Comamonas sp. 3ah48 is a dibenz[a,h]anthracene-degrading bacterium that is tolerant to heavy metals.

Industrialization often causes polycyclic aromatic hydrocarbon (PAH) and heavy metal contamination of soil and water. In this study, we isolated a bacterium from bottom mud water around a park of Kawasaki Port, Japan, that degrades the 5-ring PAH dibenz[a,h]anthracene (DBA). The strain, Comamonas sp. 3ah48, degraded 29% of DBA (30 µg ml-1 ) in 7 days, and the degradation level increased drastically, to 59%, by the addition of glutamate to the medium. The strain also degraded 40, 14, 15 and 19% of pyrene (Pyr), benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF) and benzo[g,h,i]perylene (BghiP) respectively. Benzo[a]pyrene (BaP) was degraded only when glutamate was added to the medium. Strain 3ah48 retained its degradation levels in the presence of 2 mmol l-1 Co2+ , Zn2+ or Cr2+ , at almost the same level as that without metal, and increased the DBA degradation level to 57% in the presence of 2 mmol l-1 Cu2+ , suggesting the possibility of the presence of laccase. SIGNIFICANCE AND IMPACT OF THE STUDY: Sixteen polycyclic aromatic hydrocarbons (PAHs) are listed as priority pollutants by the United States Environmental Protection Agency (USEPA). Information about the biodegradation of one of those PAHs, dibenz[a,h]anthracene (DBA), is limited. The present study focuses on DBA degradation by Comamonas sp. 3ah48 strain isolated around Kawasaki Port, Japan. Comamonas sp. 3ah48, cultured with the addition of glutamate to the medium, was found to increase the degradation level of DBA and to degrade DBA even in the presence of high concentrations of heavy metals.

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.

Cloning and expression of the hydrogenase gene from Clostridium butyricum in Escherichia coli.

Hydrogenase gene from Clostridium butyricum was cloned in Escherichia coli HK16 (Hyd-) using pBR322 and PstI. The plasmid, pCBH1, containing hydrogenase gene was 7.3 MDa and pCBH1 had 5 PstI-DNA fragments (3.9, 2.6, 0.7, 0.03-0.04, less than 0.02 MDa, respectively). The hydrogenase activity of HK16 (pCBH1) was about 3.1-3.5-times as high as those of the present strains, such as C.butyricum and E.coli C600 (Hyd+).

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.

Profile of a nonylphenol-degrading microflora and its potential for bioremedial applications.

Nonylphenol (NP) is an important intermediate in the production of various commercial and industrial materials, but is also known as a ubiquitous pollutant in urban aquatic environments. We recently studied the NP-degrading activities of microflora in several aquatic environments, and found a notable degrading activity for wastewater from a sewage treatment plant in Tokyo. This result led us to isolate NP-degrading microbes and identify biodegradation products. Using conventional plate culture techniques and molecular biological methods, Pseudomonas and Sphingomonas species, which are known for their degradation activities of many aromatic compounds, have been isolated. But it has also been found that Sphingomonas sp. (S-strain) is necessary and sufficient for the degradation of NP. Although the role of Pseudomonas sp. (P-strain) remains unclear, P-strain seems to provide some co-nutrients for the growth of S-strain. The degradation products were analyzed by GC/MS and NMR. More than 95% of NP was degraded within 10 days and aromatic compounds other than NP were not found, suggesting that the phenolic part of NP was completely degraded. We also examined the potential of S-strain for bioremedial applications. S-strain cells immobilized on chitosan or alginate beads retain their NP-degrading activity in flask-scale experiments. Furthermore, the chitosan-bound cells in a lab-scale bioreactor have been found to be persistent for repeated use, suggesting that S-strain is applicable to the treatment of NP-contaminated wastewater.

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.

Effect of osmotic stabilizers on protoplast generation of Chlorella ellipsoidea yellow/white color mutants.

The effect of osmotic stabilizers on protoplast formation of yellow/white color mutants of Chlorella ellipsoidea was studied. The UF-1 strain, one of the mutants, had the same growth rate as the parent C. ellipsoidea at 4.02 microE/m2/s and no reversion to normal cells at 0-26.8 microE/m2/s. Therefore, protoplast formation from UF-1 was carried out, and it was found that the yield of protoplasts was 93% in the medium containing 1.0 M NaCl as an osmotic stabilizer.

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.

Increase of hybrids in yeast electrofusion by mitochondrial mutations.

Yeast mitochondria were found to affect the zeta-potential of protoplasts, resulting in electrofusion of membrane behavior. For modeling purposes, two fusion systems were investigated: (1) parent yeasts of Saccharomyces cerevisiae G706 (rho(+)) x O708-11-16A (rho(+)), with zeta-potentials of -10 to -27 mV and -28 to -45 mV, depending on MgCl(2) concentration in the medium, respectively; and (2) parent yeasts of S. cerevisiae G706-1 (rho(-)) x O708-11-16A (rho(+)), with zeta-potentials of -30 to -60 mV, depending on MgCl(2). Yields of the hybrids in system (2) were over 100-fold higher than those in system (1). Thus, regulation of yeast electrofusion was found to be possible by mitochondrial mutations.

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.

Alzheimer's disease: a pathogenic role for aluminosilicate-induced phagocytic free radicals.

The occurrence of aluminosilicate deposits within the cerebral plaques in Alzheimer's senile dementia sufferers has prompted further consideration of the possible role of such materials in the aetiology and pathogenesis of the disease. We have monitored the ability of various natural and synthetic model aluminosilicate particulates of differing morphological and chemical composition to stimulate the generation of phagocyte-derived free radical reactive oxygen metabolites (ROM) using an in vitro chemiluminescent technique on purified human blood-derived polymorphonuclear leukocytes (PMN). The results indicate that an enhanced chemiluminescent response is produced by calcium-bearing fibriform particulates. It is proposed that an analogous in vivo particle-induced and phagocyte-mediated oxidative stress could provide a potential pathogenic mechanism in the development of Alzheimer's disease.

Sphingomonas cloacae sp. nov., a nonylphenol-degrading bacterium isolated from wastewater of a sewage-treatment plant in Tokyo.

A nonylphenol-assimilating bacterium isolated at a sewage-treatment plant in Tokyo was studied phenotypically, genotypically and phylogenetically. Analysis of the 16S rDNA sequence, the G+C content of the DNA (63 mol%) and the isoprenoid quinone composition, as well as the presence of sphingoglycolipid and the whole-cell fatty acid profile, revealed that the isolate is a member of the genus Sphingomonas. However, the sequence similarity of the 16S rDNA with that of known Sphingomonas spp. was found to be at most 96%, implying that the isolate is distinctive. Furthermore, the results of DNA-DNA hybridization experiments and its physiological characteristics clearly indicated that the isolate represents a new Sphingomonas species, for which the name Sphingomonas cloacae is proposed; strain S-3T (= JCM 10874T = IAM 14885) is the type strain.