Continous production of 1-kestose by β-fructofuranosidase immobilized onshirasu porous glass

Summary 1-Kestose was produced continously and selectively from 40% (w/v) sucrose solution at fast flow rate by a column packed with an immobilized -fructofuranosidase onshirasu porous glass.     

Cytokinin Activity of cis-Zeatin and Phenotypic Alterations Induced by Overexpression of Putative cis-Zeatin-O-glucosyltransferase in Rice

cis-Zeatin (cZ) is generally regarded as a cytokinin with little or no activity, compared with the highly active trans-zeatin (tZ). Although recent studies suggested possible roles for cZ, its physiological significance remains unclear. In our studies with rice (Oryza sativa), cZ inhibited seminal root elongation and up-regulated cytokinin-inducible genes, and its activities were comparable to those of tZ. Tracer experiments showed that exogenously supplied cZ-riboside was mainly converted into cZ derivatives but scarcely into tZ derivatives, indicating that isomerizations of cZ derivatives into tZ derivatives are a minor pathway in rice cytokinin metabolism. We identified three putative cZ-O-glucosyltransferases (cZOGT1, cZOGT2, and cZOGT3) in rice. The cZOGTs preferentially catalyzed O-glucosylation of cZ and cZ-riboside rather than tZ and tZ-riboside in vitro. Transgenic rice lines ectopically overexpressing the cZOGT1 and cZOGT2 genes exhibited short-shoot phenotypes, delay of leaf senescence, and decrease in crown root number, while cZOGT3 overexpressor lines did not show shortened shoots. These results propose that cZ activity has a physiological impact on the growth and development of rice.     

Galectin-8 modulates neutrophil function via interaction with integrin alphaM

The members of the galectin family are associated with diverse cellular events, including immune response. We investigated the effects of galectin-8 on neutrophil function. Human galectin-8 induced firm and reversible adhesion of peripheral blood neutrophils but not eosinophils to a plastic surface in a lactose-sensitive manner. Other human galectins, galectins-1, -3, and -9, showed low or negligible effects on neutrophil adhesion. Confocal microscopy revealed actin bundle formation in the presence of galectin-8. Cytochalasins inhibited both actin assembly and cell adhesion induced by galectin-8. Affinity purification of galectin-interacting proteins from solubilized neutrophil membrane revealed that N-terminal carbohydrate recognition domain (CRD) of galectin-8 bound promatrix metalloproteinase-9 (proMMP-9), and C-terminal CRD bound integrin alphaM/CD11b and proMMP-9. A mutant galectin-8 lacking the carbohydrate-binding activity of N-terminal CRD (galectin-8R69H) retained adhesion-inducing activity, but inactivation of C-terminal CRD (galectin-8R233H) abolished the activity. MMP-3-mediated processing of proMMP-9 was accelerated by galectin-8, and this effect was inhibited by lactose. Galectins-1 and -3 did not affect the processing. Superoxide production, an essential event in bactericidal function of neutrophils, was stimulated by galectin-8 to an extent comparable to that induced by fMLP. Galectin-8R69H but not galectin-8R233H could stimulate superoxide production. Taken together, these results suggest that galectin-8 is a novel factor that modulates the neutrophil function related to transendothelial migration and microbial killing.     

The role of intrinsically disordered C‐terminal region of FliK in substrate specificity switching of the bacterial flagellar type III export apparatus

The bacterial flagellar export switching machinery consists of a ruler protein, FliK, and an export switch protein, FlhB and switches substrate specificity of the flagellar type III export apparatus upon completion of hook assembly. An interaction between the C‐terminal domain of FliK (FliK_C) and the C‐terminal cytoplasmic domain of FlhB (FlhB_C) is postulated to be responsible for this switch. FliK_C has a compactly folded domain termed FliK_T3S4 (residues 268–352) and an intrinsically disordered region composed of the last 53 residues, FliK_CT (residues 353–405). Residues 301–350 of FliK_T3S4 and the last five residues of FliK_CT are critical for the switching function of FliK. FliK_CT is postulated to regulate the interaction of FliK_T3S4 with FlhB_C, but it remains unknown how. Here we report the role of FliK_CT in the export switching mechanism. Systematic deletion analyses of FliK_CT revealed that residues of 351–370 are responsible for efficient switching of substrate specificity of the export apparatus. Suppressor mutant analyses showed that FliK_CT coordinates FliK_T3S4 action with the switching. Site‐directed photo‐cross‐linking experiments showed that Val‐302 and Ile‐304 in the hydrophobic core of FliK_T3S4 bind to FlhB_C. We propose that FliK_CT may induce conformational rearrangements of FliK_T3S4 to bind to FlhB_C.     

Accumulation of l-Glutamic Acid from Dibasic Carboxylic Acids by a Hydrocarbon Utilizing Bacterium

In order to know the substrate specificity in a hydrocarbon utilizing bacterium, the following materials were examined: n-alkanes, n-alkenes, monohydric alcohols, aldehydes, monobasic carboxylic acids, dihydric alcohols and dibasic carboxylic acids.

It was found that dibasic carboxylic acids were well utilized, and a great deal of l-glutamic acid was accumulated from them. Then suberic acid, which is C₈ dibasic carboxylic acid, was compared with n-dodecane in the effects of thiamine, penicillin, C/N ratio and substrate concentration on l-glutamic acid accumulation and cell growth.     

Utility and Limitation of Cumulative Stone Diameter in Predicting Urinary Stone Burden at Flexible Ureteroscopy with Holmium Laser Lithotripsy: A Single-Center Experience

Purpose

To retrospectively assess the clinical utility in ureteroscopy (URS) planning of cumulative stone diameter (CSD), which does not account for stone width or depth, as a predictor of URS outcome and compare it with stone volume.

Materials and Methods

Patients with renal stones treated at a single institute by flexible URS were retrospectively evaluated. To assess the clinical utility of CSD, relationships between stone-free (SF) status and stone burden (CSD and volume) were analyzed using the area under the receiver operating characteristics (AUROC) curve. To identify stone number impact on CSD, the AUROC of CSD divided by stone number was evaluated. Correlation coefficients of CSD and stone volume were also calculated for groups by stone number.

Results

In cases with CSD <20.0 mm, CSD and stone volume revealed equal ability to predict SF status. In cases with CSD ≥20.0 mm, stone volume showed higher predictive ability. The ROC curves for cases with ≥4 stones showed that CSD was less predictive of SF status than stone volume. The correlation coefficients of CSD and stone volume by stone number were 0.922 for 1 stone, 0.900 for 2–3 stones, and 0.661 for ≥4 stones.

Conclusions

In cases with CSD ≥20.0 mm or ≥4 stones, we should evaluate stone volume for a more predictive stone burden, and pretreatment non-contrast CT seems sufficient. In cases with CSD <20.0 mm or 1–3 stones, CSD was as valid a predictor of preoperative stone burden as stone volume, so preoperative kidney-ureter-bladder (KUB) films may be sufficient.     

Chondrogenesis enhanced by overexpression of sox9 gene in mouse bone marrow-derived mesenchymal stem cells

We investigated chondrogenesis of cell-mediated sox9 gene therapy as a new treatment regimen for cartilage regeneration. pIRES2-EGFP vector containing a full-length mouse sox9 cDNA was transfected into bone marrow-derived mesenchymal stem cells (MSCs) by lipofection and chondrogenic differentiation of these cells was evaluated. In vitro high density micromass culture of these sox9 transfected MSCs demonstrated that a matrix-rich micromass aggregate with EGFP expressing MSCs was positively stained by Alcian blue and type II collagen. Next, sox9 transfected MSCs were loaded into the diffusion chamber and transplanted into athymic mice to analyze in vivo chondrogenesis. A massive tissue formation in about 2mm diameter was visible in the chamber after 4 weeks transplantation. Histological examinations demonstrated that both Alcian blue and type II collagen were positively stained in the extracellular matrix of the mass while type X collagen was not stained. These results indicated that cell-mediated sox9 gene therapy could be a novel strategy for hyaline cartilage damage.     

Production of Nucleic Acid-Related Substances by Fermentative Processes. XXVIII. Accumulation of 5′ Inosinic Acid by a Manganese-Insensitive Mutant of Brevibacterium ammoniagenes

A manganese-insensitive mutant, KY 13105, of Brevibacterium ammoniagenes which accumulates considerable amounts of 5' inosinic acid (IMP) in the presence of 100 to 1,000 mug of Mn(2+) per liter was obtained from an IMP-producing mutant of a manganese-sensitive strain, KY 13102. The effects of Mn(2+) at 0 to 30 mug/liter on IMP accumulation by KY 13105 were similar to those by KY 13102. However, the accumulation of IMP by KY 13105 was not affected by 100 to 1,000 mug of Mn(2+) per liter, showing a clear difference from KY 13102. The accumulation of IMP by KY 13105 was always accompanied by cellular morphological changes irrespective of Mn(2+) concentration. In the presence of Mn(2+), factors which affect IMP accumulation by KY 13105 were examined. Most of the nutrients tested stimulated IMP accumulation at a relatively low concentration (2 g/liter). Iron, calcium, and zinc were found to be essential for IMP accumulation and were independent of Mn(2+). Biotin regulated the growth but not the accumulation of IMP. Under limited or surplus amounts of Mn(2+), the dynamics of IMP fermentation were followed. Under both conditions, the fermentations proceeded in a similar way. The morphological changes were found to be closely related to IMP accumulation.