Molecular mechanisms of ginsenoside Rp1-mediated growth arrest and apoptosis.

Ginsenoside Rp1, a semi-synthesized ginseng saponin, was shown to have chemopreventive action and anti-metastatic potential. However, the molecular mechanisms of Rp1 on cell growth and death are not fully understood. In this study, the antiproliferative effect of Rp1 on HeLa cells in vitro was investigated. Treatment with Rp1 at 40 microM inhibited the proliferation and partial accumulation of cells at the G1 phase. Rp1-mediated G1 arrest was accompanied by decreased expression of cyclin D1, E, and A and increased expression of p21 without any significant change in p53 or phospho-p53 (Ser15). On the other hand, prolonged incubation with Rp1 at 40 microM caused apoptosis and activation of caspase-3, -8, and -9. The participation of these three caspases in apoptosis was more clearly shown in experiments using inhibitors, which markedly prevented Rp1-induced apoptosis in the case of each caspase. Cleavage of the polyADP-ribose polymerase, often used as an apoptotic marker, was also found in Rp1-induced apoptosis. Among Bcl-2 family proteins (Bad, Bax, Bid, Bcl-2), Bax and Bid were activated by Rp1 treatment, which resulted in the release of cytochrome c from mitochondria, following activation of caspase-9. These observations indicate that multiple cell cycle regulatory proteins and apoptosis-inducing proteins are regulated by Rp1 and contribute to Rp1-induced growth arrest and apoptosis.

SAFB1, an RBMX-binding protein, is a newly identified regulator of hepatic SREBP-1c gene.

Sterol regulatory element-binding protein (SREBP)-1c plays a crucial role in the regulation of lipogenic enzymes in the liver. We previously reported that an X-chromosome-linked RNA binding motif (RBMX) regulates the promoter activity of Srebp-1c. However, still unknown was how it regulates the gene expression. To elucidate this mechanism, we screened the cDNA library from mouse liver by yeast two-hybrid assay using RBMX as bait and identified scaffold attachment factor B1 (SAFB1). Immunoprecipitation assay demonstrated binding of SAFB1 to RBMX. Chromatin immunoprecipitation assay showed binding of both SAFB1 and RBMX to the upstream region of Srebp-1c gene. RNA interference of Safb1 reduced the basal and RBMX-induced Srebp-1c promoter activities, resulting in reduced Srebp-1c gene expression. The effect of SAFB1 overexpression on Srebp-1c promoter was found only in the presence of RBMX. These results indicate a major role for SAFB1 in the activation of Srebp-1c through its interaction with RBMX.

RBMX is a novel hepatic transcriptional regulator of SREBP-1c gene response to high-fructose diet.

In rodents a high-fructose diet induces metabolic derangements similar to those in metabolic syndrome. Previously we suggested that in mouse liver an unidentified nuclear protein binding to the sterol regulatory element (SRE)-binding protein-1c (SREBP-1c) promoter region plays a key role for the response to high-fructose diet. Here, using MALDI-TOF MASS technique, we identified an X-chromosome-linked RNA binding motif protein (RBMX) as a new candidate molecule. In electrophoretic mobility shift assay, anti-RBMX antibody displaced the bands induced by fructose-feeding. Overexpression or suppression of RBMX on rat hepatoma cells regulated the SREBP-1c promoter activity. RBMX may control SREBP-1c expression in mouse liver in response to high-fructose diet.

Effects of cyanide and dissolved oxygen concentration on biological Au recovery.

The number of discarded electric devices containing traces of Au is currently increasing. It is desirable to recover this Au because of its valuable physicochemical properties. Au is usually dissolved with relatively high concentrations of cyanide, which is associated with environmental risk. Chromobacterium violaceum is able to produce and detoxify small amounts of cyanide, and may thus be able to recover Au from discarded electric devices. This study investigated the effects of cyanide and dissolved oxygen concentration on biological Au recovery. Cyanide production by C. violaceum was sufficient to dissolve Au, while maintaining a high cyanide concentration did not enhance Au dissolution. Increased oxygen concentration enhanced Au dissolution from 0.04 to 0.16 mmol/l within the test period of 70 h. Electrochemical measurement clarified this phenomenon; the rest potential of Au in the cyanide solution produced by C. violaceum increased from -400 to -200 mV, while in the sterile cyanide solution, it was constant in cyanide concentrations ranging from 0 to 1.5 mmol/l and increased in dissolved oxygen concentrations ranging from 0 to 0.25 mmol/l. Therefore, it was clarified that dissolved oxygen concentration is the main factor affecting the efficiency of cyanide leaching of gold by using bacteria.

Molecular cloning of C4 gene and identification of the class III complement region in the shark MHC.

To clarify the evolutionary origin of the linkage of the MHC class III complement genes with the MHC class I and II genes, we isolated C4 cDNA from the banded hound shark (Triakis scyllium). Upon phylogenetic tree analysis, shark C4 formed a well-supported cluster with C4 of higher vertebrates, indicating that the C3/C4 gene duplication predated the divergence of cartilaginous fish from the main line of vertebrate evolution. The deduced amino acid sequence predicted the typical C4 three-subunits chain structure, but without the histidine residue catalytic for the thioester bond, suggesting the human C4A-like specificity. The linkage analysis of the complement genes, one C4 and two factor B (Bf) genes, to the shark MHC was performed using 56 siblings from two typing panels of T. scyllium and Ginglymostoma cirratum. The C4 and one of two Bf genes showed a perfect cosegregation with the class I and II genes, whereas two recombinants were identified for the other Bf gene. These results indicate that the linkage between the complement C4 and Bf genes, as well as the linkage between these complement genes and the MHC class I and II genes were established before the emergence of cartilaginous fish >460 million years ago.