A cryptic melibiose transporter gene possessing a frameshift from Citrobacter freundii

Wild-type Citrobacter freundii cannot grow on melibiose as a sole source of carbon. The melibiose transporter gene melB was cloned from a C. freundii mutant M4 that could utilize melibiose as a sole carbon source. Although the cloned melB gene is closely similar to the melB genes of other bacteria, it is cryptic because of a frameshift mutation. Site-directed mutagenesis was used to construct a functional melB gene by deleting one nucleotide, resulting in the production of an active melibiose transporter. The active MelB transporter could utilize Na(+) and H(+) as coupling cations to melibiose transport. The amino acid sequence of the C. freundii MelB was found to be most similar to those of Salmonella typhimurium and Escherichia coli MelB. These facts are consistent with the phylogenetic relationship of bacteria and the cation coupling properties of the melibiose transporters.     

Asialoglycoprotein receptor deficiency in mice lacking the major receptor subunit. Its obligate requirement for the stable expression of oligomeric receptor

The asialoglycoprotein receptor is an abundant hetero-oligomeric endocytic receptor that is predominantly expressed on the sinusoidal surface of the hepatocytes. A number of physiological and pathophysiological functions have been ascribed to this hepatic lectin (HL), the removal of desialylated serum glycoproteins and apoptotic cells, clearance of lipoproteins, and the sites of entry for hepatotropic viruses. The assembly of two homologous subunits, HL-1 and HL-2, is required to form functional, high affinity receptors on the cell surface. However, the importance of the individual subunits for receptor transport to the cell surface is controversial. We have previously generated HL-2-deficient mice and showed that the expression of HL-1 was significantly reduced, and the functional activity as the asialoglycoprotein receptor was virtually eliminated. However, we failed to detect phenotypic abnormalities. To explore the significance of the major HL-1 subunit for receptor expression and function in vivo, we have disrupted the HL-1 gene in mice. Homozygous HL-1-deficient animals are superficially normal. HL-2 expression in the liver is virtually abrogated, indicating that HL-1 is strictly required for the stable expression of HL-2. Although these mice are almost unable to clear asialo-orosomucoid, a high affinity ligand for asialoglycoprotein receptor, they do not accumulate desialylated glycoproteins or lipoproteins in the plasma.     

Evaluation of the effect of 90Sr beta-radiation on human blood cells by chromosome aberration and single cell gel electrophoresis (comet assay) analysis

Among various environmental genotoxins, ionizing radiation has received special attention because of its mutagenic, carcinogenic and teratogenic potential. In this context and considering the scarcity of literature data, the objective of the present study was to evaluate the effect of 90Sr beta-radiation on human cells. Blood cells from five healthy donors were irradiated in vitro with doses of 0.2-5.0Gy from a 90Sr source (0.2Gy/min) and processed for chromosome aberration analysis and for comet assay. The cytogenetic results showed that the most frequently found aberration types were acentric fragments, double minutes and dicentrics. The alpha and beta coefficients of the linear-quadratic model, that best fitted the data obtained, showed that 90Sr beta-radiation was less efficient in inducing chromosome aberrations than other types of low linear energy transfer (LET) radiation such as 3H beta-particles, 60Co gamma-rays, 137Cs and 192Ir and X-rays. Apparently, 90Sr beta-radiation in the dose range investigated had no effect on the modal chromosome number of irradiated cells or on cell cycle kinetics. Concerning the comet assay, there was an increase in DNA migration as a function of radiation dose as evaluated by an image analysis system (tail moment) or by visual classification (DNA damage). The dose-response relation adequately fitted the non-linear regression model. In contrast to the cytogenetic data, 90Sr beta-radiation induced more DNA damage than 60Co gamma-radiation when the material was analyzed immediately after exposures. A possible influence of selective death of cells damaged by radiation was suggested.     

Ceramide generation in nitric oxide-induced apoptosis. Activation of magnesium-dependent neutral sphingomyelinase via caspase-3

Sodium nitroprusside (SNP), a NO donor, has been recognized as an inducer of apoptosis in various cell lines. Here, we demonstrated the intracellular formation of ceramide, a lipid signal mediator, in SNP-induced apoptosis in human leukemia HL-60 cells and investigated the mechanisms of ceramide generation. The levels of intracellular ceramide increased to, at most, 160% of the control level in a time- and dose-dependent manner when the cells were treated with 1 mM SNP. SNP also decreased the sphingomyelin level to approximately 70% of the control level and increased magnesium-dependent neutral sphingomyelinase (N-SMase) activity to 160% of the control activity 2 h after treatment. Neither acid SMase nor magnesium-independent N-SMase was affected by SNP. Caspases are thought to be key enzymes in apoptotic cell death. Acetyl-Asp-Glu-Val-Asp-aldehyde, a synthetic tetrapeptide inhibitor of caspases, inhibited magnesiumdependent N-SMase, ceramide generation, and apoptosis. Moreover, recombinant purified caspase-3 increased magnesium-dependent N-SMase in a cell-free system. These results suggest that the findings that SNP increased ceramide generation and magnesium-dependent N-SMase activity via caspase-3 are interesting to future study to determine the relation between caspases and sphingolipid metabolites in NO-mediated signaling.     

Age-independent constancy of mineral contents in human ribs

On age relationships of mineral contents in human bones, the contents of the sixth rib and a piece of its compact bone were determined by inductively coupled plasma atomic emission spectrometry (ICPS). The ribs were resected from 21 subjects (14 men and 7 women) who died in age ranging from 65 to 93 yr. There were no age-dependent decreases in Ca and P contents of the ribs in the age range on ICPS. It was found that there were no age-dependent decreases in Ca and P in compact bones of ribs.     

Development of damage function of acidification for terrestrial ecosystems based on the effect of aluminum toxicity on net primary production

Background  Acidification is one of the important impact categories for life cycle impact assessment. Although its characterization has progressed during this decade through the employment of midpoint approaches, only limited studies of endpoint approaches have been performed. Objective. This study aimed at developing damage function of acidification for terrestrial ecosystems in Japan. Damage function expresses a quantitative relationship between the inventory and endpoint damage. Methods  The geographical boundary was limited in Japan both for emission and impact. In this study, sulfur dioxide (SO₂), nitrogen monoxide (NO), nitrogen dioxide (NO₂) (NO and NO₂ collectively mean NO_x), hydrogen chloride (HC1), and ammonia (NH₃) were considered as major causative substances of acidification. Net primary production (NPP) of existing vegetation was adopted as an impact indicator of terrestrial ecosystems. The aluminum toxicity was adopted as the major factor of effect on terrestrial ecosystems due to acidification. The leachate concentration of monomeric inorganic aluminum ions was selected to express the plant toxicity of aluminum. Results and Discussion  The results of damage function gave utilizable factors both for a midpoint approach and an endpoint approach; Atmospheric Deposition Factor (ADF) and Damage Factor (DF) applicable to the former and the latter, respectively. The ADF indicates an increase of H⁺ deposition per unit area to an additional emission of causative sustance. The additional emission corresponds to some alternatives in industry, not the baseline emission. The DF indicates the total NPP damage in all of Japan due to the additional emission of causative substances. The derived NPP damage is on the order of one millionth of the NPP itself. HC1 and NH3 showed larger ADFs and DFs than that of SO₂ and NO_x. The reason was ascribed to the relatively large source-receptor relationships (SRR) of HC1 and NH₃. However, since the method applied to determine the SRR of HC1 and NH₃ has larger uncertainties than that of SO₂ and NO_x, attention is needed to handle the difference. Conclusion  The damage function easily defines the concrete NPP damage due to an additional emission. The impact indica tor, NPP, also has an advantage in its mass unit that is directly summable through the entire impact categories. Expansion of endpoints, such as in aquatic ecosystems, material degradation, human health, and biodiversity aspects of terrestrial ecosystems, is an important subject for future work. Further, uncertain analyses for major parameters will provide helpful information on the reliability of damage function.     

A novel protein-conjugating system for Ufm1, a ubiquitin-fold modifier

Several studies have addressed the importance of various ubiquitin-like (UBL) post-translational modifiers. These UBLs are covalently linked to most, if not all, target protein(s) through an enzymatic cascade analogous to ubiquitylation, consisting of E1 (activating), E2 (conjugating), and E3 (ligating) enzymes. In this report, we describe the identification of a novel ubiquitin-fold modifier 1 (Ufm1) with a molecular mass of 9.1 kDa, displaying apparently similar tertiary structure, although lacking obvious sequence identity, to ubiquitin. Ufm1 is first cleaved at the C-terminus to expose its conserved Gly residue. This Gly residue is essential for its subsequent conjugating reactions. The C-terminally processed Ufm1 is activated by a novel E1-like enzyme, Uba5, by forming a high-energy thioester bond. Activated Ufm1 is then transferred to its cognate E2-like enzyme, Ufc1, in a similar thioester linkage. Ufm1 forms several complexes in HEK293 cells and mouse tissues, revealing that it conjugates to the target proteins. Ufm1, Uba5, and Ufc1 are all conserved in metazoa and plants but not in yeast, suggesting its potential roles in various multicellular organisms.     

Prediction of the coding sequences of mouse homologues of FLJ genes: the complete nucleotide sequences of 110 mouse FLJ-homologous cDnas identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse KIAA-homologous genes since 2001. As an extension of this project, we also started to accumulate mouse cDNA clones homologous to the human FLJ cDNA clones which are another long cDNA resource produced in our institute. We have isolated the cDNA clones from size-fractionated cDNA libraries derived from five different mouse tissues and natural killer T-cells. Although the human FLJ cDNA clones were originally derived from human spleen libraries, one-third of their mouse homologues were obtained from the brain library. We designated these homologues "mFLJ" plus a 5-digit number and herein characterized 110 mFLJ cDNA clones. We assigned an integrity of the CDSs from the comparison of the 110 cDNA clones with the corresponding human FLJ cDNA clones. The average size of the 110 mouse cDNA sequences was 3.8 kb and that of the deduced amino acid sequences from their longest CDS in each cDNA was 663 amino acid residues. Homology and/or motif search against public databases revealed new domains and/or motifs in 26 mFLJ gene products which provide additional speculation regarding the function of FLJ genes.     

A comprehensive approach for establishment of the platform to analyze functions of KIAA proteins: generation and evaluation of anti-mKIAA antibodies

Since December 2001 we have been conducting a project to isolate and determine entire sequences of mouse KIAA cDNA clones which encode polypeptides corresponding to human KIAA proteins. The ultimate goal of this project is the elucidation of the functions of KIAA proteins. A critical step in this project is the generation of antibodies based on the cDNA sequence information. Although antibodies are the most optimal tools for biological analysis, the production and isolation of multiple recombinant proteins for an antigen is a rate-limiting step in antibody production. To address this problem, we established a system utilizing the in vitro recombination-assisted method and shotgun clones that were generated during the sequencing of mouse KIAA cDNAs (DNA Res. 2003, 10, 129-136). The authenticity of the expressed proteins was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Another critical step for antibody production is the evaluation of the antibodies. Thus, we also made efforts to develop a systematic approach for evaluation of the titer and the specificity of the antibodies. Using these systems, we have produced and evaluated more than 500 antibodies raised against mouse KIAA proteins to date. We are currently generating antibody arrays for analysis of protein expression profiles. We will verify protein-protein interactions using immunoprecipitation and tandem mass spectrometry analysis.