An attenuated plasmid-cured strain of Aeromonas hydrophila elicits protective immunity in Clarias batrachus L

Wild type Aeromonas hydrophila (Strain AO1) isolated from the lesions of ulcerative disease syndrome (UDS) affected fish bears a 21 kb virulence plasmid. With plasmid curing the isolates became attenuated and failed to induce fatal haemorrhagic ulcers in fish. The objective of the present work was to check the immunogenicity of these plasmid-cured derivatives and determine whether such strains could be used as candidate antigens for eliciting protective immunity to A. hydrophila infections in the Indian catfish Clarias batrachus L. It was observed that the plasmid-cured strains were immunogenic since infection with live plasmid-cured AO1 isolates generated effective T cell responses and led to increase in serum antibacterial agglutinin titres in C. batrachus. Plasmid-cured AO1 strains injected into C. batrachus could disseminate into head kidney (HK) and spleen but never attained the same bacterial loads obtained with wild type AO1 and were cleared rapidly from the host. Immunisation with plasmid-cured bacteria prevented systemic spread and conferred protection against lethal challenge (10 x LD(50)) with wild type A. hydrophila as well as other pathogenic strains of Aeromonas sp. These results demonstrate the potentials of plasmid-cured A. hydrophila derivatives as candidate antigens for eliciting protective immunity in fish and the possibility of using such isolates as shuttle vectors in aquaculture.     

Structural Basis of Inactivation of Thiol Protease by N-Acetyl-p-benzoquinone Imine (NAPQI). A Knowledge-Based Molecular Modeling of the Adduct of NAPQI with Thiol Protease of the Papain Family

Inhibition of hepatic cysteine proteases by non-steroidal anti-inflammatory drug (NSAID) metabolites is implicated in several pathological conditions. It has been reported in the literature that N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen (APAP) can quickly arylate and oxidize thiol (cysteine) protease of the papain family to form an adduct in the pathogenesis of acetaminophen-induced hepatotoxicity. It was also clarified by earlier NMR studies that the 3-position of the aromatic ring (C-3) is the only site of conjugation with cysteinyl thioethers for protein arylation. In a recent study, the adduct of NAPQI has been identified and characterized by LC/MS/MS, LC/NMR and UV spectroscopy, and two possible covalent binding modes corresponding to the 2-position (model-1) and the 3 -position (model-2) of the aromatic ring of NAPQI have been proposed. The work presented here has been initiated to check the structural viability of inhibition for the two proposed adducts at the atomic level. Results of our investigation by computer-assisted molecular modeling structurally demonstrate why model-2 would be more applicable to the static x-ray structure of the complex at physiological pH. This coordinated computational and molecular biology experiment can be used for metabolic screening of NSAIDs. A combinatorial approach of this kind alleviates the doubts in interpreting the results of metabolic function and enhances our insights obtained from either computational or experimental studies alone.     

Monoamine oxidase in adult Ascaridia galli

Monoamine oxidase (MAO), catalysing oxidative deamination of biogenic monoamines, has been detected in adult Ascaridia galli. MAO was present in mitochondria and deaminated noradrenaline at the maximal rate, although serotonin, adrenaline, tyramine and dopamine were also degraded but more slowly. Of the organs studied, the body wall, female reproductive organ and intestine, the body wall (containing neuronal structures) showed highest MAO activity. Km value for chick ascarid mitochondrial MAO using tyramine as substrate was 1.66 X 10(-3) M and it was most active at 2.5 mM tyramine concentration, pH 7.5 and 40 degrees C. MAO of A. galli appeared to be thermolabile as nearly 80% of its activity was lost when the incubation temperature was increased 5 degrees above optimum.     

Polypeptides with known repeating sequences of amino acids. Comparison of several methods used for the synthesis of poly-gamma-D- and L-glutamylglycine and investigation of its serological reaction with antianthrax immune serum

To make a comparison of some of the various polymerization methods, poly-γ-D -and L -glutamylglycine were synthesized through the mixed anhydride, carbodiimide, and pentachlorophenyl active ester methods. This last method proved to be best, on the basis of weight-average molecular weights (M?_w). A detailed study was also carried out to determine the optimum conditions for this method. The M?_w, values of the polymers were determined by the sedimentation equilibrium method; it was found that the pentachlorophenyl active ester gave M?_w values of up to 11, 500 but the mixed anhydride and carbodiimide methods gave M?_w, values of only about 1000. Tests with rabbit antianthrax immune serum showed that neither D - nor L -isomer precipitated with the antiserum, but that, both were able to inhibit the precipitation of the antiserum by anthrax polypeptide. In addition, the D -polymer gave more inhibition than the L , which would indicate a specificity of the antibody for D -glutamic acid residues.     

Hyaluronan constitutively regulates ErbB2 phosphorylation and signaling complex formation in carcinoma cells

Hyaluronan is enriched in many types of human cancers, and manipulations of hyaluronan expression or interactions have a major influence on tumor progression in animal models. Increased ErbB2 activity is characteristic of several cancers and is responsible for many aspects of malignant cell behavior in these cancers. In this study we show that constitutively high levels of active, i.e. autophosphorylated, ErbB2 in HCT116 colon carcinoma cells and TA3/St mammary carcinoma cells are dependent on endogenous hyaluronan-CD44 interaction. Dependence on hyaluronan-CD44 interaction was demonstrated by the administration of hyaluronan oligomers, experimentally induced expression of soluble CD44, and small interfering RNA knockdown of CD44 expression. On the other hand, increasing hyaluronan production by overexpression of hyaluronan synthase 2 or emmprin causes elevated ErbB2 phosphorylation in MCF-7 mammary carcinoma cells, which normally exhibit low levels of ErbB2 activity. Furthermore, in HCT116 and TA3/St cells, inhibition of endogenous hyaluronan-CD44 interaction causes disassembly of a constitutive, lipid raft-associated, signaling complex containing phosphorylated ErbB2, CD44, ezrin, phosphoinositide 3-kinase, and the chaperone molecules, Hsp90 and cdc37. Stimulation of hyaluronan production in MCF-7 cells induces assembly of this complex. We conclude that hyaluronan regulates ErbB2 activity and its interactions with other signaling factors in carcinoma cells.     

Alkaloids of Haemanthus kalbreyeri

The roots of Haemanthus kalbreyeri contain a new phenanthridone alkaloid, kalbretorine, and a new glucosyloxy alkaloid, kalbreclasine. Additionally, six known alkaloids, viz. haemanthamine, haemanthidine, hippadine, lycorine, narciclasine and pratorimine, previously reported from other Amaryllidaceous plants have now been isolated also from this species. Kalbretorine produced marked inhibition of growth and viability of S-180 tumour cells. Kalbreclasine caused significant mitogenic activation of splenic lymphocytes characteristic of immuno stimulants.