Action of nitrofurans on E. coli: mutation and induction and repair of daughter-strand gaps in DNA.

The antibacterial and mutagenic potency of 9 nitrofurans in "treat and plate" experiments varied over almost 5 orders of magnitude. The relative toxicities were as follows: FANFT greater than AF2 greater than ANFT greather than furazolidone greater than furagin greater than nitrofurantoin greater than nitrofurazone greater than methylnitrofuroate greater than nitrofuroic acid. In general, mutagenic activity paralleled toxicity. The compounds at concentrations corresponding to their LD50's, induced mutations at frequencies which ranged from 2.5/10(6) survivors for FANFT to 130/10(6) survivors for furagin (NF416). The observed differences in antibacterial and mutagenic activity are unlikely to be due to lack of activation of the weaker agents since the two most potent agents were reduced somewhat more slowly than many of the less active agents. The relative sensitivities to the antibacterial effects of AF2 of strains WP2, WP2 uvrA, CM561 (lexA) and CM571 (recA) were 1 : 1.6 : 3 : 7 and to nitrofurazone 1 : 1 : 25 : 50. The wvrA strain was 6--7-fold more mutable with both these agents than was WP2. No increase over the spontaneous mutation frequency was observed when recA or lexA strains were exposed to either AF2 or nitrofurazone in these experiments. When wild-type of wvrA bacteria containing nitrofuran-induced lesions replicated their DNA in drug-free medium in the presence of [3H]thymidine for 5 min, the label was found in low molecular weight DNA indicating that daughter-strand gaps were formed. During subsequent incubation in nonradioactive medium the molecular weight of the DNA increased to the control value. A recA strain (which was very sensitive to the lethal effects of AF2 and nitrofurazone) lacked the ability to repair daughter-strand gaps caused by nitrofuran-induced lesions.     

Inhibition of deoxyribonucleic acid repair in Escherichia coli by caffeine and acriflavine after ultraviolet irradiation.

The effects of caffeine and acriflavine on cell survival, single-strand deoxyribonucleic acid break formation, and postreplication repair in Escherichia coli wild-type WP2 and WP2 uvrA strains after ultraviolet irradiation was studied. Caffeine (0.5 mg/ml) added before and immediately after ultraviolet irradiation inhibited single-strand deoxyribonucleic acid breakage in wild-type WP2 cells. Single-strand breaks, once formed, were no longer subject to repair inhibition by caffeine. At 0.5 to 2 mg/ml, caffeine did not affect postreplication repair in uvrA strains. These data are consistent with the survival data of both irradiated WP2 and uvrA strains in the presence and absence of caffeine. In unirradiated WP2 and uvrA strains, however, a high caffeine concentration (greater than 2 mg/ml) resulted in gradual reduction of colony-forming units. At a concentration insufficient to alter survival of unirradiated cells, acriflavine (2 microgram/ml) inhibited both single-strand deoxyribonucleic acid breakage and postreplication repair after ultraviolet irradiation. These data suggest that although the modes of action for both caffeine and acriflavine may be similar in the inhibition of single-strand deoxyribonucleic acid break formation, they differ in their mechanisms of action on postreplication repair.     

Effects of sodium arsenite on single-strand DNA break formation and post-replication repair in E. coli following UV irradiation

A non-lethal dose of sodium arsenite is found to inhibit the formation of single-strand DNA breaks in Escherichia coli WP2 wild-type and WP6 polA strains after UV irradiation. Inhibition of single-strand breakage follows a dose-dependent relationship with respect to increasing sodium arsenite concentration. ATP level in WP2 cells is decreased in the presence of sodium arsenite and therefore the inhibition of DNA break formation may be mediated through lowered ATP levels in the irradiated cells. In the presence of a non-lethal dose of sodium aresenite, post-replication repair in WP2 uvrA strains after UV irradiation is also inhibited.     

A comparative analysis of the time-dependent antiproliferative effects of daunorubicin and WP631.

Jurkat T lymphocytes were treated with daunorubicin and WP631, a daunorubicin-based DNA binding agent, in experiments aimed to analyze cellular uptake of these drugs and their effect on cell viability. WP631 was taken up more slowly than daunorubicin, but laser confocal microscopy and spectrofluorometric quantification showed that the drug accumulated in the cells. Despite the slow uptake rate, the antiproliferative capacity of WP631 (measured as IC50 after a 72-h continuous treatment) was greater than that of daunorubicin. The propensities of daunorubicin and WP631 to promote apoptosis were compared. Our results indicate that the major effect of WP631 was a G2/M arrest followed, after about 72 h of treatment, by polyploidy and mitotic (reproductive) death. In contrast, daunorubicin induced a rapid response with classic features of apoptosis.     

The superiority of hamster liver microsomal fraction for activating nitrosamines to mutagens in Salmonella typhimurium

46 chemicals of various classes and structures, including 30 known animal carcinogens, were evaluated for genotoxic effects using the Escherichia coli rec assay with strains WP2 (wild-type) and WP100 (uvrA- recA-) in qualitative and quantitative spot tests and in quantitative suspension tests. The rec assay detected 17 of 30 known carcinogens as genotoxic agents, including mitomycin C and diethylnitrosamine, both negative in the Salmonella/Ames test as utilized in these studies. The rec assay in conjunction with the Salmonella/Ames test detected 20 of 30 known carcinogens as genotoxic agents. Azo/aminoazo carcinogens showed little gentoxicity, and the aromatic amine 2-acetylaminofluorene was non-genotoxic in the rec assay. The rec assay was more effective than pol tests with E. coli strains W3110/p3478 and strains WP2/WP67. Effectiveness of the rec assay was related to the DNA repair-defective nature of the uvrA- recA- genotype of strain WP100.     

Biological effects of dyes on bacteria. VI. Mutation induction by acridine orange and methylene blue in the dark with special reference to Escherichia coli WP6 (polA1)

Acridine orange (AO) and methylene blue (MB) in the dark were shown to be weak to moderate mutagens (induction of resistance to T5 phage) in repair-deficient strains of Escherichia coli B/r. However, strain WP2 (wild-type) was not mutated by AO in the dark, in confirmation of earlier data. The presence of 2 microM AO reduced by 41% the spontaneous mutation rate in strain WP2, from 4.1 to 2.4 mutants/10(8) cells/generation. In the polymerase I-deficient strain WP6 (polA1), 2 microM AO increased the mutation rate in the dark 14-fold. We propose that both spontaneous and AO-induced mutagenesis in the absence of light occur at the site of semiconservative DNA replication. If the intercalation mechanism for the effects in the absence of light is valid, the wild-type strain (WP2) may be resistant to frameshift mutagenesis induced by intercalated compounds, while the polymerase I-deficient strain (WP6) may be highly suceptible to the presence of an intercalated dye such as AO at the DNA-replication fork. MB and AO likely act through different mechanisms since MB is only a moderate mutagen in strain WP6 and the other repair-deficient strains tested.     

Mutation induction by monochromatic 254-nm and 365-nm radiation in strains of Escherichia coli that differ in repair capability

Mutation to tryptophan independence after exposure to radiation at the monocrhomatic wavelengths of 254 and 365 nm was studied and compared in 7 strains of Escherichia coli B/r that differ in repair capability. Efficient mutation induction was obtained with both 254-nm and 365-nm radiation with strains WP2 (wild-type), WP2s (uvrA), WP6s (polA uvrA). Mutants were not induced at either wavelength in the lexA strain WP5 or the recA strains WP10 and WP100. These results support the induction of mutants with 365-nm radiation through the error-prone (SOS) pathway of postreplication repair. Log-log plots of tryptophan revertant data at 254 nm showed the expected slopes of approximately 2.0 over the entire influence range tested. In contrast, similar plots of revertant data at 365 nm were complex in all cases tested: at low fluence values (survival greater than 0.5) in all cases where reversion occurred the slopes were approximately 1.0, while at higher fluences (survival less than 0.5) the slopes of the log-log plots were approximately 3.0 with strains WP2s and WP6s, approximately 4.0 with strain WP6 and approximately 6.0 with strain WP2. Differential sensitivity of components of excision and postreplication repair systems to 365-nm radiation may account for the 2-part mutation curves obtained with uvr⁺ rec⁺ lex⁺ strains. It is proposed that efficient error-free repair of mutational lesions occurs at 365-nm fluences below 2–4×10⁵ J m²⁻; at greater 365-nm fluences, error-free excision repair may be selectively inhibited, forcing a greater fraction of mutational lesions to be processed by the error-prone component of the postreplication repair system. The similarity of the mutational responses of WP2s and WP6 at 365 nm supports the selective inhibition of error-free excision repair.     

Mutagenic characteristics of formaldehyde on bacterial systems

The mutagenic characteristics of formaldehyde on bacteria were examined. All the tester strains of Escherichia coli deficient in DNA-repair enzymes tested in the present study were significantly more sensitive to the killing effect of formaldehyde than the corresponding wild-type strain. Among the E. coli B strains, H/r30R (wild-type) and Hs30R (uvrA) were mutable, whereas NG30 (recA) and O16 (polA) were not. There is no appreciable difference in mutation frequency of E. coli B between the wild-type and the uvrA strains in a dose range below 4 mM. However, the mutation frequency of the wild-type strain started to decrease in a higher concentration range, whereas that of the uvrA strain continued to increase linearly. This was confirmed with the E. coli B/r tester strains. The decrease in mutation frequency may be produced by prolongation of the lag period before entering the S-phase so as to give the cells a greater chance for DNA repair through the excision mechanism. In fact, it was evidenced that formaldehyde retarded to a remarkable extent the initiation of DNA synthesis of the cells at the higher dose range used for mutation assay. Some discrepancies found between the results obtained in this study and those previously reported by Nishioka (1973) were pointed out.     

Variation in interferon sensitivity and induction among strains of eastern equine encephalitis virus

Eastern equine encephalitis virus (EEEV) causes human encephalitis in North America (NA), but in South America (SA) it has rarely been associated with human disease, suggesting that SA strains are less virulent. To evaluate the hypothesis that this virulence difference is due to a greater ability of NA strains to evade innate immunity, we compared replication of NA and SA strains in Vero cells pretreated with interferon (IFN). Human IFN-alpha, -beta, and -gamma generally exhibited less effect on replication of NA than SA strains, supporting this hypothesis. In the murine model, no consistent difference in IFN induction was observed between NA and SA strains. After infection with most EEEV strains, higher viremia levels and shorter survival times were observed in mice deficient in IFN-alpha/beta receptors than in wild-type mice, suggesting that IFN-alpha/beta is important in controlling replication. In contrast, IFN-gamma receptor-deficient mice infected with NA and SA strains had similar viremia levels and mortality rates to those of wild-type mice, suggesting that IFN-gamma does not play a major role in murine protection. Mice pretreated with poly(I-C), a nonspecific IFN inducer, exhibited dose-dependent protection against fatal eastern equine encephalitis, further evidence that IFN is important in controlling disease. Overall, our in vivo results did not support the hypothesis that NA strains are more virulent in humans due to their greater ability to counteract the IFN response. However, further studies using a better model of human disease are needed to confirm the results of differential human IFN sensitivity obtained in our in vitro experiments.     

Role of UV-inducible proteins in repair of various wild-type Escherichia coli cells

3 wild-type strains of E. coli, namely K12 AB2497, B/r WP2 and 15 555-7v proficient in excision and post-replication repair, differ markedly in their UV resistance. To elucidate this difference, the influence was investigated of induction by application of inducing fluence (IF) before lethal fluence (LF) on repair processes after LF. In cells distinguished by low UV resistance (E. coli 15 555-7; E. coli B/r WP2), dimer excision was less complete in cultures irradiated with IF + LF than in cultures irradiated with LF only. The highly resistant E. coli K12 AB2497 performed complete excision both after IF + LF or after LF alone. All 3 types of cell survived better after IF + LF than after LF only. Because, in most strains so far investigated, the application of IF reduced dimer excision and increased survival, dimer excision per se does not appear important for survival.We conclude that the rate and completeness of dimer excision can serve as a measure of efficiency of the excision system whose action is necessary for repair of another lesion. Cells of all investigated strains could not resume DNA replication and died progressively when irradiated with LF and post-incubated with chloramphenicol (LF CAP⁺). Thus, it appears that inducible proteins are necessary for repair in all wild-type E. coli cells give with potentially lethal doses of UV irradiation.     

Fusion-Related Host Proteins Are Actively Regulated by NA during Influenza Infection as Revealed by Quantitative Proteomics Analysis

Three recombinant influenza A viruses with different neuraminidases (NAs) in the background of A/PR/8/34 (PR8), named rPR8-H5N1NA, rPR8-H9N2NA, and rPR8-H1N1NA, derived from H5N1, H9N2, H1N1 (swine) viruses, respectively, were constructed. We performed a quantitative proteomics analysis to investigate differential protein expression in Madin-Darby canine kidney (MDCK) cells infected with recombinant and wild-type influenza viruses to determine whether NA replacement would alter host cell gene expression. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-TOF MS) and two-dimensional gel electrophoresis (2-DE), we identified 12 up-regulated and 49 down-regulated protein spots, including cytoskeletal proteins, molecular biosynthesis proteins, ubiquitin-proteasome pathway proteins, and heat shock proteins. The most significant changes in infected cells were observed for molecular biosynthesis proteins. We found more differentially expressed protein spots in cells infected with rPR8-H5N1NA or rPR8-H9N2NA viruses than cells infected with wild-type virus. Many of those proteins are postulated to be involved in cell-cell fusion, but the full mechanism remains to be explored. Meanwhile, our data demonstrate that the wild-type virus has evolutionary advantages over recombinant viruses.     

Evolutional analysis of human influenza A virus N2 neuraminidase genes based on the transition of the low-pH stability of sialidase activity

The 1957 and 1968 human pandemic influenza A virus strains as well as duck viruses possess sialidase activity under low-pH conditions, but human H3N2 strains isolated after 1968 do not possess such activity. We investigated the transition of avian (duck)-like low-pH stability of sialidase activities with the evolution of N2 neuraminidase (NA) genes in human influenza A virus strains. We found that the NA genes of H3N2 viruses isolated from 1971 to 1982 had evolved from the side branches of NA genes of H2N2 epidemic strains isolated in 1968 that were characterized by the low-pH-unstable sialidase activities, though the NA genes of the 1968 pandemic strains preserved the low-pH-stable sialidase. These findings suggest that the prototype of the H3N2 epidemic influenza strains isolated after 1968 probably acquired the NA gene from the H2N2 low-pH-unstable sialidase strain by second genetic reassortment in humans.     

Radioadaptive enhancement of the repair of UV-induced postreplication gaps in Escherichia coli cells deficient in DNA repair

In UV-irradiated E. coli WP2 uvrA, deficient in excision repair of DNA with pyrimidine dimers, gamma-irradiation in low doses (radioadaptation) before UV-irradiation leads to the intensification of postreplication repair of DNA. This process in WP2 uvrA polA and uvrA lexA mutants is less than in WP2 uvrA cells, but in WP2 uvrA recA both postreplication repair and its radioadaptive intensification are absent. In E. coli AB1157 excising pyrimidine dimers the radioadaptive intensification of postreplication repair of DNA is expressed almost to the same extent as in WP2 uvrA. In GW2100 umuC mutant, deficient in DNA polymerase V, postreplication repair of DNA is expressed, but its radioadaptive intensification is absent, while in AB2463 recA13 both postreplication repair of DNA and radioadaptive intensification of postreplication repair of DNA are absent. The above data suggest that DNA polymerase I and LexA protein are needed for radioadaptive intensification of postreplication repair of DNA in uvrA strain, and DNA polymerase V is needed for radioadaptive intensification in E. coli AB1157, and that RecA protein is required for postreplication repair and radioadaptive intensification of postreplication repair of DNA.     

Relationship among oxidative stress, growth cycle, and sporulation in Bacillus subtilis.

The sensitivity of Bacillus subtilis to hydrogen peroxide (oxidative stress) was found to vary with the position of the culture in the growth cycle. The most dramatic change occurred at the stationary phase, when the cells became totally resistant to 10 mM H2O2, in contrast to the loss of 3 to 4 log units of viability when treated at the early log phase. Two of the eight proteins induced by a protective concentration of H2O2 (50 muM) were also induced (in the absence of oxidative stress) on entry into the late log phase of growth. The response of five isogenic spo0 mutants (spo0B, spo0E, spo0F, spo0H, and spo0J) to oxidative stress was identical to that of the wild-type parental strain. In an isogenic spo0A strain, mid-log-phase cells were 100-fold less sensitive to 10 mM H2O2 than was the wild type. Pretreatment with 50 microM H2O2 induced little further protection, suggesting that the response is constitutive in this strain. By comparison of proteins induced by 50 microM H2O2 in the wild-type, spo0A, spo0H, and spo0J strains, four proteins were identified that may be essential for protection against lethal concentrations of H2O2. The presence of multiple copies of the spo0H gene in a spo0A background converted the stress phenotype of the spo0A mutant to that of the wild type but left the sporulation phenotype unaltered.     

Characterization of a temperature-sensitive influenza B virus mutant defective in neuraminidase.

ts5, a temperature-sensitive mutant of influenza B virus, belongs to one of seven recombination groups. When the mutant infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating and enzymatic activities were undetectable. However, viral protein synthesis and transport of hemagglutinin (HA) and neuraminidase (NA) to the cell surface were not affected. The NA was found as a monomer within cells even at 32 degrees C, in contrast to wild-type virus NA, existing mostly as an oligomer, but the mutant had oligomeric NA, like the wild-type virus. Its enzymatic activity was more thermolabile than that of wild-type virus. Despite the low yield, large aggregates of progeny virus particles were found to accumulate on the cell surface at the nonpermissive temperature, and these aggregates were broken by treatment with bacterial neuraminidase, with the concomitant appearance of hemagglutinating activity, suggesting that NA prevents the aggregation of progeny virus by removal of neuraminic acid from HA and cell receptor, allowing its release from the cells. Further treatment with trypsin resulted in the recovery of infectivity. When bacterial NA was added to the culture early in infection, many hemagglutinable infectious virus was produced. We also suggest that the removal of neuraminic acid from HA by NA is essential for the subsequent cleavage of HA by cellular protease. Nucleotide sequence analysis of RNA segment 6 revealed that ts5 encoded five amino acid changes in the NA molecule but not in NB.     

Impact of the H275Y and I223V Mutations in the Neuraminidase of the 2009 Pandemic Influenza Virus In Vitro and Evaluating Experimental Reproducibility

The 2009 pandemic H1N1 (H1N1pdm09) influenza virus is naturally susceptible to neuraminidase (NA) inhibitors, but mutations in the NA protein can cause oseltamivir resistance. The H275Y and I223V amino acid substitutions in the NA of the H1N1pdm09 influenza strain have been separately observed in patients exhibiting oseltamivir-resistance. Here, we apply mathematical modelling techniques to compare the fitness of the wild-type H1N1pdm09 strain relative to each of these two mutants. We find that both the H275Y and I223V mutations in the H1N1pdm09 background significantly lengthen the duration of the eclipse phase (by 2.5 h and 3.6 h, respectively), consistent with these NA mutations delaying the release of viral progeny from newly infected cells. Cells infected by H1N1pdm09 virus carrying the I223V mutation display a disadvantageous, shorter infectious lifespan (17 h shorter) than those infected with the wild-type or MUT-H275Y strains. In terms of compensating traits, the H275Y mutation in the H1N1pdm09 background results in increased virus infectiousness, as we reported previously, whereas the I223V exhibits none, leaving it overall less fit than both its wild-type counterpart and the MUT-H275Y strain. Using computer simulated competition experiments, we determine that in the presence of oseltamivir at doses even below standard therapy, both the MUT-H275Y and MUT-I223V dominate their wild-type counterpart in all aspects, and the MUT-H275Y outcompetes the MUT-I223V. The H275Y mutation should therefore be more commonly observed than the I223V mutation in circulating H1N1pdm09 strains, assuming both mutations have a similar impact or no significant impact on between-host transmission. We also show that mathematical modelling offers a relatively inexpensive and reliable means to quantify inter-experimental variability and assess the reproducibility of results.     

The inhibitory performance of flavonoid cyanidin-3-sambubiocide against H274Y mutation in H1N1 influenza virus

Oseltamivir (Tamiflu) is the most accepted antiviral drug that targets the neuraminidase (NA) protein to inhibit the viral release from the host cell. Few H1N1 influenza strains with the H274Y mutation creates drug resistance to oseltamivir. In this study, we report that flavonoid cyanidin-3-sambubiocide (C3S) compound acts as a potential inhibitor against H274Y mutation. The drug resistance mechanism and inhibitory activity of C3S and oseltamivir against wild-type (WT) and H274Y mutant-type (MT) have been studied and compared based on the results of molecular docking, molecular dynamics, and quantum chemical methods. Oseltamivir has been found less binding affinity with MT. C3S has more binding affinity with WT and MT proteins. From the dynamical study, the 150th loop of the MT protein has found more deformation than WT. A single H274Y mutation induces the conformational changes in the 150th loop which leads to produce more resistance to oseltamivir. The 150th cavity is more attractive target for C3S to stop the conformational changes in the MT, than 430th cavity of NA protein. The C3S is stabilized with MT by more number of hydrogen bonds than oseltamivir. The electrostatic interaction energy shows a stronger C3S binding with MT and this compound may be more effective against oseltamivir-resistant virus strains.     

Influenza Virus Neuraminidases with Reduced Enzymatic Activity That Avidly Bind Sialic Acid Receptors

Influenza virus neuraminidase (NA) cleaves off sialic acid from cellular receptors of hemagglutinin (HA) to enable progeny escape from infected cells. However, NA variants (D151G) of recent human H3N2 viruses have also been reported to bind receptors on red blood cells, but the nature of these receptors and the effect of the mutation on NA activity were not established. Here, we compare the functional and structural properties of a human H3N2 NA from A/Tanzania/205/2010 and its D151G mutant, which supports HA-independent receptor binding. While the wild-type NA efficiently cleaves sialic acid from both α2-6- and α2-3-linked glycans, the mutant exhibits much reduced enzymatic activity toward both types of sialosides. Conversely, while wild-type NA shows no detectable binding to sialosides, the D151G NA exhibits avid binding with broad specificity toward α2-3 sialosides. D151G NA binds the 3′ sialyllactosamine (3′-SLN) and 6′-SLN sialosides with equilibrium dissociation constant (K_D) values of 30.0 μM and 645 μM, respectively, which correspond to much higher affinities than the corresponding affinities (low mM) of HA to these glycans. Crystal structures of wild-type and mutant NAs reveal the structural basis for glycan binding in the active site by exclusively impairing the glycosidic bond hydrolysis step. The general significance of D151 among influenza virus NAs was further explored by introducing the D151G mutation into three N1 NAs and one N2 NA, which all exhibited reduced enzymatic activity and preferential binding to α2-3 sialosides. Since the enzymatic and binding activities of NAs are not routinely assessed, the potential for NA receptor binding to contribute to influenza virus biology may be underappreciated.