Immobilization ofβ-fructofuranosidase fromAureobasidium on DEAE-cellulose

Summary -Fructofuranosidase, which produces fructo-oligosaccharides (1-kestose and nystose) from sucrose, was purified fromAureobasidium and immobilized on DEAE-cellulose at especially high efficiency (95%). The enzymatic profiles of the immobilized enzyme were almost identical to those of the native form except that the stability was slightly improved. The immobilized enzyme was stable during long-term continuous reaction for up to 360 h.     

Redox Buffer Capacity in Water-Rock-Microbe Interaction Systems in Subsurface Environments

An incubation experiment was conducted to estimate redox buffer capacity of “water-rock-microbe” interaction systems in sedimentary rocks. The water chemistry, microbial growth and community structure were analyzed during the incubations. The dissolved oxygen (DO) concentrations and oxidation-reduction potential (ORP) values decreased notably in the presence of active microorganisms, whereas abiotic reactions did not lead to reducing conditions during incubation. The change in microbial community structure suggests that nitrate-reducing and sulfate-reducing bacteria played an important role in reduction of water by using lignite-derived organic matter. These results show that the microbial role is extremely important for the redox buffering capacity in sedimentary rock environments.     

K252a, an indrocarbazole derivative, causes the membrane of myoblasts to enter a fusion-capable state

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in plants and microorganisms. ALS is the target of several structurally diverse classes of herbicides, including sulfonylureas, imidazolinones, and triazolopyrimidines. The roles of three well-conserved histidine residues (H351, H392, and H487) in tobacco ALS were determined using site-directed mutagenesis. Both H487F and H487L mutations abolished the enzymatic activity as well as the binding affinity for the cofactor FAD. Nevertheless, the mutation of H487F did not affect the secondary structure of the ALS. The K(m) values of H351M, H351Q, and H351F are approximately 18-, 60-, and fivefold higher than that of the wild-type ALS, respectively. Moreover, the K(c) value of H351Q for FAD is about 137-fold higher than that of wALS. Mutants H351M and H351Q showed very strong resistance to Londax (a sulfonylurea) and Cadre (an imidazolinone), whereas mutant H351F was weakly resistant to them. However, the secondary structures of mutants H351M and H351Q appeared to be different from that of wALS. The mutation of H392M did not have any significant effect on the kinetic parameters nor the resistance to ALS-inhibiting herbicides. These results suggest that the His487 residue is located at the active site of the enzyme and is likely involved in the binding of cofactor FAD in tobacco ALS. Mutational analyses of the His351 residue imply that the active site of the ALS is probably close to its binding site of the herbicides, Londax and Cadre.     

Peptides binding to a Gb3 mimic selected from a phage library

Peptides binding to a Gb3 mimic were selected from 12-mer peptide library. The self-assembled monolayer (SAM) of a Gb3 mimic was formed on the gold surface, and biopanning was carried out with the phage display peptide library. After three rounds of biopanning, four individual sequences were obtained from 10 phage clones, and the selected peptides having the specific 7-mer sequence (FHENWPS) showed affinities to the Gb3 mimic as strong as to RCA120. Molecular dynamics calculations suggested that the peptides bound to the Gb3 mimic by hydrophobic interaction and hydrogen bonding formation, and the cooperative interactions played an important role in the recognition. The Stx-1 binding was inhibited by the peptides.     

Decrease in Epstein-Barr virus-positive AIDS-related lymphoma in the era of highly active antiretroviral therapy

Recent introduction of highly active antiretroviral therapy (HAART) is reported to have reduced the incidence of lymphoma among HIV-infected individuals. A clinicopathological study was performed on 86 AIDS-related lymphoma patients who were treated in Tokyo area from 1987 to 2005. The incidence of lymphoma detected by autopsy was 27% (53 cases/198 autopsies). Diffuse large B cell lymphoma was the most predominant histological subtype throughout the period (78%). Burkitt's lymphoma (BL) increased from 2% in the pre-HAART era (before end-1997) to 13% in the HAART era, whereas incidence of BL did not vary between HAART users and non-users. Epstein-Barr virus (EBV)-positive lymphoma decreased from 88% in the pre-HAART era to 58% in the HAART era, but did not differ significantly between HAART users (73%) and non-users (74%). Nodal involvement of lymphoma increased from 14% in the pre-HAART era to 50% in the HAART era; however, central nervous system involvement decreased from 62 to 38%. Kaposi's sarcoma-associated herpesvirus infection was rare (4%) among all cases. These data suggest that HAART might play a partial role in these changes, and the alteration in immunological backgrounds, such as EBV prevalence, is suggested as another leading cause of these changes in Japanese AIDS-related lymphoma.     

Immunohistochemical analysis of two stem cell markers of α-smooth muscle actin and STRO-1 during wound healing of human dental pulp

Recent studies have employed two markers, alpha-smooth muscle actin (α-SMA) and STRO-1, to detect cells with mesenchymal stem cell properties in dental pulp. The present study aimed to explore the expression profile of α-SMA and STRO-1 in intact dental pulp as well as during wound healing in adult dental pulp tissue. Healthy pulps were mechanically exposed and capped with the clinically used materials MTA (ProRoot White MTA) or Ca(OH)(2) to induce a mineralized barrier at the exposed surface. After 7-42?days, the teeth were extracted and processed for immunohistochemical analysis using antibodies against α-SMA, STRO-1 and nestin (a neurogenic cytoskeletal protein expressed in odontoblasts). In normal pulp, α-SMA was detected in vascular smooth muscle cells and pericytes. Double immunofluorescent staining with STRO-1 and α-SMA showed that STRO-1 was localized in vascular smooth muscle cells, pericytes and endothelial cells, in addition to nerve fibers. During the process of dental pulp healing, numerous α-SMA-positive cells emerged at the wound margin at 14?days, and the initially formed mineralized barrier was lined with α-SMA-positive cells similar in appearance to reparative odontoblasts, some of which co-expressed nestin. STRO-1 was abundant in nerve fibers. In the advanced stage of mineralized barrier formation at 42?days, cells lining the barrier were stained with nestin, and no staining of α-SMA was detected in those cells. These observations indicate that α-SMA-positive cells temporarily appear along the wound margin during the earlier phase of mineralized barrier formation and STRO-1 is confined in vascular and neuronal elements.     

An FGF1:FGF2 chimeric growth factor exhibits universal FGF receptor specificity, enhanced stability and augmented activity useful for epithelial proliferation and radioprotection

Structural instability of wild-type fibroblast growth factor (FGF)-1 and its dependence on exogenous heparin for optimal activity diminishes its potential utility as a therapeutic agent. Here we evaluated FGFC, an FGF1:FGF2 chimeric protein, for its receptor affinity, absolute heparin-dependence, stability and potential clinical applicability. Using BaF3 transfectants overexpressing each FGF receptor (FGFR) subtype, we found that, like FGF1, FGFC activates all of the FGFR subtypes (i.e., FGFR1c, FGFR1b, FGFR2c, FGFR2b, FGFR3c, FGFR3b and FGFR4) in the presence of heparin. Moreover, FGFC activates FGFRs even in the absence of heparin. FGFC stimulated keratinocytes proliferation much more strongly than FGF2, as would be expected from its ability to activate FGFR2b. FGFC showed greater structural stability, biological activity and resistance to trypsinization, and less loss in solution than FGF1 or FGF2. When FGFC was intraperitoneally administered to BALB/c mice prior to whole body gamma-irradiation, survival of small intestine crypts was significantly enhanced, as compared to control mice. These results suggest that FGFC could be useful in a variety of clinical applications, including promotion of wound healing and protection against radiation-induced damage.     

Fibronectin Combined with Stem Cell Factor Plays an Important Role in Melanocyte Proliferation,Differentiation and Migration in Cultured Mouse Neural Crest Cells

Stem cell factor (SCF) is essential to the migration and differentiation of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, KIT, result in defects in coat pigmentation in mice. Using a neural crest cell (NCC) primary culture system from wild‐type mice, we previously demonstrated that KIT‐positive and/or L ‐3, 4‐dihydroxyphenylalanine (DOPA)‐positive melanocyte precursors proliferate following the addition of SCF to the culture medium. Extracellular matrix (ECM) proteins are considered to play a role in the migration and differentiation of various cells including melanocytes. We cultured mouse NCCs in the presence of SCF in individual wells coated with ECM; fibronectin (FN), collagen I (CLI), chondroitin sulphate, or dermatan sulphate. More KIT‐positive cells and DOPA‐positive cells were detected in the presence of SCF on ECM‐coated wells than on non‐coated wells. A statistically significant increase in DOPA‐positive cells was evident in FN and CLI wells. In contrast, in the absence of SCF, few DOPA‐positive cells and KIT‐positive cells were detected on either the ECM‐coated or non‐coated wells. We concluded that ECM affect melanocyte proliferation and development in the presence of SCF. To determine the key site of FN function, RGDS peptides in the FN sequence, which supports spreading of NCCs, were added to the NCC culture. The number of DOPA‐positive cells decreased with RGDS concentration in a dose‐dependent fashion. Immunohistochemical staining revealed the presence of integrin a5, a receptor of RGDS, in NCCs. These results suggest the RGDS domain of FN plays a contributory role as an active site in the induction of FN function in NCCs. In addition, we examined the effect of FN with SCF on the NCC migration by measuring cluster size, and found an increase in size following treatment with FN.     

Vacuolar processing enzyme is up-regulated in the lytic vacuoles of vegetative tissues during senescence and under various stressed conditions

Vacuolar processing enzyme (VPE) has been shown to be responsible for maturation of various seed proteins in protein-storage vacuoles. Arabidopsis has three VPE homologues; betaVPE is specific to seeds and alphaVPE and gammaVPE are specific to vegetative organs. To investigate the activity of the vegetative VPE, we expressed the gammaVPE in a pep4 strain of the yeast Saccharomyces cerevisiae and found that gammaVPE has the ability to cleave the peptide bond at the carbonyl side of asparagine residues. An immunocytochemical analysis revealed the specific localization of the gammaVPE in the lytic vacuoles of Arabidopsis leaves that had been treated with wounding. These findings indicate that gammaVPE functions in the lytic vacuoles as the betaVPE does in the protein-storage vacuoles. The betaVPE promoter was found to direct the expression of the beta-glucuronidase reporter gene in seeds and the root tip of transgenic Arabidopsis plants. On the other hand, both the alphaVPE and gammaVPE promoters directed the expression in senescent tissues, but not in young intact tissues. The mRNA levels of both alphaVPE and gammaVPE were increased in the primary leaves during senescence in parallel with the increase of the mRNA level of a senescence-associated gene (SAG2). Treatment with wounding, ethylene and salicylic acid up-regulated the expression of alphaVPE and gammaVPE, while jasmonate slightly up-regulated the expression of gammaVPE. These gene expression patterns of the VPEs were associated with the accumulation of vacuolar proteins that are known to respond to these treatments. Taken together, the results suggest that vegetative VPE might regulate the activation of some functional proteins in the lytic vacuoles.