Production, recovery and immunogenicity of the protective antigen from a recombinant strain of Bacillus anthracis

The protective antigen (PA) is one of the three components of the anthrax toxin. It is a secreted nontoxic protein with a molecular weight of 83 kDa and is the major component of the currently licensed human vaccine for anthrax. Due to limitations found in the existing vaccine formulation, it has been proposed that genetically modified PA may be more effective as a vaccine. The expression and the stability of two recombinant PA (rPA) variants, PA-SNKE-ΔFF-E308D and PA-N657A, were studied. These proteins were expressed in the nonsporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-ΔFF-E308D, which lacks two proteolysis-sensitive sites, is more stable than PA-N657A. Process development was conducted to establish an efficient production and purification process for PA-SNKE-ΔFF-E308D. pH, media composition, growth strategy and protease inhibitors composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only source of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time was 14–18 h after inoculation, and EDTA (5 mM) was added upon harvest for proteolysis control. Recovery of the rPA was performed by expanded-bed adsorption (EBA) on a hydrophobic interaction chromatography (HIC) resin, eliminating the need for centrifugation, microfiltration and diafiltration. The EBA step was followed by ion exchange and gel filtration. rPA yields before and after purification were 130 and 90 mg/l, respectively. The purified rPA, without further treatment, treated with small amounts of formalin or adsorbed on alum, induced, high levels of IgG anti-PA with neutralization activities. Journal of Industrial Microbiology & Biotechnology (2002) 28, 232–238 DOI: 10.1038/sj/jim/7000239 Received 28 August 2001/ Accepted in revised form 20 December 2001     

Total soluble protein profiles of Beauveria bassiana and their relationship with virulence against Helicoverpa armigera

Intracellular total soluble proteins of Beauveria bassiana are believed to play an important role in virulence against insect hosts. Thirty B. bassiana isolates collected from different geographical regions and host ranges were characterised by total soluble proteins present in cells, using the SDS–PAGE technique to differentiate the isolates based on virulence and host insect origin. In vitro analysis of total soluble protein profiles of 30 isolates was studied to understand the relationship of isolates with their host of origin and virulence against Helicoverpa armigera. There was a positive relationship between virulence and host origin. All the non-virulent isolates are grouped together. Similarly, highly virulent isolates against H. armigera were grouped together. The relationship between total soluble proteins and pathogenicity was positively correlated. Thirty isolates shared only 22% similarity in their protein profiles.     

Oxidative stress triggered by aluminum in plant roots

Aluminum (Al) is a major growth-limiting factor for plants in acid soils. The primary site of Al accumulation and toxicity is the root meristem, and the inhibition of root elongation is the most sensitive response to Al. Al cannot catalyze redox reactions but triggers lipid peroxidation and reactive oxygen species (ROS) production in roots. Furthermore, Al causes respiration inhibition and ATP depletion. Comparative studies of Al toxicity in roots with that in cultured plant cells suggest that Al causes dysfunction and ROS production in mitochondria, and that ROS production, but not lipid peroxidation, seems to be a determining factor of root-elongation inhibition by Al.     

Chemical Composition,Cytotoxic and Antibacterial Activities of Essential Oils of Cultivated Clones of Juniperus communis and Wild Juniperus Species

Needles of seven cultivated clones (C1 – C7) of Juniperus communis at lower altitude and three wild Juniperus species (J. communis, J. recurva and J. indica) at higher altitudes were investigated comparatively for their essential oils (EOs) yields, chemical composition, cytotoxic and antibacterial activities. The EOs yields varied from 0.26 to 0.56% (v/w) among samples. Sixty‐one volatile components were identified by gas chromatography‐mass spectrometry (GC/MS) and quantified using gas chromatography GC (FID) representing 82.5 – 95.7% of the total oil. Monoterpene hydrocarbons (49.1 – 82.8%) dominated in all samples (α‐pinene, limonene and sabinene as major components). Principal component analysis (PCA) of GC data revealed that wild and cultivated Juniperus species are highly distinct due to variation in chemical composition. J. communis (wild species) displayed cytotoxicity against SiHa (human cervical cancer), A549 (human lung carcinoma) and A431 (human skin carcinoma) cells (66.4 ± 2.2%, 74.4 ± 1.4% and 57.4 ± 4.0%), respectively, at 200 μg/ml. EOs exhibited better antibacterial activity against Gram‐positive bacteria than against Gram‐negative bacteria with the highest zone of inhibition against Staphylococcus aureus MTCC 96 (19.2 ± 0.7) by clone‐7. As per the conclusion of the findings, EOs of clone‐2, clone‐5 and clone‐7 can be suggested to the growers of lower altitude, as there is more possibility of uses of these EOs in food and medicinal preparations.     

Characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain DOR4 Toxic to Castor Semilooper <Emphasis Type="Italic">Achaea janata</Emphasis>: Proteolytic Processing and Binding of Toxins to Receptors

We have isolated a strain of Bacillus thuringiensis (Bt) from Indian soil samples that was shown to be toxic to Achaea janata larvae. The isolate, named B. thuringiensis DOR4, serotypically identified with the standard subspecies kurstaki (H3a3b3c) and produced bipyramidal inclusions along with an amorphous type. Although the plasmid pattern of DOR4 was different from that of the reference strain, a crystal protein profile showed the presence of two major bands (130 and 65 kDa) similar to those of Bt subsp. kurstaki HD-1. To verify the cry gene content of DOR4, triplex PCR analysis was performed; it showed amplification of the cry1C gene in addition to cry1Aa, cry1Ac, cry2A, and cry2B genes, but not the cry1Ab gene. RT-PCR analysis showed the expression of cry1Aa and cry1Ac genes. In vitro proteolysis of DOR4 protoxin with midgut extract generated products of different sizes. Zymogram analysis of DOR4 protoxin as substrate pointed to a number of distinct proteases that were responsible for activation of protoxins. Furthermore, toxin overlay analysis revealed the presence of multiple toxin-binding proteins in midgut epithelium. Based on all these characterizations, we suggest that the Bt DOR4 strain can be exploited for an A. janata control program.     

Synthesis and biological evaluation of trans 6-methoxy-1,1-dimethyl-2-phenyl-3-aryl-2,3-dihydro-1H-inden-4-yloxyalkylamine derivatives against drug susceptible,non-replicating M. tuberculosis H37Rv and clinical multidrug resistant strains

Synthesis of a library of novel trans 6-methoxy-1,1-dimethyl-2-phenyl-3-aryl-2,3-dihydro-1H-inden-4-yloxy alkyl amines and their antimycobacterial activity against drug sensitive and multidrug resistant strains of Mycobacterium tuberculosis have been reported. All the new compounds in the series exhibited MIC between 1.56 and 6.25 μg/ml. Two compounds 1i and 1j with low MIC and low cytotoxicity showed significant reduction in CFU in infected mouse macrophages at 1× MIC concentration. The compound 1i inhibited the growth of M. tuberculosis in mice at 100 mg/kg dose with 1.35 log₁₀ reduction of CFU in lungs tissue and was active against non-replicating Mycobacterium tuberculosis under anaerobic condition.     

Production of haploids and doubled haploids in oil palm

Background

Studies on host-pathogen interactions in a range of pathosystems have revealed an array of mechanisms by which plants reduce the efficiency of pathogenesis. While R-gene mediated resistance confers highly effective defense responses against pathogen invasion, quantitative resistance is associated with intermediate levels of resistance that reduces disease progress. To test the hypothesis that specific loci affect distinct stages of fungal pathogenesis, a set of maize introgression lines was used for mapping and characterization of quantitative trait loci (QTL) conditioning resistance to Setosphaeria turcica, the causal agent of northern leaf blight (NLB). To better understand the nature of quantitative resistance, the identified QTL were further tested for three secondary hypotheses: (1) that disease QTL differ by host developmental stage; (2) that their performance changes across environments; and (3) that they condition broad-spectrum resistance.

Results

Among a set of 82 introgression lines, seven lines were confirmed as more resistant or susceptible than B73. Two NLB QTL were validated in BC₄F₂ segregating populations and advanced introgression lines. These loci, designated qNLB1.02 and qNLB1.06, were investigated in detail by comparing the introgression lines with B73 for a series of macroscopic and microscopic disease components targeting different stages of NLB development. Repeated greenhouse and field trials revealed that qNLB1.06 _Tx303 (the Tx303 allele at bin 1.06) reduces the efficiency of fungal penetration, while qNLB1.02 _B73 (the B73 allele at bin 1.02) enhances the accumulation of callose and phenolics surrounding infection sites, reduces hyphal growth into the vascular bundle and impairs the subsequent necrotrophic colonization in the leaves. The QTL were equally effective in both juvenile and adult plants; qNLB1.06 _Tx303 showed greater effectiveness in the field than in the greenhouse. In addition to NLB resistance, qNLB1.02 _B73 was associated with resistance to Stewart's wilt and common rust, while qNLB1.06 _Tx303 conferred resistance to Stewart's wilt. The non-specific resistance may be attributed to pleiotropy or linkage.

Conclusions

Our research has led to successful identification of two reliably-expressed QTL that can potentially be utilized to protect maize from S. turcica in different environments. This approach to identifying and dissecting quantitative resistance in plants will facilitate the application of quantitative resistance in crop protection.     

Environmental and spatial characterisation of an unknown fauna using DNA sequencing – an example with Himalayan Hydropsychidae (Insecta: Trichoptera)

相似文献   

Mitochondrial import and accumulation of alpha-synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson disease brain

Alpha-synuclein, a protein implicated in the pathogenesis of Parkinson disease (PD), is thought to affect mitochondrial functions, although the mechanisms of its action remain unclear. In this study we show that the N-terminal 32 amino acids of human alpha-synuclein contain cryptic mitochondrial targeting signal, which is important for mitochondrial targeting of alpha-synuclein. Mitochondrial imported alpha-synuclein is predominantly associated with the inner membrane. Accumulation of wild-type alpha-synuclein in the mitochondria of human dopaminergic neurons caused reduced mitochondrial complex I activity and increased production of reactive oxygen species. However, these defects occurred at an early time point in dopaminergic neurons expressing familial alpha-synuclein with A53T mutation as compared with wild-type alpha-synuclein. Importantly, alpha-synuclein that lacks mitochondrial targeting signal failed to target to the mitochondria and showed no detectable effect on complex I function. The PD relevance of these results was investigated using mitochondria of substantia nigra, striatum, and cerebellum of postmortem late-onset PD and normal human brains. Results showed the constitutive presence of approximately 14-kDa alpha-synuclein in the mitochondria of all three brain regions of normal subjects. Mitochondria of PD-vulnerable substantia nigra and striatum but not cerebellum from PD subjects showed significant accumulation of alpha-synuclein and decreased complex I activity. Analysis of mitochondria from PD brain and alpha-synuclein expressing dopaminergic neuronal cultures using blue native gel electrophoresis and immunocapture technique showed the association of alpha-synuclein with complex I. These results provide evidence that mitochondrial accumulated alpha-synuclein may interact with complex I and interfere with its functions.     

Evaluation of the antimicrobial potency of silver nanoparticles biosynthesized by using an endophytic fungus,Cryptosporiopsis ericae PS4

In the present study, silver nanoparticles (AgNPs) with an average particle size of 5.5 ± 3.1 nm were biosynthesized using an endophytic fungus Cryptosporiopsis ericae PS4 isolated from the ethno-medicinal plant Potentilla fulgens L. The nanoparticles were characterized using UV-visible spectrophotometer, transmission electron microscopy (TEM), scanning electron microscopy (SEM), selective area electron diffraction (SAED), and energy dispersive X-ray (EDX) spectroscopy analysis. Antimicrobial efficacy of the AgNPs was analyzed singly and in combination with the antibiotic/antifungal agent chloramphenicol/fluconazole, against five pathogenic microorganisms-Staphylococcus aureus MTCC96, Salmonella enteric MTCC735, Escherichia coli MTCC730, Enterococcus faecalis MTCC2729, and Candida albicans MTCC 183. The activity of AgNPs on the growth and morphology of the microorganisms was studied in solid and liquid growth media employing various susceptibility assays. These studies demonstrated that concentrations of AgNPs alone between 10 and 25 μM reduced the growth rates of the tested bacteria and fungus and revealed bactericidal/fungicidal activity of the AgNPs by delaying the exponential and stationary phases. Examination using SEM showed pits and ruptures in bacterial cells indicating fragmented cell membrane and severe cell damage in those cultures treated with AgNPs. These experimental findings suggest that the biosynthesized AgNPs may be a potential antimicrobial agent.