An Effective Method for Raising Antisera Against β-defensins: Double-copy Protein Expression of mBin1b in E. coli

Defensins are a family of evolutionarily relatedvertebrate antimicrobial peptides with a characteristicβ-sheet-rich fold and a framework of six disulphide-linkedcysteines. The two main defensin subfamilies, α- andβ-defensin, differ in the length of pep…     

Secretory/releasing proteome-based identification of plasma biomarkers in HBV-associated hepatocellular carcinoma

For successful therapy, hepatocellular carcinoma (HCC) must be detected at an early stage. Herein, we used a proteomic approach to analyze the secretory/releasing proteome of HCC tissues to identify plasma biomarkers. Serum-free conditioned media (CM) were collected from primary cultures of cancerous tissues and surrounding noncancerous tissues. Proteomic analysis of the CM proteins permitted the identification of 1365 proteins. The enriched molecular functions and biological processes of the CM proteins, such as hydrolase activity and catabolic processes, were consistent with the liver being the most important metabolic organ. Moreover, 19% of the proteins were characterized as extracellular or membrane-bound. For validation, secretory proteins involved in transforming growth factor-β signaling pathways were validated in plasma samples. Alphafetoprotein (AFP), metalloproteinase (MMP)1, osteopontin (OPN), and pregnancy-specific beta-1-glycoprotein (PSG)9 were significantly increased in HCC patients. The overall performance of MMP1 and OPN in the diagnosis of HCC remained greater than that of AFP. In addition, this study represents the first report of MMP1 as a biomarker with a higher sensitivity and specificity than AFP. Thus, this study provides a valuable resource of the HCC secretome with the potential to investigate serological biomarkers. MMP1 and OPN could be used as novel biomarkers for the early detection of HCC and to improve the sensitivity of biomarkers compared with AFP.     

Atomic force microscopy imaging of live mammalian cells

Atomic force microscopy (AFM) was used to examine the morphology of live mammalian adherent and suspended cells. Time-lapse AFM was used to record the locomotion dynamics of MCF-7 and Neuro-2a cells. When a MCF-7 cell retracted, many small sawtooth-like filopodia formed and reorganized, and the thickness of cellular lamellipodium increased as the retraction progressed. In elongated Neuro-2a cells, the cytoskeleton reorganized from an irregular to a parallel, linear morphology. Suspended mammalian cells were immobilized by method combining polydimethylsiloxane-fabricated wells with poly-L-lysine electrostatic adsorption. In this way, the morphology of a single live lymphoma cell was imaged by AFM. The experimental results can improve our understanding of cell locomotion and may lead to improved immobilization strategies.     

Detection and Genetic Characterization of Rabies Virus from Human Patients

Saliva and blood were collected from two patients who had not received post exposure prophylaxis in the cities of Wenzhou and Xinning respectively. Both patients were confirmed as positive for rabies by detection of rabies virus specific nucleoprotein antibodies in the sera by Western Blot. However, rabies virus specific RNA was only identified in the saliva collected from the patient in Wenzhou. Furthermore, the isolate Zhejiang Wz0 (H) was obtained by inoculating one-day-old suckling mice. Both nucleoprotein (N) and glycoprotein (G) genes from the isolate were amplified by RT-PCR and sequenced. Phylogenetic analysis indicated that the isolate belonged to classic rabies virus, and shared a higher homology with the street viruses from dogs in the main endemic areas in China and the street virus from dogs in Indonesia than with other known strains. Further comparison of the deduced amino acid sequences between the isolate and the vaccine strains used in China showed that the virus had a higher level of homology with the vaccine strain CTN than with the other vaccine strains (3aG, PV, PM and ERA). In particular, amino acid residues substitutions located in antigenic site Ⅲ in the G protein, which could react with the neutralizing antibodies, were observed. These results suggested that the virus belonged to the classic rabies virus, and both N and G genes diverged from the current vaccine strains used in China at either the nucleotide or the amino acid level.     

Effects of epoxyeicosatrienoic acids on levels of eNOS phosphorylation and relevant signaling transduction path-ways involved

Vascular endothelial cells play crucial roles in regulating cardiovascular function, maintaining car-diovascular homeostasis and preventing the occur-rence of cardiac and cerebral vascular diseases. All these protective effects are fulfilled through various vasoactive products secreted by endothelium including nitric oxide (NO), prostacyclin (PGI2) and endothe-lium-derived hyperpolarizing factor (EDHF). NO, pro-duced from L-arginine by endothelial nitric-oxide synthase (eNOS), is an impor…     

Effects of temperature acclimation on body mass and energy budget in the Chinese bulbul Pycnonotus sinensis

Chinese bulbuls(Pycnonotus sinensis) are small passerine birds that inhabit areas of central, southern and eastern China. Previous observations suggest that free–living individuals of this species may change their food intake in response to seasonal changes in ambient temperature. In the present study, we randomly assigned Chinese bulbuls to either a 30 °C or 10 °C group, and measured their body mass(BM), body temperature, gross energy intake(GEI), digestible energy intake(DEI), and the length and mass of their digestive tracts over 28 days of acclimation at these temperatures. As predicted, birds in the 30 °C group had lower body mass, GEI and DEI relative to those in the 10 °C group. The length and mass of the digestive tract was also lower in the 30 °C group and trends in these parameters were positively correlated with BM, GEI and DEI. These results suggest that Chinese bulbuls reduced their absolute energy demands at relatively high temperatures by decreasing their body mass, GEI and DEI, and digestive tract size.     

Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer

Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs.Conventional gene targeting in pig somatic cells is extremely inefficient.Zinc-finger nuclease(ZFN)technology has been shown to be a powerful tool for efficiently inducing mutations in the genome.However,ZFN-mediated targeting in pigs has rarely been achieved.Here,we used ZFNs to knock out the porcineα-1,3-galactosyl-transferase(GGTA1)gene,which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation.Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1.Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4%and 5.2%,respectively.The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer(SCNT).Three GGTA1 null piglets were born,and one knockout primary fibroblast cell line was established from a cloned fetus.Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane.Functionally,GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum.This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs.GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.     

一种新的cDNA 末端快速扩增获取全长cDNA的方法

为克隆精子发生相关基因的全长cDNA,根据mRNA差异显示获得的ESTs设计引物,利用一种新的cDNA末端快速扩增方法(SMARTRACE)扩增该EST的5′末端,并进行克隆测序,与mRNA差异显示获得ESTs拼接后,获得了三个新的全长cDNA.结果表明,SMARTRACE是一种简便、有效的克隆cDNA5′末端未知序列的技术. Abstract:To clone the full-length cDNAs of genes related to spermatogenesis,ESTs obtained by mRNA differential display were used to design gene-specific primer.Then SMART RACE was performed to obtain the 5′ region of these ESTs.After cloning,sequencing and splicing with ESTs obtained by mRNA differential display,three full-length cDNAs were obtained.The results indicate that SMART RACE is a simple and an effective technique for cloning 5′-end unknown sequence of gene.     

Isolation and functional analysis of a Brassica juncea gene encoding a com-ponent of auxin efflux carrier

Polar auxin transport plays a divergent role in plant growth and developmental processes including root and embryo development, vascular pattern formation and cell elongation. Recently isolated Arabidopsis pin gene family was believed to encode a component of auxin efflux carrier (G(?)lweiler et al, 1998). Based on the Arabidopsis pin1 sequence we have isolated a Brassica juncea cDNA (designated Bjpin1), which encoded a 70-kDa putative auxin efflux carrier. Deduced BjPIN1 shared 65% identities at protein level with AtPINl and was highly homologous to other putative PIN proteins of Arabidopsis (with highest homology to AtPIN3). Hydrophobic analysis showed similar structures between BjPINl and AtPIN proteins. Presence of 6 exons (varying in size between 65 bp and 1229 bp) and 5 introns (sizes between 89 bp and 463 bp) in the genomic fragment was revealed by comparing the genomic and cDNA sequences. Northern blot analysis indicated that Bjpin1 was expressed in most of the tissues tested, with a relatively h     

家禽(鹅、鸭、鸡)血清酯酶多态性比较血型学初步研究

鸡的酯酶在两个区域出现了变异, 靠近阳极的Es-1区共有3条带,表现出7种表型组合(AA、 BB、CC、AB、AC、BC和无带O型),分别受控于常染色体上相同基因座位上3个复等位基因(Es-1^A|、Es-1^B|、Es-1^C|)和1个隐性基因(Es-1^O|)。在原点和Es-1之间的Es-2区表现出有带(+)和无带(-)两种类型。鹅的血清酯酶与鸡截然不同。在鸡的Es-2区域,鹅的带谱信号弱(无)。在与鸡Es-1区域相应位置,鹅存在着8-9条谱带,其中的1号、2 -7号存在着个体水平的多态现象。鸭血清酯酶的聚丙烯酰胺谱带与鹅非常相似。鹅、鸭血清酯酶的遗传机制有待交配实验确定。