Predictive indicators for the therapeutic effect of OK-432 in patients with chronic active type B hepatitis

Twenty-two patients with chronic type B hepatitis were treated with OK-432. Immunological parameters were serially measured to find predictive indicators for the seroconversion from hepatitis B envelope antigen(HBe Ag) to anti-HBe. In patients who achieved the disappearance of HBe Ag associated with or without the appearance of anti-HBe, the numbers of CD8⁺DR⁺ and CD4⁺DR⁺T cells in peripheral blood increased gradually during OK-432 therapy and then reduced subsequently to the seroconversion from HBe Ag positive to anti-HBe positive. Increases of DR-positive T cells in numbers were significantly correlated with increased amounts of IFN- produced in response toin vitro OK-432 stimulation.In vitro OK-432-stimulated IFN- production and the increase of CD8⁺DR⁺T cells in number in peripheral blood could be proposed as predictive indicators for the disappearance of HBe Ag.     

Does Tn10 transpose via the cointegrate molecule?

Summary It has been well established that Tn3 and its relatives transpose from one replicon to another by two successive reactions: formation of the cointegrate molecule and resolution from it. Whether or not the 9300 base pair tetracycline resistance transposon Tn10 transposes in the same manner as Tn3 was investigated by two methods.In the first method, 55, a lambda phage carrying Tn10 was lysogenized in an Escherichia coli strain carrying a Tn10 insertion; the phage has a deletion in attP, hence it was lysogenized in a Tn10 sequence in the E. coli chromosome by reciprocal recombination. The chromosomal structure in these lysogens is equivalent to the Tn10-mediated cointegrate molecule of lambda and the E. coli chromosomal DNA. The stability of the cointegrate molecule was examined by measuring the rate of excision of lambda from the host chromosome, and was found to be stable, especially in a Rec^- strain. Because of this stability, the cointegrate molecule should be accumulated if Tn10 transposes via the cointegrate molecule. Then, we examined the configuration of products made by transposition of Tn10 from 55 to the E. coli chromosome. The cointegrate molecule was found in products of Tn10 transposition in a Rec⁺ strain at a frequency of 5% per Tn10 transposition, but this molecule could not be found in a Rec^- strain. Since transposition of Tn10 was recA-independent, absence of the cointegrate molecule formed in a RecA^- strain strongly suggested that the cointegrate molecule is not an obligatory intermediate of transposition of Tn10.In the second method, mobilization of pACYC177 by R388 and by R388:: Tn10 was examined. The pACYC177 plasmid was mobilized by R388::Tn10 at a frequency of 10^-4 per donor but not by R388. It occurred, in most cases, by inverse transposition of R388::Tn10 to pACYC177 forming plasmids such as pACYC177::IS10-R388-IS10. Mobilization of pACYC177 by a Tn10-mediated cointegrate in the form of pACYC177::Tn10-R388-Tn10 was not observed in crosses using a Rec^- donor. These observations also suggested that transposition of Tn10 in Rec^- cells does not occur via the cointegrate molecule.     

Induction of anti-EBNA-1 protein by 12-O-tetradecanoylphorbol-13-acetate treatment of human lymphoblastoid cells.

Binding of the Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) to BamHI-C DNA was studied by affinity column chromatography followed by immunoblotting with human serum specific for EBNA-1. Two species of EBNA-1 (68 and 70 kilodaltons) were identified in nuclear extracts of the EBV-positive Burkitt's lymphoma cell line Raji and not in nuclear extracts of the EBV-negative Burkitt's lymphoma cell line BJAB. Both EBNA-1s bound specifically to the region required for EBV plasmid DNA maintenance (oriP) located in the BamHI-C fragment. Upon treatment with 12-O-tetradecanoylphorbol-13-acetate, which activates latent EBV genome in Raji cells, the 68-kilodalton EBNA-1 was uncoupled from binding to EBV oriP. Nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated BJAB cells also uncoupled the binding of both EBNA-1s to oriP. DNA-cellulose column chromatography identified two protein species which competed for and uncoupled the binding of EBNA-1 to oriP. The two cellular competitors we called anti-EBNA-1 proteins had molecular masses of 60 and 40 kilodaltons, respectively. They were not found in nuclear extracts of BJAB cells not activated by 12-O-tetradecanoylphorbol-13-acetate.     

Comparison of the nucleotide sequences of the meta-cleavage pathway genes of TOL plasmid pWW0 from Pseudomonas putida with other meta-cleavage genes suggests that both single and multiple nucleotide substitutions contribute to enzyme evolution

TOL plasmid pWW0 from Pseudomonas putida mt-2 encodes catabolic enzymes required for the oxidation of toluene and xylenes. The structural genes for these catabolic enzymes are clustered into two operons, the xylCMABN operon, which encodes a set of enzymes required for the transformation of toluene/xylenes to benzoate/toluates, and the xylXYZLTEGFJQKIH operon, which encodes a set of enzymes required for the transformation of benzoate/toluates to Krebs cycle intermediates. The latter operon can be divided physically and functionally into two parts, the xylXYZL cluster, which is involved in the transformation of benzoate/toluates to (methyl)catechols, and the xylTEGFJQKIH cluster, which is involved in the transformation of (methyl)catechols to Krebs cycle intermediates. Genes isofunctional to xylXYZL are present in Acinetobacter calcoaceticus, and constitute a benzoate-degradative pathway, while xylTEGFJQKIH homologous encoding enzymes of a methylphenol-degradative pathway and a naphthalene-degradative pathway are present on plasmid pVI150 from P. putida CF600, and on plasmid NAH7 from P. putida PpG7, respectively. Comparison of the nucleotide sequences of the xylXYZLTEGFJQKIH genes with other isofunctional genes suggested that the xylTEGFJQKIH genes on the TOL plasmid diverged from these homologues 20 to 50 million years ago, while the xylXYZL genes diverged from the A. calcoaceticus homologues 100 to 200 million years ago. In codons where amino acids are not conserved, the substitution rate in the third base was higher than that in synonymous codons. This result was interpreted as indicating that both single and multiple nucleotide substitutions contributed to the amino acid-substituting mutations, and hence to enzyme evolution. This observation seems to be general because mammalian globin genes exhibit the same tendency.     

Strain differences to effects of aging on concentrations of amino acids in cerebrospinal fluid between Sprague Dawley rat and Wistar Kyoto rat.

Age-related alterations and differences of weights and those of amino acid concentrations in the cerebrospinal fluid (CSF) were evaluated between Sprague Dawley (SD) rats and Wistar Kyoto (WKY) rats from eight to twenty weeks of age. The weights of SD rats were heavier than WKY rats at all ages. The age-related alterations of the CSF concentration of many amino acids within each strain were significant but showed no significant trend with age. Between the strains, the concentration differences of excitatory and inhibitory amino acids were not frequent although the concentrations of arginine, alanine and threonine were significantly higher in SD rats than in WKY rats. These results suggest that the different CSF concentrations of amino acids may relate to characteristics of rat strains.     

Transposon-insertion mutants of Escherichia coli K12 defective in a component common to galactose and ribose chemotaxis.

Summary From a collection of 8,000 transposon-insertion mutants of Escherichia coli K12 we identified two mutations, trg-1::Tn5 and trg-2::Tn10, that simultaneously eliminate chemotactic response to ribose and galactose, two attractants recognized by independent receptors. We show that these transposon-insertions confer a Trg phenotype, indicating that this specific pattern of tactic defects is a null phenotype. The two mutation sites are cotransductionally linked to an extend consistent with placement in the same gene. The Trg phenotype of a family of deletion mutants produced by curing trg-2::Tn10 implies that trg is a single gene. Experiments with appropriate F-primes and Hfr's locate the trg locus at approximately 31 min on the linkage map, with a marker order: pyrF-rac-(P.O. 43)-trg-man.We also found one trg mutant whose Trg phenotype was not linked to a transposon-insertion but is probably the result of a mutator activity in the parent strain. Selection of transposon-insertions near, but not in trg allowed demonstration of a very close linkage between the spontaneous trg-3 and the transposon-generated trg's, indicating all three mutations are probably in the same gene. In our manipulations of transposon-insertions we found that Tn5 had a tendency to translocate from its initial site of insertion while Tn10 was relatively stable.The trg-product is probably a chemotactic signal transducer, which interacts directly with two independent receptor proteins and transmits information to the central chemotactic machinery.     

Role of various carrageenans in autologous and allogeneic mixed lymphocyte reaction

Carrageenans (CGNs) are extracted from cell walls of certain algae of the Rhodophyta and are gel-forming polysaccharides. There are three kinds of purified carrageenans available—kappa, lambda, and iota CGN. All of them stimulated responding T lymphocytes in both autologous and allogeneic mixed lymphocyte reaction, lambda CGN being the most effective. Silica particles abrogated proliferation of responding cells in autologous and allogeneic mixed lymphocyte reaction by killing human monocytes. However, when 1 mg/ml of CGN was added to silica-added assay system, CGN could induce proliferation of responding T cells even in the absence of monocytes. It was concluded that CGN was a monocyte-independent T-lymphocyte mitogen that was also toxic for human monocytes.     

Cosmid cloning and transposon mutagenesis in Salmonella typhimurium using phage lambda vehicles

Summary We have constructed a strain of Salmonella typhimurium which contains the malB region from Escherichia coli and carries the bacteriophage receptor protein in its outer membrane. Phage adsorbs to this strain but cannot grow, thus providing a very useful system for transposon mutagenesis of S. typhimurium using vehicles carrying transposons. This system can also be used for cosmid cloning.