Occurrence of 3-epi-22-deoxy-20-hydroxyecdysone and its phosphoric ester in diapause eggs of the silkworm,Bombyx mori

Ecdysteroids in diapause eggs of the silkworm,Bombyx mori, were analyzed using high-performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA). A relatively large amount of an unidentified free ecdysteroid and its phosphoric ester (conjugated form) were detected. These two compounds were isolated by a combination of column chromatography on silicic acid, thin-layer chromatography (TLC), and HPLC using a reverse-phase (RP) column. The purified compounds were identified as 3-epi-22-deoxy-20-hydroxyecdysone (22d20E) and 3-epi-22-deoxy-20-hydroxyecdysone 2-phosphate (22d20E2P) by means of mass spectrometry and nuclear magnetic resonance spectroscopy. to our knowledge, this is the first report of 22d20E and 22d20E2P.     

Blocking by picrotoxin of nigra-evoked inhibition of neurons of ventromedial nucleus of the thalamus

Summary The transmitter substance released by nigro-thalamic fibres is proposed to be -aminobutyric acid, since picrotoxin blocked nigra-evoked monosynaptic inhibition of thalamic neurons.Supported by grants Nos 212009 and 311410 from Ministry of Education of Japan.The authors wish to thank Dr Marjorie Anderson, University of Washington, Seattle, for improving the English.     

Trans-4-hydroxy-3-methoxycinnamic acid (ferulic acid) inhibits the effect of androgens on the rat prostate

Summary Trans-4-hydroxy-3-methoxycinnamic acid (ferulic acid, FA) antagonized the effect of exogenous androgens on the ventral prostate (VP) in castrated rats as well as the effect of endogenous androgens in intact rats. FA, however, had no effect on the seminal vesicles (SV) and levator ani muscle (LAM), nor oestrogenic effect in female rats and mice. FA did not antagonize the receptor binding of testosterone nor inhibit the conversion of testosterone into 5-dihydrotestosterone (DHT).Supported in part by the project fund (grant 53-403) of Kawasaki Medical School.The authors would like to thank the Central Laboratory, Sankyo Pharmaceutical Co., Tokyo, for supplying 19-nortestosterone-17-ferulate.     

Improving the thermal stability of lactate oxidase by directed evolution

Lactate oxidase is used in biosensors to measure the concentration of lactate in the blood and other body fluids. Increasing the thermostability of lactate oxidase can significantly prolong the lifetime of these biosensors. We have previously obtained a variant of lactate oxidase from Aerococcus viridans with two mutations (E160G/V198I) that is significantly more thermostable than the wild-type enzyme. Here we have attempted to further improve the thermostability of E160G/V198I lactate oxidase using directed evolution. We made a mutant lactate oxidase gene library by applying error-prone PCR and DNA shuffling, and screened for thermostable mutant lactate oxidase using a plate-based assay. After three rounds of screening we obtained a thermostable mutant lactate oxidase, which has six mutations (E160G/V198I/G36S/T103S/A232S/F277Y). The half-life of this lactate oxidase at 70 °C was about 2 times that of E160G/V198I and about 36 times that of the wild-type enzyme. The amino acid mutation process suggests that the combined neutral mutations are important in protein evolution. Received 15 September 2006; received after revision 21 October 2006; accepted 2 November 2006     

Macropinocytosis,mTORC1 and cellular growth control

The growth and proliferation of metazoan cells are driven by cellular nutrient status and by extracellular growth factors. Growth factor receptors on cell surfaces initiate biochemical signals that increase anabolic metabolism and macropinocytosis, an actin-dependent endocytic process in which relatively large volumes of extracellular solutes and nutrients are internalized and delivered efficiently into lysosomes. Macropinocytosis is prominent in many kinds of cancer cells, and supports the growth of cells transformed by oncogenic K-Ras. Growth factor receptor signaling and the overall metabolic status of the cell are coordinated in the cytoplasm by the mechanistic target-of-rapamycin complex-1 (mTORC1), which positively regulates protein synthesis and negatively regulates molecular salvage pathways such as autophagy. mTORC1 is activated by two distinct Ras-related small GTPases, Rag and Rheb, which associate with lysosomal membranes inside the cell. Rag recruits mTORC1 to the lysosomal surface where Rheb directly binds to and activates mTORC1. Rag is activated by both lysosomal luminal and cytosolic amino acids; Rheb activation requires phosphoinositide 3-kinase, Akt, and the tuberous sclerosis complex-1/2. Signals for activation of Rag and Rheb converge at the lysosomal membrane, and several lines of evidence support the idea that growth factor-dependent endocytosis facilitates amino acid transfer into the lysosome leading to the activation of Rag. This review summarizes evidence that growth factor-stimulated macropinocytosis is essential for amino acid-dependent activation of mTORC1, and that increased solute accumulation by macropinocytosis in transformed cells supports unchecked cell growth.     

ITPKC functional polymorphism associated with Kawasaki disease susceptibility and formation of coronary artery aneurysms

Kawasaki disease is a pediatric systemic vasculitis of unknown etiology for which a genetic influence is suspected. We identified a functional SNP (itpkc_3) in the inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) gene on chromosome 19q13.2 that is significantly associated with Kawasaki disease susceptibility and also with an increased risk of coronary artery lesions in both Japanese and US children. Transfection experiments showed that the C allele of itpkc_3 reduces splicing efficiency of the ITPKC mRNA. ITPKC acts as a negative regulator of T-cell activation through the Ca2+/NFAT signaling pathway, and the C allele may contribute to immune hyper-reactivity in Kawasaki disease. This finding provides new insights into the mechanisms of immune activation in Kawasaki disease and emphasizes the importance of activated T cells in the pathogenesis of this vasculitis.     

灰色理论在化学研究中用作建模与预测新的方法

研究了灰色理论作为一种有用的化学计量学多元建模与定量预测新方法的可行性，将其应用于某些不确定关系如电泳迁移率，色谱保留值和农药需求量等建模，定量预测结果良好。     

Functional identification of gene cluster for the aniline metabolic pathway mediated by transposable element

A convenient and widely applicable method has been developed to clone aniline metabolic gene cluster in this study. Three positive recombinant plasmids pDA1, pDB2 and pDB11 were cloned from genomic library of aniline degradation strain AD9. The result of aniline dioxygenase （AD） activity and catechol 2,3-oxygenase （C230） activity assay showed that pDA1 and pDB11 contain aniline dioxygenase genes and catechol 2,3-dioxygenase genes, respectively. The sequence analysis of the total 24.7-kb region revealed that this region contains 25 ORFs, of which 17 genes involve metabolism of aniline. In the gene cluster, the first five genes （tadQTA1A2B） and the subsequent gene （tadR1） were predicted to encode a multi-component aniline dioxygenase and a LysR-type regulator, respectively, while the others （tadD1C1D2C2EFGIJKL） were expected to encode metacleavage pathway enzymes for catechol degradation. The gene cluster was surrounded by two IS1071 sequences.     

Mechanically driven ATP synthesis by F1-ATPase

ATP, the main biological energy currency, is synthesized from ADP and inorganic phosphate by ATP synthase in an energy-requiring reaction. The F1 portion of ATP synthase, also known as F1-ATPase, functions as a rotary molecular motor: in vitro its gamma-subunit rotates against the surrounding alpha3beta3 subunits, hydrolysing ATP in three separate catalytic sites on the beta-subunits. It is widely believed that reverse rotation of the gamma-subunit, driven by proton flow through the associated F(o) portion of ATP synthase, leads to ATP synthesis in biological systems. Here we present direct evidence for the chemical synthesis of ATP driven by mechanical energy. We attached a magnetic bead to the gamma-subunit of isolated F1 on a glass surface, and rotated the bead using electrical magnets. Rotation in the appropriate direction resulted in the appearance of ATP in the medium as detected by the luciferase-luciferin reaction. This shows that a vectorial force (torque) working at one particular point on a protein machine can influence a chemical reaction occurring in physically remote catalytic sites, driving the reaction far from equilibrium.