Effects of exogenous stress protein 70 on the functional properties of human promonocytes through binding to cell surface and internalization.

The presence of antibodies against the major stress protein, Hsp70, in patients with autoimmune diseases led us to hypothesize that Hsp70 may occur extracellularly, and could exert chaperoning and regulatory effects on various cells. We examined the action of pure Hsp/Hsc70 on the main physiological functions of human promonocytic U-937 cells. The protein was isolated from calf muscle and was shown to be a mixture of inducible Hsp70 (60%) and constitutive Hsc70 (40%) isoforms. It was observed that the addition of the protein up-regulated two major monocyte/macrophage differentiation markers, CD11c and CD23, by 20-35%, while it had no effect on CD14. The experiments performed to investigate the influence of Hsp/Hsc70 on the reaction of U-937 cells to differentiation stimuli demonstrated that the addition of the protein prior to PMA was able to inhibit binding of proper transcription factors to double-symmetry and cAMP-response elements of the c-fos early response gene promoter. Administration of exogenous Hsp/Hsc70 prior to treatment with the tumor necrosis factor-alpha significantly lowered the number of apoptotic and necrotic cells. In no case did the control protein, ovalbumin, taken in the same concentration give a comparable effect on U-937 cells. Since the Hsp/Hsc70 effects occurred within the first hour of co-incubation, and therefore they might be explained by its interaction with the cell surface, we assayed binding of the biotinylated protein to U-937 cells by immunoenzyme assay, flow cytometry and indirect immunofluorescence. Using these three techniques we were able to detect Hsp/Hsc70 bound to cells after a 20 min incubation. According to flow cytometry data, at this time 32% of cells were positively stained with streptavidin-FITC. Immunofluorescence studies demonstrated Hsp/Hsc70 bound to the cell surface after a 20 min incubation followed by induction of patch and cap-like structures. One hour later, the majority of the protein had been internalized by U-937 cells.

Accumulation of major stress protein 70kDa protects myeloid and lymphoid cells from death by apoptosis.

The major heat shock protein, hsp70, is known to contribute to the mechanisms of cell protection against a variety of stress and cytotoxic factors, providing an increase of cell survival. Whether hsp70 could be implicated in the rescue of cells from stress-induced death proceeding on apoptotic pathway is not well established. Here we report that susceptibility of myeloid and lymphoid cell lines to apoptosis induced by heat shock or ethanol coincides with hsp70 content and can be modulated by changes in expression of this protein. Cells of lymphoid and myeloid lines differing in basal and inducible level of the protein were tested. The cells containing higher amounts of hsp70 (U937, Jurkat, Molt4) were more resistant to the apoptosis-inducing stimuli then cells which accumu-late lower amounts of the protein (HL60) and especially those lacking the protein (NSO). Inhibition of hsp70 accumulation by quercetin made cells more susceptible to the same apoptotic inducer. Enhancement of hsp70 expression by previous heating or by liposomal delivery of the exogenic protein to the cells lacking hsp70 made them more resistant to apoptosis. The possible mechanisms of the hsp70 protective effect in apoptosis are discussed.

[The physical mapping of human chromosomes. II. The production of unique chromosome-specific DNA fragments using the polymerase chain reaction with oligonucleotide primers to conservative regions of Alu repeats]. / Fizicheskoe kartirovanie khromosom cheloveka. II. Poluchenie unikal'nykh khromosom-spetsificheskikh fragmentov DNK s pomoshch'iu polimerzanoi tsepnoi reaktsii s oligonukleotidnymi zatravkami na konservativnye uchastki Alu-povtorov.

Unique DNA fragments localised between Alu-repeats have been produced by PCR. The reaction was carried out with oligonucleotide primers to conservative regions of Alu-repeats. The DNA fragments from different pulls, individual clones, chromosome-specific clonotecs derived from phage lambda, cosmids and individual human chromosomes served as matrixes. The possibilities are discussed of Alu-primer applying in production of exceptional physical features of DNA molecules, suitable for constructing clone couple groups and for direct physical mapping on the DNA of isolated chromosomes, missing the stage of cloning.

[The structure of Alu-like DNA nucleotide sequences in cyclostomes and fishes]. / Struktura Alu-podobnykh nukleotidnykh posledovatel'nostei DNK u ryboobraznykh i ryb.

By methods of DOT hybridization and polymerase chain reaction (PCR) structures similar to Alu repeats were shown in the genome of fish and lamprey. The fact of the presence of such sections in representatives of many orders of fishes, and also our experimental data allow to suppose that Alu-like structures, similar to 3' region of Alu repeats on mammals, have been already formed in these. The evolutionary significance of described structures has been supposed.

[The physical mapping of human chromosomes. The use of the polymerase chain reaction on unique DNA sequences with a known location on chromosome 3]. / Fizicheskoe kartirovanie khromosom cheloveka. Ispol'zovanie polimeraznoi tsepnoi reaktsii na unikal'nykh posledovatel'nostiakh DNK s izvestnoi lokalizatsiei v khromosome 3.

16 pairs of oligonucleotide primers, complementary to unique DNA sequences of human chromosome 3, were synthesized. For 10 of these, fragments of expected length were generated in polymerase chain reaction (PCR). These fragments may be used as markers for detailed physical mapping of this chromosome. The above primers were used in PCR in order to analyse a hybrid mice-human cell line which contained presumably a fragment of human chromosome 3. The presence of human DNA in the hybrid line has been shown, but no ultimate evidence was received to confirm its location in human chromosome 3. By means of primers, complementary to the butyrylcholinesterase gene (BCHE), pools of clones from the yeast total library of human DNA were analysed, and then the pool and later the individual [correction of undividual] clone, containing a fragment of BCHE gene, were identified.

[Extracellular DNA in the blood of pregnant women]. / Vnekletochnaia DNK v krovi beremennykh zhenshchin.

The level of extracellular DNA increases in the blood of women during pregnancy. By means of PCR, the full-size Alu repeats were observed among extracellular blood DNA repeats of pregnant women. Furthermore, with Tc65 type primer the PCR method allowed to observe in the blood DNA fragments flanked by inverted Alu repeats (inter Alu repeats). The presence of such a type of inter Alu repeats was estimated in the blood of women being in the first trimester of pregnancy only, but was not estimated among blood DNA fragments of women of the last trimester of pregnancy. It is discussed which types of cells may serve as a source of extracellular blood DNA (either trophoblast cells, lymphocytes, or decidual cells), the significance of such DNA for pregnancy being appreciated.

Differential binding of human nuclear proteins to Alu subfamilies.

Several diagnostic differences that distinguish human Alu subfamilies are clustered just downstream from the B box of the RNA polymerase III promoter; we tentatively refer to this diagnostic region as the DB box. Assuming that this region might determine the relative transcriptional activity of Alu subfamilies, we examined the interaction of nuclear proteins with DB box sequences representing different Alu subfamilies. Gel mobility shift assays suggest the existence of two factors which discriminate among the DB boxes of different Alu subfamilies: 1) An abundant, ca. 50 kd, protein binds more stably to a young 'PV' Alu subfamily (PVS) than to the older major subfamily (MS). 2) Methylation of CpG dinucleotides stimulates the binding of a less abundant, ca. 70 kd, protein to the DB boxes of younger Alu subfamilies.

Rat DNA sequence associated with a complex form of DNA polymerase alpha in nonregenerating liver interacts with a ubiquitous transcription/replication factor Oct-1.

A ubiquitous mammalian transcription factor, Oct-1 (also known as OTF-1, NF-A1, OBP100, or NFIII), stimulates the initiation of replication of adenovirus DNA, and may also be involved in the activation of some chromosomal replication origins. If this is true, binding sites for Oct-1 should be present within regions responsible for the initiation of DNA replication. In this study such a binding site has been identified within a 340bp fragment that was originally isolated from a minor fraction of DNA associated with a complexed form of DNA polymerase alpha from nonregenerating rat liver, and which shows autonomous replication sequence activity in a transient transfection assay. Northern blot analysis was used to show that Oct-1 mRNA is induced in regenerating rat liver 6-14 h after hepatectomy.

[Several copies of angiogenin gene are present in the mammalian genome]. / Gen angiogenina predstavlen neskol'kimi kopiiami v genomakh mlekopitaiushchikh.

Human cDNAs coding for angiogenin were isolated from Li7 hepatoma. Analysis of the nucleotide sequences of isolated cDNA clones and its comparison with recently published sequences of cDNA and human angiogenin gene permitted to suggest that an intron is present in the 5' region of the gene, dividing the coding and 5' untranslating regions. The size of the intron exceeds 1700 b.p. Up to now it has been known that the angiogenin gene is a single copy gene. However our results of blot-hybridization with genomic DNA of some mammals showed that there are 2-3 copies of the gene in their genomes. According to our results the angiogenin gene is conserved in mammalian genomes.

Human nuclear protein interacting with a conservative sequence motif of Alu-family DNA repeats.

Human retrotransposons, Alu-family DNA repeats (AFRs), have variable nucleotide sequence but conservative short elements, which may have important functions, are also present. In our previous reports we have described human nuclear DNA-binding protein interacting with AFRs and evidence was presented that the protein recognizes sequence motif 5'-GGAGGC-3' which is conserved in the spacer of RNA polymerase III promoter of AFRs and in the SV40 T-antigen-dependent replication origin of AFRs. In this study it was found that double-stranded synthetic oligonucleotides containing indicated conservative sequences of AFRs actually have high-affinity binding site for HeLa nuclear protein. The data suggest that non-infected human cells contain nuclear DNA-binding protein which recognizes the conservative sequence motif of AFRs - GGAGGC.

[Cloning of human erythropoietin gene]. / Klonirovanie gena éritropoétina cheloveka.

The 17-mer oligonucleotide probe homologous to the fragment of the gene for human erythrocyte differentiation factor erythropoietin was used to screen the human genomic library for this gene. Restriction analysis and partial sequencing of one of the identified clones have confirmed that the clone does contain the human erythropoietin gene. We are planning to use the cloned human erythropoietin gene for developing a stably transfected mammalian cell line that should secrete erythropoietin.

[Several enzymic properties of the DNA-polymerase alpha complexes with antibodies]. / Nekotorye fermentativnye svoistva kompleksa DNK-polimerazy al'fa s antitelami.

A highly specific rabbit antiserum against DNA polymerase alpha from regenerating rat liver (antigen AG 1) and an antiserum against the preparation of the enzyme proteolytic fragments possessing catalytic activity (antigen AG 2) were obtained. The enzyme neutralization test revealed that antibodies against AG 2 inhibit the DNA polymerase activity in a much stronger degree, than those against AG 1. Data from a kinetic analysis of the enzyme complexed with the antibodies against AG 1 suggest that the catalytic and binding sites for dNTP and free Mg2+ are altered. The value of apparent Km for activated DNA is unchanged in the DNA polymerase complexes with antibodies both against AG 1 and AG 2.

[Cellular DNA repair, proliferative activity and biochemical characteristics in the human premature aging syndrome (progeria)]. / Reparatsiia DNK, proliferativnaia aktivnost' i biokhimicheskie osobennosti kletok pri sindrome prezhdevremennogo stareniia (progeriia).

Fibroblasts of a patient with the Hutchinson-Gilford progeria show a decreased proliferative activity in culture and run through no more than 19 subcultivations. Progeria cells rejoin gamma-induced single strand DNA breaks to the same extent and with the same rate as do normal cells. Spontaneous and induced by X-ray chromosome aberration frequency in progeria cells does not differ from that in normal cells. The activity of DNA-polymerases alpha, beta and gamma in different way depends on culture conditions in progeria and normal cells, but no expressed deficiency of these enzymes was found in progeria cells. The activity of easy-soluble arginine-specific proteases in progeria fibroblasts is sharply decreased, and sensitivity of proteins in these cells to trypsin-catalyzed hydrolysis is significantly increased compared to that of normal cells. Defect in the turnover of intracellular proteins is considered to be a reason of a rapid accumulation of altered proteins in cycling cells and a possible reason of accelerated ageing of progeria cells in vitro.