Yasuo Kikuchi

TWO NEW MEMBERS OF THE MURINE SIM GENE FAMILY ARE TRANSCRIPTIONAL REPRESSORS AND SHOW DIFFERENT EXPRESSION PATTERNS DURING MOUSE EMBRYOGENESIS

From a cDNA library of mouse skeletal muscle, we have isolated mouse Sim1 (mSim1) cDNA encoding a polypeptide of 765 amino acids with striking amino acid identify in basic helix-loop-helix (89% identify) and PAS (89 % identify) domains to previously identified mSim2, although the carboxy-terminal third of the molecule did not show any similarity to mSim2 or Drosophila Sim (dSim). Yeast two-hybrid analysis and coimmunoprecipitation experiments demonstrated that both of the mSim gene products interacted with Arnt even more efficiently than AhR, a natural partner of Arnt, suggesting a functional cooperativity with Arnt. In sharp contrast with dSim having transcriptional-enhancing activity in the carboxy-terminal region, the two mSims possessed a repressive activity toward Arnt in the heterodimer complex. This is the first example of bHLH-PAS proteins with transrepressor activity, although some genetic data suggest that dSim plays a repressive role in gene expression (Z. Chang, D. Price, S. Bockheim, M. J. Boedigheimer, R. Smith, and A. Laughon, Dev. Biol. 160:315-322, 1993; D. M. Mellerick and M. Nirenberg, Dev. Biol. 171:306-316, 1995). Whole-mount in situ hybridization showed restricted and characteristic expression patterns of the two mSim mRNAs in various tissues and organs during embryogenesis, such as those for the somite, the nephrogenic cord, and the mesencephalon (for mSim1) and those for the diencephalon, branchial arches, and limbs (for mSim2). From sequence similarity and chromosomal localization, it is concluded that mSim2 is an ortholog of hSim2, which is proposed to be a candidate gene responsible for Downs syndrome. The sites of mSim2 expression showed an overlap with the affected regions of the syndrome, further strengthening involvement of mSim2 in Downs syndrome.

Regulation of CYP1A1 expression.

CYP1A1 is considered to be involved mainly in oxidative metabolism of exogenous chemicals and drugs. Synthesis of this hemoprotein is induced in livers, lungs, and other tissues of experimental animals by the administration of these chemicals. Regulatory mechanisms of the induction process of the protein have been investigated by the DNA transfer method using the isolated genomic DNA. At least two kinds of cis‐acting regulatory DNA sequences are localized 5′ upstream of the gene. One is distributed five times in a relatively wide range from –0.5 to –3.5 kb and functions as an inducible enhancer‐designated xenobiotic responsive element or XRE. The other is localized just upstream of the TATA sequence and acts as a regulatory element for the constitutive expression. The two DNA elements are required for a high level of the inducible expression. Their cognate DNA binding factors are recognized in the nuclear extracts of Hepa‐1 cells and rat liver cells which show the inducible expression of CYP1A1 in response to the inducer. This paper discusses the regulatory mechanisms of CYP1A1 gene expression by summarizing the present state of knowledge about properties of the DNA regulatory elements and their cognate DNA‐binding factors.—Fujii‐Kuriyama, Y.; Imataka, H.; Sogawa, K.; Yasumoto, K.‐i.; Kikuchi, Y. Regulation of CYP1A1 expression. FASEB J. 6: 706‐710; 1992.

Transactivation mechanisms of mouse clock transcription factors, mClock and mArnt3.

The Arnt3 (also termed as BMAL1 or MOP3)/Clock heterodimer is a positive regulator of circadian rhythm and activates the transcription of target genes such as per1 and vasopressin.

HIF-1α-prolyl hydroxylase: molecular target of nitric oxide in the hypoxic signal transduction pathway

Abstract We have investigated inhibitory mechanisms of hypoxic activation of HIF-1α by nitric oxide (NO). Using a Hep3B cell-derived cell line, HRE7 cells, we found that the inhibition of HIF-1α activity by NO requires a substantial amount of oxygen, albeit at a lower level. We further investigated the effect of NO on the binding activity of the von Hippel–Lindau tumor suppressor protein (pVHL) to the N-terminal activation domain (NAD) overlapping the oxygen-dependent degradation domain (ODD) of HIF-1α, because this reaction involves prolyl hydroxylation in NAD that requires oxygen. Although we could not detect any binding activity when NAD was incubated with whole cell extracts from cells treated with CoCl 2 or desferrioxamine, the binding capacity was manifested when Hep3B cells were treated together with NO. This activation was also observed when whole cell extracts from CoCl 2 -treated cells were incubated with NO. The prolyl hydroxylase from Hep3B cells treated with CoCl 2 was partially purified about 80-fold, and several enzymatic properties were examined. The enzyme required ferrous ion and 2-oxoglutaric acid. Strong activation of the prolyl hydroxylase by NO was observed without further addition of ferrous ion.

Synthetic studies on spider neurotoxins (I): total synthesis of nephilatoxins (NPTX-9) and NPTX-11), new neurotoxins of Joro spider (Nephila clavata)

Abstract The first and efficient synthesis of Nephilatoxins (NPTX-9 and NPTX-11), new neurotoxins of Joro spider ( Nephila clavata ), has been achieved by employing a key azide intermediate as the polyamine unit.

Syntheses and enzymic hydroxylation of protocollagen model peptides containing glutamyl or leucyl residue.

Protocollagen-proline hydroxylase converts the prolyl residues in protocollagen to hydroxyprolyl residues by an oxygenase mechanism [ l-3] . ProtocolIagen model peptides, (Pro-Cly-Pro), [4] or (ProPro-Gly), _-20 [5], were used as synthetic substrates of this enzyme. Studies on the enzymic hydroxylation of (Pro-Pro-Gly), with defined molecular weight (n = I-20) suggested that the prolyl residues in nonterminal -Pro--Pro-Glyunits were more easily hydroxylated than those at the terminal and that the triple stranded conformation in larger peptides was unfavourable for the hydroxylation [5). As natural protocollagen has a molecular weight of about 140 000 161, most of the pro&y1 residues to be hydroxylated are at the non-ternlinal -X-Pro-Glysequence. The amino acid residues at the X-position of the above sequence may also affect the hydroxylation. No hydroxylation was observed with the repeating tripeptides(Gly-Pro-Gly)i_4, however, synthetic (Ala-Pro-Gly), _ s were reported to be hydroxylated [7]. The present work was carried out to examine the effect of glutamyl and leucyl residues at the X-position of -X-Pro-Glysequence on the enzymic hydroxylation.

Hydroxylation of poly(l-prolyl-l-prolylglycyl) of defined molecular weights by protocollagen proline hydroxylase

Hydroxyproline in collagen is synthesized by the hydroxylation of proline residues in protocollagen, a collagen precursor [l] . The hydroxylating enzyme is an oxygenase [2,3] and is purified from chick embryos [4]. The latter authors studied the hydroxylation of polypeptides of various molecular weights, which were synthesized by polymerization of a tripeptide, Lprolylglycyl-Lproline, and fractionated by gel filtration. The molecular weight of the polymerization products had a distribution range. Recently, Sakakibara et al. (including one of the authors) synthesized sequential polypeptides with defined molecular weights by stepwise addition of t-amyloxycarbonyl-L-prolyl-Lprolylglycine on the Merrifield resin [5] . The products, (Lprolyl-L-prolylglycyl), or (Pro-Pro-Gly), , with n values bigger than ten showed a temperature-dependent transition in optical rotation and apparent molecular weight. The phenomenon was not observed with the above nondefined polymers. The transition was ascribed to the conformational change between single chain and triple stranded structures, similar to those observed with collagen [6]. The present paper describes the enzymatic hydroxylation of the products.

Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Epstein-Barr Virus-Associated Nuclear Antigen-1 Using a Synthetic Oligopeptide

Epstein-Barr virus (EBV) is a human lymphotropic herpes virus and a causative agent of infectious mononucleosis. EBV is also closely associated with Burkitts lymphoma and nasopharyngeal carcinoma (1) . EBV infection is generally latent and is accompanied by the synthesis of EBV-associated nuclear antigen (EBNA) (14). EBNA, therefore, serves as an indicator of the presence of the latent EBV genome in the cells, and demonstration of antibodies to EBNA is important for the diagnosis of EBV-related diseases (1) . Recently, several types of EBNA (EBNA-1 or -K, EBNA-2 or -M, and EBNA-3) have been identified by transfection of mouse L cells with cloned fragments of EBV-DNA (4, 5, 17). Among these different types of EBNA, EBNA-1 is a chromosome-binding protein and is the serologically dominant antigen since essentially all anti-EBNA positive human sera reacted with EBNA-1 (12). The EBNA-1 protein has been shown to contain a unique irregular glycinealanine copolymer region (6). A fusion protein composed of bacterial ƒA-galacto

Methionine sulfoxide in the hinge-ligament protein of molluscan bivalve species

Methionine sulfoxide, an oxidized form of methionine, was detected at extremely high levels (15–30 mol% of total amino acids) in the protein from the elastic hinge-ligaments of molluscan bivalve species. The presence of methionine sulfoxide in the intact protein was confirmed by amino acid analysis of the NaOH-hydrolysate of the protein, non-destructive analyses of the ligament with solid-state 13C-NMR and IR-spectrometries and by the observation that the protein was resistant to the BrCN treatment. The conversion of methionine into its sulfoxide is almost complete. The oxidation process may be non enzymatic because two diastereomers, (5R)-L- and (5S)-L-methionine sulfoxide, were detected in the protein. The methionine sulfoxide residues might contribute to keep the protein highly hydrophilic and to promote the swelling of the ligaments.