Identification of molecular target of AMP-activated protein kinase activator by affinity purification and mass spectrometry

We show an efficient method to identify molecular targets of small molecular compounds by affinity purification and mass spectrometry. Binding proteins were isolated from target cell lysate using affinity columns, which immobilized the active and inactive compounds. All proteins bound to these affinity columns were eluted by digestion using trypsin and then were identified by mass spectrometry. The specific binding proteins to the active compound, a candidate for molecular targets, were determined by subtracting the identified proteins in an inactive compound-immobilized affinity column from that in an active compound-immobilized affinity column. This method was applied to identification of molecular targets of D942, a furancarboxylic acid derivative, which increases glucose uptake in L6 myocytes through AMP-activated protein kinase (AMPK) activation. To elucidate the mechanism of AMPK activation by D942, affinity columns that immobilized D942 and its inactive derivative, D768, were prepared, and the binding proteins were purified from L6 cell lysate. NAD(P)H dehydrogenase [quinone] 1 (complex I), which was shown as one of the specific binding proteins to D942 by subtracting the binding proteins to D768, was partially inhibited by D942, not D768. Because inhibition of complex I activity led to a decrease in the ATP/AMP ratio, and the change in the ATP/AMP ratio triggered AMPK activation, we identified complex I as a potential protein target of AMPK activation by D942. This result shows our approach can provide crucial information about the molecular targets of small molecular compounds, especially target proteins not yet identified.     

Identification of sialyltransferases of Streptococcus agalactiae

Group B streptococci, Streptococcus agalactiae, produce high-molecular-weight polysaccharides containing N-acetylneuraminic acid. Although the type-specific capsular polysaccharide (CP) synthesis (cps) genes of several S. agalactiae strains have been extensively analyzed, to date, no sialyltransferase activity has been detected from any gene product of the cps gene cluster. Among the cps genes, the cpsK gene products of S. agalactiae types la and Ib showed weak similarity to several bacterial sialyltransferases. In this study, the cpsIaK and cpsIbK gene products were found to show sialyltransferase activity specific for lacto-N-neotetraose and lacto-N-tetraose, respectively. This acceptor specificity seems to reflect the respective CP structure, since the repeating unit of type la CP is sialyllacto-N-neotetraose and that of type Ib CP is sialyllacto-Ntetraose. We also found that the C-terminal regions of CpsKs were almost completely conserved in various S. agalactiae strains.     

Imaging saponin-induced structural changes in neural processes with atomic force microscopy

Temporal changes in the structure of neuronal processes in the presence of saponin were studied by atomic force microscopy in a fluid medium. After saponin treatment, concavities were formed on the surface of some neurites and fibrous structures in other neurites were splayed. The vertical height of these splayed fibrils or fibrillar bundles ranged from 13 to 370 nm, and the horizontal width was less than 500 nm. These findings suggest that formation of concavities and separation of bundled fibrils occurred simultaneously in saponin-treated neurites.     

Simultaneous measurement of current and voltage by use of one bismuth germanate crystal

An optical fiber sensor is presented that allows current and voltage to be measured simultaneously by use of only one block of bismuth germanate crystal. The polarized light from the sensing crystal is split into two light beams: One beam is utilized for current measurement based on the Faraday effect, and the other one is utilized for voltage measurement based on the Pockels effect. Compared with the existing optical sensors that can measure current and voltage simultaneously, this sensor is simple and inexpensive and allows measurement of electric power. The simultaneous measurements of ac electric current from 0.05 to 10 A, voltage from 1 to 235 V, and power from 2 to 1000 W have been achieved with good linear-response characteristics. The input characteristics and measurement uncertainties that are due to the nonlinear error of the sensing system are also discussed.     

High-tilt-angle boron implantation into polycrystalline Si gates

We ion-implanted B into 400-nm-thick polycrystalline Si and evaluated the profiles. The ion-implanted profiles were almost the same for tilt angles of 0/spl deg/ and 7/spl deg/. The channeling tail was more significant than the profile in crystalline Si with a tilt of 0/spl deg/. The channeling phenomenon was significantly suppressed by high-tilt angles because ions cross grain boundaries. We succeeded in expressing these prominent profiles through using an analytical model.     

Degradation of Azo Compounds by Aeromonas hydrophila var. 24B

A bacterium which has the ability to degrade azo dyes has been isolated from a polluted environment and identified as Aeromonas hydrophilia. The specificity of azo dyes to the bacterium was examined. p-Acetamidoazobenzene, p-aminoazobenzene, acetanilide, aniline and p-phenylenediamine, which are produced as metabolites, were also isolated.     

An approach for the regularization of a power flow solution aroundthe maximum loading point

A method which is capable of greatly widening the range where the power flow solution is regular is presented. This is achieved by giving some modifications to the conventional power flow solution method and eliminating the singular point or shifting it to region where the voltage is lower than that of the maximum loading point. Then, the continuous execution of V-P curves including the maximum loading point is realized. The efficiency and effectiveness of the method were tested in a practical 598-node system are compared with the conventional method     

Growth of AIGalnP in a high-speed rotating disk OMVPE reactor

In an OMVPE reactor with a high speed rotating disk, growth of a quaternary material AIGalnP is carried out after a basic investigation on the GaAs growth rate uniformity. Experimental growth rate and growth efficiency results are compared with the theoretical results obtained from an infinite diameter rotating disk model. Distributions of the lattice mismatch, PL spectra and carrier concentration were measured to clarify the disk rotation effect on these properties. The main reason for non-uniformities is not the disk rotation but the temperature distribution on a substrate carrier.