Antiviral activity of berberine and related compounds against human cytomegalovirus

Berberine chloride (1) and the structurally related compounds were assessed for the anti-human cytomegalovirus (HCMV) activity using the plaque assay. The anti-HCMV activity (IC(50) 0.68 microM) of 1 was equivalent to that (IC(50) 0.91 microM) of ganciclovir (GCV). The mechanism of action by which 1 inhibits the replication of HCMV is presumed to be different from that of GCV; 1 would interfere with intracellular events after virus penetration into the host cells and before viral DNA synthesis.     

Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization

Thermolysin is a representative zinc metalloproteinase derived from Bacillus thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. Extracellular production of thermolysin has been achieved in Bacillus, but not in Escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. In this study, we expressed thermolysin as a single polypeptide pre-proenzyme in E. coli under the original promoter sequences in the npr gene, the gene from B. thermoproteolyticus, which encodes thermolysin. Active mature thermolysin (34.6 kDa) was secreted into the culture medium. The recombinant thermolysin was purified to homogeneity by sequential column chromatography procedures of the supernatant with hydrophobic-interaction chromatography followed by affinity chromatography. The purified recombinant product is indistinguishable from natural thermolysin from B. thermoproteolyticus as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-asparatyl-L-phenylalanine methyl ester. The results demonstrate that our expression system should be useful for structural and functional analysis of thermolysin.     

Effects of introducing negative charges into the molecular surface of thermolysin by site-directed mutagenesis on its activity and stability

Thermolysin is remarkably activated and stabilized by neutral salts, and surface charges are suggested important in its activity and stability. The effects of introducing negative charge into the molecular surface on its activity and stability are described. Seven serine residues were selected, and each of them was changed for aspartate by site-directed mutagenesis in a thermolysin mutant. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide, the k(cat)/K(m) values of all mutants were almost similar to that of the wild-type enzyme (WT). However, those of six out of seven mutants were enhanced 17-19 times with 4 M NaCl, being slightly higher than WT. The remaining casein-hydrolyzing activities of the S53D and S65D mutants (Ser53 and Ser65 are replaced with Asp, respectively) after 30-min incubation with 10 mM CaCl(2) at 85 degrees C were 78 and 63%, being higher than those of WT (51%) and the other mutants (35-53%). S53D was stabilized with increase in the enthalpy change of activation for thermal inactivation while S65D was with decrease in the entropy change of activation. The stability of WT was enhanced by CaCl(2) and reached the level of S53D and S65D at 100 mM, suggesting that S53D and S65D might be stabilized by reinforcement of the Ca(2+)-binding structures.     

Utilization of Polyphenyl and Polyphenyl-related Compounds by Microorganisms. Part I

Two hundreds and fifty eight strains of microorganisms have been isolated from 526 samples (soil, leaf and river water gathered from 17 prefectures) by repeating liquid enrichment culture techniques in the medium containing biphenyl, diphenylmethane, diphenylethane or terphenyl, as the sole source of carbon.

In the course of investigation, several strains were found to produce a large amount of γ-benzoylbutyric acid from biphenyl. Furthermore these strains utilized p-Cl-biphenyl and produced p-Cl-benzoic acid in good yield.

Microorganisms obtained were almost short rod, motile bacteria, and fungi were also found from the screening medium of diphenylethane.     

Thiol-disulfide Transhydrogenase from Baker’s Yeast and a New Method for the Direct Assay of an Enzyme-catalyzed Thiol-disulfide Interchange Activity

This paper presents a study on the enzyme reduction of the disulfide bond and the following results have been found.

In enzyme preparation, antioxidants showed a stability effect and EDTA appeared to have both enzyme stabilization and solubilization. On the distribution of the enzyme activity in subcellular fractions, the water soluble fraction appeared to contain the major released enzyme activity. The enzyme was inhibited with several metals. Hg²⁺ and transition metals were the most toxic. The substrate specificity of this enzyme was wide for the low molecular substrates, but the protein disulfide reducing activity was not detected in this preparation. It was assumed that the thiol-disulfide transhydrogenase was coupled with glutathione reductase and the disulfide substrates were reduced by the system involving the two enzymes. A new method for the direct recording of an enzyme-catalyzed thiol-disulfide interchange using diphenyl disulfide and p,p-dinitro diphenyl disulfide was devised.     

Coexistence of different pathways in the metabolism of n-propylbenzene by Pseudomonas sp.

Pseudomonas desmolytica S449B1 and Pseudomonas convexa S107B1 grown on n-propylbenzene oxidized n-propylbenzene to beta-phenylpropionic acid and benzoic acid by initial oxidation of the n-propyl side chain and the following beta-oxidation, respectively. The same strains also oxidized n-propylbenzene to 3-n-propylcatechol by initial oxidation of positions 2 and 3 of the aromatic nucleus. A ring fission product, 2-hydroxy-6-oxononanoic acid, was also isolated from the culture broth. Together with the results of oxygen uptake experiments, the data obtained suggested not only the existence of a reductive step to form 2-hydroxy-6-oxononanoic acid, but also the coexistence of two different pathways in the metabolism of n-propylbenzene by the strains used.