Anti-CD8 antibodies can inhibit or enhance peptide-MHC class I (pMHCI) multimer binding: this is paralleled by their effects on CTL activation and occurs in the absence of an interaction between pMHCI and CD8 on the cell surface

Cytotoxic T lymphocytes recognize short peptides presented in association with MHC class I (MHCI) molecules on the surface of target cells. The Ag specificity of T lymphocytes is conferred by the TCR, but invariable regions of the peptide-MHCI (pMHCI) molecule also interact with the cell surface glycoprotein CD8. The distinct binding sites for CD8 and the TCR allow pMHCI to be bound simultaneously by both molecules. Even before it was established that the TCR recognized pMHCI, it was shown that CTL exhibit clonal heterogeneity in their ability to activate in the presence of anti-CD8 Abs. These Ab-based studies have since been interpreted in the context of the interaction between pMHCI and CD8 and have recently been extended to show that anti-CD8 Ab can affect the cell surface binding of multimerized pMHCI Ags. In this study, we examine the role of CD8 further using point-mutated pMHCI Ag and show that anti-CD8 Abs can either enhance or inhibit the activation of CTL and the stable cell surface binding of multimerized pMHCI, regardless of whether there is a pMHCI/CD8 interaction. We further demonstrate that multimerized pMHCI Ag can recruit CD8 in the absence of a pMHCI/CD8 interaction and that anti-CD8 Abs can generate an intracellular activation signal resulting in CTL effector function. These results question many previous assumptions as to how anti-CD8 Abs must function and indicate that CD8 has multiple roles in CTL activation that are not necessarily dependent on an interaction with pMHCI.     

干旱胁迫下牛心朴子幼苗相对含水量、质膜透性及保护酶活性变化

牛心朴子(Cynanchum komarovii)幼苗用不同浓度PEG-6000(聚乙二醇)模拟干旱处理,测定处理12d和复水24h中根、叶的RWC、质膜相对透性、MDA含量及几种保护酶(SOD、CAT、POD)活性变化情况,结果表明：高浓度胁迫后期,RWC明显下降,MDA含量增大,致使膜脂过氧化引起膜损伤;低、中浓度胁迫下,RWC下降程度低,细胞膜脂过氧化及膜透性影响小,且可能对膜脂过氧化起到一定的防御作用。复水后,低、中度胁迫下,各项指标能迅速恢复到CK水平,而高浓度胁迫下,除叶质膜透性、MDA含量、SOD活性不能恢复外,根、叶的其余指标均能达到CK水平。保护酶系统的作用,在一段时间内,可能是通过它们之间的相互协调而保持一个稳定的平衡态进行的。     

A modular, fully integrated ultra-high-throughput screening system based on confocal fluorescence analysis techniques

The rapid increase in size of compound libraries, as well as new targets emerging from the Human Genome Project, require progress in ultra-high-throughput screening (uHTS) systems. In a joint effort with scientists and engineers from the biotech and the pharmaceutical industry, a modular, fully integrated system for miniaturized uHTS was developed. The goal was to achieve high data quality in small assay volumes (1-4 microL) combined with reliable and unattended operation. Two new confocal fluorescence readers have been designed. One of the instruments is a 4-channel confocal fluorescence reader, measuring with 4 objectives in parallel. The fluorescence readout is based on single-molecule detection methods, allowing high sensitivity at low tracer concentrations and delivering an information-rich output. The other instrument is a confocal fluorescence imaging reader, where the images are analyzed in terms of generic patterns and quantified in units of intensity per pixel. Both readers are spanning the application range from assays with isolated targets in homogenous solution or membrane vesicle-based assays (4-channel reader) to cell-based assays (imaging reader). Results from a comprehensive test on these assay types demonstrate the high quality and robustness of this screening system.     

Enhanced heterologous polyketide production in<Emphasis Type="Italic"> Streptomyces</Emphasis> by exploiting plasmid co-integration

A plasmid named pSMALL was discovered in a Streptomyces coelicolor strain that significantly enhanced the levels of production of 15-methyl-6-deoxyerythronolide B, a polyketide lactone normally produced in low amounts by engineered polyketide synthase (PKS) genes. It is a co-integrate between a conventional SCP2*-derived Streptomyces expression plasmid, pJRJ2, and SCP2@, a variant of the parental SCP2* plasmid. SCP2@ has a 45-bp deletion 35 bp upstream of the start codon of the repI gene in the replication region; and this correlated with an enhanced plasmid copy number and polyketide overproduction by its derivatives. This discovery was exploited to construct pBOOST, a high-copy-number cloning vector that can be used to achieve greatly elevated (at least 25-fold), stable metabolite production by PKS genes cloned in SCP2*-derived vectors by forming co-integrates with them.     

Quantification of the O- and N-demethylated metabolites of hydrocodone and oxycodone in human liver microsomes using liquid chromatography with ultraviolet absorbance detection

High-performance liquid chromatographic assays for the O- and N-demethylated oxidative metabolites of hydrocodone and oxycodone formed in human liver microsomes are described. A solvent-solvent extraction/re-extraction procedure followed by reversed-phase HPLC with UV detection at 210 nm allows for the quantification of hydromorphone, norhydrocodone, oxymorphone and noroxycodone. Calibration curve concentration ranges were 0.63-400 microM (0.18-114 microg/ml) and 1.25-400 microM (0.36-114 microg/ml) for hydromorphone and norhydrocodone, respectively and 0.13-20 microM (0.04-6.03 microg/ml) and 1-200 microM (0.30-60 microg/ml) for oxymorphone and noroxycodone, respectively. Assay performance was determined by intra- and inter-assay precision and inaccuracies for quality control samples and was <15% for all metabolites at each quality control concentration. These methods provide good precision, accuracy and sensitivity for use in in vitro kinetic studies investigating the oxidative metabolism of hydrocodone and oxycodone in human liver microsomes.     

Sequential localization of two herpes simplex virus tegument proteins to punctate nuclear dots adjacent to ICP0 domains

The subcellular localization of herpes simplex virus tegument proteins during infection is varied and complex. By using viruses expressing tegument proteins tagged with fluorescent proteins, we previously demonstrated that the major tegument protein VP22 exhibits a cytoplasmic localization, whereas the major tegument protein VP13/14 localizes to nuclear replication compartments and punctate domains. Here, we demonstrate the presence of a second minor population of VP22 in nuclear dots similar in appearance to those formed by VP13/14. We have constructed the first-described doubly fluorescence-tagged virus expressing VP22 and VP13/14 as fusion proteins with cyan fluorescent protein and yellow fluorescent protein, respectively. Visualization of both proteins within the same live infected cells has indicated that these two tegument proteins localize to the same nuclear dots but that VP22 appears there earlier than VP13/14. Further studies have shown that these tegument-specific dots are detectable as phase-dense bodies as early as 2 h after infection and that they are different from the previously described nuclear domains that contain capsid proteins. They are also different from the ICP0 domains formed at cellular nuclear domain 10 sites early in infection but, in almost all cases, are located in juxtaposition to these ICP0 domains. Hence, these tegument proteins join a growing number of proteins that are targeted to discrete nuclear domains in the herpesvirus-infected cell nucleus.     

Antitumour benzothiazoles. Part 20: 3'-cyano and 3'-alkynyl-substituted 2-(4'-aminophenyl)benzothiazoles as new potent and selective analogues

The synthesis of a new series of antitumour 2-(4-aminophenyl)benzothiazole analogues, substituted in the 3'-position by cyano or alkynyl groups, is described. Several of the analogues, notably the 5-fluorinated compounds 7c and 9c, were found to possess potent in vitro activity against MCF-7 and MDA 468 human cancer cell lines. More comprehensive in vitro analysis (NCI 60-cell line) established compound 7c as a particularly potent and selective 2-(4-aminophenyl)benzothiazole analogue.     

Nonpeptide alpha V beta 3 antagonists. Part 9: Improved pharmacokinetic profile through the use of an aliphatic, des-amide backbone

A series of alphaVbeta3 receptor antagonists lacking the amide bond of previously-reported 'chain-shortened' compounds is described. Replacement of the lone amide bond with two methylene groups in this series yields more lipophilic compounds that have longer half-lives, lower clearance, and greater oral bioavailability when administered to dogs.     

Dipyridyl amines: potent metabotropic glutamate subtype 5 receptor antagonists

Modulation of the metabotropic glutamate subtype 5 (mGlu5) receptor may be useful in the treatment of a variety of central nervous system disorders. Herein, we report on the discovery, synthesis, and biological evaluation of dipyridyl amines as small molecule mGlu5 antagonists.     

Substantial genetic substructuring in southeastern and alpine Australia revealed by molecular phylogeography of the Egernia whitii (Lacertilia: Scincidae) species group

Palaeoclimatic events and biogeographical processes since the mid-Tertiary are believed to have strongly influenced the evolution and distribution of the terrestrial vertebrate fauna of southeastern Australia. We examined the phylogeography of the temperate-adapted members of the Egernia whitii species group, a group of skinks that comprise both widespread low- to mid-elevation (E. whitii) and montane-restricted species (Egernia guthega, Egernia montana), in order to obtain important insights into the influence of past biogeographical processes on the herpetofauna of southeastern Australia. Sequence data were obtained from all six temperate-adapted species within the E. whitii species group, and specifically from across the distributional ranges of E. whitii, E. guthega and E. montana. We targeted a fragment of the ND4 mitochondrial gene (696 bp) and analysed the data using maximum likelihood and Bayesian methods. Our data reveal a deep phylogeographical break in the east Gippsland region of Victoria between 'northern' (Queensland, New South Wales, Australian Capital Territory) and 'southern' (Victoria, Tasmania, South Australia) populations of E. whitii. This divergence appears to have occurred during the late Miocene-Pliocene, with the Gippsland basin possibly forming a geographical barrier to dispersal. Substantial structuring within both the 'northern' and the 'southern' clades is consistent with the effects of Plio-Pleistocene glacial-interglacial cycles. Pleistocene glacial cycles also appear to have shaped the phylogeographical patterns observed in the alpine species, E. guthega and E. montana. We used our results to examine the biogeographical process that led to the origin and subsequent diversification of the lowland and alpine herpetofauna of southeastern Australia.