Nitroreductase-activated nitric oxide (NO) prodrugs

Due to the involvement of nitric oxide (NO) in numerous and diverse physiological processes, site-directed delivery of therapeutic NO in order to minimize unwanted side-effects is necessary. O²-(4-Nitrobenzyl) diazeniumdiolates are designed as substrates for Escherichia coli nitroreductase (NTR), an enzyme that is frequently used to facilitate directed delivery of cytotoxic species to cancers. O²-(4-Nitrobenzyl) diazeniumdiolates are found to be stable in aqueous buffer but are metabolized by NTR to produce NO. A cell viability assay revealed that cytotoxic effects of O²-(4-nitrobenzyl)1-(2-methylpiperidin-1-yl)diazen-1-ium-1,2-diolate (4b) towards two cancer cell lines is significantly enhanced in the presence of NTR suggesting the potential for use of this compound in nitric oxide-based directed prodrug therapy.     

Targeted enzyme prodrug therapy for metastatic prostate cancer – a comparative study of L-methioninase,purine nucleoside phosphorylase,and cytosine deaminase

Background

Enzyme prodrug therapy shows promise for the treatment of solid tumors, but current approaches lack effective/safe delivery strategies. To address this, we previously developed three enzyme-containing fusion proteins targeted via annexin V to phosphatidylserine exposed on the tumor vasculature and tumor cells, using the enzymes L-methioninase, purine nucleoside phosphorylase, or cytosine deaminase. In enzyme prodrug therapy, the fusion protein is allowed to bind to the tumor before a nontoxic drug precursor, a prodrug, is introduced. Upon interaction of the prodrug with the bound enzyme, an anticancer compound is formed, but only in the direct vicinity of the tumor, thereby mitigating the risk of side effects while creating high intratumoral drug concentrations. The applicability of these enzyme prodrug systems to treating prostate cancer has remained unexplored. Additionally, target availability may increase with the addition of low dose docetaxel treatment to the enzyme prodrug treatment, but this effect has not been previously investigated. To this end, we examined the binding strength and the cytotoxic efficacy (with and without docetaxel treatment) of these enzyme prodrug systems on the human prostate cancer cell line PC-3.

Results

All three fusion proteins exhibited strong binding; dissociation constants were 0.572 nM for L-methioninase-annexin V (MT-AV), 0.406 nM for purine nucleoside phosphorylase-annexin V (PNP-AV), and 0.061 nM for cytosine deaminase-annexin V (CD-AV). MT-AV produced up to 99% cell death (p < 0.001) with limited cytotoxicity of the prodrug alone. PNP-AV with docetaxel created up to 78% cell death (p < 0.001) with no cytotoxicity of the prodrug alone. CD-AV with docetaxel displayed up to 60% cell death (p < 0.001) with no cytotoxicity of the prodrug alone. Docetaxel treatment created significant increases in cytotoxicity for PNP-AV and CD-AV.

Conclusions

Strong binding of fusion proteins to the prostate cancer cells and effective cell killing suggest that the enzyme prodrug systems with MT-AV and PNP-AV may be effective treatment options. Additionally, low-dose docetaxel treatment was found to increase the cytotoxic effect of the annexin V-targeted therapeutics for the PNP-AV and CD-AV systems.     

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.     

Phospholipid fatty acid and lipopolysaccharide fatty acid signature lipids in methane-utilizing bacteria

The effect of human bile juice and bile salts (sodium cholate, sodium taurocholate, sodium glycochenodeoxycholate and sodium chenodeoxycholate) on growth, sporulation and enterotoxin production by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens was determined. Each bile salt inhibited growth to a different degree. A mixture of bile salts completely inhibited the growth of enterotoxin-positive strains of this organism. Human bile juice completely inhibited the growth of all the strains at a dilution of 1:320. A distinct stimulatory effect of the bile salts on sporulation was observed in the case of C. perfringens strains NCTC 8239 and NCTC 8679. The salts also increased enterotoxin concentrations in the cell extracts of the enterotoxin-positive strains tested. No effect on enterotoxin production was detected when an enterotoxin-negative strain was examined.     

Purification and amino acid sequence of two small, acid-soluble proteins from Clostridium bifermentans spores

Clostridium bifermentans spores contain two major small, acid-soluble, proteins (SASP) termed SASP-alpha and beta. The amino acid sequences of SASP-alpha and beta are almost identical, and are very similar to those of alpha/beta-type SASP from spores of C. perfringens and various Bacillus species. However, the C. bifermentans proteins contain an extra five amino acids in the middle of their sequence. Surprisingly, no gamma-type SASP were found in C. bifermentans or C. perfringens spores, although these are the most prominent SASP in spores of Bacillus species.     

Understanding the Broad Substrate Repertoire of Nitroreductase Based on Its Kinetic Mechanism

The oxygen-insensitive nitroreductase from Enterobacter cloacae (NR) catalyzes two-electron reduction of nitroaromatics to the corresponding nitroso compounds and, subsequently, to hydroxylamine products. NR has an unusually broad substrate repertoire, which may be related to protein dynamics (flexibility) and/or a simple non-selective kinetic mechanism. To investigate the possible role of mechanism in the broad substrate repertoire of NR, the kinetics of oxidation of NR by para-nitrobenzoic acid (p-NBA) were investigated using stopped-flow techniques at 4 °C. The results revealed a hyperbolic dependence on the p-NBA concentration with a limiting rate of 1.90 ± 0.09 s⁻¹, indicating one-step binding before the flavin oxidation step. There is no evidence for a distinct binding step in which specificity might be enforced. The reduction of p-NBA is rate-limiting in steady-state turnover (1.7 ± 0.3 s⁻¹). The pre-steady-state reduction kinetics of NR by NADH indicate that NADH reduces the enzyme with a rate constant of 700 ± 20 s⁻¹ and a dissociation constant of 0.51 ± 0.04 mm. Thus, we demonstrate simple transient kinetics in both the reductive and oxidative half-reactions that help to explain the broad substrate repertoire of NR. Finally, we tested the ability of NR to reduce para-hydroxylaminobenzoic acid, demonstrating that the corresponding amine does not accumulate to significant levels even under anaerobic conditions. Thus E. cloacae NR is not a good candidate for enzymatic production of aromatic amines.     

Molecular genetics and the initiation of solventogenesis in Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052

Abstract: A physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum ) NCIMB 8052 chromosome has been constructed, encompassing about 90 rare restriction sites. The 14 rrn operons together with 40 genes have been assigned positions on the map. Genetic analysis and gene transfer have been developed in this organism to enable in vivo analysis of the roles of cloned genes using marker replacement technology. Experiments using the available genetic tools have shown that spo0A plays a cardinal role in controlling several aspects of the transition from exponential growth to stationary phase in C. beijerinckii . These include initiation of sporulation, accumulation of the storage polysaccharide, granulose, and production of acetone and butanol. Several C. beijerinckii and C. acetobutylicum genes concerned with fermentative metabolism, whose expression is modulated at the onset of solventogenesis, contain sequence motifs resembling 0A boxes in their 5' regulatory regions. This invites the speculation that they are under direct control of Spo0A, and additional data are now required to test this prediction.     

Enzyme electrophoresis combined with serogrouping for improved differentiation of Clostridium difficile strains

Abstract We used enzyme electrophoresis to study a set of epidemiologically related and unrelated isolates of Clostridium difficile . The 53 strains belonged to the most frequent serogroups (A1, C, G, H and K). Nine electrophoretic profiles were defined on the basis of five enzymes, and two were characteristic of a single strain. Each serogroup was resolved into two or three different enzyme patterns. By combining the two methods we were able to resolve the strains into 12 types. There was an excellent correlation between enzyme electrophoresis and serogrouping data. This method may be of use in investigating nosocomial transmission.     

肠道梭菌属与2型糖尿病的相关性研究进展

摘要：随着经济社会的发展和人类生活水平的提高,2型糖尿病及其慢性并发症不仅严重影响了患者自身的生活质量,而且也给其家庭和社会带来了沉重的负担,已逐渐成为全球突出的社会问题。如果人体内肠道菌群的结构和功能发生紊乱,则可能引起2型糖尿病的发生,因此肠道菌群成为备受关注的研究对象。相关文献指出肠道菌群中的梭菌属与肠黏膜完整性、内脏脂肪、免疫稳态、能量代谢、肠蠕动有关。本文总结了梭菌属的生物学特性、在机体内的生理功能及与2型糖尿病之间的相关性,旨在为进一步研究肠道梭菌属与2型糖尿病间的关系提供参考。     

Control of the Diadenylate Cyclase CdaS in Bacillus subtilis: AN AUTOINHIBITORY DOMAIN LIMITS CYCLIC DI-AMP PRODUCTION*

The Gram-positive bacterium Bacillus subtilis encodes three diadenylate cyclases that synthesize the essential signaling nucleotide cyclic di-AMP. The activities of the vegetative enzymes DisA and CdaA are controlled by protein-protein interactions with their conserved partner proteins. Here, we have analyzed the regulation of the unique sporulation-specific diadenylate cyclase CdaS. Very low expression of CdaS as the single diadenylate cyclase resulted in the appearance of spontaneous suppressor mutations. Several of these mutations in the cdaS gene affected the N-terminal domain of CdaS. The corresponding CdaS mutant proteins exhibited a significantly increased enzymatic activity. The N-terminal domain of CdaS consists of two α-helices and is attached to the C-terminal catalytically active diadenylate cyclase (DAC) domain. Deletion of the first or both helices resulted also in strongly increased activity indicating that the N-terminal domain serves to limit the enzyme activity of the DAC domain. The structure of YojJ, a protein highly similar to CdaS, indicates that the protein forms hexamers that are incompatible with enzymatic activity of the DAC domains. In contrast, the mutations and the deletions of the N-terminal domain result in conformational changes that lead to highly increased enzymatic activity. Although the full-length CdaS protein was found to form hexamers, a truncated version with a deletion of the first N-terminal helix formed dimers with high enzyme activity. To assess the role of CdaS in sporulation, we assayed the germination of wild type and cdaS mutant spores. The results indicate that cyclic di-AMP formed by CdaS is required for efficient germination.     

肿瘤导向治疗研究进展

特异性地杀伤肿瘤细胞并降低对正常细胞的毒副作用是各种导向治疗方法的共同目标。本文综述了前体药物疗法,免疫毒素,基因疗法,以肿瘤增殖转移过程中的关键分子为靶的导向治疗,生物素－亲和素系统等导向治疗的研究进展。     

Production of novel pullulanases at high concentrations by two newly isolated thermophilic clostridia

Two thermophilic bacteria, which are capable of growing on starch at 60-70 degrees C under anaerobic conditions, were isolated from a sugar refinery in Uelzen and from Solar lake in Israel. On the basis of their physiological characteristics they were identified as Clostridium thermohydrosulfuricum Uel 1 and C. thermohydrosulfuricum Sol 1, respectively. The product pattern of glucose polymer hydrolysis showed that both strains secreted enzymes that possess amylolytic and pullulytic activities. The major product formed was maltose. In addition, alpha-glucosidase activity could be detected in the supernatants of Uel 1 strain. Compared to most anaerobes investigated these isolates secreted extremely high concentrations of pullulanases in batch culture. Up to 85% of the total enzyme synthesized was detected in the culture fluid. Unlike the pullulanases of type I, which can only attack the alpha-1,6-glycosidic linkages, the pullulanases of both clostridial strains were also capable of hydrolyzing alpha-1,4-linkages. The enzyme system of both bacteria was found to be highly thermoactive; optimal activity was detected at pH 5.0 and 85 degrees C. Even at 95 degrees C and without the addition of metal ions still 15% to 25% of enzymatic activity was detectable.     

The diverse sporulation characteristics of Clostridium difficile clinical isolates are not associated with type

Clostridium difficile causes diarrhoeal diseases ranging from asymptomatic carriage to a fulminant, relapsing, and potentially fatal colitis. Endospore production plays a vital role in transmission of infection, and in order to cause disease these spores must then germinate and return to vegetative cell growth. Type BI/NAP1/027 strains of C. difficile have recently become highly represented among clinical isolates and are associated with increased disease severity. It has also been suggested that these 'epidemic' types generally sporulate more prolifically than 'non-epidemic' strains, although the few existing reports are inconclusive and encompass only a small number of isolates. In order to better understand any differences in sporulation rates between epidemic and non-epidemic C. difficile types, we analysed these characteristics using 14 C. difficile clinical isolates of a variety of types. Sporulation rates varied greatly between individual BI/NAP1/027 isolates, but this variation did not appear to be type-associated. Furthermore, a number of BI/NAP1/027 spores appeared to form colonies with a lower frequency than specific non-BI/NAP1/027 strains. The data suggest that (i) careful experimental design is required in order to accurately quantify sporulation; and (ii) current evidence cannot link differences in sporulation rates with the disease severity of the BI/NAP1/027 type.     

Sporulation studies in Clostridium difficile

Clostridium difficile is a leading cause of healthcare-associated diarrhoea. In recent years, certain C. difficile types have become highly represented among clinical isolates and are associated with outbreaks of increased disease severity, higher relapse rates and an expanded repertoire of antibiotic resistance. Endospores, produced during sporulation, play a pivotal role in infection and disease transmission and it has been suggested in the literature that these so-called ‘hypervirulent’ C. difficile types are more prolific in terms of sporulation in vitro. However, work in our laboratory has provided evidence to the contrary suggesting that although there is significant strain-to-strain variation in C. difficile sporulation characteristics this variation does not appear to be type-associated. On analysis of the literature, it is apparent that the methods used to quantify sporulation in previous studies have varied greatly and sample sizes have remained small. The conflicting data in the literature may, therefore, not necessarily be generally representative of C. difficile sporulation. Instead, these inconsistencies may reflect differences in the experimental design of each study. In this review, the need for further investigations of C. difficile sporulation rates is highlighted. Specifically, the advantages and disadvantages of the different experimental approaches previously used are discussed and a standard set of principles for measuring C. difficile sporulation in the future is proposed.     

NDV CN株对几种人肿瘤细胞的杀伤作用及其机理的分析

本文研究了NDV-CN株对5株不同的人肿瘤细胞的体外杀伤作用。结果表明5株肿瘤细胞对NDV-CN敏感,于感染早期出现细胞收缩变圆,失去贴壁性,感染第5天时细胞存活率低于10％。其中尤以HEP3B细胞最敏感。但NDV-CN株对人二倍体细胞2BS有弱杀伤性。病毒感染早期可检测到感染细胞中有病毒核酸的复制、感染细胞表面有病毒蛋白的表达,脑浆内有病毒粒子存在。NDV-CN株主要诱导HEP3B及T24细胞产生凋亡,主要诱导Hep2、Hela及A549细胞产生坏死。