Masatada Aoki

Buprenorphine hydrochloride induces apoptosis in NG108-15 nerve cells

A morphine alkaloid derivative, buprenorphine hydrochloride, induces apoptosis in NG108-15 cells. Apoptosis was detected mainly by apoptosis-specific DNA fragmentation and morphological changes. This apoptosis was dose-dependent and the time-course experiment indicated that DNA fragmentation occurred within 4 h after administration of buprenorphine hydrochloride. Specific inhibitors of the previously characterized apoptotic signal cascade as well as antagonists for opioid receptors were tested. Zn2+, herbimycin A, caspase inhibitors YVAD (Ac-Tyr-Val-Ala-Asp-CHO) and DEVD (Ac-Asp-Glu-Val-Asp-CHO), naloxone and naltrindole had no effect on apoptosis-specific DNA fragmentation. The serine protease inhibitor TPCK (N-tosyl-L-phenylalanyl chloromethyl ketone) specifically inhibited apoptosis-specific DNA fragmentation induced by buprenorphine hydrochloride; however, cell viability measurements revealed that cell death still occurred in NG108-15 cells. Thus TPCK pretreatment before buprenorphine hydrochloride administration induced apoptosis-independent cell death, presumably necrosis, in NG108-15 cells. This suggests that an unidentified serine protease, presumably functioning in the buprenorphine hydrochloride-specific death-signal cascade, could be pivotal for the rapid apoptosis observed in NG108-15 cells upon treatment with buprenorphine hydrochloride.

Purification and characterization of 3α/β-hydroxysteroid dehydrogenase from mature porcine testicular cytosol

Abstract NADPH-dependent 3α/β-hydroxysteroid dehydrogenase (3α/β-HSD) was purified to apparent homogeneity from testicular cytosol of mature pigs. The purified enzyme catalyzes the conversion of 5α-dihydrotestosterone (5α-DHT) to both 5α-androstane-3α,17β-diol and 5α-androstane-3β,17β-diol. The molecular weight of the enzyme was estimated to be 31 kDa by SDS-polyacrylamide gel electrophoresis and 40 kDa by gel filtration chromatography indicating that the native 3α/β-HSD is a monomer. The isoelectric point of the purified enzyme was found to be 6.2 by density gradient isoelectric focusing and 6.4 by chromatofocusing. The enzyme reduced both 5α- and 5β-DHT, 5α- and 5β-dihydroprogesterone, 5α- and 5β-dihydrocortisol, prostaglandin E2, 13,14-dihydro-15-keto-prostaglandin E2 and 13,14-dihydro-15-keto-prostaglandin F2α. Moreover, the enzyme caused rapid reduction of other carbonyl compounds including aldehydes, ketones and quinones. The rates of reduction of these compounds are fast relative to the rates of reduction of steroids and prostaglandins. The purified enzyme was inhibited by AgNO3, SH-reagent, quercetin, hexesterol, stilbestrol, disulfiram and divalent cation such as Cu2+, Hg2+ and Cd2+. The two enzymes show certain similarities (e.g. molecular weight, cross-reactivity to a common antibody) and certain striking differences (e.g. pI, effects of various inhibitors and greater enzyme activity towards steroids (neonatal form) or prostaglandins (mature form). Reasons are give for suggesting that these enzymes are closely related to carbonyl reductase.

Expression of the Polyubiquitin Gene Early in the Buprenorphine Hydrochloride-induced Apoptosis of NG108-15 Cells

To clone genes expressed early in the buprenorphine hydrochloride (Bph)-induced apoptosis of NG108-15 nerve cells, we adopted a previously reported rapid and simple differential display (DD) cloning procedure. Complementary DNA was generated from differentially expressed mRNAs by reverse transcription (RT) using a fully degenerate 6-mer oligonucleotide as the primer. PCR amplification was then conducted using a combination of three arbitrary but defined 10-bp nucleotide primers. The differentially generated DNA fragments were detected by agarose gel electrophoresis, and 9 were excised from the gel and subcloned into a sequencing vector. Three DNAs that were specifically expressed upon Bph-induced apoptosis of NG108-15 cells were sequenced. Their specific expression was then confirmed by reverse-transcription PCR. One was identified as the mouse polyubiquitin gene c, and the others remain unidentified. Northern and western blots indicated the transcription and translation of polyubiquitin early in Bph-induced apoptosis. The polyubiquitination of apoptotic cellular proteins was also confirmed.