Integration of mechanical cell disruption and fluidised bed recovery of G3PDH from unclarified disrupted yeast: a comparative study of the performance of unshielded and polymer shielded dye-ligand chromatography systems

The development of a simplified process for the simultaneous disruption and direct selective purification of intracellular proteins from unclarified yeast disruptate has been investigated. The recovery of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from baker's yeast was selected as a potential demonstration of the generic applicability and practical feasibility of this integrated technique. The application of an adsorbent characterised by high density (UpFront steel-agarose; rho=2.65g ml(-1)) facilitated the combining of cell disruption operation (bead milling of 50% ww/v of yeast suspension at 7.2 lh(-1)) with fluidised bed dye-ligand (Cibacron Blue 3GA) adsorption operated immediately downstream of the disrupter. The adoption of a polymer shielded, dye-ligand technique advanced recovery efficiency. It was demonstrated that G3PDH could be recovered with a yield of 67.5% bound activity and a specific activity of 40.2IU mg(-1), after a single step elution with 0.15M NaCl. The generic application of this approach has been evaluated.     

BRCTx is a novel, highly conserved RAD18-interacting protein

The BRCT domain is a highly conserved module found in many proteins that participate in DNA damage checkpoint regulation, DNA repair, and cell cycle control. Here we describe the cloning, characterization, and targeted mutagenesis of Brctx, a novel gene with a BRCT motif. Brctx was found to be expressed ubiquitously in adult tissues and during development, with the highest levels found in testis. Brctx-deficient mice develop normally, show no pathological abnormalities, and are fertile. BRCTx binds to the C terminus of hRAD18 in yeast two-hybrid and immunoprecipitation assays and colocalizes with this protein in the nucleus. Despite this, Brctx-deficient murine embryonic fibroblasts (MEFs) do not show overt sensitivity to DNA-damaging agents. MEFs from Brctx-deficient embryos grow at a similar rate to wild-type MEF CD4/CD8 expressions, and the cell cycle parameters of thymocytes from wild-type and Brctx knockout animals are indistinguishable. Intriguingly, the BRCT domain of BRCTx is responsible for mediating its localization to the nucleus and centrosome in interphase cells. We conclude that, although highly conserved, Brctx is not essential for the above-mentioned processes and may be redundant.     

纳米管和镉对蚕豆幼苗根系的毒害效应

以蚕豆幼苗为实验材料,采用水培法研究不同浓度(0、2、4、8、16 mg·L~(-1))羟基化多壁碳纳米管(MWCNTs-OH)与10μmol·L~(-1)镉(Cd)单一和联合作用对蚕豆幼苗根系生长、生理及Cd含量的影响。结果表明:(1)单一MWCNTs-OH处理下,随着MWCNTs-OH浓度(2～8 mg·L~(-1))的增加,蚕豆的根长度增加,且■产生速率增加的同时会伴随着SOD、POD活性升高。(2)单一Cd处理的幼苗根系MDA含量和■产生速率均高于对照,其中■产生速率较对照增加了1.98倍。(3)MWCNTs-OH联合处理诱导了蚕豆根■的产生和MDA含量增加,16 mg·L~(-1) MWCNTs-OH和Cd共同作用对蚕豆幼苗根的损伤最大,会促使部分根尖细胞受损脱落,Cd含量明显下降。研究发现,低浓度MWCNTs-OH可促进蚕豆的根生长,降低Cd的毒性;高浓度MWCNTs-OH(16 mg·L~(-1))和Cd对蚕豆幼苗根的致毒性表现为协同效应。     

A single dose of recombinant VSV-RABVG vaccine provides full protection against RABV challenge

Highlights:
1. A replication-competent recombinant VSV with RABV-G protein replacement was generated.
2. Single dose of VSV-RABVG immunization induce potent antigen-specific humoral immune response, especially the virus neutralizing antibodies.
3. Mice intranasally immunized with single dose of VSV-RABVG were 100% protected upon RABV challenge.     

Characterization of 12 microsatellite loci of the human MHC in a panel of reference cell lines

 The human genome contains a large number of interspersed microsatellite repeats which exhibit a high degree of polymorphism and are inherited in a Mendelian fashion, making them extremely useful genetic markers. Several microsatellites have been described in the HLA region, but allele nomenclature, a set of broadly distributed controls, and typing methods have not been standardized, which has resulted in discrepant microsatellite data between laboratories. In this report we present a detailed protocol for genotyping microsatellites using a semi-automated fluorescence-based method. Twelve microsatellites within or near the major histocompatibility complex (MHC) were typed in the 10th International Histocompatibility Workshop homozygous typing cell lines (HTCs) and alleles were designated based on size. All loci were sequenced in two HTCs providing some information on the level of complexity of the repeat sequence. A comparison of allele size obtained by genotyping versus that obtained by direct sequencing showed minor discrepancies in some cases, but these were not unexpected given the technical differences in the methodologies. Fluorescence-based typing of microsatellites in the MHC described herein is highly efficient, accurate, and reproducible, and will allow comparison of results between laboratories. Received: 10 May 1997 / Revised: 1 August 1997