Purification and characterization of N-acetylglucosamine 6-phosphate deacetylase with activity against N-acetylglucosamine from Vibrio cholerae non-O1

An enzyme that deacetylates N-acetylglucosamine to glucosamine from Vibrio cholerae non-O1 was purified to homogeneity by sequential procedures. The native enzyme had a molecular mass of 190,000 Da and was predicted to be composed of four identical subunits with molecular masses of 45,000 Da. The purified enzyme hydrolyzed N-acetylglucosamine, N-acetylglucosamine 6-phosphate, and N-acetylglucosamine 6-sulfate, but not chitin oligosaccharides, and N-acetylgalactosamine. The deacetylase activity was completely abolished by N-ethylmaleimide, p-chloromercuribenzoate, EDTA, and Cu2+. On the other hand, the activity was activated by Co2+. The amino-terminal amino acids of the purified enzyme were sequenced. Among the 22 N-terminal amino acid residues, 12 residues of Vibrio deacetylase were identical with that of Escherichia coli GlcNAc 6-phosphate deacetylase.     

Development of RYUGA for three-dimensional dynamic visualization of molecular dynamics results

We have developed a new computer graphics (CG) code RYUGA for three-dimensional dynamic visualization of molecular dynamics (MD) results. The applicability of RYUGA for visualizing and analyzing the dynamics of atomic motions in various materials was demonstrated. RYUGA supports various functions, such as solid-modeling CG pictures (called the CPK model), CG animation of the MD results, Miller plane cutting of crystal structures, building graphs, etc., similar to other CG codes for MD simulation. In addition, RYUGA has a number of advantages as follows: (i) a perspective is employed for drawing CG pictures, (ii) three-dimensional trajectories of atoms can be constructed, (iii) an operator can travel inside the materials, (iv) graphic speed and animation speed are enhanced because of the specific algorithms, and (v) it works on any workstations, moreover even personal computers with a UNIX operating system and an X window system are available.     

Vacuum electrical breakdown characteristics and surface condition of Ti electrodes with oxidation conditions

Outgassing from an electrode surface is regarded as a major factor leading to electrical breakdowns in vacuum. Recently oxidation treatment at 200/spl deg/C was reported as an effective means of reducing Ti outgassing. In this paper, we report our measurement and comparison of the electrical breakdown characteristics of Ti electrodes with different oxidation conditions (without oxidation, oxidation at 200/spl deg/C, oxidation at 450/spl deg/C). In addition, we analyzed electrode surfaces before and after breakdown experiments in situ with X-ray photoelectron spectroscopy (XPS). Before oxidation, we machined the electrode's surfaces to the roughness of 0.8 /spl mu/m Rmax with diamond turning. Breakdown experiments demonstrated that the breakdown field is highest at the first application of voltage to electrodes with oxidized at 200/spl deg/C. Before breakdown experiment, surface analysis revealed that all the sample electrodes had a large amount of carbon originating from the hydrocarbons of contaminants, and after the experiments, they revealed that the carbons had disappeared. To obtain breakdown characteristics of electrodes with smoother surfaces, we conducted experiments on electrodes with a surface roughness of 0.05 /spl mu/m Ra. For these electrodes, the breakdown field was higher at first breakdown; the repetitions required to achieve saturated breakdown fields were significantly fewer, and the amount of carbon on electrode surfaces before breakdown was less.     

Increase in capacitance and tan δ between conductors onprinted circuit board at low frequency due to ionic migration

An endurance test for a printed circuit board to ionic migration was carried out for 2000 h in a chamber controlled at 85°C and 85%RH. The capacitance C and tan δ between the conductors on the board were measured to find their correlation with ionic migration in the board. The measurement frequency of C and tan δ ranged from 1 to 1000 Hz. The configuration of the test circuit was designed according to IPC-SM-840. The material of the insulating board was fiberglass-reinforced epoxy resin. The distance between the Cu conductors was 0.165 mm, and 70 V dc was applied continuously. In the low frequency region (<30 Hz), C and tan δ gradually increase after ~800 h voltage application. During the gradual increase, steeply transient increases in C and tan δ are detected at about the same time when a deposit was seen to have formed on the board between the conductors, although no significant decrease in insulating resistance between the conductors is observed. It was confirmed that the deposit could be detected early from the steep increases in C and tan δ in the low frequency region. In the high frequency region (>30 Hz), on the other hand, gradual slight increases are observed from ~1100 h after voltage application, but no steep increases. The dependence of C and tan δ on frequency at the measurement is due to the low mobility of Cu ions in the water film on the insulating board     

Functional expression of the Erwinia uredovora carotenoid biosynthesis gene crtl in transgenic plants showing an increase of beta-carotene biosynthesis activity and resistance to the bleaching herbicide norflurazon

The possibility that extracellular matrix-cell surface interaction might lead to signalling was examined in human mononuclear cells. In mononuclear cells loaded with Indo-1 acetoxymethylester, collagen binding elicited a 2-3 fold increase in cytosolic free calcium concentration within seconds. Plasma fibronectin, serum albumin and heparin did not cause any significant change in intracellular calcium levels. Collagen I, collagen IV and glucosylated collagen I evoked an increase in intracellular Ca++ both in the presence and absence of extracellular calcium. However, in the presence of 2mM EDTA, the increase in cytosolic free calcium concentration caused by collagen I and collagen IV was partly abolished, suggesting the requirement of a cation-dependent interaction of collagen with mononuclear cells. Glucosylated collagen induced intracellular calcium mobilization even in the presence of EDTA suggesting a cation-independent interaction. These results indicate that collagen binding induces rapid mobilization of calcium in human mononuclear cells, apparently from intracellular sources.     

The estimation of parameters of the hypergeometric distribution andits application to the software reliability growth model

Six ways to estimate parameters of the hypergeometric distribution are investigated, and their accuracies are examined comparatively. It is demonstrated that the least-squares sum method is the best one among those tried, and can be applied to real test/debug data for estimating the number of faults still resident in a program after test/debugging. By this method the estimation time can be reduced greatly     

The role of the destruction box and its neighbouring lysine residues in cyclin B for anaphase ubiquitin-dependent proteolysis in fission yeast: defining the D-box receptor

Programmed proteolysis of proteins such as mitotic cyclins and Cut2/Pds1p requires a 9-residue conserved motif known as the destruction box (D-box). Strong expression of protein fragments containing destruction boxes, such as the first 70 residues of Cdc13 (N70), inhibits the growth of Schizosaccharomyces pombe at metaphase. This inhibition can be overcome either by removal of all lysine residues from N70 using site-directed mutagenesis (K0-N70) or by raising the concentration of intracellular ubiquitin. Consistent with the idea that competition for ubiquitin accounts for some of its inhibitory effects, wild-type N70 not only stabilized D-box proteins, but also Rum1 and Cdc18, which are degraded by a different pathway. The K0-N70 construct was neither polyubiquitinated nor degraded in vitro, but it blocked the growth of strains of yeast in which anaphase-promoting complex/cyclosome (APC/C) function was compromised by mutation, and specifically inhibited proteolysis of APC/C substrates in vivo. Both K0-N70 and 20-residue D-box peptides blocked polyubiquitination of other D-box-containing substrates in a cell-free ubiquitination assay system. These data suggest the existence of a D-box receptor protein that recognizes D-boxes prior to ubiquitination.     

Trifluoromethyl Dihydrothiazine-Based β-Secretase (BACE1) Inhibitors with Robust Central β-Amyloid Reduction and Minimal Covalent Binding Burden

The β-site amyloid precursor protein cleaving enzyme 1 (BACE1, also known as β-secretase) is a promising target for the treatment of Alzheimer's disease. A pK_a lowering approach over the initial leads was adopted to mitigate hERG inhibition and P-gp efflux, leading to the design of 6-CF₃ dihydrothiazine 8 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-cyanopicolinamide). Optimization of 8 led to the discovery of 15 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-(fluoromethoxy)pyrazine-2-carboxamide) with an excellent balance of potency, hERG inhibition, P-gp efflux, and metabolic stability. Oral administration of 8 elicited robust Aβ reduction in dog even at 0.16 mg/kg. Reflecting the reduced hERG inhibitory activity, no QTc prolongation was observed at high doses. The potential for reactive metabolite formation of 15 was realized in a nucleophile trapping assay using [¹⁴C]-KCN in human liver microsomes. Utilizing covalent binding (CVB) in human hepatocytes and the maximum projected human dosage, the daily CVB burden of 15 was calculated to be at an acceptable value of below 1 mg/day. However, hepatotoxicity was observed when 15 was subjected to a two-week tolerance study in dog, which prevented further evaluation of this compound.