Materials for Chinese Ainsliaea (Compositae)

The genus, Ainsliaea DC. from China is revised in this paper. Three species, A. nana Y. C. Tseng, A. pingbianensis Y. G. Tseng and A. trinervis Y. C. Tseng, are newly described; two species, A. chapaensis Merr. and A. angustifolia Hook. f. et Thoms. ex C. B. Clarke are new records for China and two new combinations, A. apteroides (Chang) Y. C. Tseng and A. macrocephala (Mattf.) Y. C. Tseng, are made. In addition, one species, A. hypoleuca Diels ex Limpr. and four varieties, A. bonatii Beauverd var. arachnoidea Beauverd, A. pteropoda DC. var. leiophylla Franch., A. elegans Hemsl. var. tomentosa Mattf. and A. glabra Hemsl. var. tenuiculis (Mattf.)Chang, are reduced to synonyms.     

构建多顺反子表达载体的有效工具——FMDV 2A

近年来肿瘤多基因治疗倍受关注,然而目前缺少一种有效的构建多顺子的工具。传统的构建多顺反子的工具,如IRES等,由于存在结构大,上下游基因表达差异显著等缺陷,严重限制了其应用。FMDV2A,因其具有结构小、剪切效率高等优点为多基因治疗带来了新的希望。主要介绍了FMDV2A的特性、"剪切"活力及其在构建多顺反子载体中的应用。     

SAR study of bisamides as cyclophilin a inhibitors for the development of host-targeting therapy for hepatitis C virus infection

The therapy of chronic hepatitis C virus infections has significantly improved with the development of direct-acting antivirals (DAAs), which contain NS3/4A protease, NS5A, and NS5B polymerase inhibitors. However, mutations in specific residues in these viral target genes are associated with resistance to the DAAs. Especially inhibitors of NS3/4A protease and NS5A, such as grazoprevir and velpatasvir, have a low barrier to resistant mutations. As a result, the mutations influence the virological outcomes after DAA treatment. CypA inhibitors, as host-targeted agents, act on host factors to inhibit HCV replication, exhibiting a high resistance barrier and pan-genotype activities against HCV. Therefore, they can be developed into alternative, more effective anti-HCV agents. However, CypA inhibitors are natural products and analogs. Based on previous studies, bisamide derivatives were designed and synthesized to develop a novel class of CypA inhibitors. Bisamide derivative 7c is a promising compound with potent anti-HCV activity at subtoxic concentrations. Surface plasmon resonance experiments revealed that 7c directly binds to CypA. All these studies indicated that the derivative 7c is a potent CypA inhibitor, which can be used as a host-targeted agent in combination with other antiviral agents for anti-HCV treatment.     

检测A组轮状病毒的微阵列技术初探

采用荧光染料TAMRA标记上游引物,经RT semi nestedPCR扩增A组轮状病毒的保守区域,并将扩增产物与自行研制的玻片微阵列进行杂交。经杂交信号扫描分析,可简便快速地检出A组轮状病毒,并能达到高灵敏性,为下步进入临床打下了基础。     

Microscale high-performance liquid chromatography–electrospray tandem mass spectrometry assay for cyclosporin A in blood

To facilitate quantitative analysis of cyclosporin A in low volume blood samples we developed a sensitive and specific microscale reversed-phase HPLC–electrospray tandem mass spectrometry assay. Blood samples (100 μl) were prepared by acetonitrile precipitation and C₁₈ solid-phase extraction. Detection was by multiple-reactant monitoring. The method was linear over the range 5–1000 μg/l (r≥0.997) with accuracy between 95.4 and 102.0% over this range. Total imprecision was 11.1% at 10 μg/l and 2.8% at 800 μg/l. Absolute recovery of cyclosporin A and internal standard was 72.5 and 73.3%, respectively. When this method was evaluated against a conventional HPLC with UV detection, in patient samples, they were interchangeable (y=0.988x+10.0, r=0.996). This HPLC–ESI-MS–MS method will be applicable to therapeutic monitoring in paediatric transplant patients and multiple point pharmacokinetic studies in animals and humans.     

Nutrition of Acetobacter rancens S3 and F11 Isolated from Tanks for Vinegar Production

The strains S3 and F11 which were isolated respectively from static and submerged tanks for vinegar production were identified as Acetobacter rancens. Neither strain grew in an ammonium defined medium containing ethanol, glucose, glycerol or organic acids as the sole carbon source. When casamino acids were added, they grew luxuriantly with lactate, ethanol or glycerol as the carbon source and less well with acetate or glucose. They grew, forming much acetic acid, in defined ethanol medium when alanine was supplied in place of casamino acids, but strain S3 showed a longer lag time than strain Fl1. This lag time could be shortened by addition of aspartate and glutamate. These amino acids could be replaced by succinate, fumarate, malate, lactate, pyruvate or propionate but not by glucose. Both strains required lactate or pyruvate in defined glucose medium but many other organic acids, which were effective in defined ethanol medium, were ineffective or slightly effective in glucose medium.     

A surface plasmon resonance-based assay for small molecule inhibitors of human cyclophilin A

A simple protocol for generating a highly stable and active surface plasmon resonance (SPR) sensor surface of recombinant human hexahistidine cyclophilin A (His-CypA) is described. The sensor surface was sensitive and stable enough to allow, for the first time, the screening and ranking of several novel small-molecule (Mr approximately 250-500 Da) ligands in a competition binding assay with cyclosporin A (CsA). It also allowed us to accurately determine the kinetic rate constants for the interaction between His-CypA and CsA. His-CypA was first captured on a Ni2+-nitrilotriacetic acid (NTA) sensor chip and was then briefly covalently stabilized, coupling via primary amines. The significant baseline drift observed due to dissociation of weakly bound His-CypA from the Ni2+-NTA moiety was eliminated, resulting in a surface that was stable for at least 36 h. In addition, immobilized protein activity levels were high, typically between 85 and 95%, assayed by the interaction between His-CypA and CsA. The mean equilibrium dissociation constant for CsA (K(dCsA)) binding to the immobilized His-CypA was 23+/-6 nM, with on and off rates of 0.53+/-0.1 microM(-1) s(-1) and 1.2+/-0.1 (x 10(-2)) s(-1), respectively. These values agree well with the values for the corresponding binding constants determined from steady-state and kinetic fluorescence titrations in solution.     

Structural Basis for the Activity and Substrate Specificity of Fluoroacetyl-CoA Thioesterase FlK

The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr⁴², His⁷⁶, and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg¹²⁰ located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity.     

Evidence for a quaternary structure change in the cooperative binding of cyanide to ferrihemoglobin A.

At pH 6.5 in a 0.05 $\text{M}$ bis-Tris-0.1 $\text{M}$ Cl^? buffer, tetra aquo ferrihemoglobin A (HbA⁺) binds CN^? with a Hill coefficient of n = 1.4. The Hill coefficient increases slightly and the average CN^? affinity decreases in the presence of excess spin labeled triphosphate (SLTP). This is probably the result of the finding that the SLTP exhibits a twofold higher affinity for HbA⁺ than for tetra cyano HbA⁺. Over the course of heme saturation with CN^?, a certain fraction of the SLTP is specifically released. This shows linkage between organic phosphate binding and heme ligation. These findings bear a marked resemblance to the ligand binding phenomena in hemoglobin A (HbA) and provide good evidence that under these experimental conditions, HbA⁺ is undergoing a quaternary conformation change as the hemes become saturated.     

Characterization of human autoantibodies specific for lamin A

We have characterized human autoimmune polyclonal antibodies reactive with lamin A, a 74 kDa peripheral protein of the nuclear envelope. Unlike other known antibodies to lamin A, the antibodies described here do not crossreact with the structurally related lamin C. These antibodies feature only chi light chains suggesting that their specificity is restricted to a limited number of epitopes. Based on the known amino acid sequence of human lamins A and C, the epitope(s) are most likely located in the 80 amino acid carboxyl tail of mature lamin A.     

Fluorescence quenching method for the determination of bleomycins A5 and A2 with halofluorescein dyes

In weak acidic medium, the anticancer antibiotics bleomycin A₅ (BLMA₅) and bleomycin A₂ (BLMA₂) bind with halofluorescein dyes, such as erythrosin (Ery), eosin Y (EY) and eosin B (EB), to form ion‐association complexes, which causes fluorescence quenching of halofluorescein dyes. The quenching values (ΔF) are directly in proportional to the concentrations of bleomycins over the range 0.09–2.5 µg/mL. Based on this, a fluorescence quenching method for the determination of BLMA₅ and BLMA₂ has been developed. The dynamic range is 0.12–2.5 µg/mL for the determination of BLMA₅ and 0.09–2.0 µg/mL for BLMA₂, with detection limits (3σ) of 0.04 µg/mL for BLMA₅, 0.03 µg/mL for BLMA₂, respectively. It has been applied to determine the two antibiotics in human serum, urine and rabbit serum samples. The recovery is in the range 90–102%. In this work, the optimum reaction conditions and the spectral characteristics of the fluorescence are investigated. The reasons for fluorescence quenching are discussed, based on the fluorescence theory. Copyright © 2007 John Wiley & Sons, Ltd.     

SRp30a (ASF/SF2) regulates the alternative splicing of caspase-9 pre-mRNA and is required for ceramide-responsiveness

Two splice variants are derived from the caspase-9 gene, proapoptotic caspase-9a and antiapoptotic caspase-9b, by either the inclusion or exclusion of an exon 3, 4, 5, and 6 cassette. Previous studies from our laboratory have shown that the alternative splicing of caspase-9 and the phosphorylation status of SR proteins, a conserved family of splicing factors, are regulated by chemotherapy and ceramide via the action of protein phosphatase-1. In this study, a link between ceramide, SR proteins, and the alternative splicing of caspase-9 was established. The downregulation of SRp30a in A549 cells by RNA interference technology resulted in an increase in the caspase-9b splice variant, with a concomitant decrease in the caspase-9a splice variant, thereby significantly decreasing the caspase-9a/9b ratio from 1.67 +/- 0.11 to 0.56 +/- 0.08 (P < 0.005). The specific downregulation of SRp30a also inhibited the ability of exogenous ceramide treatment to induce the inclusion of the exon 3, 4, 5, and 6 cassette. Therefore, we have identified SRp30a as an RNA trans-acting factor that functions as a major regulator of caspase-9 pre-mRNA processing and is required for ceramide to mediate the alternative splicing of caspase-9.     

中国铁角蕨属(铁角蕨科)一新名称

提出新名称 Asplenium dulongjiangense Y.F.Deng以代替晚出同名 Asplenium qiujiangense Chingex S.H.Wu non Nakaike,1 986。     

Components of plastic: experimental studies in animals and relevance for human health

Components used in plastics, such as phthalates, bisphenol A (BPA), polybrominated diphenyl ethers (PBDE) and tetrabromobisphenol A (TBBPA), are detected in humans. In addition to their utility in plastics, an inadvertent characteristic of these chemicals is the ability to alter the endocrine system. Phthalates function as anti-androgens while the main action attributed to BPA is oestrogen-like activity. PBDE and TBBPA have been shown to disrupt thyroid hormone homeostasis while PBDEs also exhibit anti-androgen action. Experimental investigations in animals indicate a wide variety of effects associated with exposure to these compounds, causing concern regarding potential risk to human health. For example, the spectrum of effects following perinatal exposure of male rats to phthalates has remarkable similarities to the testicular dysgenesis syndrome in humans. Concentrations of BPA in the foetal mouse within the range of unconjugated BPA levels observed in human foetal blood have produced effects in animal experiments. Finally, thyroid hormones are essential for normal neurological development and reproductive function. Human body burdens of these chemicals are detected with high prevalence, and concentrations in young children, a group particularly sensitive to exogenous insults, are typically higher, indicating the need to decrease exposure to these compounds.     

Effect of aging on induction of rat liver messenger RNA activity for malic enzyme

Rats were fasted and then refed a high carbohydrate-fat free diet, and the activities of the mRNA coding for liver malic enzyme [EC 1.1.1.40] in 6-week-old and 10-month-old male rats were determined by in vitro translation of the liver cytoplasmic poly(A)-containing RNA in a rabbit reticulocyte lysate. After refeeding the mRNA activities of the young rats were about 7-fold of those of the aged rats, and roughly parallel to the enzyme activities. This suggests that the age-dependent impairment of the enzyme induction [Iritani, N. et al. (1981) Biochim. Biophys. Acta 665, 636] can be ascribed to the decrease of mRNA activity.     

Identification of Ski as a target for Aurora A kinase

Ski is a negative regulator of the transforming growth factor-β and other signalling pathways. The absence of SKI in mouse fibroblasts leads to chromosome segregation defects and genomic instability, suggesting a role for Ski during mitosis. At this stage, Ski is phosphorylated but to date little is known about the kinases involved in this process. Here, we show that Aurora A kinase is able to phosphorylate Ski in vitro. In vivo, Aurora A and Ski co-localized at the centrosomes and co-immunoprecipitated. Conversely, a C-terminal truncation mutant of Ski (SkiΔ491-728) lacking a coiled-coil domain, displayed decreased centrosomal localization. This mutant no longer co-immunoprecipitated with Aurora-A in vivo, but was still phosphorylated in vitro, indicating that the Ski–Aurora A interaction takes place at the centrosomes. These data identify Ski as a novel target of Aurora A and contribute to an understanding of the role of these proteins in the mitotic process.     

Evidence for the glycoprotein nature of radish beta-fructosidase.

Concanavalin A (Con A) was utilized free, bound to Sepharose 4 B or cross-linked to glutaraldehyde to investigate the possibility of binding this lectin to radish beta-fructosidase (E.C.3.2.1.26). The choice of cross-linked Con A as affinoadsorbent is discussed and standard conditions for binding are defined. Specificity of precipitation of this enzyme by the lectin was especially investigated. Thus, the possibility of binding was tested in the presence of high ionic strength, ethylene glycol, alpha-methyl mannoside, alpha-methyl glucoside and during periodate oxidation of the enzyme. Based on the interactions observed between beta-fructosidase and Con A under these conditions it is concluded that the saccharide binding site of the lectin is primarily involved with a secondary contribution from the hydrophobic site. The specificity of binding and the complete precipitation of beta-fructosidase activity by the insolubilized lectin imply that all beta-fructosidase activity measured in Raphanus sativus seedling extracts is linked to (a) glycoprotein form(s) of this enzyme.     

Inflammation: a novel mechanism for the transport of extracellular nucleotide-induced arachidonic acid by S100A8/A9 for transcellular metabolism

Extracellular nucleotides cause neutrophil degranulation by activating the purinergic receptor subtype P2Y. However, the molecular mechanism involved in the signal pathway remains unknown. A hypothetical scheme suggesting that leukotriene(s) and leukotriene receptor(s) activation is required for extracellular nucleotide-mediated neutrophil degranulation is presented here. Subsequent to the extracellular nucleotide binding to its receptors, intracellular arachidonic acid (AA) levels are elevated. Although AA is a known substrate of the lipoxygenase pathway mediated by 5-lipoxygenase, excess AA could form a complex with S100A8/A9 for transport to the extracellular milieu. Extracellular availability of the S100A8/A9+AA complex could potentially be used for transcellular metabolism by resting and/or activated leukocytes (PMN, MN), vascular endothelium and smooth muscle cells at the inflammatory foci. Once imported into the resting and/or activated leukocytes, AA derived from the S100A8/A9+AA complex could serve as a substrate in the 5-lipoxygenase-mediated leukotriene pathway. Essentially, in addition to extracellular nucleotide-induced leukotrienes, AA derived from the S100A8/A9+AA complex could also be utilized for the synthesis of inflammatory mediators such as leukotriene B(4)(LTB(4)), which in turn could trigger leukocyte degranulation, as well as cellular damage to vascular endothelium and smooth muscle cells, thereby exacerbating inflammation.