Cloning and expression of the L-1-amino-2-propanol dehydrogenase gene from Rhodococcus erythropolis, and its application to double chiral compound production.

The gene encoding NADP(+)-dependent L-1-amino-2-propanol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154 was cloned and sequenced. A 780-bp nucleotide fragment was confirmed to be the gene encoding AADH by agreement of the N-terminal and internal amino acid sequences of the purified AADH. The gene (aadh) codes a total of 259 amino acid residues, and the deduced amino acid sequence shows similarity to several short-chain dehydrogenase/reductase family proteins. An expression vector, pKKAADH, which contains the full length aadh was constructed. Escherichia coli cells possessing pKKAADH exhibited a 10.4-fold increase in specific activity as to catalysis of the reduction of (S)-1-phenyl-2-methylaminopropan-1-one (MAK), as compared with that of R. erythropolis MAK154 induced by 1-amino-2-propanol (1 mg/ml). Coexpression of aadh with a cofactor regeneration enzyme (glucose dehydrogenase) gene was also performed, and a system for sufficient production of d-pseudoephedrine from racemic MAK was constructed.

A novel NADP+-dependent L-1-amino-2-propanol dehydrogenase from Rhodococcus erythropolis MAK154: a promising enzyme for the production of double chiral aminoalcohols.

AIM: A novel NADP(+)-dependent L-1-amino-2-propanol dehydrogenase was isolated from Rhodococcus erythropolis MAK154, and characterized. METHODS AND RESULTS: The enzyme was inducibly produced on cultivation with aminoalcohols such as 1-amino-2-propanol, 1-amino-2-butanol and 2-aminocyclohexanol. The enzyme catalyses the NADP(+)-dependent oxidation of several aminoalcohols, and also the NADPH-dependent asymmetric reduction of an aminoketone compound to a double chiral aminoalcohol, d-pseudoephedrine. Amino acid sequence analysis showed that the enzyme might belong to the short-chain dehydrogenase/reductase family. CONCLUSIONS: NADP(+)-dependent L-1-amino-2-propanol dehydrogenase isolated from R. erythropolis MAK154 reversibly catalysed dehydrogenation of aminoalcohols, and exhibited a unique sterospecifity for the reduction reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: The enzyme is a promising catalyst for the production of double chiral compound, d-pseudoephedrine, from prochiral substrate.

A case of basal cell carcinoma of the nipple and areola with intraductal spread.

We report an 82-year-old Japanese woman with basal cell carcinoma of the left nipple and areola extending into the lactiferous duct. The patient developed a small papular lesion of the left areola about 1 year before admission. The lesion, which had slowly progressed to involve the nipple, had become symptomatic showing weeping and bleeding. Mammography revealed microcalcification in the nipple. Although Paget's disease was suspected from these clinical features, histologically basal cell carcinoma was diagnosed. There was no axillary lymphadenopathy, and no evidence of distant metastasis. The lesion of the nipple and areola was resected with a 2 cm free margin along with the underlying mammary tissue. The patient has remained well without signs of recurrence for 2 years after surgery. We reviewed cases of basal cell carcinoma of the nipple or areola and discuss considerations and problems of this rare tumor.

Crocidolite-induced reactive oxygen metabolites generation from human polymorphonuclear leukocytes.

In order to study the mechanism of carcinogenicity of crocidolite asbestos, we have investigated the species of reactive oxygen metabolites (ROM) induced by crocidolite from human polymorphonuclear leukocytes (PMN) utilizing both an electron spin resonance (ESR) spin trapping method with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and a luminol-dependent chemiluminescence (CL) method. The present study confirms the generation of OH. from human peripheral blood PMN stimulated by UICC crocidolite utilizing ESR. In addition, PMN incubated with 25-400 micrograms/ml of crocidolite produced CL, the intensity of CL increasing in a dose-dependent manner. Superoxide dismutase, catalase, and dimethyl sulfoxide, which are scavengers of O2-, H2O2, and OH., respectively, inhibited the production of crocidolite-stimulated CL from PMN, also in a dose-dependent manner. Sodium azide, an inhibitor of myeloperoxidase (MPO) to produce OCl-, also inhibited CL production. These results suggest the involvement of O2-, H2O2, OH., and OCl- in the production of CL by crocidolite-stimulated PMN. In conclusion, it is proposed that OH. is a key ROM species in the mechanism of crocidolite-induced carcinogenesis.

Reactive oxygen metabolites produced by the carcinogenic fibrous mineral erionite.

Erionite, a fibrous mineral and the causative agent of the endemic outbreak of mesothelioma in Turkey, has been shown to generate reactive oxygen metabolites (ROM) from polymorphonuclear leukocytes (PMN). In order to investigate the mechanism of the production of ROM by erionite from PMN, a luminol-dependent chemiluminescence (CL) method was utilized. Human peripheral blood PMN were incubated with 50-800 micrograms/ml of erionite. PMN CL was produced immediately after the addition of erionite; the maximal CL production was reached within 2 to 6 min and the CL response increased with the dose of erionite. Superoxide dismutase, catalase, and dimethyl sulfoxide were utilized as scavengers of O2-, H2O2, and OH., respectively. These scavengers inhibited the production of erionite-stimulated PMN CL dose dependently, thus indicating the production of O2-, H2O2, and OH. by erionite-stimulated PMN. The less phagocytically active cells, namely, mononuclear cells and erythrocytes, produced CL immediately after the addition of erionite or phorbol myristate acetate and displayed a significant delay period after the addition of zymosan. Therefore, the direct interaction between the cell surface membrane and erionite would appear to be more important than phagocytosis, per se, for the production of ROM by erionite.

Possible role of hepatic glutathione in transport of methylmercury into mouse kidney.

The mechanism of the renal uptake of methylmercury was studied in mice. Preadministration of 1,2-dichloro-4-nitrobenzene (DCNB), which is a reagent that depletes hepatic glutathione (GSH) without affecting the renal GSH level, 30 min before injection of methylmercury significantly decreased the renal accumulation of mercury. The renal accumulation of mercury in mice receiving methylmercury-GSH intravenously was significantly higher than that in mice receiving methylmercuric chloride. These results suggest the possibility that hepatic GSH, as a source of extracellular GSH, plays an important role in the renal accumulation of methylmercury. No significant difference in renal mercury accumulation between bile duct-cannulated mice and normal mice was observed, indicating that the enterohepatic circulation of methylmercury is not an important factor in the renal accumulation of methylmercury in mice. Pretreatment of mice with acivicin, a potent inhibitor of gamma-glutamyl transpeptidase (gamma-GTP), significantly depressed the renal uptake of methylmercury and increased the urinary excretion of GSH and methylmercury. In in vitro reactions, methylmercury-GSH was degraded into methylmercury-cysteinylglycine by gamma-GTP, and this product was then converted to methylmercury-cysteine by dipeptidase. These results suggest that methylmercury is transported into the kidney as a complex with GSH, and then incorporated into the renal cells after degradation of the GSH moiety by gamma-GTP and dipeptidase, although the methylmercury bound to extracellular GSH can be reversibly transferred to plasma proteins in the bloodstream.