Yoshikazu Sukenaga

Impact of chymase inhibitor on cardiac function and survival after myocardial infarction

OBJECTIVES Recent studies have demonstrated that hamsters, like humans, possess both angiotensin converting enzyme (ACE)- and chymase-dependent angiotensin (Ang) II-forming pathways in cardiovascular tissues. We recently found that, after myocardial infarction (MI) in hamsters, cardiac chymase was significantly activated. In order to determine whether suppression of cardiac chymase activity could provide prognostic benefit after MI, we examined the effects of NK3201, a novel, orally active and specific chymase inhibitor, on cardiac function and survival during the acute phase of MI in hamsters. METHODS Two hundred and ten male Syrian hamsters were used in the present study. The left coronary artery of each hamster was ligated to induce MI. NK3201 at a dose of 30 mg/kg per day was administered orally by gastric gavage, starting either 3 days before or 1 day after MI. RESULTS ACE and chymase activities were significantly increased in the infarcted left ventricle 3 days after MI. NK3201 treatment starting 3 days before MI significantly inhibited the increase in cardiac chymase activity, while it did not affect ACE activity either in plasma or in heart 3 days after MI. A significant improvement in cardiac function was observed 3 and 14 days after MI in the group receiving NK3201. Compared with vehicle treatment, NK3201 treatment initiated either 3 days before or 1 day after MI significantly reduced the mortality rate during the 14 days of observation following MI. CONCLUSIONS These findings indicate that cardiac chymase plays an important role after MI and this finding may provide a novel therapeutic target in post-MI treatment.

Oral administration of a specific chymase inhibitor, NK3201, inhibits vascular proliferation in grafted vein.

Chymase may play an important role in vascular proliferation, as shown by in-vitro experiments, but the role of chymase in vivo has been unclear. In this study, we investigated the effect of a novel chymase inhibitor, NK3201, on this proliferation in dog grafted veins. NK3201 inhibited human and dog chymases, but not rabbit ACE. NK3201 suppressed the Ang I-induced vascular contraction in isolated dog arteries in the presence of an ACE inhibitor, and the IC50 value of chymostatin and NK3201 in dog artery was 320 nM. In dog, the concentration of NK3201 in blood was about 10 microM at 24 h after oral administration of the drug (5 mg/kg). In the group treated with NK3201, each dog was administered orally 5 mg/kg per day from 5 days before to the day before the removal of the grafted veins. Each dog underwent right common carotid artery bypass grafting with the ipsilaterial external jugular vein. By 28 days after grafting, a significant vascular proliferation was observed in the grafted veins and the chymase activity was also increased significantly. Treatment with chymase inhibitor significantly suppressed the proliferation of the grafted veins and the increased chymase activity. In this study, we demonstrate for the first time that oral administration of a specific chymase inhibitor, NK3201, appears useful for preventing vascular proliferation.

Construction of a runaway vector and its use for a high-level expression of a cloned human superoxide dismutase gene

SummaryA runaway cloning vector pR3 from plasmid pBEU17 has been constructed by introducing three deletions (ΔI, ΔH and ΔEHi), multiple cloning sites and transcription terminator sequences. Introduction of the deletion ΔH restored a drastic plasmid amplification at high temperature (37° C). This suggested the presence of unknown sequences around the ΔH region which repressed runaway replication. To examine the usefulness of a pR3 runaway vector on the high-level expression of a cloned gene, a pRT8 plasmid containing a cloned human superoxide dismutase (SOD) gene in the pR3 was constructed. A host with tolerance to high copy numbers of pRT8 was selected, and the cultural conditions required for high-level expression were investigated. It was shown that the copy number of pRT8 increased in the stationary phase even at 30° C, and that cells carrying an excess number of plasmid did not support the runaway replication, yielding less SOD protein. SOD synthesis in the production culture lasted for about 24 h after the thermal shift, though viability was soon lost. The maximum yield was determined as approximately 20% of total cellular protein. This was 12-fold higher than that of a vector having the ColE1 plasmid replicon.

The 5′ untranslated region of the human cellular glutathione peroxidase gene is indispensable for its expression in COS-7 cells

We studied the expression of the human cellular glutathione peroxidase (GPx) gene, from which a key enzyme containing selenocysteine (Scy) at the active site is produced. Expression of some human GPx gene mutants in COS‐7 cells revealed that the 5′ untranslated region (utr) was necessary for expression of the GPx gene, since mutant genes having 10 base pairs (bps) at the 5′utr (the complete had 311 bps) expressed GPx at very low levels. The genes with 311 or 408 bps at the 5′utr were better expressed than those having 257 bps. The GPx gene having 133 bps at the 3′utr (80 bps shorter than the entire length) was highly expressed. This deletion did not influence expression. We constructed some mutants in which 3 bases were altered at the upstream region of the Scy UGA codon in the frame of the GPx gene, by site‐directed mutagenesis. GPx expression decreased but the expression was restored. Therefore, the upstream region of the in‐frame Scy codon was not essential in the Scy decoding mechanisms. Finally, the 5′utr was essential for the expression of GPx gene. However, the deletion of a part of the 3′utr and the site‐directed mutation upstream of the Scy codon did not show drastic effects on the expression.

Purification and molecular cloning of chymase from human tonsils

A chymotrypsin‐like protease was purified to homogeneity from human tonsils by a series of Chromatographie procedures. The purified enzyme gave a single protein band with an apparent molecular mass of 30 kDa on SDS‐PAGE. The sequence of the first 21 amino acids at the N‐terminus of the enzyme was determined. A cDNA for the enzyme was cloned by PCR amplification from extracted tonsillar mRNA using a supposed N‐terminal oligonucleotide primer and a conserved C‐terminal primer of the chymase family. The deduced amino acid sequence of the isolated clone was identical to that of human chymase in connective tissue‐type mast cells from heart except for a Ser instead of a Cys at the N‐terminal 7th position.

Purification and characterization of a ubenimex (Bestatin)-sensitive aminopeptidase B-like enzyme from K562 human chronic myeloid leukemia cells

A ubenimex‐sensitive aminopeptidase B‐like enzyme was purified from the non‐membrane‐bound fraction of K562 cells by a series of chromatographic procedures and slab‐gel electrophoresis. The apparent molecular mass of the enzyme was estimated to be 73 kDa by SDS‐PAGE. The aminopeptidase activity was activated by chloride ions and inhibited by Zn2+, Cu2+, Cd2+, and p‐chloromercuribenzoic acid. Ubenimex was a potent inhibitor of this aminopeptidase in the nanomolar range. The sequence of the N‐terminus of the protein was not determined. Partial amino acid sequencing revealed that the N‐terminus of this aminopeptidase B‐like enzyme was blocked by acylation. The partial sequences of the two fragments produced by CNBr cleavage and an acylamino acid‐releasing reaction showed this enzyme to be a new aminopeptidase.

The detection of natural opal suppressor seryl-tRNA in Escherichia coli by the dot blot hybridization and its phosphorylation by a tRNA kinase

It was believed that there was no natural suppressor tRNA in Escherichia coli, however, it has been suggested that selC, relating to the synthesis of formate dehydrogenase of a selenoprotein [(1988) Nature 331, 723–725], codes for tRNA, even though the presence of tRNA has not yet been demonstrated. We detected the product of selC in the tRNA preparation of the E. coli MC 4100 strain by the dot blot hybridization method with a DNA probe (ACCGCTGGCGGC) corresponding to the extra arm of selC tRNA. Two hybridization peaks were found in the chromatographic pattern from Sephadex A50. The amount of tRNA was estimated to be about 0.03% of the total tRNA. The suppressor [3H]seryl‐tRNA was phosphorylated by a tRNA kinase in E. coli B. These results suggest that the opal suppressor seryl‐tRNA in E. coli should be converted to selenocysteyl‐tRNA through phosphoseryl‐tRNA, and occurs in vertebrates as a general phenomenon.