Biological and Molecular Characterization of Three Isolates of Tomato aspermy virus Infecting Chrysanthemums in India

Severe mosaic, chlorotic ringspots and flower deformation were observed during the winter of November 2006–February 2007 on chrysanthemums ( Chrysanthemum morifolium ) at three locations in India: Lucknow (UP), Dhanbad (MP) and Kolkata (WB). Tomato aspermy virus (TAV) was detected in affected plants by ELISA and by RT-PCR using TAV specific primers. These TAV isolates were mechanically transmitted to test plant species and also by aphids ( Aphis gossypii ) to Lycopersicon esculentum . The complete RNA 3 of each TAV isolate was cloned and sequenced and determined to be 2386 nucleotides (nt) long, and to encode two open reading frames (ORFs): the movement protein (MP) of 741 nt and the coat protein (CP) of 657 nt translating in to 246 and 218 amino acid (aa), respectively. When RNA 3 sequences of the Indian isolates were multiple aligned with seven other strains of TAV occurring worldwide, Indian isolates shared 98–99% identities among themselves and with the KC, V, P, B, I and C strains of TAV. In phylogenetic analysis, the Lucknow and Kolkata isolates of TAV clustered together and showed a close relationship with the KC-TAV strain from South Korea, whereas the Dhanbad isolate formed an independent cluster and showed closeness with the V-TAV strains from Spain and Australia. Recombination events were also observed in the CP region of the Dhanbad isolate, supporting its diverse behaviour. This is the first report of the complete RNA 3 sequence of these three Indian TAV isolates.     

Detection of Hepatitis B Virus (HBV) DNA at femtomolar concentrations using a silica nanoparticle-enhanced microcantilever sensor

We report Hepatitis B Virus (HBV) DNA detection using a silica nanoparticle-enhanced dynamic microcantilever biosensor. A 243-mer nucleotide of HBV DNA precore/core region was used as the target DNA. For this assay, the capture probe on the microcantilever surface and the detection probe conjugated with silica nanoparticles were designed specifically for the target DNA. For efficient detection of the HBV target DNA using silica nanoparticle-enhanced DNA assay, the size of silica nanoparticles and the dimension of microcantilever were optimized by directly binding the silica nanoparticles through DNA hybridization. In addition, the correlation between the applied nanoparticle concentrations and the resonant frequency shifts of the microcantilever was discussed clearly to validate the quantitative relationship between mass loading and resonant frequency shift.HBV target DNAs of 23.1 fM to 2.31 nM which were obtained from the PCR product were detected using a silica nanoparticle-enhanced microcantilever. The HBV target DNA of 243-mer was detected up to the picomolar (pM) level without nanoparticle enhancement and up to the femtomolar (fM) level using a nanoparticle-based signal amplification process. In the above two cases, the resonant frequency shifts were found to be linearly correlated with the concentrations of HBV target DNAs. We believe that this linearity originated mainly from an increase in mass that resulted from binding between the probe DNA and HBV PCR product, and between HBV PCR product and silica nanoparticles for the signal enhancement, even though there is another potential factor such as the spring constant change that may have influenced on the resonant frequency of the microcantilever.     

Gold nanoparticles based dipstick immunoassay for the rapid detection of dichlorodiphenyltrichloroethane: An organochlorine pesticide

Gold nanoparticles (GNPs) based dipstick competitive immunoassay was developed to detect organochlorine pesticide such as DDT at nanogram level (ppb). GNPs of definite size were synthesized and conjugated to anti-DDT antibodies (IgY), which served as the detecting reagent. DDA-BSA conjugate (antigen) was immobilized on to nitro cellulose (NC) membrane containing strip. GNPs conjugated anti-DDT antibodies were treated with different concentrations of free DDT ranging from 0.7 ng mL⁻¹ to 1000 ng mL⁻¹ to form an immunocomplex. This immunocomplex solution was further reacted with DDA-BSA conjugate immobilized NC membrane containing strips by dipping the strip in the immunocomplex solution. The free GNPs conjugated anti-DDT antibodies present in the immunocomplex solution were targeted for competitive binding with immobilized DDA-BSA on NC membrane containing strip. Depending on the concentration of free DDT in the sample the binding of GNPs conjugated anti-DDT antibodies to the immobilized DDA-BSA varied and was detected by the development of red color (due to gold nanoparticles) in the detection zone of NC membrane containing strips. The intensity of color development was inversely proportional to the DDT concentration with maximum intensity at zero DDT concentration. The lowest detection limit of DDT was determined to be 27 ng mL⁻¹ with the optimized conditions. The dipstick technique based on GNPs is suitable for the detection of several toxins in food and environmental samples and can be applied for rapid on-site testing of pesticides.     

Highly hydroxylated or γ-cyclodextrin-bicapped water-soluble derivative of fullerene: The antioxidant ability assessed by electron spin resonance method and β-carotene bleaching assay

Antioxidant ability of the water-soluble derivative of fullerene (C60), prepared by high-degree hydroxylation [C60-(OH)₃₂·8H₂O] or C60/γ-cyclodextrin (1:2 mol/mol) clathrate formation [C60/(γ-CD)₂], was assessed by electron spin resonance method and β-carotene bleaching assay. These C60 derivatives have an ability to diminish a 1:2:2:1 quartet ESR spectrum attributed to hydroxyl radicals (OH) as shown by DMPO-spin trap/ESR method. Meanwhile, a singlet radical-signal different from OH-attributed signals increased in a manner dependent on concentrations of C60-(OH)₃₂·8H₂O. This might suggest that C60-(OH)₃₂·8H₂O scavenges OH owing to dehydrogenation of C60-(OH)₃₂·8H₂O, and is simultaneously oxidized to a stable radical species, which may be a dehydrogenated fullerenol radical (C60-O). Furthermore, these water-soluble derivatives of C60 suppressed fading of yellowish color characteristic of intact β-carotene in β-carotene bleaching assay. Antioxidant abilities of these derivatives were assessed as retention of yellowish color (viz absorbance at 470 nm) for 180 min. Namely, β-carotene-attributed chromaticity (% relative absorbance at 470 nm compared with the control) after 180 min was 69% for C60-(OH)₃₂·8H₂O (400 μM: C60-eq.), and 32% for C60/(γ-CD)₂ (400 μM: C60-eq.), whereas it was 6% for l(+)-ascorbic acid (400 μM) which is hydrophilic, and 85% for (±)-α-tocopherol (400 μM) which is lipophilic, respectively. Thus C60-(OH)₃₂·8H₂O and C60/(γ-CD)₂ can scavenge OH, and have a distinct antioxidative activity in the aqueous system containing linoleic acid which is abundantly contained in the cell membrane together with other unsaturated lipids. These C60 derivatives have a potential to protect the cell membrane from oxidative stress due to OH.     

Adaptive response in different mitotic cycles after irradiation

The frequency of cells with chromosome aberrations and the number of aberrations per cell have been studied by metaphase analysis in the nonirradiated progeny of irradiated human blood lymphocytes. DNA fragmentation (DNA double-stranded breaks) has been investigated by DNA comet assay. To study the adaptive response (AR), PHA-stimulated lymphocytes were irradiated by the adaptive dose (0.05 Gy) in 24 h and by challenge dose (1 Gy) in 48 h after stimulation. The first through fourth mitoses were identified by 5-bromodeoxyuridine. It was found that the frequency of chromosome aberrations and double-strand breaks were increased in all mitotic cycles after the challenge irradiation. In most individuals, the adaptive response is induced by adaptive and challenge irradiations in the first and the second mitotic cycles (48 and 72 h after stimulation, respectively); however, it is absent in the third and the fourth mitoses. In the first mitosis (1Gy in 48 h after stimulation), only chromatid aberrations are observed; chromosome aberrations were registered in subsequent mitoses. DNA comet assay showed that the adaptive response was obvious at 48–72 h, but not 96 h, after stimulation. It can be concluded that the nonirradiated progeny of irradiated lymphocytes have genomic instability. The adaptive response is manifested up to the third mitosis and is explained by the decreasing number of chromatid and chromosome aberrations and DNA fragmentation. We suppose that double-stranded DNA breaks may be damage signals for the induction of adaptive response.     

重金属增强核因子与水稻金属硫蛋白基因启动子的结合

植物金属硫蛋白(metallothioneins, MT)被认为在植物应答重金属胁迫方面发挥了重要作用,但该基因的转录调控机制目前仍不清楚.我们以前的研究初步鉴定出位于-331/-194的水稻MT基因(ricMT)启动子区对于金属激活ricMT的表达是必需的.为了明确 -331/-194启动子序列在控制ricMT表达中的作用,本课题开展了该序列 与核因子的结合特性研究.从2周龄水稻嫩叶中提取了几乎不含叶绿体污染的细胞核,并制备 了核蛋白用于凝胶阻滞实验,发现核因子能够与-331/-194启动子序列特异 结合.本研究还进一步考察了重金属对核因子结合活性的影响,发现在结合体系中去除重金 属离子,核因子与-331/-194序列的结合能力会丧失,而在结合体系中加入重金属离子, 结合能力则会随着外加的离子浓度提高而增强,证明这种结合确实依赖于重金属.这些证据 结合以前的结果表明,某些金属响应的核因子可能通过结合-331/-194启动子区域来调控 ricMT基因表达.     

NetMHCpan,a method for MHC class I binding prediction beyond humans

Binding of peptides to major histocompatibility complex (MHC) molecules is the single most selective step in the recognition of pathogens by the cellular immune system. The human MHC genomic region (called HLA) is extremely polymorphic comprising several thousand alleles, each encoding a distinct MHC molecule. The potentially unique specificity of the majority of HLA alleles that have been identified to date remains uncharacterized. Likewise, only a limited number of chimpanzee and rhesus macaque MHC class I molecules have been characterized experimentally. Here, we present NetMHCpan-2.0, a method that generates quantitative predictions of the affinity of any peptide–MHC class I interaction. NetMHCpan-2.0 has been trained on the hitherto largest set of quantitative MHC binding data available, covering HLA-A and HLA-B, as well as chimpanzee, rhesus macaque, gorilla, and mouse MHC class I molecules. We show that the NetMHCpan-2.0 method can accurately predict binding to uncharacterized HLA molecules, including HLA-C and HLA-G. Moreover, NetMHCpan-2.0 is demonstrated to accurately predict peptide binding to chimpanzee and macaque MHC class I molecules. The power of NetMHCpan-2.0 to guide immunologists in interpreting cellular immune responses in large out-bred populations is demonstrated. Further, we used NetMHCpan-2.0 to predict potential binding peptides for the pig MHC class I molecule SLA-1*0401. Ninety-three percent of the predicted peptides were demonstrated to bind stronger than 500 nM. The high performance of NetMHCpan-2.0 for non-human primates documents the method’s ability to provide broad allelic coverage also beyond human MHC molecules. The method is available at . Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Advantage of using PSIRB over PSRB and IRB to improve plant health of tomato

Twenty-one isolates of phosphate solubilizing-indole acetic acid producing rhizobacteria (PSIRB), 20 isolates of phosphate solubilizing rhizobacteria (PSRB) and 42 isolates of indole acetic acid producing rhizobacteria (IRB) were isolated from 49 rhizospheric soil samples of tomato (Lycopersicon esculentum Mill.) collected from tomato growing regions of Karnataka. A method combining Pikovskaya’s and Bric’s technique was developed to isolate PSRIB, PSRB and IRB’s. The selected isolates were further analyzed for their ability to solubilize calcium phytate. Based on the root colonization assays and the abilities of bacterial isolates to increase the seed germination and seedling vigor under laboratory conditions, five isolates were selected from each group for further studies. Under greenhouse conditions, all the selected rhizobacteria isolates significantly increased root length, shoot length, fresh weight, dry weight and total phosphorus content of 30-day-old-seedlings with respect to control. Isolate PSIRB1 and IRB36 significantly reduced the Fusarium wilt incidence over other isolates of same and other group, and the control. On the basis of results from laboratory and greenhouse studies, one bacterial isolate from each group was selected for plant growth and yield analysis studies. Isolate PSIRB2 showed increased plant height, fresh weight, number of fruits per plant and average weight of fruit over PSRB9, IRB36 and untreated controls. Studies on the nature of protection offered by these bacterial isolates following split-root technique revealed that the isolates PSIRB2 and PSRB9 had the ability to induce systemic resistance. One isolate, IRB36 appeared to protect the tomato seedlings through direct antagonism.     

Interaction of α-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7 nAChR-targeting α-conotoxin ImI, blocked α7 and muscle nAChRs without displacing α-bungarotoxin ( Ellison et al. 2003, 2004 ), suggesting binding at a different site. We synthesized α-conotoxin ImII, its ribbon isomer (ImII iso ), 'mutant' ImII(W10Y) and found similar potencies in blocking human α7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [¹²⁵I]-α-bungarotoxin from human α7 nAChRs in the cell line GH₄C₁ (IC₅₀ 17 and 23 μM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC₅₀ 2.0–9.0 μM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized α-bungarotoxin ( K _d and IC₅₀ 2.5–8.2 μM). On Torpedo nAChR, α-conotoxin [¹²⁵I]-ImII(W10Y) revealed specific binding ( K _d 1.5–6.1 μM) and could be displaced by α-conotoxin ImII, ImII iso and ImII(W10Y) with IC₅₀ 2.7, 2.2 and 3.1 μM, respectively. As α-cobratoxin and α-conotoxin ImI displaced [¹²⁵I]-ImII(W10Y) only at higher concentrations (IC₅₀≥ 90 μM), our results indicate that α-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.