UDP-Gal: <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine β 1–4 galactosyltransferase expressing live attenuated parasites as vaccine for visceral leishmaniasis

As compared to cutaneous leishmaniasis, vaccination against visceral leishmaniasis (VL) has received limited attention. In this study, we demonstrate for the first time that an UDP-Galactose: N-acetylglucosamine β 1–4 galactosyltransferase (GenBank Accession No. EF159943) expressing attenuated LD clonal population (A-LD) is able to confer protection against the experimental challenge with the virulent LD AG83 parasite. A-LD was also effective in established leishmania infection. The vaccinated animals showed both cell mediated (in vitro T-cell proliferation, and DTH response) and humoral responses (Th1 type). These results demonstrate the potential of the attenuated clones as an immunotherapeutic and immunoprophylactic agent against visceral leishmaniasis.     

Identification of a Genetic Locus in Pseudomonas aureofaciens Involved in Fungal Inhibition

In iron-rich conditions, Pseudomonas aureofaciens PA147-2 produces an antibiotic-like compound that inhibits the growth of a plant fungal pathogen, Aphanomyces euteiches. To contribute to the potential use of PA147-2 as a biocontrol organism, we report the identification of a genetic locus important for antibiotic biosynthesis. Mutants defective for fungal inhibition (Af^-) were generated by Tn5 mutagenesis. Southern hybridization of total DNAs from three Af^- mutants indicated that loss of fungal inhibition was due to a single Tn5 insertion in each mutant. Restriction mapping of the mutation points showed that in two mutants the Tn5 insertions were in the same 16.0-kb EcoRI fragment and were separated by 2.1 kb. A genomic library of PA147-2 was constructed and screened by using a region of DNA flanking the Tn5 insertion in one mutant (PA109) as a probe to recover complementing cosmids. Three cosmids containing a 16.0-kb EcoRI fragment complementary to the two mutants were recovered. Allele replacement by homologous recombination with putative complementing cosmids restored one mutant to antifungal activity against A. euteiches. Southern analysis of the complemented mutants confirmed that allele replacement had occurred between cosmid DNA and Tn5. The wild-type 16.0-kb EcoRI fragment was cloned from the cosmid and complemented the two mutants to antifungal activity. An antifungal compound was isolated from PA147-2 grown on solid medium. Antifungal activity correlated to a peak on high-pressure liquid chromatography analysis. Under the same growth and extraction conditions, the antifungal activity seen in PA147-2 was absent in two Af^- mutants. Furthermore, absence of an antifungal compound in each mutant correlated to the absence of the wild-type “antifungal” peak on high-pressure liquid chromatography analysis.     

Characterization of Serratia entomophila Bacteriophages and the Phage-Resistant Mutant Strain BC4B

Successful large-scale fermentations of the bacterium Serratia entomophila for use in biological control of the soil-dwelling insect Costelytra zealandica has required the development of a phage-resistant mutant, BC4B. We report our investigations into S. entomophila phages and the nature of the phage resistance mechanism of strain BC4B. The parental strain of BC4B, A1MO2, was found to contain two previously unidentified prophages, (phi)9A and (phi)9B, which were UV inducible and also released spontaneously in large numbers. BC4B was shown to be completely cured of (phi)9A. Single lysogens of (phi)9A and (phi)9B were not homoimmune to any other S. entomophila phages. However, on the basis of DNA-DNA homology, all S. entomophila phages except (phi)CW3 were shown to have significant regions of homology and also packaged their DNA via pac-like mechanisms. The failure of phage particles to adsorb was identified as the basis of phage resistance in BC4B. In addition, it was demonstrated that all known S. entomophila phages are naturally temperature sensitive.     

Parallel regulation of IL-4 and IL-5 in human helminth infections.

To investigate the relationship between cytokine production and the increased levels of serum IgE and peripheral eosinophilia commonly accompanying human helminth infections, we studied the ability of PBMC of normal (N1) (n = 18) and eosinophilic individuals with helminth infections (H1) (n = 9) to produce IL-3, IL-4, IL-5, granulocyte-macrophage-CSF, and IFN-gamma in vitro after stimulation with PMA (50 ng/ml) and ionomycin (1 microgram/ml). The two groups differed in both the levels of serum IgE and eosinophilia. For mitogen-induced production of granulocyte-macrophage-CSF and IFN-gamma, there was no difference in cytokine production between the two groups. In marked contrast, supernatants from PBMC of infected individuals had significantly higher levels of IL-4 (mean = 213 pg/ml for N1 and 944 pg/ml for H1, p less than 0.02), IL-5 (mean = 180 pg/ml for N1 and 1118 pg/ml for HL, p less than 0.001), and IL-3 (mean = 13900 pg/ml for N1, 28029 pg/ml for H1, p less than 0.05). In addition, helminth-infected patients had approximately 5-fold greater numbers of T cells capable of producing IL-5 and 2.5-fold greater frequency of IL-4-secreting cells than did normal individuals; GM-CSF- and IFN-gamma-producing T cell numbers were not significantly different in the two groups. IL-3-producing cell frequencies could not be evaluated by this method. There was a direct correlation between IL-4 production and IL-5 production at the level of both protein production and frequency of T cells capable of producing these cytokines. These data indicate that individuals with reactive eosinophilia and elevated serum IgE have an expanded population of lymphocytes producing IL-4 and IL-5 and the association of the two suggests that the regulation of IL-4 and IL-5 may be linked.     

1H, 13C, 15N assignments of the dimeric regulatory subunit (ilvN) of the E. coli AHAS I

Acetohydroxyacid synthase (AHAS) is an enzyme involved in the biosynthesis of the branched chain amino acids viz, valine, leucine and isoleucine. The activity of this enzyme is regulated through feedback inhibition by the end products of the pathway. Here we report the backbone and side-chain assignments of ilvN, the 22 kDa dimeric regulatory subunit of E. coli AHAS isoenzyme I, in the valine bound form. Detailed analysis of the structure of ilvN and its interactions with the catalytic subunit of E. coli AHAS I will help in understanding the mechanism of activation and regulation of the branched chain amino acid biosynthesis.     

Regulatory networks that function to specify flower meristems require the function of homeobox genes PENNYWISE and POUND-FOOLISH in Arabidopsis

Flowering is a major developmental phase change that transforms the fate of the shoot apical meristem (SAM) from a leaf-bearing vegetative meristem to that of a flower-producing inflorescence meristem. In Arabidopsis, floral meristems are specified on the periphery of the inflorescence meristem by the combined activities of the FLOWERING LOCUS T (FT)–FD complex and the flower meristem identity gene, LEAFY ( LFY ). Two redundant functioning homeobox genes, PENNYWISE ( PNY ) and POUND-FOOLISH ( PNF ), which are expressed in the vegetative and inflorescence SAM, regulate patterning events during reproductive development, including floral specification. To determine the role of PNY and PNF in the floral specification network, we characterized the genetic relationship of these homeobox genes with LFY and FT . Results from this study demonstrate that LFY functions downstream of PNY and PNF. Ectopic expression of LFY promotes flower formation in pny pnf plants, while the flower specification activity of ectopic FT is severely attenuated. Genetic analysis shows that when mutations in pny and pnf genes are combined with lfy , a synergistic phenotype is displayed that significantly reduces floral specification and alters inflorescence patterning events. In conclusion, results from this study support a model in which PNY and PNF promote LFY expression during reproductive development. At the same time, the flower formation activity of FT is dependent upon the function of PNY and PNF.     

Prognostic significance of head-and-neck cancer biomarkers previously discovered and identified using iTRAQ-labeling and multidimensional liquid chromatography-tandem mass spectrometry

Diagnostic oncoproteomics is an emerging field; at present, studies on evaluation of prognostic utility of potential biomarkers identified using proteomic techniques are limited. Analysis with isobaric mass tags (iTRAQ) by multidimensional liquid chromatography-mass spectrometry (LC-MS/MS) to identify proteins that are differentially expressed in human head-and-neck/oral squamous cell carcinomas (HNOSCCs) versus noncancerous head-and-neck tissues has led to the discovery, identification, and verification of consistently increased expression of a panel of proteins, including stratifin (14-3-3sigma) and YWHAZ (14-3-3zeta), that may serve as potential cancer biomarkers. Herein, we describe the prognostic utility of these two candidate biomarkers for head-and-neck/oral squamous cell carcinoma (HNOSCC). To determine the clinical significance of stratifin and YWHAZ in head-and-neck tumorigenesis, the expressions of these two proteins were analyzed in HNOSCCs (51 cases) and nonmalignant tissues (39 cases) using immunohistochemistry. Significant increase in stratifin expression was observed in the HNOSCCs as compared to the nonmalignant mucosa [p=0.003, Odd's Ratio (OR)=3.8, 95% CI=1.6-9.2]. Kaplan-Meier survival analysis reveals correlation of stratifin overexpression with reduced disease-free survival of HNOSCC patients (p=0.06). The most intriguing finding is the significant decrease in median disease-free survival (13 months) in HNOSCC patients showing overexpression of both stratifin and YWHAZ proteins, as compared to patients that did not show overexpression of these proteins (median disease-free survival=38 months, p=0.019), underscoring their utility as adverse prognosticators for HNOSCCs. Co-immunoprecipitation assays show the formation of stratifin-YWHAZ heterodimers in HNOSCC cells and tissue samples, and interactions with NFkappaB, beta-catenin, and Bcl-2 proteins. These results suggest the involvement of these proteins in the development of head-and-neck cancer and their association with adverse disease outcome.     

In vitro morphogenesis in Selaginella microphylla (Kunth.) Spring

Selaginella, an extant genus of primitive vascular plants, has survived over 400 million years of evolution. In vitro morphogenesis in Selaginella microphylla is considered for the first time to establish a well-documented aseptic culture on half- strength Murashige and Skoog’s basal medium with 2ip (4.92–49.21 μM), or Kn (4.65–46.47 μM) or GA₃ (2.89–28.90 μM) for shoot multiplication, and with different concentrations of IBA (4.9–49 μm) to initiate root cultures. GA₃ was instrumental for shoot multiplication as well as induction of reproductive structures in each and every leaf axil. On the other hand, it is observed that IBA alone in S. microphylla can act as signal molecules for induction of enormous numbers of root masses from a few existing roots. An interesting pattern of re-differentiation has also been observed where apical portions of large numbers of roots were converted to green shoot apical meristems. Further differentiation produced tiny green shoots. Distinct bipolarity was noted in shoots when they were isolated from root masses and appeared as embryo-like structures. Chromosome analysis from in vitro sporophytic plants revealed 2n = 16 chromosomes, indicating chromosomal stability. The interesting in vitro pattern of morphogenesis obtained in S. microphylla may provide new insights into totipotency of plants.     

Comparative physico-kinetic properties of a homogenous purified β-glucosidase from Withania somnifera leaf

β-glucosidase from Withania somnifera (Solanaceae) leaf has been purified to homogeneity and characterized for its physico-kinetic properties. The enzyme purification was achieved through a sequence of gel filtration and ion-exchange column chromatography, and PAGE revealed the homogeneity purification status of the enzyme. The properties of the enzyme included an acidic pH optima (4.8), alkaline pI (8.7), meso-thermostabity, monomeric structure with subunit molecular weight of about 50 kDa, high affinity for substrate (K _m) for pNPG (0.19 mM) and high (105,263 M^?1 s^?1) catalytic efficiency (K _cat/K _m). The mesostable enzyme had a stringent substrate specificity restricted only to β-linked gluco-conjugate. The enzyme is optimally active at 40 °C with 12.4 kcal Mol^?1 activation energy, and was highly sensitive to d-gluconic acid lactone inhibition (94 % at 1 mM) with an apparent K _i 0.21 mM. The enzyme could catalyze transglucosylation of geraniol with pNPG as glucosyl donor, but not with cellobiose. Some of the physico-kinetic properties were noted to be novel when comprehensively compared with its counterparts from plant, animal and microbial counterparts. Nevertheless, the catalytic and other features of the enzyme were relatively closer to Oryza sativa among plants and Talaromyces thermophillus among fungi. Significance of building-up of a library of novel plant β-glucosidases for structural investigation to understand naturally evolved mechanistics of catalysis has been indicated.