Dimethylarsine likely acts as a mouse-pulmonary tumor initiator via the production of dimethylarsine radical and/or its peroxy radical

AimsRecent animal experiments have indicated that dimethylarsinic acid (DMA), a main metabolite of inorganic arsenic, is a complete carcinogen in the lung of mice and the urinary bladder of rats, nevertheless, no ultimate-active metabolite from DMA has been identified thus far. We have proposed that dimethylarsine ((CH₃)₂AsH), an ultimate reductive metabolite of DMA, is excreted in the expired air of mice administered DMA, and furthermore, was easily converted into dimethylarsine radical ((CH₃)₂As?) and dimethylarsine peroxy radical ((CH₃)₂AsOO?) by its reaction with O₂. The aim of the present study was to elucidate the possible mode of the tumorigenic action by dimethylated arsenic.Main methodsIn vitro experiments using GSH reductase as a two-electron donor of dimethylarsenic-glutathione conjugate ((CH₃)₂As-SG) and DNA adduct assay via a photochemical approach were performed. A lung tumorigenicity assay of (CH₃)₂AsH suspended in argon-atmospheric olive oil for 5 days was also conducted in mice.Key findingsThe results indicated that (CH₃)₂AsH was easily produced enzymatically from (CH₃)₂As-SG and showed tumor-initiating action in mouse lung via the production of (CH₃)₂As? and (CH₃)₂AsOO? by its reaction with O₂, and that these radicals have the ability to form DNA adducts.SignificanceThe carcinogenicity of DMA, at least in mouse lung, could be explained based on the proposal that oral administration of DMA induces pulmonary tumors in mice, and arises from the arsine radicals produced through (CH₃)₂AsH, which was enzymatically reduced from (CH₃)₂As-SG.     

Messenger RNA quantification after fluorescence activated cell sorting using intracellular antigens

Recent studies using stem cells or cancer stem cells have revealed the importance of detecting minor populations of cells in blood or tissue and analyzing their biological characteristics. The only possible method for carrying out such procedures is fluorescence activated cell sorting (FACS). However, FACS has the following limitations. First, cells without an appropriate cell surface marker cannot be sorted. Second, the cells have to be kept alive during the sorting process in order to analyze their biological characteristics. If an intracellular antigen that was specific to a particular cell type could be stained with a florescent dye and then the cells can be sorted without causing RNA degradation, a more simple and universal method for sorting and analyzing cells with a specific gene expression pattern could be established since the biological characteristics of the sorted cells could then be determined by analyzing their gene expression profile. In this study, we established a basic protocol for messenger RNA quantification after FACS (FACS-mQ) targeting intracellular antigens. This method can be used for the detection and analysis of stem cells or cancer stem cells in various tissues.     

Distribution and properties of novel deglycating enzymes for fructosyl peptide in fungi

Our fungal culture collection was screened for fructosyl peptide oxidase, an enzyme that could be used for the determination of glycated hemoglobin in diabetic subjects with hyperglycemia. Fructosyl peptide oxidases were found in strains of eight genera: Achaetomiella, Achaetomium, Chaetomium, Coniochaeta, Eupenicillium, Gelasinospora, Microascus and Thielavia. By their substrate specificity toward N-fructosyl valyl-histidine (-keto-amine) and N-fructosyl lysine (-keto-amine), fructosyl peptide oxidases could be categorized into two groups: (1) enzymes that oxidize both -keto-amine and -keto-amine, and (2) enzymes that preferably oxidize -keto-amine. A fructosyl peptide oxidase from Achaetomiella virescens ATCC 32393, active toward both N-fructosyl valyl-histidine and N-fructosyl lysine, was purified to homogeneity and characterized. The enzyme was monomeric (M_r=50,000), was most active at 40 °C and pH 8.0, and had a covalently bound flavin as a prosthetic group. Apparent K_m values for N-fructosyl valyl-histidine and N-fructosyl lysine were 2.30 and 1.69 mM, respectively. N-fructosyl valyl-histidine was consumed and the same molar amount of valyl-histidine was produced by the fructosyl peptide oxidase reaction. This enzyme could be useful for the measurement of hemoglobin A_1C, the N-terminal valine residue of the -subunit of which is glycated.Abbreviations HbA_1C Hemoglobin A_1C - FPOX Fructosyl peptide oxidase - FAOX Fructosyl amino acid oxidase - Fru-ValHis N-fructosyl valyl-histidine - Fru-Val N-fructosyl valine - Fru-Lys N-fructosyl lysine - Fru-Gly Fructosyl glycine - TOOS N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt     

Reduced N-acetylaspartate levels in mice lacking aralar, a brain- and muscle-type mitochondrial aspartate-glutamate carrier

Aralar is a mitochondrial calcium-regulated aspartate-glutamate carrier mainly distributed in brain and skeletal muscle, involved in the transport of aspartate from mitochondria to cytosol, and in the transfer of cytosolic reducing equivalents into mitochondria as a member of the malate-aspartate NADH shuttle. In the present study, we describe the characteristics of aralar-deficient (Aralar-/-) mice, generated by a gene-trap method, showing no aralar mRNA and protein, and no detectable malate-aspartate shuttle activity in skeletal muscle and brain mitochondria. Aralar-/- mice were growth-retarded, exhibited generalized tremoring, and had pronounced motor coordination defects along with an impaired myelination in the central nervous system. Analysis of lipid components showed a marked decrease in the myelin lipid galactosyl cerebroside. The content of the myelin lipid precursor, N-acetylaspartate, and that of aspartate are drastically decreased in the brain of Aralar-/- mice. The defect in N-acetylaspartate production was also observed in cell extracts from primary neuronal cultures derived from Aralar-/- mouse embryos. These results show that aralar plays an important role in myelin formation by providing aspartate for the synthesis of N-acetylaspartate in neuronal cells.     

Photosynthetic Characteristics and Carbonic Anhydrase Activity in Cells Cultured Photoautotrophically and Mixotrophically and Cells Isolated from Leaves

Cultured green cells of Nicotiana tabacum var. Samsun, Cytisusscoparius Link and Hyoscyamus niger which were grown photoautotrophicallyunder a stream of air enriched with 1% CO₂ or mixotrophicallyin the presence of 3% sucrose and ordinary air showed very lowcarbonic anhydrase activity, which was only 0–9% of thatin the respective intact leaves. The CO₂ compensation pointfor photosynthesis of autotrophically and mixotrophically culturedgreen cells of tobacco was higher than 0.3 mM NaHCO₃ at pH 7.8,but that of the cells isolated from tobacco leaves was lowerthan 0.1 mM NaHCO₃ at pH 7.8. The fact that the cultured cellscannot grow photoautotrophically under ordinary air is due toa high CO₂ compensation point in photosynthesis. The dark respiratoryactivity in both photoautotrophically and mixotrophically culturedtobacco cells was more than 7-fold that in the cells isolatedfrom tobacco leaves. We therefore could not conclude whetherthe high CO₂ compensation point in the cultured cells is dueto the low carbonic anhydrase activity or simply reflects thehigh respiratory activity. (Received July 10, 1980; Accepted November 25, 1980)     

Effects of woody debris on the habitat of juvenile masu salmon (Oncorhynchus masou) in northern Japanese streams

1. The effects of woody debris on stream habitat of juvenile masu salmon ( Oncorhynchus masou ) were examined at two spatial scales, stream reach and channel unit, for first to thirdorder tributaries of the Teshio River in northern Hokkaido, Japan. The fortyeight study reaches were classified into three distinct types: coarsesubstrate steppool (CSP), coarsesubstrate poolriffle (CPR) and finesubstrate poolriffle (FPR) reaches. Each reach type included reaches with different riparian settings, broadly classified as forest (relatively undisturbed forest and secondary forest after fires) or grassland (bamboo bushland and pasture).
2. The reachscale analyses showed that neither total pool volume nor pooltopool spacing was correlated with woody debris abundance in any of the three reach types. Masu salmon density was positively correlated with both woodydebris cover area and total cover area, but not with total pool volume in the reaches.
3. Channelunitscale analyses revealed that woody debris reduced nonpool velocity, increased pool depth and retained fine sediment in pools in FPR reaches, where the size of woody debris was very large relative to the substrate material size. However, woody debris did not influence any of the hydraulic variables (depth, velocity, substrate) in either nonpools or pools of CSP and CPR reaches. Habitat use by masu salmon in nonpools or pools was affected by woodydebris cover area or total cover area rather than by hydraulic variables in any of the reach types.
4. The effects of woody debris on habitat at the reach and channelunit scales in the study area were less than those indicated by previous work in the Pacific Northwest, North America, owing to the relatively small size of the riparian trees. However, the overall results suggested that woody debris in the study area contributed to masu salmon habitat by providing cover at the smaller, microhabitat scale.     

A Novel Isoenzyme of Ascorbate Peroxidase Localized on Glyoxysomal and Leaf Peroxisomal Membranes in Pumpkin

A novel isoenzyme of ascorbate peroxidase with molecular massof 31 kDa was found to be localized on membranes of microbodies.Intact microbodies had no latent ascorbate peroxidase activity,an indication that the active site of the ascorbate peroxidasewas exposed to the cytosol and the peroxidase would scavengehydrogen peroxide leaked from microbodies. (Received June 28, 1995; Accepted July 24, 1995)     

Vesicle transport and processing of the precursor to 2S albumin in pumpkin

Cell fractionation of pulse-chase-labeled developing pumpkin cotyledons demonstrated that proprotein precursor to 2S albumin is transported from the endoplasmic reticulum to dense vesicles and then to the vacuoles, in which pro2S albumin is processed to the mature 2S albumin. Immunocytochemical analysis showed that dense vesicles of about 300 nm in diameter mediate the transport of pro2S albumin to the vacuoles.
The primary structure of the precursor (16 578 Da) to pumpkin 2S albumin has been deduced from the nucleotide sequence of an isolated cDNA insert. The presence of a hydrophobic signal peptide at the N-terminus indicates that the precursor is a preproprotein that is converted into pro2S albumin after cleavage of the signal peptide. N-terminal sequencing of the pro2S albumin in the isolated vesicles revealed that the signal peptide is cleaved off co-translationally on the C-terminal side of alanine residue 22 of prepro2S albumin. By contrast, post-translational cleavages occur on the C-terminal sides of asparagine residues 35 and 74, which are conserved among precursors to 2S albumin from different plants. Hydropathy analysis revealed that the two asparagine residues are located in the hydrophilic regions of pro2S albumin. These findings suggest that a vacuolar processing enzyme can recognize exposed asparagine residues on the molecular surface of pro2S albumin and cleave the peptide bond on the C-terminal side of each asparagine residue to produce mature 2S albumin in the vacuoles.