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In the Finnerty pathway for n-alkane oxidation in Acinetobacter sp., n-alkanes are postulated to be attacked by a dioxygenase and the product, n-alkyl hydroperoxide, is further metabolized to the corresponding aldehyde via the peroxy acid [W. R. Finnerty, p. 184-188, in A. H. Applewhite (ed.), Proceedings of the World Conference on Biotechnology for the Fats and Oil Industry, 1988]. However, no biochemical evidence regarding the first-step reaction is available. In this study, we found a novel n-alkane-oxidizing enzyme that requires only molecular oxygen, i.e., not NAD(P)H, in our isolate, Acinetobacter sp. strain M-1, and purified it to apparent homogeneity by gel electrophoresis. The purified enzyme is a homodimeric protein with a molecular mass of 134 kDa, contains 1 mol of flavin adenine dinucleotide per mol of subunit, and requires Cu 2؉ for its activity. The enzyme uses n-alkanes ranging in length from 10 to 30 carbon atoms and is also active toward n-alkenes (C 12 to C 20 ) and some aromatic compounds with substituted alkyl groups but not toward a branched alkane, alcohol, or aldehyde. Transient accumulation of n-alkyl hydroperoxide was detected in the course of the reaction, and no oxygen radical scavengers affected the enzyme activity. From these properties, the enzyme is most probably a dioxygenase that catalyzes the introduction of two atoms of oxygen to the substrate, leading to the formation of the corresponding n-alkyl hydroperoxide. The enzymatic evidence strongly supports the existence of an n-alkane oxidation pathway, which is initiated by a dioxygenase reaction, in Acinetobacter spp.Alkanes, ranging from methane to compounds with chain lengths of 40 or more carbon atoms, have generally been found to be degraded in both laboratory cultures and the natural environment (14,25,36). Some n-alkanes are recognized as feasible carbon sources for microbial production, such as that of biodegradable polymers (2), biosurfactants (17), and so on. Recently, much attention has been paid to the application of n-alkane-using microorganisms to bioremediation for oil spill environments (1). Three pathways for the metabolic attack on n-alkanes are known, and the participating enzyme reactions have been elucidated: (i) the monoterminal oxidation pathway (RCH 3 3 RCH 2 OH 3 RCHO 3 RCOOH), which is the most representative one in Pseudomonas spp. (20); (ii) biterminal oxidation (H 3 CRCH 3 3 H 3 CRCH 2 OH 3 HOCH 2 RCH 2 OH 3 HOOCRCOOH) of several types of bacteria and fungi (29), in which both terminal methyl groups of n-alkanes are sequentially hydroxylated; and (iii) subterminal oxidation [RCH 2 CH 3 3 RCH(OH)CH 3 3 RC(O)CH 3 ], which has been recognized in Nocardia spp. and so on (19). In all cases, an n-alkane is initially attacked by hydroxylases (monooxygenases) to produce the corresponding primary or secondary alcohol, although the enzymes participating in the catalytic mechanism differ. On the other hand, Finnerty (9) postulated a unique pathway [RCH 3 3 RCH 2 ⅐ OOH 3 RCO(O)OH 3 RCHO RCOOH] for long-chain n-al...

IntroductionAngiogenesis is an important factor in the development of osteoarthritis (OA). We investigated the efficacy of bevacizumab, an antibody against vascular endothelial growth factor and an inhibitor of angiogenesis, in the treatment of OA using a rabbit model of anterior cruciate ligament transection.MethodsFirst, we evaluated the response of gene expression and histology of the normal joint to bevacizumab treatment. Next, in a rabbit model of OA induced by anterior cruciate ligament transection, we used macroscopic and histological evaluations and real-time polymerase chain reaction (PCR) to examine the responses to intravenous (systemic) administration of bevacizumab (OAB IV group). We also investigated the efficacy of intra-articular (local) administration of bevacizumab in OA-induced rabbits (OAB IA group).ResultsHistologically, bevacizumab had no negative effect in normal joints. Bevacizumab did not increase the expression of genes for catabolic factors in the synovium, subchondral bone, or articular cartilage, but it increased the expression of collagen type 2 in the articular cartilage. Macroscopically and histologically, the OAB IV group exhibited a reduction in articular cartilage degeneration and less osteophyte formation and synovitis compared with the control group (no bevacizumab; OA group). Real-time PCR showed significantly lower expression of catabolic factors in the synovium in the OAB IV group compared with the OA group. In articular cartilage, expression levels of aggrecan, collagen type 2, and chondromodulin-1 were higher in the OAB IV group than in the OA group. Histological evaluation and assessment of pain behaviour showed a superior effect in the OAB IA group compared with the OAB IV group 12 weeks after administration of bevacizumab, even though the total dosage given to the OAB IA group was half that received by the OAB IV group.ConclusionsConsidering the dosage and potential adverse effects of bevacizumab, the local administration of bevacizumab is a more advantageous approach than systemic administration. Our results suggest that intra-articular bevacizumab may offer a new therapeutic approach for patients with post-traumatic OA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-014-0427-y) contains supplementary material, which is available to authorized users.

An integrative transformation system was established for an asporogenous methylotrophic yeast, Candida boidinii. This system uses a uracil auxotrophic mutant of C. boidinii as the host strain in combination with its URA3 gene as the selectable marker. First, the C. boidinii URA3 gene coding for orotidine-5'-phosphate decarboxylase (ODCase) was cloned by using complementation of the pyrF mutation of Escherichia coli. Next, the host ODCase-negative mutant strains (ura3 strains) were isolated by mutagenesis and selection for 5-fluro-orotic acid (5-FOA) resistance. Five ura3 host strains that exhibited both a low reversion rate and good methylotrophic growth were obtained. All of these strains could be transformed to Ura+ phenotype with a C. boidinii URA3-harboring plasmid linearized within the Candida DNA. The transformants had a stable Ura+ phenotype after nonselective growth for 10 generations. These results and extensive Southern analysis indicated that the linearized plasmid was integrated into the host chromosomal DNA by homologous recombination at the URA3 locus in C. boidinii.