ROCK1 induces ERK nuclear translocation in PDGF-BB-stimulated migration of rat vascular smooth muscle cells

It has been known that Rho-associated protein kinase (ROCK) signaling regulates the migration of vascular smooth muscle cells (VSMCs). However, the isoform-specific roles of ROCK and its underlying mechanism in VSMC migration are not well understood. The current study thus aimed to investigate the roles of ROCK1/2 and their relationship to the MAPK signaling pathway in platelet-derived growth factor (PDGF)-induced rat aorta VSMC migration by manipulating ROCK gene expression. The results revealed that ROCK1 small interfering ribonucleic acid (siRNA) rather than ROCK2 siRNA decreased PDGF-BB-generated VSMC migration, and upregulation of ROCK1 expression via transfection of constructed pEGFP-C1/ROCK1 plasmid further increased the migration of PDGF-BB-treated VSMCs. In PDGF-treated VSMCs, ROCK1 siRNA did not affect the phosphorylation levels of ERK and p38 in the cytoplasm, but decreased the level of ERK phosphorylation in the nucleus. These findings demonstrate that activated ROCK1 can promote VSMC migration through facilitating phosphorylation and nuclear translocation of ERK protein.     

Neighborhood Regularized Logistic Matrix Factorization for Drug-Target Interaction Prediction

In pharmaceutical sciences, a crucial step of the drug discovery process is the identification of drug-target interactions. However, only a small portion of the drug-target interactions have been experimentally validated, as the experimental validation is laborious and costly. To improve the drug discovery efficiency, there is a great need for the development of accurate computational approaches that can predict potential drug-target interactions to direct the experimental verification. In this paper, we propose a novel drug-target interaction prediction algorithm, namely neighborhood regularized logistic matrix factorization (NRLMF). Specifically, the proposed NRLMF method focuses on modeling the probability that a drug would interact with a target by logistic matrix factorization, where the properties of drugs and targets are represented by drug-specific and target-specific latent vectors, respectively. Moreover, NRLMF assigns higher importance levels to positive observations (i.e., the observed interacting drug-target pairs) than negative observations (i.e., the unknown pairs). Because the positive observations are already experimentally verified, they are usually more trustworthy. Furthermore, the local structure of the drug-target interaction data has also been exploited via neighborhood regularization to achieve better prediction accuracy. We conducted extensive experiments over four benchmark datasets, and NRLMF demonstrated its effectiveness compared with five state-of-the-art approaches.     

Combining Untargeted and Targeted Proteomic Strategies for Discrimination and Quantification of Cashmere Fibers

Cashmere is regarded as a specialty and luxury fiber due to its scarcity and high economic value. For fiber quality assessment, it is technically very challenging to distinguish and quantify the cashmere fiber from yak or wool fibers because of their highly similar physical appearance and substantial protein sequence homology. To address this issue, we propose a workflow combining untargeted and targeted proteomics strategies for selecting, verifying and quantifying biomarkers for cashmere textile authentication. Untargeted proteomic surveys were first applied to identify 174, 157, and 156 proteins from cashmere, wool and yak fibers, respectively. After marker selection at different levels, peptides turned out to afford much higher selectivity than proteins for fiber species discrimination. Subsequently, parallel reaction monitoring (PRM) methods were developed for ten selected peptide markers. The PRM-based targeted analysis of peptide markers enabled accurate determination of fiber species and cashmere percentages in different fiber mixtures. Furthermore, collective use of these peptide makers allowed us to discriminate and quantify cashmere fibers in commercial finished fabrics that have undergone heavy chemical treatments. Cashmere proportion measurement in fabric samples using our proteomic approach was in good agreement with results from traditional light microscopy, yet our method can be more readily standardized to become an objective and robust assay for assessing authenticity of fibers and textiles. We anticipate that the proteomic strategies presented in our study could be further implicated in discovery of quality trait markers for other products containing highly homologous proteomes.     

18F-EF5 PET Is Predictive of Response to Fractionated Radiotherapy in Preclinical Tumor Models

We evaluated the relationship between pre-treatment positron emission tomography (PET) using the hypoxic tracer ¹⁸F-[2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3- pentafluoropropyl) acetamide] (¹⁸F-EF5) and the response of preclinical tumor models to a range of fractionated radiotherapies. Subcutaneous HT29, A549 and RKO tumors grown in nude mice were imaged using ¹⁸F-EF5 positron emission tomography (PET) in order to characterize the extent and heterogeneity of hypoxia in these systems. Based on these results, 80 A549 tumors were subsequently grown and imaged using ¹⁸F-EF5 PET, and then treated with one, two, or four fraction radiation treatments to a total dose of 10–40 Gy. Response was monitored by serial caliper measurements of tumor volume. Longitudinal post-treatment ¹⁸F-EF5 PET imaging was performed on a subset of tumors. Terminal histologic analysis was performed to validate ¹⁸F-EF5 PET measures of hypoxia. EF5-positive tumors responded more poorly to low dose single fraction irradiation relative to EF5-negative tumors, however both groups responded similarly to larger single fraction doses. Irradiated tumors exhibited reduced ¹⁸F-EF5 uptake one month after treatment compared to control tumors. These findings indicate that pre- treatment ¹⁸F-EF5 PET can predict the response of tumors to single fraction radiation treatment. However, increasing the number of fractions delivered abrogates the difference in response between tumors with high and low EF5 uptake pre-treatment, in agreement with traditional radiobiology.     

鲈鱼IgM重链基因cDNA的克隆及其原核表达

采用同源克隆和RACE技术扩增到鲈鱼 (Lateolabrax japonicus) 免疫球蛋白M (Immunoglobulin M,IgM) 重链 (Heavy chain,H) 基因全长cDNA序列。鲈鱼IgM cDNA全长为1 901 bp,开放阅读框包含1 749 bp,编码582个氨基酸。根据鲈鱼IgM和其他硬骨鱼免疫球蛋白重链恒定区的氨基酸序列构建的系统发育树表明IgM、IgZ和IgD分别聚为一枝,其中IgM与IgZ分支的进化关系较近,而与IgD分支的进化关系较远。RT-PCR检测IgM在鲈鱼各组织器官的表达情况,其中在头肾及脾脏中表达量最高,心脏、肌肉及脑中几乎不表达。利用已获得的鲈鱼IgM cDNA序列,构建原核表达载体pQE30-IgM,并在M15大肠杆菌中成功诱导表达了分子量为63kD的重组蛋白His-IgM,Western-blotting显示鲈鱼IgM重组蛋白能与鼠源抗6×His的单克隆抗体特异性结合,说明已经获得了基因工程表达IgM重链蛋白。     

Distinct functions of two FabA-like dehydratase domains of polyunsaturated fatty acid synthase in the biosynthesis of very long-chain polyunsaturated fatty acids

Thraustochytrium is a unicellular marine protist for the commercial production of very long-chain polyunsaturated fatty acids (VLCPUFAs). Biosynthesis of these VLCPUFAs in the protist is catalysed by a PUFA synthase comprising three subunits, each with multiple catalytic domains. Among these domains, two tandem FabA-like dehydratase domains (DH1 and DH2) in subunit-C together are responsible for introducing double bonds in VLCPUFAs. Domain swapping analysis in yeast showed that the defective phenotype of a Scfas1 mutant could be complemented by expressing an engineered ScFAS1 gene in which the DH domain was replaced by a single DH1 or mutated DH2 of the two. Heterologous expression of the PUFA synthase in E. coli showed that the mutation of DH1 of the two or deletion of DH1 or substitution of DH1 with DH2 resulted in the complete loss of activity in the biosynthesis of VLCPUFAs. Mutation of DH2 of the two or deletion of the DH2 domain produced a small amount of DPA, but not docosahexaenoic acid (DHA). These results indicate that each of the two FabA-like domains of the PUFA synthase possesses distinct function. DH1 domain is essential for the biosynthesis of VLCPUFAs, but DH2 domain is required for the biosynthesis of DHA.     

Configurations of germinal vesicle (GV) chromatin in the goat differ from those of other species

Configuration of germinal vesicle (GV) chromatin has been studied and found correlated with the developmental competence of oocytes in several mammalian species. A common feature in the configuration of GV chromatin in the species studied so far is that the diffuse chromatin (the so called "NSN" pattern) condenses into a perinucleolar ring (the so called "SN" configuration) with follicular growth. However, no study has been published on the configuration of GV chromatin in the goat. Nor is it known whether the perinucleolar ring of condensed chromatin (CC) in an oocyte represents a step toward final maturation or atresia. Changes in configurations of GV chromatin and RNA synthesis during goat oocyte growth, atresia and maturation in vivo and in vitro were investigated in this study. Based on both the size of nucleoli and the degree of chromatin condensation, the GV chromatin of goat oocytes was classified into GV1 characterized by large nucleoli and diffuse chromatin, GV2 with medium-sized nucleoli and condensed net-like (GV2n) or clumped (GV2c) chromatin, GV3 with small nucleoli and net-like (GV3n) or clumped (GV3c) chromatin, and GV4 with no nucleolus but clumped chromatin. The results showed that (i) the configurations of GV chromatin in the goat differ from those of other species in that the chromatin did not condense into a perinucleolar ring; (ii) most of the goat oocytes are synchronized at the GV3n configuration before GVBD; (iii) the GVn pattern might represent a healthy state, but the GVc an atretic state; (iv) in both goats and mice, the GC-specific (Chromomycin A3, CMA3) and the AT-specific (Hoechst 33342) fluorochromes followed the same pattern of distribution in GV chromatin; (v) the nucleolar size decreased significantly with oocyte growth and maturation in vivo and in vitro; and (vi) goat oocytes began GVBD at 8 hr and had completed it by 20 hr after onset of estrus. The peculiar configuration of GV chromatin of goat oocytes can be a useful model for studies of morphological and functional changes of different nuclear compartments during the cell cycle and cell differentiation, and the functional differentiation between GV3n and GV3c might be used for reference to the question whether the "SN" configuration in other species inclines toward ovulation or atresia.     

人新基因hbrp的染色体定位及其对TPK活性的抑制作用（简报）

hbrp(Human BSP-Related Protein)是我们实验室最近在睾丸组织中克隆的一个人与BSP（bovine seminal plasma)蛋白相关的新基因。为了将有关该新基因信息与现有人类基因组转录图相整合,我们应用荧光原位杂交(fluorescent in situ hybridizationFISH)法进行了该基因的人染色体基因定位,结果成功地将hbrp基因定位在人19号染色体长臂1区3带上。hbrp基因是在对BSP蛋白功能的研究过程中发现并克隆的,其同源性分析发现,与其序列最相近     

转梅PGIP基因增强菊花抗病性研究

通过农杆菌介导法将梅多聚半乳糖醛酸酶抑制蛋白基因(PGIP)转入盆栽小菊品种'05-44-2'中,以提高对真菌病害的抗性.经Hyg抗性筛选,获得232株抗性苗.对其中40株进行PCR检测,有8株扩增出目的条带;对这8株进行RT-PCR检测,发现其中有5个株系有目的条带出现,且发生基因转录.转基因菊花的抗病性检测证实,与对照相比,转基因株系对黑斑病有不同程度的抗性,表现为发病延迟,病情指数降低;株系7抗性最强,其苗期病情指数仅为33.