Epidemiological study of respiratory syncytial virus infection in adults during the pandemic of COVID-19.

INTRODUCTION: 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. PATIENTS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Carbonic anhydrase inhibitor induces otic hair cell apoptosis via an intrinsic pathway and ER stress in zebrafish larvae.

Carbonic anhydrase (CA) catalyzes reversible hydration of CO2 to HCO3 - to mediate pH and ion homeostasis. Some chemical pollutants have been reported to have inhibitory effects on fish CA. In this study, we investigated effects of a CA inhibitor ethoxyzolamide (EZA) on neuromasts development during zebrafish embryogenesis, since embryogenesis in aquatic organisms can be particularly sensitive to water pollution. EZA caused alteration of pH and calcium concentration and production of reactive oxygen species (ROS) in larvae, and induced apoptosis in hair cells especially in the otic neuromast, in which CA2 was distributed on the body surface. mRNA levels of apoptotic genes and caspase activities were increased by EZA, whereas anti-oxidants and apoptotic inhibitors, Bax, NF-κB, and p53 inhibitors significantly relieved the induction of hair cell death. Also, mRNA levels of Bip and CHOP, which are induced in response to ER stress, were upregulated by EZA, suggesting that EZA induces otic hair cell apoptosis via the intrinsic mitochondrial pathway and ER stress. Our results demonstrated an essential role of CA in neuromast development via maintenance of ion transport and pH, and that the CA, which is directly exposed to the ambient water, shows marked sensitivity to EZA.

Carbonic Anhydrase Inhibitors Induce Developmental Toxicity During Zebrafish Embryogenesis, Especially in the Inner Ear.

In vertebrates, carbonic anhydrases (CAs) play important roles in ion transport and pH regulation in many organs, including the eyes, kidneys, central nervous system, and inner ear. In aquatic organisms, the enzyme is inhibited by various chemicals present in the environment, such as heavy metals, pesticides, and pharmaceuticals. In this study, the effects of CA inhibitors, i.e., sulfonamides [ethoxyzolamide (EZA), acetazolamide (AZA), and dorzolamide (DZA)], on zebrafish embryogenesis were investigated. In embryos treated with the sulfonamides, abnormal development, such as smaller otoliths, an enlarged heart, an irregular pectoral fin, and aberrant swimming behavior, was observed. Especially, the development of otoliths and locomotor activity was severely affected by all the sulfonamides, and EZA was a consistently stronger inhibitor than AZA or DZA. In the embryos treated with EZA, inner ear hair cells containing several CA isoforms, which provide HCO3- to the endolymph for otolith calcification and maintain an appropriate pH there, were affected. Acridine orange/ethidium bromide staining indicated that the hair cell damage in the inner ear and pectral fin is due to apoptosis. Moreover, RNA measurement demonstrated that altered gene expression of cell cycle arrest- and apoptosis-related proteins p53, p21, p27, and Bcl-2 occurred even at 0.08 ppm with which normal development was observed. This finding suggests that a low concentration of EZA may affect embryogenesis via the apoptosis pathway. Thus, our findings demonstrated the importance of potential risk assessment of CA inhibition, especially regarding the formation of otoliths as a one of the most sensitive organs in embryogenesis.

[Measurement of Nicotine, Tobacco-Specific Nitrosamines, and Additives in the Filler and Gas from Japanese Brand Snuff].

OBJECTIVE: The World Health Organization (WHO) Framework Convention on Tobacco Control (FCTC) reported that understanding the use and impact of smokeless tobacco (SLT) products is complicated by product diversity. Many different SLT products with different characteristics are used worldwide. ZERO STYLE STIX(TM) (sold by Japan Tobacco Inc.) is a brand of snuff, a type of smokeless tobacco. Our objective was to determine the constituents of the gas from SLT and analyze the ingredients in tobacco fillers. METHODS: ZERO STYLE STIX smokeless tobacco was released in the Japanese market in 2010. Nicotine, menthol, and tobacco-specific nitrosamines in the smokeless tobacco fillers were determined by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS). The gaseous compounds were collected by a smoking machine using two smoking protocols, i.e., the ISO and Health Canada Intense methods. Nicotine and menthol in the gas were determined by GC/MS. RESULTS: Nicotine, menthol, and the total tobacco-specific nitrosamines were detected in the tobacco fillers. The level of menthol in the snuff was more than ten times that of nicotine. The determined levels of the two components of the gas from the snuff were higher when using the Health Canada Intense protocol than when using the ISO protocol. In addition, flavors other than menthol were emitted from the smokeless tobacco. CONCLUSION: The new type of snuff introduced in the Japanese market in 2010 contained added flavors, and was attractive smokeless tobacco. Flavors in tobacco products need to regulate on the basis of FCTC 9 and 10 in JAPAN.

Analysis of spatiotemporal expression and function of the single-minded homolog in the branchiopod crustacean Daphnia magna.

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.

In vivo evidence that phenylalanine 171 acts as a molecular brake for translesion DNA synthesis across benzo[a]pyrene DNA adducts by human DNA polymerase κ.

Humans possess multiple specialized DNA polymerases that continue DNA replication beyond a variety of DNA lesions. DNA polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) DNA adducts in an almost error-free manner. In the previous work, we changed the amino acids close to the adducts in the active site and examined the bypass efficiency. The substitution of alanine for phenylalanine 171 (F171A) enhanced by 18-fold in vitro, the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N(2)-dG. In the present study, we established human cell lines that express wild-type Pol κ (POLK+/-), F171A (POLK F171A/-) or lack expression of Pol κ (POLK-/-) to examine the in vivo significance. These cell lines were generated with Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, which has high efficiency for gene targeting. Mutations were analyzed with shuttle vectors having (-)- or (+)-trans-anti-BPDE-N(2)-dG in the supF gene. The frequencies of mutations were in the order of POLK-/->POLK+/->POLK F171A/- both in (-)- and (+)-trans-anti-BPDE-N(2)-dG. These results suggest that F171 may function as a molecular brake for bypass across BPDE-N(2)-dG by Pol κ and raise the possibility that the cognate substrates for Pol κ are not BP adducts in DNA but may be lesions in DNA induced by endogenous mutagens.

Ay allele promotes azoxymethane-induced colorectal carcinogenesis by macrophage migration in hyperlipidemic/diabetic KK mice.

The incidence of colorectal cancer has been increasing and is associated with obesity and diabetes. We have found that type 2 diabetes model KK-Ay/TaJcl (KK-Ay) mice develop tumors within a short period after treatment with azoxymethane (AOM). However, factors that contribute to the promotion of carcinogenesis have not been clarified. Therefore, we looked at the genetic background of KK-Ay, including two genetic characteristics of KK/TaJcl (KK) mice and C57BL/6J-Ham-Ay/+ (Ay) mice, compared with other non-obese and non-diabetic mouse strains C57BL/6J and ICR, and induced colorectal premalignant lesions, aberrant crypt foci (ACF), and tumors using AOM (150 µg/mouse/week for 4 weeks and 200 µg/mouse/week for 6 weeks, respectively). The mice with a diabetes feature, KK-Ay and KK, developed significantly more ACF, 67 and 61 per mouse, respectively, whereas ICR, Ay, and C57BL/6J mice developed 42, 24, and 18 ACF/mouse, respectively, at 17 weeks of age. Serum insulin and triglyceride levels in KK-Ay and KK mice were quite high compared with other non-diabetic mouse strains. Interestingly, KK-Ay mice developed more colorectal tumors (2.7 ± 2.3 tumor/mouse) than KK mice (1.2 ± 1.1 tumor/mouse) at 25 weeks of age, in spite of similar diabetic conditions. The colon cancers that developed in both KK-Ay and KK mice showed similar activation of ß-catenin signaling. However, mRNA levels of inflammatory factors related to the activation of macrophages were significantly higher in colorectal cancer of KK-Ay mice than in KK. These data indicate that factors such as insulin resistance and dyslipidemia observed in obese and diabetic patients could be involved in susceptibility to colorectal carcinogenesis. In addition, increase of tumor-associated macrophages may play important roles in the stages of promotion of colorectal cancer.

Miscoding properties of 8-chloro-2'-deoxyguanosine, a hypochlorous acid-induced DNA adduct, catalysed by human DNA polymerases.

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.

Deinococcus radiodurans YgjD and YeaZ are involved in the repair of DNA cross-links.

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 (H(2)O(2)), 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 H(2)O(2) 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.

Chemopreventive effects of silymarin against 1,2-dimethylhydrazine plus dextran sodium sulfate-induced inflammation-associated carcinogenicity and genotoxicity in the colon of gpt delta rats.

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.

An alkyltransferase-like protein from Thermus thermophilus HB8 affects the regulation of gene expression in alkylation response.

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.

Mutational specificities of brominated DNA adducts catalyzed by human DNA polymerases.

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.

Role of alkyltransferase-like (ATL) protein in repair of methylated DNA lesions in Thermus thermophilus.

Thermus thermophilus is an extremely thermophilic eubacterium that grows optimally at 70-75°C. It does not have a gene encoding O(6)-alkylguanine-DNA alkyltransferase (AGT) for the repair of O(6)-methylguanine (O(6)-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 O(6)-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 O(6)-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 O(6)-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 O(6)-meG.

Phenylalanine 171 is a molecular brake for translesion synthesis across benzo[a]pyrene-guanine adducts by human DNA polymerase kappa.

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.

Critical amino acids in human DNA polymerases eta and kappa involved in erroneous incorporation of oxidized nucleotides.

Oxidized DNA precursors can cause mutagenesis and carcinogenesis when they are incorporated into the genome. Some human Y-family DNA polymerases (Pols) can effectively incorporate 8-oxo-dGTP, an oxidized form of dGTP, into a position opposite a template dA. This inappropriate G:A pairing may lead to transversions of A to C. To gain insight into the mechanisms underlying erroneous nucleotide incorporation, we changed amino acids in human Poleta and Polkappa proteins that might modulate their specificity for incorporating 8-oxo-dGTP into DNA. We found that Arg61 in Poleta was crucial for erroneous nucleotide incorporation. When Arg61 was substituted with lysine (R61K), the ratio of pairing of dA to 8-oxo-dGTP compared to pairing of dC was reduced from 660:1 (wild-type Poleta) to 7 : 1 (R61K). Similarly, Tyr112 in Polkappa was crucial for erroneous nucleotide incorporation. When Tyr112 was substituted with alanine (Y112A), the ratio of pairing was reduced from 11: 1 (wild-type Polkappa) to almost 1: 1 (Y112A). Interestingly, substitution at the corresponding position in Poleta, i.e. Phe18 to alanine, did not alter the specificity. These results suggested that amino acids at distinct positions in the active sites of Poleta and Polkappa might enhance 8-oxo-dGTP to favor the syn conformation, and thus direct its misincorporation into DNA.

The steric gate amino acid tyrosine 112 is required for efficient mismatched-primer extension by human DNA polymerase kappa.

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 kappa (hPolkappa) is a member of the Y-family of DNA Pols and a direct counterpart of DinB in Escherichia coli. hPolkappa is characterized by its ability to bypass several DNA adducts [e.g., benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) and thymine glycol] and efficiently extend primers with mismatches at the termini. hPolkappa is structurally distinct from E. coli DinB in that it possesses an approximately 100-amino acid extension at the N-terminus. Here, we report that tyrosine 112 (Y112), the steric gate amino acid of hPolkappa, 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., k(cat), of the extending mismatched primers and lowered the efficiency, i.e., k(cat)/K(m), of this process by approximately 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-N(2)-dG in template DNA, nor did it affect the ability of hPolkappa to bind strongly to template-primer DNA with BPDE-N(2)-dG/dCMP. We conclude that the steric gate of hPolkappa is a major fidelity factor that regulates extension reactions from mismatched primer termini.

Mutagenesis of uracil-DNA glycosylase deficient mutants of the extremely thermophilic eubacterium Thermus thermophilus.

Thermus thermophilus is an extremely thermophilic, aerobic, and gram-negative eubacterium that grows optimally at 70-75 degrees 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 degrees C when it was monitored by beta-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 degrees C were about 2, 12, and 117 His(+)/10(8)/generation, respectively. At 70 degrees 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.

Mutagenicity of UV-irradiated maltol in Salmonella typhimurium.

We investigated the photomutagenicity of maltol (3-hydroxy-2-methyl-4H-pyran-4-one) in bacterial cells. Maltol has a caramel-butterscotch odour and is used as a food additive to impart flavour to bread and cakes. Unirradiated maltol was not mutagenic up to 5 mg/plate in the Ames test. When maltol was irradiated with either UVA (a black light, 320-400 nm, 230 microW/cm(2)) for 5-30 min or UVC (a germicidal lamp, 610 microW/cm(2)) for 3 min in sodium phosphate buffer (pH 7.4) prior to the exposure of bacterial cells, it was mutagenic to Salmonella typhimurium TA100, TA104 and TA97. Mutagenic activation of maltol by UVA-irradiation was more evident in neutral and alkaline conditions (pH 7.0-9.0) than in acidic conditions. On the other hand, photomutagenicity was not observed when maltol was irradiated with UVA in 100 mM NaCl solution or water. The mutagenic photoproduct was stable for at least 60 min after UVA-irradiation. However, addition of thiol compounds (cysteine or glutathione) to the UVA-irradiated maltol diminished the mutagenicity. Mutational spectrum analysis revealed that the predominant base-substitutions induced were G:C-->T:A transversions and G:C-->A:T transitions. An increase of 8-hydroxydeoxyguanosine formation in salmon sperm DNA exposed to maltol and UVA in vitro was detected by HPLC-ECD, but it was too small to explain the photomutagenicity. We are considering the formation of DNA adducts as the photomutagenic mechanism.

beta-Glucosidase as a reporter for the gene expression studies in Thermus thermophilus and constitutive expression of DNA repair genes.

Thermus thermophilus is an extremely thermophilic eubacterium that grows optimally at 70-75 degrees C. Because the frequency of DNA damage, such as deamination, depurination and single-strand breaks, increases as the temperature rises, the regulation of expression as well as the specificities and activities of T.thermophilus DNA repair systems are of particular interest. To study those systems, we developed a gene expression vector using the T.thermophilus beta-glucosidase gene (bgl) with host strain JOS9 (Deltabgl) derived from the T.thermophilus wild-type strain HB27. Since HB27 has two putative beta-galactosidase genes, the use of a single bgl gene as a reporter in combination with a Deltabgl host strain permits the study of gene expression against a low background level. We assayed Bgl activity with 2-nitrophenyl-beta-d-glucopyranoside as the substrate at 80 degrees C. We measured the expression of seven genes involved in DNA repair--three nucleotide excision repair genes (uvrA, uvrB and uvrC) and four recombinational repair genes (recA, ruvA, ruvB and ruvC). Expression levels of uvrA and uvrB were about three times those of uvrC, while those of ruvA, ruvB and ruvC were almost equal. Both ruvA and ruvC formed an operon with their adjacent 5'-upstream gene paaG and ftsQAZ, respectively. recA was transcribed as an operon of four genes, amt-cinA-ligT-recA. All seven DNA repair genes were expressed constitutively, and the DNA damaging agent mitomycin C did not increase their expression.

Tissue-specific expression of a bHLH-PAS protein homologous to ARNT during the development of crustacean Daphnia magna.

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-1alpha 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.