膽汁酸ノ生物學的研究 (第二報) 枯草菌ニヨル「グリセリン」醗酵ニ就テ

余ハ前ニ大腸菌ノ作用ニヨル「Cholsäure」ノ新分解産物ニ就テ報告セリ. 次デ同ジ目的ノ下ニ「Cholsäure」竝ニ「Desoxybiliansäure」ニ枯草菌ヲ作用セシメント企テタリ. 本實驗ニ於テ余ハ所期ノ分解産物ヲ得ル事能ハザリシモ, 其際培養液トシテ用ヒタル「グリセリン」ノ醗酵ニ於テ稍興味アル結果ヲ觀タルガ故ニココニ報告セントス. 即余ハ「Desoxybiliansäure」ノ分解ヲ企テタル「グリセリン」ヲ含ム培養液中ニ「メチルアセチルカルビノル」「アセトアルデヒド」「醋酸」「蟻酸」「乳酸」竝ニ「エチルアルコホレ」ノ生成セシコトヲ證明シ得タリ. 而シテ其際「ヂアセチルフエニルオサツオン」トシテ分離セシ「メチルアセチルカルビノル」ノ量ハ甚ダ多量ニシテ, コレガ主要所産物ナルコトハ疑ナシ. コノ「メチルアセチルカルビノル」ハ「グリセリン」醗酵ニ由リテ生ジタル「アセトアルデヒド」ノ二分子ノ結合ニヨリテ生ジタルモノト見ルベク, コレニヨリテNeubergノ所謂「カルボリガアゼ酵素」ハ尚或條件ノ下ニ於テ枯草菌ニヨリテモ産出シ得ルモノナル事ヲ明ラカニセリ. コノ「メチルアセチルカルビノル」ノ生成ニ對シテ膽汁酸ノ存在ガ決定的ニ關與スルモノナリヤ, 又ハ枯草菌其自體ノ單獨ナル動作ニ因スルモノナリヤニ就テハ尚決定シ得ズ. 余ノ觀察シタルトコロニヨレバ枯草菌ノ發育ハ, 0.2%「Cholsäure曹達鹽」ニヨリテ妨ゲラルルモ, 0.2%「Desoxybiliansaure曹達鹽」ニヨリテハ妨ゲラレザルガ如シ

Activation of Stat1 and subsequent transcription of inducible nitric oxide synthase gene in C6 glioma cells is independent of interferon-γ-induced MAPK activation that is mediated by p21ras

AbstractRat C6 glioma cells have been used to characterize molecular events involved in the regulation of inducible nitric oxide synthase (iNOS) gene expression stimulated by interferon-γ (IFN-γ) plus lipopolysaccharide (LPS). IFNs induce a signaling event which involves activation of Stat1 transcription factor. Previous studies have shown that IFNs also induce extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activation. However, the mechanisms by which IFNs stimulate MAPK activation remain elusive. Here we show that in C6 glioma cells, transiently expressing the dominant-negative form of c-Ha-Ras (Asn-17) abrogated IFN-γ-induced ERK1 and ERK2 activation. Furthermore, PD98059, a specific MEK1 inhibitor, also blocked this activation. These results indicate that p21ras and MEK1 are required for IFN-γ-induced ERK1 and ERK2 activation. Recent studies have reported that MAPK is responsible for serine phosphorylation of Stat1 which is required for Stat1's DNA binding and maximal transcriptional activity. Thus, we examined the role of the Ras-MAPK pathway in Stat1 activation and subsequent iNOS induction in C6 glioma cells. Further experiments showed that neither Asn-17 Ras expression nor concentrations of PD98059, which completely abrogated IFN-γ-induced ERK1 and ERK2 activation, affected Stat1 DNA binding activity or iNOS induction, indicating that the Ras-MAPK pathway does not appear to be involved in the activation of Stat1 and subsequent iNOS induction in C6 glioma cells

A unique tRNA recognition mechanism of Caenorhabditis elegans mitochondrial EF-Tu2

Nematode mitochondria expresses two types of extremely truncated tRNAs that are specifically recognized by two distinct elongation factor Tu (EF-Tu) species named EF-Tu1 and EF-Tu2. This is unlike the canonical EF-Tu molecule that participates in the standard protein biosynthesis systems, which basically recognizes all elongator tRNAs. EF-Tu2 specifically recognizes Ser-tRNA(Ser) that lacks a D arm but has a short T arm. Our previous study led us to speculate the lack of the D arm may be essential for the tRNA recognition of EF-Tu2. However, here, we showed that the EF-Tu2 can bind to D arm-bearing Ser-tRNAs, in which the D–T arm interaction was weakened by the mutations. The ethylnitrosourea-modification interference assay showed that EF-Tu2 is unique, in that it interacts with the phosphate groups on the T stem on the side that is opposite to where canonical EF-Tu binds. The hydrolysis protection assay using several EF-Tu2 mutants then strongly suggests that seven C-terminal amino acid residues of EF-Tu2 are essential for its aminoacyl-tRNA-binding activity. Our results indicate that the formation of the nematode mitochondrial (mt) EF-Tu2/GTP/aminoacyl-tRNA ternary complex is probably supported by a unique interaction between the C-terminal extension of EF-Tu2 and the tRNA

Proteins Interacting With Caenorhabditis elegans Gα Subunits

To identify novel components in heterotrimeric G-protein signalling, we performed an extensive screen for proteins interacting with Caenorhabditis elegans Gα subunits. The genome of C. elegans contains homologues of each of the four mammalian classes of Gα subunits (Gs, Gi/o, Gq and G12), and 17 other Gα subunits. We tested 19 of the GGα subunits and four constitutively activated Gα subunits in a largescale yeast two-hybrid experiment. This resulted in the identification of 24 clones, representing 11 different proteins that interact with four different Gα subunits. This set includes C. elegans orthologues of known interactors of Gα subunits, such as AGS3 (LGN/PINS), CalNuc and Rap1Gap, but also novel proteins, including two members of the nuclear receptor super family and a homologue of human haspin (germ cell-specific kinase). All interactions were found to be unique for a specific Gα subunit but variable for the activation status of the Gα subunit. We used expression pattern and RNA interference analysis of the G-protein interactors in an attempt to substantiate the biological relevance of the observed interactions. Furthermore, by means of a membrane recruitment assay, we found evidence that GPA-7 and the nuclear receptor NHR-22 can interact in the animal

Rapid kinetics of G protein subunit association: A rate-limiting conformational change?

G protein subunit association and dissociation are thought to play an important role in signal transduction. We measured [alpha][beta][gamma] heterocomplex formation using resonance energy transfer. Fluorescein-labelled [alpha](F-[alpha]) emission was quenched ~ 10% on mixing with eosin-labelled [beta][gamma](E-[beta][gamma]). Unlabelled [beta][gamma] did not quench F-[alpha] fluorescence. Stopped-flow kinetics showed a t1/2 ranging from 2.5 s to 0.25 s for 50 nM to 1200 nM E-[beta][gamma]. The rate saturated at high E-[beta][gamma] concentrations consistent with a two-step mechanism. We report the first rapid-mix studies of G protein subunit association kinetics which suggest that [alpha] and [beta][gamma] combine by a two-step process with a maximal rate of 4.1 +/- 0.4 s-1.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31136/1/0000033.pd

G-proteins coupled to phosphoinositide hydrolysis in the cochlear and vestibular sensory epithelia of the rat are insensitive to cholera and pertussis toxins

In the cochlear (CSE) and vestibular sensory epithelia (VSE), phosphoinositides are hydrolyzed in response to stimulation of phospholipase C (PLC) by cholinergic muscarinic and purinergic P2y agonists. Such receptor-mediated activation of PLC is expected to be coupled through guanine nucleotide-binding proteins (G-proteins). Although several classes of G-proteins have been identified in the inner ear, nothing is known about the type of G-proteins associated with the phosphoinositide second messenger system in CSE and VSE. Phosphoinositide hydrolysis was determined by the release of radiolabeled inositol phosphates (InsPs). Ten mM NaF plus 10 [mu]M AlCl3 increased basal InsPs accumulation 2-fold in both CSE and VSE of the rat. Release of InsPs was also enhanced by guanosine 5'-O-(3-thiotriphosphate) (GTP-[gamma]-S) in saponin-permeabilized tissues. Furthermore, release of InsPs stimulated by both carbamylcholine (CCh) and adenosine 5'-O-[3-thiotriphosphate](ATP-[gamma]-S) was significantly inhibited by 100 [mu]M guanosine 5'-O-[2-thiodiphosphate](GDP-[beta]-S). These results strongly suggest the involvement of G-proteins in the receptor-PLC coupling in CSE and VSE. ADP-ribosylation in membrane fractions of CSE and VSE in the presence of cholera toxin (CTX) or pertussis toxin (PTX) indicated the existence of Gs- and Gi-type G-proteins. However, neither CTX nor PTX affected basal or agonist-stimulated release of InsPs. These observations suggest that muscarinic and P2y purinergic receptors are coupled to PLC via CTX- and PTX-insensitive G-proteins in CSE and VSE.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31661/1/0000595.pd

A Computational Study of Elongation Factor G (EFG) Duplicated Genes: Diverged Nature Underlying the Innovation on the Same Structural Template

BACKGROUND: Elongation factor G (EFG) is a core translational protein that catalyzes the elongation and recycling phases of translation. A more complex picture of EFG's evolution and function than previously accepted is emerging from analyzes of heterogeneous EFG family members. Whereas the gene duplication is postulated to be a prominent factor creating functional novelty, the striking divergence between EFG paralogs can be interpreted in terms of innovation in gene function. METHODOLOGY/PRINCIPAL FINDINGS: We present a computational study of the EFG protein family to cover the role of gene duplication in the evolution of protein function. Using phylogenetic methods, genome context conservation and insertion/deletion (indel) analysis we demonstrate that the EFG gene copies form four subfamilies: EFG I, spdEFG1, spdEFG2, and EFG II. These ancient gene families differ by their indispensability, degree of divergence and number of indels. We show the distribution of EFG subfamilies and describe evidences for lateral gene transfer and recent duplications. Extended studies of the EFG II subfamily concern its diverged nature. Remarkably, EFG II appears to be a widely distributed and a much-diversified subfamily whose subdivisions correlate with phylum or class borders. The EFG II subfamily specific characteristics are low conservation of the GTPase domain, domains II and III; absence of the trGTPase specific G2 consensus motif "RGITI"; and twelve conserved positions common to the whole subfamily. The EFG II specific functional changes could be related to changes in the properties of nucleotide binding and hydrolysis and strengthened ionic interactions between EFG II and the ribosome, particularly between parts of the decoding site and loop I of domain IV. CONCLUSIONS/SIGNIFICANCE: Our work, for the first time, comprehensively identifies and describes EFG subfamilies and improves our understanding of the function and evolution of EFG duplicated genes

Elevation of tissue coenzyme Q (ubiquinone) and cytochrome c concentrations by endurance exercise in the rat

Six months of enforced and voluntary endurance training of young female Wistar rats resulted in significant decreases of body weight and gastrocnemius muscle wet weight and protein content, and increases in heart weight and protein content, and liver protein content. The coenzyme Q and cytochrome c concentrations of cardiac, gastrocnemius, and deep red region of the vastus lateralis muscles were increased, while small or nonsignificant trends toward increases in cytochrome c and coenzyme Q were seen in kidney, brain, lung, liver, internal + external oblique muscles, and the superficial white region of the vastus lateralis muscle. These results are discussed with regard to several roles for coenzyme Q in cellular function.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24652/1/0000065.pd