Effect of cellular glutathione content on the induction of DNA double strand breaks by 25 MeV electrons

The effect of endogenous glutathione (GSH) on the induction of DNA double strand breaks (dsb) by 25 MeV electrons was investigated using stationary haploid yeast cells defective in gamma-glutamyl-cysteine-synthetase (gsh 1) containing less than 5 per cent of the normal GSH content. In gsh 1 cells the induction of dsb is increased by a factor of 1.5 under oxic and 1.8 under anoxic irradiation conditions: whereas the oxygen enhancement ratio was only slightly decreased (1.9) compared to wild-type cells (2.4).     

The effect of the anoxic radiosensitizing agent TAN on induction of revertants by gamma-rays and helium ions in Salmonella tester strains.

The modification effect of the anoxic radiosensitizer TAN on the mutagenesis in various Salmonella tester strains after gamma-ray and helium ion irradiation was studied. The oxygen enhancement ratios (OER) for all 3 strains on the lethal assay after gamma-irradiation are approximately equal to 2. The induction of reversions in TA98 and TA100 does not modify under anoxia. The value of OER on the mutagenic assay in TA102 equals 1.6. The OER after helium ion irradiation on the lethal and mutagenic assays was less than after gamma-irradiation. The mutagenesis in 3 strains after irradiation under anoxia is enhanced by TAN. The value of the TAN modification effect after gamma-irradiation increases from 2.1 +/- 0.2 for TA102 to 5.2 +/- 0.4 for TA100. However, the TAN influence on mutagenesis in TA100 after helium ion irradiation decreases to 3.1 +/- 0.3. We conclude that peculiarities of mutagenesis in various tester strains under anoxia with TAN can be explained by considering the nature of premutational DNA damages.     

Role of Thin Fimbriae in Adherence and Growth of Acinetobacter calcoaceticus RAG-1 on Hexadecane

Acinetobacter calcoaceticus RAG-1, a hydrocarbon-degrading bacterium which adheres avidly to hydrocarbons and other hydrophobic surfaces, possesses numerous thin fimbriae (ca. 3.5-nm diameter) on the cell surface. MR-481, a nonadherent mutant of RAG-1 which is unable to grow on hexadecane under conditions of limited emulsification and low initial cell density, lacks these fimbriae. Prolonged incubation of MR-481 in hexadecane medium enriched for partial adherence revertants. The reappearance of thin fimbriae was observed in all such revertant strains. RAG-1 cells and partial revertant strains were agglutinated in the presence of antibody, whereas MR-481 cells were not. Another mutant, AB15, which was previously isolated on the basis of its nonagglutinability in the presence of antibody, also lacked thin fimbriae and was conditionally nonadherent. Furthermore, strain AB15 was unable to grow on hexadecane medium. Adherence of RAG-1 cells to hexadecane was considerably reduced after shearing treatment. The material removed from the cell surface by shearing of RAG-1 and the partial revertant strains yielded a single antigenic band in RAG-1 and partial revertant strains, as observed by crossed immunoelectrophoresis. This band was absent in both fimbriae-less mutants, MR-481 and AB15. The data demonstrate that the thin fimbriae of RAG-1 (i) are a major factor in adherence to polystyrene and hydrocarbon, (ii) may be crucial in enabling growth of cells on hexadecane, and (iii) constitute the major cell surface agglutinogen.     

Free radicals induced by adriamycin-sensitive and adriamycin-resistant cells: a spin-trapping study

The radicals generated by adriamycin-sensitive (CHO-AB) and adriamycin-resistant (CHO-C5) Chinese hamster ovary cells as well as by adriamycin-sensitive and -resistant human breast cancer cells (MCF7-WT and MCF7-ADR) have been studied with spin-trapping and ESR spectroscopy. During anoxic exposure to adriamycin (ADR) both pairs of cell lines produced the broad ESR singlet characteristic of ADR semiquinone (AQ.). By use of tris(oxalato)chromate (CrOx) as an extracellular line-broadening agent, the distribution of AQ. between the intra- and extracellular compartments was studied. For cell densities of (1-3) X 10(7) cells/mL, CrOx eliminated most, though not all, of the ESR signal, indicating that the AQ. radicals freely diffuse and partition between the intra- and extracellular compartments proportionally to their respective volumes. Similar behavior was exhibited by all four cell lines studied. Upon introduction of oxygen to anoxic cells in the presence of the spin trap 5,5-dimethylpyrroline N-oxide (DMPO), the AQ. signal was replaced by that of the DMPO-OH spin adduct. Metal chelators such as desferrioxamine had no effect on DMPO-OH or AQ. formation. Superoxide dismutase, not catalase, totally eliminated the ESR signal, indicating that DMPO-OH produced by ADR-treated cells originates from superoxide rather than from .OH produced from H2O2. In the presence of CrOx, the DMPO-OH signal was not distinguishable from the background noise, thus excluding any contribution to the signal by intracellular spin adducts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tumor-associated neutrophils (TAN) develop pro-tumorigenic properties during tumor progression

The role and characteristics of tumor-associated neutrophils (TAN) in cancer are poorly defined. We have recently shown that TAN can have anti-tumorigenic (N1) or pro-tumorigenic (N2) functions. An interesting unanswered question is how the phenotype of TAN is influenced by the ongoing evolvement of tumor microenvironment. We therefore studied the phenotype and effects of TAN at different time points during tumor progression. We used two models of murine tumor cancer cell lines—Lewis lung carcinoma (LLC) and AB12 (mesothelioma). Neutrophils were studied at early and late stages and compared to each other and to neutrophils from bone marrow/periphery of naïve mice. Although there was no difference in the number of neutrophils entering the tumor, we found that at early stages of tumor development, neutrophils were almost exclusively at the periphery of the tumor. Only at later stages, neutrophils were also found scattered among the tumor cells. We further found that TAN from early tumors are more cytotoxic toward tumor cells and produce higher levels of TNF-α, NO and H₂O₂. In established tumors, these functions are down-regulated and TAN acquire a more pro-tumorigenic phenotype. In line with this phenotype, only depletion of neutrophils at later stages of tumor development inhibited tumor growth, possibly due to their central location in the tumor. Our work adds another important layer to the understanding of neutrophils in cancer by further characterizing the changes in TAN during time. Additional research on the functional role of TAN and differences between subsets of TAN is currently underway.     

Mutation W284L of the human delta opioid receptor reveals agonist specific receptor conformations for G protein activation

Intrinsic activities of different delta opioid agonists were determined in a [35S]GTPgammaS binding assay using cell membranes from Chinese hamster ovary (CHO) cells stably expressing the wild type (hDOR/CHO) or W284L mutant human delta opioid receptor (W284L/CHO). Agonist binding affinities were regulated more robustly by sodium and guanine nucleotide in W284L/CHO than in hDOR/ CHO cell membranes. The W284L mutation selectively reduced the affinity of SNC 80 while having moderate effect ((-) TAN 67) or no effect (DPDPE) on the affinities of other delta selective agonists. The mutation had opposite effects on the intrinsic activities of agonists belonging to different chemical classes. The effects of the mutation on agonist affinities and potencies were independent from its effects on the intrinsic activities of the agonists. Maximal stimulation of [35S]GTPgammaS binding by SNC 80 was 2-fold higher in W284L mutant cell membranes than in wild type hDOR/CHO cell membranes, despite lower receptor expression levels in the W284L/CHO cells. The binding affinity of SNC 80 however, was significantly reduced (15-fold and 30-fold in the absence or presence of sodium+GDP respectively) in W284L/CHO cell membranes relative to wild type hDOR/CHO membranes. Conversely, the Emax of (-)TAN 67 in the [35S]GTPgammaS binding assay was markedly reduced (0.6-fold of that of the wild type) with only a slight (6-fold) reduction in its binding affinity. The affinity and intrinsic activity of DPDPE on the other hand remained unchanged at the W284L mutant hDOR. The mutation had similar effects on the affinities potencies and intrinsic activities of (-)TAN 67 and SB 219825. The results indicate that delta opioid agonists of different chemical classes use specific conformations for G protein activation.     

Anabaena sp. strain PCC 7120 gene devH is required for synthesis of the heterocyst glycolipid layer

In response to deprivation for fixed nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 provides a microoxic intracellular environment for nitrogen fixation through the differentiation of semiregularly spaced vegetative cells into specialized cells called heterocysts. The devH gene is induced during heterocyst development and encodes a product with characteristics of a trans-acting regulatory protein. A devH mutant forms morphologically distinguishable heterocysts but is Fox(-), incapable of nitrogen fixation in the presence of oxygen. We demonstrate that rearrangements of nitrogen fixation genes take place normally in the devH mutant and that it is Fix(+), i.e., has nitrogenase activity under anoxic conditions. The Fox(-) phenotype was shown by ultrastructural studies to be associated with the absence of the glycolipid layer of the heterocyst envelope. The expression of glycolipid biosynthetic genes in the mutant is greatly reduced, and heterocyst glycolipids are undetectable.     

Effects of 3-aminobenzamide on the rejoining of DNA-strand breaks in mammalian cells exposed to methyl methanesulphonate; role of poly(ADP-ribose) polymerase

The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase, on DNA-repair processes has been investigated after treating V79 hamster cells with methyl methanesulphonate (MMS). Repair activity was observed as changes in DNA-strand break levels. MMS induces transient strand breaks, the level of which slowly decreases with time. Addition of 3AB leads to a rapid increase in the number of breaks. The level of breaks increases linearly with time until it suddenly levels off. Increasing the concentration of 3AB does not change the slope of this curve, but the steady-state level of breaks increases. The incision-rejoining kinetics indicates that 3AB induces a delay in the strand-break rejoining process. In the absence of 3AB the breaks have a lifetime of 1-2 min and this is increased by a factor of 5 in the presence of 5 mM 3AB.     

砂质潮间带自由生活海洋线虫对缺氧的响应——微型受控生态系研究

以青岛砂质潮间带自由生活海洋线虫为研究对象,建立微型受控生态系,研究缺氧对海洋线虫群落结构和垂直分布的影响,以及环境复氧后海洋线虫群落的恢复能力。研究结果显示,海洋线虫是耐低氧的小型底栖动物类群,可通过垂直迁移来耐受缺氧造成的不利条件。但是,海洋线虫通过主动迁出而耐受缺氧条件的特性具有种的区别。研究中Pseudosteineria sp1、Rhynchonema sp1等海洋线虫通过向有氧环境的主动迁移耐受缺氧条件;Thalassironus sp1却可通过自身耐受机制抵御缺氧条件,在缺氧生境中仍能保持较高的丰度。此外,研究结果显示,当表层海洋线虫暴露于缺氧环境时,其总丰度显著降低,种类组成发生改变。Pseudosteineria sp1对缺氧环境较为敏感,可暂时性地离开沉积物进入水层;而沉积物溶解氧恢复正常后,该种可以重新回到沉积物中。Daptonema sp1成熟个体及其幼龄个体对缺氧均具有较高的耐受性,是缺氧群落的绝对优势种。D.sp3则表现出对缺氧环境较高的敏感性。环境恢复正常,线虫群落丰度及多样性增加,Neochromadora sp1和Spilophorella sp1等具有机会种的特点,首先表现出丰度和繁殖能力的增加。但是线虫群落种类组成在受测时间内并未能完全恢复,群落结构的恢复需要更长的时间。     

Sensitization of neuronal cells to oxidative stress with mutated human alpha-synuclein

Linkage of alpha-synuclein (alpha-SN) mutations to familial Parkinson's disease (PD) and presence of alpha-SN as a major constituent of Lewy body in both sporadic and familial PD implicate alpha-SN abnormality in PD pathogenesis. Here we demonstrate that overexpression of wild-type or mutant alpha-SN does not cause any deleterious effect on the growth or continued propagation of transfected human cells, but overproduction of mutant alpha-SN heightens their sensitivity to menadione-induced oxidative injury. Such enhanced vulnerability is more pronounced in neuronal transfectants than in their nonneuronal counterparts and is associated with increased production of reactive oxygen species. The data suggest that mutated alpha-SN, especially with an alanine-to-proline substitution at residue 30, sensitizes neuronal cells to oxidative damage.     

A mutant in the ADH1 gene of Chlamydomonas reinhardtii elicits metabolic restructuring during anaerobiosis

The green alga Chlamydomonas reinhardtii has numerous genes encoding enzymes that function in fermentative pathways. Among these, the bifunctional alcohol/acetaldehyde dehydrogenase (ADH1), highly homologous to the Escherichia coli AdhE enzyme, is proposed to be a key component of fermentative metabolism. To investigate the physiological role of ADH1 in dark anoxic metabolism, a Chlamydomonas adh1 mutant was generated. We detected no ethanol synthesis in this mutant when it was placed under anoxia; the two other ADH homologs encoded on the Chlamydomonas genome do not appear to participate in ethanol production under our experimental conditions. Pyruvate formate lyase, acetate kinase, and hydrogenase protein levels were similar in wild-type cells and the adh1 mutant, while the mutant had significantly more pyruvate:ferredoxin oxidoreductase. Furthermore, a marked change in metabolite levels (in addition to ethanol) synthesized by the mutant under anoxic conditions was observed; formate levels were reduced, acetate levels were elevated, and the production of CO(2) was significantly reduced, but fermentative H(2) production was unchanged relative to wild-type cells. Of particular interest is the finding that the mutant accumulates high levels of extracellular glycerol, which requires NADH as a substrate for its synthesis. Lactate production is also increased slightly in the mutant relative to the control strain. These findings demonstrate a restructuring of fermentative metabolism in the adh1 mutant in a way that sustains the recycling (oxidation) of NADH and the survival of the mutant (similar to wild-type cell survival) during dark anoxic growth.     

Endonuclease III and endonuclease IV protect Escherichia coli from the lethal and mutagenic effects of near-UV irradiation.

In contrast to the DNA damage caused by far-UV (lambda < 290 nm), near-UV (290 < lambda < 400 nm) induced DNA damage is partially oxygen dependent, suggesting the involvement of reactive oxygen species. To test the hypothesis that enzymes that protect cells from oxidative DNA damage are also involved in preventing near-UV mediated DNA damage, isogenic strains deficient in one or more of exonuclease III (xthA), endonuclease IV (nfo), and endonuclease III (nth) were exposed to increasing levels of far-UV and near-UV. All strains, with the exception of the nth single mutant, were found to be hypersensitive to the lethal effects of near-UV relative to a wild-type strain. A triple mutant strain (nth nfo xthA) exhibited the greatest sensitivity to near-UV-mediated lethality. The triple mutant was more sensitive than the nfo xthA double mutant to the lethal effects of near-UV, but not far-UV. A forward mutation assay also revealed a significantly increased sensitivity for the triple mutant compared to the nfo xthA deficient strain in the presence of near-UV. However, the triple mutant was no more sensitive to the mutagenic effects of far-UV than a nfo xthA double mutant. These data suggest that exonuclease III, endonuclease IV, and endonuclease III are important in protection against near-UV-induced DNA damage.     

Dithiothreitol pretreatment and inducible repair in UV-irradiated Escherichia coli K12 cells

The UV radiation survival of several Escherichia coli K12 strains was measured after pretreatment of the cells with dithiothreitol (DTT). In DNA repair-competent cells (AB1157), UV survival was enhanced (ER = 1.2) after pretreating cells for 1.0 h using 10 mmol dm-3 DTT and then incubating the cells for 1.5 h in buffer before UV irradiation. Similar experiments using the excision repair mutant, AB1886uvrA6, or the recombination repair and SOS-deficient mutant, AB2462recA, strains did not show enhanced UV survival. None of the E. coli strains tested were protected against UV killing by simultaneous treatment with DTT (10 mmol dm-3). These results, and the fact that incubation in chloramphenicol removed the wild-type response in DTT-pretreated, UV-irradiated cells, suggest that the observed UV radioprotection was a result of inducible enzymatic repair processes such as recA-dependent repair. The proposed stimulus for inducible repair in these cells is DNA damage caused by intracellular hydroxyl radicals arising from thiol oxidation. The involvement of oxygen radicals in the induction pathway is supported by results that showed superoxide dismutase and catalase could inhibit a portion (one-third) of the inducible repair.     

The effect of nitroimidazole and nitroxyl radiosensitizers on the post-irradiation synthesis of DNA.

The modification of DNA damage by three radiosensitizing drugs, present during gamma-irradiation of hypoxic Chinese hamster cells, was investigated. Both 2-methyl-5-nitroimidazole-1-ethanol (metronidazole) and 1-(2-nitro-1-imidazole)-3-methoxy-2-propranol (Ro-07-0582) were found to cause large increases in the yield of DNA single-strand breaks (SSB); triacetoneamine-N-oxyl (TAN) was found to have only a small effect on SSB production. The three drugs tested did not inhibit the rejoining of SSB. A pulse label and chase procedure was used to examine post-irradiation DNA synthesis. TAN present during irradiation under hypoxia was found to cause interruptions in subsequent DNA synthesis. Metronidazole and Ro-07-0582 had no effect on post-irradiation DNA synthesis. In addition, the effects of pre- and post-irradiation exposure to TAN were investigated, since these treatments have shown increased cell-killing in survival studies. TAN pre- and post-treatments were found to have no significant effect on subsequent DNA synthesis.     

Constitutive inhibition of DNA degradation due to the enzyme RecBCD in the radiation-resistant Escherichia coli K-12 mutant Gam(r)444]

Exonucleolytic degradation of [3]H-labeled DNA was examined in partially purified fractions of lysates obtained from nonirradiated RecBCD enzyme-containing cells of Escherichia coli and in the radiation-resistant mutant Gamr444. The degradative activity was shown to be lowered in these cells to the same extent as in the recBC mutant. The efficiency of plating of the mutant phage T4 2-, DNA of which can be degraded by exonuclease V, was 400-fold higher on the strain Gamr444 than on the wild-type strain AB1157. This value was shown to be only twice as low as that on the recB mutant or on the strain AB1157 carrying plasmid pGam26 with a radiation-resistance allele gam26 cloned from mutant Gamr444. The data obtained confirmed the hypothesis that the Gamr444 mutant contains a constitutive inhibitor of exonucleolytic activity of the RecBCD enzyme in nonirradiated cells. This inhibitor was shown to be encoded by the gam26 allele that had previously been mapped at 56.8 min of the E. coli chromosome. A possible mechanism of the involvement of this inhibitor in enhanced radiation resistance of the mutant Gamr444 is considered.     

The donor side of photosystem II is impaired in a Cd-tolerant mutant strain of the unicellular green algaChlamydomonas reinhardtii

Growth of a cadmium-tolerant mutant strain of the unicellular green alga Chlamydomonas reinhardtii was found to be impaired under photoautrotrophic, but not under mixotrophic conditions. As compared to wild-type cells, oxygen evolution by the photoautotrophically grown mutant was considerably decreased and higher photon fluence rates were required both for light compensation of oxygen consumption/production and maximal oxygen evolution. The capability for oxygen production was decreased in Chlamydomonas reinhardtii cells when grown in the presence of acetate without aeration. Wild-type cells grown under these conditions showed a rather low but significant oxygen evolution immediately after transfer to photoautotrophic conditions. This residual oxygen production was completely suppressed in the presence of acetate, obviously due to acetate inhibition of the water-splitting complex. In the case of our cadmium-tolerant mutant strain, however, residual oxygen production was measured even in the presence of acetate. After removal of acetate, oxygen evolution by the cadmium-tolerant mutant strain was increased to higher rates than measured for wild-type cells, but considerably higher photon fluence rates were required both for light compensation of oxygen consumption/production and maximal oxygen evolution. The conclusion that the donor side of photosystem II is affected in our cadmium-tolerant mutant strain was further corroborated by a stronger decrease of the fluorescence level caused by hydroxylamine.     

Protection of nonmodified phageλ againstEcoK restriction mediated byrecA protein

A study was conducted to establish whether the EcoK-specific restriction, which is alleviated in E. coli cells after UV induction of the SOS response (Day 1977), is also alleviated under the influence of an increased level of recA protein without induction of other SOS functions. The host cells used were E. coli K-12, strain AB2497, and its derivatives; the nonmodified phage lambda was a mutant b2b5(vir). An increase of the recA protein level was induced using the plasmid pX02, which is a recombinant of pBR322 carrying the recA gene of E. coli. AB2497(pX02) cells were found to exhibit a lower level of restriction than those without plasmid. The results indicate that the recA protein protects phage DNA during the process of restriction. A further factor affecting restriction is the growth phase of the culture of the restricting host: cells in the late stationary phase exhibit lower restriction than those in the exponential phase of growth. By a combination of these two factors (presence of plasmid pX02 and stationary growth phase) one can reduce the restriction of nonmodified phage about 300 times.     

Gene responsible for protecting Escherichia coli from sodium dodecyl sulfate and toluidine blue plus light.

Escherichia coli cells were killed by visible light irradiation in the presence of the photosensitizing dye, toluidine blue. Two uvrB mutant strains of E. coli K-12 (AB1885 and N3-1) were much more sensitive than the isogenic uvrA and uvrC strains to treatment with toluidine blue plus light, suggesting that the uvrB+ gene product was involved in repair of DNA damage induced by the treatment. The uvrB+ gene cloned in a high- or low-copy-number plasmid was transformed into the uvrB strain (AB1885). Although all the transformants showed the same resistance as its wild-type strain (AB1157) to UV irradiation, they were as sensitive as AB1885 was to treatment with toluidine blue plus light. The two uvrB strains were more sensitive to sodium dodecyl sulfate than the other strains, suggesting that these strains had a defect in the cell surface. A sodium dodecyl sulfate-resistant revertant obtained from AB1885 was more resistant than AB1885 was to treatment with toluidine blue plus light. The two uvrB strains (AB1885 and N3-1) appear to have a defective gene (tentatively called dvl) different from uvrB. Its map position was around 7 min on the E. coli map.