Multiple shoot induction by benzyladenine and complete plant regeneration from seed explants of chickpea (Cicer arietinum L.)

The efficacy of benzyladenine (BA) to induce multiple shoots from seed explants of chickpea (Cicer arietinum L.) was assessed. Shoot differentiation was influenced by the type of seed explant, genotype and concentration of BA. Orientation of the explant also strongly influenced the shoot regeneration response. The optimum BA concentration for shoot/shoot bud regeneration was genotype dependent. Two types of BA-induced response were observed: (1) at less than 7.5 gm BA, direct shoot differentiation (2 to 4-cm-long shoots) was observed within 30 days; (2) at higher BA concentrations (75–100 m), shoot/shoot bud differentiation was achieved in 45–90 days. A high BA concentration inhibited subsequent rooting of shoots. Roots, however, could be easily induced on shoots derived from <12.5 m BA. Following transfer to soil, 80% of the regenerants developed into morphologically normal and fertile plants.Abbreviations BA Benzyladenine     

Type 1 fimbriae of insecticidal bacterium Xenorhabdus nematophila is necessary for growth and colonization of its symbiotic host nematode Steinernema carpocapsiae

Xenorhabdus nematophila produces type 1 fimbriae on the surface of Phase I cells. Fimbriae mediate recognition and adhesion of the bacteria to its target cell. To investigate the role of fimbriae in the biology of X. nematophila , we have produced a fimbrial mutant strain by insertional inactivation of the mrx A gene, encoding the structural subunit of type 1 fimbriae. Phenotypic characterization of the mutant revealed loss of fimbriae on the cell surface. Cell surface characteristics like dye absorption, biofilm formation, red blood cell agglutination remained unaltered. The mrx A mutant was defective in swarming on soft agar, although swimming motility was not affected. Flagellar expression was suppressed in the mrxA strain under swarming conditions, but not swimming conditions. Agglutination and cytotoxicity of the mutant to larval haemocytes was also reduced. When the mutant cells were injected in the haemocoel of the fourth instar larvae of Helicoverpa armigera , an increase in the LT₅₀ of 9–12 h was observed relative to the wild-type strain. The nematode growth was slow on the lawn of the fimbrial mutant. The mrxA negative strain was unable to colonize the nematode gut efficiently. This study demonstrates importance of type 1 fimbriae in establishment of bacteria-nematode symbiosis, a key to successful pest management program.     

CHPVDB ‐ a sequence annotation database for Chandipura Virus

Databases containing proteomic information have become indispensable for virology studies. As the gap between the amount of sequence information and functional characterization widens, increasing efforts are being directed to the development of databases. For virologist, it is therefore desirable to have a single data collection point which integrates research related data from different domains. CHPVDB is our effort to provide virologist such a one‐step information center. We describe herein the creation of CHPVDB, a new database that integrates information of different proteins in to a single resource. For basic curation of protein information, the database relies on features from other selected databases, servers and published reports. This database facilitates significant relationship between molecular analysis, cleavage sites, possible protein functional families assigned to different proteins of Chandipura virus (CHPV) by SVMProt and related tools.     

Use of Pseudomonas fluorescens-based formulations for management of tomato spotted wilt virus (TSWV) and enhanced yield in tomato

Strains of Pseudomonas fluorescens were investigated for biocontrol efficacy against tomato spotted wilt virus (TSWV) in tomato both alone and in mixtures. P. fluorescens strains applied to seed, soil and foliage or as a seedling dip significantly reduced TSWV, with a concomitant increase in growth promotion in both the glasshouse and field. Two native strains (CoP-1 and CoT-1) and one foreign strain (CHAO) reduced TSWV. In P. fluorescens-treated tomato plants, increased activity of polyphenol oxidase, β-1,3-glucanase and chitinase was observed, and induction of chitinase was confirmed by western blot analysis. Induction of new protein (18 kDa) detected by SDS-PAGE in P. fluorescens-treated tomato plants was not found in healthy and P. fluorescens-untreated virus inoculated control plants. Indirect ELISA clearly showed a reduction in viral antigen concentration in P. fluorescens-treated tomato plants corresponding to reduced disease ratings. All the P. fluorescens-treated tomato plants also showed enhanced growth and yield compared to control plants. Hence, plant growth promoting rhizobacteria (PGPR) could play a major role in reducing TSWV and increasing yield in tomato plants.     

Establishment of an efficient and rapid method of multiple shoot regeneration and a comparative phenolics profile in in vitro and greenhouse-grown plants of psophocarpus tetragonolobus (L.) DC

An in vitro method of multiple shoot induction and plant regeneration in Psophocarpus tetragonolobus (L.) DC was developed. Cotyledons, hypocotyls, epicotyls, internodal and young seedling leaves were used as explants. MS media supplemented with various concentrations of either thidiazuron (TDZ) or N6-benzylaminopurine (BAP) along with NAA or IAA combinations were used to determine their influence on multiple shoot induction. MS media supplemented with TDZ induced direct shoot regeneration when epicotyls and internodal segments were used as explants. TDZ at 3 mg L⁻¹ induced highest rate (89.2 ± 3.28%) of regeneration with (13.4 ± 2.04) shoots per explant. MS media supplemented with BAP in combination with NAA or IAA induced callus mediated regeneration when cotyledons and hypocotyls were used as explants. BAP (2.5 mg L⁻¹) and IAA (0.2 mg L⁻¹) induced highest rate (100 ± 2.66%) of regeneration with (23.2 ± 2.66) shoots per explant. Mature plants produced from regenerated shoots were transferred successfully to the greenhouse. In a comparative study, the phenolics contents of various parts of greenhouse-grown plants with that of in vitro-raised plants showed significant variations.     

Genetic Characterization of Hepatitis B Virus in Peripheral Blood Leukocytes: Evidence for Selection and Compartmentalization of Viral Variants with the Immune Escape G145R Mutation

The compartmentalization of viral variants in distinct host tissues is a frequent event in many viral infections. Although hepatitis B virus (HBV) classically is considered hepatotropic, it has strong lymphotropic properties as well. However, unlike other viruses, molecular evolutionary studies to characterize HBV variants in compartments other than hepatocytes or sera have not been performed. The present work attempted to characterize HBV sequences from the peripheral blood leukocytes (PBL) of a large set of subjects, using advanced molecular biology and computational methods. The results of this study revealed the exclusive compartmentalization of HBV subgenotype Ae/A2-specific sequences with a potent immune escape G145R mutation in the PBL of the majority of the subjects. Interestingly, entirely different HBV genotypes/subgenotypes (C, D, or Aa/A1) were found to predominate in the sera of the same study populations. These results suggest that subgenotype Ae/A2 is selectively archived in the PBL, and the high prevalence of G145R indicates high immune pressure and high evolutionary rates of HBV DNA in the PBL. The results are analogous to available literature on the compartmentalization of other viruses. The present work thus provides evidence in favor of the compartment-specific abundance, evolution, and emergence of the potent immune escape mutant. These findings have important implications in the field of HBV molecular epidemiology, transmission, transfusion medicine, organ transplantation, and vaccination strategies.Hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae family and classically has been described to be hepatotropic, causing a wide range of clinical and subclinical manifestations of liver disease (57). Nevertheless, studies of HBV-infected human subjects and woodchucks infected with Woodchuck hepatitis virus (WHV; an animal model of hepadnaviral infection) have reported different molecular forms of replicative intermediates in the lymphatic cells and have established that hepadnaviruses are strongly lymphotropic in nature (29). Moreover, the results of studies of human subjects as well as with animal models have revealed that the life-long occult persistence of replication- and transmission-competent viruses in lymphatic cells is a strict consequence of hepadnaviral infections (29).More interestingly, in animal models, lymphatic system-restricted occult hepadnaviral infection has been found to be transmissible vertically as an asymptomatic, serologically occult infection exclusively confined to the lymphatic system (29). Earlier we provided evidence that occult HBV persisting in the lymphatic cells are transmissible, specifically to the PBL through horizontal intrafamilial modes (9). These observations clearly indicate important immunological, pathogenic, and epidemiological implications of lymphatic system-restricted hepadnaviral infections. Although the involvement of specific viral variants has been suggested to explain this lymphatic system-restricted hepadnaviral infection and transmission (29), the classical belief that hepatocytes are the primary target and only reservoir of HBV has precluded the genetic characterization of hepadnaviruses from extrahepatic sites.Fascinatingly, despite being classically considered a hepatotropic virus, hepatitis C virus (HCV), belonging to the family Flaviviridae, also shows occult persistence and lymphotropism very similar to that of hepadnaviruses (37). Similarly to WHV, HBV, and HCV, other viruses, including HIV (human immunodeficiency virus), small ruminant lentivirus, and Epstein-Barr virus, also have been shown to infect and persist in different anatomical compartments of the body in addition to their classical target cells (38, 40, 43, 45, 50). Furthermore, recent molecular evolutionary analyses based on envelope sequences of these viruses (e.g., HIV, HCV, small ruminant lentivirus, Epstein-Barr virus, etc.) have established clearly that these viruses undergo selection and independent evolution in diverse tissues, leading to the tissue-specific compartmentalization of viral populations (38, 40, 43, 45, 50). In contrast to other viruses, to the best of our knowledge, methodical molecular evolutionary studies to characterize HBV sequences isolated from extrahepatic sites of HBV-infected subjects have not been reported in the literature.We hypothesized that similar to other viruses, HBV also undergo independent evolution in different compartments of the body under the influence of differential immune pressure. To examine our hypothesis, we used the most easily available lymphatic cells, the peripheral blood leukocytes (PBL), determined the HBV envelope sequences from HBV DNA isolated from these cells, and performed advanced genetic, phylogenetic, and mutational analysis. The results of this work demonstrate a highly compartment-specific preponderance of HBV genetic variants in serum and PBL of the same study population, providing evidence in favor of the compartmentalization of HBV genetic variants. The results and important implications of these findings are discussed in this work.     

Diversity and polymorphism in AHL-lactonase gene (aiiA) of Bacillus

To explore bacterial diversity for elucidating genetic variability in acylhomoserine lactone (AHL) lactonase structure, we screened 800 bacterial strains. It revealed the presence of a quorum quenching (QQ) AHL-lactonase gene (aiiA) in 42 strains. These 42 strains were identified using rrs (16S rDNA) sequencing as Bacillus strains, predominantly B. cereus. An in silico restriction endonuclease (RE) digestion of 22 AHL lactonase gene (aiiA) sequences (from NCBI database) belonging to 9 different genera, along with 42 aiiA gene sequences from different Bacillus spp. (isolated here) with 14 type II REs, revealed distinct patterns of fragments (nucleotide length and order) with four REs; AluI, DpnII, RsaI, and Tru9I. Our study reflects on the biodiversity of aiiA among Bacillus species. Bacillus sp. strain MBG11 with polymorphism (115Alanine > Valine) may confer increased stability to AHL lactonase, and can be a potential candidate for heterologous expression and mass production. Microbes with ability to produce AHL-lactonases degrade quorum sensing signals such as AHL by opening of the lactone ring. The naturally occurring diversity of QQ molecules provides opportunities to use them for preventing bacterial infections, spoilage of food, and bioremediation.     

Evaluation of genus <Emphasis Type="Italic">Cenchrus</Emphasis> based on malondialdehyde,proline content,specific leaf area and carbon isotope discrimination for drought tolerance and divergence of species at DNA level

Cenchrus (family Poaceae) is an important component of major grass covers of the world. Largely it is apomictic and both annual and perennial species exist in nature. Variations in contents of malondialdehyde, proline, specific leaf area and carbon isotope discrimination for drought tolerance were estimated among eight prominent species of Cenchrus. Simultaneously, genetic variations were also estimated by employing 187 RAPD primers. Of these, 23 primers did not react, 2 performed poorly and 7 produced many non-scorable bands and one primer yielded a single monomorphic band. Rest of the 154 primers generated one or more unambiguously scorable fragments. Twelve hundred and four of the 1,296 putative loci were polymorphic (93%) between at least one pair-wise comparisons among eight species. Dice coefficient and neighbor-joining algorithm analyses showed clustering patterns that fit with the known habitat of the species except perennial, C. myosuroides which formed a node between two annuals species. When these species were subjected to water stress tolerance test, a correlation (r = 0.612) between specific leaf area (SLA) and carbon isotope discrimination (CID) and difference in levels of drought tolerance based parameters among eight species were observed. Of the eight species investigated two annuals viz., C. biflorus and C. echinatus showed highest level of genetic similarity which was also evident from the similar levels of SLA, MDA, proline contents and carbon isotope discrimination values observed in these two species.     

Characterization of an extracellular keratinase of Trichophyton simii and its role in keratin degradation

The ability of Trichophyton simii HN 50, isolated from the Ghana Bird Sanctuary, Bharatpur, India, to produce extracellular keratinase was studied. Enzyme was produced on a keratin salt broth medium at pH7 and a temperature of 28 ± 1 °C. Enzyme secretion was best at 15 days of incubation. Asparagine and keratin were repressive to enzyme yield in comparison to gelatin. No relationship was observed between enzyme release and biomass. Exogenous sugars suppressed keratinase production in descending order as follows: glucose > mannose > maltose > arabinose > fructose. The enzyme showed ability to degrade all of the 3 keratin substrates. Buffalow skin was best degraded in the absence of glucose while chicken feathers were the least degraded in its presence.This revised version was published online in October 2005 with corrections to the Cover Date.     

Caveolin-1 null (-/-) mice show dramatic reductions in life span

Caveolae are 50-100 nm flask-shaped invaginations of the plasma membrane found in most cell types. Caveolin-1 is the principal protein component of caveolae membranes in nonmuscle cells. The recent development of Cav-1-deficient mice has allowed investigators to study the in vivo functional role of caveolae in the context of a whole animal model, as these mice lack morphologically detectable caveolae membrane domains. Surprisingly, Cav-1 null mice are both viable and fertile. However, it remains unknown whether loss of caveolin-1 significantly affects the overall life span of these animals. To quantitatively determine whether loss of Cav-1 gene expression confers any survival disadvantages with increasing age, we generated a large cohort of mice (n = 180), consisting of Cav-1 wild-type (+/+) (n = 53), Cav-1 heterozygous (+/-) (n = 70), and Cav-1 knockout (-/-) (n = 57) animals, and monitored their long-term survival over a 2 year period. Here, we show that Cav-1 null (-/-) mice exhibit an approximately 50% reduction in life span, with major declines in viability occurring between 27 and 65 weeks of age. However, Cav-1 heterozygous (+/-) mice did not show any changes in long-term survival, indicating that loss of both Cav-1 alleles is required to mediate a reduction in life span. Mechanistically, these dramatic reductions in life span appear to be secondary to a combination of pulmonary fibrosis, pulmonary hypertension, and cardiac hypertrophy in Cav-1 null mice. Taken together, our results provide the first demonstration that loss of Cav-1 gene expression and caveolae organelles dramatically affects the long-term survival of an organism. In addition, aged Cav-1 null mice may provide a new animal model to study the pathogenesis and treatment of progressive hypertrophic cardiomyopathy and sudden cardiac death syndrome.