Interleukin 1 (IL-1) has been obtained from the Epstein-Barr virus-infected B-lymphoblastoid cell line 3B6 and shown to be involved in autocrine growth of 3B6 B cells. Independently, adult T-cell leukemia-derived factor (ADF) was purified from human T-lymphotropic virus I-infected leukemic T-cell line (ATL-2) and reported as an interleukin 2 (IL-2) receptor-inducing factor. We have previously reported the same molecular mass, pI, and NH2-terminal amino acid sequence for both 3B6-derived IL-1 and ADF. cDNA cloning of ADF demonstrated high homology with the prokaryotic disulfide reducing enzyme thioredoxin. We show here that ADF and 3B6-derived IL-1 are identical. By RNA blot, 3B6 and ATL-2 cells were shown to contain high levels of 0.6-kilobase mRNA corresponding to ADF. Such message was not detected in resting peripheral blood lymphocytes but could be weakly induced by lymphocyte activation. Antibodies have been raised against synthetic peptides corresponding to the NH2 terminus and the COOH terminus of ADF. Immunoblotting and sequential immunoprecipitation with these antibodies revealed the same 13-kDa protein in 3B6 and ATL-2 cells. Recombinant ADF could sustain growth of 3B6 and ATL-2 cells at low cellular concentration without fetal calf serum; ADF, thus, appears involved in their autocrine growth. Similarly, recombinant ADF could enhance growth of other B-cell lines, including the Epstein-Barr virus-negative Burkitt lymphoma line BL41 and the lymphoblastoid cell lines CRAG8, CRB95, and 1G8. Finally, recombinant ADF exhibits marked synergism with other cytokines, such as IL-1 and IL-2, allowing virally infected lymphocytes to respond to suboptimal amounts of a variety of growth factors.

No abstract

The ability of HIV-1 to evolve resistance to antiretroviral drugs leads to treatment failure. By nucleotide sequencing of HIV-1 subtype B isolates, amino acids responsible for drug resistance have been identified. Less information is available, however, on the extent and distribution of these amino acids in HIV-1 nonsubtype B viruses circulating mainly in developing countries. More HIV-infected patients in the developing world are now using antiretroviral drugs, and hence there is a need to monitor drug resistance mutations in HIV-1 non-subtype B viruses. This study examines the prevalence of drug resistance mutations in 28 antiretroviral drug-naive HIV-1-infected Zambians. HIV-1 proviral DNA was extracted from peripheral blood mononuclear cells. The region encompassing gag p17 to env C2-V3-C3 was amplified by the polymerase chain reaction followed by direct sequencing. Sequence analyses for drug resistance-associated mutations in th e protease and reverse transcriptase genes, and HIV-1 subtyping, were done. Overall, 92.8% of the generated sequences were HIV-1 subtype C. The generated sequences revealed only secondary associated, but no primary, drug-resistance mutations The most frequent secondary mutations in the protease and RT genes were, respectively, I93L(91.7%), L89M (79.2%), M3611V (79%, 4.2%), and R211K (70.8%), S48T (62.5%). The atypical residues M41N (3.6%) and D67A (3.6%) were detected in the RT gene. This study reveals many naturally occurring polymorphisms in HIV-1 subtype C isolates from antiretroviral drug-naive individuals. Such polymorphisms could lead to rapid treatment failure and development of drug-resistant HIV-1 mutants in individuals undergoing antiretroviral therapy.

Background

In the course of our screening program for inhibitors of thioredoxin (TRX) system, a strain of Actinoplanes, A40644, isolated from a soil sample collected at Ohita Prefecture, Japan, was found to produce the first potent inhibitor of TRXsystem from a natural source. The structure of this novel compound, BE-40644, was elucidated as shown in Fig. 1. BE-40644 showed strong inhibitory activity against TRXsystem, while it showed weak inhibitory activity against glutathione reductase. This paper describes the strain, production, isolation, physico-chemical properties, structure elucidation and activity of this new inhibitor of TRXsystem. Assay ofTRX system essentially followed the modified method of Krause et al.1] and was carried out in the reaction mixture of 470/il of 0.1 m Tris-HCl (pH 7.5) containing 2mM EDTA, 15 jA of0.25 mg/ml E. coli TRX (IMCO Co.), 20jA of0.023 mg/ml E. coli TRX reductase (IMCO Co.), 10jal of 10mg/ml bovine insulin (Sigma Chem. Co.) as substrate of TRX and 5jA of dimethyl sulfoxide with or without test sample. Thereaction was started by adding 15/A of 0.2mM NADPHsolution at room temperature and after 30minutes optical density decrease of NADPH consumption was read at 340nm. Assay of human TRXsystem was carried out by the same method described above but the reaction mixture consisted of S0/A of 0.1 m Tris-HCl (pH 7.5) containing 2mMEDTA, 10/A of 75mg/ml human TRX produced by expressing as a fusion protein with glutathione S-transferase in E. coli, 10 jA of0.214mg/ml human TRX reductase (IMCO Co.), 2/A of 10mg/ml bovine insulin (Sigma Chem. Co.) as substrate of TRXand 1^1 of dimethyl sulfoxide with or without test sample and 3 jA of NADPHsolution. To investigate the selectivity of test samples, inhibition against glutathione/glutathione reductase system was carried out. This counter assay was performed by the modified method of Brehe et al.2) in a reaction mixture comprised of 750 jA of 0.1 m Tris-HCl (pH 7.5), 30 fi\ of oxidative dimer of glutathione (GSSG, 30.6 mg/ml), 30 jA of water, 10 /d of yeast GSSG reductase (Sigma Chem. Co., 5IU/ml) and 20^1 of dimethyl sulfoxide with or without test sample. Twominutes after addition of 80^1 of 0.8mg/ml NADPH solution, inhibitory activity was measured in the same way described above in TRXassay system. Characterization of the strain followed the method adopted by the International Streptomyces Project (ISP)3). Strain A40644 had a brownish vegetative mycelium and bottle shaped sporangia on the surface of the agar media which had hair-like structures on their surfaces (Fig. 2). The spores were rod shaped or ellipsoidal and motile by a polar tuft of flagella. Chemotaxonomic analysis of strain A40644 revealed raestf-diaminopimelic acid and glycine as distinguishing components of the cell wall. Xylose and arabinose were the major sugars in the whole-cell hydrolysate. These results indicated that the strain A40644 has a type IID