Degradation pathways of cyclic alkanes in Rhodococcus sp. NDKK48.

The degradation pathways for cyclic alkanes (c-alkanes) in Rhodococcus sp. NDKK48 were investigated. Strain NDKK48 used dodecylcyclohexane as a sole carbon and energy source, and five metabolites in the dodecylcyclohexane degradation pathway were detected by gas-chromatography/mass spectra. The metabolites were identified as cyclohexanecarboxylic acid, cyclohexylacetic acid, 1-cyclohexene-1-acetic acid, 4-dodecylcyclohexanol, and 4-dodecylcyclohexanone. The strain degrades dodecylcyclohexane via a ring oxidation pathway and an alkyl side chain oxidation pathway. Cyclohexanecarboxylic acid was further oxidized to muconic acid via 1-cyclohexene-1-carboxylic acid and benzoic acid, and the muconic acid was finally used by strain NDKK48 for growth. Methylcyclohexane and cyclohexane were co-oxidized with hexadecane by strain NDKK48. Methylcyclohexane was degraded via a ring oxidation pathway, and the degradation pathway contained part of the Baeyer-Villiger oxidation for ring cleavage. Cyclohexane was also degraded by the same pathway as methylcyclohexane. Thus, strain NDKK48 has two pathways for the complete degradation of c-alkanes.

Biodegradation of long-chain n-paraffins from waste oil of car engine by Acinetobacter sp.

Microorganisms that degrade long-chain n-paraffins from used car engine oil were isolated from soil. For the screening, a fraction of n-paraffin prepared from car engine oil was applied as the sole carbon source. The strain was identified as Acinetobacter sp. The ability of the strain to assimilate long-chain n-paraffins was assessed and characterized. The strain mineralized long-chain n-paraffins (0.1% w/v) in the minimal medium after cultivation for 96 h and also reduced the weight of the waste oil added (1% w/v) by 20% after 72 h without an extracellular biosurfactant. When n-hexadecane was fed as substrate, 1-hexadecanol and 1-hexadecanoic acid were detected as the intermediates by gas chromatography/mass spectrometry. This indicates that the long-chain n-paraffins were metabolized via the terminal oxidation pathway of n-alkane.

Synthesis of alanine and leucine by reductive amination of 2-oxoic acid with combination of hydrogenase and dehydrogenase.

Alanine synthesis by reductive amination of pyruvate was performed by the combination of NADH regeneration system and alanine dehydrogenase (AlaDH). The conversion of pyruvate to alanine was 99% after 1 h. Leucine synthesis was also carried out by the combination of NADH regeneration system and leucine dehydrogenase (LeuDH). The conversion of 4-methyl-2-oxovalerate to leucine was 60% after 1.5 h.

Isolation and characterization of soybean waste-degrading microorganisms and analysis of fertilizer effects of the degraded products.

Two microorganisms which could degrade soybean lees efficiently were isolated and identified as Bacillus circulans and B. stearothermophilus. These two strains secreted thermostable proteases into the medium and could digest soybean lees rapidly and completely at 50 degrees C. Initially, the soybean lees were degraded to proteins in approximately 20 h by these two strains, after which time the concentrations of peptides in the medium gradually increased. The degraded products from soybean lees contained abundant nitrogen compounds, such as peptides, amino acids, and amides. Approximately 10 times more fresh plant weight was obtained (in the case of Brassica campestris) when these degraded products were applied than when water was applied for 42 days. These stimulatory effects of the soybean lees products were almost equal to those of a chemically synthesized fertilizer.

Regeneration of NADH and ketone hydrogenation by hydrogen with the combination of hydrogenase and alcohol dehydrogenase. Scientific note.

The regeneration of nicotinamide-adenine dinucleotide (reduced form, NADH) by the reaction of NAD with hydrogen gas was carried out in the presence of the hydrogenase from Alcaligenes eutrophus. And the formations of alcohol, CO2, and 6-phospho-gluconate were observed by a combination of the above system and corresponding dehydrogenases. NADH was regenerated by hydrogen gas with the hydrogenase and recycled in these reactions.

Changes in glycosaminoglycan synthesis during the differentiation of HL-60 cells induced by 12-O-tetra-decanoylphorbol-13-acetate.

The changes in glycosaminoglycan (GAG) synthesis during the differentiation of HL-60 cells to macrophage-like cells by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) were studied by using 35S-sulfate or 3H-glucosamine as a tracer. The incorporation of 35S-sulfate into GAGs of HL-60 cells treated with TPA (TPA-treated HL-60 cells) decreased by 30% compared with untreated HL-60 cells (HL-60 cells). The profiles of the Sepharose CL-6B chromatography showed that the molecular weight size of proteoglycans (PGs) in the medium fraction of TPA-treated HL-60 cells was larger than that of HL-60 cells although the PGs in the cell fractions of both HL-60 and TPA-treated HL-60 cells were of the same molecular weight size. On the other hand, the molecular weight sizes of GAGs in both the medium and the cell fractions of TPA-treated HL-60 cells were larger than those of HL-60 cells. In order to examine the synthesis of GAG chains, HL-60 and TPA-treated HL-60 cells were incubated with 35S-sulfate in the presence of 4-methylumbelliferyl-beta-D-xyloside, an exogenous initiator. The incorporation of 35S-sulfate into total GAGs of the TPA-treated HL-60 cells exposed to the beta-D-xyloside increased 3-fold over that of HL-60 cells. HL-60 cells synthesized core protein-initiated GAGs and beta-D-xyloside-initiated GAGs but TPA-treated HL-60 cells synthesized beta-D-xyloside-initiated GAGs only. beta-D-Xyloside-initiated GAGs synthesized by both cells were obtained in the same fraction by Sepharose CL-6B chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the biosynthesis of glycosaminoglycans in polymorphonuclear leukocytes in relation to their induction into the peritoneal cavity.

The synthesis and function of dermatan sulfate in peritoneal polymorphonuclear (PMN) leukocytes were studied. The peritoneal PMN leukocytes were obtained at 4,8, and 16 h after guinea pigs were injected intraperitoneally with caseinate solution, and were incubated with [35S] sulfate or [3H] glucosamine on plastic. The total glycosaminoglycan synthesis and the proportion of dermatan sulfate to total glycosaminoglycans linearly increased with time. In order to clarify the function of the increased dermatan sulfate, peritoneal PMN leukocytes were cultured with [35S] sulfate on plastic or collagen gel. The total glycosaminoglycan synthesis on the collagen gel culture increased 1.5 times compared with that on the plastic culture, and especially, dermatan sulfate synthesis increased twofold. In addition, 65% of dermatan sulfate on the collagen gel culture was found in the cell and the collagenase-soluble fractions. These results indicate that proteodermatan sulfate synthesized by PMN leukocytes interacts with collagen in vitro, and suggest that PMN leukocytes, which migrated to the inflammatory locus, lastly adhere to the injured tissue through the interaction of proteodermatan sulfate synthesized by themselves with collagen fibers exposed in the inflammatory locus.

Collagen and glycosaminoglycans in the human ovarian capsule with polycystic ovarian disease.

Connective tissue of human ovarian capsule with polycystic ovarian disease (PCO) is analyzed. The connective tissue components of the capsule were found to mainly consist of type I collagen and acid glycosaminoglycans, such as dermatan sulfate, heparan sulfate, and chondroitin 4- and 6-sulfate. Although their concentration and constituent ratio in the PCO capsule are found to be similar to those in the normal, their total amounts in whole capsule with PCO are higher than in normal ones, because of the enlarged ovary and the thickened capsule. Furthermore, collagen solubility for pepsin in the PCO capsule is larger than that in the normal one. The results suggest that the activation of connective tissue metabolism in the ovarian capsule is one of the factors of anovulation in PCO.

Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs.

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.

Biosynthesis of glycosaminoglycans in peritoneal macrophages from the guinea pig.

The majority of glycosaminoglycans synthesized in peritoneal macrophages from the guinea pig in vitro were secreted into culture medium. The secreted glycosaminoglycans were reduced in size with alkali treatment indicating that the glycosaminoglycans existed in the form of proteoglycans. After the glycosaminoglycans were digested with chondroitinase AC and ABC, the high voltage paper electrophoretic analysis and the descending paper chromatographic analysis indicated the presence of a considerable amount of unsaturated disulfated disaccharides. Based on the enzymatic assay with chondro-4- and 6-sulfatase, the positions of sulfation in the disulfated disaccharide have been identified as the 4- and 6-position of N-acetylgalactosamine. Moreover, the results of the ion-exchange chromatography and the chondroitinase AC and ABC digestion indicated that delta Di-diSE derived from dermatan sulfate. This suggests that peritoneal macrophages are capable of synthesizing oversulfated proteodermatan sulfate as main component. The proportion of synthesized oversulfated dermatan sulfate to the total glycosaminoglycans was independent of the incubation time, and the distribution of oversulfated dermatan sulfate in cell and incubation medium also did not change. After exposure of macrophages to Escherichia coli for 15 min, the incorporation of [35S]sulfate and [3H]glucosamine into the glycosaminoglycans was increased by about 40% with no significant change in the proportion of synthesized oversulfated dermatan sulfate, but the release of glycosaminoglycans into the culture medium remains essentially unchanged. The difference of the existence of oversulfated dermatan sulfate is not yet understood.