[Dust collection efficiency of commercial gas collection tubes].

Objectives: Gas sampling tubes are essential tools for the evaluation of air quality in work environments. It adsorbs toxic gaseous matters onto the surface of various granular adsorbents, such as silica gel or activated carbon packed in a thin glass tube, for quantitative analysis by gas chromatography. Currently, most of the semi-volatile matters are evaluated via aerosol filtration or solid-phase gas adsorption depending on the main phase of the substances; however, only a few substances have a sampling protocol regarding both solid and gaseous phases.

Flow cytometric quantitation of EpCAM-positive extracellular vesicles by immunomagnetic separation and phospholipid staining method.

Extracellular vesicles (EV) have attracted attention as circulating biomarkers for many diseases, particularly cancer. Conventional immunofluorescence staining has been used for the detection of target antigens on EV by flow cytometry. However, the staining intensity depends on the amount of antigen expressed on the vesicles and is often only around the noise level.

Functional Complementation Assay for 47 MUTYH Variants in a MutY-Disrupted Escherichia coli Strain.

MUTYH-associated polyposis (MAP) is an adenomatous polyposis transmitted in an autosomal-recessive pattern, involving biallelic inactivation of the MUTYH gene. Loss of a functional MUTYH protein will result in the accumulation of G:T mismatched DNA caused by oxidative damage. Although p.

Cysteine-poor region-specific EpCAM monoclonal antibody recognizing native tumor cells with high sensitivity.

EpCAM is a ∼40 kDa transmembrane glycoprotein. EpCAM overexpression is a popular trait of almost all carcinomas and is considered as a targeted cancer immunotherapy as well as a practical marker for circulating tumor cells (CTC). Its extracellular part (EpEx) consists of an N-terminal EGF-like (EGF) domain, a TY-like (TY) domain, and an uncharacterized cysteine-poor (CP) region.

A DNA oligomer containing 2,2,4-triamino-5(2H)-oxazolone is incised by human NEIL1 and NTH1.

The nucleobase derivative, 2,2,4-triamino-5(2H)-oxazolone (Oz), is an oxidation product of guanine or of 8-oxo-7,8-dihydroguanine that causes G-to-C transversions in DNA. Human NEIL1 (hNEIL1) and NTH1 (hNTH1) are homologues of two prokaryotic base excision repair enzymes, FPG/NEI and NTH, respectively. Here, we demonstrated that hNEIL1 and hNTH1 cleave Oz sites as efficiently as 5-hydroxyuracil sites.

Enumeration, characterization, and collection of intact circulating tumor cells by cross contamination-free flow cytometry.

Circulating tumor cells (CTC) are an important biomarker for several solid cancers. Most of the commercially available systems for enumeration of CTC are based on immunomagnetic enrichment of epithelial cell adhesion molecule (EpCAM/CD326)-expressing CTC before microscopic cell imaging or reverse-transcription PCR (RT-PCR). The aim of this study was to establish a practical method for enumeration of CTC using a novel flow cytometer that has a disposable microfluidic chip, which is designed to realize absolute cross contamination-free measurements and to collect the analyzed cell sample.

Cells with pathogenic biallelic mutations in the human MUTYH gene are defective in DNA damage binding and repair.

Inherited biallelic mutations in the human MUTYH gene are responsible for the recessive syndrome--adenomatous colorectal polyposis (MUTYH associated polyposis, MAP)--which significantly increases the risk of colorectal cancer (CRC). Defective MUTYH activity causes G:C to T:A transversions in tumour APC and other genes thereby altering genomic integrity. We report that of the four established cell lines, derived from patients with the MAP phenotype and containing biallelic MUTYH mutations, three contain altered expressions of MUTYH protein (MUTYH Y165C(-/-), MUTYH 1103delC/G382D and MUTYH Y165C/G382D but not MUTYH G382D(-/-)), but that all four cell lines have wild type levels of MUTYH mRNA.

DNA glycosylase activities for thymine residues oxidized in the methyl group are functions of the hNEIL1 and hNTH1 enzymes in human cells.

Bacteria and eukaryotes possess redundant activities that recognize and remove oxidatively damaged bases from DNA through base excision repair. DNA glycosylases excise damaged bases to initiate the base excision repair pathway. hOgg1 and hNTH1, homologues of E.

In situ analysis of repair processes for oxidative DNA damage in mammalian cells.

Oxidative DNA damage causes blocks and errors in transcription and replication, leading to cell death and genomic instability. Although repair mechanisms of the damage have been extensively analyzed in vitro, the actual in vivo repair processes remain largely unknown. Here, by irradiation with an UVA laser through a microscope lens, we have conditionally produced single-strand breaks and oxidative base damage at restricted nuclear regions of mammalian cells.

UV light-induced DNA damage and tolerance for the survival of nucleotide excision repair-deficient human cells.

DNA damage can cause cell death unless it is either repaired or tolerated. The precise contributions of repair and tolerance mechanisms to cell survival have not been previously evaluated. Here we have analyzed the cell killing effect of the two major UV light-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs), in nucleotide excision repair-deficient human cells by expressing photolyase(s) for light-dependent photorepair of either or both lesions.

Functional and physical interactions between ERCC1 and MSH2 complexes for resistance to cis-diamminedichloroplatinum(II) in mammalian cells.

Bulky DNA lesions are mainly repaired by nucleotide excision repair (NER), in which the interaction of ERCC1 with XPA protein recruits the ERCC1-XPF complex, which acts as a structure-specific endonuclease in the repair process. However, additional functions besides NER have been suggested for the ERCC1-XPF complex, because ERCC1- or XPF-deficient rodent cells are significantly more sensitive to DNA interstrand cross-linking (ICL) agents such as cis-diamminedichloroplatinum(II) (CDDP) than any other NER-deficient cells and because ERCC1-deficient mice suffer a more severe phenotype than XPA-deficient mice. By using RNA interference we show here that suppression of ERCC1 expression increases the sensitivity of xeroderma pigmentosum group A (XPA)-deficient human cells to CDDP but not to UV.

[DNA damage, repair and aging].

Oxidative DNA damage has been shown to accumulate with age in the nuclear and mitochondrial genome and cause cancer. Among DNA lesions produced by reactive oxygen species, base lesions and single-strand breaks are most frequently produced and cause mutation and cell death. However, these lesions are effectively repaired by base excision repair, which is very well conserved from bacteria to human.

A back-up glycosylase in Nth1 knock-out mice is a functional Nei (endonuclease VIII) homologue.

Thymine glycol, a potentially lethal DNA lesion produced by reactive oxygen species, can be removed by DNA glycosylase, Escherichia coli Nth (endonuclease III), or its mammalian homologue NTH1. We have found previously that mice deleted in the Nth homologue still retain at least two residual glycosylase activities for thymine glycol. We report herein that in cell extracts from the mNth1 knock-out mouse there is a third thymine glycol glycosylase activity that is encoded by one of three mammalian proteins with sequence similarity to E.

Identification of 5-formyluracil DNA glycosylase activity of human hNTH1 protein.

5-formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. The elucidation of repair mechanisms for 5-foU will yield important insights into the biological consequences of the lesion. Recently, we reported that 5-foU is recognized and removed from DNA by Escherichia coli enzymes Nth (endonuclease III), Nei (endonuclease VIII) and MutM (formamidopyrimidine DNA glycosylase).

Anti-bovine rhodopsin monoclonal antibody recognizing light-dependent structural change.

The antigenic structure of the bovine rhodopsin molecule was investigated by using a bovine rhodopsin-specific monoclonal antibody designated Rh 29. Competition assay with sealed intact disks and broken disks indicated that the antibody-binding region was localized in the intradiscal surface. An antigenic peptide obtained by a cyanogene bromide cleavage of rhodopsin was purified and determined as residues 2-39 in the amino acid sequence.

Novel nuclear and mitochondrial glycosylases revealed by disruption of the mouse Nth1 gene encoding an endonuclease III homolog for repair of thymine glycols.

Endonuclease III, encoded by nth in Escherichia coli, removes thymine glycols (Tg), a toxic oxidative DNA lesion. To determine the biological significance of this repair in mammals, we established a mouse model with mutated mNth1, a homolog of nth, by gene targeting. The homozygous mNth1 mutant mice showed no detectable phenotypical abnormality.