Purification and characterization of an extracellular alpha-neoagarooligosaccharide hydrolase from Bacillus sp. MK03

An agarolytic bacterium was isolated from soil in Gifu prefecture, Japan, and identified as Bacillus sp. strain MK03. The strain secreted neoagarooligosaccharide hydroluse into the culture medium. The enzyme was purified 49.7-fold from the culture fluid by ammonium sulfate precipitation and anion-exchange and gel-filtration column chromatographic methods. The purified enzyme appeared as a single band on polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. Estimations of the molecular mass by gel filtration and SDS-PAGE gave values of 320 kDa and 42 kDa, respectively, indicating that the enzyme is octametric. The enzyme cleaved the alpha-1,3 linkage in neoagarobiose to produce 3,6-anhydro-L-galactose and D-galactose. It also selectively cleaved the alpha-1,3 linkage at the nonreducing end in neoagarotetraose or neoagarohexaose to give 3,6-anhydro-L-galactose and agarotriose or agaropentaose. The optimum temperature and pH for the enzyme were 30 degrees C and 6.1, respectively. The N-terminal amino acid sequence showed no homology to sequences of other known neoagarooligosaccharide hydrolases and agarases.     

Design of a high-temperature and high-pressure liquid flow cell for x-ray absorption fine structure measurements under catalytic reaction conditions

The design and performance of a new high-pressure and high-temperature cell for measurement of x-ray absorption fine structure (XAFS) spectra of solid catalysts working in a flowing liquid are presented. The cell has flat, high-purity sintered cubic boron nitride (c-BN) windows which can tolerate high temperature (900 K) and high pressure (10 MPa). The c-BN is a new material which has the highest tensile strength, second only to diamond, and is also chemically and thermally stable. The use of the cell is demonstrated for measurements of PtPdAl(2)O(3) and Ni(2)PSiO(2) hydrodesulfurization catalysts at reaction conditions. A technique called delta chi (Deltachi), involving determining the difference between XAFS spectra of the sample at reaction conditions and the bare sample, is introduced.     

Multivariate evaluation of prognostic determinants for renal cell carcinomas which are metastatic at initial diagnosis

To clarify the relative importance of clinicopathological factors potentially affecting the survival of individuals initially diagnosed as having metastatic renal cell carcinoma (RCC), univariate and multivariate analyses using Cox's proportional hazards model were performed for 111 RCC patients who were admitted to four hospitals in Nagoya, Japan, between 1978 and 1993. Survival time was calculated from the date of diagnosis to the date of death or the end of May, 1995. Nineteen factors (17 clinically applicable before treatment and 2 histological in nature) were included for assessment. Univariate analysis revealed that 10 of the total 19 factors were significantly associated with patient survival. Multivariate analysis found 7 factors to be statistically significant determinants. Survival was further evaluated by categorizing the patients into three groups according to the number of the 7 factors. These groups showed clearly different survivals (log-rank test, p < 0.0001), confirming the importance of the 7 factors as definite determinants. They should therefore prove to be of advantage for classifying metastatic RCC patients when designing clinical prospective studies of immunotherapy and chemotherapy.     

Dual-Function Semaphorin 4D Released by Platelets: Suppression of Osteoblastogenesis and Promotion of Osteoclastogenesis

Effects of the antiosteoblastogenesis factor Semaphorin 4D (Sema4D), expressed by thrombin-activated platelets (TPs), on osteoblastogenesis, as well as osteoclastogenesis, were investigated in vitro. Intact platelets released both Sema4D and IGF-1. However, in response to stimulation with thrombin, platelets upregulated the release of Sema4D, but not IGF-1. Anti-Sema4D-neutralizing monoclonal antibody (mAb) upregulated TP-mediated osteoblastogenesis in MC3T3-E1 osteoblast precursors. MC3T3-E1 cells exposed to TPs induced phosphorylation of Akt and ERK further upregulated by the addition of anti-sema4D-mAb, suggesting the suppressive effects of TP-expressing Sema4D on osteoblastogenesis. On the other hand, TPs promoted RANKL-mediated osteoclastogenesis in the primary culture of bone-marrow-derived mononuclear cells (BMMCs). Among the known three receptors of Sema4D, including Plexin B1, Plexin B2 and CD72, little Plexin B2 was detected, and no Plexin B1 was detected, but a high level of CD72 mRNA was detected in RANKL-stimulated BMMCs by qPCR. Both anti-Sema4D-mAb and anti-CD72-mAb suppressed RANKL-induced osteoclast formation and bone resorptive activity, suggesting that Sema4D released by TPs promotes osteoclastogenesis via ligation to a CD72 receptor. This study demonstrated that Sema4D released by TPs suppresses osteogenic activity and promotes osteoclastogenesis, suggesting the novel property of platelets in bone-remodeling processes.     

Complex formation, thermal properties, and in-vitro digestibility of gelatinized potato starch-fatty acid mixtures

Effects of the type and amount of fatty acid (0-2.0 mmol/g-starch of lauric, myristic, palmitic, stearic, oleic, and linoleic acids) on the complex formation, thermal properties, and in-vitro digestibility of gelatinized potato starch-fatty acid mixtures were investigated. Complex index (CI) evaluated by the reduction in the iodine binding capacity of starch increased with an increase in the amount of fatty acids, and reached a plateau depending on the type of fatty acid. The maximum CI value was higher in the order of lauric (49.9%), linoleic (47.6%), myristic (42.4%), oleic (36.7%), stearic (35.3%), and palmitic acid (30.9%). From the calorimetric study, it was demonstrated that melting temperature of the complexes was higher in the order of stearic (96.7 °C) > lauric, myristic, palmitic, and oleic (89.6-92.1 °C) > linoleic acid (78.3 °C). Melting enthalpy for complexes was roughly related to the CI value (R² = 0.667). From the in-vitro digestibility measurement, it was found that a certain amount of fatty acid reduced the starch content hydrolyzed at a given condition. Among them, 0.50 mmol/g-starch lauric and oleic acid samples showed the largest reduction in the hydrolyzed starch content. This result was related to the extent of complex formation characterized by CI value and its helical order characterized by melting temperature. In addition, there was a possibility of the complex formation between amylose and unsaturated fatty acid during the hydrolysis of gelatinized starch.     

Application of nonlinear fitting and selection of the most fitted equation by AIC in stability test of pharmaceutical ingredients

We proposed a method for objectively selecting the most fitted rate equation among candidate rate equations based on chemical kinetics by both nonlinear regression analysis and Akaike's information criterion (AIC), to express the decrease of pharmaceutical ingredient as an appropriate equation. Pseudo-zero, first, and second-order rate equations were prepared as candidates beforehand, and AIC was introduced for selecting the most fitted rate equation among the candidates. We compared the proposed method to the Weibull method that expressed any decrease patterns as a single equation.

We quantified the contents of thiamine nitrate (VB1) and taurine after storage for one, three, and six months under 40°C-75% relative humidity by high-pressure liquid chromatography. Decrease patterns of each sample were most fitted to one of the candidate rate equations, that is, pseudo-zero, first, and second-order rate equations, respectively, and the degree of fit in the most fitted equation was superior to that of the Weibull method, except for the pseudo-first-order rate equation.

Moreover, we confirmed that the proposed method was considerably precise for estimating the stability of pharmaceuticals by comparing estimated residual rates and confidence intervals to experimental data.     

Computational dynamics method for evaluating the mobility of charged biomolecules passing through the electrical potential field within the ion selective channel on an excitable membrane

A mathematical method is introduced to characterize the electrokinetic behavior (electrophoresis) of a biomolecular particle which passes through a specific channel pore on an excitable biological membrane. The basic approach was first proposed by Booth (1950). The system was described by an equation of continuity and an equation of motion in which the driving force involves the diffusion effect, the hydrostatic pressure, and the electrostatic potential. By assuming linear relations between the velocity and the applied electrical field, solutions for the potential, pressure, and velocity were given by a series expansion of the charges on the particle. To examine the influence of ions surrounding the particle and forming an ionic cloud, the Debye–Huckel parameter was introduced. As the thickness of the double layer around the particle increased, the potential, velocity, pressure, and viscosity were changed significantly. The maximum influence was obtained when the radius of the particle became equal to the thickness of the double layer. Although this theory is valid for a charged, spherical, nonconducting particle only, the method is available for evaluating the kinetic behavior of a biomolecule that passes through a channel pore on a cellular membrane.This work was presented, in part, at the 8th International Symposium on Artificial Life and Robotics, Oita, Japan, January 24–26, 2003     

Parallel implementation of the projector augmented plane wave method for charged systems

A parallel implementation of the projector augmented plane wave (PAW) method with the applications to several transition metal complexes is presented. A unique aspect of our PAW code is that it can treat both charged and neutral cluster systems. We discuss how this is achieved via accurate numerical treatment of the Coulomb Green's function with free space boundary conditions. The strategy for parallelizing the PAW code is based on distributing the plane wave basis across processors. This is a versatile approach and is implemented using a parallel three-dimensional Fast Fourier Transformation (FFT). We report parallel performance analysis of our program and of the three-dimensional FFT's and discuss large-scale parallelization issues of the PAW code. Using a series of transition metal monoxides and dioxides, as well as two iron aqueous complexes, it is shown that a free space PAW code can give structural parameters and energies in good accord with Gaussian based methods.     

Magnetic Torque Measurements on Bi2201 Single Crystal in High Magnetic Fields

The magnetic field direction dependence of the magnetic torque in the superconducting state of Bi ₂ Sr _1.65 La _0.35 CuO ₆₊ (Bi2201) single crystal has been measured in several static magnetic fields up to 200 kOe. The applied field dependence of the anisotropy parameter, , defined by the square root of the effective mass ratio has been obtained by fitting the magnetic torque curves at 17 K by means of the effective mass model. For Bi ₂ Sr ₂ CaCu ₂ O ₈₊ (Bi2212), the values up to 210 kOe were reported previously. At each field, for Bi2201 is larger than that for Bi2212. Since the value decreases as the field increases both for Bi2201 and Bi2212, the reduction of by the application of the high magnetic field is considered to be common for Bi-based layered superconductors.     

Adaptive control of surviving symbol replica candidates in QRM-MLD for OFDM MIMO multiplexing

This paper proposes adaptive control of the number of surviving symbol replica candidates, S/sub m/ (m denotes the stage index), based on the minimum accumulated branch metric of each stage in maximum-likelihood detection employing QR decomposition and the M-algorithm (QRM-MLD) in orthogonal frequency-division multiplexing with multiple-input-multiple-output (MIMO) multiplexing. In the proposed algorithm, S/sub m/ at the mth stage (1/spl les/m/spl les/N/sub t/, N/sub t/ is the number of transmission antenna branches) is independently controlled using the threshold value calculated from the minimum accumulated branch metric at that stage and the estimated noise power. We compared the computational complexity of QRM-MLD employing the proposed algorithm with that of conventional methods at the same average packet error rate assuming the information bit rate of 1.048 Gb/s in a 100-MHz channel bandwidth (i.e., frequency efficiency of approximately 10 bit/s/Hz) using 16QAM modulation and turbo coding with the coding rate of 8/9 in 4-by-4 MIMO multiplexing. Computer simulation results show that the average computational complexity of the branch metrics, i.e., squared Euclidian distances, of the proposed adaptive independent S/sub m/ control method is decreased to approximately 38% that of the conventional adaptive common S/sub m/ control and to approximately 30% that of the fixed S/sub m/ method (S/sub m/=M=16), assuming fair conditions such that the maximum number of surviving symbol replicas at each stage is set to M/spl circ/=16.