Influence of different inhibitors on the activity of theRAD54 dependent step of DNA repair inSaccharomyces cerevisiae

Summary The recombinagenic pathway of DNA repair in yeast was characterized by the effect of different inhibitors on the temperature-dependent survival after-irradiation in haploid cells of the thermoconditional mutantrad54-3. Blocking protein synthesis with cycloheximide in replicating cells caused partial inhibiton of theRAD54 dependent function but some repair activity remained detectable. This indicates that-rays can induceRAD54 activity above some constitutive level of function. Inhibition of DNA replication by hydroxyurea efficiently blocked theRAD54 dependent function in stationary-phase cells but not in logarithmic-phase cells. In logarithmic-phase cells, we found a strong inhibitory effect of caffeine on theRAD54 mediated repair process.     

DNA profile of the spore of blastocladiella emersonii: Evidence for γ-particle DNA

Summary Procedures were developed for isolating DNA from zoospores of Blastocladiella emersonii, critical steps being the use of non-frozen cells, enzymatic elimination of polysaccharide, and a combined treatment with T₁RNAse and pancreatic RNAse for complete removal of RNA. Methods for isolating -particles from spores were also established. Extraction of DNA directly from purified -particles substantiated previous assumptions that they contain DNA. Heat denaturation and preparative CsCl equilibrium sedimentation studies of whole spore DNA revealed the presence of four distinct components, I–IV, with buoyant densities of 1.731, 1.715, 1.705, and 1.687 g/cm³. The major species appeared to be nuclear chromatin. Nucleolar and mitochondrial sites were tentatively assigned to species II and III, respectively. Evidence was presented that species IV resided in -particles. Some possible roles for -particle DNA were discussed.Abbreviations EGTA ethyleneglycol-bis (B-aminoethyl ether)-N,N-tetracetic acid - EDTA ethylenediaminetetracetic acid - TCA trichloroacetic acid - SDS sodium dodecyl sulfate - SSC 0.15 M NaCl and 15 mM Na citrate     

Influence of SOS repair on the specificity of radiation mutagenesis in bacteriophage ?X174

Summary The ochre mutant oc9 of bacteriophage X174 was irradiated with -rays and the revertants were assayed on unirradiated and UV-irradiated host bacteria carrying an amber suppressor. The yield of revertants (amber+wild type) was higher on UV-irradiated than on unirradiated bacteria, showing that -irradiated X174 was subjected to W-mutagenesis.For oc9 -irradiated in the presence of oxygen the fraction of amber mutants among the revertants was lower when mutants were scored on UV-irradiated bacteria than when assayed on unirradiated indicator cells. The same fraction of ambers was obtained when mutants were assayed on unirradiated and UV-irradiated samples of a recA indicator strain. UV-irradiated X174 showed a similar phenomenon. These results suggest that the specificity with regard to insertion of bases opposite radiation damage in X174 DNA is different for host cells in which SOS repair has been induced and cells in which SOS repair is not operative.     

Diversity of the mouse T cell receptor Cγ1 gene: structural analysis in C57BL/Ka

We have isolated an unusual T cell receptor chain cDNA clone (7.1) from a library made from RNA derived from adult thymus of C57BL/Ka mice. This cDNA clone corresponds to the appropriately processed C1 constant region exons preceded by 1.5 kb of J-C1 intron. The 7.1 coding region is extremely homologous to the C1 gene of BALB/c mice, differing at the protein level by a single deletion (alanine 139) and a single substitution. This latter change eliminates the sole N-linked sugar attachment site, providing a basis for strain-specific glycosylation patterns. The J-C1 intronic region contains two DNA segments (termed J1 and J2) that are highly reminiscent of joining (J) segments; both have potentially functional recombination and donor splice sequences flanking an open reading frame. Northern analysis suggests that 7.1 may be derived from a large, variable region-containing precursor.     

γδ T-cell receptor repertoire in human peripheral blood and thymus

T-cell clones expressing the T-cell receptor (Tcr) were generated from peripheral blood lymphocytes (PBLs) and from a thymus sample. In the panel of ten thymus-derived clones, four Tcr phenotypes [as defined by the reaction of monoclonal antibodies (mAbs) directed against known V and V regions] were identified. All the clones lacked expression of the V3 V region, while seven clones were V1+ . V1 was found in combination with V9 or with undefined VVregions. In addition, two other Tcr phenotypes were identified on these clones: V9⁺ V1^– V3^– and V9^– V1^– V3^– One of the clones expressed CD4 and another was CD8positive. The remaining clones were CD4^– CD8^–. In the panel of 76 PBL-derived, Tcr-bearing clones, five Tcr phenotypes could be identified. In contrast to the thymus-derived clones, 30% of the clones were V3⁺ whereas V1 was expressed by a minority of the clones only. One clone was CD4-positive and approximately 30% of the clones were CD8-positive. Four of the five mAb-defined Tcr phenotypes could be identified on both thymus and PBL-derived T-cell clones. However, biochemical analysis of the Tcrs demonstrates differences in the usage of Ct- and C2-encoded y chains by T cells derived from the thymus and PBLs. The results therefore indicate that, at the clonal level, similarities and differences exist between the Tcr repertoires expressed in the thymus and by PBLs. Furthermore, they indicate that combinatorial Tcr heterogeneity is larger than has so far been described. The receptor diversity, combined with the potential of Tcr+ cells to express CD4 or CD8, indicates that these cells are a heterogeneous population that might mediate a number of immune functions.     

The Effect of γ-Radiation and Desiccation on the Viability of the Soil Bacteria Isolated from the Alienated Zone around the Chernobyl Nuclear Power Plant

Methylobacterium extorquens, M. mesophilicum, and Bacillus subtilis strains were found to be resistant to -radiation, irrespective of whether they were isolated from the alienated zone around the Chernobyl Nuclear Power Plant or outside this zone. The LD₉₀ of Methylobacterium and B. subtilis strains with respect to -radiation was 2.0–3.4 and 3.7–4.4 kGy, respectively, whereas their LD_99.99 values were 4.5–6.9 and more than 10 kGy, respectively. The high threshold levels of -radiation for Methylobacterium and B. subtilis imply the efficient functioning of DNA repair systems in these bacteria. Unlike Bacillus polymyxa cells, the cells of M. extorquens, M. mesophilicum, and B. subtilis were also resistant to desiccation. Pseudomonas sp., Nocardiasp., and nocardioform actinomycetes were sensitive to both -radiation and desiccation. Similar results were obtained when the bacteria studied were exposed to hydrogen peroxide and ultraviolet radiation. The results obtained indicate that the bacteria that are resistant to -radiation are also resistant to desiccation, UV radiation, and hydrogen peroxide. The possibility of using common laboratory tests (such as the determination of bacterial resistance to UV light and desiccation) for the evaluation of bacterial resistance to -radiation is discussed.     

Gamma carbonic anhydrase like complex interact with plant mitochondrial complex I

We report the identification by two hybrid screens of two novel similar proteins, called Arabidopsis thaliana gamma carbonic anhydrase like1 and 2 (AtCAL1 and AtCAL2), that interact specifically with putative Arabidopsis thaliana gamma Carbonic Anhydrase (AtCA) proteins in plant mitochondria. The interaction region that was located in the N-terminal 150 amino acids of mature AtCA and AtCA like proteins represents a new interaction domain. In vitro experiments indicate that these proteins are imported into mitochondria and are associated with mitochondrial complex I as AtCAs. All plant species analyzed contain both AtCA and AtCAL sequences indicating that these genes were conserved throughout plant evolution. Structural modeling of AtCAL sequences show a deviation of functionally important active site residues with respect to CAs but could form active interfaces in the interaction with AtCAs. We postulate a CA complex tightly associated to plant mitochondrial complex.     

Induction of c-mutations in extracellular phage lambda by gamma-rays

Summary Mutagenic action of ⁶⁰Co -rays on extracellular phages red ⁺ and red1 13 after irradiation in 4% nutrient broth in the absence or in the presence of 0.1 M cysteamine or in dried samples was studied. The yield of c mutations was almost independent of the repair genotype of the host cells (uvrA6, polA1, recA13, lexA102, uvrE502, uvrD3 or xthA9), of the phage Red function and of the conditions of -irradiation and was 1·10^-12 per base pair and 1 rad. When the SOS-repair system of the host cells was induced by moderate UV irradiation, the yield of c-mutations was drastically enhanced in phage irradiated in broth, but not in phage irradiated in the dried state. These data allow us to suppose that the direct action of -rays induces, in phage DNA, premutational lesions that are fixed into mutations by replication. On the other hand after -irradiation in broth, when indirect radiation effects are only partially suppressed, about 85% of premutational lesions are converted into mutations by means of the inducible, errorprone SOS-repair system.     

β-Glucuronidase activity in human T and B lymphocytes and the Tμ and Tγ subpopulations

Summary The -glucuronidase staining characteristics of isolated T cell populations and the T and T enriched fractions derived of them were studied. T lymphocytes were obtained from purified T lymphocytes by ox-IgG rosette sedimentation. The rosette-forming cells in the pellet were referred to as T lymphocytes, whereas the lymphocytes in the interface were referred to as T depleted or T lymphocytes. B cells were studied on rosetted mononuclear cells with either mouse erythrocytes or with Staphylococcus Aureus (Cowan I) bacteria, after a preceeding polyvalent anti-human Ig treatment of the cells. While B cells showed mostly no reactivity, T and T cells were respectively characterised by a dot-like and granular pattern of reactivity. These findings are in agreement with those observed by others after -naphthyl-acetate esterase or acid phosphatase staining. Within the T lymphocyte fraction, the T non-, non lymphocytes seemed to have a granular pattern of reactivity. The same staining pattern was found in non-B, non-T lymphocytes.     

The human genes for the α and γ subunits of the mast cell receptor for immunoglobulin E are located on human chromosome band 1823

The receptor with high affinity for immunoglobulin E (FcERI) is a key molecule in triggering the allergic reaction. It is tetrameric complex of one subunit, one subunit, and two disulfide-linked subunits. This receptor is present exclusively on mast cells and basophils. Molecules identical to the subunit of FcRI also form cell surface complex with other Fc receptors such as mouse FcRIIa in macrophages and most probably with human FcRIII (CD16) in natural killer (NK) cells. Here we show by in situ hybridization that the human genes for the (FCER1A) and subunits (FCER1 G) of FcERI and the gene for FcRIII (FCGR3, CD16) are located on human chromosome band 1823.     

Immunocytochemical analysis of phosphatidylinositol-specific phospholipase C in PC12 cells: predominance of the δ isoform during neural differentiation

The rat pheochromocytoma PC12 cell line, which differentiates into sympathetic neurons under nerve growth factor (NGF) treatment, contains at least three phosphoinositidase C (PIC) isozymes, PIC , PIC , PIC . These isozymes have been previously shown to display a different subcellular localization. To determine whether or not NGF induces changes in the presence and/or distribution of PIC isozymes during PC12 neural differentiation, studies were carried out by means of in situ immunocytochemistry. After NGF administration the proliferative activity was progressively reduced to very low levels, as measured by bromodeoxyUridine incorporation, and a neuron-like morphology was displayed by almost all cells. In unstimulated PC12 cells, PIC was detected in the nucleus whereas PIC was only cytoplasmic; PIC was found in both cell compartments. In cells treated with NGF for 3 days, neural processes extended to twice the diameter of the cell body; the isoform was concentrated near the nucleus, while the immunoreactivity of the form remained constant and the form was increased. After 10 days of treatment with NGF, PIC was hardly detectable and PIC immunostaining was considerably decreased. On the contrary, PIC progressively increased and, after 14 days of NGF exposure, fully differentiated cells displayed an intense labelling of cell body and neurites. In the same cells, PIC and PIC were almost negative. These results suggest that NGF dependent neural differentiation is related to the selective down regulation of PIC and and the increase of PIC isozyme associated with the decrease of cell proliferation.     

Restriction fragment length polymorphisms associated with immunoglobulin heavy chain gamma genes in Tunisians

Summary DNA polymorphisms in the human immunoglobulin gamma () region have been studied in random Arabo-Berber Tunisians and in a large Tunisian Berber kindred. Haplotypes have then been designated, based on variation in the BamHI restriction fragments containing the C1, C2, C4, and C genes. Two new haplotypes, in addition to the four previously described, have been observed. These new haplotypes, designated H5 and H6, were confirmed by family studies. The H5 haplotype was associated with black African Gm haplotypes · (Gm^{1,17;..;5,6,11} and Gm^1,17;..;5,11) (Gm^{a,z;..;blc3bo} and Gm^a,z;..;blbo) and probably represents a common haplotype in the black population. The haplotype H6 may be derived from H5. One of 39 random Tunisians was homozygous for a multigene deletion. DNA polymorphisms of the C genes, in conjuction with Gm markers, provide highly variable genetic markers important for the characterization of human populations.     

Double staining of intracellular cytokine proteins and T-lymphocyte subsets. Evaluation of the method in blood and bronchoalveolar lavage fluid

An immunocytochemical staining method has been developed for simultaneous staining of both cell surface markers (CD4 and CD8) and intracellular cytokine proteins IFN-, IL-4 and IL-5. Cell surface molecules were visualized with alkaline phosphatase, which was developed by Fast Blue BB. Intracellular cytokine proteins were detected by amino-ethyl carbazole. We applied this technique to T cells from T-cell lines and T-cell clones, peripheral blood mononuclear cells and broncho-alveolar lavage fluid cells. Cells were used either unstimulated or stimulated for 4h with 1ng/ml PMA and 1g/ml ionomycin, which proved to be an optimal stimulus taking cytokine staining, cell recovery and cell viability into account. We studied peripheral blood mononuclear cells from healthy subjects and found that without in vitro stimulation on average 0.4% of the cells were IFN- positive cells. In unstimulated broncho-alveolar lavage fluid cells of the 2 allergic asthmatic subjects studied so far we found higher numbers of cytokine-positive cells (up to 22% of the lymphocytes being IL-4⁺ cells). By in vitro stimulation, the numbers of cytokine-positive peripheral blood mononuclear cells from the healthy subjects were increased to maximally 5% IFN-⁺ cells. In stimulated lavage fluid cells from allergic asthmatic subjects maximally 34% of the lymphocytes became IFN-⁺. We conclude that this method allows detection of intracellular cytokine proteins in both CD4⁺ and CD8⁺ T cells without the need for stimulating the cells in vitro. In vitro stimulation may change the cytokine profile detected.     

Comparative study of the γ-butyrolactone and pantolactone contents in cells and musts during vinification by three Saccharomyces cerevisiae races

Summary Changes in the -butyrolactone and pantolactone contents in yeast cells and musts during fermentation and subsequent flor veil formation of Sherry wines were studied. Saccharomyces cerevisiae race cerevisiae, S. cerevisiae race bayanus and S. cerevisiae race capensis were used. During the alcoholic fermentation (first 31 days), -butyrolactone contents in musts and yeast cells were similar for the three yeast races tested. In this period, pantolactone was excreted to the must by bayanus and capensis races, and it was not detected in cerevisiae race cells. During flor veil formation (31 to 134 days), bayanus and capensis races yield higher -butyrolactone and pantolactone contents than cerevisiae race in the wines. In the final wines, pantolactone contents were always lower than those of -butyrolactone.     

Activation of peritoneal macrophages in patients with gynecological malignancies by sizofiran and recombinant interferon-γ

We investigated the influence of the combined use of sizofiran, a-1,3-glucan and a recombinant interferon- (rIFN-) upon biological activities of peritoneal macrophages (M). The number of peritoneal M and the production of cytokines (interleukin-1, interferon- and tumor necrosis factor) was increased by the combined treatment. Fully activated peritoneal M based on the increased number of elongated pseudopods were observed by electromicroscope. Sizofiran seems to assure a sufficient supply of M to kill tumor cells in the peritoneal cavity and co-administered rIFN- seems to directly stimulate the accumulated M in addition to its direct cytotoxicity against tumor cells. This combination therapy may be a step to the prevention of the recurrence of gynecological malignancies including ovarian cancer, after a negative second-look laparotomy.Abbreviations rIFN- recombinant interferon- - IL-1 interleukin-1 - TNF tumor necrosis factor - SLL second look laparotomy     

RecA-ssDNA interaction: Induced strand cleavage by hydroxyl radical at a defined distance from the 5′ end

Summary Interaction of the RecA protein with single-stranded DNA (ssDNA) was analyzed by challenge with the hydroxyl radical, which can cleave the DNA backbone. We found that RecA protein induces cleavage by the radical at a defined distance from the 5 end. The cleavage was at the 11th nucleotide in many oligodeoxynucleotides. Cleavage may be intermittent since a second cleavage was induced at the 22nd or 21st site. This specific cleavage was observed under optimal conditions for filament formation, homologous pairing and strand exchange. Specificity in cleavage was, however, decreased by replacement of ATP by adenosine 5-(-thio)triphosphate (ATPS), replacement of RecA protein by a mutant (RecAl) protein, or an increase in Mg^2– concentration. We propose that RecA protein induces a special structural alteration, such as bending, perhaps sequentially, on ssDNA and that this altered site plays an important role in homologous pairing and strand exchange.Abbreviations ssDNA single-stranded DNA - dsDNA doublestranded DNA - ATPS adenosine 5-(-thio)triphosphate Deceased     

Concurrent effect of visible light on γ-particles,chitin synthetase,and encystment capacity in zoospores ofBlastocladiella emersonii

Summary Zoospores derived from ordinary colorless plants ofBlastocladiella emersonii grown under 240 W/cm² of visible light contain an average of ca. 5 -particles and numerous aggregates of cytoplasmic granules which resemble the -matrix, while spores from dark-grown plants contain ca. 12 -particles but none of the granules. Correspondingly, the amount of chitin synthetase associated with -particles is approximately proportional to the number of -particles in the two spore types. The foregoing light effects, known to be accompanied by an increased capacity for encystment, probably take place before sporogenesis. Conversely, zoospores derived from dark-grown plants do not encyst appreciably over an 8 h period unless they are illuminated, but the number of -particles/spore decreases from 12 to ca. 6 whether or not light is present. Arguments are presented for considering the latter disappearance of -particles as an essential ark reaction which precedes a photochemical reaction, both being needed for light-induced encystment.     

A fluorometric assay for γ-glutamyl transpeptidase: Demonstration of enzymatic activity in cultured cells of neural origin

A new fluorometric assay was developed for the measurement of -glutamyl transpeptidase (-GTP). The assay utilizes as a substrate the synthetic compound 7--glutamylamido-4-methyl coumarin which is cleaved by -GTP to yield the highly fluorescent product 7-amino-4-methyl coumarin. Optimal excitation and emission wavelengths for the assay are 345 nm and 470 nm, respectively, and the sensitivity of the assay is greatly enhanced by the high-pressure liquid chromatographic separation of the product from the substrate. The assay is minimally 25 times more sensitive than the conventional spectrophotometric assay and permits analysis of as little as 5000 cultured cells of neuronal and glial origin. Analysis of a variety of cultured cells of neuronal and glial origin with this assay suggests that -GTP is largely present in glia and to a lesser extent in neurons.     

ATP synthesis associated with the conversion of hexachlorocyclohexane related compounds

Clostridium rectum strain S-17 converts -1,2,3,4,5,6-hexachlorocyclohexane (HCH) related compounds to chlorobenzenes. The metabolites from -1,2,3,4,5,6-hexachlorocyclohexene and -1,3,4,5,6-pentachlorocyclohexene are identified as 1,2,4-trichlorobenzene and 1,4-dichlorobenzene, respectively. ATP synthesis, converting these chlorinated compounds, is observed in the cell suspension of C. rectum as indicated by luciferase-luciferin reaction and phosphorylation of ³²P-labeled phosphate. These observation lead to the conclusion that HCH and related compounds serve as artificial electron acceptors of the Stickland reaction, and therefore, the reductive dechlorination is associated with ATP synthesis.Abbreviations HCH -1,2,3,4,5,6-hexachlorocyclohexane - HCCH -1,2,3,4,5,6-hexachlorocyclohexene - PCCH -1,3,4,5,6-pentachlorocyclohexene - TCCH -3,4,5,6-tetrachlorocyclohexene - 1,2,4-TCB 1,2,4-trichlorobenzene - 1,4-DCB 1,4-dichlorobenzene - MCB monochlorobenzene - DTT 1,4-dithiothreitol - IAA monoiodoacetic acid     

Characterization of γ-glutamyl transpeptidase activity of cultured endothelial cells from porcine brain capillaries

Summary Endothelial cells were isolated with high viability (>93%) from porcine brain capillaries by Percoll gradient centrifugation after purely enzymatic digestion. Primary cultures were grown to confluent cell monolayers and quantitated for the activity of -glutamyl transpeptidase. The -glutamyl transpeptidase activity starts from a high enzymatic level, decreases with time in culture to about 15% of the initial value, and remains constant at this level after day 10 in culture. The activity progression depends on surface conditions. In the presence of collagen, an exponential decrease starts immediately after seeding, with a time constant of 70±10h. In the absence of collagen, -glutamyl transpeptidase activity first decreases on day 1 after plating, recovers to the initial value on day 2 and 3 and afterwards declines exponentially to a low and constant activity level. Ethanol added to the cell culture at a time when low constant activity is reached, reactivates the -glutamyl transpeptidase to 30% of the initial value.