[Preparation of T-lymphoblastoid cell lines from patients with T-cell leukemia and asymptomatic carriers of HTLV-I: analysis of the viral genome status]. / Poluchenie T-limfoblastoidnykh kletochnykh linii ot bol'nogo T-kletochnym leikozom i bessimptomnykh nositelei HTLV-I: analiz statusa virusnogo genoma.

Three HTLV-I-infected partially interleukin-2-dependent lymphoid cell lines were derived from a patient with T-cellular leukemia (ATL) from Georgia and a carrier of HTLV-I from Sakhalin. The strains cultured in the presence of 3-5% interleukin-2 were designated as NBK-1, NBK-2, and YE-1, respectively. Immunoblotting analysis showed typical HTLV-I proteins in them except NBK-2 which expressed nontypical proteins p40K and p28-29K. Unexpectedly, leukemic cell fraction ATL/NBK from a patient contained gag proteins p19 (CA), p24 (MA), Pr53, and unidentified protein p29. Southern blot analysis of primary leukemic cells NBK showed one full-length non-defective provirus with restriction sites Sacl in both LTRs. Limited restriction map of the provirus virtually did not differ from previously described HTLV-I prototypes. Although the mechanism of abnormal protein expression remains to be determined, this event can be explained by defective provirus formation in NBK-2 cell line during coculturing of leukemic cells with human umbilical cord blood lymphocytes.

[Serological and molecular-biological study of T-cell leukemia virus in Turkmenistan]. / Serologicheskoe i molekuliarno-biologicheskoe izuchenie virusa T-kletochnogo leikoza (HTLC-1) v Turkmenistane.

Seroepidemiological and molecular-biological screening of 1510 donor blood samples, collected from the residents of the town of Ashgabat (Turkmenistan), for lymphotropic virus of human T-cellular leukemia (HTLV) virus revealed one donor with a high level of immune response to a wide spectrum of viral proteins. Three donors were serologically assessed as dubious, for their sera contained antibodies to gag gene protein but no antibodies to env gene protein. Screening of family members of the donor infected with HTLV-1 revealed four more highly reactive carriers of HTLV-1 virus. The presence of proviral sequences of HTLV-1 in the lymphocyte DNA of infected donor and her relatives was confirmed by polymerase chain reaction and subsequent Southern-blot hybridization of specific amplification products. Proviral sequences of gag, pol, and LTR genes were detected in all the cases. Short-term culturing of peripheral blood lymphocytes of all seropositive subjects was associated with expression of HTLV-1 structural proteins. Analysis of the possible routes of transmission of HTLV-1 isolated in Turkmenistan permits us to hypothesize an Iranian origin of the isolated virus strain.

[Cloning cDNA for the ha-SDGF gene from a Syrian hamster cell line with increased metastatic potential using subtractive hybridization]. / Klonirovanie s pomoshch'iu vychitaiushchei gibridizatsii kDNK gena ha-SDGF iz linii kletok siriiskogo khomiachka s povyshennym potentsialom metastazirovaniia.

Using subtractive hybridization, a cDNA library containing over 50% of clones specific for a highly metastatic cell line was obtained from two hamster embryo fibroblast lines with different metastatic potentials. Most of the clones (83%) contained new sequences. One clone contained the ha-SDGF gene cDNA homologous to SDGF cDNA from rodents. The level of ha-SDGF mRNA expression was considerably higher in the highly metastatic cell line.

[Regulation of the cycle of phospholipid turnover in hamster fibroblasts transformed by v-src and N-ras oncogenes]. / Reguliatsiia tsikla obrashcheniia fosfolipidov v fibroblastakh khomiaka, transformirovannykh onkogenami v-src i N-ras.

The phospholipid turnover has been studied in two lines of golden hamster cells: in cells transformed by the Rous sarcoma virus (line HET-SR) and in cells additionally transfected with the activated oncogene N-ras (line HET-SR-N-ras, clone 6). It has been found that HET-SR cells are distinguished by a high level of phosphatidylcholine turnover and a relatively low level of phosphoinositide turnover. Transfection of cells with the activated N-ras (line HET-SR-N-ras) leads to the inhibition of phosphatidylcholine synthesis and activation of phosphoinositide metabolism. Both cell lines preserve their sensitivity to serum growth factors stimulating the rate of phospholipid turnover. In both cell lines dexamethasone decreases the rate of DNA synthesis and inhibits the phosphatidylcholine and phosphoinositide turnover. At the same time, dexamethasone does not influence the predominant activation of phosphatidylcholine synthesis in HET-SR cells or the activation of phosphoinositide synthesis characteristic of HET-SR-N-ras cells. The data obtained suggest that the transmission of the mitogenic signal from growth factor in HET-SR and HET-SR-N-ras cells occurs via the activation of the phospholipid turnover and is controlled by steroid hormones. The role of v-src and N-ras oncogens in the transmission of the mitogenic signal seems to be insignificant; their activity is not controlled by dexamethasone.

[The isolation and characteristics of the green monkey lentivirus]. / Vydelenie i kharakteristika lentivirusa zelenykh martyshek.

We characterized the simian immunodeficiency virus isolated from Cercopithecus aethiops (subspecies C. a. pygerythrus) originating from Kenya. SIV was isolated and continuously produced with the MOLT4 clone 8 cell line and was designated as SIV-SU1. SIV-SU1 isolate replicated with high efficiency in MOLT4 clone 8, MT-2 with moderate efficiency in CEM x 174 and with poor efficiency in HUT-78, U937, C8166. The infection of MT-2, C8166 and HUT-78 resulted in extensive cell killing. Western blotting of purified preparations of SIV-SU1 revealed viral proteins of 130, 68, 55, 41, 24, 17 kDa. Cross-reactivity of SIV-SU1 proteins with HIV-1, HIV-2, SIVmac, SIVsm, SIVmnd was studied by radioimmunoprecipitation assay. The most extensive cross-reactivity was observed with SIVmac. Total cellular DNA from chronically infected cells was hybridized to SIVagm266 DNA probes. Detection of cross-hybridizing DNA sequences required very low stringency, and the restriction endonuclease fragmentation pattern of SIV-SU1 differed from other SIVs.

Modulation of pp60v-src and pp60c-src expression in Rous sarcoma virus-transformed hamster fibroblasts transfected with activated N-ras.

Three phenotypically different hamster cell lines transformed with Rous sarcoma virus (RSV) were transfected with plasmid DNA containing an activated N-ras oncogene, and nine clones expressing various levels of p21N-ras were characterized. We examined the effects of p21N-ras on expression and kinase activity of resident src proteins by using a variety of assays that allowed us to discriminate between viral and cellular src proteins. In eight clones with a 10- to 20-fold increase in p21N-ras levels relative to the endogenous protein, we observed a marked reduction in the synthesis and kinase activity of p60v-src. This decrease correlated with transcriptional downregulation of RSV genomic and v-src subgenomic mRNAs. In the same cells, we found a concomitant accumulation of p60c-src and, accordingly, an increase in its protein kinase activity without an apparent increase in c-src mRNA levels. Therefore, modulation of viral and cellular src proteins in cells overexpressing p21N-ras appeared to result from two distinct effects: a downregulation of long terminal repeat-driven transcription and a more complex interaction with cellular effectors that control the stability of p60c-src. Such modulation also seemed to depend on the levels of p21N-ras and, possibly, on host-cell factors, since it was not observed in the third cell line, in which the relative increase in p21N-ras was only 2.5-fold to fivefold.