the stimulatory effect of PMA on superoxide anion production by the neutrophils. Therefore, we conclude that PMA-induced respiratory burst in bovine neutrophils is [Ca2+]i- but not [Ca2+]0-dependent, and also requires PKC activation.     

Indole-3-carbinol prevents H(2)O(2)-induced inhibition of gap junctional intercellular communication by inactivation of PKB/Akt

Indole-3-carbinol (I3C) is a phytochemical found in cruciferous vegetables and possesses a variety of biological and biochemical effects. Despite a wealth of data about the chemopreventive properties of I3C, its effects on gap junctional intercellular communication (GJIC), which is associated with the promotion and progression phases of the multi-stage process of carcinogenesis, has not been studied. In this study, we examined the ability of I3C to prevent H(2)O(2)-induced inhibition of GJIC in WB-F344 rat liver epithelial cells (WB cells). The cells were preincubated with I3C for 48 hr, and then treated with 1 mM H(2)O(2) for 1 hr. We found that I3C could prevent the H(2)O(2)-induced inhibition of GJIC through prevention of the phosphorylated state of gap junction protein connexin 43 (Cx43) phosphorylation. Prevention of GJIC by I3C was dependent upon inactivation of Akt, but not MAPK, although inhibition of GJIC by H(2)O(2) leads to activation of both. Similar to I3C, modulation of Akt activation through the phosphoinositide-3 kinase inhibitor, LY294002, could also prevent H(2)O(2)-induced inhibition of GJIC and phosphorylation of Cx43. Our results suggest that I3C might exert its dietary chemopreventive effects by interfering with the Akt signaling pathway, which appears to be linked to modulating GJIC, a cellular mechanisms regulating cell proliferation, differentiation and apoptosis.     

Effect of incorporation of additives in tris-based egg yolk extender on buffalo (Bubalus bubalis) sperm tyrosine phosphorylation during cryopreservation

Phosphorylation of tyrosine residues on sperm protein is a known indicator of capacitation and a major intracellular signalling event. There is evidence that sperm cryopreservation promotes tyrosine phosphorylation and is associated with reduced fertility of spermatozoa. Under this study, cryoprotective role of different additives namely taurine, trehalose, catalase and 4-bromophenacyl bromide on buffalo sperm quality was evaluated. Buffalo semen was cryopreserved in tris-based egg yolk extender supplemented with additives like taurine (50 mm) or trehalose (100 mm) or 4-bromophenacyl bromide (200 μm) or catalase (100 U/ml) and used for assessment of levels of tyrosine phosphorylation in frozen-thawed spermatozoa. The results obtained were compared with the level of protein tyrosine phosphorylation of semen cryopreserved in tris-based egg yolk extender without additives. Proteins were extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random and analysed for tyrosine phospho-proteins using SDS-PAGE followed by immunoblotting. Monoclonal anti-phosphotyrosine antibody (Clone pT-154) was used as primary antibody followed by treatment with HRP-conjugated secondary antibody. Signals were detected on X-ray film using chemiluminescence. Nine proteins (p20, p30, p32, p38, p49, p56, p59, p72 and p86) were found to be tyrosine phosphorylated in cryopreserved spermatozoa. Supplementation of additives significantly (p<0.05) reduced the level of protein tyrosine phosphorylation in spermatozoa. Moreover, this study showed improved (p<0.05) post-thaw motility, viability and membrane integrity of spermatozoa on addition of these additives. The results obtained clearly indicate reduced level of capacitation like changes on supplementation of additives in terms of protein tyrosine phosphorylation.