Strain-specific fingerprints of Rhizobium galegae generated by PCR with arbitrary and repetitive primers

Strain-specific genomic patterns of Rhizobium galegae were generated by PCR using both arbitrary and repetitive (BOX, ERIC and REP) primers. The identification of the strains was achieved also by RFLP analysis. However, the PCR genomic fingerprinting has significant advantages: it is not only simpler and faster, but it is also much more discriminative because it deals with the full bacterial genome and not only with parts of it as is the case with RFLP. In addition, both kinds of PCR fingerprinting (using arbitrary or repetitive primers) generated highly specific and reproducible patterns when parallel reactions with total bacterial DNA, extracted from independent liquid cultures were performed. The latter shows that AP- and rep-PCR are convenient for controlling the production and application of Rhizobium inoculants.     

Functional Expression and Properties of Sec14p-Like Protein with Molecular Mass 45 kD from Rat Olfactory Epithelium

cDNA of Sec14p-like water-soluble protein with molecular mass 45 kD from rat olfactory epithelium was expressed in Escherichia coli Rosetta cells. The expression product was purified by a two-step chromatographic procedure on DEAE-Sepharose and Sephacryl S-200. The identity of structural and functional characteristics of the recombinant and native proteins was demonstrated by CD, mass spectrometry, and Western blotting. Using several lipids immobilized on nitrocellulose membranes, it was shown that phosphatidylinositol-3,4,5-triphosphate is the specific ligand for the studied protein.     

兔阑尾中一种新的21kD的钙结合蛋白的纯化与鉴定

纯化与鉴定了Ｂ淋巴细胞中一种新的分子量为２１ｋＤ的钙结合蛋白（ＣａＢＰ２１）。兔阑尾淋巴细胞匀浆经热变性,Ｐｈｅｎｙｌ－Ｓｅｐｈａｒｏｓｅ与ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱层析,自每１ｋｇ细胞沉积物中获得ＳＤＳ－ＰＡＧＥ均一的ＣａＢＰ２１５．３ｍｇ。ＨＣｌ水解后的酸性氨基酸（Ａｓｐ＋Ｇｌｕ）含量为２６％。如同大多数钙结合蛋白一样,Ｎ末端封闭阻止其进行Ｅｄｍａｎ降解。ＣａＢＰ２１中疏水性氨基酸（计Ｇｌｙ,不计Ｔｒｐ）约占４６％,碱性氨基酸１０％,酸性氨基酸与极性氨基酸约４４％。ＣａＢＰ２１有较高的Ｓｅｒ、Ｔｙｒ含量。肽谱分析等确证ＣａＢＰ２１为２个相同或相似亚基二聚体。以ＡｒｓｅｎａｚｏⅢ作Ｃａ２＋结合分析表明每分子ＣａＢＰ２１可结合４分子Ｃａ２＋,对Ｃａ２＋的结合常数约为１０－５ｍｏｌ／Ｌ。各种性质表明ＣａＢＰ２１是一种不同于其他已知钙结合蛋白的新钙结合蛋白。     

Purification of Maize Cytosolic 70 kD Stress Protein: a Calmodulin-binding Protein in Plants

The maize cytosolic 70 kD stress protein (HSC70) has been purified by a two-step procedure employing affinity chromatography on ATP-agarose followed by DEAE52 ion-exchange chromatography. Using a biotinylated cauliflower calmodulin (CAM) gel-overlay technique in the presence of 1 mmol/L Ca2+ , the HSCT0 could bind to CAM. No band was shown on sodium dodecyl sulfate-polyacrylamide gel overlayed with biotinylated cauliflower CaM when 1 mmoL/L Ca2+ was replaced by 5 mmol/L EGTA. It indicated that the binding of HSC70 to CaM was dependent on Ca2+. The purified HSC70 inhibited the activity of CaM-dependent NADK and the degree of inhibition increased with augmentation of the HSC70, which appeared to be typically characteristic to CaM- binding protein.     

Analysis on Amino-Terminal Sequence and Purification of a 61 kD Protein from Photoperiod-Sensitive Genic Male-Sterile Rice

The specific protein P2,one of the three specific proteins(P1,P2 and P3)in chloroplasts from photoperiod-sensitive genlc malesterile rice previously reported was purlfied through preparative two dimensional gel electrophoresis and preparatwe isoelectric 10-CUSlng(1EF) and from which an uniform P2,checked with SDS-PAGE and IEF,was obtained．The molecular weight and isoelectric point was 61 kD and 5,8,respectively．Therefore P2 was referred as P61．A search in databases revealed that the aminoterminal sequence Of P61 was identical to that of perb unit of chloroplast ATP ase from barley and rice     

神经诱向生长因子18kD蛋白的纯化

提取与纯化诱向生长因子是神经生物化学研究中的一个重要课题．采用自然系统凝胶电泳,分离周围神经损伤过程中产生的具有诱向生长作用的１８ｋＤ蛋白,再以等电聚焦凝胶电泳与神经组织联合培养,揭示了ｐＩ为５．２的１８ｋＤ蛋白具有诱神经生长的作用．经高效液相色谱仪分析获得较纯的１８ｋＤ诱向因子．蛋白／多肽测序仪检测,１８ｋＤ蛋白Ｎ端氨基酸序列为：ＰＥＰＡＷＳＡＰＡＰ．     

鹰嘴豆蛋白降解产物的抗氧化作用

旨在分析鹰嘴豆分离蛋白(CPI)及其不同分子量短肽的抗氧化活性。用碱性蛋白酶(Alcalase)处理CPI,得到其降解产物,该降解产物经超滤离心,得到分子量分别为3 k D、3-5 k D、5-10 k D及10 k D的短肽。结果表明,与其它大分子肽段相比,3 k D的短肽具有较强的自由基清除活性;与谷胱甘肽(GSH)相比,CPI及其不同分子量短肽具有显著的金属离子螯合活性;CPI及其肽段具有一定的三价铁离子还原能力。上述结果表明,CPI及其肽段的显著抗氧化活性,使得其具有制备抗氧化功能食品和保健品的应用前景。     

刺山柑70 kD热休克蛋白基因克隆及原核表达

以干旱区特有的抗逆性植物刺山柑为材料,利用RT-PCR结合cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)方法,克隆了一个HSP70基因,将该基因进行原核表达,通过对大肠杆菌进行温度胁迫实验,并计算存活率来验证该基因对细胞的保护作用.结果表明:该基因全长2 202 bp,开放阅读框共1 950 bp,编码649个氨基酸,表达产物分子量为71.044 6 kD;序列分析表明,该基因属于HSP70基因家族,将该基因命名为CsHSP70,并提交到GenBank,登录号为EU574936.构建了CsHSP70基因的原核表达载体,并使重组质粒pGEX-4T-2-CsHSP70在大肠杆菌中异源表达,对大肠杆菌进行温度胁迫实验结果显示,在高温(50℃)和低温(4℃)条件下,转重组质粒的大肠杆菌在两种胁迫下存活率均高于对照,说明在温度胁迫下CsHSP70对大肠杆菌细胞具有保护作用.     

An Antibody to the Castor Bean Glyoxysomal Lipase (62 kD) also Binds to a 62 kD Protein in Extracts from Many Young Oilseed Plants

An antibody raised against purified glyoxysomal lipase (triacylglycerol hydrolase EC 3.1.1.3.) from castor bean (relative molecular weight of 62,000) also binds to a protein with a relative molecular weight of 62,000 in extracts of food reserve tissues from many young oilseed plants. These plants include Brassica napus L., Zea mays L., Arachis hypogaea L., Glycine max L., Gossipium hirsutum L., Cucurbita pepo L., Helianthus annuus L., Pisum sativum L., and Cicer arietinum L. The antibody caused inhibition of triacylglycerol hydrolysis by the lipases in extracts from seedlings of corn, oilseed rape, castor bean, soybean, and peanut. The pattern of antilipase binding to the 62 kilodalton protein in subcellular fractions from these other seedlings was consistent with the patterns of lipase activity reported in the literature and it is suggested that lipases from these oil seeds all have a subunit with a molecular weight of 62,000. The protein was only found in the food reserve tissues and was not present in extracts of roots and leaves of mature plants. In addition, the immunoreactive 62 kilodalton polypeptide was not detectable in lima beans and only at very low levels in kidney beans. Both these seeds are known to contain very little storage lipid and would not be expected to contain lipase. With the exception of the acid lipase of castor bean, ungerminated seeds do not generally contain active lipases. The immunoreactive 62 kilodalton protein could not be detected in the ungerminated seeds of most plants and only at very low low levels in others.     

Isolation and Identification of a Novel Antioxidant Peptide from Chickpea (Cicer arietinum L.) Sprout Protein Hydrolysates

International Journal of Peptide Research and Therapeutics - Chickpea (Cicer arietinum L.) is the second most widely cultivated leguminous plant in the world. In this study, the chickpea sprout...     

植物28kD蛋白的纯化及部分性质研究

经丙酮粉、硫酸铵分级沉淀和阴离子交换层析等方法,首次从玉米花粉细胞质粗提物中纯化了一种具有ＡＴＰａｓｅ活性的可溶性蛋白。ＳＤＳ－ＰＡＧＥ测得分子量为２８ｋＤ,ＩＥＦ－ＰＡＧＥ测得等电点为８．３。免疫印迹鉴定结果表明该酶蛋白与抗牛脑动蛋白或动力蛋白的抗体无免疫交叉反应。最大紫外吸收波长为２７８ｎｍ,并作了ＣＤ谱分析。底物特异性研究表明：ＡＴＰａｓｅ水解活性最高。药理学研究表明：该酶蛋白可被矾酸钠强烈抑制,但对ＮＥＭ基本不敏感,氟化钠可使酶活力丧失５０％左右,寡霉素、硝酸钾及乌本苷对酶活力没有抑制作用．     

Evidence for genetic exchange and recombination of Rhizobium symbiotic plasmids in a soil population

A soil population of 16 Rhizobium leguminosarum bv. trifolii isolates was characterized by using three Sym (for symbiotic) plasmid-specific DNA hybridization probes: (i) an R. leguminosarum bv. trifolii-specific, repeated-sequence probe; (ii) a nifHDK gene probe, and (iii) a nod gene probe. A predominant Sym plasmid family was identified among the isolates. Three other unrelated Sym plasmid families were also identified. The isolates were also classified either by using a chromosomal DNA hybridization probe or by serological relatedness to 25 different R. leguminosarum bv. trifolii antisera. With either method, it was possible to group the 16 soil isolates into identical or related families. However, the correlation between the two techniques was not high. Irrespective of the means used to classify the bacterial host strain, it was possible to identify the same Sym plasmids in unrelated strains, as well as unrelated Sym plasmids in identical host strains. These data indicate that, within this soil population, there has been genetic exchange of Sym plasmids, and in one instance the hybridization pattern indicates that in vivo recombination of two different Sym plasmids may have occurred. Symbiotic effectiveness tests on red, strawberry, and subterranean clovers clearly differentiated the isolates. In general, the pattern of response was similar within groupings on the basis of Sym plasmid and chromosomal profiles but different between such groups.     

霜霉菌或水分胁迫诱导的黄瓜叶片胞间隙27kD蛋白为几丁质酶

蛋白含量测定和十二烷基硫酸钠．聚丙烯酰胺凝胶电泳（SDS-PAGE）分析结果表明,水分胁迫或霜霉菌接种处理均使黄瓜（Cucumis sativus L.）叶片胞间隙总蛋白含量升高,27kD蛋白积累。通过基体辅助激光解析电离飞行时间质谱0VIALDI-TOFMS）分析纯化的27kD蛋白,将所得的PMF（肽指纹图谱）在NCBInr蛋白质数据库中比对,发现水分胁迫和霜霉菌接种所诱导的27kD蛋白是同一种蛋白,均为一种酸性的几丁质酶。其酶活性测定结果表明,水分胁迫或霜霉菌接种处理的叶片胞间隙液几丁质酶活性均高于对照。     

玉米花粉32kD蛋白的纯化及性质

利用丙酮粉、硫酸铵分段盐析及离子交换层析技术,从玉米花粉细胞质中分离纯化了一种３２ｋＤ的可溶性蛋白,其ＧＴＰａｓｅ活性大于ＡＴＰａｓｅ。ＳＤＳ－ＰＡＧＥ表明达银染电泳纯。等电聚焦电泳测得等电点为５．２５。免疫印迹鉴定表明该蛋白与抗牛脑动蛋白或动力蛋白的抗体无免疫交叉反应。最大紫外吸收波长为２８２ｎｍ,ＣＤ谱分析说明具有球蛋白特征。     

Behaviour of a sym plasmid from Rhizobium 'hedysari' in different Rhizobium species

Abstract The symbiotic plasmid pRHc1J of Rhizobium 'hedysari' has been transferred to different Rhizobium species. It expression and incompatibility with the recipient resident plasmids as well as the effect of the host plants on the selection of Rhizobium symbiotic information has been studied. When the symbiotic plasmid pRHc1J was transferred to Nod⁺Fix⁺ Rhizobium species, it underwent specific deletions, either spontaneously or after the passage of transconjugants through plants, leading to the loss of some essential nod genes.     

Protein Complex Identification by Integrating Protein-Protein Interaction Evidence from Multiple Sources

Background

Understanding protein complexes is important for understanding the science of cellular organization and function. Many computational methods have been developed to identify protein complexes from experimentally obtained protein-protein interaction (PPI) networks. However, interaction information obtained experimentally can be unreliable and incomplete. Reconstructing these PPI networks with PPI evidences from other sources can improve protein complex identification.

Results

We combined PPI information from 6 different sources and obtained a reconstructed PPI network for yeast through machine learning. Some popular protein complex identification methods were then applied to detect yeast protein complexes using the new PPI networks. Our evaluation indicates that protein complex identification algorithms using the reconstructed PPI network significantly outperform ones on experimentally verified PPI networks.

Conclusions

We conclude that incorporating PPI information from other sources can improve the effectiveness of protein complex identification.     

脊神经节感觉神经35kD特异蛋白的纯化

为纯化和鉴定感觉神经特异蛋白,以兔脊髓背根神经节及背根纤维组织为材料,通过制备匀浆、离子交换层析 Ｄ Ｅ Ａ Ｅ Ｓｅｐｈａｃｅｌ,高压液相凝胶过滤层析分离纯化了脊神经感觉神经元 ３５ ｋ Ｄ蛋白,将其作为抗原制备抗 ３５ ｋ Ｄ 多克隆抗体． Ｗ ｅｓｔｅｒｎ ｂｌｏｔ 的结果表明,该蛋白特异地存在于脊感觉神经而不存在于脊运动神经．并初步观察到它对鸡胚背根节有神经营养作用．     

Co-purification of 130 kD nitric oxide synthase and a 22 kD link protein from human neutrophils.

The synthesis of nitric oxide (NO) from L-arginine has been demonstrated in several cell types. Both constitutive and inducible forms of NO synthase have been described in different cells. We purified the constitutive form of NO synthase enzyme in human neutrophils using a two-column procedure. Crude 100,000g supernatant of human neutrophils was passed through a 2'-5'-ADP-agarose column followed by a DEAE-Bio-Gel A anion exchange column. NO synthase enzyme migrated as a single band (MW approximately 130,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its activity was dependent upon nicotinamide adenine dinucleotide phosphate (NADPH) and (6R)-tetrahydro-L-biopterin (BH4). In addition, flavin adenine dinucleotide (FAD) was also found to be essential for its maximal activity. A second NADPH, FAD-dependent component (MW approximately 22kD) was also found consistently on the SDS-PAGE gel. These observations suggest co-regulation between NO synthase enzyme and this NADPH, FAD-dependent component, which may be associated with the superoxide radical generating system.