Branched-chain amino acids promote albumin synthesis in rat primary hepatocytes through the mTOR signal transduction system

The administration of branched-chain amino acids (BCAAs) to cirrhosis patients increases serum albumin levels and improves the blood Fischer's ratio. Although it has been reported that albumin synthesis in rat primary hepatocytes is diminished under lower Fisher's ratio conditions compared to normal Fischer's ratio conditions, the mode of action at the molecular level for these effects is still uncertain. It has been reported recently that the triggering signal for protein synthesis is transmitted through mTOR (mammalian target of rapamycin). We have had an interest in the mTOR signal transduction system. In the present study, we analyzed the mode of action of BCAA-induced albumin synthesis using rat primary hepatocytes. The BCAA mixture dose-dependently promoted the production of albumin, with leucine being the major effector half of which was inhibited by the mTOR inhibitor rapamycin. We also showed that only leucine induces P70 S6 kinase activation and 4E-BP1 phosphorylation which are mTOR's downstream translational effectors. These activations were completely inhibited by rapamycin. Our results suggest that BCAAs, especially leucine, promote the production of albumin in rat primary hepatocytes through an mTOR signal transduction system.     

A method for determination of iododeoxyuridine substitution of thymidine using reversed-phase high-performance liquid chromatography

An assay for thymidine substitution by iododeoxyuridine (IdUrd) using reversed-phase high-performance liquid chromatography (HPLC) has been developed. Three principal steps in this procedure are: extraction of DNA from cell or tissues, hydrolysis of DNA into deoxynucleosides and separation using HPLC. Approximately 1 microgram of DNA was recovered from 10(5) cells by phenol extraction, and subjected to hydrolysis into deoxynucleosides which required a three-stage DNA digestion using enzymes DNAse I. phosphodiesterase I and alkaline phosphatase. The deoxynucleosides were separated on the Microsorb C18 column with isocratic elution; 90-100% of the DNA was recovered as deoxynucleosides on the column. The method was used to determine quantitatively the percent IdUrd substitution of thymidine in Chinese hamster lung cells in vitro and BA1112 rhabdomyosarcoma in WAG/Rij rats perfused with IdUrd. It was possible to determine the thymidine substitution by IdUrd as small as 1% using a few micrograms of DNA. The close correspondence between the percent substitutions determined by HPLC and those determined by radioactive assay using [125I]-labelled IdUrd, confirmed the accuracy of our HPLC method. The HPLC analysis is especially suitable for the determination of percent IdUrd substitution of thymidine in tissue biopsies from animals used in in vivo experiments or humans undergoing radiation treatment.     

Mechanism of stimulation of polyphenylalanine synthesis by spermidine.

It is shown that the stimulation of polyphenylalanine synthesis by spermidine is due mainly to the stimulation of initiation of polypeptide synthesis by following reasons: 1) the binding of poly(U) to ribosomes was stimulated more by spermidine than the binding of Phe-tRNA to ribosomes, and 2) the number of polyphenylalanine chains was increased more by spermidine than the extension of the chain length. In addition, it is shown that 30S ribosomal subunits are responsible for the stimulation of polyphenylalanine synthesis by spermidine.     

Multistep fluorescence resonance energy transfer in sequential chromophore array constructed on oligo-DNA assemblies

Sequential arrays of chromophores at regulated distances were constructed on a noncovalent DNA molecular assembly system in aqueous media. Photoinduced fluorescence resonance energy transfer (FRET) behaviors were then observed. We designed a number of chromophore/oligo-DNA conjugates with varying sequences. The chromophores eosin (Eo), TexasRed (TR), and tetramethylrhodamine (Rho) were employed as the energy donor, acceptor, and mediator, respectively, based on overlapping excitation and emission spectra. The chromophores were attached via aminolinkers to the 5'-terminals of 10mer oligo-DNAs consisting of AT rich sequences. The arrangement of Eo-Rho or Rho-TR with 10-residue (1 pitch of duplex) distances was ensured by duplex formation of the conjugates with a 20mer matrix oligo-DNA composed of complementary sequences to the conjugates. Single-step FRET from Eo to Rho and from Rho to TR was confirmed on the duplex. The three chromophore conjugates were then mixed with longer matrix oligo-DNAs (30 or 40mer) consisting of complementary sequences to the conjugates, producing Eo-(Rho)(n)-TR (n = 1 or 2) arrays with 10-residue distances. Multistep FRET from Eo to TR through the Rho mediator(s) was observed on the molecular assemblies. This photoenergy transmission system offers a good model for a photoenergy transmission system mimicking photosynthetic systems.     

Lateral transfer of eukaryotic ribosomal RNA genes: an emerging concern for molecular ecology of microbial eukaryotes

Ribosomal RNA (rRNA) genes are widely utilized in depicting organismal diversity and distribution in a wide range of environments. Although a few cases of lateral transfer of rRNA genes between closely related prokaryotes have been reported, it remains to be reported from eukaryotes. Here, we report the first case of lateral transfer of eukaryotic rRNA genes. Two distinct sequences of the 18S rRNA gene were detected from a clonal culture of the stramenopile, Ciliophrys infusionum. One was clearly derived from Ciliophrys, but the other gene originated from a perkinsid alveolate. Genome-walking analyses revealed that this alveolate-type rRNA gene is immediately adjacent to two protein-coding genes (ubc12 and usp39), and the origin of both genes was shown to be a stramenopile (that is, Ciliophrys) in our phylogenetic analyses. These findings indicate that the alveolate-type rRNA gene is encoded on the Ciliophrys genome and that eukaryotic rRNA genes can be transferred laterally.     

Crystal structure of the N-terminal RecA-like domain of a DEAD-box RNA helicase, the Dugesia japonica vasa-like gene B protein

The Dugesia japonica vasa-like gene B (DjVLGB) protein is a DEAD-box RNA helicase of a planarian, which is well known for its strong regenerative capacity. DjVLGB shares sequence similarity to the Drosophila germ-line-specific DEAD-box RNA helicase Vasa, and even higher similarity to its paralogue, mouse PL10. In this study, we solved the crystal structure of the DjVLGB N-terminal RecA-like domain. The overall fold and the structures of the putative ATPase active site of the DjVLGB N-terminal RecA-like domain are similar to those of the previously reported DEAD-box RNA helicase structures. In contrast, the surface structure of the side opposite to the putative ATPase active site is different from those of the other DEAD-box RNA helicases; the characteristic hydrophobic pockets are formed with aromatic and proline residues. These pocket-forming residues are conserved in the PL10-subfamily proteins, but less conserved in the Vasa orthologues and not conserved in the DEAD-box RNA helicases. Therefore, the structural features that we found are characteristic of the PL10-subfamily proteins and might contribute to their biological roles in germ-line development.     

Terminal Nerve GnRH3 Neurons Mediate Slow Avoidance of Carbon Dioxide in Larval Zebrafish

1. Download high-res image (139KB)
2. Download full-size image

     

Glucose-sensing carbon paste electrode containing polyethylene glycol-modified glucose oxidase

Polyethylene glycol-modified glucose oxidase (PEG-GOD) was prepared. Carbon paste (CP) containing PEG-GOD retained enzyme activity of 0·02 U cm⁻². Anodic and cathodic peak currents of modified GOD in CP matrix were observed on the differential pulse voltammograms at the potential of −0·36 and −0·36 V vs. Ag/AgCl, respectively. The addition of glucose to a test solution brought about an increase in the anodic current on the PEG-GOD-based electrode at the potential as low as 0·0 V vs. Ag/AgCl. The current increase was proportional to the concentration of glucose up to 50 mM.     

FABP3 Protein Promotes α-Synuclein Oligomerization Associated with 1-Methyl-1,2,3,6-tetrahydropiridine-induced Neurotoxicity

α-Synuclein (αSyn) accumulation in dopaminergic (DA) neurons is partly regulated by long-chain polyunsaturated fatty acids. We found that fatty acid-binding protein 3 (FABP3, H-FABP), a factor critical for arachidonic acid (AA) transport and metabolism in brain, is highly expressed in DA neurons. Fabp3 knock-out (Fabp3^−/−) mice were resistant to 1-methyl-1,2,3,6-tetrahydropiridine-induced DA neurodegeneration in the substantia nigra pars compacta and showed improved motor function. Interestingly, FABP3 interacted with αSyn in the substantia nigra pars compacta, and αSyn accumulation following 1-methyl-1,2,3,6-tetrahydropiridine treatment was attenuated in Fabp3^−/− compared with wild-type mice. We confirmed that FABP3 overexpression aggravates AA-induced αSyn oligomerization and promotes cell death in PC12 cells, whereas overexpression of a mutant form of FABP3 lacking fatty-acid binding capacity did not. Taken together, αSyn oligomerization in DA neurons is likely aggravated by AA through FABP3 in Parkinson disease pathology.