Enamel matrix serine proteinase 1: stage-specific expression and molecular modeling

Enamel proteins are cleaved by proteinases soon after their secretion by ameloblasts. Intact proteins concentrate in the outer enamel at or near the growing tips of the enamel crystallites while cleavage products accumulate in the deeper enamel. In the transition and early maturation stages there is a dramatic increase in proteolytic activity. This activity, coupled with the diminished secretory and increased reabsorptive functions of ameloblasts, leads to a precipitous fall in the amount of enamel protein in the matrix. Recently we have cloned and characterized an mRNA encoding a tooth-specific serine proteinase designated enamel matrix serine proteinase 1 (EMSP1) [Simmer et al., JDR (1998) 77: 377]. EMSP1 can be detected in the inner enamel during the secretory stage and its activity increases sharply during the transition stage. Stage-specific Northern blot analysis demonstrates this increase is accompanied by a parallel increase in the amount EMSP1 mRNA. A 3-dimensional computer model of EMSP1, based upon the crystal structure of bovine trypsin, has been generated and analyzed. All six disulfide bridges as well as the active site are conserved. Changes in the peptide binding region and the specificity pocket suggest that interaction of the proteinase with protein substrates is altered, potentially causing a shift in substrate specificity. The calcium binding region of trypsin is thoroughly modified suggesting that the calcium independence of EMSP1 activity is due to an inability to bind calcium. The three potential N-linked glycosylation sites, N104, N139 and N184, are in surface accessible positions away from the active site.     

基质金属蛋白酶与食管癌相关性的研究进展

MMPs(Matrix metallo proteinase,MMPs)是一类依赖金属离子锌(Zn2+)并以细胞外基(Extracellular matrix,ECM)作为水解底物的蛋白水解酶。MMPs能降解ECM中的各种蛋白成分,在肿瘤侵袭转移中起关键性作用,本文就近年来的基质金属蛋白酶与食管癌的相关性研究进展进行综述。     

脓毒症大鼠心脏基质金属蛋白酶及其抑制物基因的表达变化

目的：探讨脓毒症大鼠心脏中基质金属蛋白酶（MMP）及其组织抑制物（TIMP）的基因表达谱变化及其与脓毒症导致的心脏损伤的关系。方法：雄性3月龄Wistar大鼠20只，随机均分为2组，分别以盲肠结扎针刺法建立脓毒症动物模型或行假手术作为对照组。术后24 h快速摘取心脏，以Langendorff 离体鼠心灌注法测量大鼠心功能参数，其后做心脏病理检查，并以寡核苷酸基因芯片检测基质金属蛋白酶及其抑制物基因在2组大鼠心脏组织中的表达差异。结果：脓毒症大鼠心脏无明显病理改变，但心功能明显下降，表明大鼠心脏处于抑制状态；基因芯片结果显示：20个MMP基因中14个表达上调3倍以上，包括胶原酶MMP8、明胶酶（MMP2和MMP9）、基质溶解素（MMP3、MMP7及MMP10）和膜型金属蛋白酶（MMP15、MMP17和MMP24）等；4个TIMP基因中仅TIMP3基因表达上调。结论：脓毒症时心脏组织中多种MMP基因表达上调、MMP/TIMP基因表达失衡，可能导致心肌抑制和ECM重构，是脓毒症诱发心脏损伤的重要分子机制。     

Heart failure alters matrix metalloproteinase gene expression and activity in rat skeletal muscle

Heart failure is associated with a skeletal muscle myopathy with cellular and extracellular alterations. The hypothesis of this investigation is that extracellular changes may be associated with enhanced mRNA expression and activity of matrix metalloproteinases (MMP). We examined MMP mRNA expression and MMP activity in Soleus (SOL), extensor digitorum longus (EDL), and diaphragm (DIA) muscles of young Wistar rat with monocrotaline-induced heart failure. Rats injected with saline served as age-matched controls. MMP2 and MMP9 mRNA contents were determined by RT-PCR and MMP activity by electrophoresis in gelatin-containing polyacrylamide gels in the presence of SDS under non-reducing conditions. Heart failure increased MMP9 mRNA expression and activity in SOL, EDL and DIA and MMP2 mRNA expression in DIA. These results suggest that MMP changes may contribute to the skeletal muscle myopathy during heart failure.     

细胞外基质因子在兔软骨细胞损伤模型中的表达变化

目的:研究细胞外基质因子在兔关节软骨细胞损伤后不同时间点的表达变化水平。方法:通过机械划伤制备关节软骨细胞损伤模型,观察软骨细胞在划伤后的形态变化。采用实时荧光定量PCR检测软骨细胞在划伤前与划伤后1、3、5 d和7 d共5个时间点的基质金属蛋白酶13(MMP13)及基质金属蛋白酶抑制物1(TIMP1)、透明质酸和Ⅱ型胶原(ColⅡ)基因mRNA表达水平。结果:成功建立了兔关节软骨细胞损伤模型。与划伤前相比,MMP13基因水平在细胞损伤第1天表达明显下降,随后出现升高趋势,第5天达到最高后逐渐下降。TIMP1基因表达量损伤后先上升至第1天然后下降,在第3天降至最低,随后呈现升高趋势。透明质酸和Col-Ⅱ基因水平第1天表达明显降低,随后呈上升趋势,透明质酸mRNA表达量至第5天达到最高,然后逐渐下降。结论:MMP13、TIMP1、透明质酸和Col-Ⅱ基因在细胞损伤后的表达规律相异,为软骨细胞外基质因子表达与软骨细胞之间关系提供实验依据。     

人Bcl-2蛋白的基因克隆、表达及初步活性分析

目的：以特定形式对高度疏水性的人Bcl-2(hBcl-2)蛋白进行可溶性表达，获得非疏水性hBcl-2蛋白并具备生物学结合活性，为进一步研究hBcl-2三维结构并在此基础上研发特异性小分子药物提供充足的蛋白样品。方法：用PCR方法对hBcl-2基因进行克隆重组，插入原核表达载体pET28a( )中。用Western Blot和MALDI-TOF生物质谱鉴定，采用荧光极化方法进行活性测定。结果：重组hBcl-2在E．Coli中实现了高效、可溶性的融合表达，重组蛋白经纯化至电泳纯，具备了与Bcl-2家族Bak蛋白BH3功能域特异结合的能力，表明原核表达的重组hBcl-2具有较好的结合活性。结论：成功地对重组hBcl-2蛋白进行了可溶性表达和活性测定。     

抗人CD86单链抗体的基因克隆、表达及结合活性分析

目的 构建并在CHO细胞中表达抗人CD86单链抗体(CD86-scFv),研究该抗体与抗原的结合特性及介导的生物学功能.方法 从分泌鼠源CD86抗体的杂交瘤细胞中克隆出VH和VL基因,构建含有前导肽L-VH-Linker-VL抗体编码基因的表达载体并转染CHO细胞.经筛选高分泌株并扩大培养后收集上清进行纯化.流式细胞术分析CD86-scFv对不同细胞膜型CD86分子的识别及与鼠源亲本抗体1D1的竞争抑制效应.MTT法分析CD86-scFv与Raji细胞表达的CD86结合后对细胞生长的影响.结果 获得了稳定表达抗人CD86-scFv的CHO细胞株及相应抗体.CD86-scFv与CD86基因转染细胞株L929-CD86、天然表达CD86分子的人B系淋巴瘤细胞Raji和Daudi细胞的阳性结合率分别为67.0％、72.3％和80.5％.CD86-scFv对鼠源亲本抗体1D1具有竞争结合能力,将CD86-scFv(终浓度20μg/mL)加入Raji细胞共同培养72 h时的细胞生长抑制率为28.3％.结论 成功构建了稳定分泌抗人CD86-scFv的CHO细胞株(命名为SA-IV).该抗体能够识别CD86分子并具有良好的生物学活性.     

Extracellular proteinase activity of Cryptococcus neoformans.

Extracellular proteinase activity was studied for eight strains of Cryptococcus neoformans var. neoformans and two strains of Cryptococcus neoformans var. gattii. Proteinase activity was measured by protein agar clearance, azoalbumin hydrolysis, gelatin liquefaction, and protein substrate polyacrylamide gel electrophoresis. All strains of C. neoformans produced extracellular proteolytic activity. Maximal extracellular proteinase activity in supernatants of C. neoformans cultures was associated with late logarithmic- and stationary-phase cultures. C. neoformans was able to utilize murine immunoglobulin G1, bovine immunoglobulin G, and human complement factor 5 for growth in media containing these proteins as the sole sources of carbon and nitrogen, suggesting a capacity to degrade immunologically important proteins. Protein substrate polyacrylamide gel electrophoresis revealed several bands with proteolytic activity at apparent molecular masses of 200, 100, and 50 kDa. The results confirm the existence of extracellular proteinase activity for C. neoformans.     

Thiol proteinase expression and pathogenicity of Entamoeba histolytica.

Expression of the 56-kilodalton (kDa) neutral thiol proteinase has been shown to correlate with the potential of clinical isolates of Entamoeba histolytica to produce invasive disease. A 56-kDa band was identified by gelatin substrate gel electrophoresis in 10 of 10 isolates from patients with colitis or amebic liver abscesses, but in only 1 of 10 isolates from asymptomatic patients. Pathogenic isolates appear capable of releasing significantly larger quantities of the proteinase, as measured by cleavage of a synthetic peptide substrate, ZRR-AMC (benzyloxy-carbonyl-arginine-arginine-4-amino-7-methylcoumarin). We have also shown that the proteinase is released during the course of clinical invasive amebic disease, as demonstrated by the presence of circulating antibodies detectable by enzyme-linked immunosorbent assay. These studies support the importance of the 56-kDa thiol proteinase in the pathogenesis of invasive amebiasis.     

重组的脂联素调节肝细胞系L02基质金属蛋白酶-9表达及活性

目的 观察重组脂联素对肝细胞系L02基质金属蛋白酶-9表达及活性的影响,探讨脂联素在肝纤维化中的作用.方法 细胞分未转染对照组、转染空载体组、整合了adi-pEGFP-C1的L02细胞3组.体外扩增脂联素adiponectin mRNA全长,通过双酶切、插入及连接等基因克隆方法构建真核表达载体adi-pEGFP-C1.然后通过基因转染方法,将adi-pEGFP-C1转染到肝细胞系L02细胞中.通过G418压力筛选出adi-pEGFP-C1/L02稳定细胞系,用基因、蛋白以及蛋白酶谱等技术检测基质金属蛋白酶MMP-9的基因水平、蛋白水平以及酶的活性水平.结果 adi-pEGFP-C1构建成功,并成功筛选到L02稳定细胞系adi-pEGFP-C1/L02,与空白对照组相比,MMP-9mRNA表达水平上调(P＜0.01),MMP-9蛋白水平和活性均上调(P＜0.05).结论 脂联素通过调节MMP-9的表达及活性可能影响肝纤维化进程.     

Glycosaminoglycans and the control of cell surface proteinase activity

It is postulated that the metabolically variable fine structure of pericellular heparan glycosaminoglycans affects the ability of these molecules to influence cell proliferation-associated proteinase-catalysed reactions occurring at cell surfaces. Evidence suggesting the possibility of a wide repertoire of glycosaminoglycan-mediated positive and negative effects on such reactions is reviewed. It is suggested that clinical administration of compounds related chemically to heparins might usefully modulate cell proliferation-associated proteinase activity.