Modulation by sphingosine of phosphorylation of substrate proteins by protein kinase C in nuclei from cow mammary gland

Protein kinase C (PKC) is an enzyme activated by diacylglycerols such as 1-oleoyl-2-acetyl-sn-glycerol (OAG), phospholipids (in particular phosphatidylserine; PS) and Ca2+, which regulate a wide variety of intracellular functions by phosphorylating multiple substrate proteins and enzymes. The effect of sphingosine, the backbone moiety of sphingolipids, on PKC activity and phosphorylation of endogenous proteins catalyzed by PKC was investigated in nuclei of cow mammary gland. Sphingosine inhibited nuclear PKC activity when lysine-rich histone was used as the substrate. The sphingosine inhibition of the PKC activity was reversed by the excess addition of PS, but not by OAG or Ca2+. Several nuclear proteins, including 56-kDa, 43-kDa, 38-kDa and 36-kDa proteins, were shown to be substrates for PKC. Of the substrate proteins, the 38-kDa and 36-kDa proteins were identified as annexin I, the Ca2+/phospholipid-binding protein; the 56-kDa and 43-kDa proteins have not yet been identified. Sphingosine inhibited phosphorylation of the 56-kDa protein and the 36-kDa annexin I, whereas it enhanced that of the 43-kDa protein. The 38-kDa annexin I species was unaffected by sphingosine. As with the PKC activity, inhibition by sphingosine of phosphorylation of the 56-kDa protein and 36-kDa annexin I was reversed by the excess addition of PS, but not by OAG or Ca2+. In addition, by the excess addition of PS and not by OAG or Ca2+, the sphingosine-enhanced phosphorylation of the 43-kDa protein was reversed and returned to near the level in the absence of sphingosine. It is suggested that sphingosine is involved in the regulation of PKC-dependent phosphorylation in the nucleus by modulating the association of PKC or its substrates, particularly annexin I, 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.     

Signal transduction systems mediated by protein kinase-C and estradiol receptors in cow placenta and caruncle.

Characteristics of signal transduction systems mediated by protein kinase-C (Ca2+/phospholipid-dependent protein kinase) and estradiol receptors in cow placenta and caruncle were conducted by evaluating protein kinase-C activity and estradiol receptor concentrations and by exploring substrate proteins for the enzyme. The enzyme activity was detected in cytosolic and total particulate fractions of both tissues. The activity levels in these fractions were comparable to those in other cow tissues such as liver and mammary gland. The enzyme activity was inhibited by palmitoylcarnitine, gossypol and adriamycin, known phospholipid-interacting inhibitors of the enzyme. Phosphorylation by the enzyme and subsequent autoradiography revealed that only 125K protein in placental cytosol and two low molecular weight proteins in caruncular cytosol were found to be substrates for protein kinase-C. Ca2+ acts as inhibitor of the phosphorylation of several phosphoproteins other than the substrates. Estradiol receptor concentrations in cytosolic and nuclear fractions were similar in both tissues, and the cytosolic concentrations were also comparable to those in pregnant uterus. However, the nuclear concentrations were extremely low when compared to those in the uterus. The signal transduction systems mediated by protein kinase-C and by estradiol receptors seem to be concertedly suppressed in cow placenta and caruncle.     

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     

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.     

蛋氨酸对奶牛乳腺上皮细胞亚细胞蛋白质组的影响研究

为寻找奶牛乳腺上皮细胞亚细胞结构中与泌乳相关的重要蛋白质、从蛋白质水平揭示乳蛋白合成的调控机制,应用双向凝胶电泳技术分离体外培养的蛋氨酸处理组与正常组奶牛乳腺上皮细胞蛋白质,利用Image Maser 2D软件对图谱进行对比分析,基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)和数据库检索鉴定,并采用实时荧光定量PCR技术在mRNA水平上验证2-DE结果。质谱鉴定出8个表达上调的差异蛋白质,大多数差异蛋白质的功能涉及细胞骨架构成、能量代谢等过程。这些差异点的发现为研究奶牛泌乳机理提供了有益的线索。     

Parathyroid hormone-related protein (PTHrP) and Ca levels in the milk of lactating cows

To investigate the roles of mammary PTHrP in calcium uptake and/or release in the mammary gland of cows, plasma PTHrP and Ca levels, and their arterial-venous differences were examined in a Jersey cow during the periparturient period. Levels of Ca in both abdominal aorta and abdominal subcutaneous vein blood slightly decreased around the parturition and at 24 days after the parturition, however, no remarkable arterial-venous differences were observed. Plasma PTHrP levels in both arterial and venous samples were below the detection limit (0.57 pmol/l) during the experimental period. Milk PTHrP and Ca levels were measured in 9 Holstein dairy cows. Although plasma PTHrP levels in all arterial and venous samples were also below the detection limit, milk PTHrP and Ca levels were remarkably high, ranging from 14,900 pmol/l to 41,200 pmol/l and from 772 mg/l to 1,200 mg/l, respectively. In addition, a significant positive correlation (P<0.01) was observed between milk PTHrP and Ca levels. These results suggested that mammary PTHrP is closely related to Ca concentration in the milk.     

Effects of antimicrobials and weaning on porcine serum insulin-like growth factor binding protein levels

The effects of subtherapeutic antimicrobial supplementation and weaning on serum levels of IGF-I and insulin-like growth factor binding proteins (IGFBP)-2, -3 and -4 were determined in crossbred weanling pigs. At weaning, pigs were allotted to a diet containing 21.8% crude protein and 1.15% lysine with or without Aureozol (110 mg/kg of Aureomycin chlortetracycline, 110 mg/kg of sulfathiazole, and 55 mg/kg of penicillin) for 4 wk. Insulin-like growth factor-binding proteins and IGF-I analyses were performed on blood samples that were drawn weekly. Weaning decreased serum IGFBP-3 levels in both control and Aureozol-treated groups on d 6 and d 14 (P < 0.05) relative to preweaning levels. The IGFBP-3 values returned to preweaning levels by d 21. Although the circulating levels of both the 43-kDa and the 39-kDa glycosylation variants of IGFBP-3 were affected by weaning, the level of the 39-kDa IGFBP-3 was affected relatively more than that of the 43-kDa IGFBP-3 (P < 0.05). Compared with circulating IGFBP-3 levels in control pigs, Aureozol-treated pigs had higher circulating IGFBP-3 levels on d 21 (43%, P < 0.05) and d 27 (46%, P < 0.05). In direct contrast to the effect of weaning on serum IGFBP-3 level, serum IGFBP-2 levels increased on d 6 and d 14 after weaning (P < 0.05) and decreased to preweaning levels by d 21. The IGFBP-2 levels continued to decline and were less than preweaning levels by d 27 (P < 0.05). Aureozol treatment had no effect on serum IGFBP-2 levels at any time. Serum levels of nonglycosylated IGFBP-4 were not affected by either weaning or Aureozol supplementation. Weaning decreased circulating IGF-I concentration on d 6 in both control and Aureozol-treated pigs (76 and 73%, respectively, P < 0.05) and on d 14 (62%, P < 0.05) and d 21 (32%, P < 0.05) in control pigs. Aureozol-supplemented pigs had higher serum IGF-I concentrations than control pigs on d 14 (82%, P < 0.05), d 21 (55%, P < 0.05), and d 27 (36%, P < 0.05). The Aureozol-fed pigs had a 14.2% increase in BW gain (P < 0.05) and a 59.6% increase in ADG (P < 0.05) compared with pigs fed the control diet. Both Aureozol-supplementation and weaning cause changes in serum IGFBP levels and IGF-I concentrations that might be involved in regulating rate and efficiency of growth.     

Expression of parathyroid hormone-related protein (PTHrP) mRNA in mammary gland of periparturient cows

The expression of parathyroid hormone-related protein (PTHrP) mRNA was examined in mammary gland with or without lactation, and during periparturient period in a Holstein cow and a Jersey cow. In the lactating mammary gland, PTHrP was detected in alveolar epithelial cells and the lumen by immunohistochemical analysis. The relative expression levels of PTHrP mRNA in mammary gland from lactating cows were significantly higher than those from non-lactating cows (P<0.05). During periparturient period, relative PTHrP mRNA level was remarkably low before the parturition in a Jersey and a Holstein cow, however, both levels were gradually increased and reached a peak level at 5-6 weeks after the parturition. In addition, the peak level in a Jersey cow was approximately 3-fold higher than that in a Holstein cow. From these results, PTHrP was synthesized and secreted in alveolar epithelial cells in mammary gland and increased subsequently with the lactation, suggesting a possible mechanism for the regulation of local calcium homeostasis.     

IGF-1 stimulates protein synthesis by enhanced signaling through mTORC1 in bovine mammary epithelial cells

Using the MAC-T cell line as a model, the effects of insulin-like growth factor (IGF)-1 on the regulation of protein synthesis through the mammalian target of rapamycin complex 1 (mTORC1) signaling in bovine mammary epithelial cells were evaluated. Global rates of protein synthesis increased by 47% within 30 min of IGF-1 treatment. The effect of IGF-1 on protein synthesis was associated with enhanced association of the eukaryotic initiation factor (eIF) 4E with eIF4G and a concomitant reduction of eIF4E association with eIF4E-binding protein-1 (4E-BP1). There was a progressive increase in the phosphorylation state of ribosomal protein S6 kinase-1, a downstream target of mTORC1 in response to IGF-1. In addition, IGF-1 stimulated mTORC1 kinase activity toward 4E-BP1 in vitro. Phosphorylation on Ser473 of Akt was induced by IGF-1 within 5 min and remained elevated throughout a 30-min time course. The effect of IGF-1 on Akt phosphorylation was also concentration dependent. Activation of Akt by IGF-1 led to increased phosphorylation of tuberous sclerosis complex 2 on Thr1426, without any change in its association with tuberous sclerosis complex 1. Phosphorylation of proline-rich Akt substrate of 40-kDa (PRAS40) at Thr246 was stimulated by IGF-1. The amount of PRAS40 associated with mTORC1 decreased in response to IGF-1, and PRAS40 binding to mTORC1 was inversely related to its phosphorylation level. Overall, these results suggest that activation of the PI3K-Akt pathway by IGF-1 stimulated global protein synthesis in bovine mammary epithelial cells through changes in the phosphorylation and association state of components of the mTORC1 signaling pathway.     

植物中钙依赖蛋白激酶(CDPK)的研究进展

Ca²⁺是植物细胞信号转导中重要的第二信使,在植株受到外界逆境胁迫时,细胞中的钙信号经钙传感蛋白(CaMs、CaMLs、CBLs和 CDPKs)传递到下游组分引起相关基因的表达,从而响应环境的各种变化。钙依赖性蛋白激酶(CDPK)是Ca²⁺传感器,可以将细胞内Ca²⁺信号传递到可以与14-3-3蛋白相互作用的靶蛋白从而刺激靶蛋白发生磷酸化。CDPK在植物生长的诸多方面发挥关键作用,如花粉管的伸长、植物的生长发育以及对生物胁迫、非生物胁迫的应激反应。大多数CDPK具有明显的亚细胞分布,使它们能够“感受”局部Ca²⁺浓度的变化并与其靶细胞发生特异性作用。14-3-3蛋白是一类通过磷酸化激活的真核蛋白,它可以与不同靶蛋白相互作用,调控植物重要的生理生化过程。近年来研究发现,14-3-3蛋白作为CDPK的靶蛋白和CDPK交叉磷酸化形成CDPK/14-3-3复合物,进一步调节植物初级代谢、开花和激素合成。将从CDPK的结构、亚细胞定位、靶蛋白、生物学功能,特别是CDPK与14-3-3之间的交叉调节及其在植物信号通路中的协同作用方面进行全面的综述,旨在为未来CDPK研究提供参考和新的方向。     

Experience with the use of Chronicin foam and Masticort PS in the nonselective treatment of milk cows during the dry period

The effect of the non-selective treatment of dairy cows with antibiotics (Chronicin foam--Galena, and Masticort PS--Poland) and the effect of teat disinfection (Jodonal A--Lachema) at the last milking in lactation was studied as exerted on the reduction in the occurrence of infection processes in mammary gland after calving. The trials were performed with 180 cows in three groups of 60 animals. At the last milking the mammary secretion of each cow was sampled for bacteriological examination. Two cow groups were treated at the same time. The occurrence of infection processes in the mammary gland in the period after calving can be characterized as follows: a) in the period after calving the control group had an increased proportion of cows with an infection process in their udders (from 58.3% at the end of lactation to 68.3% in the period of 14 days following calving); b) in the group treated with Chronicin foam the infection was reduced by 3/5, i. e. from 71.7% of cows infected at the start of drying off to 28.3% with infection process after calving; c) in the group treated with Masticort PS the infection after calving was reduced by 3/4 as compared with the infection at the onset of drying off, i. e. from 80% to 20%. In the control group 20% of the cows recovered spontaneously. The preparation Chronicin foam showed 79.1% therapeutic effectiveness. Therapy with Masticort PS was successful in 81.3% of the animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of enrofloxacin with breast cancer resistance protein (BCRP/ABCG2): influence of flavonoids and role in milk secretion in sheep

The ATP-binding cassette (ABC) transporter breast cancer resistance protein (BCRP)/ABCG2 is a high-capacity efflux transporter with wide substrate specificity located in apical membranes of epithelia, which is involved in drug availability. BCRP is responsible for the active secretion of clinically and toxicologically important substrates to milk. The present study shows BCRP expression in sheep and cow by immunoblotting with MAb (BXP-53). Vanadate-sensitive ATPase activity with specific BCRP substrates and inhibitors was measured in bovine mammary gland homogenates. To assess the role of BCRP in ruminant mammary gland we tested the fluoroquinolone enrofloxacin (ENRO). In polarized cell lines, ENRO was transported by Bcrp1/BCRP with secretory/absorptive ratios of 6.5 and 2 respectively. The efflux was blocked by the BCRP inhibitor Ko143. ENRO pharmacokinetics in plasma and milk was studied in sheep after co-administration of drug (2.5 mg/kg, i.v.) and genistein (0.8 mg/kg, i.m.) or albendazole sulfoxide (2 mg/kg, i.v) as BCRP inhibitors. Concomitant administration of BCRP inhibitors with ENRO had no significant effect on the plasma disposition kinetics of ENRO but decreased ENRO concentrations in milk.     

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.     

王不留行增乳活性单体对奶牛乳腺上皮细胞增殖及泌乳性能的影响

应用CASY细胞分析仪及HPLC分别检测王不留行邻苯二甲酸二丁酯对乳腺上皮细胞增殖、细胞活力及乳腺上皮细胞分泌β-酪蛋白、乳糖的影响,研究王不留行增乳活性单体邻苯二甲酸二丁酯对奶牛泌乳中期乳腺上皮细胞增殖和泌乳性能的影响。试验结果表明,王不留行增乳活性单体成份邻苯二甲酸二丁酯对奶牛乳腺上皮细胞的增殖和细胞活力提高均显著(P0.05);能显著提高乳腺上皮细胞β-酪蛋白的表达(P0.05),也能提高乳糖的分泌(P0.05,P0.05)。因此,王不留行增乳活性单体邻苯二甲酸二丁酯可显著提高乳腺上皮细胞的增殖和泌乳能力。     

'In vitro' capacitation and further 'in vitro' progesterone-induced acrosome exocytosis are linked to specific changes in the expression and acrosome location of protein phosphorylation in serine residues of boar spermatozoa

The aim of this study is to determine changes in the expression and location of protein serine phosphorylation (pSer) during 'in vitro' capacitation (IVC) and 'in vitro' acrosome exocytosis (IVAE) in boar spermatozoa. This was performed in both mono- and bi-dimensional analyses of protein expression through Western blot, as well as through immunocytochemistry. Furthermore, IVC was induced through incubation in an IVC medium, and afterwards, progesterone-induced IVAE was performed. The mono-dimensional Western blot analysis showed the presence of a predominant pSer band of approximately 70-75 kDa, which was accompanied by fainter bands, especially three with molecular weights of approximately 50, 35 and 32 kDa. Neither IVC nor IVAE significantly modified this pattern. Bi-dimensional analyses showed a more complex pattern, with at least five protein clusters. The attainment of IVC caused the disappearance of the proteins with the highest molecular weight concomitantly with the appearance of pSer proteins of 75-kDa/pI 9.5 and 80-kDa/pI 10. The induction of IVAE caused the appearance of new pSer proteins of a 75-kDa/pI 6.5-7.5 and 75-kDa/pI 10. Immunocytochemistry showed that the main pSer expression in boar expression before the attainment of IVC was located at the midpiece. The IVC induced the appearance of acrosomal pSer, which was greatly increased during IVAE. Our results indicate that the changes in serine protein phosphorylation associated with IVC and IVAE comprise not only the appearance of specific phosphorylated proteins, such as the pSer-75 kDa, but also changes in pI and displacements in the sperm location of phosphorylated proteins, like the specific acrosomal pSer signal induced during IVC.     

Expression of MAP kinases and connexins in the differentiation of rat mammary epithelial cells

Gap junctional intercellular communication (GJIC) is involved in the regulation of many cellular processes. MAP kinases are known to affect GJIC and phosphorylation of connexin (Cx). MAP kinases can also be a regulator of cell proliferation and growth. This study was undertaken to show the relevance between expression patterns of Cxs and MAP kinases in rat mammary epithelial cells (RMECs). In order to characterize the RMECs, they were stained with Peanut lectin, which indicates most alveolar epithelial cells, and Thy-1.1 was used as a marker of luminal epithelial cells or myoepithelial cells, respectively. We studied the expression patterns of major gap junction proteins, Cx26, 32, and 43 in RMECs. Western blot analysis demonstrated that Cx26 gradually decreased from day 2, while Cx32 was expressed constantly from day 1 to 14. Cx43 dramatically increased on day 5 and decreased thereafter. The expression patterns and phosphorylation of ERK1/2 and JNK were similar to Cx43, but expression of p38 was like that of Cx32. These results showed that the MAP kinases that comprise ERK1/2, p38, and JNK were involved in regulation of Cxs. Our data suggests that GJIC plays an important role during rat mammary differentiation and that MAP kinases may be closely related functionally to regulate the gap junction.     

健康奶牛与临床型乳腺炎奶牛乳腺核蛋白组差异表达分析

亚细胞蛋白质组的优点在于对低丰度蛋白的研究,运用亚细胞蛋白质组学的研究策略,可以提高低丰度蛋白质在双向电泳中的检出数量.通过分析奶牛乳腺炎乳腺与正常乳腺核蛋白组差异表达情况,为奶牛乳腺炎发病机理研究寻找尽可能多的生物标记分子.经超速离心法分离细胞核,双向凝胶电泳分离蛋白,用PDQuest7.4软件分析寻找差异蛋白质斑点,高效液相色谱串联离子阱质谱鉴定蛋白质.从核蛋白组2-DE图谱中筛选出22个差异表达的蛋白质斑点,质谱鉴定出17个差异表达的蛋白质,2个蛋白质在乳腺炎发病过程中下调,7个上调,5个只在正常情况下表达,3个只表达在乳腺炎组织中,筛选出的差异表达蛋白质涉及细胞骨架构成、代谢调节及凋亡调控等许多方面.表明奶牛乳腺炎发生时乳腺组织核结构和代谢状态都发生了的变化.