Search

A highly-selective three-dimensional high-performance liquid chromatographic (3D-HPLC) system was developed for the determination of serine (Ser), threonine (Thr) and allo-threonine (aThr) enantiomers in human plasma to screen the new biomarker of chronic kidney disease (CKD). d-Ser has been reported to be the candidate biomarker of CKD, however, multiple biomarkers are still required. Therefore, Ser analogs of hydroxy amino acids are the focus in the present study. For the sensitive analysis, the amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and detected by their fluorescence. The 3D-HPLC system consisted of a reversed-phase column (Singularity RP18, 1.0 × 250 mm), an anion-exchange column (Singularity AX, 1.0 × 150 mm) and a Pirkle-type chiral stationary phase (Singularity CSP-013S, 1.5 × 250 mm). The developed method was validated and applied to the human plasma samples obtained from 15 healthy volunteers and 165 CKD patients. The concentrations of the d-forms were 1.13-2.26 (Ser), 0.01-0.03 (Thr) and 0.04-0.10 μM (aThr) for the healthy volunteers and 0.95-19.0 (Ser), 0-0.57 (Thr) and 0.04-1.02 μM (aThr) for the CKD patients. The concentrations and the %d values of all the target d-amino acids were increased along with the decreasing of renal function and further investigation for clinical applications are expected., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Coastal seagrass meadows are essential in blue carbon and aquatic ecosystem services. However, this ecosystem has suffered severe eutrophication and destruction due to the expansion of aquaculture. Therefore, methods for the flourishing of seagrass are still being explored. Here, data from 49 public coastal surveys on the distribution of seagrass and seaweed around the onshore aquaculture facilities are revalidated, and an exceptional area where the seagrass Zostera marina thrives was found near the shore downstream of the onshore aquaculture facility. To evaluate the characteristics of the sediment for growing seagrass, physicochemical properties and bacterial ecological evaluations of the sediment were conducted. Evaluation of chemical properties in seagrass sediments confirmed a significant increase in total carbon and a decrease in zinc content. Association analysis and linear discriminant analysis refined bacterial candidates specified in seagrass overgrown- and nonovergrown-sediment. Energy landscape analysis indicated that the symbiotic bacterial groups of seagrass sediment were strongly affected by the distance close to the seagrass-growing aquaculture facility despite their bacterial population appearing to fluctuate seasonally. The bacterial population there showed an apparent decrease in the pathogen candidates belonging to the order Flavobacteriales. Moreover, structure equation modeling and a linear non-Gaussian acyclic model based on the machine learning data estimated an optimal sediment symbiotic bacterial group candidate for seagrass growth as follows: the Lachnospiraceae and Ruminococcaceae families as gut-inhabitant bacteria, Rhodobacteraceae as photosynthetic bacteria, and Desulfobulbaceae as cable bacteria modulating oxygen or nitrate reduction and oxidation of sulfide. These observations confer a novel perspective on the sediment symbiotic bacterial structures critical for blue carbon and low-pathogenic marine ecosystems in aquaculture., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: Although essential oils exhibit antimicrobial properties, their application is limited, owing to their strong volatility and poor water solubility. Emulsification is a valid strategy for improving chemical stability. In this study, we prepared a mustard oil (MO) emulsion with egg yolk lecithin and evaluated its antimicrobial activity against Listeria monocytogenes in vitro and in cheese curd. The particle size of the MO emulsion was approximately 0.19 μm and remained stable for 30 days of storage. The MO emulsion showed strong antimicrobial activity against L. monocytogenes in vitro. Moreover, 40 ppm of MO was sufficient to inhibit the growth of L. monocytogenes in culture, and the addition of 160 ppm of MO decreased the population of L. monocytogenes. When 50 ppm of emulsified MO was added to milk during cheese curd production and it was stored at 10°C for 10 days, the growth of L. monocytogenes was suppressed. When the cheese curd with MO emulsion was stored at 4°C, the bacterial count was significantly decreased (P < 0.05), and no bacterial growth was observed after 14 days of storage. Furthermore, the sensory characteristics of cheese curd with the MO emulsion were acceptable. These results indicate MO emulsions may be useful in controlling the growth of L. monocytogenes in fresh cheese., (Copyright ©, International Association for Food Protection.)

Bird death is often caused by renal lesions induced by chemicals. The avian kidney has a renal portal system with significant blood flow that is sensitive to many chemicals. However, early avian biomarkers for kidney injury are yet to be identified. This study aimed to identify novel renal biomarkers. Acute kidney injury (AKI) can be divided into acute interstitial nephritis (AIN) and acute tubular necrosis (ATN). A chicken model of kidney damage was created by an injection of diclofenac or cisplatin, which caused either AIN or ATN, respectively. Microarray analysis was performed to profile the gene expression patterns in the chickens with nephropathy. A gene enrichment analysis suggested that the genes related to responses to external stimuli showed expression changes in both AIN and ATN. However, hierarchical clustering analyses suggested that gene expression patterns differed between AIN and ATN, and the number of biomarkers relating to renal damage was low. To identify early biomarkers for nephropathy, we focused on genes that were induced at various levels of renal damage. The gene, vanin-1 (VNN1) was highly induced in the early stages of renal damage. A quantitative real-time PCR analysis supported this finding. These results suggest VNN1 could be a useful early biomarker of kidney injury in avian species., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Effective biological utilization of wood biomass is necessary worldwide. Since several insect larvae can use wood biomass as a nutrient source, studies on their digestive microbial structures are expected to reveal a novel rule underlying wood biomass processing. Here, structural inferences for inhabitant bacteria involved in carbon and nitrogen metabolism for beetle larvae, an insect model, were performed to explore the potential rules. Bacterial analysis of larval feces showed enrichment of the phyla Chroloflexi, Gemmatimonadetes, and Planctomycetes, and the genera Bradyrhizobium, Chonella, Corallococcus, Gemmata, Hyphomicrobium, Lutibacterium, Paenibacillus, and Rhodoplanes, as bacteria potential involved in plant growth promotion, nitrogen cycle modulation, and/or environmental protection. The fecal abundances of these bacteria were not necessarily positively correlated with their abundances in the habitat, indicating that they were selectively enriched in the feces of the larvae. Correlation and association analyses predicted that common fecal bacteria might affect carbon and nitrogen metabolism. Based on these hypotheses, structural equation modeling (SEM) statistically estimated that inhabitant bacterial groups involved in carbon and nitrogen metabolism were composed of the phylum Gemmatimonadetes and Planctomycetes, and the genera Bradyrhizobium, Corallococcus, Gemmata, and Paenibacillus, which were among the fecal-enriched bacteria. Nevertheless, the selected common bacteria, i.e., the phyla Acidobacteria, Armatimonadetes, and Bacteroidetes and the genera Candidatus Solibacter, Devosia, Fimbriimonas, Gemmatimonas Opitutus, Sphingobium, and Methanobacterium, were necessary to obtain good fit indices in the SEM. In addition, the composition of the bacterial groups differed depending upon metabolic targets, carbon and nitrogen, and their stable isotopes, δ 13 C and δ 15 N, respectively. Thus, the statistically derived causal structural models highlighted that the larval fecal-enriched bacteria and common symbiotic bacteria might selectively play a role in wood biomass carbon and nitrogen metabolism. This information could confer a new perspective that helps us use wood biomass more efficiently and might stimulate innovation in environmental industries in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A two-dimensional (2D) HPLC system focusing on the determination of phenylalanine (Phe) enantiomers in mammalian physiological fluids has been developed. ᴅ-Phe is indicated to have potential values as a disease biomarker and therapeutic molecule in several neuronal and metabolic disorders, thus the regulation of ᴅ-Phe in mammals is a matter of interest. However, the precise determination of amino acid enantiomers is difficult in complex biological samples, and the development of an analytical method with practically acceptable sensitivity, selectivity and throughput is expected. In the present study, a 2D-HPLC system equipped with a reversed-phase column in the 1st dimension and an enantioselective column in the 2nd dimension has been designed, following the fluorescence derivatization of the target amino acid enantiomers with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The analytical method was validated using both plasma and urine samples, and successfully applied to human, rat and mouse fluids. Trace levels of ᴅ-Phe were determined in the plasma, and the %ᴅ values were around 0.1% for all species. In the urine, relatively large amounts of ᴅ-Phe were observed, and the %ᴅ values for humans, rats and mice were 3.99, 1.76 and 5.25%, respectively. The relationships between the enzymatic activity of ᴅ-amino acid oxidase (DAO) and the amounts of intrinsic ᴅ-Phe have also been clarified, and high ᴅ-Phe amounts were observed (around 0.3% in the plasma and around 50% in the urine) in the DAO deficient rats and mice., (Copyright © 2020 Elsevier B.V. All rights reserved.)

d-Amino acids, enantiomers of l-amino acids, are increasingly recognized as physiologically active molecules as well as potential biomarkers for diseases. d-Amino acid oxidase (DAO) catalyzes the oxidative deamination of d-amino acids and is present in a wide variety of organisms from yeasts to humans. Previous studies indicated that LEA rats lacked DAO activity, and levels of d-Ser and d-Ala were markedly increased in their tissues, suggesting a mutated locus responsible for the lack of Dao activity (ldao) existed in the LEA genome. Sequence analysis identified deletion breakpoints located in intron 4-5 of the Dao gene and intron 1-2 of the Svop gene, resulting in a 54.1-kb deletion which encompassed exons 5-12 of the Dao gene and exons 2-16 of the Svop gene. We developed a novel congenic rat strain, F344-Dao ldao , harboring the Dao ldao mutation from LEA rats delivered onto the F344 genetic background. Compared to the parental F344 strain, in F344-Dao ldao rats d-Ala was markedly increased in both cerebrum and cerebellum, while d-Ser content was increased in cerebellum but not cerebrum. d-Ala, d-Ser, d-Pro and d-Leu levels were also elevated in F344-Dao ldao plasma. F344-Dao ldao rats represent a novel model system that will aid in elucidating the physiological functions of d-amino acids in vivo. (203 words)., Competing Interests: Declaration of Competing Interest There authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: METastasis Reporting and Data System for Prostate Cancer (MET-RADS-P) has been proposed as a standard of data acquisition and interpretation for whole-body diffusion-weighted magnetic resonance imaging (WB-DWI) performed in men with advanced prostate cancer. The aim of this study is to demonstrate the clinical significance of the scores in castration-resistant prostate cancer (CRPC)., Materials and Methods: We retrospectively evaluated WB-DWI obtained from 72 patients with CRPC between 2014 and 2017, when disease progression was suspected at the time of starting a new line of anticancer therapy. Twenty-five (35%) and 30 (42%) patients had a treatment history that included taxane-based chemotherapy and new hormonal drugs, respectively., Results: Active bone metastases were identified in 60 patients (83%; number of bone metastasis = 0, 1-2, 3-5, 6-10, and > 10: n = 12 [17%], 20 [28%], 11 [15%], 1 [1%], and 28 [39%], respectively). Progressive lymph node and visceral metastases were identified in 10 (14%) and 4 (6%), respectively. During the median follow-up period of 24 months, 36 (50%) died of prostate cancer. Cancer-specific survival (CSS) was significantly stratified according to the MET-RADS-P scores of osseous metastatic burden and the presence of visceral metastasis (P < .0001). Multivariate analysis revealed that high osseous metastatic burden (> 10) and the presence of visceral metastasis were significant indicators of shorter CSS (P = .0036 and P = .0017, respectively)., Conclusions: The extent of bone metastasis and the presence of visceral metastasis on WB-DWI were associated with a shorter CSS in CRPC. MET-RADS-P score can be a prognostic imaging biomarker for CRPC., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Engulfment and cell motility (ELMO) proteins bind to Dock180, a guanine nucleotide exchange factor (GEF) of the Rac family, and regulate GEF activity. The resultant ELMO/Dock180/Rac module regulates cytoskeletal reorganization responsible for the engulfment of apoptotic cells, cell migration, and neurite extension. The expression and function of Elmo family proteins in the nervous system, however, are not yet fully understood. Here, we characterize the comparative gene expression profiles of three Elmo family members (Elmo1, Elmo2, and Elmo3) in the brain of C57BL/6J mice, a widely used inbred strain, together with reeler mutant mice to understand gene expression in normal laminated brain areas compared with abnormal areas. Although all three Elmo genes showed widespread mRNA expression over various mouse tissues tested, Elmo1 and Elmo2 were the major types expressed in the brain, and three Elmo genes were up-regulated between the first postnatal week (infant stage) and the third postnatal week (juvenile, weaning stage). In addition, the mRNAs of Elmo genes showed distinct distribution patterns in various brain areas and cell-types; such as neurons including inhibitory interneurons as well as some non-neuronal cells. In the cerebral cortex, the three Elmo genes were widely expressed over many cortical regions, but the predominant areas of Elmo1 and Elmo2 expression tended to be distributed unevenly in the deep (a lower part of the VI) and superficial (II/III) layers, respectively, which also changed depending on the cortical areas and postnatal stages. In the dentate gyrus of the hippocampus, Elmo2 was expressed in dentate granule cells more in the mature stage rather than the immature-differentiating stage. In the thalamus, Elmo1 but not the other members was highly expressed in many nuclei. In the medial habenula, Elmo2 and Elmo3 were expressed at intermediate levels. In the cerebellar cortex, Elmo1 and Elmo2 were expressed in differentiating-mature granule cells and mature granule cells, respectively. In the Purkinje cell layer, Elmo1 and Elmo2 were expressed in Purkinje cells and Bergmann glia, respectively. Disturbed cellular distributions and laminar structures caused by the reeler mutation did not severely change expression in these cell types despite the disturbed cellular distributions and laminar structures, including those of the cerebrum, hippocampus, and cerebellum. Taken together, these results suggested that these three Elmo family members share their functional roles in various brain regions during prenatal-postnatal development., (Copyright © 2019 Elsevier B.V. All rights reserved.)

An automated two-dimensional HPLC-MS/MS system was designed and developed for the highly selective determination of trace levels of d-amino acids. As the targets, frequently observed ones in mammalian physiological fluids, Ala, Asp, Glu, Leu, Pro and Ser, were selected because these d-amino acids are the potential biomarkers for the early and sensitive diagnoses of various diseases. The target analytes were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), then isolated by a reversed-phase column in the first dimension. The NBD-amino acid fractions were automatically collected into the multi-loop device, and introduced into the enantioselective column in the second dimension to separate the d- and l-forms followed by detection by an MS/MS. The obtained resolution values of the enantiomers were 1.87-5.17 and the calibration lines, precision and accuracy were practically sufficient. By using the present 2D HPLC-MS/MS system, trace levels of d-amino acids in complex biological matrices were determined without disturbance by the intrinsic interfering compounds, and successfully applied to the analysis of the human clinical samples (plasma and urine)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Avian species have a unique renal structure and abundant blood flow into the kidneys. Although many birds die due to nephrotoxicity caused by chemicals, there are no early biomarkers for renal lesions. Uric acid level in blood, which is generally used as a renal biomarker, is altered when the kidney function is damaged by over 70%. Therefore, early biomarkers for kidney injury in birds are needed. In humans, glycomics has been at the forefront of biological and medical sciences, and glycans are used as biomarkers of diseases, such as carcinoma. In this study, a glycomics approach was used to screen for renal biomarkers in chicken. First, a chicken model of kidney damage was generated by injection of diclofenac or cisplatin, which cause acute interstitial nephritis (AIN) and acute tubular necrosis (ATN), respectively. The nephrotoxicity levels were determined by a blood chemical test and histopathological analysis. The plasma N-glycans were then analyzed to discover renal biomarkers in birds. Levels of 14 glycans increased between pre- and post administration in kidney-damaged chickens in the diclofenac group, and some of these glycans had the same presumptive composition as those in human renal carcinoma patients. Glycan levels did not change remarkably in the cisplatin group. It is possible that there are changes in glycan expression due to AIN, but they do not reflect ATN. Although further research is needed in other species of birds, glycans are potentially useful biomarkers for AIN in avian species.

There are currently very few English reports about Japanese pediatric human immunodeficiency virus (HIV). In this study, we introduce our experience with pediatric HIV in a single hospital, and review the present status of HIV infections in children in Japan. In Japan, the main infection routes of HIV include sexual activity, mother-to-child transmission (MTCT), blood or blood product transfusion, and drug use. Most pediatric HIV patients have been infected by MTCT in recent years. One survey showed that in Japan, 52 babies were infected by MTCT between 1984 and 2011. Only 2 cases of pediatric HIV infection have been reported since 2010. The MTCT rate has decreased to 0.5% owing to several preventive interventions. In addition, the HIV antibody test is now performed in more than 98.3% of pregnant women in Japan., (Copyright © 2014. Published by Elsevier Ltd.)

DS-8587 is a novel broad-spectrum fluoroquinolone with extended antimicrobial activity against both Gram-positive and Gram-negative pathogens. In this study, we evaluated the in vitro and in vivo antibacterial activity of DS-8587 against multidrug-resistant (MDR) Acinetobacter baumannii. The MIC range of DS-8587 against MDR A. baumannii was 0.25-2 mg/L. These DS-8587 MICs were a minimum of 16-fold or 8-fold more potent than ciprofloxacin or levofloxacin, respectively. Bactericidal activity, a 3 log10 reduction from the initial bacterial counts, was observed within 2 h for 1593644 and 4 h for 1593684 after exposure to DS-8587. Therapeutic efficacy of DS-8587 in the murine calf muscle model was observed at 256 mg/kg. The analysis of the pharmacokinetic and pharmacodynamic index revealed that the AUC/MIC ratio showed the best correlation with efficacy. The total and free drug AUC/MIC value required for a static effect was 29.4 and 14.1, respectively. These data indicate DS-8587 would be an effective agent against MDR A. baumannii infection., (Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling. In this process, Fbxw7α, the F-box protein of the SCF complex, binds to c-Myb via its C-terminal WD40 domain, and induces the ubiquitination of c-Myb. Here, we report that Fbxw5, another F-box protein, enhances sumoylation of nuclear c-Myb. Fbxw5 enhanced c-Myb sumoylation via the DDB1-Cul4A-Rbx1 complex. Since the Fbxw5-DDB1-Cul4A-Rbx1 complex was shown to act as a ubiquitin ligase for tumor suppressor TSC2, our results suggest that this complex can function as a dual SUMO/ubiquitin ligase. Fbxw5, which is localized to both nucleus and cytosol, enhanced sumoylation of nuclear c-Myb and induced the localization of c-Myb to nuclear dot-like domains. Co-expression of Fbxw5 suppressed the trans-activation of c-myc promoter by wild-type c-Myb, but not by v-Myb, which lacks the sumoylation sites. These results suggest that multiple E3 ligases suppress c-Myb activity through sumoylation or ubiquitination, and that v-Myb is no longer subject to these negative regulations., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Gene targeting induced by homologous integration of a foreign DNA segment into a chromosomal target sequence enables precise disruption or replacement of genes of interest and provides an effective means to analyze gene function, and also becomes an useful technique for breeding. But, integration of introduced DNA fragments is predominantly non-homologous in most species. However, we presented high-efficient homologous integration in disruptants of non-homologous end joining (NHEJ), that is, the Ku70-, Ku80- or Lig4-homologs deficient strain, in a model fungus Neurospora crassa. When the effect of NHEJ-defective plants for gene targeting was therefore examined in a model plant Arabidopsis (Arabidopsis thaliana), the efficiencies of gene targeting in the Atlig4/Atlig4 plant were 2/7 (28.6%) against calli obtained a selection-marker gene, 2/16 (12.5%) against selected calli, and about 2/540 (0.004%) against total cell particles at the starting point for transformation. The results of this paper show that the NHEJ-deficient system might cause a decrease in the efficiency of transformation but gives true targeted transformants with high efficiency in plant cell., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

DNA damage checkpoint is an important mechanism for organisms to maintain genome integrity. In Neurospora crassa, mus-9 and mus-21 are homologues of ATR and ATM, respectively, which are pivotal factors of DNA damage checkpoint in mammals. A N. crassa clock gene prd-4 has been identified as a CHK2 homologue, but its role in DNA damage response had not been elucidated. In this study, we identified another CHK2 homologue and one CHK1 homologue from the N. crassa genome database. As disruption of these genes affected mutagen tolerance, we named them mus-59 and mus-58, respectively. The mus-58 mutant was sensitive to hydroxyurea (HU), but the mus-59 and prd-4 mutants showed the same HU sensitivity as that of the wild-type strain. This indicates the possibility that MUS-58 is involved in replication checkpoint and stabilization of stalled forks like mammalian CHK1. Phosphorylation of MUS-58 and MUS-59 was observed in the wild-type strain in response to mutagen treatments. Genetic relationships between those three genes and mus-9 or mus-21 indicated that the mus-9 mutation was epistatic to mus-58, and mus-21 was epistatic to prd-4. These relationships correspond to two signal pathways, ATR-CHK1 and ATM-CHK2 that have been established in mammalian cells. However, both the mus-9 mus-59 and mus-21 mus-58 double mutants showed an intermediate level between the two parental strains for CPT sensitivity. Furthermore, these double mutants showed severe growth defects. Our findings suggest that the DNA damage checkpoint of N. crassa is controlled by unique mechanisms.

The mutagen sensitive uvs-3 and mus-9 mutants of Neurospora show mutagen and hydroxyurea sensitivity, mutator effects and duplication instability typical of recombination repair and DNA damage checkpoint defective mutants. To determine the nature of these genes we used cosmids from a genomic library to clone the uvs-3 gene by complementation for MMS sensitivity. Mutation induction by transposon insertion and RIP defined the coding sequence. RFLP analysis confirmed that this sequence maps in the area of uvs-3 at the left telomere of LG IV. Analysis of the cDNA showed that the UVS-3 protein contains an ORF of 969 amino acids with one intron. It is homologous to UvsD of Aspergillus nidulans, a member of the ATRIP family of checkpoint proteins. It retains the N' terminal coiled-coil motif followed by four basic amino acids typical of these proteins and shows the highest homology in this region. The uvsD cDNA partially complements the defects of the uvs-3 mutation. The uvs-3 mutant shows a higher level of micronuclei in conidia and failure to halt germination and nuclear division in the presence of hydroxyurea than wild type, suggesting checkpoint defects. ATRIP proteins bind tightly to ATR PI-3 kinase (phosphatidylinositol 3-kinase) proteins. Therefore, we searched the Neurospora genome sequence for homologues of the Aspergillus nidulans ATR, UvsB. A uvsB homologous sequence was present in the right arm of chromosome I where the mus-9 gene maps. A cosmid containing this genomic DNA complemented the mus-9 mutation. The putative MUS-9 protein is 2484 amino acids long with eight introns. Homology is especially high in the C-terminal 350 amino acids that correspond to the PI-3 kinase domain. In wild type a low level of constitutive mRNA is present for both genes. It is transiently induced upon UV exposure.

The level of CYP24 mRNA in cultured human fibroblasts increases up to 20,000-fold in response to 1,25-dihydroxyvitamin D(3). Two vitamin D-responsive elements (VDREs) located immediately upstream of the CYP24 gene are primarily responsible for the induction. We studied roles of other regions in the 5'-flanking sequence of this gene. A series of deletion constructs between nucleotides -1918 and +209 of the gene were examined for their promoter activities employing the luciferase reporter assay. We found that the VDREs were not sufficient to account for the extent of induction. The sequence between nucleotides -548 and -294, which is located immediately upstream of the VDREs and includes three potential Sp1 sites, acted synergistically with the VDREs for the induction. Further upstream sequence and the 5'-untranslated region did not appear to play a major role in the vitamin D response.

The progression of replication forks is often impeded by obstacles that cause them to stall or collapse, and appropriate responses to replication-associated DNA damage are important for genome integrity. Here we identified a new gene, mus7(+), that is involved in the repair of replication-associated DNA damage in the fission yeast Schizosaccharomyces pombe. The Deltamus7 mutant shows enhanced sensitivity to methyl methanesulfonate (MMS), camptothecin, and hydroxyurea, agents that cause replication fork stalling or collapse, but not to ultraviolet light or X-rays. Epistasis analysis of MMS sensitivity indicates that Mus7 functions in the same pathway as Mus81, a subunit of the Mus81-Eme1 structure-specific endonuclease, which has been implicated in the repair of the replication-associated DNA damage. In Deltamus7 and Deltamus81 cells, the repair of MMS-induced DNA double-strand breaks (DSBs) is severely impaired. Moreover, some cells with either mutation are hyper-elongated or enlarged, and most of these cells accumulate in late G2 phase. Spontaneous Rad22 (recombination mediator protein RAD52 homolog) foci increase in S phase to late G2 phase in Deltamus7 and Deltamus81 cells. These results suggest that replication-associated DSBs accumulate in these cells and that Rad22 foci form in the absence of Mus7 or Mus81. We also found that the rate of spontaneous conversion-type recombination is reduced in mitotic Deltamus7 cells, suggesting that Rhp51- (RAD51 homolog) dependent homologous recombination is disturbed in this mutant. From these data, we propose that Mus7 functions in the repair of replication-associated DSBs by promoting RAD51-dependent conversion-type recombination downstream of Rad22 and Mus81.

In a previous paper, we reported that the Neurospora crassa upr-1 gene is a homolog of the yeast gene REV3, which encodes the catalytic subunit of DNA polymerase zeta (polzeta). Characterization of the upr-1 mutant indicated that the UPR1 protein plays a role in DNA repair and mutagenesis. To help understand the mechanisms of mutagenic DNA repair in the N. crassa more extensively, we identified N. crassa homologs of yeast REV1 and REV7 and obtained mutants ncrev1 or ncrev7, which had similar phenotypes to the upr-1 mutant. Mutant carrying ncrev7 was more sensitive to UV and 4NQO, and slightly sensitive to MMS than the wild-type. The sensitivity to UV and MMS of the ncrev1 mutant was moderately higher than that of the wild-type, but the sensitivity to 4NQO of the mutant was similar to that of the wild-type. In reversion assay using testers with base substitution or frameshift mutation at the ad-3A locus, each of ncrev1 and ncrev7 mutants showed lower induced-mutability than the wild-type. Expression of ncrev1 and ncrev7 was found to be UV-inducible like the case of upr-1. Genetic analyses showed that the ncrev7 was identical to mus-26, which belongs to the upr-1 epistasis group, and that the ncrev1 was a newly identified DNA repair gene and designated as mus-42. Interestingly, all three mutants have a normal CPD photolyase gene, however, they showed a partial photoreactivation defect (PPD) phenotype, not completely defective but inefficient in photoreactivation. These results suggest that N. crassa REV homolog genes function in DNA repair and UV mutagenesis through the bypass of (6-4) photoproducts.

The crystal structure of congerin II, a galectin family lectin from conger eel, was determined at 1.45A resolution. The previously determined structure of its isoform, congerin I, had revealed a fold evolution via strand swap; however, the structure of congerin II described here resembles other prototype galectins. A comparison of the two congerin genes with that of several other galectins suggests acceralated evolution of both congerin genes following gene duplication. The presence of a Mes (2-[N-morpholino]ethanesulfonic acid) molecule near the carbohydrate-binding site in the crystal structure points to the possibility of an additional binding site in congerin II. The binding site consists of a group of residues that had been replaced following gene duplication suggesting that the binding site was built under selective pressure. Congerin II may be a protein specialized for biological defense with an affinity for target carbohydrates on parasites' cell surface.

In an attempt to understand the relationship between photorepair and dark repair in Neurospora crassa, a new mutant was isolated, which showed defects in both repair processes. The new mutant, mus-38, is moderately sensitive to UV and shows imperfect photoreactivation following UV irradiation. DNA was purified from this mutant and the other UV-sensitive mutants, and analyzed for the removal of cyclobutane pyrimidine dimers (CPDs). UV-specific endonuclease-sensitive sites (ESS) completely disappeared with 1 h of photoreactivation in mus-38 DNA, although the survival recovery with photoreactivation was greatly reduced in this mutant. This suggests that the insufficient survival recovery with photoreactivation in mus-38 does not result from a failure of photo-reversal of CPDs. Removal of ESS during liquid holding (dark repair) was slower in mus-38 compared to wild type. To test the possibility that this mutant was involved in excision repair, the double mutant was made between mus-38 and mus-18, which encodes a UV-damage-specific endonuclease. CPD excision in the mus-18 null mutant was severely affected but not completely inhibited. The double mutant showed a complete loss of the excision activity and was super sensitive to UV. These results indicate that mus-38 participates in an excision pathway that is different from the mus-18 pathway. The mus-38 mutant was sensitive not only to UV but also to some chemical mutagens which make adducts on DNA. Thus, mus-38 is possibly involved in an excision-repair pathway that is related to the Saccharomyces cerevisiae RAD3 pathway.

To understand the possible roles of the Neurospora meiotic genes mei-2 and mei-3 in DNA repair, the frequencies of spontaneous and UV-induced mutation at the ad-3 loci were investigated and double mutants between mei mutants and other DNA-repair mutants were analyzed for mutagen sensitivity. Spontaneous mutation frequency in mei-2 was similar to that of the wild type, while the frequencies were high in both of two mei-3 strains which contained different mei-3 alleles. In addition, the frequency of spontaneous mutation in mei-3 varied greatly from experiment to experiment, which clearly showed a mutator phenotype for mei-3 mutation. UV irradiation increased mutation frequencies in both mei-2 and mei-3. The mutagen sensitivity of the double mutant, mei-2 mei-3, was no greater than that of the single mutants when treated with UV and methyl methanesulfonate (MMS). Thus, both mei genes appear to be involved in the same repair group. When analyzed in combination with other mutations, both mei mutations showed an epistatic relationship to uvs-6 and a synergistic relationship to mus-18 and uvs-2. However, uvs-3 was epistatic to mei-2, but the relationship of uvs-3 to mei-3 could not be tested directly, because the double mutant was barely viable. These results indicate that mei-2 and mei-3 are involved in the same repair group as uvs-6, and uvs-3 is possibly involved in the same group. Alternatively, the mei genes, or uvs-3 itself, may be related to more than one DNA repair group.

Seven different mutants that show high sensitivity to MMS killing were isolated and mapped at different loci. One group, mms-(SA1), mms-(SA2) and mms-(SA6), showed high sensitivity to MMS but not to UV or gamma-rays. Another group, mms-(SA4) and mms-(SA5), showed extremely high sensitivity to UV and MMS. And mms-(SA3) and mms-(SA7) were moderately sensitive to both UV and MMS. Mms-(SA4) and mms-(SA1) were identified as alleles of uvs-2 and mus-7, respectively, which had been previously isolated. The mms-(SA1), mms-(SA6) and mms-(SA7) strains were barren in homozygous crosses, and the mms-(SA5) strain was barren in heterozygous crosses. The mms-(SA1), mms-(SA3) and mms-(SA5) strains showed high sensitivity to histidine. In summary, at least two new loci involved in the repair of MMS damage have been identified. The possibility that some of these new mutants are in new repair pathways is suggested.

A mutant, uvs-(SA3B), which shows high sensitivity to UV light segregated among the progeny in a back-cross of a presumptive MMS-sensitive mutant to a wild-type strain. At 37% survival, this mutant was approximately 5 times more sensitive to UV and also 6 times more sensitive to 4-NQO than the wild type. But it was only slightly sensitive to gamma-ray, MMS, MNNG, MTC and histidine. It showed an unusual photoreactivation response. Its time course of photorecovery was similar to the photoreactivation-defective strain upr-1 of Neurospora crassa. Mutation induction by UV at the ad-3 loci in this mutant strain was lower than that at the same loci in the wild-type strain. The uvs-(SA3B) mutant maps between met-1 and col-4 in linkage group IV, and it was not allelic with the mutagen-sensitive mutant mus-8 which is located in this area. We have concluded, therefore, that uvs-(SA3B) has resulted from mutation in a new DNA-repair gene. This new mutant was barren in homozygous crosses.

Double mutants were constructed combining mus-26, formerly designated uvs-(SA3B), with other UV-sensitive mutants. Tests of sensitivity of these double mutants to UV and to chemical mutagens revealed that mus-26 and upr-1 belong to the same epistatic group. The UV dose-response curve of mus-26 showed a characteristic plateau in the range of 100-200 J/m2. The same characteristic was also shown in the dose-response curves of upr-1 and the double mutant, upr-1 mus-26. Photoreactivation of UV damage in mus-26, upr-1 and upr-1 mus-26 was defective but not null. Assays were made of the reversion rate of ad-8 in strains that also carried UV-sensitive mutations. The reversion frequencies of the strains with upr-1 and upr-1 mus-26 were very low for the UV dose range below 300 J/m2, similarly to mus-26. Previously reported homozygous sterility of mus-26 was not caused by the mus-26 locus itself, and fertile strains were obtained among progeny. The results of this study suggest that mus-26 and upr-1 have similar properties in DNA repair.

Preheating of Neurospora conidia increased their susceptibility to mutation induction by chemical mutagens. Optimal conditions of heat shock for enhanced mutagenesis were determined in 2.5 X 10(7) conidia/ml 0.067 M KH2PO4-Na2HPO4 (pH 7.0) buffer to be treatment at 43 degrees C for 60 min. When protein synthesis during heat stock was eliminated by cycloheximide or by use of the temperature-sensitive mutation psi-1, induction of thermotolerance was inhibited while induction of the enhanced state of mutability was not. Therefore, inducible protein synthesis is not involved in this process. To discover whether DNA-repair systems are altered by heat shock and, as a result, whether reversion frequencies increase, DNA-repair mutants (upr-1, uvs-2, uvs-3, uvs-6, mus-7, mus-16) were heated and their reversion frequencies at the ad-8 locus were measured. All the DNA-repair mutants showed higher reversion frequencies with MNNG treatment after heat shock than in non-heated control. It therefore seems that DNA repair is not involved in the enhancement of chemical mutagenesis by heat shock. Heat shock does not increase frequencies of reversion induced by ultraviolet light, and heat shock after treatment with chemical mutagens does not affect reversion frequencies. These results suggest that heat shock may change the structure and function of cellular membranes and thereby increase the influx of mutagens into cells.

A prospective study has shown that aspiration treatment of ganglion cysts of the wrist and hand can safely remove 85% of these tumors if one, two or three separate treatments are administered. There is a decided cost savings when this treatment is compared with surgical excision, even when the expense of surgical excision for aspiration treatment failures is added to the equation.