Stimulation of maize invertase activity following infection by Ustilago maydis

The effect of U. maydis infection on the invertase activity of maize leaves has been studied. Infection causes a specific stimulation of an acid invertase in soluble and pellet fractions of homogenized tissue. Invertase activity is stimulated within one day after infection, and is maintained at a high level until 10 days after infection, in contrast to the progressive decline in activity with age, in healthy leaves. Analyses of soluble extracts by gel electrophoresis suggest that the invertase showing this increase is derived from the host and not the pathogen.     

Physiological activators of invertase from Hevea brasiliensis latex

Potassium or sodium nitrates or phosphates, and thiols such as reduced glutathione or cysteine, stimulate the activity of invertase from Hevea brasiliensis latex. These activators raise the V_max but do not affect the K_m of the enzyme for sucrose. The action of these effectors is additive. Their efficiency is pH dependent, being higher below pH 7.0 and markedly decreasing above it.     

单克隆抗体在淋病奈瑟氏菌研究和感染诊断中的应用

淋病是全球性疾病,对于淋球菌的研究已进入分子水平,本文从以下３个方面综述了单克隆体抗体在淋球菌研究和感染诊断中的应用：１．对于脂寡糖的研究２．对于淋球菌外膜蛋白Ⅰ的研究３．单克隆抗体在淋病流行病学和诊断中的应用。     

酶联免疫吸附试验检测A型产气荚膜梭菌肠毒素

本实验建立了双抗体夹心ＥＬＩＳＡ检测产气荚膜梭菌肠毒素的实验体系。以家兔单特异ＩｇＧ包被酶标板,辣根过氧化物酶标记的绵羊ＩｇＧ作为指示物,可检测出１．２５ｎｇ／ｍｌ（０．１２５ｎｇ）的产气荚膜梭菌肠毒素,线性范围大,重复性及稳定性好,对培养上清及粪便滤液检查无非特异反应。是产气荚膜梭菌食物中毒实验室诊断的一种快速可靠的检测方法。     

A regulatory invertase from sugar cane leaf-sheaths

A soluble β-fructofuranosidase was isolated from sugar cane leaf-sheaths. The enzyme attacks sucrose with an activation energy of 5700 cal/mol above 30° and 17 000 cal/mol below 30°. The enzyme was inhibited by the reaction products. Glucose is a simple non-competitive inhibitor, but fructose is a competitive inhibitor. Kinetic studies using double reciprocal plots and replots of 1/K_i, slope vs inhibitor concentration showed that fructose binds to two interacting sites of the enzyme. Per cent residual activity plotted against inhibitor concentration, and Hill plots confirmed the regulatory properties of the invertase. n was found to be close to 2, the number of binding sites established with the double reciprocal method. The tissue and cellular levels of sucrose, fructose and glucose were measured. Fructose was found at inhibitory concentrations confirming that the activity of the enzyme is probably modulated by the hexose pool of the leaf-sheaths.     

一起兔产气荚膜梭菌病爆发流行及病原体的分离鉴定

黑山县某兔场出现仔兔突然发病、大量死亡现象,根据临床症状和病理剖检变化疑似细菌感染,采取肝脏等样本进行病原分离、培养特性观察、菌落形态学观察、生化反应试验、致病性试验、药敏试验。结果从组织样本中分离获得1株病原菌,其培养特性、菌落形态、生化反应特性等均符合产气荚膜梭菌特征,药敏实验结果显示,该分离菌对氟苯尼考、头孢哌酮、哌拉西林等药物敏感。说明该兔场发生了产气荚膜梭菌病的疫情,采用敏感药物治疗后,收到明显效果。     

Stimulation of growth and sporulation of Clostridium perfringens by its homologous enterotoxin

C. perfringens enterotoxin shortened the lag phase and time of onset of sporulation of the same organism in a dose-dependent manner. The toxin stimulated macromolecular synthesis of pre-exponential phase cells.     

Molecular typing of Clostridium perfringens isolated from turkey meat by multiplex PCR

Aims:  To determine the presence of toxin genes in 22 Clostridium perfringens isolated from turkey meat samples by molecular typing.
Methods and Results:  For this purpose, alpha ( cpa ), beta ( cpb ), beta 2 ( cpb2 ), epsilon ( etx ), iota ( iA ) and enterotoxin ( cpe ) toxin genes were analysed by multiplex PCR. All 22 turkey meat Cl. perfringens isolates were found to carry the cpa , gene but in none of the isolates cpb , etx, iap or cpe genes were detected. Results showed that all isolates represented type A and were cpe negative.
Conclusions:  Our results indicate that Cl. perfringens type A is the most common type in turkey meat. Also multiplex PCR is effective and rapid method for typing of Cl. perfringens .
Significance and Impact of the Study:  It is the first study about molecular typing of Cl. perfringens using multiplex PCR in turkey meat samples in Turkey.     

Utilization of 1-chloromethylsilatrane by Rhodotorula mucilaginosa

Abstract The imperfect yeast, Rhodotorula mucilaginosa utilized nitrogen of 1-chloromethylsilatrane (CMS) as a sole nitrogen source when grown on glucose, glycerol, methanol, ethanol and succinate. Under such conditions and at concentrations from 0.45 to 4.5 mM, CMS was a growth-limiting factor. Atomic absorption spectrometry revealed the accumulation of silicon compounds in the cultural liquid which were chloroform-insoluble in contrast to CMS. The following pathway of the partial decomposition of CMS is propoposed: CMS → chloromethylsilanethryol → bis(chloromethyldisiloxane) tetraol.     

Comparison of Sucrose-Hydrolyzing Enzymes Produced by Rhizopus oryzae and Amylomyces rouxii

Rhizopus oryzae produces lactic acid from glucose but not efficiently from sucrose, while Amylomyces rouxii, a species closely related to R. oryzae, ferments these sugars equally. The properties of two sucrose-hydrolyzing enzymes purified from culture filtrates of R. oryzae NBRC 4785 and A. rouxii CBS 438.76 were compared to assess lactic acid fermentation by the two fungi. The substrate specificity of the enzymes showed that the enzymes from strains NBRC 4785 and CBS 438.76 are to be classified as glucoamylase and invertase respectively. The entity of the enzyme from strain NBRC 4785 might be a glucoamylase, because eight residues of the N-terminal amino acid sequence coincided with those of the deduced protein from the amyB gene of R. oryzae. The enzyme from NBRC 4785 was more unstable than that from strain CBS 438.76 under conditions of lower pH and higher temperature. These observations mean that the culture conditions of R. oryzae for lactic acid production from sucrose should be strictly controlled to prevent inactivation of the glucoamylase hydrolyzing sucrose.     

Transformation of Bile Acids by Mixed Microbial Cultures from Human Feces and Bile Acid Transforming Activities of Isolated Bacterial Strains

Microbial transformation of cholic acid and chenodeoxycholic acid by anaerobic mixed cultures of human fecal microorganisms was investigated, and the results were examined in relation to the bile acid transforming activities of 75 bacterial strains isolated from the same fecal cultures. The reactions involved in the mixed cultures were dehydrogenation and dehydroxylation of the 7α-hydroxy group in both primary bile acids and epimerization of the 3α-hydroxy group in all metabolic bile acids. Extensive epimerization of the 7α-hydroxy group of chenodeoxycholic acid yielding ursodeoxycholic acid was also demonstrated by certain fecal samples. 7α-Dehydrogenase activity was widespread among the fecal isolates (88% of 16 facultative anaerobes and 51% of 59 obligate anaerobes), and 7α-dehydroxylase activity was revealed in one of the isolates, an unidentified gram-positive nonsporeforming anaerobic bacterium. 3α-Epimerization was effected by seven strains assigned to Eubacterium lentum, which were also active for 3α- and 7α-dehydrogenations. No microorganism accounting for 7α-epimerization was recovered among the isolates. Splitting of conjugated bile acid was demonstrated by the majority of obligate anaerobes but the activity was rare among facultative anaerobes.     

Biochemical and immunological properties of the C-terminal domain of the alpha-toxin of Clostridium perfringens

Abstract The C-terminal domain of the alpha-toxin (cpa_247–370) of Clostridium perfringens has been expressed in Escherichia coli and purified. Antiserum raised against cpa_247–370 reacted in an identical manner to anti-alpha-toxin serum when used to map epitopes in the C-terminal domain, suggesting that cpa_247–370 was immunologically and structurally identical to this region in the alpha-toxin. The isolated cpa_247–370 was devoid of sphingomyelinase activity or haemolytic activity and was not cytotoxic for mouse lymphocytes. Haemolytic activity was detected when cpa_247–370 was tested with the N-terminal domain of the alpha-toxin (cpa_1–249), confirming that cpa_247–370 confers haemolytic properties on the phospholipase C activity of the alpha-toxin. Haemolytic activity was not detected if cpa_247–370 was tested with the Bacillus cereus phosphatidylcholine phospholipase C, nor if cpa_1–249 and cpa_247–370 were incubated sequentially with erythrocytes.     

多重PCR鉴定不同毒素型的产气荚膜梭菌菌落

参照文献报道的产气荚膜梭菌α,β,ε,τ毒素基因cpa、cpb,etx及iA序列合成了针对4种毒素基因的4对特异引物,建立了一种简单的产气荚膜梭菌定型的菌落多重PCR方法.结果本所保存的A,B,c,D,E各型产气荚膜梭菌参考菌株均扩增出了相应的预期条带,而诺维氏梭菌、腐败梭菌和破伤风梭菌的扩增均为阴性;将单个菌落稀释100倍利用此菌落多重PCR仍能扩增到相应的目的片段.并利用此多重PCR对13株不同动物来源的产气荚膜梭菌进行了定型鉴定,并与毒素中和试验鉴定结果进行了比较,结果表明两种方法具有较高的符合率.本方法的建立对于产气荚膜梭菌的快速检测、定型具有十分重要的意义.     

Solubility of plant invertases

Solubilization of acid invertase associated with cell wall preparations from aged slices of Jerusalem artichoke tuber tissue was achieved at high ionic     

多重PCR鉴定不同毒素型的产气荚膜梭菌菌落

参照文献报道的产气荚膜梭菌a, b, e, t 毒素基因cpa、cpb、etx 及iA序列合成了针对4种毒素基因的4对特异引物, 建立了一种简单的产气荚膜梭菌定型的菌落多重PCR方法。结果本所保存的A, B, C, D, E各型产气荚膜梭菌参考菌株均扩增出了相应的预期条带, 而诺维氏梭菌、腐败梭菌和破伤风梭菌的扩增均为阴性; 将单个菌落稀释100倍利用此菌落多重PCR仍能扩增到相应的目的片段。并利用此多重PCR对13株不同动物来源的产气荚膜梭菌进行了定型鉴定, 并与毒素中和试验鉴定结果进行了比较, 结果表明两种方法具有较高的符合率。本方法的建立对于产气荚膜梭菌的快速检测、定型具有十分重要的意义。     

Comparison of the nucleotide sequence and development of a PCR test for the epsilon toxin gene of Clostridium perfringens type B and type D

The sequence of the epsilon toxin gene of Clostridium perfringens type D was determined and compared with that of the previously reported type B sequence. It showed two nucleotide changes in the open reading frame, giving rise to one amino acid substitution. The promoter sequences were not homologous, and different putative -35 and -10 regions have been identified in each. The sequence information was used to develop PCR primers which were specific for the epsilon toxin gene. The utility of this system for identifying type B or D strains of C. perfringens was demonstrated.     

Plasmid transformation of Clostridium perfringens by electroporation methods

Two electroporation methods were compared and modified to improve the frequencies of transfer of plasmid DNA into Clostridium perfringens. A plasmid shuttle vector, pSB92A2, containing chloramphenicol and ampicillin resistance genes and a clostridial origin of replication isolated from a cryptic C. perfringens plasmid, was constructed and successfully introduced into C. perfringens by both electrotransformation methods. Modifications which improved frequencies by 15-28 fold are described and may improve frequencies sufficiently for some vector/host combinations to consider the future use of more direct cloning strategies for the clostridia.     

Hemagglutinating activity of trypsinized Clostridium perfringens enterotoxin

Abstract The hemagglutinating activity of Clostridium perfringens enterotoxin (CPE) was studied after trypsin treatment. Untreated CPE did not show any hemagglutinating activity to human type A, B, and O, sheep, chicken, horse, guinea-pig, or rabbit erythrocytes. Trypsinized CPE resulted in a more than 100-fold increase in hemagglutinating activity with rabbit erythrocytes only. Other erythrocytes and trypsinized rabbit erythrocytes were not agglutinated at all. The hemagglutinating activity of CPE was also found on treatment with a lysine-specific proteinase. On the other hand, trypsinized CPE did not significantly increase the cytotoxic and enterotoxic activities. The binding reaction between trypsinized and rabbit erythrocytes was not inhibited by any mono-, di-, or polysaccharides, glycoproteins or ganglioside mixtures. These results suggest that the hydrolysis of bonds involving lysine residues is mainly required for hemagglutinating activity, and that the receptor for trypsinized CPE on rabbit erythrocytes is probably the protein moiety.     

Nitrate salts suppress sporulation and production of enterotoxin in Clostridium perfringens strain NCTC8239

Clostridium perfringens type A is a common source of food‐borne illness in humans. Ingested vegetative cells sporulate in the small intestinal tract and in the process produce C. perfringens enterotoxin (CPE). Although sporulation plays a critical role in the pathogenesis of food‐borne illness, the molecules triggering/inhibiting sporulation are still largely unknown. It has previously been reported by our group that sporulation is induced in C. perfringens strain NCTC8239 co‐cultured with Caco‐2 cells in Dulbecco's Modified Eagle Medium (DMEM). In contrast, an equivalent amount of spores was not observed when bacteria were co‐cultured in Roswell Park Memorial Institute‐1640 medium (RPMI). In the present study it was found that, when these two media are mixed, RPMI inhibits sporulation and CPE production induced in DMEM. When a component of RPMI was added to DMEM, it was found that calcium nitrate (Ca[NO₃]₂) significantly inhibits sporulation and CPE production. The number of spores increased when Ca(NO₃)₂‐deficient RPMI was used. The other nitrate salts significantly suppressed sporulation, whereas the calcium salts used did not. qPCR revealed that nitrate salts increased expression of bacterial nitrate/nitrite reductase. Furthermore, it was found that nitrite and nitric oxide suppress sporulation. In the sporulation stages, Ca(NO₃)₂ down‐regulated the genes controlled by Spo0A, a master regulator of sporulation, but not spo0A itself. Collectively, these results indicate that nitrate salts suppress sporulation and CPE production by down‐regulating Spo0A‐regulated genes in C. perfringens strain NCTC8239. Nitrate reduction may be associated with inhibition of sporulation.     

Cloning and characterization of the lytB gene of Campylobacter jejuni

LytB of Escherichia coli is an essential gene involved in penicillin tolerance and the stringent response. The lytB gene of Campylobacter jejuni was cloned and characterized. It could complement a temperature-sensitive E. coli lytB mutant. The C. jejuni lytB gene encodes a protein of 277 amino acids that has 34, 36 and 40% amino acid identity with the LytB proteins of E. coli, Haemophilus influenzae, and Synechocystis sp. PCC6803, respectively. The lytB gene is situated between the aroA gene and a gene that encodes ribosomal protein S1.