Inhibition of isocitrate lyase: the basis for inhibition of growth of two Arthrobacter species by pyruvate

Growth of Arthrobacter atrocyaneus and A. pyridinolis on certain growth substrates was found to be inhibited by pyruvate and compounds which can be converted to pyruvate. Growth of A. atrocyaneus on acetate, for example, was completely inhibited by 5 mm pyruvate; growth of this organism on glucose was less sensitive and growth on succinate was insensitive to inhibition by pyruvate. Growth of a third Arthrobacter species, A. crystallopoietes, on acetate and other substrates was not inhibited by pyruvate. The site of pyruvate inhibition was shown to be the isocitrate lyase reaction. Glyoxylate, which affords a bypass of this reaction, restored the ability of A. atrocyaneus to evolve (14)CO(2) from acetate in the presence of pyruvate. The isocitrate lyases from A. atrocyaneus and A. pyridinolis were competitively inhibited by concentrations of pyruvate as low as 1 mm, whereas the enzyme from A. crystallopoietes was unaffected by this concentration of pyruvate. Comparable levels of phosphoenolpyruvate did not inhibit the isocitrate lyases from any of the species. A mutant strain of A. atrocyaneus, PW11, which is deficient in isocitrate lyase activity, grew on glucose at a reduced rate that was comparable to the rate of growth of the wild-type strain on glucose plus lactate. Addition of lactate to PW11 did not further reduce its rate of growth on glucose. Thus, the glyoxylate pathway appears to be used as an anaplerotic pathway during growth of A. atrocyaneus on glucose. Two other considerations suggest that A. atrocyaneus and A. pyridinolis, but not A. crystallopoietes, may be deficient in the ability to convert pyruvate to 4-carbon acids. First, the former two species accumulate intracellular pyruvate from exogenous l-alanine to a much greater extent than does A. crystallopoietes. Moreover, A. atrocyaneus and A. pyridinolis are incapable of growth on lactate as sole source of carbon whereas A. crystallopoietes can grow on lactate.     

嗜硫色谱分离纯化碱性脂肪酶及其氨基酸序列测定

扩展青霉(Penicillium expansum)FS1884所产生的碱性脂肪酶经硫酸铵沉淀、Sephacryl S-200柱层析后,再经嗜硫色谱(Thiophilic Chromatography)柱层析,被纯化了173．8倍,最终比活为5694．9U／mg。纯化后的脂肪酶达到了SDS-PAGE电泳纯、PAGE电泳纯以及毛细管液相色谱(Capillary Liquid Chromatography)纯。该脂肪酶N-端氨基酸序列测定的结果是：A T A D A A A F P D L H R A A K L S S A,与来自青霉属其它真菌脂肪酶一级结构作了比较。     

Effect of Protein A on Adsorption of Bacteriophages to Staphylococcus aureus

Experiments were performed to determine if protein A influenced the association of bacteriophages with Staphylococcus aureus. Bacteriophage adsorption was compared in a S. aureus strain rich in protein A and mutants of this strain with very little protein A, in a strain with little protein A, and in mutants of this strain with increased protein A. In addition, the effect of growth in mannitol-salt broth and trypsin digestion (known to reduce protein A) on bacteriophage absorption was measured. There was an inverse relationship between protein A content of strains and the quantity of bacteriophage absorbed. However, no inhibition of staphylococcal phages was obtained with purified soluble protein A. Protein A as a surface component rendered the bacteria more resistant to adsorption of staphylococcal typing phages presumably by masking the phage receptor sites. When protein A-deficient mutants were incubated with bacteriophages, there was survival of staphylococci with increased protein A content probably due to a selective action.     

Characterization of a variant of beta-hexosaminidase: "hexosaminidase Paris".

A family (father and daughter) was found with a deficiency of hexosaminidase (HEX A and HEX B). Residual HEX A activity was about 30% of usual heterozygotes with very little HEX B activity. Thermostability of HEX A was decreased. No immunological cross reacting material was found for HEX A or B. The mechanism seems to be the production of abnormal, unstable beta subunits, which are still capable of combining with alpha subunits to form functional HEX A.     

Binding Properties of Bacillus thuringiensis Cry4A Toxin to the Apical Microvilli of Larval Midgut of Culex pipiens

Cry4A is a dipteran-specific δ-endotoxin produced by Bacillus thuringiensis, and toxic to Culex pipiens (mosquito) larvae. The immunohistochemical staining of the midgut sections of C. pipiens larvae revealed that Cry4A bound in vitro and in vivo to the microvilli of the epithelial cells of posterior midgut and gastric caecae. The binding of digoxigenin-labeled Cry4A (DIG-Cry4A) to the apical microvilli was almost abolished in the presence of excess unlabeled Cry4A, suggesting that the binding of Cry4A to the microvilli was specific. Several Cry4A-specific binding proteins were detected using the ligand blotting technique with DIG-Cry4A. Moreover, an insertion assay was done, where the binding of DIG-Cry4A to the BBMVs was completely irreversible and did not compete with excess unlabeled Cry4A. On the basis of these results, we propose a schematic interpretation for the binding process of Cry4A.     

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.     

Studies of the cell surface of Dictyostelium discoideum during differentiation. The binding of 125I-concanavalin A to the cell surface.

125I-concanavalin A (125I-Con A) was found to be equally effective as native Con A in binding to and agglutinating cells of Dictyostelium discoideum, suggesting that iodination of the molecule had no effect on the interaction of the protein with the cell surface. Almost all of the 125I-Con A binding to the cells was inhibited by alpha-methyl glucoside. The binding of 125I-Con A to the cells was extremely rapid, and once bound, the molecule was not readily displaced by prolonged incubation or by the addition of excess native concanavalin A (Con A). In contrast, the 125I-Con A was displaced rapidly from the cell surface by alpha-methyl glucoside. The binding of 125I-Con A to D. discoideum was identical at 22 degrees and 4 degrees, and was unaffected by metabolic inhibitors, suggesting that the protein was not subject to endocytosis. The cell surface Con A binding sites became saturated at high 125I-Con A concentrations. Scatchard plots of the data indicated that growing cells possessed 4 X 10(7) sites/cell, all of equal affinity. Similar plots for "aggregation phase" cells indicated at least two classes of binding sites. A small proportion of the sites had an affinity close to that for the sites on growing cells, but the majority of the sites had a markedly decreased affinity. The total number of binding sites increased only slightly during aggregation to 5.6 X 10(7) sites/cell.     

大豆基因型对根癌农杆菌菌株敏感性的研究

以栽培大豆［Glycine max (L.) Mer］吉林30、吉林43、绥农8、黑农35和东农42等的下胚轴为外植体,用EHA105和LBA4404 2个根癌农杆菌菌株（分别含有pGBI121S4ABC和pGBI4A2B质粒）研究大豆基因型对根癌农杆菌的敏感性,以及根癌农杆菌对大豆的侵染能力。结果表明,大豆基因型对根癌农杆菌的敏感性存在显著差异,以吉林43最敏感。根癌农杆菌菌株对大豆下胚轴侵染能力不同,含有pGBI121S4ABC质粒的LBA4404侵染能力较强,但差异未达显著水平。 Abstract:The sensitivity of genotypes in soybean to lines of Agrobacterium tumefaciens and the ability of A.tumefaciens infecting to soybean were investigated with hypocotyls of soybean (Jilin30,Jilin43,Suinong8,Heinong35 and Dongnong42) and lines of A.tumefaciens LBA4404 and EHA105 which including plasmid pGBI121S4ABC and pGBI4A2B respectively.The results showed that the sensitivity of genotypes in soybean to A.tumefaciens was significantly different.Jilin43 was the most sensitive materials to A.tumefaciens.The ability of A.tumefaciens infecting hypocotyls in soybean was different.LBA4404 including plasmid pGBI121S4ABC was easier to infect hypocotyls of soybean.     

Cross-linking of the anticodon of P and A site bound tRNAs to the ribosome via aromatic azides of variable length: involvement of 16S rRNA at the A site

The topography of the ribosomal decoding site was explored by affinity labeling from the 5'-anticodon base, 5-(carboxymethoxy)uridine-34, of P or A site bound tRNA1Val. A nitrophenyl azide was attached to the carboxyl group of this nucleotide via side chains varying in length from 18 to 24 A. Binding of acetylvalyl-tRNA to the P site was codon dependent and that of valyl-tRNA to the A site was both codon and elongation factor Tu (EFTu) dependent. Cross-linking to both A and P sites was irradiation, probe, codon, and, in the case of the A site, EFTu dependent. Putative P-site cross-linked aminoacyl-tRNA was reactive with puromycin. The yield of cross-linking was little affected by placement of the tRNA at the A or P site but varied considerably with the length and structure of the probe side chain. When the distance from the pyrimidine C-5 atom to the azide group was 23 A, 42-45% cross-linking was obtained at each site, but when the distance was decreased to 18 A, only 7-12% was found. Placing an S-S bond in the center of the 23-A leash decreased the A-site yield to about half, while insertion of a CONH group decreased A-site cross-linking about 8-fold. P-site cross-linking was more sensitive to mercaptan quenching (50% at 0.5 mM) than was that at the A site (50% at greater than 2.0 mM) but both were partially shielded from solvent.(ABSTRACT TRUNCATED AT 250 WORDS)     

The transition state in the folding-unfolding reaction of four species of three-disulfide variant of hen lysozyme: the role of each disulfide bridge

The effects of lacking a specific disulfide bridge on the transition state in folding were examined in order to explore the folding-unfolding mechanism of lysozyme. Four species of three-disulfide variant of hen lysozyme (3SS-lysozyme) were prepared by replacing two Cys residues with Ala or Ser: C6S/C127A, C30A/C115A, C64A/C80A and C76A/C94A. The recombinant hen lysozyme was studied as the standard reference containing four authentic disulfide bridges and the extra N-terminal Met: the recombinant hen lysozyme containing the extra N-terminal. Folding rates were measured by monitoring the change in fluorescence intensity associated with tri-N-acetyl-d-glucosamine binding to the active site of refolded lysozyme. It was confirmed that the folding rate of the recombinant hen lysozyme containing the extra N-terminal was the same as that of wild-type lysozyme, and that the folding rate was little affected by the presence of tri-N-acetyl-d-glucosamine (triNAG). The folding rate of C64A/C80A was found to be the fastest and almost the same as that of the recombinant hen lysozyme containing the extra N-terminal, and that of C30A/C115A the second, and that of C6S/C127A the third. The folding rate of C76A/C94A was particularly slow. On the other hand, the unfolding rates which were measured in the presence of triNAG showed the dependence on the concentration of triNAG. The intrinsic unfolding rate in the absence of triNAG was determined by extrapolation. Also in the unfolding rate, C76A/C94A was markedly slower than the others. It was found from the analysis of binding constants of triNAG to C64A/C80A during the unfolding process that the active site of C64A/C80A partly unfolds already prior to the unfolding transition. On the basis of these kinetic data, we suggest that C64A/C80A folding transition can occur with leaving the loop region around SS3 (C64-C80) flexible, while cross-linking by SS4 (C76-C94) is important for the promotion of folding, because it is an indispensable constraint on the way towards the folding transition state.     

一种新型粘多糖结构与性能的检测

粘多糖是由糖醛酸和氨基己糖交替连接成的高分子物质, 理化性质独特, 应用范围广泛。通过对突变株兽疫链球菌Streptococcus zooepidemicus BU100进行发酵, 可产一种新型粘多糖(下文用粘多糖A代替)。利用咔唑法、Elson-Morgan法、考马斯亮蓝法、红外光谱以及13C核磁共振谱测定粘多糖A的结构, 结果显示粘多糖A中糖醛酸和氨基糖的摩尔比例接近1:1, 蛋白含量符合标准(<0.1%); 粘多糖A图谱中出现的结构特征峰大部分与透明质酸相同。对粘多糖A的实用性能进行检测, 并用透明质酸做对比, 结果表明透明质酸在两种湿度下的吸湿性均要好于粘多糖A, 但粘多糖A的保湿性要好于透明质酸。粘多糖A总体的抗氧化性好于透明质酸, 并且粘多糖A耐透明质酸酶。粘多糖A可作为保湿剂、润滑剂、抗氧化剂等被更加有效地应用在医疗和化妆品等领域。     

Binding properties of Bacillus thuringiensis Cry4A toxin to the apical microvilli of larval midgut of Culex pipiens.

Cry4A is a dipteran-specific delta-endotoxin produced by Bacillus thuringiensis, and toxic to Culex pipiens (mosquito) larvae. The immunohistochemical staining of the midgut sections of C. pipiens larvae revealed that Cry4A bound in vitro and in vivo to the microvilli of the epithelial cells of posterior midgut and gastric caecae. The binding of digoxigenin-labeled Cry4A (DIG-Cry4A) to the apical microvilli was almost abolished in the presence of excess unlabeled Cry4A, suggesting that the binding of Cry4A to the microvilli was specific. Several Cry4A-specific binding proteins were detected using the ligand blotting technique with DIG-Cry4A. Moreover, an insertion assay was done, where the binding of DIG-Cry4A to the BBMVs was completely irreversible and did not compete with excess unlabeled Cry4A. On the basis of these results, we propose a schematic interpretation for the binding process of Cry4A.     

The phosphorylation of the primary gene products of alpha-crystallin

The alpha-crystallin primary gene product A2 and its post-translational modified counterpart A1 were isolated from calf lens cortex. The amino acid compositions determined from both chains were almost identical and in excellent agreement with that calculated from the reported sequence of A2. Chemical analysis of phosphate revealed 1 mol/mol of A1 and was negative in A2. Phosphoamino acid analysis demonstrated the presence of phosphoserine only in A1. Chymotryptic peptide maps of A2 and A1 resolved approximately 50 peptides and were strikingly similar. An apparent change in the relative mobility of one peptide was the only difference observed between A1 and A2. Phosphate analysis of this peptide obtained from A1 and A2 was positive only in the peptide from A1. Identical amino acid composition and the sequence Arg-Leu-Pro-Ser-Asn-Val-Asp-Gln-Ser-Ala-Leu was found for the peptide isolated from both chains, corresponding to residues 119 to 129 in the reported sequence of A2. These results indicate that the post-translational modification of A2 to A1 is the result of a phosphorylation reaction rather than a spontaneous nonenzymatic deamidation as previously suggested.     

Expression and regulation of mutant forms of cardiac TnI in a reconstituted actomyosin system: Role of kinase dependent phosphorylation

When phosphorylated, the inhibitory subunit of troponin (TnI) causes a loss in calcium sensitivity and a decrease in actomyosin ATPase. To examine this process, we bacterially expressed wild type TnI and TnI mutants in which serine 22 and 23, a putative protein kinase A (PKA) site, and threonine 143, a putative protein kinase C (PKC) site, were replaced by alanine S22A/23A and T143A. PKA dependent phosphorylation was ~90% reduced in the S22A/23A mutant and unaffected in T143A. PKC dependent phosphorylation was markedly reduced in T143A relative both to a wild type construct and to S22A/23A, although some residual phosphorylation (likely at sites other than T143) was seen. The calcium sensitivity (i.e. inhibition of actomyosin ATPase in the presence of EGTA) and regulation of the reconstituted actomyosin system was preserved in the absence of phosphorylation using wild type TnI or either mutant. Calcium sensitivity was decreased by both PKA and PKC with the wild type TnI but was unaffected by PKA when the S22A/23A mutant was employed and by PKC when the T143A mutant was reconstituted. The calcium dependency of the ATPase curve was substantially right shifted when PKC phosphorylated wild type TnI was employed for regulation, and this was markedly attenuated when T143 A was reassociated (although a slight rightward shift and a reduction in maximal ATPase activity was still seen). These data confirm that phosphorylation of TnI by regulatory kinases plays a major role in the regulation of myofibrillar ATPase. The N-terminal serines (22 and 23) appear to be uniquely important for the PKA response whereas threonine 143 is involved in the PKC response although other residues may also have functional significance.     

Purification and characterization of an inhibitor of the cysteine protease from the hemolymph of Sarcophaga peregrina larvae

The hemolymph of Sarcophaga peregrina (flesh fly) larvae was found to contain multiple inhibitors of hemocyte cysteine protease. One of them, named sarcocystatin A, was purified and found to be a mixture of the components sarcocystatin A alpha and A beta in a molar ratio of 2:1. These components can exist in either the associated or dissociated form. The apparent heterogeneity of the protease inhibitors in the hemolymph was found to be partly due to association of sarcocystatin A alpha and A beta.     

Microheterogeneity of molecular forms of arginase in mammalian tissues

Two isoforms of arginase, A1 and A2, were found in rat liver, submaxillary gland and kidney as well as beef kidney. In beef liver, however, A2 was the only detectable form. Two additional forms, A3 and A4, found only in rat kidney were probably artifactitious. A1 and A2 exhibited chromatographic and immunological microheterogeneity. While A1 in rat liver and submaxillary gland was excluded by DEAE-cellulose (pH 8.3) and retained on CM-cellulose (pH 7.5), that (A'1) in beef and rat kidneys was excluded by both ion-exchangers. A2 in all tissues was retained on DEAE-cellulose, but not on CM-cellulose. Both A1 and A2 in rat liver and beef kidney, A1 from rat submaxillary gland and A2 from beef liver were precipitated by antibodies to rat and beef liver arginases. None of the forms in rat kidney (A1, A2, A3 and A4) showed any cross-reactivity to either antibody. Rat submaxillary gland A2 was precipitated by anti-rat liver arginase, but activated by anti-beef liver arginase. While the major molecular forms were A1 in rat liver and submaxillary gland and A2 in beef liver and rat kidney, the two forms occurred in equal proportions in beef kidney. It appears that different isoforms might function as components of the urea cycle in the liver of different mammals and of the arginine catabolic pathway in different extrahepatic tissues.     

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。     

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.