Persistence of Toxic Activity and Recycling of Bacillus thuringiensis var. israelensis in Cold Water: Field Experiments Using Diffusion Chambers in a Pond

The persistence of the larvicidal activity of Bacillus thuringiensis var. israelensis (Bti) was tested over a five-month period in a low-temperature aquatic environment. Diffusion chambers filled with a suspension of Bti (100 mg l - 1) and different experimental substrates (pond water, periphyton, sediments and vegetation), but without mosquito larvae, were placed near the bottom of a large pond and removed at various intervals to measure residual toxic activity to mosquito larvae, spore concentration and proteolytic activity. Within the pond water substrate, 50% of the initial toxicity was still present after one month in cold water, while with the periphyton substrate, 30% remained in the liquid fraction after the same period of exposure. Within the vegetation substrate (blue-joint grass, Calamagrostis canadensis), an average of 30% of the initial toxicity was still present in the liquid fraction between day 84 to day 154. Solid fractions of vegetation became toxic very early and remained toxic for five months. At the end (day 154), there was still 54% of the original toxic activity put in the chambers associated with the vegetation samples. In the absence of mosquito larvae, spore recycling was observed in the chambers especially with sediments and vegetation. But spore recycling did not appear to play a major role in the observed persistence, but rather rapid absorption onto vegetation substrates was responsible for the persistence of Bti in a cold climate.     

Specific immuno capturing of the Staphylococcal superantigen toxic‐shock syndrome toxin‐1 in plasma

Toxic‐shock syndrome is primarily caused by the Toxic‐shock syndrome toxin 1 (TSST‐1), which is secreted by the Gram‐positive bacterium Staphylococcus aureus. The toxin belongs to a family of superantigens (SAgs) which exhibit several shared biological properties, including the induction of massive cytokine release and V_β‐specific T‐cell proliferation. In this study we explored the possibility to use monoclonal Variable domains of Llama Heavy‐chain antibodies (VHH) in the immuno capturing of TSST‐1 from plasma. Data is presented that the selected VHHs are highly specific for TSST‐1 and can be efficiently produced in large amounts in yeast. In view of affinity chromatography, the VHHs are easily coupled to beads, and are able to deplete TSST‐1 from plasma at very low, for example, pathologically relevant, concentrations. When spiked with 4 ng/mL TSST‐1 more than 96% of TSST‐1 was depleted from pig plasma. These data pave the way to further explore application of high‐affinity columns in the specific immuno depletion of SAgs in experimental sepsis models and in sepsis in humans. Biotechnol. Bioeng. 2009; 104: 143–151 © 2009 Wiley Periodicals, Inc.     

Operculina turpethum attenuates N-nitrosodimethylamine induced toxic liver injury and clastogenicity in rats

The root extract of Operculina turpethum (OTE) has been used as an anti-inflammatory, purgative, and hepato-protective agent. N-Nitrosodimethylamine (NDMA) is a potent hepatotoxin that induces fibrosis of the liver. In the present study, we examined the therapeutic effects of OTE root extract against NDMA-induced hepatotoxicity and clastogenicity in rats. Hepatic fibrosis was induced in adult male albino rats through serial intraperitoneal administrations of NDMA at a concentration of 10 mg/kg body weight on three consecutive days of each week over a period of three weeks. A group of rats received OTE orally in doses of 75, 150 and 200 mg/kg body weight at 5 h after the administration of NDMA. The controls and treated animals were sacrificed on days-7, 14 and 21 after the start of the administration of NDMA. The progression of hepatic fibrosis as well as the amelioration effect of OTE was evaluated through histopathologically as well as by immunohistochemical staining for the activation of hepatic stellate cells. Alterations in serum and liver biochemical parameters and LDH isoenzymes were also studied. Serial administration of NDMA resulted in well formed fibrosis in the liver and induction of micronuclei in the bone marrow cells. Staining of α-SMA demonstrated activated stellate cells from day-7 onwards which was dramatically increased on day-21. An elevation of micronuclei count, liver function enzymes, serum hydroxyproline levels and LDH isoenzymes 4 and 5 were also observed. All these changes were remarkably reduced in OTE administered animals and fibrogenesis was completely absent. Our results suggest that OTE has hepatoprotective and anti-clastogenic effects against NDMA-induced hepatic fibrosis. Therefore OTE may be used as a hepatoprotective agent against various liver diseases including toxic liver injury.     

A model of amyloid's role in disease based on fibril fracture

Although the correlative evidence relating the presence of amyloid fibrils and certain disease states (e.g. Alzheimer's disease and Type 2 Diabetes) is overwhelming, a direct causative role for amyloid has proved harder to establish. Current thinking links a narrow region of the aggregate protein size distribution, the so called ‘early aggregate’ domain to cellular toxicity. A troubling feature of this theory however is that the nucleated reaction mechanism by which amyloid formation is believed to occur results in a very low number concentration of early aggregates which are rapidly extended to form amyloid fibrils. This situation means that the concentration of early aggregates is very low at the time when they are supposedly at their most toxic. Here we adopt a novel explicit simulation strategy to examine a kinetic regime involving nucleated growth combined with fibril fragmentation under which this situation can be reversed so as to produce a high number concentration of small on-pathway toxic aggregates. Dependent upon the rate of fragmentation, the time scale for generation of toxic early aggregates may be coupled, uncoupled or disassociated from the time scale for the appearance of amyloid fibrils. Furthermore the model presents itself as a biochemical ‘switch’ transitioning between modes of amyloid induced cell death dependent upon either specific amyloid toxicity or non-specific solid mass induced tissue damage.     

Pesticide ecotoxicological effect factors and their uncertainties for freshwater ecosystems

Background, aim, and scope  Characterization factors for ecotoxicity in the Life Cycle Impact Assessment (LCIA) are used to convert emissions into ecotoxicological impacts. Deriving them involves a fate and an effect analysis step. The fate factor quantifies the change in environmental concentration per unit of emission, while the effect factor quantifies the change in impact on the ecosystem per unit of environmental concentration. This paper calculates freshwater ecotoxicological effect factors for 397 pesticides belonging to 11 pesticide-specific toxic modes of action (TMoA), such as acetylcholinesterase inhibition and photosynthesis inhibition. Moreover, uncertainties in the effect factors due to uncertain background concentrations and due to limited toxicity data are quantified. Methods  To calculate median ecotoxicological effect factors (EEFs), toxic pressure assessments were made, based on the species sensitivity distribution—and the multisubstance potentially affected fraction—concept. The EEF quantifies an estimate of the fraction of species that is probably affected due to a marginal change in concentration of a pesticide. EEFs were divided into a TMoA-specific and a chemical-specific part, which were calculated on the basis of physicochemical properties, emissions, and toxicity data. Propagation of parameter uncertainty in the EEFs and the TMoA- and chemical-specific parts was quantified by Monte Carlo simulation and results were reported as 90% confidence intervals. Results  Median EEFs range from 2·10⁻³ to 7·10⁶ l/g. Uncertainty in the TMoA-specific part is dominated by uncertainty in the TMoA-specific spread in species sensitivity and by uncertainty in the effective toxicity of a TMoA. Uncertainty in the chemical-specific part of the EEFs depends on the number of species for which toxicity data are available to calculate average toxicity (n _s) and ranges from a median uncertainty of 2.6 orders of magnitude for n _s = 2 to one order of magnitude for n _s ≥ 4. The TMoA-specific effect factor for systemic fungicides shows the largest uncertainty range. For seven TMoAs, uncertainty ranges of the TMoA-specific effect factor are less than two orders of magnitude. For the other four TMoAs, the EEF uncertainty range is between two and eight orders of magnitude. For the chemical-specific part of the EEFs, we found that variation in uncertainty readily decreases for pesticides for which toxicity data are available for at least three species. Discussion  The same parameters that contributed most to uncertainty were found for pesticides as were found before for high-production-volume chemicals. However, uncertainty in concentrations of pesticides was lower. TMoA-specific factors obtained with the applied nonlinear method differ up to nine orders of magnitude from the factor of 0.5, which is used in the linear method. With the applied method, a distinction in EEFs can be made among different TMoAs. Conclusions   Ecotoxicological effect factors are presented, including overviews of their uncertainty ranges and the main contributors to uncertainty. The applied nonlinear method provides the possibility to quantify parameter uncertainty in the TMoA-specific part of the ecotoxicological effect factor, which is helpful to get more insight in how uncertainty in ecotoxicological characterization factors can be reduced. Recommendations and perspectives  The calculated uncertainty ranges can be included in life cycle assessment (LCA) case studies, which allows for better interpretation of LCA results obtained with the EEFs. To put the uncertainty in effect factors into perspective within LCIA, more information on the uncertainty in fate factors should be derived. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biosurfactant of marine origin exhibiting heavy metal remediation properties

The present study was aimed at elucidating the role of biosurfactant product isolated from a marine bacterium in removing heavy metals from heavy metal containing solutions. In this study, metal removal was biosurfactant-mediated. Efficiency of metal removal depended on the concentration of the metal as well as that of the biosurfactant. At a concentration 5×, the critical micelle concentration (CMC), almost complete removal of 100 ppm of lead and cadmium occurred. Atomic absorption spectroscopy (AAS) studies also showed metal removal at a concentration less than the CMC in contrast to earlier findings that only micelles are involved in metal removal. Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM) equipped with energy dispersive X-ray spectroscopy (EDS) further substantiated these findings.     

Novel strategies to overcome expression problems encountered with toxic proteins: Application to the production of Lac repressor proteins for NMR studies

NMR studies of structural aspects of allosteric regulation by the Lac repressor requires overexpression and isotope labeling of the protein. The size of the repressor makes it a challenging target, putting constraints on both expression conditions and sample preparation methods to overcome problems associated with studies of larger proteins by NMR. We optimized protocols for the production of deuterated functionally active thermostable dimeric Lac repressor and its core domain mutants. The Lac repressor core domain has never been obtained as a recombinant protein, possibly due to the observed toxicity to the host cells. We overcame the core domain induced toxicity by co-expression of this domain with the full length Lac repressor, combined with a stringent control of culture conditions. Significant overexpression was only obtained if during all stages of pre-culturing the bacteria were kept in their exponential growth phase at low density. The sensitivity of NMR measurements is dramatically affected by buffer conditions; we therefore used a thermofluor buffer optimization screen to determine the optimal buffer conditions. The combined thermofluor and NMR screening method yielded thermostable fully functional Lac repressor domain samples suitable for high-resolution NMR studies. The optimized procedures to adapt Escherichia coli to growth in D₂O, to overcome toxicity, and to optimize protein sample conditions provides a broad range of universally applicable techniques for production of larger proteins for NMR spectroscopy.     

The effect of subminimal inhibitory concentrations of antibiotics on virulence factors expressed by Staphylococcus aureus biofilms

Aims: The effect of subminimal inhibitory concentrations (sub‐MICs) of cefalexin, ciprofloxacin and roxithromycin was investigated on some virulence factors [e.g. coagulase, Toxic Shock Syndrome Toxin 1 (TSST‐1) and biofilm formation] expressed by Staphylococcus aureus biofilms. Methods and Results: Biofilms were grown with and without the presence of 1/16 MIC of antibiotics on Sorbarod filters. Eluate supernatants were collected, and coagulase and TSST‐1 production were evaluated. Coagulase production was reduced in eluates exposed to roxithromycin when compared to control, while TSST‐1 production was reduced in biofilms exposed to cefalexin and to a lesser extent, ciprofloxacin. In addition, the ability of Staph. aureus to produce biofilm in microtitre plates in the presence of sub‐MIC antibiotics indicated that cefalexin induced biofilm formation at a wide range of sub‐MICs. TSST‐1 produced from the challenged and control biofilms was purified, and its proliferative activity was studied on single cell suspension of mouse splenocytes using MTS/PMS assay. No significant difference in the activity between the treated toxin and the control has been observed. Conclusions: Antibiotics at sub‐MIC levels interfere with bacterial biofilm virulence expression depending on the type and concentration of antibiotic used. Significance and Impact of the Study: The establishment of sub‐MICs of antibiotics in clinical situations may result in altered virulence states in pathogenic bacteria.     

三峡库区藻类水华调查及其毒理学研究

三峡水库一期工程蓄水近两年(2003年7月—2005年4月)期间,库区发生过多次藻类“水华”,尤其在阳光充足、气温回升的春季。经鉴定形成水华的藻类主要有拟多甲藻(Peridiniopsissp1,sp2),新星形冠盘藻(Stephanodiscusneoastraea),里海小环藻(Cyclotella caspia),湖沼红胞藻(Rhodomonas lacustris),实球藻(Pandorina morum)和空球藻(Eudorina elegans);其中以拟多甲藻水华涉及面积最大,持续时间最久,污染程度最为严重。通过对拟多甲藻水华水及其藻细胞提取液的生物毒性测试,表明其对其他水生生物和小白鼠没有明显的生物毒性。     

水稻新基因OsRwp34在大肠杆菌中表达的毒害作用

在水稻高温诱导cDNA文库中克隆了一个新的水稻基因O sRwp34,同源性分析表明O sRwp34是一典型的孤儿基因。为了研究O sRwp34的功能,构建了O sRwp34的表达载体pET-28(a)OsRwp34,并转化大肠杆菌菌株BL21(DE3),用IPTG诱导并定时从培养液中取样以检测OD600值和用平板记数法测定活菌数,结果发现在诱导30 m in后宿主菌开始大量死亡,表明O sRwp34的表达产物使宿主菌死亡。随后构建了1个N末端缺失15个氨基酸残基和2个C末端分别缺失12和47个氨基酸残基的O sRwp34缺失体,IPTG诱导后都没有出现毒性作用,推测N和C末端氨基酸残基的同时存在是OsRwp34的毒性作用所必须的。其详细机理有待进一步研究。