Protection against aflatoxin-B1-induced liver mutagenesis by Scutellaria baicalensis

We have measured the inhibition of the mutagenicity of the mycotoxin aflatoxin-B(1) in the liver of the rat by plant material of Scutellaria baicalensis, or Huang-qin. The addition of one percent dried Huang-qin to the feed of the animals reduced the mutant frequency of a subsequent administration of aflatoxin-B1 by approximately 60 and 77%, respectively, for two different batches of the plant material. The addition of Huang-qin also increased the expression of the gene for glutathione S-transferase A5 subunit by 2.5-3.0-fold, and decreased expression of P450 cytochrome 3A2 by 1.8-2.0-fold. The greater increase of the expression of the GST gene may result in the protection shown by Huang-qin. The sensitivity of the hepatic mitochondria to swelling, a measure of the mitochondrial permeability transition, is increased significantly in animals that are on a diet containing Huang-qin. This may lead to increased sensitivity to apoptosis on treatment with toxic compounds. The two batches of Huang-qin material show differences in both chemical composition and preventive potential. This study demonstrates how a combination of generating and analysis of plant varieties together with a mammalian assay for efficacy may improve the search for better plant-based prevention of cancer initiation.     

Preservation of Neisseria genus microbes by a deep freezing method]

A method of meningococcus and of vaccine gonococcus strains preservation under conditions of deep freezing with polyethyleneoxide of mol mass 400 was developed. Peculiarities of phasic transitions were studied by the method of differential thermal analysis; hydration properties of polyethyleneoxide were investigated by nuclear magneticresonance (spin-echo). The main cultural and biochemical properties after freezing and after one year of storage in 8 strains of the microbes under study remained unchanged; their survival constituted not less than 90%.     

黄曲霉毒素B1降解菌的分离鉴定及其降解特性

【目的】黄曲霉毒素是一类强毒、致癌的真菌次级代谢产物。本文旨在筛选出能高效降解黄曲霉毒素B1(AFB1)的细菌。【方法】以AFB1结构类似物香豆素为惟一碳源进行AFB1降解菌株初筛,得到的活性菌株的培养液分别与AFB1标准品(2.5μg/mL)共同作用,以AFB1降解率为指标进行复筛。对降解活性最好的菌株通过形态、生理生化特性以及16S rRNA序列分析进行初步鉴定;并对细胞浓度、pH、温度、金属离子等对菌株降解活性的影响进行考察。【结果】初筛获得了10株在香豆素培养基上生长良好的细菌,复筛发现这些菌均具有良好的AFB1降解活性,其中从金毛羚牛粪便中筛选出的菌株F4降解活性最好,去除AFB1能力达到90.03%。根据F4菌株16S rRNA序列同源性分析,结合形态、生理生化特性,初步确定菌株F4为施氏假单胞菌(Pseudomonas stutzeri)。F4的降解活性与细胞浓度呈正相关。当pH 7.0,35℃,菌细胞作用72 h后毒素降解率达到82.91%。Mg2+可增强F4的降解活性,降解率提高7.68%,而Cu2+可抑制其降解活性,降解率降低51.1%。【结论】筛选到能高效降解AFB1的施氏假单胞菌(Pseudomonas stutzeri)F4,F4降解毒素的活性物质主要存在于菌体细胞,其作用受到温度、pH等的影响,可能是一种胞内酶。     

Distribution of microbes producing antimicrobial epsilon-poly-L-lysine polymers in soil microflora determined by a novel method

We developed a simple and sensitive screening method to investigate the distribution of microbes producing an antimicrobial poly(amino acid), epsilon-poly-L-lysine (epsilon-PL), in microflora. An acidic dye, Poly R-478, incorporated in an agar plate detected epsilon-PL producers by electrostatic interaction with the secreted basic polymers. All epsilon-PL producers, isolated after careful and sufficient screening of soil microflora, belonged exclusively to two groups of bacteria of the family Streptomycetaceae and ergot fungi. They were characterized based on the density and diameter of the concentric zone formed by the secreted polymers. The density depended on each isolate. The increase in the diameter of the concentric zone per unit of time varied among isolates and was negatively correlated with the molecular weight. Although the distribution of epsilon-PL producers was extremely limited, their products were structurally varied. The molecular masses of the secreted polymers among the isolates ranged from 0.8 to 2.0 kDa. There were also isolates producing unknown polymers inconsistent with the correlation or producing a mixture of polymers with original and modified structures. A chemically modified polymer was an epsilon-PL derivative, as determined by mass spectrometry. Since the structural variations had no relation to the phylogenetic position of the isolates, it is possible that enzymes involved in the synthesis diversified after putative horizontal transfers of relevant genes.     

Development of a simple cultivation method for isolating hitherto-uncultured cellulase-producing microbes

Although enrichment culture is typically employed to isolate cellulolytic microbes, this approach tends to favor fast-growing species and discriminates against all others. Therefore, efforts to prevent the overgrowth of fast-growing species are necessary to isolate novel cellulase-producing strains. In this study, we developed a simple culture method for isolating hitherto-uncultured microbes that possess cellulase activity, particularly exocellulase. In this method, the microbial source (a forest soil) was suspended in sterilized water and inoculated onto a mineral salts agar medium, which was then overlaid with filter paper to sandwich the microbial suspension between the agar surface and paper. The filter paper fibers served to immobilize the microbial cells and were the dominant carbon source. Following cultivation at 30°C for 2 weeks, emerging colonies were isolated based on their morphology and were then subjected to phylogenetic and enzyme analyses. Using this method, 2,150 CFUs/g dry soil were obtained, and the ratio of fungal to bacterial isolates was approximately 4:1. Phylogenetic analyses revealed that most fungal and bacterial isolates belong to ten and two genera, respectively. Notably, all isolates possessed exocellulase activity, and several strains showed strong activity that was comparable to Trichoderma cellulase. Many isolates also exhibited cellulase and xylanase activity, and several strains possessed laccase activity. It is expected that the culture method described here will be useful for the isolation of hitherto-uncultured cellulolytic microbes and the identification of novel cellulases.     

Elaboration of a method of stabilizing the protective fraction of pertussis microbes

A method for stabilizing adsorbed preparations of the protective fraction of B. pertussis has been developed; according to this method, a colloid suspension of protective protein in phosphate buffer is obtained, the protein is then adsorbed on aluminium hydroxide gel and lyophilized with 10% of sucrose. If stored at 4 degrees C, these dried preparations have been found to retain their immunogenicity for 1 year.     

一种提取小鼠肠道中活体微生物的方法

目的 建立一种有效的肠道微生物提取方法,用于基于培养方法研究肠道微生物.方法 研究NaCl溶液体积、内容物振荡时间、离心转速和高速离心次数对肠道微生物提取数量、质量及其活性的影响.结果 在盛有适量玻璃珠的三角瓶中,将肠道内容物与0.85％的NaCl溶液按质量体积比(g∶mL)不大于1∶10进行混合,120 r/min漩涡振荡30 min,500 r/min离心5 min去除沉淀,将上清液分装于EP管中12 000 r/mim高速离心1次,弃去上清,在EP管中加入0.85％ NaCl溶液进行漩涡振荡使菌体充分分散,即可得到提取完全、质量高的小鼠肠道微生物.结论 该方法能够提取足够多的肠道微生物,并且保持其活性.     

Oxidative killing of microbes by neutrophils

Neutrophils and other phagocytic leukocytes contain a phagocyte NADPH oxidase enzyme that generates superoxide after cell activation. Reactive oxygen species derived from superoxide, together with proteases liberated from the granules, are used to kill ingested microbes. Dysfunction of the phagocyte NADPH oxidase results in chronic granulomatous disease, with life-threatening infections.     

Protection of salvia miltiorrhiza against aflatoxin-B1-induced hepatocarcinogenesis in Fischer 344 rats dual mechanisms involved.

Extract of Salvia Miltiorrhiza (SM) has been widely used in traditional Chinese medicine for treating liver diseases. Recent experimental evidence indicates that it has anti-tumor potential. In this study, the effect of SM on alfatoxin B1 (AFB1)-induced hepatocarcinogenesis was investigated in male Fischer 344 rats. AFB1 (40 microg/100 g body wt, by gavage) was administered once a week for 24 weeks. In SM treatment group, rats were given SM (0.25g/100g body wt, 5 days/week by gavage) for a total of 28 weeks, including 4 weeks before and 24 weeks during AFB1 exposure. Results showed that the elevation of serum alanine aminotransferase and aspartate aminotransferase activities due to AFB1 dosing was almost completely abolished by the treatment of SM, indicating that SM could prevent AFB1-induced liver cell injury. It was further observed that SM substantially reduced glutathione S-transferase placenta form (GST-P) positive foci formation and GST-P mRNA expression caused by AFB1, which clearly suggests that SM is effective in preventing AFB1-induced hepatocarcinogenesis. Furthermore, the inhibition on AFB1 hepatocarcinigenesis was associated with a corresponding decrease in AFB1-DNA adducts formation as well as AFB1-induced oxidative DNA damage (8-hydroxydeoxyguanosine) in rat liver. Our results also indicate that the protective effect of SM might be mediated through dual mechanisms: (i) the enhancement of AFB1 detoxification pathway, especially the induction of GST-Yc2 mRNA expression, and (ii) the antioxidant property of SM.     

Production of artemisinin by genetically-modified microbes

Artemisinin, an endoperoxidized sesquiterpene originally extracted from the medicinal plant Artemisia annua L., is a potent malaria-killing agent. Due to the urgent demand and short supply of this new antimalarial drug, engineering enhanced production of artemisinin by genetically-modified or transgenic microbes is currently being explored. Cloning and expression of the artemisinin biosynthetic genes in Saccharomyces cerevisiae and Escherichia coli have led to large-scale microbial production of the artemisinin precursors such as amorpha-4,11-diene and artemisinic acid. Although reconstruction of the complete biosynthetic pathway toward artemisinin in transgenic yeast and bacteria has not been achieved, artemisinic acid available from these transgenic microbes facilitates the subsequent partial synthesis of artemisinin by either chemical or biotransformational process, thereby providing an attractive strategy alternative to the direct extraction of artemisinin from A.annua L. In this review, we update the current trends and summarize the future prospects on genetic engineering of the microorganisms capable of accumulating artemisinin precursors through heterologous and functional expression of the artemisinin biosynthetic genes.     

A new strain, Streptomyces venezuelae GY1, producing a poly(vinyl alcohol)-degrading enzyme

Summary An actinomycete strain, which could produce an extracellular poly(vinyl alcohol) (PVA)-degrading enzyme, was isolated from a PVA-contaminated soil sample using PVA as the sole carbon source. The strain was identified as Streptomyces venezuelae according to the whole-nucleotide-sequence analysis of 16S rDNA, the morphological and the physiological characteristics. The strain produced 120 U/l extracellular PVA-degrading enzyme when PVA was used as the sole carbon source. When glucose was used as the sole carbon source, however, the extracellular enzyme activity was very low (12 U/l). This is the first report showing that an actinomycete strain can produce a PVA-degrading enzyme.     

产黄曲霉毒素B1降解酶菌株的筛选鉴定及发酵条件优化

以香豆素为唯一碳源筛选到27株能高效降解黄曲霉毒素B1(AFB1)的微生物菌株.用高效液相色谱检测AFB1含量的方法进行AFB1降解酶活力测定.以不同菌株发酵上清液中AFB1降解酶活力高低为复筛条件,筛选到AFB1降解酶活力最高的一株菌并命名为HSD8.该菌株经形态学、生理生化及系统发育学方法鉴定为Sinomonas sp..筛选所得最优菌株的发酵上清液中酶活达443 U/mL,通过单因素试验对其产酶发酵条件进行优化,以提高酶活.优化所得最佳发酵条件为:装液量50 mL/250 mL,发酵周期48 h,初始pH 5.0,接种量8％,发酵温度37℃,摇床转速160r/min.在最佳发酵条件下,该菌株发酵上清液中酶活可达548 U/mL,比优化前提高23.7％.优选菌株HSD8在生物降解黄曲霉毒素B1方面具有应用潜力,值得进一步研究开发.     

Water is a preservative of microbes

Water is the cellular milieu, drives all biochemistry within Earth’s biosphere and facilitates microbe-mediated decay processes. Instead of reviewing these topics, the current article focuses on the activities of water as a preservative—its capacity to maintain the long-term integrity and viability of microbial cells—and identifies the mechanisms by which this occurs. Water provides for, and maintains, cellular structures; buffers against thermodynamic extremes, at various scales; can mitigate events that are traumatic to the cell membrane, such as desiccation–rehydration, freeze–thawing and thermal shock; prevents microbial dehydration that can otherwise exacerbate oxidative damage; mitigates against biocidal factors (in some circumstances reducing ultraviolet radiation and diluting solute stressors or toxic substances); and is effective at electrostatic screening so prevents damage to the cell by the intense electrostatic fields of some ions. In addition, the water retained in desiccated cells (historically referred to as ‘bound’ water) plays key roles in biomacromolecular structures and their interactions even for fully hydrated cells. Assuming that the components of the cell membrane are chemically stable or at least repairable, and the environment is fairly constant, water molecules can apparently maintain membrane geometries over very long periods provided these configurations represent thermodynamically stable states. The spores and vegetative cells of many microbes survive longer in the presence of vapour-phase water (at moderate-to-high relative humidities) than under more-arid conditions. There are several mechanisms by which large bodies of water, when cooled during subzero weather conditions remain in a liquid state thus preventing potentially dangerous (freeze–thaw) transitions for their microbiome. Microbial life can be preserved in pure water, freshwater systems, seawater, brines, ice/permafrost, sugar-rich aqueous milieux and vapour-phase water according to laboratory-based studies carried out over periods of years to decades and some natural environments that have yielded cells that are apparently thousands, or even (for hypersaline fluid inclusions of mineralized NaCl) hundreds of millions, of years old. The term preservative has often been restricted to those substances used to extend the shelf life of foods (e.g. sodium benzoate, nitrites and sulphites) or those used to conserve dead organisms, such as ethanol or formaldehyde. For living microorganisms however, the ultimate preservative may actually be water. Implications of this role are discussed with reference to the ecology of halophiles, human pathogens and other microbes; food science; biotechnology; biosignatures for life and other aspects of astrobiology; and the large-scale release/reactivation of preserved microbes caused by global climate change.     

Establishment of a lepidopteran hybrid cell line by use of a biochemical blocking method

Summary We report here on the establishment of aCydia pomonella (Cp) hybrid cell line IZD-Cp 4/13. As there have been no reports on somatic cell fusion involving lepidopteran cell lines so far, we had to develop an appropriate fusion procedure. We first tried—but without much success—to obtain HAT (hypoxanthine, aminopterin and thymidine)- or TAM (thymidine, adenine, and methotrexate)-selectable strains of the threeCydia pomonella cell lines IZD-Cp 2202, IZD-Cp 0508 and Cp 169. We then tried and succeeded in developing a fusion procedure based on the use of biochemically blocked permanent cells as one partner in the fusion. Biochemically inhibited IZD-Cp 2202 cells and embryonic Cp cells were hybridized by polyethyleneglycol treatment. The cells of the hybrid cell line IZD-Cp 4/13 differ from the permanent parental cells (cell line IZD-Cp 2202) with respect to morphology, DNA content isoenzyme patterns, and response to challenge with theChoristoneura murinana nuclear polyhedrosis virus. This work was supported by the Bundesministerium fuer Forschung und Technologie, Bonn, FRG.     

The use of a radioisotopic method for estimation of galactose-1-phosphate in galactosemia

A new, specific, and sensitive radioisotopic method for the determination of galactose-1-P in red blood cells was described. The amount of [¹⁴C]glucose-1-P formed in the reaction UDP-[¹⁴C]glucose + galactose-1-P ⇄ UDP-galactose + [¹⁴C]glucose-1-P is a measure of the amount of galactose-1-P present in the investigated material.