Biochemical Characterization of Aeromonad Strains Differing in Pathogenicity

A virulent strain of motile aeromonad (77-18) differed from an avirulent strain (78-16) in the contents of lipids and phospholipids and odd-numbered fatty acids, activity of hydrolytic enzymes, and amount of proteins with molecular weights of 47–56 kDa. It is assumed that proteins with molecular weights of 47–56 kDa, proteolytic enzymes active within a wide pH range, and odd fatty acids may act as pathogenicity factors. Each of these compounds or their combination determines a certain stage of infection.     

Characterization and toxicity to mosquito larvae of four Bacillus sphaericus strains isolated from Brazilian soils.

Four Bacillus sphaericus strains, S1, S2, S5, and L2, isolated from Brazilian soils, were found to be toxic to larvae of the mosquitoes Culex pipiens and Anopheles stephensi at a level similar to that of strain 2362 which is now used operationally. Like strain 2362, the four strains belonged to the serotype H5 and produced major proteins of apparent molecular weights of 125, 110, 56, and 43 kDa. These latter two proteins were immunologically related to toxins of the same molecular weight as B. sphaericus 2362. Although the four Brazilian strains were very similar to strain 2362, gas chromatography analysis of the fatty acids revealed that these strains were different from strain 2362 and from each other, except for a possible similarity between strains S1 and S5.     

Spectrum of antibodies to iron-regulated proteins in the blood sera of patients with meningococcal infection

55 paired sera from 25 patients with meningococcal infection (meningitis, meningococcemia) were studied with the use of immunoblotting. In these sera antibodies to 15 iron-regulated proteins (IRP) were detected. In the process of the development of meningococcal infection an increase in the content of specific antibodies to IRP with molecular weights of 35 kDa (38%), 43 kDa (52%) and 47 kDa (38%) was found to occur. The induction of antibodies did not depend on the group of the infecting strain, as well as on the patient's age.     

Purification and characterization of a protein capable of binding to fatty acids and bile salts in Giardia lamblia

A specific fatty acid binding protein was isolated from Giardia lamblia, using an affinity column with butyric acid acting as a ligand in place of stearic acid. This method has proved to be more efficient than the one previously described using stearic acid as ligand. The purified fraction showed 8 electrophoretic bands of proteins, with molecular weights ranging between 8 and 80 kDa. This pattern is a consequence of the aggregation of a protein with a molecular weight of 8,215 Da, corresponding to the lower molecular weight band, the only one capable of binding to fatty acids. The labeled oleic acid bound to these purified proteins was replaced by a 100-fold greater concentration of taurocholate, glycocholate, deoxycholate, palmitic acid, and arachidonic acid, having a greater displacement of the bile salts than the free fatty acids.     

Phosphorylation of both 47 and 49kDa proteins accompanies superoxide release by neutrophils

Neutrophils stimulated with activators of protein kinase C (i.e., 4 beta-phorbol 12-myristate 13-acetate; sn-1,2-dioctanoylglycerol) exhibit a dramatic, dose-dependent incorporation of 32P[Pi] into two proteins with molecular weights of ca. 47 and 49kDa. Proteins of the same molecular weights are also labelled when the cells are stimulated with a chemotactic peptide. However, with the latter stimulus, labelling of the 47kDa species is transient whereas that of the 49kDa entity persists. Labelling of both proteins always accompanied the release of O2-stimulated by these agents. The kinetics of labelling are compatible with the involvement of both phosphoproteins in the stimulation of these cells.     

Purification and cloning of nicosulfuron-degrading enzymes from Bacillus subtilis YB1

The nicosulfuron-degrading enzymes from Bacillus subtilis strain YB1 were purified and their genes were cloned. The proteins of bacterial culture filtrate were precipitated with ammonium sulfate or acetone. The extracellular proteins concentrated by acetone were purified from DEAE-Sepharose Fast Flow chromatography. The four protein peaks eluted from DEAE-column were separated and purified by native PAGE. Three components (P1-1, P3-2, P4-3) had nicosulfuron-degrading activity, and component P4-3 degradated 57.5% of this compound. The molecular weights of the components were 33.5, 54.8 and 37.0 kDa, respectively. The amino acid sequences of nicosulfuron-degrading enzymes from B. subtilis YB1 were determined by MALDI-TOF-MS, indicating these enzymes as manganese ABC transporter, vegetative catalase 1 and acetoin dehydrogenase E1, respectively. Using PCR amplification, genes 918, 1428, 1026 bp in size were detected for the enzymes studied.     

Anaerobic n-alkane metabolism by a sulfate-reducing bacterium, Desulfatibacillum aliphaticivorans strain CV2803T

The alkane-degrading, sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803T, recently isolated from marine sediments, was investigated for n-alkane metabolism. The total cellular fatty acids of this strain had predominantly odd numbers of carbon atoms (C odd) when the strain was grown on a C-odd alkane (pentadecane) and even numbers of carbon atoms (C even) when it was grown on a C-even alkane (hexadecane). Detailed analyses of those fatty acids by gas chromatography/mass spectrometry allowed us to identify saturated 2-, 4-, 6-, and 8-methyl- and monounsaturated 6-methyl-branched fatty acids, with chain lengths that specifically correlated with those of the alkane. Growth of D. aliphaticivorans on perdeuterated hexadecane demonstrated that those methyl-branched fatty acids were directly derived from the substrate. In addition, cultures on pentadecane and hexadecane produced (1-methyltetradecyl)succinate and (1-methylpentadecyl)succinate, respectively. These results indicate that D. aliphaticivorans strain CV2803T oxidizes n-alkanes into fatty acids anaerobically, via the addition of fumarate at C-2. Based on our observations and on literature data, a pathway for anaerobic n-alkane metabolism by D. aliphaticivorans is proposed. This involves the transformation of the initial alkylsuccinate into a 4-methyl-branched fatty acid which, in addition to catabolic reactions, can alternatively undergo chain elongation and desaturation to form storage fatty acids.     

西伯利亚鲟致病性嗜水气单胞菌外膜蛋白及其抗原性分析

采用十二烷基肌氨酸钠(Sarkosyl)法提取西伯利亚鲟嗜水气单胞菌(Aeromonas hydrophila)外膜蛋白,电泳显示所提取的主要外膜蛋白分子量为26～120 kDa;为比较该菌株与气单胞菌菌属其他细菌外膜蛋白组分及抗原性异同,以致病性豚鼠气单胞菌(A.caviae)、温和气单胞菌(A.sobria)和无致病力的嗜水气单胞菌为对照,电泳图谱显示4种气单胞菌外膜蛋白的分子量主要集中在26～120 kDa之间;利用抗西伯利亚鲟嗜水气单胞菌血清的免疫印迹试验表明该菌株外膜蛋白中分子量为75 kDa、52 kDa、43 kDa、40 kDa、34 kDa、28 kDa的蛋白条带呈现阳性反应,其他3种气单胞菌外膜蛋白中均有与该抗血清反应的条带,且分子量为28 kDa、34 kDa的反应条带为4株菌共有;43 kDa与75 kDa反应条带为部分菌株共有.为进一步筛选和研究致病性气单胞菌的共同保护抗原提供参考.     

Stability of liver nuclear proteins in inbred strains of mice

1. A remarkable similarity in the gel patterns of liver nuclear proteins between four inbred strains of mice (A.CA, B10.A, CBA and DBA/2) was observed. 2. Only a very few quantitative differences were detected in the protein spot patterns of nucleoplasmic (spot of about 41 kDa) and chromatin (spot of about 37 kDa) non-histone proteins between those strains of mice. 3. Comparison of two-dimensional gel patterns of non-histone proteins from males and females revealed a few sex-linked spots. Nucleoplasmic protein with molecular weight of about 59 kDa and chromatin proteins with molecular weights of approximately 47 and 57 kDa were more abundant in liver nuclei of male mouse.     

Evaluation of immunogenic potential of 75kDa and 56kDa proteins of newcastle disease virus (NDV)

The R2B strain of virus of new castle disease virus (NDV) was propagated in 9-11 day old embryonated chicken eggs via allantoic cavity route and after seven serial passages virus was purified from allantoic fluid. Purified virus was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis which yielded six major polypeptides ranging from 38-200 kDa. Protein fractions, corresponding to 75 and 56kDa, resembling haemagglutinin-neuraminidase (HN) and fusion (F) proteins were used to ascertain their immunization potential. Immunization of viral proteins was compared with the whole virus vaccine. Among different group of birds, highest haemagglutination inhibition (HI) and enzyme linked immunosorbent assay (ELISA) titers were obtained in birds immunized with whole virus vaccine followed by viral proteins, 75 and 56kDa in combination which was comparable with birds immunized with 56kDa protein alone. Despite lower values of HI and ELISA titers elicited by viral subunits in immunized birds, when challenged with virulent NDV strain, protection accorded by viral proteins in combination (75 +56kDa) or 56kDa alone was comparable with whole virus vaccine.     

Structural study of immunoaffinity-purified DNA polymerase alpha-DNA primase complex from calf thymus

The DNA polymerase alpha-DNA primase complex was purified over 17,000-fold to near homogeneity from calf thymus using an immunoaffinity column. Sodium dodecyl sulfate gel electrophoresis revealed three polypeptides with molecular weights of 140, 50 and 47 kDa, in a ratio of 1:2:0.25. The complex showed a sedimentation coefficient of 9.7 S, a Stokes radius of 56 A and a native molecular weight of 250-260 kDa. Taken together, the data suggest that the calf thymus dNA polymerase alpha-DNA primase complex is essentially a heterotrimer of large (140 kDa) and small (50 kDa) subunits in a ratio of 1:2, with a globular conformation. Electron-microscopic studies of the complex revealed a spherical particle of 120 A in diameter, in agreement with the physiochemical results. The binding of the complex to DNA was also demonstrated.     

Determination of the nutritional value of proteins obtained from Ulva armoricana

The in vitro digestibility of Ulva armoricana proteins by trypsin, chymotrypsin and human intestinal juice was determined to evaluate their nutritional value. The amino acid composition of the protein fraction and its changes during a sampling period from October to February were also studied. Some differences in the protein pattern shown by SDS PAGE were found in different months, such as the presence of a 54 kDa protein in February. The protein fraction is composed mainly of aspartic and glutamic acids (24–35% of protein fraction, according to season) and the essential amino acids constitute 27–36% of the total fraction. The efficiencies of trypsin and chymotrypsin in Ulva protein digestion are comparable. Only four proteins with apparent molecular weights of 86, 68, 40, and 29 KDa are digested by these proteolytic systems. The proteins from the October sample were more sensitive to chymotrypsin than those from the February sample. For instance, two proteins with apparent molecular weights of 100 and 67 kDa were weakly digested by chymotrypsin in the February extract, were fully digested in the October sample. The February sample differed from two others in the presence of glycosylated proteins, most of which have apparent molecular weights higher than 43 KDa. With the October sample, the activity of human intestinal juice was more effective than two other proteolytic systems. This is especially evident with a 27 kDa protein, which was only partially digested by the intestinal liquid and not digested by chymotrypsin or trypsin. However, human intestinal juice in the February apparently did not attack the 27 kDa protein. These data suggest a change in protein structure making it less sensitive to human intestinal juice. The glycosylation of protein extract, which was especially marked in February, could explain the differences in behaviour of U. armoricana proteins in response to the digestive action of human enzymes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Anaerobic n-Alkane Metabolism by a Sulfate-Reducing Bacterium, Desulfatibacillum aliphaticivorans Strain CV2803T

The alkane-degrading, sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803^T, recently isolated from marine sediments, was investigated for n-alkane metabolism. The total cellular fatty acids of this strain had predominantly odd numbers of carbon atoms (C odd) when the strain was grown on a C-odd alkane (pentadecane) and even numbers of carbon atoms (C even) when it was grown on a C-even alkane (hexadecane). Detailed analyses of those fatty acids by gas chromatography/mass spectrometry allowed us to identify saturated 2-, 4-, 6-, and 8-methyl- and monounsaturated 6-methyl-branched fatty acids, with chain lengths that specifically correlated with those of the alkane. Growth of D. aliphaticivorans on perdeuterated hexadecane demonstrated that those methyl-branched fatty acids were directly derived from the substrate. In addition, cultures on pentadecane and hexadecane produced (1-methyltetradecyl)succinate and (1-methylpentadecyl)succinate, respectively. These results indicate that D. aliphaticivorans strain CV2803^T oxidizes n-alkanes into fatty acids anaerobically, via the addition of fumarate at C-2. Based on our observations and on literature data, a pathway for anaerobic n-alkane metabolism by D. aliphaticivorans is proposed. This involves the transformation of the initial alkylsuccinate into a 4-methyl-branched fatty acid which, in addition to catabolic reactions, can alternatively undergo chain elongation and desaturation to form storage fatty acids.     

Carbohydrate, protein and amino acid status of Glycine-Glomus-Bradyrhizobium symbioses

Soybean [ Glycine max (L.) Merr. cv. Amsoy 71] plants were inoculated with either the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum , with a strain of Bradyrhizobium japonicum . or with both endophytes together. Non-inoculated plants were fertilized with levels of N and P that resulted in plant growth similar to that following infection by Bradyrhizobium or Glomus , respectively. In general, plants colonized by Glomus contained more pinitol (1 d -3-O-methyl- chiro -inositol) but less starch than corresponding P-fertilized soybeans, while nodulated soybeans contained more pinitol and sucrose in the roots than corresponding N-fertilized plants. Compared to nutrient-amended soybeans, increases or decreases in free amino acids in symbiotically grown plants tended to follow transient changes in N content and endophyte activity. The exception to this was the elevated levels of methionine in nodulated roots or aspartate and arginine in Glomus -inoculated roots regardless of N level. There were both quantitative and qualitative changes in the proteins found in inoculated soybeans, especially in infected roots where nodulins (nodule-specific proteins) and endomycorrhizins (mycorrhiza-specific proteins) were detected by polyacrylamide gel eletrophoresis. Five nodulins (11.8% of root proteins) were detected with molecular weights of 14, 34, 40, 41 and 100 kDa. Five endomycorrhizins (5.1% of root proteins) were detected with molecular weights of 16, 17, 18, 22 and 30 kDa. Differences in nutrient uptake and allocation in symbiotic plants could not be duplicated by fertilizer input alone, although nutrient addition could lead to similar growth rates and dry weight accumulation. These findings suggest that the quality and quantity of plant carbohydrates, proteins and amino acids shift in response to the physiological changes resulting from infection by N₂-fixing bacteria or endomycorrhizal fungi.     

The seed proteins of white spruce and their mobilization following germination

Buffer-soluble proteins that have subunit molecular weights, in the presence of sodium dodecyl sulphate (SDS), of 47, 31 and 27 kilodaltons (kDa) form the major storage proteins in the mature white spruce [ Picea glauca (Moench) Voss] seed. These proteins were found mainly in the megagametophyte. but smaller amounts were identified in the embryo. Following the completion of germination, this reserve was rapidly hydrolyzed in both tissues and probably plays a major nutritional role in the germinated seed. Buffer-insoluble proteins were also found in megagametophytes and embryos from the mature seed. These proteins were soluble in buffer only if SDS was present. Predominant in this class of proteins were several that have a subunit molecular weight and structure that is characteristic of seed crystalloid storage proteins; the subunits were shown to be heterodimers with polypeptide molecular weights in the 33 kDa to 37 kDa and 23.5 kDa to 25 kDa ranges. This reserve was rapidly hydrolyzed in the germinated seed. Storage protein hydrolysis was accompanied by a significant increase in the soluble amino acid pool in both megagametophytes and embryos. Cell-free extracts of mature seed megagametophytes and embryos contained leucine-naphthylamidase (leuNAase) activity. Following germination. this activity was maintained at a constant level in megagametophytes but increased substantially in embryos.     

（R）与（S）-羰基还原酶偶联一步法制备（S）-苯乙二醇

【目的】通过 (R) - 和(S) -羰基还原酶在大肠杆菌中偶联,实现了一步法制备（S）-苯乙二醇的生物转化过程。【方法】将来源于近平滑假丝酵母(Candida parapsilosis CCTCC M203011)的(R)- 羰基还原酶基因（rcr）和(S) -羰基还原酶基因（scr）串联于共表达载体pETDuetTM-1上。重组质粒pETDuet-rcr-scr转化稀有密码子优化型菌株Escherichia coli Rosetta,获得酶偶联重组菌株E. coli Rosetta / pETDuet-rcr-scr。当重组菌体培养至OD600 0.6-0.8时,添加终浓度1 mmol/L IPTG,30℃诱导蛋白表达10 h。【结果】SDS-PAGE结果表明(R)- 和(S) -羰基还原酶均明显表达,它们的相对分子质量分别为37 kDa和30 kDa。重组菌生物转化结果表明：在pH7.0的磷酸缓冲液中,添加5 mmol/L Zn2+时,获得产物(S)-苯乙二醇,产物光学纯度为91.3% e.e.,产率为75.9%。【讨论】采用分子重组技术成功整合了两种氧化还原酶的催化功能,实现了(S)- 苯乙二醇的一步法转化,为简化手性醇制备途径提供了一条崭新的思路。     

Protein and lipid composition of a vitellin isolated from eggs of Sparus aurata

The protein and lipid composition of a vitellin isolated from eggs of Sparus aurata were characterized by SDS PAGE, N-terminal sequence analysis and lipid analysis by thin layer chromatography and gas chromatography. The lipoprotein complex contains proteins with apparent molecular weights of 69, 59, 23, 21 and 12 kDa and were characterized as vitellinogenin fragments by N-terminal sequencing. Lipid extraction and analysis indicate an association of cholesterol and phospholipids with the protein subunits. The phospholipids contain fatty acids with 14, 16 and 18 carbon atoms as determined by GC/MS.     

Suppression of phaseolin and lectin in seeds of common bean, Phaseolus vulgaris L.: increased accumulation of 54 kDa polypeptides is not associated with higher seed methionine concentrations

Suppression of phaseolin and lectin accumulation in common bean resulted in higher concentrations of bean seed polypeptides with apparent molecular weights of 54 kDa and from 70 to 84 kDa on SDS-polyacrylamide gel electrophoresis. Polypeptides of 54 and 56 kDa segregated as products of different alleles. Genes for the 54/56 kDa bands and phaseolin were estimated to be 26.2±3.7 map units apart. The 54 kDa band phenotype manifested by SDS-PAGE consisted of from one to three polypeptides of 54 kDa MW on 2D gels, and the 56 kDa phenotype consisted of one polypeptide of 56 kDa plus two minor polypeptides of 54-54.5 kDa molecular weight. The pK_I of these polypeptides was approximately 5.25. The methionine content of the 54 kDa polypeptides of the cultivar Great Northern Star was 1.6±0.1 g/100 g protein, which was not statistically different from the value (1.5±0.1%) obtained for phaseolin isolated by the same procedure. F₂ seeds deficient for phaseolin and lectin contained as much total N per g as wild-type seeds and were not shrunken, but contained 50% more free amino acids. F₂ seeds from two of the three populations contained from 8 to 13% less methionine per mg total N.     

Suppression of phaseolin and lectin in seeds of common bean,Phaseolus vulgaris L.: increased accumulation of 54 kDa polypeptides is not associated with higher seed methionine concentrations

Suppression of phaseolin and lectin accumulation in common bean resulted in higher concentrations of bean seed polypeptides with apparent molecular weights of 54 kDa and from 70 to 84 kDa on SDS-polyacrylamide gel electrophoresis. Polypeptides of 54 and 56 kDa segregated as products of different alleles. Genes for the 54/56 kDa bands and phaseolin were estimated to be 26.2±3.7 map units apart. The 54 kDa band phenotype manifested by SDS-PAGE consisted of from one to three polypeptides of 54 kDa MW on 2D gels, and the 56 kDa phenotype consisted of one polypeptide of 56 kDa plus two minor polypeptides of 54-54.5 kDa molecular weight. The pK_I of these polypeptides was approximately 5.25. The methionine content of the 54 kDa polypeptides of the cultivar Great Northern Star was 1.6±0.1 g/100 g protein, which was not statistically different from the value (1.5±0.1%) obtained for phaseolin isolated by the same procedure. F₂ seeds deficient for phaseolin and lectin contained as much total N per g as wild-type seeds and were not shrunken, but contained 50% more free amino acids. F₂ seeds from two of the three populations contained from 8 to 13% less methionine per mg total N.     

Lipid and fatty acid composition of Eisenia foetida

It is found out that the content of lipids in the biomass of the studied populations of Eisenia foetida is rather high: 2.5-5.2% of the wet mass. The content of phospholipids is 40-55%, C27-sterols--1.5-3.4% of the mass of coarse extracts of lipids. Lipids of tissues contain also 47-54% of saturated (C10-C24) fatty acids as well as to 23% of monoene and to 13% of polyene unsaturated (C14-C22) fatty acids. The acids with the odd number of carbon atoms compose about 25% and acids with a branched carbon chain about 23% of the above percentage. Considerable content of lipids and biologically active fatty acids in tissues of the studied object permits considering it as a promising source of raw materials for production of valuable pharmacological preparations.