高糖对体外培养Schwann细胞生长及细胞外信号调节激酶1／2磷酸化的影响

目的:探讨高糖对体外培养Schwann细胞生长及细胞外信号调节激酶(ERK)磷酸化的影响.方法:按照培养液中葡萄糖浓度的小同,分为对照组与高糖组.用MTT法检测Schwann细胞生长情况;用ELISA法检测对照组与高糖组ERK1/2磷酸化的程度,以及加入神经源性一氧化氮合成酶(nNOS)抑制剂后ERK1/2磷酸化的程度.结果:高糖浓度下,细胞虽有增殖但幅度及时程明显低于对照组,高糖抑制Schwann细胞生长;随着精浓度的升高.ERK1/2磷酸化的程度逐渐增加,并与加入nNOS抑制剂有相似的表现.结论:高糖抑制Schwann细胞生长,并且降低nNOS的量,减弱一氧化氮(NO)对ERK1/2的抑制作用,导致ERK1/2磷酸化水平升高.     

Glycosaminoglycans and proteoglycans in the skin of aneuploid fetuses with increased nuchal translucency

BACKGROUND: First trimester increased fetal nuchal translucency is associated with fetal aneuploidies. One of the mechanisms of pathophysiology could be an abnormal extracellular matrix facilitating the formation of an interstitial edema. A previous study investigating interstitial edema in first trimester fetuses found large amounts of hyaluronan in the skin of fetuses with trisomy 21. The aim of this study was to establish distribution patterns for a number of other glycosaminoglycans-dermatan, heparan and keratan sulphate, chondroitin-6-sulphate and chondroitin-4-sulphate proteoglycan-in the nuchal skin of normal and chromosomally abnormal fetuses at 11-14 weeks. We also investigated whether biglycan (BGN), which is located on chromosome X, is underexpressed in fetuses with Turner syndrome. Decorin (DCN), a similar-sized proteoglycan located on chromosome 12, was taken as a control. METHODS: We studied the distribution and concentration of various extacellular matrix components using immunohistochemistry, a double staining technique, in-situ hybridization, Northern and Western blot analysis. RESULTS: Chondroitin-6-sulphate and chondroitin-4-sulphate proteoglycan were increased in Turner syndrome fetuses and BGN seemed to be underexpressed compared with normal controls, while DCN was not. Dermatan, heparan and keratan sulphate showed no significant abnormal distribution in trisomies 21, 18, 13, or in Turner syndrome, compared with normal. Western and immunohistochemical analysis revealed that absence of a second X chromosome, as is the case in Turner syndrome, affects BGN protein pattern. CONCLUSIONS: An abnormal amount of glycosaminoglycans and proteoglycans presumably contributes to increased nuchal translucency.     

Significance of anti-nuclear and anti-extracellular matrix autoantibodies for albuminuria in murine lupus nephritis; a longitudinal study on plasma and glomerular eluates in MRL/l mice

The relationship between autoantibody reactivities and nephritis in systemic lupus erythematosus (SLE) is unclear. We studied MRL/l mice which developed a considerable albuminuria (either mice with short (< 1 week) or heavy and prolonged (3 weeks) albuminuria) and compared them with non-albuminuric age-matched controls, with young (12 weeks old) non-albuminuric mice and with mice which were followed for 36 weeks and did not develop albuminuria. In a longitudinal prospective study on plasma samples we correlated a variety of anti-nuclear reactivities and reactivities against extracellular matrix (ECM) components, with the onset of albuminuria. We found that at the onset of albuminuria, anti-DNA was higher while anti-nucleosome and anti-H2A/H2B-DNA subnucleosome reactivities were lower compared with age-matched non-albuminuric mice. We also studied glomerular eluates of these mice in ELISA and in indirect immunofluorescence (IF). In the eluates we found with IF that anti-glomerular basement membrane (GBM)-tubular basement membrane (TBM) antibodies were already present in 12-week-old non-albuminuric mice. These eluates showed no anti-nuclear antibodies. In eluates of albuminuric mice more immunoglobulin was deposited, and anti-ECM, anti-DNA and anti-nucleosome reactivities were higher than in eluates of age-matched non-albuminuric mice. The deposition of anti-nucleosome antibodies preceded the deposition of anti-DNA antibodies since they were deposited to a greater extent in mice with a short albuminuria. We conclude that anti-GBM-TBM antibodies are the first autoantibodies that deposit in glomeruli of MRL/l mice at an early age. The onset of albuminuria is associated with additional deposition of both anti-ECM and anti-nuclear (anti-nucleosome and anti-DNA) antibodies, but the difference with non-albuminuric mice seems to be more quantitative than qualitative.     

Matrix metalloproteinases (MMP), EMMPRIN (extracellular matrix metalloproteinase inducer) and mitogen-activated protein kinases (MAPK): Co-expression in metastatic serous ovarian carcinoma

Activation or suppression of intracellular signaling via the mitogen-activated protein kinase (MAPK) family has been linked to expression of matrix metalloproteinases (MMP) in experimental models, but this association has not been demonstrated in clinical material. The objective of this study was to investigate the possible association between expression and activity of MMP, expression of the MMP inducer EMMPRIN, and the expression (level) and phosphorylation status (activity) of the extracellular-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK) and high osmolarity glycerol response kinase (p38) in effusions from patients diagnosed with serous ovarian carcinoma. MAPK level and activity were studied in 55 effusions using immunoblotting. MMP-1, MMP-2, MMP-9 and EMMPRIN expression was studied using immunocytochemistry (ICC) and mRNA in situ hybridization (ISH). The gelatinolytic activity of MMP-2 and MMP-9 was measured by zymography. ERK and phospho-ERK (p-ERK) were detected in 54/55 (98%) and 50/55 (91%) specimens, respectively. JNK and p-JNK were detected in 53/55 (96%) and 38/55 (69%) specimens, respectively. p38 was expressed in 54/55 (98%) specimens, and its phosphorylated form was found in 51/55 (92%). MMP-2 mRNA expression (P=0.048), protein expression (P=0.046) and gelatinolytic activity (P=0.039) correlated with ERK phosphorylative activity. MMP-2 activity also correlated with p38 activity (P=0.017). MMP-9 protein expression correlated with phosphorylation of p38 (P=0.046), but enzyme activity showed inverse relationship with both p-ERK (P=0.05) and p-p38 (P=0.033) expression. EMMPRIN expression correlated with MMP-1 (P<0.001), MMP-2 (P=0.042) and MMP-9 (P=0.029) expression, as well as with ERK activity (P=0.001). Our results present the first evidence of a possible link between MAPK signaling and MMP expression and activity in vivo. These data may expand our understanding regarding the mechanisms by which MMP synthesis is regulated in effusions and possibly affect treatment strategies for this form of malignancy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of integrin chains in normal and neoplastic human pancreas.

Integrins are a complex family of non-covalently linked heterodimeric glycoproteins which function as cell adhesion molecules, interacting with extracellular matrix molecules such as laminin, fibronectin, vitronectin, and collagen, and also having a role in intercellular adhesion. Each integrin subfamily is characterized by a common beta chain associated with variable alpha chains. We have examined, using immunohistological methods, the expression of the VLA (very late activation) family comprising beta 1 in association with alpha 1-6, and also alpha 6 in association with beta 4, the LFA beta chain beta 2, and the vitronectin receptor, in association with beta 1 or beta 5 and as the complex alpha v beta 3. Cryostat sections of normal pancreas, pancreatic adenocarcinomas, and ampullary tumours were studied together with six pancreatic carcinoma cell lines. Normal pancreas showed expression of beta 1 in all parenchyma. alpha 2 and alpha 6 had a similar distribution whereas alpha 3 expression was confined to ducts, including the very smallest radicles. Staining along the basement membranes of ducts was seen with beta 4 and the anti-vitronectin alpha v chain receptor antibody 13C2. Islet cells failed to stain with any antibody. No staining of epithelial components was seen with antibodies to alpha 1, alpha 4, alpha 5, or to the alpha v beta 3 form of the vitronectin receptor (beta 3 and alpha v beta 3 using the antibody 23C6). Pancreatic adenocarcinomas and ampullary tumours showed expression of alpha 2, alpha 3, alpha 6, beta 1, beta 4, and the vitronectin receptor (alpha v associated with beta 1 or beta 5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of radioprotectors on cyclic AMP-dependent protein phosphorylation

The ability of radioprotectors (serotonin, aminoethylisothiouronium) in radioprotective doses to stimulate cyclic AMP-dependent phosphorylation of mouse liver cytosol and nuclear and spleen cytosol proteins in vivo was demonstrated. In experiments in vitro, the radioprotectors had no direct action on protein kinase activity or its stimulation by cyclic AMP. It is postulated on the basis of these results and those of previous investigations that activation of cyclic AMP-dependent phosphorylation is due to an increase in the intracellular cyclic AMP concentration under the influence of the radioprotectors.Laboratory of Radiation Biophysics, Department of Biophysics, Biological Faculty, Moscow State University. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 87, No. 3, pp. 230–232, March, 1979.     

Cellular characterization of blastocysts derived from rabbit 4-, 8- and 16-cell embryos and isolated blastomeres cultured in vitro

The purpose of this study was to investigate the developmental potential of isolated rabbit blastomeres under various culture conditions to gain insight into their ability to form the two cell lineages of a viable blastocyst. Intact embryos at the 4-cell, 8-cell, 16-cell stages and blastomeres isolated from 4-, 8- and 16-cell rabbit embryos (1/4, 1/8 or 1/16 blastomeres respectively) were cultured in drops of one of three different media, each supplemented with either fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA). The effects of the extracellular matrix fibronectin (FN) on the development of isolated rabbit blastomeres were also investigated. Supplementation of the medium with FCS yielded a higher (P < 0.05) proportion of blastocysts than BSA or PVA, predominantly from 1/4 blastomeres. No major differences were found between the three basic culture media. In 1/4, 1/8 or 1/16 blastomeres, blastocyst formation rates were greater (P < 0.05) in groups cultured in matrix-free (54.5, 59.6 and 54.6% respectively) than in FN-coated groups (35.4, 46.0 and 26.1% respectively). Only in blastocysts derived from 1/4 blastomeres, were the numbers of inner cell mass (ICM) and total cells of blastocysts higher (P < 0.05) in FN-coated groups than in matrix-free groups (12.7 +/- 1.1 versus 8.5 +/- 0.7 ICM, 73.8 +/- 3. 7 versus 57.8 +/- 3.3 total cells). The percentage of blastocysts derived from single blastomeres with ICM cells decreased with increasing cell stage of the parent embryos in FN-coated (93.6, 78.3 and 44.0%, respectively) as well as matrix-free groups (96.2, 69.3 and 55.2%). In FN-coated groups, after 96 h (1/4) or 72 h (1/8 and 1/16) of culture, approximately 20-30% of blastomeres did not develop into normal blastocysts but formed sheets with 30-50 cells attached to the bottom of the dishes. These results indicate that the development of rabbit blastomeres shares important characteristics with those from mouse and domestic species and may thus aid in developing an efficient culture system for blastomeres, derived from human embryos.     

不同年龄高血压大鼠血管平滑肌中ERK和MKP-1的表达

目的：研究不同年龄的自发性高血压大鼠（SHR）和Wistar Kyoto大鼠（WKY）主动脉平滑肌中丝裂原活化蛋白激酶（MAPK）及其磷酸酶（MKP-1）的表达及其与高血压的关系。 方法： 用tail-cuff测量大鼠尾动脉血压；分别用Western blotting法和RT-PCR法半定量测定血管平滑肌中磷酸化细胞外信号调节激酶（p-ERK）和MKP-1的蛋白表达以及MKP-1 mRNA的含量。 结果： （1）SHR的血压自8周龄起明显高于WKY（P<0.01），且随年龄增长而升高（P<0.05）至14周以后趋于稳定；（2）SHR主动脉平滑肌中的p-ERK表达明显高于同年龄的WKY（P<0.01），随年龄增长而递增（P<0.05），与血压呈正相关；（3）SHR主动脉平滑肌中MKP-1蛋白明显高于同龄WKY，而mRNA的表达在5周龄时明显高于WKY，之后均随年龄的增长而递减（P<0.05），与血压和ERK呈负相关，而WKY下降不明显。 结论： MKP-1在高血压的发生和发展过程中起重要作用，其表达逐渐下降可能是导致ERK激活增加，从而导致血管平滑肌细胞增殖、血压升高的重要原因。     

Role of mitogen-activated protein kinases in influenza virus induction of prostaglandin E2 from arachidonic acid in bronchial epithelial cells

BACKGROUND: Influenza virus (IV) infection causes airway inflammation; however, it has not been determined whether IV infection could catabolize arachidonic acid cascade in airway epithelial cells. In addition, the responsible intracellular signalling molecules that catabolize arachidonic acid cascade have not been determined. OBJECTIVE: In the present study, to clarify these issues, we examined the cyclooxygenase (COX) expression, cytosolic phospholipase A2 (cPLA2) phosphorylation and prostaglandin E2 (PGE2) release in human bronchial epithelial cells (BEC) upon IV infection, and the role of mitogen-activated protein kinase (MAPK) including extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun-NH2-terminal kinase (JNK) in catabolizing arachidonic acid cascade in BEC. METHODS: COX-2 expression, phosphorylation of cPLA2 and phosphorylation of ERK, JNK and p38 MAPK were determined by Western blot. The concentrations of PGE2 were determined by ELISA. PD 98059 as a specific inhibitor of MAPK kinase-1 (MEK-1), an up-stream kinase of ERK, SB 203580 as a specific inhibitor of p38 MAPK and CEP-11004 as a specific inhibitor of JNK cascade were used to investigate the role of ERK, p38 MAPK and JNK in catabolizing arachidonic acid cascade in BEC. RESULTS: The results showed that (1) IV infection increases COX-2 expression, cPLA2 phosphorylation and PGE2 release, (2) ERK, p38 MAPK and JNK were phosphorylated, (3) CEP-11004 and PD 98059 predominantly attenuated COX-2 expression and cPLA2 phosphorylation, respectively, (4) SB 203580 did not remarkably affect COX-2 expression and cPLA2 phosphorylation, and (5) each inhibitor dose-dependently attenuated PGE2 release by various extents. CONCLUSION: These results indicate that IV infection activates three distinct MAPKs, ERK, p38 MAPK and JNK, to participate to various extents in the induction of PGE2 synthesis from arachidonic acid in BEC.     

肺组织β-AR和GRK2与重症急性胰腺炎肺损伤的关系以及甲强龙的影响

目的：探讨重症急性胰腺炎（SAP）致急性肺损伤（ALI）大鼠肺组织β-AR和G蛋白偶联受体激酶2（GRK2）的变化规律以及甲强龙的影响。方法：SD大鼠36只，随机分为3组，每组12只。对照组，仅做十二指肠翻动；模型组，胰胆管逆行注射5%牛磺胆酸钠（1 mL/kg），建立SAP模型；干预组，建立SAP模型后1 h给予甲强龙（30 mg/kg）。于6 h和12 h分别处死大鼠6只取肺组织，放射配基结合实验测量肺组织β-AR最大结合容量B_max和平衡解离常数K_d，免疫荧光法检测肺组织GRK2表达。结果：模型组和干预组胰腺组织损伤评分显著高于对照组，模型制备成功；模型组和干预组肺组织损伤评分显著高于对照组，发生SAP肺损伤；模型组β-AR B_max显著低于对照组和干预组，K_d显著高于对照组和干预组；模型组GRK2的表达显著高于对照组和干预组。结论：SD大鼠肺组织有丰富的GRK2表达，SAP肺损伤大鼠肺组织GRK2表达显著增加，这可能是β-AR下调的重要机制。