Monitoring of Ralstonia eutropha KT1 in groundwater in an experimental bioaugmentation field by in situ PCR.

Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 x 10(5) cells/ml and the amount of Ralstonia and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 x 10(7) cells/ml 1 h after injection, and the concentration of Ralstonia detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR.     

Suppression of UVC-induced cell damage and enhancement of DNA repair by the fermented milk, Kefir

An aqueous extract of Kefir, fermented milk originally produced in the Caucasus mountains, suppressed morphological changes of human melanoma HMV-1 and SK-MEL cells and human normal fibroblastTIG-1 cells caused by UVC-irradiation, suggesting that UV damage can be suppressed by the Kefir extract. The addition of the Kefir extract after UVC-irradiation of HVM-1 cells resulted in a remarkable decrease in intracellular reactive oxygen species (ROS) which had been increased by UVC irradiation. The Kefir extract also stimulated unscheduled DNA synthesis and suppressed UVC-induced apoptosis of HMV-1 cells. A colony formation assay revealed that the Kefir extract rescued HMV-1 cells from cell death caused by UVC irradiation. The Kefir extract, as well as methyl methanethiosulfonate which is known to enhance the nucleotide excision repair (NER) activity, exhibited strong thymine dimer repair-enhancing activity. Epigalocatechin exhibited a weak NER activity but vitamins A, C, and E and catechin showed no NER activity. The thymine dimer repair-enhancing factors in the Kefir extract were heat-stable and assumed to be molecules with a molecular weight of less than 5000. The treatment of HMV-1 cells with the Kefir extract during or before UVC- irradiation also prevented the generation of ROS and thymine dimmer, and suppressed the apoptosis of HMV-1 cells, suggesting that application of Kefir can prevent UV damage.     

Protective mechanism of reduced water against alloxan-induced pancreatic β-cell damage: Scavenging effect against reactive oxygen species

Reactive oxygen species (ROS) cause irreversible damage to biological macromolecules, resulting in many diseases. Reduced water (RW) such as hydrogen-rich electrolyzed reduced water and natural reduced waters like Hita Tenryosui water in Japan and Nordenau water in Germany that are known to improve various diseases, could protect a hamster pancreatic β cell line, HIT-T15 from alloxan-induced cell damage. Alloxan, a diabetogenic compound, is used to induce type 1 diabetes mellitus in animals. Its diabetogenic effect is exerted via the production of ROS. Alloxan-treated HIT-T15 cells exhibited lowered viability, increased intracellular ROS levels, elevated cytosolic free Ca²⁺ concentration, DNA fragmentation, decreased intracellular ATP levels and lowering of glucose-stimulated release of insulin. RW completely prevented the generation of alloxan-induced ROS, increase of cytosolic Ca²⁺ concentration, decrease of intracellular ATP level, and lowering of glucose-stimulated insulin release, and strongly blocked DNA fragmentation, partially suppressing the lowering of viability of alloxan-treated cells. Intracellular ATP levels and glucose-stimulated insulin secretion were increased by RW to 2–3.5 times and 2–4 times, respectively, suggesting that RW enhances the glucose-sensitivity and glucose response of β-cells. The protective activity of RW was stable at 4 °C for over a month, but was lost by autoclaving. These results suggest that RW protects pancreatic β-cells from alloxan-induced cell damage by preventing alloxan-derived ROS generation. RW may be useful in preventing alloxan-induced type 1-diabetes mellitus. This revised version was published online in August 2006 with corrections to the Cover Date.     

Establishment of hybridomas producing cancer specific human antibodies from B cell line derived from PBL of a patient with adult T cell leukemia

Adult T cell leukemia (ATL) is a malignant disease characterized by tumorous proliferation of CD4⁺ T cells infected with retrovirus human T cell leukemia virus Type-I (HTLV-I) and concurs with an autoimmune disease and cancer due to attenuated immune response. In this study, we established ATL patient derived B-cell line TM-1 producing cancer-specific IgM antibodies, and further characterized its antigen specificity by establishing hybridomas fused with human-mouse origin hetero-myeloma cell line RF-S1. We established three hybridoma cell lines termed 2E12, 3E9, and 3E10, which continuously secreted human IgM antibodies. Immunohistochemical staining of formalin-fixed tissue section using antibodies secreted from these hybridomas showed that these antibodies specifically recognized tumor sites of human colon adenocarcinomas. Antibody produced from hybridoma 3E9 bound to some of leukemic cell lines, but not to normal human PBL, which was evidenced by the flow cytometric analysis, indicating that antibody produced from 3E9 recognizes cell surface antigen specifically expressed in the leukemic cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of a microbioreactor for glycoconjugate synthesis

A microbioreactor immobilized with a synthase-type mutant enzyme, Endo-M-N175Q (glycosynthase) of endo-β-N-acetylglucosaminidase derived from Mucor hiemalis (Endo-M), was constructed and used for glycoconjugate synthesis. The transglycosylation was performed with a reaction mixture containing an oxazoline derivative of sialo complex-type glycoside (SG), which was prepared from a sialo complex-type glycopeptide SGP derived from hen egg yolk, as a glycosyl donor and N-Fmoc-N-acetylglucosaminyl-l-asparagine [Fmoc-Asn(GlcNAc)-OH] as an acceptor. The reaction mixture was injected into a glycosynthase microbioreactor at a constant flow rate. Highly efficient and nearly stoichiometric transglycosylation occurred in the microbioreactor, and the transglycosylation product was eluted from the other end of the reactor. The glycosynthase microbioreactor was stable and could be used repeatedly for a long time.     

Accumulation of Nucleic Acid-related Substances by Microorganisms

The induction of adenosine-producing mutants from an inosine-producing mutant previously derived from a Bacillus strain was attempted, and it was found out that the xanthine-requiring mutants lacking of adenase produce a large amount of adenosine.

The outline of the processes for the derivation of these mutants was described. Main product of these mutants was adenosine, and the culture broth contained a little amount of adenine as a by-product.

The culture conditions optimal for the production of adenosine were investigated, and the yield of adenosine in the culture broth was more than 16 mg/ml.     

Effect of biodegradable chelating ligands on Fe uptake in and growth of marine microalgae

Iron (Fe) is an important nutrient for phytoplankton. The low solubility of Fe in oxic waters can be a growth-limiting factor for phytoplankton. Synthetic aminopolycarboxylates (APCs) such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) are widely used as Fe complexing agents for microalgae culture. However, the presence of these non-ready biodegradable APC_S in aquatic systems may have serious environmental consequences. In the present study, the effects of biodegradable chelating ligands (hydroxyiminodisuccinic acid (HIDS), methylglycinediacetic acid (MGDA), and iminodisuccinate (IDS)) on Fe uptake in and growth of three coastal microalgae (Heterosigma akashiwo, Prymnesium parvum, and Skeletonema marinoi-dohrnii complex) were investigated, and the results were compared with those of non-ready biodegradable APCs (EDTA, ethylenediamine tetra-methylene phosphonic acid (EDTMP), and DTPA). The biodegradable chelating ligands did not have significant growth inhibition effect on the phytoplankton. Although the growth of the algae (except S. marinoi-dohrnii complex) was not affected substantially by 1.5 and 7.5 μM of DTPA, growth inhibition occurred by 7.5 μM of EDTMP and 150 μM of EDTA, DTPA, and EDTMP. The effect of chelating ligands on microalgal growth was likely to be associated with the intracellular Fe uptake influenced by the chelating ligands. On average, intracellular Fe concentrations for biodegradable chelating ligands were substantially higher than those for non-ready biodegradable APCs. Except H. akashiwo, the ratio of intra/extracellular Fe concentrations was highest for MGDA followed by IDS and HIDS. The results indicate that biodegradable chelating ligands are more efficient than non-ready biodegradable APCs in intracellular Fe uptake and algal growth.     

Monitoring of Ralstonia eutropha KT1 in Groundwater in an Experimental Bioaugmentation Field by In Situ PCR

Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 × 10⁵ cells/ml and the amount of Ralstonia and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 × 10⁷ cells/ml 1 h after injection, and the concentration of Ralstonia detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR.     

Improved segmental isotope labeling of proteins and application to a larger protein

A new isotope labeling technique for peptide segments in a protein sample was recently established using the protein splicing element intein [Yamazaki et al. (1998) J. Am. Chem. Soc., 120, 5591–5592]. This method makes it possible to observe signals of a selected amino (N-) or carboxyl (C-) terminal region along a peptide chain. However, there is a problem with the yield of the segmentally labeled protein. In this paper, we report an increase in the yield of the protein that enables the production of sufficient amounts of segmentally ¹³C/¹⁵N-labeled protein samples. This was achieved by improvement of the expression level of the N-terminal fragment in cells and the efficiency of refolding into the active splicing conformation. The N-terminal fragment was expressed as a fused protein with the cellulose binding domain at its N-terminus, which was expressed as an insoluble peptide in cells and the expression level was increased. Incubation with 2.5 M urea and 50% glycerol increased the efficiency of the refolding greatly, thereby raising the final yields of the ligated proteins. The feasibility of application of the method to a high-molecular-weight protein was demonstrated by the results for a maltose binding protein consisting of 370 amino acids. All four examined joints in the maltose binding protein were successfully ligated to produce segmentally labeled protein samples.