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%.     

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.     

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.     

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.     

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.     

Production of isoprenoid pharmaceuticals by engineered microbes

Throughout human history, natural products have been the foundation for the discovery and development of therapeutics used to treat diseases ranging from cardiovascular disease to cancer. Their chemical diversity and complexity have provided structural scaffolds for small-molecule drugs and have consistently served as inspiration for medicinal design. However, the chemical complexity of natural products also presents one of the main roadblocks for production of these pharmaceuticals on an industrial scale. Chemical synthesis of natural products is often difficult and expensive, and isolation from their natural sources is also typically low yielding. Synthetic biology and metabolic engineering offer an alternative approach that is becoming more accessible as the tools for engineering microbes are further developed. By reconstructing heterologous metabolic pathways in genetically tractable host organisms, complex natural products can be produced from inexpensive sugar starting materials through large-scale fermentation processes. In this Perspective, we discuss ongoing research aimed toward the production of terpenoid natural products in genetically engineered Escherichia coli and Saccharomyces cerevisiae.     

Rapid method of determining the dry mass of whooping cough microbes

A colorimetric method for the rapid determination of the quantitative content of microbial mass in B. pertussis suspensions has been developed. The method is based on the indirect determination of carbon in microbial suspensions by its oxidation with the mixture consisting of potassium bichromate in concentrated sulfuric acid and the subsequent colorimetric analysis of the products of this reaction. The method ensures sufficient accuracy, the determination procedure is simple, takes not more than 2 hours and requires no complex reagents. The results thus obtained are well comparable with those obtained by the classical gravimetric method. The new method permits the determination of microbial mass in B. pertussis suspensions with a minimum concentration of 0.5 mg/ml. The method is recommended for the determination of dry microbial mass in B. pertussis suspensions.     

Development of a rapid method for simultaneous recovery of diverse microbes in drinking water by ultrafiltration with sodium polyphosphate and surfactants

The ability to simultaneously concentrate diverse microbes is an important consideration for sample collection methods that are used for emergency response and environmental monitoring when drinking water may be contaminated with an array of unknown microbes. This study focused on developing a concentration method using ultrafilters and different combinations of a chemical dispersant (sodium polyphosphate [NaPP]) and surfactants. Tap water samples were seeded with bacteriophage MS2, Escherichia coli, Enterococcus faecalis, Cryptosporidium parvum, 4.5-microm microspheres, Salmonella enterica serovar Typhimurium, Bacillus globigii endospores, and echovirus 1. Ten-liter tap water samples were concentrated to approximately 250 ml in 12 to 42 min, depending on the experimental condition. Initial experiments indicated that pretreating filters with fetal bovine serum or NaPP resulted in an increase in microbe recovery. The addition of NaPP to the tap water samples resulted in significantly higher microbe and microsphere recovery efficiencies. Backflushing of the ultrafilter was found to significantly improve recovery efficiencies. The effectiveness of backflushing was improved further with the addition of Tween 80 to the backflush solution. The ultrafiltration method developed in this study, incorporating the use of NaPP pretreatment and surfactant solution backflushing, was found to recover MS2, C. parvum, microspheres, and several bacterial species with mean recovery efficiencies of 70 to 93%. The mean recovery efficiency for echovirus 1 (49%) was the lowest of the microbes studied for this method. This research demonstrates that ultrafiltration can be effective for recovering diverse microbes simultaneously in tap water and that chemical dispersants and surfactants can be beneficial for improving microbial recovery using this technique.     

Modulation of host immunity by beneficial microbes

In nature, plants abundantly form beneficial associations with soilborne microbes that are important for plant survival and, as such, affect plant biodiversity and ecosystem functioning. Classical examples of symbiotic microbes are mycorrhizal fungi that aid in the uptake of water and minerals, and Rhizobium bacteria that fix atmospheric nitrogen for the plant. Several other types of beneficial soilborne microbes, such as plant-growth-promoting rhizobacteria and fungi with biological control activity, can stimulate plant growth by directly suppressing deleterious soilborne pathogens or by priming aboveground plant parts for enhanced defense against foliar pathogens or insect herbivores. The establishment of beneficial associations requires mutual recognition and substantial coordination of plant and microbial responses. A growing body of evidence suggests that beneficial microbes are initially recognized as potential invaders, after which an immune response is triggered, whereas, at later stages of the interaction, mutualists are able to short-circuit plant defense responses to enable successful colonization of host roots. Here, we review our current understanding of how symbiotic and nonsymbiotic beneficial soil microbes modulate the plant immune system and discuss the role of local and systemic defense responses in establishing the delicate balance between the two partners.     

A novel method for measuring the charge distribution of airborne microbes

A novel method for measuring bioaerosol charge distribution was investigated using electrostatic sampling and quantitative polymerase chain reaction (qPCR). Here, Bacillus subtilis var niger and Pseudomonas fluorescens were aerosolized and precipitated into different regions of two 96-well plates placed inside an electrostatic sampler. The concentrations of negatively charged bacteria in the air samples collected were quantified using qPCR, and the relevant bioaerosol charge levels were calculated using an equation developed in this study. Experimental results showed that B. subtilis var niger bioaerosols had wider charge distribution than P. fluorescens. For B. subtilis var niger, 93% of them carried a charge level of 13–42 units, 2% carried a charge level of 8–13 or lower, and 5% carried a charge level of 42–195 or higher. While for P. fluorescens, 99% of P. fluorescens aerosols carried a charge level of 14–34 elementary units. The indicative charge levels of bioaerosols collected at a certain point in the electrostatic sampling line largely depends on the particle size, bacterial species, electrostatic field strength, and the sampling flow rates for a given electrostatic sampler. In general, the trends for the charge levels here were similar to those obtained in previous studies. Use of qPCR technology in this study can distinguish between biological and non-biological particles collected. Thus, this avoided counting the non-biological particles in traditional bioaerosol charge measurements in which an optical particle counter is used. The developed technique here can be also used to analyze the charge levels for other types of bioaerosols.