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Abstract: Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2′-deoxyguanosine (8-Br-dG), 8-bromo-2′-deoxyadenosine (8-Br-dA), and 5-bromo-2′-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols α, κ, and η. When 8-Br-dG-modified template was used, pol α primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol κ also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol η, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation. [Copyright &y& Elsevier]

Abstract: Thiabendazole (TBZ) is a post-harvest fungicide commonly used on imported citrus fruits. We recently found that TBZ showed photomutagenicity with UVA-irradiation in the Ames test using plate incorporation method. In the present study, potential of DNA-damaging activity, mutagenicity, and clastogenicity were investigated by short pulse treatment for 10min with TBZ (50–400μg/ml) and UVA-irradiation (320–400nm, 250μW/cm2) in bacterial and human cells. UVA-irradiated TBZ caused DNA damage in Escherichia coli and human lymphoblastoid WTK1 cells assayed, respectively, by the umu-test and the single cell gel electrophoresis (comet) assay. In a modified Ames test using Salmonella typhimurium and E. coli, strong induction of −1 frameshift mutations as well as base-substitution mutations were detected. TBZ at 50–100μg/ml with UVA-irradiation significantly induced micronuclei in WTK1 cells in the in vitro cytochalasin-B micronucleus assay. Pulse treatment for 10min with TBZ alone did not show any genotoxicity. Although TBZ is a spindle poison that induces aneuploidy, we hypothesize that the photogenotoxicity of TBZ in the present study was produced by a different mechanism, probably by DNA adduct formation. We concluded that UVA-activated TBZ is genotoxic in bacterial and human cells in vitro. [Copyright &y& Elsevier]

While respiratory syncytial virus (RSV) is one of the most common pathogens in adults admitted to the ICU due to respiratory diseases, no reports regarding the occurrence rate of RSV infections in adults in Japan during the COVID-19 pandemic exist. We conducted this retrospective study to examine the exact occurrence rate of RSV infections in adults. We reviewed all patients (≥18 years) with any respiratory symptoms who received quantitative polymerase chain reaction (PCR) using nasopharyngeal samples for respiratory viruses by GeneLEAD at the Aichi Medical University Hospital between November 2022 and November 2023. A total of 541 adult patients who underwent PCR test were enrolled in this study. RSV was identified in 18 cases (3.3 %); 8 (1.5 %) upper and 10 (1.8 %) lower respiratory tract infections. Influenza A and SARS-CoV-2 were found in 10 (1.8 %) and 61 (11.3 %), respectively. Patients with RSV infections and COVID-19 had more comorbidities than those with Influenza virus infections. As for RSV-associated with lower respiratory tract infection cases, 10 developed acute respiratory failure, resulting in 1 fatal case due to pneumonia and 1 died of septic shock due to ileus. The 30-, 90-day mortality rates were 1 (6 %) and 2 (11 %) respectively. About 3 % of adults had RSV infections during the COVID-19 pandemic. The outcomes of RSV infections in adults were similar to those by COVID-19. Those with comorbidities should have a preventive method against RSV infections, the same as for COVID-19. [ABSTRACT FROM AUTHOR]

Presents information on a study which adopted an alternative strategy to examine the mutations affecting the ability of Escherichia coli umuD protein to participate in SOS mutagenesis. Materials and methods used on the study; Results and discussion.

Discusses a monocysteine approach for probing the structure and protein-protein interactions of the UmuD protein. Role in ultraviolet mutagenesis; Chemistry of thiol group in each derivative; Proposed model for the interactions of the homodimer.

Orthologs of Escherichia coli ygjD and yeaZ genes are highly conserved in various organisms. The genome of the radioresistant bacterium Deinococcus radiodurans possesses single orthologs of ygjD (DR_0382) and yeaZ (DR_0756). Complete loss of either one or both genes did not result in any significant changes in cell growth efficiency, indicating that both genes are not essential for cell viability in D. radiodurans, unlike the case with other species such as E. coli, Bacillus subtilis and Saccharomyces cerevisiae. Survival rates following DNA damage induced by hydrogen peroxide (HO), N-methyl- N′-nitro- N-nitrosoguanidine (MNNG), ultra violet (UV) radiation, γ-rays, cisplatin and mitomycin C (MMC) were compared among the wild-type strain and D. radiodurans ygjD/ yeaZ null mutants. Cell viability of the null mutants did not decrease following exposure to HO or MNNG. In addition, the reduction in cell viability following exposure to γ-rays, UV radiation or cisplatin was marginal in the null mutants compared to the wild-type strain. Interestingly, the null mutants exhibited high sensitivity to MMC, which mainly causes interstrand DNA cross-links. The sensitivity of the null mutants to MMC was restored to that of the wild type by transformation with plasmids expressing these genes. These results suggest that D. radiodurans ygjD and yeaZ genes are involved in DNA repair and play a role in the repair of DNA cross-links. [ABSTRACT FROM AUTHOR]

A cDNA encoding vitellogenin (VTG), a precursor of a major yolk protein, vitellin (VTN), was isolated from cladoceran crustacean Daphnia magna. The deduced amino acid sequence of DmagVTG1, the polypeptide encoded by the cDNA, contained a possible signal peptide sequence of 16 amino acid (aa) residues. The possible mature form of DmagVTG1 consists of 1985 aa residues with a calculated molecular mass of 223,070 Da. The large lipid transfer (LLT) module and a part of the von Willebrand factor D (VWD) module found in the aa sequences of VTGs of many other organisms are well conserved in DmagVTG1. Phylogenetic analysis suggested that the LLT module of DmagVTG1 is more closely related to those of insect VTGs than those of decapodan crustaceans. A unique feature of DmagVTG1 is that it has a superoxide dismutase (SOD)-like domain at its NH2-terminus. Antisera against the SOD-like domain, the NH2-terminal part of the VTG domain and the COOH-terminal part of the VTG domain, respectively, were prepared and used for analysis of D. magna yolk proteins. Six species (I to VI) of major protein complexes were found in D. magna parthenogenetic eggs isolated immediately after ovulation. Complexes IV and V were the most abundant. DmagVTG1 was a component of Complexes III, IV and V, and the most abundant polypeptide in D. magna eggs. The protein complexes underwent gradual proteolysis during development. One of the primary sites of cleavage was between the two successive Arg residues located at the 1454th and 1455th positions of DmagVTG1. [Copyright &y& Elsevier]

In insect Drosophila melanogaster , ventral midline cells are crucial to formation of the central nervous system (CNS) and have roles in the specification of ectodermal neuroblasts. Notably, midline cells also have more recently recognized roles in the formation of the higher crustacean Parhyale dorso-ventral axis. The single-minded is a master regulator of ventral midline cells and is required for these functions. Recently sim expression patterns have been reported in various arthropods. These results suggest that the midline precursors evolved from ventral neuroectoderm of common ancestor Mandibulata. However, sim function has been only analyzed in few organisms. To investigate whether these functions of sim , the gene encoding Single-minded, are conserved among insects and crustaceans, we examined the embryonic expression pattern of a lower crustacean Daphnia sim homolog ( dma sim ) and analyzed the function of dma sim during embryonic development. The Dma Sim protein was expressed in the ventral neuroectoderm (like in onychophoran and chelicerate) and midline (like in mandibulatan). In addition to this conserved ventral neuroectoderm and midline expression, Dma Sim was expressed outside the ventral midline; it was expressed in maxilla 2, presumptive shell glands, and other tissues. To investigate dma sim function, we used RNA interference (RNAi) to inhibit dma sim in Daphnia embryos. Embryos subjected to dma sim RNAi exhibited improper axon tract formation and abnormal limb and ventral development. Furthermore, RNAi-mediated knockdown of dma slit , a putative Dma Sim target gene, resulted in similar embryonic phenotypes. These results indicated that dma sim might be required for proper dma slit -mediated ventral development in addition to being required for a conserved role in the ventral midline. Our findings indicated that sim homologs might have provided different developmental functions to ventral midline cells during metazoan evolution. [ABSTRACT FROM AUTHOR]

Highlights: [•] Human cell lines that express a variant form of Pol κ (F171A) were established. [•] The cells exhibited lower mutation frequency toward benzo[a]pyrene DNA adducts. [•] Phenylalanine 171 is a molecular brake for the translesion DNA synthesis by Pol κ. [•] Pol κ is not well tuned to bypass benzo[a]pyrene DNA adducts. [•] The cognate substances for Pol κ may be lesions induced by endogenous mutagens. [Copyright &y& Elsevier]

Many chronic inflammatory conditions are associated with an increased risk of cancer development. At the site of inflammation, cellular DNA is damaged by hypochlorous acid (HOCl), a potent oxidant generated by myeloperoxidase. 8-Chloro-2′-deoxyguanosine (8-Cl-dG) is a major DNA adduct formed by HOCl and has been detected from the liver DNA and urine of rats administered lipopolysaccharide in an inflammation model. Thus, the 8-Cl-dG lesion may be associated with the carcinogenesis of inflamed tissues. In this study, we explored the miscoding properties of the 8-Cl-dG adduct generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotide containing a single 8-Cl-dG was prepared and used as a template in primer extension reactions catalysed by human pol α, ĸ or η. Primer extension reactions catalysed by pol α and ĸ in the presence of all four dNTPs were slightly retarded at the 8-Cl-dG site, while pol η readily bypassed the lesion. The fully extended products were analysed to quantify the miscoding frequency and specificity of 8-Cl-dG using two-phased polyacrylamide gel electrophoresis (PAGE). During the primer extension reaction in the presence of four dNTPs, pol ĸ promoted one-base deletion (6.4%), accompanied by the misincorporation of 2′-deoxyguanosine monophosphate (5.5%), dAMP (3.7%), and dTMP (3.5%) opposite the lesion. Pol α and η, on the other hand, exclusively incorporated dCMP opposite the lesion. The steady-state kinetic studies supported the results obtained from the two-phased PAGE assay. These results indicate that 8-Cl-dG is a mutagenic lesion; the miscoding frequency and specificity varies depending on the DNA polymerase used. Thus, HOCl-induced 8-Cl-dG adduct may be involved in inflammation-driven carcinogenesis. [ABSTRACT FROM PUBLISHER]

Silymarin, a natural flavonoid from the seeds of milk thistle, is used for chemoprevention against various cancers in clinical settings and in experimental models. To examine the chemopreventive mechanisms of silymarin against colon cancer, we investigated suppressive effects of silymarin against carcinogenicity and genotoxicity induced by 1,2-dimethylhydrazine (DMH) plus dextran sodium sulfate (DSS) in the colon of F344 gpt delta transgenic rats. Male gpt delta rats were given a single subcutaneous injection of 40 mg/kg DMH and followed by 1.5% DSS in drinking water for a week. They were fed diets containing silymarin for 4 weeks, starting 1 week before DMH injection and samples were collected at 4, 20 and 32 weeks after the DMH treatment. Silymarin at doses of 100 and 500 p.p.m. suppressed the tumor formation in a dose-dependent manner and the reduction was statistically significant. In the mutation assays, DMH plus DSS enhanced the gpt mutant frequency (MF) in the colon, and the silymarin treatments reduced the MFs by 20%. Silymarin also reduced the genotoxicity of DMH in a dose-dependent manner in bacterial mutation assay with Salmonella typhimurium YG7108, a sensitive strain to alkylating agents, and the maximum reduction was >80%. These results suggest that silymarin is chemopreventive against DMH/DSS-induced inflammation-associated colon carcinogenesis and silymarin might act as an antigenotoxic agent, in part. [ABSTRACT FROM AUTHOR]

Alkylation is a type of stress that is fatal to cells. However, cells have various responses to alkylation. Alkyltransferase-like (ATL) protein is a novel protein involved in the repair of alkylated DNA; however, its repair mechanism at the molecular level is unclear. DNA microarray analysis revealed that the upregulation of 71 genes because of treatment with an alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine was related to the presence of TTHA1564, the ATL protein from Thermus thermophilus HB8. Affinity chromatography showed a direct interaction of purified TTHA1564 with purified RNA polymerase holoenzyme. The amino acid sequence of TTHA1564 is homologous to that of the C-terminal domain of Ada protein, which acts as a transcriptional activator. These results suggest that TTHA1564 might act as a transcriptional regulator. The results of DNA microarray analysis also implied that the alkylating agent induced oxidation stress in addition to alkylation stress. [ABSTRACT FROM PUBLISHER]

Thermus thermophilus is an extremely thermophilic eubacterium that grows optimally at 70–75°C. It does not have a gene encoding O6-alkylguanine-DNA alkyltransferase (AGT) for the repair of O6-methylguanine (O6-meG), but it has a homologous gene atl encoding alkyltransferase-like (ATL) proteins in which the cysteine residue in the active site of the PCHR motif conserved in AGT is replaced by alanine (i.e. lack of methyltransferase activity). To investigate the role of ATL protein in the repair of O6-meG, we isolated atl deletion mutants and measured specific G:C→A:T transition mutations induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) by a His+ reversion system at the hisD3110 locus. MNNG caused an increased mutation frequency in the atl-deficient mutant but a significantly higher frequency increase in a uvrA mutant, which is deficient in nucleotide excision repair (NER), indicating that both ATL protein and NER played an important role in preventing G:C→A:T transitions. We observed no difference in MNNG sensitivity between the uvrA atl double mutant and the parent uvrA strain. Our results support a recently proposed repair model in which ATL protein acts as a sensor of O6-meG damage and recruits UvrA protein to repair the lesion via an NER system. In addition, the finding that the uvrA atl strain mutated with greater frequency than the single atl strain suggests that O6-meG is repaired by NER in the absence of ATL protein. We also discuss the possible association of a transcription-repair coupling factor in a transcription-coupled repair pathway and of MutS protein in a mismatch repair pathway with ATL/NER-mediated repair of O6-meG. [ABSTRACT FROM AUTHOR]

Abstract: Human cells possess multiple specialized DNA polymerases (Pols) that bypass a variety of DNA lesions which otherwise would block chromosome replication. Human polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N 2-deoxyguanine (BPDE-N 2 -dG) DNA adducts in an almost error-free manner. To better understand the relationship between the structural features in the active site and lesion bypass by Pol κ, we mutated codons corresponding to amino acids appearing close to the adducts in the active site, and compared bypass efficiencies. Remarkably, the substitution of alanine for phenylalanine 171 (F171), an amino acid conserved between Pol κ and its bacterial counterpart Escherichia coli DinB, enhanced the efficiencies of dCMP incorporation opposite (−)- and (+)-trans-anti-BPDE-N 2-dG 18-fold. This substitution affected neither the fidelity of TLS nor the efficiency of dCMP incorporation opposite normal guanine. This amino acid change also enhanced the binding affinity of Pol κ to template/primer DNA containing (−)-trans-anti-BPDE-N 2-dG. These results suggest that F171 functions as a molecular brake for TLS across BPDE-N 2-dG by Pol κ and that the F171A derivative of Pol κ bypasses these DNA lesions more actively than does the wild-type enzyme. [Copyright &y& Elsevier]

Human DNA is continuously damaged by exogenous and endogenous genotoxic insults. To counteract DNA damage and ensure the completion of DNA replication, cells possess specialized DNA polymerases (Pols) that bypass a variety of DNA lesions. Human DNA polymerase κ (hPolκ) is a member of the Y-family of DNA Pols and a direct counterpart of DinB in Escherichia coli. hPolκ is characterized by its ability to bypass several DNA adducts [e.g., benzo[a]pyrene diolepoxide-N²-deoxyguanine (BPDE-N²-dG) and thymine glycol] and efficiently extend primers with mismatches at the termini. hPolκ is structurally distinct from E. coli DinB in that it possesses an ~100-amino acid extension at the N-terminus. Here, we report that tyrosine 112 (Y112), the steric gate amino acid of hPolκ, which distinguishes dNTPs from rNTPs by sensing the 2'-hydroxy group of incoming nucleotides, plays a crucial role in extension reactions with mismatched primer termini. When Y112 was replaced with alanine, the amino acid change severely reduced the catalytic constant, i.e., kcat, of the extending mismatched primers and lowered the efficiency, i.e., kcat/Km, of this process by ~400-fold compared with that of the wild-type enzyme. In contrast, the amino acid replacement did not reduce the insertion efficiency of dCMP opposite BPDE-N²-dG in template DNA, nor did it affect the ability of hPolκ to bind strongly to template-primer DNA with BPDE-N²-dG/dCMP. We conclude that the steric gate of hPolκ is a major fidelity factor that regulates extension reactions from mismatched primer termini. [ABSTRACT FROM AUTHOR]

Abstract: Thermus thermophilus is an extremely thermophilic, aerobic, and gram-negative eubacterium that grows optimally at 70–75°C, pH 7.5. In extremely high temperature environment, DNA damages in cells occur at a much higher frequency in thermophiles than mesophiles such as E. coli. When temperature rises, the deamination of cytosine residues in double-strand DNA is expected to increase greatly. T. thermophilus HB27 has two putative uracil-DNA glycosylase genes (udgA and udgB). Expression level of udgA gene was 2–3 times higher than that of udgB at 70, 74, and 78°C when it was monitored by β-glucosidase reporter assay. We developed hisD 3110 , hisD 3113 , hisD 3115 , and hisD 174 marker allele that can specifically detect G:C→A:T, C:G→A:T, T:A→A:T, and A:T→G:C base-substitutions, respectively, by His+ reverse mutations. We then disrupted udgA and udgB by thermostable kanamycin-resistant gene (htk) or pyrE gene insertion in each hisD background, and their spontaneous His+ reversion frequencies were compared. A udgA,B double mutant showed a pronounced increase in G:C→A:T reversion frequency compared with each single udg mutant, udgA or udgB. Estimated mutation rates of the udgA,B mutant cultured at 60, 70, and 78°C were about 2, 12, and 117 His+/108/generation, respectively. At 70°C culture, increased ratio of the mutation rate compared with the udg + strain was 12-fold in udgA, 3-fold in udgB, and 56-fold in udgA,B mutant. On the other hand, no difference was observed in other mutations of C:G→A:T, T:A→A:T, and A:T→G:C between udgA,B double mutant and the parent udg + strain. The present results indicated that gene products of udgB as well as udgA functioned in vivo to remove uracil in DNA and prevent G:C→A:T transition mutations. [Copyright &y& Elsevier]

Abstract: cDNAs encoding a Daphnia magna homolog of aryl hydrocarbon receptor nuclear translocator (ARNT) were isolated and the structural and functional features as well as the expression pattern of their product, DmagARNT, were analyzed. Among the known bHLH-PAS proteins, the deduced amino acid sequences of DmagARNT showed the highest degree of identity to that of Drosophila ARNT (TGO). Expression of DmagARNT in ARNT-lacking mouse Hepa-c4 cells resulted in the compensation for the loss of hypoxia response, suggesting the formation of a dimer with mouse HIF-1α and that the resulting heterodimer binds to the hypoxia-responsive elements (HRE), leading to transcription of the downstream luciferase gene. Expression of D. magna ARNT was evident at the middle to late stages of embryonic development (about 25 h to 48 h after ovulation) in several tissues, including a pair of the 1st antenna, 2nd antenna, 2nd maxilla, five pairs of the thoracic limbs, the central nerve system, anus, dorsal organ, maxillary gland, and carapace. As observed in other species, the D. magna ARNT is likely to function broadly as an expressed dimerization partner in developmental processes. In contrast, expression of ARNT in adult D. magna was limited to the epipodites of thoracic limbs, suggesting that ARNT plays a role solely in hypoxia response in adult Daphnia. [Copyright &y& Elsevier]

Abstract: Two Daphnia magna vitellogenin (VTG) genes in neighboring but opposite orientations were identified. One was the gene for DmagVTG1, a previously characterized VTG polypeptide with a superoxide dismutase (SOD)-like domain at its NH2-terminus [Kato et al., Gene 334 (2004) 157–165]. Both genes had a 17-exon and 16-intron structure in the same configuration. DmagVTG2, a polypeptide encoded by the other gene, also had a SOD-like domain at its NH2-terminus. The amino acid sequences of the two VTG domains were highly homologous (95.5% identity), while those of the SOD-like domains were less homologous (62.4% identity). The VTG domains are phylogenetically related to insect VTGs while the SOD-like domains are related to viral and bacterial SODs. The intergenic region of 2.6kb between the two genes contains sequences resembling known juvenile hormone (JH)-responsive and ecdysone-responsive elements. JH agonists, pyriproxyfen and fenoxycarb, strongly repressed the expression of VTG genes in neonate daphnids. [Copyright &y& Elsevier]