Yuki Fujimori

Expression of survivin mRNA in dog tumors

Survivin, a member of the inhibitor of apoptosis (IAP) gene family, overexpresses in various human tumors. Recently this protein has attracted strong interest as a potential prognostic marker because it promotes malignancy through anti-apoptotic activity and is associated with a more aggressive phenotype. To explore the utility of survivin as a veterinary marker of tumor malignancy, we performed molecular cloning of dog survivin cDNA and studied survivin mRNA expression in a variety of naturally occurring dog tumors. The dog cDNA contains a 426-bp open reading frame encoding 142 amino acids of polypeptide, in which a structure termed the baculovirus IAP repeat (BIR) domain, commonly observed in IAPs, is found, as it is in other mammalian survivin protein. The transcript was detected in many adult normal organs including heart, lung, liver, stomach, duodenum, colon, spleen, kidney and testis. As a result of quantitative expression analysis by real-time PCR undertaken for benign and malignant tumors, overexpression of the survivin gene was found in 3 of 18 malignant tumors and in none of the benign tumors, suggesting that survivin overexpression is associated with tumor malignancy in dog.

Expression of endothelin-1 and vasoactive intestinal contractor genes in mouse organs during the perinatal period

In an attempt to understand the significance of endothelin-1 (ET-1) and vasoactive intestinal contractor (VIC)/ET-2 peptides in organs during perinatal development, we performed quantitative analysis of ET-1 and VIC gene expression in mouse organs obtained from embryos at days 14 and 17 (E-14 and E-17) of pregnancy, neonates at days 0, 1, 3 and 7 after birth (N-0, -1, -3 and -7), and adult mice (10 weeks old). In intestine, VIC gene expression progressively increased between E-14 and N-1 (approximately 10-fold) and then remained constant into adulthood. ET-1 gene expression exhibited a one-step increase between E-17 and N-0, subsequently remaining constant. In lung, a sharp increase in ET-1 mRNA level (approximately 10-fold) was noticed between E-14 and N-0. The gene expression pattern of VIC, with a peak at N-0, was similar to that of ET-1 although the expression level of VIC was two to three orders of magnitudes lower than that of ET-1. Gene expression patterns of ET-1 and VIC remained nearly constant in brain, heart, liver and kidney throughout the period examined. Considering that the intestinal and pulmonary gene expression levels of both genes reached almost the same level as observed in adult soon after birth, we suggest that these peptides may be involved in the emergence and maintenance of intestinal and pulmonary functions vital after birth.

Anti-muscarinic actions of mitoxantrone in isolated heart muscles of guinea pigs

A hypotheses that mitoxantrone is a competitive antagonist at muscarinic cholinergic receptors was examined in guinea-pig hearts. In isolated left atrial muscle preparations, electrically paced at 2 Hz, the muscarinic agonist, carbachol, caused a concentration-dependent decrease in developed tension. Mitoxantrone caused a parallel right-ward shift of the concentration-response curve for carbachol. Schild plots for the effect of mitoxantrone on the carbachol concentration-response relationship were linear with a slope of 0.88 which was not significantly different from the unity. The right-ward shift of the carbachol concentration-response relationship by mitoxantrone significantly reversed after an additional incubation with a mitoxantrone-free solution, although the reversal was incomplete after a 2-h incubation in the mitoxantrone-free solution. Mitoxantrone caused a concentration-dependent displacement of specific [3H]quinuclidinyl benzilate binding to membrane preparations obtained from ventricular muscles of guinea-pig hearts. These results indicate that mitoxantrone acts as a competitive antagonist for the muscarinic receptors.

cDNA cloning, sequence analysis and organ distribution of horse preproendothelin-2.

We cloned and characterized horse preproendothelin-2 (PPET-2) cDNA from intestinal tissue. The cDNA encoded 178 amino acids of the PPET-2 polypeptide, in which a 21-amino-acid mature endothelin-2 peptide and a 16-amino acid endothelin-2-like peptide were found. For the open reading frame the correspondence of horse PPET-2 cDNA with those of the ferret, human, dog, mouse and rat was 85.1%, 84.9%, 82.1%, 77.8% and 77.2%, respectively. Analysis of the organ distribution of PPET-2 mRNA by reverse transcription-polymerase chain reaction demonstrated that the kidney, stomach and small intestine are major sites of expression of the PPET-2 gene. Surprisingly, the mRNA is not detected in the large intestine, where high expression is demonstrated in the mouse and rat. This difference may result from the underlying functional differences of the large intestine between a herbivore (horse) and an omnivore (mouse and rat).

Cloning of bovine preproendothelin-2 cDNA and organ distribution of transcripts.

Endothelin-2 (ET2), which was originally identified in human, is a bioactive peptide of 21 amino acids with strong vasoconstrictive and pressor effects. Here we report the cDNA cloning and characterization of bovine preproendothelin-2 (PPET2), the precursor form of ET2. The bovine cDNA encodes 177 amino acids of the PPET2 polypeptide, in which a 21-amino acid mature ET2 peptide and a 16-amino acid ET2-like peptide as well as a 23-amino acid putative signal peptide were found. The bovine ET2-like peptide sequence was missing a dibasic amino acid pair at the C-terminal, in contrast to human, mouse and rat, for which the ET2-like sequence is flanked by dibasic pairs at both the N- and C-terminals. Gene expression analysis by RT-PCR showed that the transcript is expressed in various organs including heart, lung, liver, kidney, gastrointestinal tract, uterus and ovary, but not in spleen. Within the gastrointestinal tract, gene expression was detected in rumen, a ruminant-specific digestive organ, as well as stomach, duodenum and colon.

Primary structure of dog preproendothelin-3 and elevated gene expression in kidney affected with interstitial nephritis.

To compare the structure of the precursor polypeptide of dog endothelin-3, preproendothelin-3 (PPET-3), with the PPET-3 of other mammals, we cloned dog cDNA from lung tissue using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. An open reading frame encoding a 198-amino-acid polypeptide was found in the cDNA. Regions corresponding to a bioactive mature endothelin-3 peptide, an intermediate form known as big-endothelin-3 and an endothelin-3- like peptide were observed in the putative PPET-3. Comparative analysis showed that the similarity of the dog open reading frame sequence with those from human hypothalamus, mouse intestine, and rat eye is 76.2%, 69.5% and 66.3%, respectively, and that the similarity at the amino acid level is 65.6%, 59.8% and 58.8%, respectively. RT-PCR demonstrated significant elevated expression of PPET-3 mRNA in the kidney of dog with interstitial nephritis.

cDNA cloning of hamster angiotensin-converting enzyme and mRNA expression

Angiotensin-converting enzyme (ACE; EC 3.4.15.1), a dipeptidyl carboxypeptidase, converts angiotensin I to angiotensin II, the central product of the renin–angiotensin system. We here report molecular cloning of the complete open reading frame (ORF) of hamster somatic-type ACE and its expression in hamster organs. The cloned cDNA comprises an ORF of 3942 bp, which encodes 1314 amino acids of the precursor protein of hamster somatic ACE. On the deduced amino acid sequence a putative signal peptide and a transmembrane segment are predicted at the N-terminus and near the C-terminus, respectively. Two homologous domains, referred to as N- and C-domains, are present within somatic ACE, and within each of the homologous domains a putative active center is found, as has been the case in human, mouse, rat and rabbit. The similarity of the hamster sequence with the sequences of these other mammals at both the nucleotide and amino acid levels is high (above 83%). mRNA expression analysis by conventional polymerase chain reaction (PCR) shows wide distribution of the transcript, with dominant expression in lung and kidney. Quantitative analysis of mRNA expression demonstrates that levels in lung and kidney are 100–1000 times higher than in the other organs, suggesting that these organs are important in the hamster renin–angiotensin system, as they are for other mammals.