C型产气荚膜梭菌β1、β2毒素基因的融合

利用PCR技术 ,从C型产气荚膜梭菌染色体DNA中扩增出 β1 和 β2 毒素基因 ,构建了含 β1 - β2 融合基因表达质粒的重组菌株BL2 1(DE3) (pETXB1_2 )。经酶切鉴定和序列测定证实 ,构建的重组质粒pETXB1_2含有 β1 - β2 融合基因 ,且基因序列和阅读框架正确。经ELISA检测 ,重组菌株表达的 β1 - β2 融合蛋白能够被 β1 、β2 毒素抗体识别。免疫实验结果表明 ,用β1 - β2 融合蛋白免疫的小鼠可以抵抗 1MLD的C型产气荚膜梭菌C5 9_4 4毒素攻击 ,表明构建的重组菌株可以作为预防仔猪红痢基因工程亚单位苗的候选菌株。     

Virulence studies on chromosomal α-toxin and Θ-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α-toxin in Clostridium perfringens-mediated gas gangrene

The pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasion and necrosis has not been determined. To enable genetic approaches to be used to study C. perfringens pathogenesis we developed an allelic exchange method which involved the transformation of C. perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin-resistance determinant. The frequency with which double reciprocal crossover events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene. Allelic exchange was used to isolate mutations in the‘chromosomal pfoA gene, which encodes an oxygen-labile haemolysin known as Θ-toxin or perfringolysin O. and in the chromosomal pic gene, which encodes the α-toxin or phospholipase C. The resultant mutants failed to produce detectable Θ-toxin or α-toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild-type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The results showed that the pic mutants had demonstrably reduced virulence and therefore provided definitive genetic evidence for the essential role of α-toxin in gas gangrene or clostridial myonecrosis.     

Adjusting for time-varying confounders in survival analysis using structural nested cumulative survival time models

Accounting for time-varying confounding when assessing the causal effects of time-varying exposures on survival time is challenging. Standard survival methods that incorporate time-varying confounders as covariates generally yield biased effect estimates. Estimators using weighting by inverse probability of exposure can be unstable when confounders are highly predictive of exposure or the exposure is continuous. Structural nested accelerated failure time models (AFTMs) require artificial recensoring, which can cause estimation difficulties. Here, we introduce the structural nested cumulative survival time model (SNCSTM). This model assumes that intervening to set exposure at time $t$ to zero has an additive effect on the subsequent conditional hazard given exposure and confounder histories when all subsequent exposures have already been set to zero. We show how to fit it using standard software for generalized linear models and describe two more efficient, double robust, closed-form estimators. All three estimators avoid the artificial recensoring of AFTMs and the instability of estimators that use weighting by the inverse probability of exposure. We examine the performance of our estimators using a simulation study and illustrate their use on data from the UK Cystic Fibrosis Registry. The SNCSTM is compared with a recently proposed structural nested cumulative failure time model, and several advantages of the former are identified.     

Development of an In Vitro Bioassay for Clostridium botulinum Type B Neurotoxin in Foods That Is More Sensitive than the Mouse Bioassay

A novel, in vitro bioassay for detection of the botulinum type B neurotoxin in a range of media was developed. The assay is amplified by the enzymic activity of the neurotoxin’s light chain and includes the following three stages: first, a small, monoclonal antibody-based immunoaffinity column captures the toxin; second, a peptide substrate is cleaved by using the endopeptidase activity of the type B neurotoxin; and finally, a modified enzyme-linked immunoassay system detects the peptide cleavage products. The assay is highly specific for type B neurotoxin and is capable of detecting type B toxin at a concentration of 5 pg ml⁻¹ (0.5 mouse 50% lethal dose ml⁻¹) in approximately 5 h. The format of the test was found to be suitable for detecting botulinum type B toxin in a range of foodstuffs with a sensitivity that exceeds the sensitivity of the mouse assay. Using highly specific monoclonal antibodies as the capture phase, we found that the endopeptidase assay was capable of differentiating between the type B neurotoxins produced by proteolytic and nonproteolytic strains of Clostridium botulinum type B.     

Naloxone prolongs the survival time of mice treated with neuroblastoma

The effect of naloxone on tumor growth and survival time was studied in mice with neuroblastoma tumors. Daily s.c. injections of either 5, 10, 15, or 20 mg/kg naloxone were initiated either 2 weeks prior to tumor cell inoculations (pre-treated groups) or one week after tumor transplantation (post-treated groups). The S20Y cell line, cloned from A/Jax murine C1300 neuroblastoma, was utilized and each animal was inoculated with 10⁶ cells. All mice in the saline- tumor and naloxone post-treated groups, developed tumors within 3 weeks after tumor cell inoculation. In the naloxone pre-treated groups, 4 of 12 mice exposed to 20 mg/kg, 2 of 12 mice exposed to 15 mg/kg, and 1 of 12 mice exposed to 10 mg/kg, did not develop tumors within the 91-day post-inoculation period. Three animals in the 20 mg/kg naloxone pre-treated group developed tumors between 43 and 63 days after tumor cell inoculation. Tumor dimensions were often reduced in naloxone-treated animals but a dose-response relationship was not found in regard to the magnitude of alterations in tumor size. At the time of death, tumor sizes of control and naloxone-exposed mice were similar. In general, tumor-bearing mice receiving naloxone lived longer than saline-tumor controls, with animals receiving higher drug dosages surviving for the longest time. In contrast to a mean survival time of 27 days for controls, naloxone pre-treated groups had increases in survival times of 25–61%, whereas naloxone post- treated groups exhibited increases of 20–40%. The median day of death for all mice exposed to naloxone was prolonged by 21–75%, occuring 6–21 days after the 28-day median for saline-tumor controls. These results suggest that naloxone, a non-addictive compound, is an effective agent in modulating neoplasia.     

Detection of neutralizing antibodies against α-toxin of different Clostridium septicum strains in cell culture

Clostridium septicum, a ubiquitious organism, is the pathogen which causes the classical malignant edema after injuries. Because of its strong cytotoxic alpha-toxin, infections are often lethal. To prevent losses in animals, vaccination with alpha-toxoid vaccines is carried out. Quality control of the vaccines is done by a neutralization test in mice. A cytotoxin test and as an alternative method to detect neutralizing antibodies, a cytotoxin inhibition test was standardized. In the studies, alpha-toxin of the C. septicum reference strain (NC 547) from the National Collection of Type Cultures was compared with alpha-toxin of a field strain from an outbreak in Germany. Sera from five heterologous polyvalent and three monovalent vaccines from eight rabbit groups were available. Each vaccination had been carried out according to the procedure of the German Pharmacopoeia. In three out of the five sera of the groups vaccinated with the heterologous polyvalent vaccine, cytotoxin neutralizing antibodies were detected. High antibody titers were observed in sera of rabbits vaccinated with a vaccine of strain NC 547, lower titers in the sera of rabbits vaccinated with a vaccine of the field strain. No cytotoxin neutralizing antibodies could be found in the sera of rabbits vaccinated with the monovalent C. chauvoei vaccine. The toxins of all strains showed the same ranking of the vaccines. Vaccines which caused high antibody titers in the animals were detected by all toxins as such, as well as vaccines which had medium or low antibody inducing capacity. The results were independent of the C. septicum strain used for the production of alpha-toxin.     

Effect of storage time and temperature on the survival of Clostridium botulinum spores in acid media.

Clostridium-botulinum type A and type B spores were stored in tomato juice (pH 4.2) and citric acid-phosphate buffer (pH 4.2) at 4, 22, and 32 degrees C for 180 days. The spore count was determined at different intervals over the 180-day storage period. There was no significant decrease in the number of type A spores in either the tomato juice or citric acid-phosphate buffer stored for 180 days at 4, 22, and 32 degrees C. The number of type B spores did not decrease when storage was at 4 degrees C, but there was an approximately 30% decrease in the number of spores after 180 days of storage at 22 and 32 degrees C.     

Effect of storage time and temperature on the survival of Clostridium botulinum spores in acid media.

Clostridium-botulinum type A and type B spores were stored in tomato juice (pH 4.2) and citric acid-phosphate buffer (pH 4.2) at 4, 22, and 32 degrees C for 180 days. The spore count was determined at different intervals over the 180-day storage period. There was no significant decrease in the number of type A spores in either the tomato juice or citric acid-phosphate buffer stored for 180 days at 4, 22, and 32 degrees C. The number of type B spores did not decrease when storage was at 4 degrees C, but there was an approximately 30% decrease in the number of spores after 180 days of storage at 22 and 32 degrees C.     

Splenectomy before tumor inoculation prolongs the survival time of cachectic mice

The effects of splenectomy on the development of cachexia, tumor growth and animal survival were studied in tumor-bearing CDF1 mice. Mice were inoculated with two subclones of colon 26 adenocarcinoma, clone 20 (with a potent capacity to induce cachexia) and clone 5 (without such activity), and underwent splenectomy before or after tumor inoculation. Splenectomy significantly prolonged the survival of mice bearing clone 20 when it was performed prior to tumor inoculation, although the progression of cachexia and tumor growth were not affected. The survival rate was higher in splenectomized than it was in nonsplenectomized mice 20–40 days after tumor inoculation. Such effects on survival were not observed, however, in mice splenectomized after inoculation with clone 20 or in mice that underwent splenectomy either before or after inoculation with clone 5. The decrease of peripheral blood lymphocyte count observed in mice bearing clone 20 was magnified when splenectomy was performed before tumor inoculation, but the serum levels of tumor necrosis factor and interleukin-6 were comparable. These results indicate that cancer death from cachexia is not directly attributable to enhanced catabolism. The mechanism by which splenectomy ameliorates the survival of cachectic mice remains to be studied, although several changes observed in the splenectomized mice after inoculation, including decreases in the peripheral blood L3T4⁺ cells and Lyt-2⁺ cells on the 9th day and 15th day respectively, and increase in the L3T4⁺/Lyt-2⁺ cell ratio on the 15th day suggest the involvement of the modified host's immune response.     

Bioassay for specific DNA sequences using a non-radioactive probe

A novel method for detecting specific DNA sequences is described. The method uses a non-radioactive DNA probe, called a probe-vector, that can transform competent Escherichia coli cells at high efficiency only when it has hybridized to a specific DNA target, thus forming a circular, double-stranded, plasmid-like molecule. The probe-vector carries a plasmid origin of replication and a gene that confers antibiotic resistance on transformed E. coli. The output of the assay--colored bacterial colonies on an agar plate--is quantitative and proportional over a wide range of target concentrations. The utility of the probe-vector method for detecting hepatitis B virus (HBV) DNA in human serum is demonstrated. The assay can detect as little as 0.1 pg HBV DNA. The presence of an internal standard monitors DNA recovery and E. coli transformation efficiency for each sample. The assay has the potential to simultaneously measure the DNA of two or more pathogens within the same clinical sample.     

Regression models for the mean of the quality-of-life-adjusted restricted survival time using pseudo-observations

In this research we develop generalized linear regression models for the mean of a quality-of-life-adjusted restricted survival time. Parameter and standard error estimates could be obtained from generalized estimating equations applied to pseudo-observations. Simulation studies with moderate sample sizes are conducted and an example from the International Breast Cancer Study Group Ludwig Trial V is used to illustrate the newly developed methodology.     

Sequencing and analysis of the gene encoding the α-toxin of Clostridium novyi proves its homology to toxins A and B of Clostridium difficile

A library of total Clostridium novyi DNA was established and screened for the α-toxin gene (tcnα) by hybridization with oligonucleotides derived from a partial N-terminal sequence and by using specific antisera. Overlapping subgenic tcnα fragments were isolated and subsequently the total sequence of tcnα was determined. The 6534 nucleotide open reading frame encodes a polypeptide of M_r 250 166 and pI 5.9. The N-terminal α-toxin (Tcnα) sequence MLITREQLMKIASIP determined by Edman degradation confirmed the identity of the reading frame and the assignment of the translation start point. The toxin is not modified posttranslationally at its N-terminus nor does it consist of different subunits. Overall the amino acid sequence shows 48% homology between the Tcnα and both toxins A (TcdA) and B (TcdB) of Clostridium difficile. The C-terminal 382 residues of Tcnα constitute a repetitive domain similar to those reported for TcdA and TcdB of C. difficile. The individual repeat motifs of these three toxins consist of oligopeptides some 19–52 amino acids in length, arranged in four to five different groups. Genetic, biochemical and pharmacological data thus confirm that the three toxins belong to one subgroup, designated large clostridial cytotoxins (LCT). Further definition of their structure and detailed molecular action should allow the LCTs to be used tools for the analysis of microfilament assembly and function.     

黄瓜香等对调整腹水瘤小鼠肠道菌群并延长荷瘤生存时间的影响

目的研究黄瓜香等在调节肠道菌群和改善荷瘤小鼠生存质量,提高生存率方面的作用。方法用肝癌腹水瘤H22细胞株注射小鼠造荷瘤小鼠模型,然后口服黄瓜香提取液治疗。观察治疗前后菌群变化、腹水量、荷瘤生存时间等。结果中药组与阴性对照组比较,肠道菌群趋于平衡、腹水出现时间延迟、腹水量降低、荷瘤生存时间延长。结论黄瓜香等能调节小鼠肠道菌群,改善荷瘤小鼠生存质量、提高生存率。