Active transport of glucose-1-phosphate in Agrobacterium tumefaciens

The presence of an active transport system for glucose-1-phosphate in Agrobacterium tumefaciens was demonstrated from the following observations. (i) The bacterium could grow on a medium containing glucose-1-phosphate as carbon source; (ii) the entry of glucose-1-phosphate into the resting cells occurred against concentration gradient obeying Michaelis-Menten kinetics; and (iii) the entry reaction was energy-dependent. The transport system for glucose-1-phosphate was formed inducibly by growing the organism on a glucose-1-phosphate or sucrose medium. From the inhibition and kinetics studies it was found that the transport system had a high specificity for glucose-1-phosphate with a high affinity, K(m) value of 4.5 x 10(-6)m at pH 8.2. The existence of glucose-1-phosphate binding factor was proved in the shock fluid which was prepared from the cells grown on both glucose-1-phosphate and sucrose media by osmotic shock.     

Quantitative evaluation of predation by spiders on the green rice leafhopper,Nephotettix cincticeps Uhler,by a sight-count method

A sight-count method for evaluation of predation by spiders on the green rice leafhopper, Nephotettix cincticeps was proposed and its applicability was tested under natural conditions. The number (n) of leafhoppers preyed on by spiders per rice hill per day was estimated by the formula: (1) where F is the frequency of predation observed per hill:P is given by dividing the time spent feeding on prey by 24 hours; and C refers to the total amount of feeding activity expressed in terms of the activity during the standard time interval. The total number (N) of prey attacked during the specified period can be given as follows: (2) With this method, the role of paddy-inhabiting spiders, Lycosa pseudoannulata, Oedothorax insecticeps, Tetragnatha spp, and Enoplognatha japonica, as predator of N. cincticeps was evaluated with reference to life tables of the prey. The advantages and limitation of the sight-count method were discussed as compared with other methods so far proposed.     

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.     

Characterization of multimetric variants of ubiquitin carboxyl-terminal hydrolase L1 in water by small-angle neutron scattering

Here, we illustrated that the morphological structures of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) variants and Parkinson's disease (PD) exhibit good pathological correlation by a small-angle neutron scattering (SANS). UCH-L1 is a neuro-specific multiple functional enzyme, deubiquitinating, ubiquityl ligase, and also involved in stabilization of mono-ubiquitin. To examine the relationship between multiple functions of UCH-L1 and the configuration of its variants [wild-type, I93M (linked to familial Parkinson's disease), and S18Y (linked to reduced risk of Parkinson's disease)], in this report, we proposed that these were all self-assembled dimers by an application of a rotating ellipsoidal model; the configurations of these dimers were quite different. The wild-type was a rotating ellipsoidal. The globular form of the monomeric component deformed by the I93M mutation. Conversely, the S18Y polymorphism promoted the globularity. Thus, the multiple functional balance is closely linked to the intermolecular interactions between the UCH-L1 monomer and the final dimeric configuration.     

Production of (R)-3-amino-3-phenylpropionic acid and (S)-3-amino-3-phenylpropionic acid from (R,S)-N-acetyl-3-amino-3-phenylpropionic acid using microorganisms having enantiomer-specific amidohydrolyzing activity

(R)-3-Amino-3-phenylpropionic acid ((R)-beta-Phe) and (S)-3-amino-3-phenylpropionic acid ((S)-beta-Phe) are key compounds on account of their use as intermediates in synthesizing pharmaceuticals. Enantiomerically pure non-natural amino acids are generally prepared by enzymatic resolution of the racemic N-acetyl form, but despite the intense efforts this method could not be used for preparing enantiomerically pure beta-Phe, because the effective enzyme had not been found. Therefore, screening for microorganisms capable of amidohydrolyzing (R,S)-N-acetyl-3-amino-3-phenylpropionic acid ((R,S)-N-Ac-beta-Phe) in an enantiomer-specific manner was performed. A microorganism having (R)-enantiomer-specific amidohydrolyzing activity and another having both (R)-enantiomer- and (S)-enantiomer-specific amidohydrolyzing activities were obtained from soil samples. Using 16S rDNA analysis, the former organism was identified as Variovorax sp., and the latter as Burkholderia sp. Using these organisms, enantiomerically pure (R)-beta-Phe (>99.5% ee) and (S)-beta-Phe (>99.5% ee) with a high molar conversion yield (67%-96%) were obtained from the racemic substrate.     

MbIDGF, a novel member of the imaginal disc growth factor family in Mamestra brassicae, stimulates cell proliferation in two lepidopteran cell lines without insulin

Imaginal disc growth factor (IDGF) is a soluble polypeptide growth factor that was first identified from the conditioned medium of Drosophilia imaginal disc C1.8+ cells. Working with insulin, IDGF stimulated the growth of cultured imaginal disk cells, which suggested that IDGF might function as a cofactor of Drosophila insulin or insulin like peptide. Here we report a new member of the IDGF family, named MbIDGF, from the cabbage armyworm, Mamestra brassicae. Using a cloned cDNA of MbIDGF, recombinant MbIDGF protein was expressed in baculovirus-infected Sf9 cells and purified. Without insulin, the recombinant MbIDGF protein stimulated cell growth of SES-MaBr-4 and NIAS-MaBr-93 cell lines that were derived from the fat bodies and hemocytes of M. brassicae, in a dose-dependent manner. The saturation of growth stimulation by MbIDGF was attained for the two types of cells at 80 ng/ml (0.8 nM) and 300 ng/ml (6 nM), respectively. The results suggest that MbIDGF may stimulate the growth of lepidopteran cells by a new mechanism without associating with the insulin pathway.     

Ultrafiltration-based assay for heparanase activity

Heparanase, a mammalian endoglycosidase that specifically cleaves heparan sulfate (HS), has been found in many tissues. Platelet, liver, and placenta have been abundant sources for the study of the enzyme. Notably, certain malignant cells also have been found to produce large amounts of the enzyme, the levels of which often correlate with their invasive and metastatic properties. To study roles of heparanase in various biological situations, a reliable method measuring the enzyme activity is indispensable. In the past, measurement of heparanase enzyme activity was done either by the detection of the degradation of fluorescent or radiolabeled HS chains by gel filtration procedures or by the use of radiolabeled substrate conjugated to solid matrices for the easy separation of degraded HS chains. A newly developed procedure, presented in this article, measures degradation of radiolabeled HS chains in the aqueous buffer by detecting their degradation products using an ultrafiltration device, the Centricon 30. This procedure has several advantages over previous assay procedures that involved tedious processing such as gel filtration chromatography of each sample or the preparation of substrate HS proteoglycans conjugated to a solid matrix. The simplicity of the new procedure allows a short setup time and a rapid processing of a large number of samples. Furthermore, the enzymatic reaction during the aqueous phase allows kinetic analyses in standard conditions.     

15-deoxy-delta 12,14-prostaglandin J2 and laminar fluid shear stress stabilize c-IAP1 in vascular endothelial cells

Laminar shear stress strongly inhibits vascular endothelial cell apoptosis by unknown mechanisms. We reported that shear stress stimulates endothelial cells to produce 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) by elevating the expression level of lipocalin-type prostaglandin D synthase. To investigate the role of 15d-PGJ2 produced in the vascular wall, we examined the effect of 15d-PGJ2 on endothelial cell apoptosis. We induced apoptosis in human umbilical vein endothelial cells (HUVECs) by growth factor deprivation. 15d-PGJ2 strongly inhibited DNA ladder formation, nuclear fragmentation, and caspase-3-like activity in HUVECs. To elucidate the mechanism by which 15d-PGJ2 inhibits endothelial cell apoptosis, we examined expression of the inhibitor of apoptosis proteins (IAP) cellular-IAP1 (c-IAP1), c-IAP2, x-linked IAP, and survivin in HUVECs. In parallel with the inhibition of apoptosis, 15d-PGJ2 elevated the expression level of c-IAP1 protein in a dose- and time-dependent manner without changing the mRNA level. Laminar shear stress also induced c-IAP1 expression. Chase experiments with the use of cycloheximide revealed that 15d-PGJ2 and shear stress both inhibited the proteolytic degradation of c-IAP1 protein. These results suggested that 15d-PGJ2 inhibits endothelial cell apoptosis through, at least in part, c-IAP1 protein stabilization. This mechanism might be involved in the antiapoptotic effect of laminar shear stress.     

The most appropriate position and number for absolute anchorages for orthodontic tooth movements

Absolute anchorages proved to be very effective for orthodontic tooth movements. We used a 3D digitizer to record each tooth on pre-treatment diagnostic and post-treatment predictive setup models and then 3D coordinate system conversion was performed to make the coordinate values comparable. An arithmetic calculation of vector and moment based on the orthodontic forces and the tooth displacement under preliminary premises undertaken to decide the most favorable position and number for absolute anchorages. Position--For two-dimensional and three-dimensional calculations, the most appropriate positions for absolute anchorages should theoretically be on the line of resultant force (2D) and the plane (3D) where the total moment effect tends to be zero. Number--As for the number of the absolute anchorages needed, it depends on the number of target teeth. Different combinations of target teeth provide different sets of results.