Ken-ichi Saguchi

Bradykinin-potentiating peptides and C-type natriuretic peptides from snake venom.

Cloning of cDNAs encoding bradykinin-potentiating peptides (BPPs)-C-type natriuretic peptide (CNP) precursor or its homologue was performed for cDNA libraries of Bothrops jararaca (South American snake), Trimeresurus flavoviridis, Trimeresurus gramineus and Agkistrodon halys blomhoffi (Asian snakes), all belonging to Crotalinae subfamily. Each cDNA library was constructed from the venom glands of a single snake to preclude ambiguity by intraspecies variation in venom components. Thirteen positive clones derived from B. jararaca were divided into two types depending on restriction sites. Differences in the nucleotide sequence arise at three locations and two of them accompanied amino acid conversions. Despite the differences, both types of cDNA clones encode the BPP-CNP precursor of 256 amino acid residues. Sequence analysis demonstrated that cDNA clones from three Asian snakes encode homologues of the BPP-CNP precursor from B. jararaca. In a precursor polypeptide, a signal sequence (approximately 25 aa) at the N-terminus is followed by sequences of BPP or the analogue (5-13 aa) with flanking spacer sequences (indefinite number of aa), an intervening linker sequence (approximately 144 aa) with unidentified function, and a CNP sequence (22 aa) with a preceding processing signal sequence (10 aa). cDNA clones from A. halys blomhoffi encode two distinct peptides in place of BPP, and T. flavoviridis and T. gramineus were shown to have considerably different sequences in the BPP domain from those known as BPP sequences. The present results provide evidence for a wide distribution of the orthologous gene expressing a series of bioactive peptides among Crotalinae subfamily.

Molecular cloning and expression analysis of the aryl hydrocarbon receptor of Xenopus laevis

The aryl hydrocarbon receptor (AHR) is a member of the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors. Although this receptor has been known to mediate the toxic effects of environmental pollutants, its physiological functions remain elusive. Here, we describe the isolation and expression pattern of the Xenopus AHR gene. The predicted amino acid sequence contained regions characteristic of other vertebrate AHRs. However, in line with previously described fish AHR genes, no distinct Q-rich domain was found. Phylogenetic analysis demonstrated that Xenopus AHR was clustered within the AHR1 clade. As in the case of mammalian AHR genes, the Xenopus AHR gene was expressed in all the adult tissues tested. Xenopus AHR was also expressed during early development, in parallel with expression of the CYP1A7 gene, which is thought to be regulated by AHR. These results suggest that while frogs are relatively tolerant to TCDD toxicity, the AHR of frogs has characteristics similar to those of other vertebrate AHRs.

Two N-terminally truncated forms of C-type natriuretic peptide from habu snake venom

Two N-terminally truncated forms of the C-type natriuretic peptide (CNP) were isolated from the venom of habu snake, Trimeresurus flavoviridis, and their structures were determined by EMI-MS spectrometry and amino acid sequencing. Tf-CNP(6-22), the shorter peptide retaining the 17-membered ring structure formed by an intra-molecular disulfide bridge, has a vasorelaxant activity in rat aortic strips and a diuretic potency in anesthetized rats. Tf-CNP(3-22), the other 20 amino acid residues peptide, also comprised the 17- membered ring with a short N-terminal extension of 3 amino acid residues. Tf-CNP(6-22), the ring, is the shortest naturally occurring CNP peptide identified so far, and as potent as Tf-CNP(1-22), the supposedly intact CNP of 22 amino acid residues.

Mapping of human inter-alpha-trypsin inhibitor family heavy chain-related protein gene (ITIHL1) to human chromosome 3p21→p14

Inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) is a novel glycoprotein isolated from human plasma. The cDNA encoding IHRP has recently been cloned from human liver cDNA libraries. We report the mapping of this gene (ITIHL1) by fluorescence in situ hybridization using a 2.5-kb cDNA fragment as a probe. ITIHL1 was localized to chromosome region 3p21-->p14 where the genes of heavy chain 1 and 3 of inter-alpha-trypsin inhibitor are located. This result, together with significant homology between the nucleotide sequences of ITIHL1 and the heavy chain genes, supports ITIHL1 as being a member of an evolutionary related gene family of ITI heavy chains. Northern blot analysis indicated that IHRP was predominantly synthesized in liver. From Southern blot analysis, it was tentatively concluded that ITIHL1 is a single copy gene.

Cloning and expression analysis of a cDNA coding for a dexamethasone‐inducible cytochrome P450 in Xenopus laevis liver

We previously purified a cytochrome P450 (P450) from liver microsomes of adult female Xenopus laevis. In this study, we screened a cDNA library of Xenopus liver to isolate the cDNA clone coding for this P450. The 5′‐end of the resultant cDNA was truncated at the N‐terminal region and extended by method of rapid amplification of eDNA end to give the complete coding sequence. Amino acid sequence showed this clone to be 36% to 55% identical to members of the CYP2 family and less than 31% identical to members of other gene famines, and to belong to the CYP2Q subfamily. This gene is expressed constitutively in the livers of adult male and female frogs, and is significantly induced by dexamethasone administration.