Involvement of cytochrome P450 in hydroxylation of propylbenzene by Fusarium moniliforme strain MS31

Fusarium moniliforme strain MS31 can oxidize propylbenzene to (R)-1-phenylpropanol with what may be a cytochrome P450. Hydroxylation of propylbenzene needed molecular oxygen, and NADPH as a coenzyme gave a higher yield than NADH. The hydroxylation proceeded further when FAD and FMN were added than in their absence, suggesting that the enzyme was a flavo-protein. Carbon monoxide inhibited the hydroxylation, as did other cytochrome P450 inhibitors such as SKF 525A and miconazole. These characteristics matched those of a microsomal cytochrome P450 monooxygenase system that contained NADPH-cytochrome P450 reductase.     

Defective bone remodelling in osteoprotegerin-deficient mice

Previous studies have reported enhanced osteoclastogenesis, increased bone resorption and osteoporosis in osteoprotegerin (OPG)-deficient mice. In the present study, we show that the tibial epiphyses contain abundant, thin trabeculae lined with numerous osteoclasts and cuboidal osteoblasts. The increase in osteoblasts and osteoclasts was associated with a dramatic increase in calcein labelling of the mineralization fronts and replacement of much of the intertrabecular marrow with numerous alkaline phosphatase-positive preosteoblasts. Furthermore, the discrete, linear cement lines seen in wild-type mice were replaced by a randomly oriented meshwork of cement lines that were stained intensely for tartrate-resistant acid phosphatase and osteopontin in the OPG-/- mice. These indices of accelerated bone remodelling in mutant bone were associated with irregular trabecular surfaces, a disorganized collagen matrix interspersed with amorphous ground substance and numerous fissures between old and new bone. In total, these observations indicate that enhanced osteoclastic activity in OPG-/- epiphyses led to a coupled increase in osteoblast differentiation and activity and an increase in bone remodelling. The high bone turnover, disorganized matrix and impaired attachment of new to old bone in the cement lines in OPG-/- mice appear to cause bone fragility.     

High-temperature formation phases and crystal structure of hot-pressed thermoelectric compounds with chalcopyrite-type structure

In this study, we introduced the temperaturedependent formation phases and crystallographic parameters of hot-pressed silver gallium telluride AgGaTe_2 and copper gallium telluride CuGaTe_2 with chalcopyrite structure from 300 to 800 K. These two compounds are potential thermoelectric materials in the intermediate temperature range; however, the temperature-dependent formation phases and crystallographic parameters of hotpressed samples have not yet been analyzed in detail. The crystal structure analysis based on synchrotron X-ray diffraction(SXRD) measurements clarifies that impurity phases such as Te and Ag_2 Te in the AgGaTe_2 matrix and Te and CuTe in the CuGaTe_2 matrix appear at some temperature regions above 300 K. The existence of such impurity phases could be correlated with the increases of the electrical resistivity and Seebeck coefficient of the samples after multiple measurement cycles of the temperature-dependent transport properties from 300 to 800 K.The tetragonal lattice parameters a and c, tetragonal lattice volume, thermal expansion coefficients, tetragonal distortion, anion displacement parameter, and isotropic displacement parameter of the hot-pressed AgGaTe_2 and CuGaTe_2 were also analyzed. These crystallographic parameters are expected to substantially affect the thermoelectric properties of AgGaTe_2 and CuGaTe_2. Our results provide prospect of the long-term high-temperature stability and clues of the detailed analysis on the transport properties of hot-pressed AgGaTe_2 and CuGaTe_2, which should aid their development for thermoelectric applications.     

Liposome‐Based in Vitro Evolution of Aminoacyl‐tRNA Synthetase for Enhanced Pyrrolysine Derivative Incorporation

Methanosarcina species pyrrolysyl‐tRNA synthetase (PylRS) attaches Pyl to its cognate amber suppressor tRNA. The introduction of two mutations (Y384F and Y306A) into PylRS was previously shown to generate a mutant, designated LysZ‐RS, that was able to attach N‐benzyloxycarbonyl‐L ‐lysine (LysZ) to its cognate tRNA. Despite the potential of LysZ derivatives, further LysZ‐RS engineering has not been performed; consequently, we aimed to generate LysZ‐RS mutants with improved LysZ incorporation activity through in vitro directed evolution. Using a liposome‐based in vitro compartmentalization (IVC) approach, we screened a randomly mutagenized gene library of LysZ‐RS and obtained a mutant that showed increased LysZ incorporation activity both in vitro and in vivo. The ease and high flexibility of liposome‐based IVC should enable the evolution of not only LysZ‐RS that can attach various LysZ derivatives but also of other enzymes involved in protein translation.     

Evidence for Altered Phosphoinositide Signaling-Associated Molecules in the Postmortem Prefrontal Cortex of Patients with Schizophrenia

Phosphoinositides (PIs) play important roles in the structure and function of the brain. Associations between PIs and the pathophysiology of schizophrenia have been studied. However, the significance of the PI metabolic pathway in the pathology of schizophrenia is unknown. We examined the expression of PI signaling-associated proteins in the postmortem brain of schizophrenia patients. Protein expression levels of phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C), phosphatidylinositol 4-kinase alpha (PIK4CA, also known as PIK4A), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), protein kinase B (Akt), and glycogen synthase kinase 3β (GSK3β) were measured using enzyme-linked immunosorbent assays and multiplex fluorescent bead-based immunoassays of the prefrontal cortex (PFC) of postmortem samples from 23 schizophrenia patients and 47 normal controls. We also examined the association between PIK4CA expression and its genetic variants in the same brain samples. PIK4CA expression was lower, whereas Akt expression was higher, in the PFC of schizophrenia patients than in that of controls; PIP5K1C, PTEN, and GSK3β expression was not different. No single-nucleotide polymorphism significantly affected protein expression. We identified molecules involved in the pathology of schizophrenia via this lipid metabolic pathway. These results suggest that PIK4CA is involved in the mechanism underlying the pathogenesis of schizophrenia and is a potential novel therapeutic target.     

Analysis of human error in nursing care

Analysis of reports about incidental and accidental events in nursing care were made using a reliability engineering method. Unnatural working hours, such as evening duty, night duty falling next to a holiday, two consecutive night-duty shifts, and two consecutive evening-duty shifts were major factors in the occurrence of errors. In a mixed-division ward (a ward containing patients belonging to different divisions), rule-based errors happened more frequently than in a single-division ward. Also, less experienced nursing staffs made errors more frequently than experienced nursing staffs.     

A Method to Identify Steady Creep Strain from Indentation Creep Using a New Reference Area of Indentation

For the design of high-density electronic packages, finite element method (FEM) analyses to evaluate strength reliabilities of solder joints should be conducted by employing the material parameters which can precisely reflect the creep properties of solder joints in actual electronic equipment. To obtain accurate results of the structural analyses of the solder joints, a method to evaluate the steady-state creep deformation in situ must be developed. The indentation creep test is an effective method to evaluate the creep properties of the solder joints in situ; however, the creep properties obtained by this method do not give the same results as those obtained by tensile creep tests using bulk specimens. In this paper, the indentation creep test at 1 N loading for 9,000 s duration was experimentally conducted to confirm that the steady-state creep deformation obtained by the indentation creep test did not coincide with that by the tensile creep tests using bulk specimens. To identify the reason, the indentation creep simulation was conducted by FEM analysis. As a result, it was found that the reference area used to obtain the creep strain from the indentation creep test should be modified. A method to obtain the new reference area is proposed from comparisons of experiments with simulations. Finally, this paper shows that the creep properties obtained by the indentation creep test using the new reference area coincided with those obtained by tensile creep tests using bulk specimens.     

Determination of preservative and antimicrobial compounds in fish from Manila Bay, Philippines using ultra high performance liquid chromatography tandem mass spectrometry, and assessment of human dietary exposure

Ultra high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination of four paraben preservatives (methyl, ethyl, propyl and butyl) and two antimicrobial agents (triclosan and triclocarban) belonging to personal care products (PCPs) in 20 species of fish from Manila Bay (Philippines) was performed. Detection of PCPs with greater frequency indicates the ubiquitous contamination of Manila Bay. Concentrations of total paraben were one order of magnitude higher than the antimicrobials in almost all fish, except in Stolephorus indicus and Leiognathus equulus. A positive correlation was observed between parabens concentration and fish length (r = 0.31-0.49; p<0.05 to <0.001) and fish weight (r = 0.28-0.49; p<0.05 to <0.001), but not for the antimicrobials. The estimated dietary exposure values of the four parabens in the Philippines through fish is four orders of magnitude lower than the acceptable daily intake (ADI) of 10mg/kg/day, but the values of antimicrobials are just half of the ADI of TCS. To our knowledge, this is the first report of PCPs contamination in fish from Philippines.     

Evaluation of drug toxicity with hepatocytes cultured in a micro-space cell culture system

A micro-space cell culture system was recently developed in which cells such as hepatocytes can be cultured and formed into a multicellular three-dimensional (3D) architecture. In this study, we assessed the performance of HepG2 cells cultured in this micro-space cell culture system in a drug toxicity test, and evaluated the effects of micro-space culture on their hepatocyte-specific functions. The micro-space cell culture facilitated the formation of 3D HepG2 cell architecture. HepG2 cells cultured in a micro-space culture plate exhibited increased albumin secretion and enhanced mRNA expression levels of cytochrome P450 (CYP) enzyme compared to those cultured in a monolayer culture. When the cells were exposed to acetaminophen, a hepatotoxic drug, the damage to the HepG2 cells grown in micro-space culture was greater than the damage to the HepG2 cells grown in monolayer culture. In addition, human primary hepatocytes grown in micro-space culture also exhibited increased albumin secretion, enhanced CYP mRNA expression levels and increased sensitivity to acetaminophen compared to those grown in monolayer culture. These results suggest that this micro-space culture method enhances the hepatocyte-specific functions of hepatocytes, including drug-metabolizing enzyme activities, making hepatocytes grown in the micro-space culture system a useful tool for evaluating drug toxicity in vitro.