The role of a single N-linked glycosylation site for a functional epitope of herpes simplex virus type 1 envelope glycoprotein gC

A monoclonal antibody, B1C1, binding to an epitope of antigenic site II of the herpes simplex virus type 1 (HSV-1) glycoprotein gC-1, is a potent inhibitor of two important biological functions of gC-1: its binding to cell surface heparan sulfate and its binding to the receptor for complement factor C3b. Here, we have analyzed a B1C1-resistant HSV- 1 variant (HSV-12762/B1C1B4.2), obtained after passage of wild type HSV- 1 (HSV-12762) in the presence of high concentrations of B1C1. The transport of newly synthesized mutant gC-1 to the cell surface was comparable to that of wild type glycoprotein, but no binding of surface- associated mutant gC-1 to B1C1 was detected. However, mutant and wild type gC-1 bound equally well to other site II Mabs. Attachment of wild type but not mutant virus was inhibited by B1C1. Sequencing of the mutant gC-1 gene revealed only one nucleotide change, resulting in replacement of Thr150 by an Ile, in turn destroying an N-glycosylation site at Asn148. Loss of one complex type N-linked glycan was confirmed by endoglycosidase digestion and subsequent SDS-polyacrylamide gel electrophoresis. Circular dichroism analysis of purified gC-1 from cells infected with mutant or wild type virus did not reveal any difference in secondary structure between mutant and wild type gC-1. It was not possible to obtain a B1C1-resistant phenotype by nucleotide- directed mutagenesis of gC-1 where Asn148 was changed to a glutamine. These data demonstrated that the threonine of the glycosylation site and not the N-linked glycan in itself was essential for B1C1 binding      

Effect of mating system, flushing procedure, progesterone dose and donor ewe age on the yield and quality of embryos within a MOET program in sheep

Multiple ovulation and embryo transfer (MOET) has the potential to increase the rates of genetic improvement in sheep. However, better realization of this potential requires a higher yield of transferable embryos. Thus we investigated some factors that may contribute to high embryo yield and quality under field conditions, as part of an ongoing MOET program. Comparison of the effects of 2 breeding systems (natural service plus laparoscopic intrauterine AI vs natural service only) on embryo yield and quality indicated that while AI did not affect embryo recovery, it significantly (P < 0.05) improved fertilization rate and embryo quality, and increased (P < 0.05) embryo survival rate after transfer to recipients. Two flushing procedures (the original semi-laparoscopic and a modified version) were compared for effects on embryo recovery. The modifications made to the original collection method increased (P < 0.001) embryo recovery from 69.0 +/- 2.4 to 83.2 +/- 0.6%. The effects of the progestagen priming dosage during superovulatory treatment and ewe age on MOET outcome were also investigated. Donor ewes primed with 30-mg progestagen sponges came into estrus 1.9 h earlier (P < 0.05) than those primed with 45-mg sponges, but there was no difference in ovulation rate or embryo recovery, or in embryo survival after transfer between the 2 regimens. However, Chi-square analysis indicated a significant benefit in favor of the higher progesterone dose on both fertilization (P < 0.01) and embryo quality (P < 0.001). Age of donor ewe did not significantly affect the timing of estrus, fertilization rate or embryo survival after transfer. While adult ewes had higher (P < 0.05) ovulation rates and embryo yields, shearling ewes produced a much higher proportion of Grade 1 embryos (P < 0.05).     

Peripherally inserted central venous catheters. Low-risk alternatives for ongoing venous access.

We prospectively evaluated the use of peripherally inserted central venous catheters to provide ongoing venous access in general medical and surgical patients in a Department of Veterans Affairs medical center. Between 1985 and 1988 trained nurses successfully inserted 393 catheters in 460 suitable patients (an 85.4% success rate). Correct catheter tip placement in the superior vena cava was documented in 359 of the 393 (91.3%) catheter insertions, but an additional 30 catheters were in a position deemed adequate for the intended use. The mean duration of catheter use was 27.6 +/- 5.2 (1 standard deviation) days (median 20 days, range 1 to 370 days). A total of 65 patients left the hospital with catheters in place, with the mean length of catheter use at home being 36.2 +/- 6.0 days (range 2 to 266). In all, 79% of the catheters were in use until the successful completion of therapy or patient death; catheter-related complications led to premature catheter removal in the remaining 21%. Catheter-related complications included bland phlebitis (8.2%), occlusion (8.2%), local infection (3.6%), bacteremia or fungemia (2.1%), mechanical failure or rupture (2.6%), venous thrombosis (0.7%), and other (3.3%). One patient required vein excision for the management of suppurative phlebitis, but no deaths were attributed to catheter use. This study illustrates the use and safety of peripherally inserted central venous catheters to provide reliable vascular access over prolonged periods in an elderly veteran population. At our facility, percutaneous central venous catheters and surgically implanted (Hickman or Broviac) catheters are now reserved for use in patients in whom peripherally inserted catheters cannot be placed.     

PKB signaling and atrogene expression in skeletal muscle of aged mice

The purpose of this study was to determine if PKB signaling is decreased and contractile protein degradation is increased in extensor digitorum longus (EDL) and soleus (SOL) muscles from middle-aged (MA) and aged (AG) mice. We also examined the effect of age on atrogene expression in quadriceps muscle. PKB activity, as assessed by Thr(308) and Ser(473) phosphorylation, was significantly higher in EDL and SOL muscles from AG than MA mice. The age-related increase in PKB activity appears to be due to an increase in expression of the kinase, as PKB-α and PKB-β levels were significantly higher in EDL and SOL muscles from AG than MA mice. The phosphorylation of forkhead box 3a (FOXO3a) on Thr(32), a PKB target, was significantly higher in EDL muscles from AG than MA mice. The rate of contractile protein degradation was similar in EDL and SOL muscles from AG and MA mice. Atrogin-1 and muscle-specific RING finger protein 1 (MuRF-1) mRNA levels did not change in muscles from AG compared with MA mice, indicating that ubiquitin-proteasome proteolysis does not contribute to sarcopenia. A significant decrease in Bcl-2 and 19-kDa interacting protein 3 (Bnip3) and GABA receptor-associated protein 1 (Gabarap1) mRNA was observed in muscles from AG compared with MA mice, which may contribute to age-related contractile dysfunction. In conclusion, the mechanisms responsible for sarcopenia are distinct from experimental models of atrophy and do not involve atrogin-1 and MuRF-1 or enhanced proteolysis. Finally, a decline in autophagy-related gene expression may provide a novel mechanism for impaired contractile function and muscle metabolism with advancing age.     

白色生物技术在可持续大规模化学生产中的应用

目前几乎所有有机化学品和塑料是从原油和天然气中生产的, 而生物技术的应用使得利用可再生资源进行大规模化工生产成为可能。以下主要综述了白色生物技术, 即利用细菌、酵母或酶将可发酵糖转化为特定的化学产品的技术。白色生物技术极大节省了不可再生能源的消耗, 减少了温室气体的排放。在有利条件下, 如果化工生产中相关技术有了发展并且可以成功以木质纤维素为原料, 那么到2050年不可再生能源的消耗将减少将近2/3 (67%)。欧洲（EU-25）地区的分析表明, 白色生物技术相关的用地在未来几年的欧洲不会受到制约, 尤其是有大量闲置资源的东欧。另外, 虽然原则上可以在白色生物技术中使用自然的细菌和酶, 但是很多专家认为, 利用经遗传改造生物体(GMO)可以达到高产量、高浓度、高效率, 这对实现经济活力是必要的。值得注意的是, 目前并不是所有的重组基因和其他物种间的相互作用所带来的后果都可预见, 因此化工生产释放的GMOs的安全失活和处理非常重要, 但是如果采取足够的预防措施, 在白色生物技术中应用GMOs的风险是可以控制的。我们认为, 生物生产过程的技术突破、下游生产过程的控制、化石燃料的高价格、可发酵糖的低价获得是生物质化学产业发展中的关键因素, 这4个因素及其他伴随策略是发展整体白色生物技术的要求。     

Influence of Diet on the Proteome of Drosophila Melanogaster as Assessed by Two-Dimensional Gel Electrophoresis and Capillary Liquid Chromatography–Mass Spectrometry: The Hamburger Effect Revisited

Proteomic biomarker discovery has been called into question. Diamandis hypothesized that seemingly trivial factors, such as eating a hamburger, may cause sufficient proteomic change as to confound proteomic differences. This has been termed the hamburger effect. Little is known about the variability of complex proteomes in response to the environment. Two methods—two-dimensional gel electrophoresis (2DGE) and capillary liquid chromatography–electrospray ionization time-of-flight mass spectrometry (LCMS)—were used to study the hamburger effect in two cross-sections of the soluble fruit fly proteome. 2DGE measured abundant proteins, whereas LCMS measured small proteins and peptides. Proteomic differences between males and females were first evaluated to assess the discriminatory capability of the methods. Likewise, wild-type and white-eyed flies were analyzed as a further demonstration that genetically based proteomic differences could be observed above the background analytical variation. Then dietary interventions were imposed. Ethanol was added to the diet of some populations without significant proteomic effect. However, after a 24-h fast, proteomic differences were found using LCMS but not 2DGE. Even so, only three of ~1000 molecular species were altered significantly, suggesting that the influence of even an extreme diet change produced only modest proteomic variability, and that much of the fruit fly proteome remains relatively constant in response to diet. These experiments suggest that proteomics can be a viable approach to biomarker discovery.