Reproductive biology of the guitarfish <Emphasis Type="Italic">Rhinobatos hynnicephalus</Emphasis> (Batoidea: Rhinobatidae) in Ariake Bay,Japan

Size at sexual maturity, reproductive cycle, and fecundity of the guitarfish, Rhinobatos hynnicephalus, were examined in Ariake Bay, Japan. Females reached sexual maturity at a larger size than males [total length (TL) at 50% sexual maturity: males, 431 mm; females, 476 mm]. Monthly gonadosomatic indices of males decreased abruptly from July to August. Histological examinations confirmed the presence of mature spermatozoa in the testes in July, with semen in seminal vesicles in July and August. Preovulatory ova were observed in females with near-term embryos in August. Parturition occurred in August, immediately followed by mating, ovulation, and fertilization. Gestation period is approximately 1 year. Fertilized uterine eggs without embryonic development were present throughout the annual reproductive cycle. Embryonic development began in June and ended in August, indicating that R. hynnicephalus undergoes embryonic diapause (9 months). Fecundity increased with TL and ranged from 1 to 9 (mean, 4.4) embryos per litter.     

A recurrent mutation in type II collagen gene causes Legg-Calvé-Perthes disease in a Japanese family

Legg-Calvé-Perthes disease (LCPD) is a common childhood hip disorder characterized by sequential stages of involvement of the capital femoral epiphyses, including subchondral fracture, fragmentation, re-ossification and healing with residual deformity. Most cases are sporadic, but familial cases have been described, with some families having multiple affected members. Genetic factors have been implicated in the etiology of LCPD, but the causal gene has not been identified. We have located a missense mutation (p.G1170S) in the type II collagen gene (COL2A1) in a Japanese family with an autosomal dominant hip disorder manifesting as LCPD and showing considerable intra-familial phenotypic variation. This is the first report of a mutation in hereditary LCPD. COL2A1 mutations may be more common in LCPD patients than currently thought, particularly in familial and/or bilateral cases.     

Site-directed mutagenesis of disulfide bridges in Aspergillus niger NRRL 3135 phytase (PhyA), their expression in Pichia pastoris and catalytic characterization

Earlier studies have established the importance of five disulfide bridges (DBs) in Aspergillus niger phytase. In this study, the relative importance of each of the individual disulfide bridge is determined by its removal by site-directed mutagenesis of specific cysteines in the cloned A. niger phyA gene. Individually, these mutant phytases were expressed in a Pichia expression system and their product purified and characterized. The removal of disulfide bridge 2 yielded a mutant phytase with a complete loss of catalytic activity. The other disulfide mutants displayed a broad array of altered catalytic properties including a lower optimum temperature from 58°C to 53°C for bridge number 1, 37°C for bridge number 3 and 4, and 42°C for bridge number 5. The pH versus activity profile was also modified in the DB mutants. The pH profile of the wild-type phytase was modified by the DB mutations. In bridge number 1, 3, and 4, the second peak at pH 2.5 was abolished, and in bridge number 5, the peak at pH 5.0 was abolished completely leaving only the pH 2.5. While the K _m was not affected drastically, the turnover number was lowered significantly in bridge number 3, 4, and 5.     

犬冠状病毒S蛋白主要抗原区基因片断的表达及免疫原性

应用Pichia pastoris酵母表达了犬冠状病毒大熊猫野毒株(CCV DXMV)S蛋白主要抗原区基因片断。用特异性引物扩增出CCV DXMV株S1基因片断,并将其克隆到pGEM-T载体中得到pTS1。用KpnI和Notl双酶切pTS1回收目的基因S1定向克隆到pPICZCαA中,构建出重组质粒pPICZCαAS1。将pPICZCαASl用SacI内切酶线性化后,电转化感受态GS115酵母细胞,用PCR法筛选阳性重组子。用1％的甲醇诱导重组酵母菌,取培养物上清进行重组蛋白的检测。结果重组酵母菌培养物上清用SDS-PAGE电泳可检测到相对分子量为106kDa大小的重组蛋白,Westem-blot证实该重组蛋白可以与CCV多克隆抗体发生特异性血清学反应。凝胶薄层扫描分析表明,3株重组酵母菌在1％甲醇诱导144h后,重组蛋白S1表达量约占培养物上清总蛋白量的6．6-8．6％左右。用重组蛋白S1免疫BALB／C小鼠3次后,小鼠血清CCV中和抗体可达1：8-1：16,表明重组S1蛋白具有一定的免疫原性。     

p38alpha mitogen-activated protein kinase plays a critical role in cardiomyocyte survival but not in cardiac hypertrophic growth in response to pressure overload

The molecular mechanism for the transition from cardiac hypertrophy, an adaptive response to biomechanical stress, to heart failure is poorly understood. The mitogen-activated protein kinase p38alpha is a key component of stress response pathways in various types of cells. In this study, we attempted to explore the in vivo physiological functions of p38alpha in hearts. First, we generated mice with floxed p38alpha alleles and crossbred them with mice expressing the Cre recombinase under the control of the alpha-myosin heavy-chain promoter to obtain cardiac-specific p38alpha knockout mice. These cardiac-specific p38alpha knockout mice were born normally, developed to adulthood, were fertile, exhibited a normal life span, and displayed normal global cardiac structure and function. In response to pressure overload to the left ventricle, they developed significant levels of cardiac hypertrophy, as seen in controls, but also developed cardiac dysfunction and heart dilatation. This abnormal response to pressure overload was accompanied by massive cardiac fibrosis and the appearance of apoptotic cardiomyocytes. These results demonstrate that p38alpha plays a critical role in the cardiomyocyte survival pathway in response to pressure overload, while cardiac hypertrophic growth is unaffected despite its dramatic down-regulation.     

澳大利亚外来入侵物种管理策略及对我国的借鉴意义

澳大利亚是一个岛状大陆,海洋运输业十分发达,通过贸易,旅游,运输等途径有意或无间引进有害外来物种的风险较大。澳大利亚政府高度重视外来入侵物种的管理工作,制定了《澳大利亚国家生物多样性保护策略》,针对外来杂草和通过压舱水载入的海洋外来入侵物种的管理制定了《国家杂草策略》,《杂草风险评价系统》和《压舱水指南》等法规和技术性文件,加强了对外来入侵物种的管理。本文简要介绍了澳大利亚外来入侵物种管理的有关策略和指南,并提出了我国在外来入侵物种管理方面的对策建议;（1）尽快建立相应的法规体系,实现外来入侵物种的依法管理;（2）加强机构建设,形成多部门的协调管理机制;（3）加强外来入侵物种管理制度的建设;（4）采取适当的引进预防,消除,控制和恢复措施;（5）开展科学研究,为外来入侵物种的管理提供科学依据;（6）制定教育和培训计划,提高公众意识。     

X-Linked inhibitor of apoptosis protein is involved in mutant SOD1-mediated neuronal degeneration

Mutations in the superoxide dismutase 1 (SOD1) gene cause the degeneration of motor neurons in familial amyotrophic lateral sclerosis (FALS). An apoptotic process including caspase-1 and -3 has been shown to participate in the pathogenesis of FALS transgenic (Tg) mouse model. Here we report that IAP proteins, potent inhibitors of apoptosis, are involved in the FALS Tg mouse pathologic process. The levels of X-linked inhibitor of apoptosis protein (XIAP) mRNA and protein were significantly decreased in the spinal cord of symptomatic G93A-SOD1 Tg mice compared with littermates. In contrast, the levels of cIAP-1 mRNA and protein were increased in symptomatic G93A-SOD1 Tg mice, whereas the levels of cIAP-2 mRNA and protein were unchanged. In situ hybridization showed that the expression of XIAP was remarkably reduced in the motor neurons of Tg mice, and the expression of cIAP-1 was strongly increased in the reactive astrocytes of Tg mice. Overexpression of XIAP markedly inhibited the cell death and caspase-3 activity in the neuro2a cells expressing mutant SOD1. Deletional mutant analysis revealed that the N-terminal domain of XIAP, the BIR1-2 domains, was essential for this inhibitory activity. These results suggest that XIAP plays a role in the apoptotic mechanism in the progression of disease in mutant SOD1 Tg mice and holds therapeutic possibilities for FALS.     

赤子爱胜蚓（Eisenia foetida）中抗肿瘤与纤溶酶原激酶活性蛋白质的分离与鉴定

采用SephadexG 75和HiPrep 1 6 / 6 0DEAE离子交换等方法从赤子爱胜蚓 (Eiseniafoetida)中提取到一组活性蛋白质成分 ,双向电泳分析证明它们为一组pI在 3 .0～ 4 .0之间的酸性蛋白质 ;利用体外K5 6 2、HeLa、SY5Y等肿瘤细胞抑制实验和纤维蛋白平板实验 ,跟踪测定活性 ,证明乙醇沉淀组分D2 (8)是既具肿瘤抑制 ,又具有激酶活性的蛋白质成分。同时应用非变性电泳对乙醇沉淀组分进行分离 ,并利用电洗脱、凝胶原位酶解和ESI MS等蛋白质组学方法 ,鉴定了其中 6种蛋白质的分子量、氨基酸组成、N末端序列和肽质量指纹图信息 ,其中条带 9与D2 (8)组分为同一种蛋白质 ;研究证明蚯蚓中含有既具抗肿瘤活性又具有激酶活性的蛋白质成分。采用的方法可适用于活性蛋白质成分的整体分离与鉴定     

DmGEN, a novel RAD2 family endo-exonuclease from Drosophila melanogaster

A novel endo-exonuclease, DmGEN (Drosophila Melanogaster XPG-like endonuclease), was identified in D.melanogaster. DmGEN is composed of five exons and four introns, and the open reading frame encodes a predicted product of 726 amino acid residues with a molecular weight of 82.5 kDa and a pI of 5.36. The gene locus on Drosophila polytene chromosomes was detected at 64C9 on the left arm of chromosome 3 as a single site. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nucleases, especially XPG. Although the XPG-N- and XPG-I-domains are highly conserved in sequence, locations of the domains are similar to those of FEN-1 and EXO-1, and the molecular weight of the protein is close to that of EXO-1. In vitro, DmGEN showed endonuclease and 3'-5' exonuclease activities with both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), but the endonuclease action with dsDNA was quite specific: 5'-3' exonuclease activity was found to occur with nicked DNA, while dsDNA was endonucleolytically cut at 3-4 bp from the 5' end. Homologs are widely found in mammals and higher plants. The data suggest that DmGEN belongs to a new class of RAD2 nuclease.