Monoclonal antibodies to the hydrogenase ofThiocapsa roseopersicina

Monoclonal antibodies were produced to electrophoretically pure hydrogenase fromThiocapsa roseopersicina. Protein immunoelectroblotting was used to identify the hydrogenase-specific antibodies. Among the 18 monoclonal antibodies selected by enzyme immunoassay, three were found to react with highly immunogenic trace contaminating proteins. One cell line produced antibody that inhibitied hydrogenase activity. This was the first specific inhibitor of the hydrogenase function. The results suggest that monoclonal antibodies could provide valuable new informations about the enzyme structure as well.     

Immunological relationship among hydrogenases.

We examined the immunological cross-reactions of 11 different hydrogenase antigens with 9 different hydrogenase antibodies. Included were antibodies and antigens of both subunits of the hydrogenases of Bradyrhizobium japonicum and Thiocapsa roseopersicina. The results showed a strong relationship among the Ni-Fe dimeric hydrogenases. The two subunits of Ni-Fe dimeric hydrogenases appeared immunologically distinct: specific interactions occurred only when antibodies to the 60- and 30-kilodalton subunits reacted with the 60- and 30-kilodalton-subunit antigens. The interspecies cross-reactions suggested that at least one conserved protein region exists among the large subunits of these enzymes, whereas the small subunits are less conserved. Antibodies to the Fe-only bidirectional hydrogenase of Clostridium pasteurianum reacted with the Desulfovibrio vulgaris bidirectional hydrogenase. Surprisingly, antibodies to the clostridial uptake hydrogenase did not react with any of the Fe-only bidirectional hydrogenases but did react with several of the Ni-Fe dimeric hydrogenases. The two hydrogenases from C. pasteurianum were found to be quite different immunologically. The possible relationship of these findings to the structure and catalytic functions of hydrogenase are discussed.     

The interrelationships between the development of ethanol-, HCl, NaOH and NaCl-induced gastric mucosal damage and the gastric mucosal superoxide dismutase activity in the rats

Gastric mucosal damage was produced by intragastric administration of 96% ethanol, 0.6 M HCl, 0.2 M NaOH or 25% NaCl. The animals were killed 1 hr later, when the number and severity of gastric lesions (ulcers) was recorded. At the time of the sacrifice of the animals gastric mucosal superoxide dismutase (SOD) activity was measured. It was found that (1) the gastric mucosal damage could be induced by the administration of any of the necrotizing agents in all animals, (2) superoxide dismutase (SOD) activity increased significantly in the damaged gastric mucosa following 96% ethanol, while its activity decreased significantly during the development of gastric mucosal damage produced by the intragastric administration of 0.6 M HCl, 0.2 M NaOH or 25% NaCl. It has been concluded that: (1) the enzyme systems necessary to generate the superoxide free radical anions can be stimulated by ethanol, and they can be inhibited by the application of 0.6 M HCl, 0.2 M NaOH and 25% NaCl: (2) the observed stimulation or inhibition of the enzyme systems to generate the superoxide free radical anions may be of pathological significance in the development of gastric mucosal damage produced by the intragastric administration of 96% ethanol, 0.6 M HCl, 0.2 M. NaOH or 25% NaCl.     

Nuclear and polyribosomal ribonucleoprotein particles containing poly(A) in rat liver cells

Poly(A) containing ribonucleoprotein particles were prepared from rat liver nuclei and polyribosomes. The particles have sedimentation coefficients of 14 S and 9 S, respectively. In Cs₂SO₄ density gradients the particles banded at densities of 1.28–1.29 g cm^-3. Both nuclear and polyribosomal poly(A)-RNP contain in addition to some minor polypeptides, two main polypeptides having molecular weights of 63 000 and 90 000 dalton, respectively indistinguishable from each other according to their electrophoretic mobilities.Abbreviations STKM 0.25 M sucrose, 0.05 M Tris-HCl, pH 7.2, 0.025 M KCl, 0.005 M MgCl₂ - TKM 0.05 M Tris-HCl, pH 7.5, 0.025 M KCl, 0.005 M MgCl₂ - STM II 0.1 M NaCl, 0.01 M Tris-HCl, pH 8, 0.001 M MgCl₂ - DTT dithiothreitol - SDS sodium dodecylsulphate     

Interrelationships between the development of the gastric cytoprotective effects of prostacyclin, atropine, cimetidine and the gastric mucosal superoxide dismutase activity in rats

Gastric mucosal damage was produced by the intragastric administration of 96% ethanol or 0.6 M HCl. The cytoprotective doses of prostacyclin (PGI2) (5 micrograms/kg), atropine (0.025 mg/kg) or cimetidine (2.5 mg/kg) were given intraperitoneally 30 min before the administration of the necrotizing agents. The animals were killed 1 hr later. The number and severity of gastric mucosal lesions (ulcer) were recorded. At the time of the sacrifice of the animals, superoxide dismutase (SOD) was prepared from the gastric fundic mucosa and its activity was measured. It was found that PGI2 (5 micrograms/kg), atropine (0.025 mg/kg) and cimetidine (2.5 mg/kg) significantly decreased the number and severity of gastric mucosal lesions (ulcers) produced by the intragastric administration of 96% ethanol a 0.6 M HCl, PGI2, atropine, cimetidine, given in cytoprotective doses, significantly mounted the ethanol-induced increase of gastric mucosal SOD activity; PGI2, atropine, cimetidine, given them in cytoprotective doses significantly shunted the HCl-induced decrease of gastric mucosal SOD activity. It has been concluded that; chemically different cytoprotective agents (PGI2, atropine, cimetidine) give rise to similar tendencies in the changes of gastric mucosal SOD activity; both the significant decrease (in the ethanol-model) and the significant increase (in the HCl-model) of this enzyme seem to be involved in the development of gastric mucosal protection by PGI2, atropine and cimetidine.     

Identification of Edg1 receptor residues that recognize sphingosine 1-phosphate

Originating from its DNA sequence, a computational model of the Edg1 receptor has been developed that predicts critical interactions with its ligand, sphingosine 1-phosphate. The basic amino acids Arg(120) and Arg(292) ion pair with the phosphate, whereas the acidic Glu(121) residue ion pairs with the ammonium moiety of sphingosine 1-phosphate. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [(35)S]GTPgammaS binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg1 receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions.     

Synthesis and pharmacological evaluation of the stereoisomers of 3-carba cyclic-phosphatidic acid

Cyclic phosphatidic acid (CPA) is a naturally occurring analog of lysophosphatidic acid (LPA) in which the sn-2 hydroxy group forms a five-membered ring with the sn-3 phosphate. Here, we describe the synthesis of R-3-CCPA and S-3-CCPA along with their pharmacological properties as inhibitors of lysophospholipase D/autotaxin, agonists of the LPA(5) GPCR, and blockers of lung metastasis of B16-F10 melanoma cells in a C57BL/6 mouse model. S-3CCPA was significantly more efficacious in the activation of LPA(5) compared to the R-stereoisomer. In contrast, no stereoselective differences were found between the two isomers toward the inhibition of autotaxin or lung metastasis of B16-F10 melanoma cells in vivo. These results extend the potential utility of these compounds as potential lead compounds warranting evaluation as cancer therapeutics.     

Identification of the plasmid and the structural gene of colicin type 7 of Shigella sonnei

Shigella sonnei colicin 7 (Scol7) is a unique bacteriocin acting only on certain dysentery-causing bacteria, like enteroinvasive Escherichia coli, S. sonnei or S. boydii. We identified a 4.2 Md plasmid (pScol7) conferring Scol7 production to the transformants. The entire plasmid was sequenced (Gene Bank Accession number AJ318075) and the structure gene of Scol7 production (sc7a) was identified. Analyzing the sequence of the plasmid revealed extensive homology to other colicin plasmids, particularly to pColE1 but only in areas not related to the bacteriocin activity gene. The similarity of the putative promoter for sc7a to the respective sequences of other colicins suggested that the production of Scol7 is under SOS regulation. Indeed, its production could be increased eightfold by mitomycin C treatment. The molecular mass of the translated polypeptide as deduced from the nucleotide sequence of sc7a (i.e. 11.2 kDa) is in good agreement with previous estimations for its subunit, but molecular filtration experiments suggest a multimeric structure of at least 50 kDa. While current data are not sufficient to predict the mode of action of Scol7, the presence of a DTLSN pentapeptide motive suggests that it could be imported to sensitive cells via the TonB transport system.