cDNA cloning of oyster matrix metalloproteinase and its possible involvement in hypoxic adaptation

ABSTRACT:   We have cloned a cDNA encoding the matrix metalloproteinase (MMP) from oyster Crassostrea gigas . The clone contains a 1797 bp open reading frame encoding a protein of 599 amino acids. Oyster MMP (Cg-MMP) has a transmembrane domain at its C-terminal and a furin/prohormone convertase cleavage site at the end of a propeptide domain, which are commonly observed in membrane-type MMPs (MT-MMPs). This suggests that Cg-MMP is an MT-MMP. The deduced amino acid sequence of oyster MMP shares approximately 30% identity with human MT4-MMP and MMPs from fruit fly and hydra. Cg-MMP mRNA was detected in the gill, mantle and adductor muscle, and more intense signals in the northern blot analysis were recognized in the gill and adductor muscle. Similar tissue distribution was observed for tissue inhibitor of metalloproteinase (Cg-TIMP) in oyster. In response to hypoxic stress, the abundance of Cg-MMP mRNA was elevated in the gill, while that of Cg-TIMP mRNA remained almost constant. These findings suggest that promotion of collagen metabolism may be implicated in the hypoxic adaptation in oyster.     

Cloning and characterization of cDNA for carp matrix metalloproteinase 9

ABSTRACT: We have cloned a cDNA encoding the MMP-9 from a carp epidermal cell (EPC) cDNA library. The clone contains a 2025-base pair (bp) open reading frame encoding a protein of 674 amino acids. The deduced amino acid sequence shares 68% and 69% identity with medaka and Japanese flounder MMP-9. The hinge domain of the carp MMP-9, like those of the other non-mammalian species, lacks a type V collagen-like region that is typical of mammalian MMP-9. Gelatin zymography and immunoblot analysis of conditioned media of EPC cells and cDNA-transfected COS-7 cells detected a 76-kDa gelatinase. The apparent molecular mass of the carp zymogen is much smaller than those of its mammalian counterparts while almost identical with that of chicken 75-kDa gelatinase B-like enzyme. Although hypo-osmotic stress induced the elevation of MMP-9 mRNA level in EPC cells, no significant change in the protein in conditioned medium was detected during hypo-osmotic stress. Northern blot analysis detected a large amount of MMP-9 mRNA in carp kidney and spleen, suggesting the high expression of MMP-9 in blood cells, neutrophils, and macrophages. The smaller amount of MMP-9 mRNA was detected in gill, heart, fin, and eye, whereas none of the mRNA was detected in the hepatopancreas, intestine, brain, muscle, and skin.     

cDNA cloning and characterization of two gelatinases from Japanese flounder

SUMMARY: Toughness is one of the most important elements that define the commercial value of the raw meat of fish. Degradation of the extracellular matrix is thought to be a cause of postmortem tenderization of fish meat. A previous study has suggested that this tenderization is caused mainly by metalloproteinases. The present study seeks to identify the proteinase(s) involved in tenderization; hence, cloned cDNA of two gelatinases from Japanese flounder, which showed high homology with mammalian matrix metalloproteinase (MMP)-2 and MMP-9, were designated as jfMMP-2 and jfMMP-9 , respectively. Northern blot analysis revealed that jfMMP-2 mRNA was expressed almost ubiquitously in adult tissues including the brain, muscle, gill, heart, gut, kidney, spleen, testis, and ovary. In contrast, the expression of jfMMP-9 mRNA was observed in those tissues which were abundant in blood cells, such as kidney, spleen, heart, and gill. Both recombinant proteins (jfMMP-2 and jfMMP-9) produced with the COS-7 cell system exhibited gelatin-degrading activity that was sensitive to 1,10-phenanthroline, a typical metalloproteinase inhibitor.     

金属蛋白酶组织抑制剂在皱纹盘鲍抗弧菌免疫中的作用

由弧菌感染引起的疾病是影响鲍存活率的主要因素,实验以皱纹盘鲍为研究对象,探讨金属蛋白酶组织抑制剂(tissue inhibitor of metalloproteinase,TIMP)在皱纹盘鲍抗弧菌免疫中的功能及其与基质金属蛋白酶1 (matrix metalloproteinase 1,MMP-1)的相互作用关系....     

海参内脏胶原降解酶的纯化及性质研究

针对海参自溶现象,研究海参内脏中与胶原降解相关蛋白酶的存在情况,以期了解内源性蛋白酶在海参自溶过程中发挥的作用,为解明自溶的内在机理提供理论依据。通过硫酸铵盐析、DEAE-Sepharose阴离子交换柱层析、Sephacryl S-200凝胶过滤柱层析、Phenyl Sepharose FF疏水柱层析及Mini Q离子交换柱层析等方法,从海参内脏中分离纯化出降解明胶的蛋白酶。利用荧光底物研究其酶学性质及其对海参体壁胶原蛋白的降解情况。结果表明,该酶最适温度为30 ℃,最适pH为7.5,可被金属蛋白酶抑制剂EDTA、EGTA完全抑制,亦可被PMSF、Pefabloc SC等丝氨酸蛋白酶抑制剂完全抑制。底物特异性表明,该酶只对金属蛋白酶荧光底物有分解作用,推测其为金属蛋白酶。该酶在4和37 ℃下均能有效地分解海参体壁胶原蛋白,表明其在海参自溶过程中起重要作用。     

Purification and characterization of a matrix shell protein from the shell of scallop <Emphasis Type="Italic">Patinopecten yessoensis</Emphasis>

ABSTRACT:   A new protein named MSP-SC (matrix shell protein from scallop) with a molecular weight of 14 kDa was isolated from the shell of a scallop, Patinopecten yessoensis , using gel filtration column chromatography, ion exchange column chromatography, and reverse phase C4 column chromatography. A comparison of the known protein sequences with the N-terminal sequence of MSP-SC showed that the protein sequence of MSP-SC was novel. Immunohistochemistry using a polyclonal antibody against MSP-SC showed that MSP-SC is expressed in the mantle pallial cell layer but not in the muscle tissue, and showed a punctate distribution along the horizontal calcified layer in the shell. The isolated MSP-SC inhibited the formation of calcium carbonate (CaCO₃) crystals in a dose-dependent manner. The CaCO₃ crystals grown in the presence of a lower concentration of MSP-SC were much larger and aggregated when compared with those formed in the absence of MSP-SC. In addition, the crystal had a radial and not cubical morphology. These results suggest that MSP-SC regulates the formation and the morphology of CaCO₃ crystals in the shell. Moreover, its ability to aggregate CaCO₃ crystals and its localization along the horizontal calcified layer in the shell suggest that MSP-SC may serve to connect the CaCO₃ layers in the scallop shell.     

气相色谱-质谱法测定渔业水质中8种除草剂残留的基质效应探讨

文章分析了气相色谱-质谱法（ GC-MS）测定渔业水质中8种除草剂残留过程中基质效应的主要来源及其影响因素：实验中目标检测物的化学性质和结构、提取溶剂、净化材料及基质的浓度和种类等。并尝试采用加入基质保护剂法、加入内标法和基质匹配标准溶液校准法等补偿方法测定过程中产生的基质效应。最终选择采用基质匹配标准溶液校准法,有效地补偿了方法测定过程中产生的基质效应,提高了方法的准确度和适用性。     

不同基质构建海水养殖系统硝化功能的比较分析

为比较不同基质构建海水养殖系统硝化功能的强弱,选取纤维毛球、陶粒、螺旋式生物绳等7种基质,其中陶粒、流化床填料、纤维毛球采取不同放置方式,共建立14个模拟海水养殖系统,比较不同基质硝化功能建立过程以及同种基质不同放置方式对氨氮和亚硝氮的去除效果。结果表明,单位体积珊瑚骨的氨氧化活性和亚硝酸盐氧化活性高于其他载体,在氨氮初始浓度20 mg/L条件下,氨氮和亚硝氮降解至检测不出分别需要3 d和11 d,而纤维毛球、陶粒、螺旋式生物绳、流化床填料、丝带内芯悬浮球、海绵内芯悬浮球硝化系统的建立分别需要18、26、30、25、27和22 d。纤维毛球100目筛绢悬挂、陶粒网兜悬挂、流化床填料100目筛绢悬挂优于其他放置方式,其中纤维毛球100目筛绢悬挂硝化功能建立时间为18 d,效果最优,流化床填料100目筛绢悬挂、陶粒网兜悬挂硝化系统建立分别需要21、24 d。     

cDNA clonings of shell matrix proteins from scallop shell

ABSTRACT:   A cDNA encoding the matrix shell protein from scallop (MSP-2) has been cloned. Comparison of the sequence of MSP-2 with the GenBank database revealed the sequence displays a high degree of homology with MSP-1, which was recently identified in the shell of the scallop Patinopecten yessoensis . Matrix shell protein-2 contains only 323 amino acids, while MSP-1 comprises 824 amino acids. The structural difference between MSP-1 and MSP-2 is mainly the presence or absence of the tandem repeat arrangement. In addition, multiple isoforms of MSP-2 were found. These isoforms share a high degree of sequence similarity to each other, suggesting that the MSP-2, MSP-2 isoforms and MSP-1 may constitute the same family. The MSP-2 isoforms in scallop shell may be responsible for the formation of molluscan shells.     

文昌鱼鳃骨条基质蛋白的氨基酸组成

以传统的分离不溶性弹性蛋白的方法——热碱提取法处理新鲜解剖出来的文昌鱼鳃组织。收集不溶解的沉淀，进行氨基酸组成分析。并将分析结果与已知的七鳃鳗软骨和盲鳗软骨的不溶性残留物以及马蹄蟹鳃软骨、猪主动脉弹性蛋白以及胶原蛋白的氨基酸组成进行比较。结果表明，文昌鱼鳃骨条的基质蛋白是不溶性的非胶原的，其不溶于热碱的残留物具有特殊的氨基酸组成，且与弹性蛋白及各种不溶性类弹性蛋白的基质蛋白的氨基酸组成具有相似的特征。     

传染性造血器官坏死病毒-Sn株基质蛋白基因克隆及生物信息学分析

基质蛋白(matrix protein,M)是传染性造血器官坏死病毒(infectious haematopoietic necrosis virus,IHNV)主要结构蛋白之一,是病毒感染后造成细胞凋亡的主要作用蛋白。为分析IHNV M蛋白的序列及结构特征,研究利用敏感细胞(EPC)培养传染性造血器官坏死病毒-Sn分离株(IHNV-Sn),根据M蛋白基因开放阅读框(open reading frame,ORF)的序列设计引物,利用RT-PCR的方法克隆得到M蛋白全长ORF,并且构建至表达载体pET27b(+)中,构建出pET27-M重组质粒。生物信息学分析结果显示,M蛋白基因的序列长度为588 bp,编码195个氨基酸残基,推导分子量约为21.88 ku,等电点为9.35;氨基酸序列分析表明,M蛋白富含丝氨酸、苏氨酸以及碱性氨基酸,存在丰富的α-螺旋、β折叠和无规则卷曲;M蛋白不含有信号肽;疏水性大于亲水性;没有跨膜区存在;抗原表位预测显示抗原性良好;结构预测显示,不存在N-糖基化位点,存在7个潜在的O-糖基化位点和15个潜在的磷酸化位点。系统进化树分析显示,IHNV-Sn株与美国分离株同为一簇。     

组合生态浮床的水体净化效果与作用机理探讨

通过在美人蕉(Canna glauca)底部悬挂生物陶粒基质构建组合生态浮床,与仅有生物陶粒的基质组和空白组比较了对室内配制富营养化污水的净化效果,分析了各组水体中的微生物数量和活性,研究组合浮床中植物、基质和微生物对水体净化的贡献率及其相互间协同作用.结果表明,经过48 d的运行,组合浮床对总氮、总磷和氨氮的去除率依次为64.03％、95.82％和96.43％,美人蕉对氮、磷的去除贡献率分别为36.03％、37.96％.组合浮床组水中的细菌总量、基质上附着的生物膜脱氢酶活性和耗氧速率均高于基质组,说明植物吸收不是组合浮床去除氮磷的主要机制,但植物对微生物的数量和活性有积极作用;组合浮床对水中污染物的去除存在着植物、基质及微生物之间的协同作用.与基质组和空白组相比,组合浮床可以有效提高对富营养化水体的净化效果.     

不同孵化基质和开口饵料对宽体金线蛭生长发育的影响

比较有机孵化基质对宽体金线蛭卵茧孵化效果和不同开口饵料对苗种生长发育的影响。孵化基质分别为土壤、牛粪有机肥、蘑菇菌渣,开口饵料分别为螺蛳、漂螺。试验结果显示,蘑菇菌渣+土壤组卵茧孵化率高达98.89%,显著高于土壤组和蘑菇菌渣组(P<0.05);幼蛭各生长指标显著高于土壤组、蘑菇菌渣组、有机肥组及有机肥+土壤组(P<0.05);混合组幼蛭各生长指标均显著高于未添加土壤组(P<0.05)。5种不同孵化基质孵化率依次为蘑菇菌渣+土壤>土壤>有机肥+土壤>有机肥>蘑菇菌渣。不同开口饵料的试验比较发现,漂螺组幼蛭成活率为72.50%,显著高于螺蛳组和螺蛳+漂螺组(P<0.05),漂螺组和螺蛳+漂螺组幼蛭体质量显著高于螺蛳组(P<0.05)。生长效果依次为漂螺组>螺蛳+漂螺组>螺蛳组。试验结果表明,蘑菇菌渣可以作为宽体金线蛭卵茧孵化基质;无厣结构的漂螺更适宜作宽体金线蛭幼蛭的开口饵料。     

Recovery potential of smalltooth sawfish,Pristis pectinata,in the United States determined using population viability models

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育珠对合浦珠母贝N19和Prismalin-14基因表达水平的影响

为探讨育珠对壳基质蛋白基因表达的影响,开展了合浦珠母贝（Pinctada fucata）N19和Prismalin-14基因在育珠贝和非育珠贝中的荧光定量表达研究。结果显示,N19和Prismalin-14在育珠贝与非育珠贝的闭壳肌、肝胰脏、性腺、肠、鳃等组织中均无表达,在外套膜均有表达;N19在珍珠囊表达,而Prismalin-14则不表达。N19勺Prismalin．14在育珠贝外套膜的表达量均大于非育珠贝,表明育珠可能对某些壳基质蛋白的表达具有一定的促进作用。其中Prismalin-14在育珠贝外套膜的表达量最高（P〈0．01）,是非育珠贝外套膜Prismalin-14的1．23倍,是育珠贝外套膜N19的25．04倍,是非育珠贝外套膜N19的41．04倍,推测在珍珠贝生长过程中,对Prismalil7-14的需求量可能比N19大。N19在育珠贝珍珠囊中表达量最高（P〈0．01）,是育珠贝N19外套膜的11．58倍,是非育珠贝外套膜的18．97倍,推测在珍珠形成的过程中,对N19的需求量可能比贝壳自身生长对N19的需求量大。     

Population viability and perturbation analyses in remnant populations of the Andean catfish Astroblepus ubidiai

Abstract – Astroblepus ubidiai (Actinopterygii; Siluriformes), which is the only native fish of the highlands of the Province of Imbabura, Ecuador, was abundant in the past in the Imbakucha watershed and adjacent drainages but currently it is restricted to a few isolated refuges. Population viability analysis (PVA) was used to detect critical aspects in the ecology and conservation biology of this unique fish. The annual population growth rate ( λ ) was estimated for six remnant populations of this Andean catfish using a deterministic matrix population model. Sensitivity and elasticity analyses complemented the PVA by providing constructive insights into vital rates affecting projections and extinction probabilities. Positive population growth rates were found in all the study populations. The high contributions of juvenile survival to the variance of λ and its high elasticity indicated that A. ubidiai population dynamics are highly sensitive to the transition values of this vital rate, which can promptly respond to management or antagonistic perturbations. Allowing fish to survive until the age of first reproduction and permitting the successful reproduction of these individuals will facilitate positive population growth rates, however the very small areas of occupancy, small extent of occurrence and severe fragmentation may still contribute to the extinction risk.     

Culture of mantle epithelial cells expressing shell matrix proteins from scallop Patinopecten yessoensis

ABSTRACT:   In molluscs, mantle epithelial cells secrete organic matrix proteins to form shells. In this study, we established a culture of mantle epithelial cells by using the mantle pallial layer of scallops. We aimed to identify the mantle epithelial cells expressing scallop shell matrix proteins and establish a culture system of epithelial cells. After the mantle pallial layer was carefully isolated from the mantle tissue, explant culture was performed at 4°C. Most cells that migrated from the explant tissue were round cells. Most of the adhered cells retained round morphology, while some of the cells adhered to the dish and showed morphology similar to that of epithelial-like and fibroblast-like cells. When the cultured cells were immunostained with a polyclonal antibody against the shell matrix protein, the antibody recognized many of the adhered cells. An estimation of the number of epithelial cells revealed that approximately 70% of the adhered cells were epithelial cells. This is the first report to describe epithelial cells in cultured mantle cells, which express shell matrix proteins. This culture system may be a useful method for characterization of the mantle epithelial cells.     

基于多源数据的东海小黄鱼资源评估与管理

为建立稳定环境和波动环境机制下预防性渔业管理生物参考点, 整合调查设计和渔捞日志等多源资源指标构建混合矩阵, 利用logistic和Fox剩余产量模型的两步分析技术, 对东海区小黄鱼(Larimichthys polyactis)渔业资源动态进行评估。模型估算参数和管理参考点显示, Fox模型对渔获量和CPUE拟合的方差贡献率高于logistic模型, 两者分别为68%和57%, 环境承载力和内禀增长率相差较大。logistic模型估算了相对较低的承载力和较高的内秉增长率、初始开发率以及MSY。稳定环境下资源状况评判结果表明: 1999―2008年间多数年份的捕捞强度超过捕捞水平限制参考点, 渔业遭受过度开发, 平均资源量保持在中位水平且未达到过度捕捞状态, 但已超过目标参考点; 波动环境条件下的判别结果显示: logistic和Fox模型拟合的渔业水平均已达到过度捕捞。采用保护性捕捞参考点可增强渔业资源稳定性, 当捕捞死亡从参考点F_MSY降至预防性参考点F_opt, logistic模型估算资源量从8.1 t上升到10.1 t, 而渔获量从13.1 t下降至12.3 t; Fox模型资源量则从11 t增加到15.9 t, 相应的捕捞产量从12.8 t下降到11.6 t。Fox模型评估结果较为保守, 适合预防性渔业管理。