Purification and properties of extracellularβ-fructofuranosidases fromAureobasidium sp. ATCC 20524

Summary Two extracellular -fructofuranosidases (E-1 andE-2) fromAureobasidium sp. ATCC 20524, producing 1-kestose (1^F--fructofuranosyl-sucrose) from sucrose, were purified to homogeneity. Molecular weights of the enzymes were estimated to be about 304000 (E-1) and 315000 (E-2) Da by gel filtration. The enzymes contained 33% (w/w) (E-1) and 27% (w/w) (E-2) carbohydrate. TheK _m values for sucrose ofE-1 andE-2 andE-2 were 0.34 and 0.28 M, respectively. were 0.34 and 0.28 M, respectively. The enzymatic profiles of these enzymes were almost identical to intracellular enzymesP-1 andP-2 except for the differences in carbohydrate content andK _m values ofE-2 andP-2.     

Effect of deglycosylation on the properties of β-fructofuranosidaseP-1 fromAureobasidium sp. ATCC 20524

Summary Most of the carbohydrate moiety of -fructofuranosidaseP-1 fromAureobasidium sp. ATCC 20524 was removed by endo--N-acetylglucosaminidase F. A subunit of 94000 Da was observed in SDS-PAGE after deglycosylation. TheK _m value for sucrose was not changed by deglycosylation but the stability at pH 4–5 and 50°C was decreased. The deglycosylated enzyme was more sensitive to proteases such as pronase E and subtilisin than the native enzyme. It is considered that the carbohydrate moiety of -fructofuranosidaseP-1 contributes to the stability of the enzyme but is not essential in its catalytic function.     

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.     

Expression and inducibility in Staphylococcus aureus of the mecA gene, which encodes a methicillin-resistant S. aureus-specific penicillin-binding protein.

A beta-lactam-sensitive strain of Staphylococcus aureus could be converted to methicillin resistance by the introduction of a plasmid carrying the 4.3-kilobase HindIII chromosomal DNA fragment which encoded the mecA gene from a methicillin-resistant S. aureus. Transformant cells produced methicillin-resistant S. aureus-specific penicillin-binding protein constitutively, and additional insertion of an inducible penicillinase plasmid caused production of the pencillin-binding protein to become inducible.     

Cirp protects against tumor necrosis factor-alpha-induced apoptosis via activation of extracellular signal-regulated kinase

Mild hypothermia shows protective effects on patients with brain damage and cardiac arrest. To elucidate the molecular mechanisms underlying these effects, we analyzed the effects of low culture temperature (32 degrees C) and cold-inducible RNA-binding protein (Cirp) expression on apoptosis in vitro. In BALB/3T3 cells treated with tumor necrosis factor (TNF)-alpha and cycloheximide, the down-shift in temperature from 37 degrees C to 32 degrees C increased the expression of Cirp and suppressed the apoptosis. Activation of caspase-8 was suppressed, and the level of phosphorylated extracellular signal-regulated kinase (ERK) was increased. Transduction of Cirp into the Cirp-deficient mouse fibroblasts increased the level of phosphorylated ERK and suppressed the TNF-alpha-induced apoptosis both at 37 degrees C and 32 degrees C. The ERK-specific inhibitor PD98059 decreased the cytoprotective effect of Cirp as well as that of low culture temperature. These data suggest that mild hypothermia protects cells from TNF-alpha-induced apoptosis, at least partly, via induction of Cirp, and that Cirp protects cells by activating the ERK pathway.     

Folate receptor-targeted novel boron compound for boron neutron capture therapy on F98 glioma-bearing rats

Radiation and Environmental Biophysics - Folic acid (FA) has high affinity for the folate receptor (FR), which is limited expressed in normal human tissues, but over-expressed in several tumor...     

Reevaluation of erythropoietin production by the nephron

Erythropoietin production has been reported to occur in the peritubular interstitial fibroblasts in the kidney. Since the erythropoietin production in the nephron is controversial, we reevaluated the erythropoietin production in the kidney. We examined mRNA expressions of erythropoietin and HIF PHD2 using high-sensitive in situ hybridization system (ISH) and protein expression of HIF PHD2 using immunohistochemistry in the kidney. We further investigated the mechanism of erythropoietin production by hypoxia in vitro using human liver hepatocell (HepG2) and rat intercalated cell line (IN-IC cells). ISH in mice showed mRNA expression of erythropoietin in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs) and cortical collecting ducts (CCDs) but not in the peritubular cells under normal conditions. Hypoxia induced mRNA expression of erythropoietin largely in peritubular cells and slightly in PCTs, DCTs, and CCDs. Double staining with AQP3 or AE1 indicated that erythropoietin mRNA expresses mainly in β-intercalated or non α/non β-intercalated cells of the collecting ducts. Immunohistochemistry in rat showed the expression of HIF PHD2 in the collecting ducts and peritubular cells and its increase by anemia in peritubular cells. In IN-IC cells, hypoxia increased mRNA expression of erythropoietin, erythropoietin concentration in the medium and protein expression of HIF PHD2. These data suggest that erythropoietin is produced by the cortical nephrons mainly in the intercalated cells, but not in the peritubular cells, in normal hematopoietic condition and by mainly peritubular cells in hypoxia, suggesting the different regulation mechanism between the nephrons and peritubular cells.