七种蒿属植物种子重量形状及萌发特性的比较研究

在实验室条件下 ,对 7种蒿属植物种子 (差巴嘎蒿、乌丹蒿、万年蒿、大籽蒿、黄蒿、野艾蒿和冷蒿 )进行重量、形状及萌发特性的比较研究。沙生先锋植物乌丹蒿和差巴嘎蒿的种子重量较大、形状扁平 ,这些特征是植物对流沙环境进化的适应机制之一。黄蒿种子小且呈圆形 ,具有持久土壤种子库 ,因此黄蒿抗干扰能力较强。 7种蒿属植物有 3种萌发格局 :大籽蒿、万年蒿、差巴嘎蒿和冷蒿的萌发前期快 ,后期平缓 ;野艾蒿和黄蒿整个萌发过程平缓 ;乌丹蒿早期和后期萌发平缓 ,中间快。乌丹蒿推迟萌发高峰是它比差巴嘎蒿更适应流沙环境的机制之一。从种子萌发格局分析 ,黄蒿种子具有生理后熟或休眠机制 ,大籽蒿种子萌发是典型的机会主义。黄蒿、野艾蒿和冷蒿种子具有风险分摊的萌发机制。种子重量和形状与发芽率之间无相关性 ,重量和形状则显著相关。     

Occurrence of indole alkaloids among secondary metabolites of soil Aspergillus

The occurrence of indole alkaloids among secondary fungal metabolites was studied in species of the genus Aspergillus, isolated from soils that were sampled in various regions of Russia (a total of 102 isolates of the species A. niger, A. phoenicis, A. fumigatus, A. flavus, A. versicolor, A. ustus, A. clavatus, and A. ochraceus). Clavine alkaloids were represented by fumigaclavine, which was formed by A. fumigatus. alpha-Cyclopiazonic acid was formed by isolates of A. fumigatus, A. flavus, A. versicolor, A. phoenicis, and A. clavatus. The occurrence of indole-containing diketopiperazine alkaloids was documented for isolates of A. flavus, A. fumigatus, A. clavatus, and A. ochraceus. No indole-containing metabolites were found among the metabolites of A. ustus or A. niger.     

Characterization of the function of the ver-1A and ver-1B genes, involved in aflatoxin biosynthesis in Aspergillus parasiticus.

The ver-1A gene was cloned and its nucleotide sequence was determined as part of a previous study on aflatoxin B1 (AFB1) biosynthesis in the filamentous fungus Aspergillus parasiticus SU-1. A second copy of this gene, ver-1B, was tentatively identified in this fungal strain. In this study, ver-1B was cloned by screening an A. parasiticus cosmid library with a ver-1A probe. The nucleotide sequence of ver-1B was determined. The predicted amino acid sequence of ver-1B had 95% identity with ver-1A. A translational stop codon, found in the ver-1B gene coding region, indicated that it encodes a truncated polypeptide. To confirm the function of the ver-1 genes in AFB1 synthesis, a plasmid (pDV-VA) was designed to disrupt ver-1A and/or ver-1B by transformation of the AFB1 producer A. parasiticus NR-1. One disruptant, VAD-102, which accumulated the pathway intermediate versicolorin A was obtained. Southern hybridization analysis of VAD-102 revealed that ver-1A but not ver-1B was disrupted. A functional ver-1A gene was transformed back into strain VAD-102. Transformants which received ver-1A produced AFB1, confirming that ver-1A is the only functional ver-1 gene in A. parasiticus SU-1 and that its gene product is involved in the conversion of versicolorin A to sterigmatocystin in AFB1 biosynthesis. A duplicated chromosomal region (approximately 12 kb) was identified upstream from ver-1A and ver-1B by Southern hybridization analysis. This duplicated region contained the aflR gene, which is proposed to be one regulator of AFB1, synthesis. A similar gene duplication was also identified in several other strains of A. parasiticus.     

17种桤木属植物的亲缘关系研究及模糊种鉴定

利用AFLP分子标记结合形态学指标,采用UPGMA法进行聚类分析,对桤木属17个种57份材料进行了亲缘关系研究及一个模糊种鉴定。结果表明：7对引物扩增出369条带,其中346个多态位点,多态位点百分率为93.77%;根据AFLP标记位点聚类分析,在相似系数为0.782时,17种桤木属植物可分为4类,第一类为日本桤木(Alnus japonica);第二类为绿桤木(A.viridis)、意大利桤木(A.cordata)、欧洲桤木(A.glutinosa)、模糊种、四川桤木(A.cremastogyne)、江南桤木(A.trabeculosa)、斑点桤木(A.incana ssp.rugosa)、东北亚灰桤木(A.hirsuta)、台湾桤木(A.formosana)、日本特有桤木(A.firma)和裂叶桤木(A.sinuata);第三类为灰桤木(A.incana)、红桤木(A.rubra)及薄叶桤木(A.tenifolia);第四类喜马拉雅灰桤(A.nitida)和尼泊尔桤木(A.nepalensis)。根据形态学聚类分析,在距离为1.4时可分为三类,意大利桤木(A.cordata)单独为一类;日本桤木(A.japonica)、台湾桤木(A.formosana)、喜马拉雅灰桤木(A.nitida)、江南桤木(A.trabeculosa)和东北亚灰桤木(A.hirsuta);第三类包括模糊种、灰桤木(A.incana)、斑点桤木(A.incana ssp.rugosa)、裂叶桤木(A.sinuata)、红桤木(A.rubra)、欧洲桤木(A.glutinosa)、绿桤木(A.viridis)、四川桤木(A.cremastogyne)、薄叶桤木(A.tenuifolia)和尼泊尔桤木(A.nepalensis)。经形态特征和AFLP分析鉴定模糊种为欧洲桤木。形态学聚类与AFLP聚类结果基本一致,但仍存在一定的差异,说明桤木属植物遗传背景丰富,种的分子分类地位和形态学分类地位具有一定的差异。     

Characterization of a novel acidic protein of 38 kDa, A0, in yeast ribosomes which immunologically cross-reacts with the 13 kDa acidic ribosomal proteins, A1/A2

A new ribosomal protein of 38 kDa, named A0, was detected in yeast ribosomes on immunoblotting. The antibody used here was that against A1/A2, 13 kDa acidic ribosomal proteins which cross-reacted with A0. Although A0 and A1/A2 share common antigenic determinants, they differ in the following biochemical properties. While A1/A2 could be extracted from ribosomes with ethanol and ammonium sulfate, A0 could not. A0 gave two protein spots in a less acidic region than for A1/A2 on two-dimensional gel electrophoresis. The heterogeneity observed for A0 was ascribable to phosphorylation because one spot disappeared after treatment of the ribosomes with phosphatase. The syntheses of A0 and A1/A2 are directed by different mRNA species, as judged with a cell-free translation system, ruling out the possibility that A0 is a precursor of A1/A2. Although a mammalian ribosomal protein equivalent to A0 has been shown to be associated with 13 kDa acidic proteins in the cytoplasm, essentially no A0 was detected on immunoblotting in the yeast cytosol, while a small but detectable amount of A1/A2 was present. The possibility that A0 is a eukaryotic equivalent of L10 of Escherichia coli is discussed.     

Regulation of S100A8/A9 (calprotectin) binding to tumor cells by zinc ion and its implication for apoptosis-inducing activity

S100A8/A9 (calprotectin), which is released by neutrophils under inflammatory conditions, has the capacity to induce apoptosis in various cells. We previously reported that S100A8/A9 induces apoptosis of EL-4 lymphoma cells via the uptake of extracellular zinc in a manner similar to DTPA, a membrane-impermeable zinc chelator. In this study, S100A8/A9-induced apoptosis was examined in several cell lines that are weakly sensitive to DTPA, suggesting S100A8/A9 is directly responsible for apoptosis in these cells. Since zinc inhibits apoptosis of MM46, one of these cells, the regulation by zinc of the capacity of S100A8/A9 to bind MM46 cells was studied. When MM46 cells were incubated with S100A8/A9 in standard or zinc-depleted medium, the amounts of S100A8/A9 bound to cells was markedly lower at 3 h than at 1 h. In contrast, when MM46 cells were incubated with S100A8/A9 in the presence of high levels of zinc, binding to cells was the same at 1 and 3 h. When the cells were permeabilized with saponin prior to analysis, a larger amount of cell-associated S100A8/A9 was detected at 3 h. The amount was further increased in cells treated with chloroquine, suggesting that S100A8/A9 was internalized and degraded in lysosomes. Although it has been reported that S100A8/A9 binds to heparan sulfate on cell membranes, the amount of S100A8/A9 bound to MM46 cells was not reduced by heparinase treatment, but was reduced by trypsin treatment. These results suggest that S100A8/A9 induces apoptosis by direct binding to MM46 cells, and that this activity is regulated by zinc.     

Development of bacterial expression system with high yield of CYP3A7, a human fetus-specific form of cytochrome P450

In an E. coli expression system for human cytochrome P450 3A7 (CYP3A7), holo-CYP3A7 was not expressed as judged by CO-difference spectra, although apo-CYP3A7 was clearly detected by Western blot analysis. Unlike CYP3A7, CYP3A4 was expressed efficiently as a hemoprotein in E. coli transformed with a CYP3A4 expression plasmid. To achieve the high yield of the holo-CYP3A7 in E. coli, we examined a causal residue(s) preventing the expression of the holo-CYP3A7 using the chimeric gene of CYP3A4 with CYP3A7. It was found that the region between residues 405 and 503 of CYP3A7 was responsible for the prevention of the holo-CYP3A7 expression in E. coli. Among amino acids examined, substitution of Thr at position 485 in CYP3A7 with Pro, which is at the corresponding position of CYP3A4, resulted in an increase in the amount of holo-CYP3A7. The Thr residue was adjacent to the heme-binding region of CYP3A7. Thus, it appeared that the incorporation of heme into CYP3A7 was possibly affected by this particular amino acid residue. Moreover, holo-CYP3A7 was expressed efficiently when CYP3A7 was co-expressed with molecular chaperone GroEL, known to assist the correct folding of unfolded proteins. Dehydroepiandrosterone 16alpha-hydroxylation was catalyzed by CYP3A7 expressed in the presence of GroEL.     

Mannose-specific lectin isolation from Canavalia ensiformis seeds by PHEMA-based cryogel

Mannose-specific lectin Concanavalin A (Con A) was purified from Canavalia ensiformis seeds. For this purpose, mannose attached poly(hydroxyethyl methacrylate) (PHEMA) cryogel was prepared by cryopolymerization. Mannose was used as the affinity ligand and was covalently attached onto the PHEMA cryogel via carbodiimide activation. The PHEMA cryogel containing 23.3 mmol mannose/g polymer were used in the binding studies. Con A binding with the mannose attached PHEMA cryogel from Con A aqueous solution was 5.2 mg/g at pH 7. Maximum binding capacity for Con A from C. ensiformis seed extract was 39 mg/g. Con A was eluted with 0.3 M galactose, and the purity of Con A was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was observed that the mannose attached PHEMA cryogel can be used without significant decrease in Con A binding capacity after six binding-elution cycles.     

Stimulation of adenylate cyclase in washed pigeon erythrocyte membrane with cholera toxin and its subunits.

Cholera toxin was found to stimulate adenylate cyclase activity in washed membrane of pigeon erythrocytes in the presence of dithiothreitol and NAD. When tested with isolated cholera toxin components, the stimulatory activity was found with subunit A or polypeptide A1 derived from this subunit, but not with A2 or subunit B. On a molar basis, polypeptide A1 was approximately four times more active than cholera toxin. Dithiothreitol was not required in the action of polypeptide A1, suggesting that the reagent was needed only to release A1 from subunit A or the holotoxin for their action on adenylate cyclase. The single SH group in polypeptide A1 was not involved in the activity of the peptide, since chemical modification of the thiol group did not alter the stimulatory activity of the peptide. The presence of NAD was, however, essential for the activation of adenylate cyclase with cholera toxin, subunit A, or polypeptide A1. Elevation of the adenylate cyclase activity was also observed when the intact pigeon erythrocytes were incubated with polypeptide A1, although a 30-fold molar excess of A1 over that of holotoxin was required for the same level of activation.     

A direct evidence for the involvement of poly(A) in protein synthesis

A radioactive polyadenylated globin mRNA was translated in either rabbit reticulocyte lysate or wheat germ extract under various conditions. When globin mRNA was translated, globin synthesis was directly proportional to the rate of loss in A units from the poly(A) tail. On the other hand, when globin poly(A) mRNA was incubated under non-translated conditions, no loss of A units was detected. The presence of ribonuclease inhibitor in the reaction mixture did not alter either the rate of globin synthesis or the loss in A units from the poly(A) tail. The present data suggests a correlation between protein synthesis and loss in A units from the poly(A) tail.     

LMP2A does not require palmitoylation to localize to buoyant complexes or for function

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is expressed constitutively in lipid rafts in latently infected B lymphocytes. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids selective for specific protein association. Lipid rafts have been shown to be necessary for B-cell receptor (BCR) signal transduction. LMP2A prevents BCR recruitment to lipid rafts, thereby abrogating BCR function. As LMP2A is palmitoylated, whether this fatty acid modification is necessary for LMP2A to localize to lipid rafts and for protein function was investigated. LMP2A palmitoylation was confirmed in latently infected B cells. LMP2A was found to be palmitoylated on multiple cysteines only by S acylation. An LMP2A mutant that was not palmitoylated was identified and functioned similar to wild-type LMP2A; unmodified LMP2A localized to lipid rafts, was tyrosine phosphorylated, was associated with LMP2A-associated proteins, was ubiquitinated, and was able to block calcium mobilization following BCR cross-linking. Therefore, palmitoylation of LMP2A is not required for LMP2A targeting to buoyant complexes or for function.     

Origin of rabbit (Oryctolagus cuniculus) in China: evidence from mitochondrial DNA control region sequence analysis

A fragment of mitochondrial DNA (mtDNA) control region (approximately 700 bp) was sequenced in 104 individuals from 20 breeds (three Chinese domestic breeds, five recently derived breeds and 12 introduced breeds) of domestic rabbits, Oryctolagus cuniculus. Nineteen sites were polymorphic, with 18 transitions and one insertion/deletion, and eight haplotypes (A1, A2, A3, A4, A5, A6, A7 and A8) were identified. Haplotype A1 was the most common and occurred in 89 individuals. In the 25 Chinese rabbits, only haplotype A1 was observed, while four haplotypes (A1, A3, A5 and A6) were found in 26 recently derived individuals. Haplotype A2 was shared by seven individuals among three introduced strains. The other six haplotypes accounted for 0.96-1.92% of the animals. Combined with the published sequences of European rabbits, a reduced median-joining network was constructed. The Chinese rabbit mtDNAs were scattered into two clusters of European rabbits. These results suggest that the (so-called) Chinese rabbits were introduced from Europe. Genetic diversity in Chinese rabbits was very low.     

沙蒿与油蒿灌丛的防风阻沙作用

沙蒿与油蒿广泛分布于我国沙漠地区,是沙地植被的重要建群种和优势种。在腾格里沙漠南缘半流动沙地,实测了两种典型固沙植物沙蒿与油蒿的防风阻沙作用,从灌丛空间构型对比分析了其防风阻沙机制。结果表明,沙蒿与油蒿灌丛均具有明显的降低风速作用,但油蒿灌丛较沙蒿灌丛具有更显著的防风作用,而且对灌丛后不同位置、近地面不同高度层风速的降低程度明显不同。在灌丛后6倍株高范围内,沙蒿灌丛对50cm高度风速降低程度显著大于20cm,而油蒿灌丛对近地面20cm高度层风速降低程度显著大于50cm。在相同风速下,油蒿灌丛后20cm高度平均风速是沙蒿灌丛的1/2,而50cm高度平均风速与沙蒿灌丛相近。同时,沙蒿灌丛阻沙作用弱,而油蒿灌丛具有明显的阻沙作用,单株积沙体积达到45.2±16.1dm3,积沙重量达到72.1±25.7kg,油蒿灌丛积沙量大小与灌丛结构间存在显著的正相关。研究表明,紧密型结构的油蒿灌丛是较松散型结构沙蒿灌丛更为理想的防风固沙植物,其灌丛分枝数多、分枝角度小、生物量大且多分布在近地面层是具有显著防风阻沙作用的根本原因,该结论可为干旱区防风固沙植被建设物种选择提供依据。     

Molecular analysis of HLA-A2.4 functional variant KLO: close structural and evolutionary relatedness to the HLA-A2.2 subtype

The structure of an HLA-A2.4 functional variant (A2.4c) expressed on donor KLO has been examined by comparative peptide mapping with other HLA-A2 antigens of known structure and radiochemical sequencing. All the peptide differences between A2.4c and A2.1 could be accounted for by five amino acid changes at positions 9, 43, 66, 95, and 156. The nature of residues 9, 43, and 95 in A2.4c was determined by sequencing to be identical to those in A2.2Y. The nature of residue 156 in A2.4c was also assigned as identical to that in A2.2Y on the basis of the identity of the corresponding peptide in its chromatographic comparison with A2.2Y. Position 66 was unique to A2.4c. It was determined to be an Asn residue instead of the Lys present in all other HLA-A2 antigens of known structure. This was the only detected amino acid difference between A2.4c and A2.2Y. The results indicate that, from a structural point of view, A2.4c is most closely related to the A2.2 subtype antigens and not to other A2.4 antigens. The data are compatible with the assumption that A2.4c was derived from A2.2Y by a single point mutation event.     

Purification, modeling, and analysis of botulinum neurotoxin subtype A5 (BoNT/A5) from Clostridium botulinum strain A661222

A Clostridium botulinum type A strain (A661222) in our culture collection was found to produce the botulinum neurotoxin subtype A5 (BoNT/A5). Its neurotoxin gene was sequenced to determine its degree of similarity to available sequences of BoNT/A5 and the well-studied BoNT/A1. Thirty-six amino acid differences were observed between BoNT/A5 and BoNT/A1, with the predominant number being located in the heavy chain. The amino acid chain of the BoNT/A from the A661222 strain was superimposed over the crystal structure of the known structure of BoNT/A1 to assess the potential significance of these differences--specifically how they would affect antibody neutralization. The BoNT/A5 neurotoxin was purified to homogeneity and evaluated for certain properties, including specific toxicity and antibody neutralization. This study reports the first purification of BoNTA5 and describes distinct differences in properties between BoNT/A5 and BoNT/A1.     

A型肉毒毒素轻链基因的克隆及其结构分析

以献报道的A型肉毒毒素基因全序列为标准,设计并合成一对引物,自肉毒梭菌基因组中扩增出肉毒毒素轻链基因片段,并将扩增产物与pGEM—T载体在体外连接,构建测序重组质粒,进行测序和基因结构分析。PCR扩增获得了产物为1 364bp的DNA片段,测序结果与DNA数据库对照检索分析证明,此基因片段与GenBank中的A型肉毒毒素LC基因的一致性达99．9％以上,可以认为克隆的基因为A型肉毒毒素LC基因。     

N-Acetyl-β-d-hexosaminidase component A. Different forms in human tissues and fluids

1. Hexosaminidase A of human serum was resolved into two components, a minor form with properties identical with those of the single hexosaminidase A component of human liver, and a major form with significantly different properties. 2. The major serum hexosaminidase A form was eluted from a DEAE-cellulose column at a lower salt concentration than that required to elute the liver form. 3. A multiple-pass technique was used to elute the major serum enzyme A from a Sephadex G-150 column before that of liver enzyme A. 4. Clostridium perfringens neuraminidase converted the major component of serum hexosaminidase A into a form that was held less tightly by DEAE-cellulose, but the minor component of the A enzyme of serum, and the A enzyme of liver were not affected. 5. The hexosaminidase A from tears was similar to the A enzyme from serum, whereas those from several human tissues and from urine and lymph were similar to the liver form. 6. The A enzyme from serum may be derived from the A enzyme from liver by glycosylation before secretion.     

Hepatitis C virus-encoded nonstructural protein NS4A has versatile functions in viral protein processing.

A transient protein expression system in COS-1 cells was used to study the role of hepatitis C virus (HCV)-encoded NS4A protein on HCV nonstructural polyprotein processing. By analyzing the protein expression and processing of a deletion mutant polypeptide, NS delta 4A, which encodes the entire putative HCV nonstructural polyprotein except the region encoding NS4A, the versatile functions of NS4A were revealed. Most of the NS3 processed from NS delta 4A was localized in the cytosol fraction and was degraded promptly. Coproduction of NS4A stabilizes NS3 and assists in its localization in the membrane. NS4A was found to be indispensable for cleavage at the 4B/5A site but not essential for cleavage at the 5A/5B site in NS delta 4A. The functioning of NS4A as a cofactor for cleavage at the 4B/5A site was also observed when 30 amino acids around this site was used as a substrate and a serine proteinase domain of 167 amino acids, from Gly-1049 to Ser-1215, was used as an enzyme protein, suggesting that possible domains for the interaction of NS4A were in those regions of the enzyme protein (NS3) and/or the substrate protein. Two proteins, p58 and p56, were produced from NS5A. For the production of p58, equal or excess molar amounts of NS4A relative to NS delta 4A were required. Deletion analysis of NS4A revealed a minimum functional domain of NS4A of 10 amino acids, from Gly-1678 to Ile-1687.     

A study on concanavalin a binding to human erythrocytes

Interactions of concanavalin A with human erythrocytes were studied using ¹²⁵I-labelled concanavalin A and a centrifugal technique with dibutyl phthalate which permitted complete separation of bound and free concanavalin A. Binding of ¹²⁵I-labelled concanavalin A to human erythrocytes was dependent on cell concentration, pH and temperature. Specificity of binding was confirmed by inhibition and dissociation studies with sugars and native concanavalin A. Positive cooperative binding of concanavalin A to human erythrocytes was observed at low concanavalin A concentrations (less than 1 μ/ml) in both buffers studied. Positive cooperativity at higher concanavalin A concentrations (more than 100 μ/ml) was seen in Tris-Hepes buffer but not in phosphate-buffered saline. Consistent with this cooperative effect was the observation that although dissociation of ¹²⁵I-labelled concanavalin A from the erythrocytes was complete in the presence of 1 mg/ml of the native lectin, release was inhibited by low concentrations (1 μ/ml). A comparison of concanavalin A binding with hemagglutination studies suggest that the amount of concanavalin A bound determines the rate of erythrocyte agglutination and the size of the aggregates formed.     

Membrane cell fusion activity of the vaccinia virus A17-A27 protein complex

Vaccinia virus enters cells by endocytosis and via a membrane fusion mechanism mediated by viral envelope protein complexes. While several proteins have been implicated in the entry/fusion event, there is no direct proof for fusogenic activity of any viral protein in heterologous systems. Transient coexpression of A17 and A27 in mammalian cells led to syncytia formation in a pH-dependent manner, as ascertained by confocal fluorescent immunomicroscopy. The pH-dependent fusion activity was identified to reside in A17 amino-terminal ectodomain after overexpression in insect cells using recombinant baculoviruses. Through the use of A17 ectodomain deletion mutants, it was found that the domain important for fusion spanned between residues 18 and 34. To further characterize A17–A27 fusion activity in mammalian cells, 293T cell lines stably expressing A17, A27 or coexpressing both proteins were generated using lentivectors. A27 was exposed on the cell surface only when A17 was coexpressed. In addition, pH-dependent fusion activity was functionally demonstrated in mammalian cells by cytoplasmic transfer of fluorescent proteins, only when A17 and A27 were coexpressed. Bioinformatic tools were used to compare the putative A17–A27 protein complex with well-characterized fusion proteins. Finally, all experimental evidence was integrated into a working model for A17–A27-induced pH-dependent cell-to-cell fusion.