Partial Characterization of Small Plasmids fromCorynebacterium renale

A naphthalene-degrading strain of corynebacteria, Corynebacterium renale, harbors multiple small plasmids designated pCR1, pCR2, pCR3, and pCR4 with sizes of 1.4, 3.2, 4.4, and 5.7 kb, respectively. Plasmid pCR1 of 1.4 kb is the smallest plasmid reported in this group of bacteria and is present in high copy number. Attempts to clone whole pCR1 in Escherichia coli were unsuccessful but two of its fragments (750 and 650 bp) could be separately cloned in it. The 4.4-kb plasmid, pCR3, bears considerable restriction pattern similarity to a 4.4-kb plasmid belonging to the pBL1 group of cryptic plasmid of corynebacteria but has no sequence homology, suggesting that pCR3 represents a new member of the 4.4-kb group of corynebacterial plasmids.     

NADPH production from NADP+ with a glucose dehydrogenase system involving whole cells and immobilized cells of Gluconobacter suboxydans

Summary A convenient and efficient method of NADPH production from NADP⁺ has been established using a glucose dehydrogenase system involving whole cells and immobilized cells of Gluconobacter suboxydans IFO 3172. Using airdried cells of the bacterium, the optimum conditions for NADPH production were examined, including the cell and glucose concentrations, NADP⁺ concentration, pH, buffer and reaction temperature. Under suitable conditions, the conversion ratio and the amount of NADPH accumulated reached about 100% and 73 mg/ml of the reaction mixture, respectively, after 1-h reaction. Intact cells of the bacterium also showed high NADPH production even in the reaction mixture without a surfactant. The addition of Triton X-100 to the reaction mixture and freeze-thawing treatment of intact cells enhanced the production. The NADPH production method was further improved by using cells of the bacterium immobilized by entrapment in a -carrageenan gel lattice. The immobilized cells had almost the same enzymatic properties as the air-dried cells. The conditions for the continuous production of NADPH with an immobilized cell column were also investigated. NADPH was produced in a good yield (about 95%) with this continuous process.     

Membrane orientation of laminin binding protein.

Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end.     

Some structural features of the d-glucan from the seed of Mirabilis jalapa

The cotyledon of the seed of Mirabilis jalapa was found to contain a d-glucan. Methylation, periodate oxidation, and graded and enzymic hydrolysis studies were conducted to elucidate its structure. For every 38 d-glucosyl residues therein, 34 are (1→4)- and 3 are (1→3)-linked; the d-glucosyl unit at the branch point is linked through O-1, O-2, and O-4. In some places in the chain, there are at least three (1→3)-linked d-glucosyl residues in a sequence. Both α- and β-d-glucosidic linkages are present in the polysaccharide, the former preponderating. The d-glucan gave with iodine a faint blue color that had λ_max 420 nm.     

Antixenosis,tolerance and genetic analysis of some rice landraces for resistance to Nilaparvata lugens (Stål.)

Twenty-six rice landraces from West Bengal, India were evaluated for antixenosis and tolerance against brown planthopper (BPH) biotype 4 at the Bidhan Chandra Krishi Viswavidyalaya (BCKV), West Bengal. High levels of resistance were observed in six landraces, namely Badshabhog, Gamra, Haldichuri, Janglijata, Kalabhat and Khara. These phenotypically resistant rice landraces including Ptb33 exhibited lowest feeding rate, fecundity, nymphal and adult preference, survival, plant dry weight loss per mg of BPH dry weight produced (PDWL), and higher functional plant loss index (FPLI), more days to wilt and unhatched eggs compared with the susceptible check Swarna. All the landraces were classified into four major clusters at 10 unit distance by the scale of similarity during genetic diversity analysis through 21 gene-linked SSR markers of BPH resistance. Some phenotypically resistant landraces were gathered under the major cluster I indicating their analogous genetic history, while some were grouped with susceptible landraces exhibiting their genetic variation. The resistant landraces can be used as potential donors in the breeding programme for the development of rice varieties with resistance to BPH.     

Morphological and molecular divergence of Indian hill trout,Barilius bendelisis (Hamilton, 1822) stocks from different Rivers in Indo‐Burma biodiversity hotspot: Does river altitude and Dam play a role?

The present study explains the intraspecific variation in Indian Hill trout (Barilius bendelisis) on the basis of image based truss network system and D‐loop region of mtDNA. A total of 210 samples were collected from three different rivers (Teesta, Kameng and Myntudu River) of North East India in Indo‐Burma Biodiversity Hotspot. By using the software applications (tpsDig version 2.1 and PAST), a total of 25 morphometric measurements were generated from 10 landmarks. The Analysis of Variance (ANOVA), Factor Analysis (FA) and Discriminate Function Analysis (DFA) showed, out of the total variations, factor 1 explained 46.74% while factor 2 and factor 3 explained 27.14% and 11.92%, respectively. Using these variables 83.33% of the cross‐validated specimens were classified into distinct groups. Analysis of Molecular Variance (AMOVA) and pairwise Fst value for D‐loop region of mtDNA also showed high to medium level of genetic variation among the stocks and within the stocks. We conclude that the observed discrete stocks might be the result of changing environmental conditions in different rivers of the hotspot as the rivers are present at different altitudinal labels. It is also believed that the variation might be due to the construction of barrages across the river which hinder the mixing among the stocks.     

Antineoplastic and antibacterial activity of some mononuclear Ru(II) complexes

These ligands (L) show a bidentate behavior, forming octahedral ruthenium complexes. The title complexes were subjected to in-vivo anticancer activity tests against a transplantable murine tumor cell line, Ehrlich's Ascitic Carcinoma (EAC) and in-vitro antibacterial activity against several Gram positive and Gram negative bacterial strains. [Ru(bpy)2(ihqs)]Cl2 and [Ru(bpy)2 (hc)]Cl2 (where bpy = 2,2'-bipyridine, ihqs = 7-iodo-8hydroxy quinoline-5-sulphonic acid and hc = 3-hydroxy coumarin) showed promising antitumor activity. Treatment with these complexes prolonged the life span of EAC bearing mice as well as decreased their tumor volume and viable ascitic cell count. All the tested complexes exhibited mild to moderate antibacterial activity.     

Dendritic cells from chronic lymphocytic leukemia patients are normal regardless of Ig V gene mutation status

Patients with B-type chronic lymphocytic leukemia (B-CLL) segregate into 2 subgroups based on the mutational status of the immunoglobulin (Ig) V genes and the patients in these subgroups follow very different clinical courses. To examine whether dendritic cells (DCs) generated from CLL patients can be candidates for immune therapy, we compared the phenotypic and functional capacities of DCs generated from patients of the 2 CLL subgroups (normal age-matched subjects [normal-DCs]). Our data show that immature DCs from B-CLL patients (B-CLL-DCs) have the same capacity to take up antigen as those from normal controls. Furthermore, B-CLL-DCs generated from the 2 CLL subgroups up-regulated MHC-II, CD80, CD86, CD83, CD40, and CD54 and down-regulated CD206 in response to stimulation with a cocktail of cytokines (CyC) and secreted increased levels of tumor necrosis factor alpha, interleukin (IL)-8, IL-6, IL-12 (p70), and RANTES in a manner typical of mature normal-DCs. Interestingly, CD54 was significantly more up-regulated by CyC in B-CLL-DCs compared with normal-DCs. Except for CD54, no significant differences in surface molecule expression were observed between normal-DCs and B-CLL-DCs. B-CLL-DCs from both subgroups, including 6 patients with VH1-69, that usually fare poorly, presented tetanus toxoid to autologous T cells in vitro similar to normal- DCs. Our data show that DCs generated from the B-CLL subgroup with unmutated Ig V genes are functionally normal. These results are very promising for the use of DCs from patients with poor prognosis for immunotherapy.     

Defining the structure-function relationships of bluetongue virus helicase protein VP6

The VP6 protein of bluetongue virus possesses a number of activities, including nucleoside triphosphatase, RNA binding, and helicase activity (N. Stauber, J. Martinez-Costas, G. Sutton, K. Monastyrskaya, and P. Roy, J. Virol. 71:7220-7226, 1997). Although the enzymatic functions of the protein have been documented, a detailed structure and function study has not been completed and the oligomeric form of the protein in solution has not been described. In this study, we have characterized VP6 activity by creating site-directed mutations in the putative functional helicase domains. Mutant proteins were expressed at high levels in an insect cell by using recombinant baculoviruses purified and analyzed for ATP binding, ATP hydrolysis, and RNA unwinding activities. UV cross-linking experiments indicated that the lysine residue in the conserved motif AXXGXGK(110)V is directly involved in ATP binding, whereas mutant R(205)Q in the arginine-rich motif ER(205)XGRXXR bound ATP at a level comparable to that of the wild-type protein. The RNA binding activity was drastically altered in the R(205)Q mutant and was also affected in the K(110)N mutant. Helicase activity was altered in both mutants. The mutation E(157)N in the DEXX sequence, presumed to act as a Walker B motif, showed an intermediate activity, implying that this motif does not play a crucial role in VP6 function. Purified protein demonstrated stable oligomers with a ring-like morphology in the presence of nucleic acids similar to those shown by other helicases. Gel filtration chromatography, native gel electrophoresis, and glycerol gradient analysis clearly indicated multiple oligomeric forms of VP6.