Purification and Properties of 2-Ketogluconate Reductase from Gluconobacter liquefaciens.

2-Ketogluconate reductase (2KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was purified about 1000-fold by a procedure involving ammonium sulfate fractionation and column chromatographies using DEAE-cellulose, hydroxylapatite, and Sephadex gel The purified enzyme gave a single band on polyacrymamide gel electrophoresis. NADP was specifically required for the oxidation reaction of gluconic acid. Using gel filtration a molecular weight of about 110,000 was estimated for the enzyme. The pH optimum for the oxidation of gluconic acid (GA) to 2-ketogluconic acid (2KGA) by the enzyme was 10.5 and for the reduction of 2KGA was 6.5. The optimum temperature of the enzyme was 50 C for both reactions of oxidation and reduction. The enzyme was stable at pH between 5.0 and 11.0 and at temperature under 50°C, The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ions, but remarkably stimulated by manganese ions (1×10^?3 m). Km value of the enzyme for GA was 1.3×10^?2 m and for 2KGA was 6.6×10^?3 _m. Km values for NADP and NADPH₂ were 1.25×10^?5 and 1.52×10^?5 m respectively.     

Cobalt chloride decreases EC-SOD expression through intracellular ROS generation and p38-MAPK pathways in COS7 cells

It is known that cells suffer a chronic hypoxic condition during the development of proximal tubulointerstitial disease. However, it is accepted that extracellular-superoxide dismutase (EC-SOD) protects the cells from oxidative stress. The purpose of this study was to elucidate the regulation of EC-SOD expression in cells under hypoxia. The results show that the expressions of EC-SOD mRNA and protein in cobalt chloride (CoCl₂)-treated COS7 cells decreased in a dose- and time-dependent manner, whereas the expressions of other SOD isoforms (Cu/Zn-SOD and Mn-SOD) were not changed. The down-regulation of EC-SOD mRNA was suppressed by pre-treatment with the antioxidant trolox and the p38 mitogen-activated protein kinase (p38-MAPK) inhibitor SB203580. It is concluded that the expression of EC-SOD is decreased through ROS and p38-MAPK signalling cascades and that the down-regulation of EC-SOD leads to a decrease in the resistance to oxidative stress of COS7 cells under hypoxia induced by CoCl₂.     

Induction of IFN-γ by a highly branched 1,3-β-d-glucan from Aureobasidium pullulans in mouse-derived splenocytes via dectin-1-independent pathways

We have previously elucidated the precise structure of a unique type of 1,3-β-D-glucan, AP-FBG (Aureobasidium pullulans-fermented β-D-glucan), from the fungus A. pullulans and found that AP-FBG strongly induced the production of various cytokines in DBA/2 mouse-derived splenocytes in vitro. However, the mechanism(s) of action of AP-FBG on in vitro mouse primary cells have not been characterized in detail. Herein, we report that the production of IFN-γ in DBA/2 mouse-derived splenocytes by AP-FBG was not inhibited following treatment with an anti-dectin-1 neutralizing antibody. In addition, AP-FBG not only failed to activate dectin-1-mediated signaling pathways, examined by a reporter gene assay but also failed to bind to dectin-1, a pivotal receptor for 1,3-β-D-glucan. Taken together, AP-FBG induced cell activation via dectin-1-independent pathways.     

Effect of tensile force on the mechanical behavior of actin filaments

Actin filaments are the most abundant components of the cellular cytoskeleton, and play critical roles in various cellular functions such as migration, division and shape control. In these activities, mechanical tension causes structural changes in the double-helical structure of the actin filament, which is a key modulator of cytoskeletal reorganization. This study performed large-scale molecular dynamics (MD) and steered MD simulations to quantitatively analyze the effects of tensile force on the mechanical behavior of actin filaments. The results revealed that when a tensile force of 200pN was applied to a filament consisting of 14 actin subunits, the twist angle of the filament decreased by approximately 20°, corresponding to a rotation of approximately -2° per subunit, representing a critical structural change in actin filaments. Based on these structural changes, the variance in filament length and twist angle was found to decrease, leading to increases in extensional and torsional stiffness. Torsional stiffness increased significantly under the tensile condition, and the ratio of filament stiffness under tensile force to that under no external force increased significantly on longer temporal scales. The results obtained from this study contribute to the understanding of mechano-chemical interactions concerning actin dynamics, showing that increased tensile force in the filament prevents actin regulatory proteins from binding to the filament.     

Vasculitis and anaphylactoid shock in mice induced by the polysaccharide fraction secreted into culture supernatants by the fungus Candida metapsilosis

The biological effects of Candida metapsilosis water-soluble fraction (CMWS), prepared using a completely synthesized medium, were examined to determine whether CMWS induces vasculitis similar to that seen in Kawasaki disease, and anaphylactoid shock, in mice. It was found that intraperitoneal injection of CMWS induces coronary arteritis and i.v. injection induces acute anaphylactoid shock in mice, similar to Candida albicans water-soluble fraction (CAWS)-induced arteritis and anaphylactoid shock. The mannan structure of the polysaccharide fraction was then analyzed by performing antiserum reactivity tests and nuclear magnetic resonance spectroscopy. The mannan structure was investigated because the present authors have recently found that the mannan moiety within the polysaccharide fraction might be responsible for these pathogenic activities. The structural analysis showed that the mannan structure within CMWS expresses α-mannan residues, but not β-mannan. In addition, the mannan structure of CMWS is quite similar to that of CAWS. The present findings indicate that the polysaccharide fraction from C. metapsilosis, which is mainly composed of mannan, contributes to coronary arteritis and acute shock, and that the mannan structure could be responsible for this pathogenicity.     

Relationship between gonadal steroid hormones and vulvar bleeding in southern tamandua, Tamandua tetradactyla

This study aimed at demonstrating the profiles of circulating gonadal steroid hormones during the estrous cycle and pregnancy in a southern tamandua (Tamandua tetradactyla). Additionally, this study clarified the relationship between vulvar bleeding and hormonal changes. The concentrations of serum progesterone (P(4)) and estradiol-17β (E(2)) were determined by enzyme immunoassays. Serum P(4) and E(2) concentrations changed cyclically and the estrous cycle length (± SD) based on the E(2) cycles was 44.3 ± 4.5 days. Vulvar bleeding started to be seen at the decreasing of P(4). The cycle length for vulvar bleeding was 43.3 ± 4.2 days. Interval from the first day of bleeding to the peak of E(2) concentration was 23.1 ± 3.1 days. Serum P(4) during pregnancy remained high and E(2) increased 8 weeks after conception and remained high until parturition. The female delivered normally after a 165 day-pregnancy period and reared the offspring well. Approximately 3 weeks after parturition, serum E(2) and P(4) cycles resumed. Visual bleeding may be useful as a real-time indicator for understanding the ovarian cycle of southern tamanduas, and estrus could be expected approximately 3 weeks after the first bleeding.     

Excretion into feces of asialo GM1 in the murine digestive tract and <Emphasis Type="Italic">Lactobacillus johnsonii</Emphasis> exhibiting binding ability toward asialo GM1. A possible role of epithelial glycolipids in the discharge of intestinal bacteria

In the digestive tract of mice (HR-1, 5 months old, ♀), asialo GM1 (GA1) exhibiting receptor activity toward several intestinal bacteria was preferentially expressed in the small intestine. Also, less than 10% of GA1 in the small intestine was converted into fucosylated and sulfated derivatives, but it was completely converted to fucosyl GA1 (FGA1) in the stomach, cecum and colon. Among the lipid components in these tissues, glycolipids other than Forssman antigen and cholesterol sulfate (CS) were present in the digestive tract contents. However, sulfated GA1, sulfatide and fucosyl GM1 in the gastro-intestinal contents were not present in the cecal and colonic contents, in which the major glycolipids were ceramide monohexoside (CMH), GA1 and FGA1. The total amount of GA1 in the whole contents was 20% of that in the tissues. Thus, glycolipids were stable during the process of digestion, and excreted from the body together with cholesterol and CS. On the other hand, Lactobacillus johnsonii (LJ), whose receptor is GA1, was detected in the cecal and colonic contents on sequential analysis of 16S-ribosomal RNA and TLC-immunostaining of antigenic glycolipids with anti-LJ antiserum. LJ was found to comprise 20% of the total bacteria cultured in the lactobacillus medium under aerobic conditions, and to be present in the cecal and colonic contents, 9.8 × 10⁷ cells versus 37 μg GA1 and 1.4 × 10⁸ cells versus 49 μg GA1, respectively. Thus, GA1 in the contents might facilitate the discharge of intestinal bacteria by becoming attached them to prevent their irregular diffusion.     

Alkali-treated collagen retained the triple helical conformation and the ligand activity for the cell adhesion via alpha2beta1 integrin

Alkaline treatment is a good method for extracting collagen with high recovery even from an aged animal specimen. However, the properties of collagen treated under alkaline conditions have not been well established yet. By the treatment with a solution of 3% sodium hydroxide and 1.9% monomethylamine, the isoelectric point of type I collagen was lowered from 9.3 to 4.8 because of the conversions of Asn and Gln to Asp and Glu. With the acidification of the pI, the denaturation temperature of the collagen was decreased from 42 to 35 degrees C after 20 d treatment, but the collagen-specific triple helical conformation was maintained. Human keratinocytes and fibroblasts adhered to the alkali-treated collagen via the collagen receptor integrin alpha2beta1. This indicates that the alkali-treated collagen maintained its property as a biological adherent molecule. Unlike acid-soluble collagen, alkali-treated collagen lost the ability to form fibrils at neutral pH under physiological conditions. This ability was lost even after 4 h of alkaline treatment, when the denaturation temperature of the collagen did not change. On the other hand, the alkali-treated collagen formed a fibrous precipitate with a uniform diameter of 50-70 nm under acidic conditions at 30 degrees C.