An n-alkane-responsive promoter element found in the gene encoding the peroxisomal protein of Candida tropicalis does not contain a C(6) zinc cluster DNA-binding motif.
(33/908)
When an asporogenic diploid yeast, Candida tropicalis, is cultivated on n-alkane, the expression of the genes encoding enzymes of the peroxisomal beta-oxidation pathway is highly induced. An upstream activation sequence (UAS) which can induce transcription in response to n-alkane (UAS(ALK)) was identified on the promoter region of the peroxisomal 3-ketoacyl coenzyme A (CoA) thiolase gene of C. tropicalis (CT-T3A). The 29-bp region (from -289 to -261) present upstream of the TATA sequence was sufficient to induce n-alkane-dependent expression of a reporter gene. Besides n-alkane, UAS(ALK)-dependent gene expression also occurred in the cells grown on oleic acid. Several kinds of mutant UAS(ALK) were constructed and tested for their UAS activity. It was clarified that the important nucleotides for UAS(ALK) activity were located within 10-bp region from -273 to -264 (5'-TCCTGCACAC-3'). This region did not contain a CGG triplet and therefore differed from the sequence of the oleate-response element (ORE), which is a UAS found on the promoter region of 3-ketoacyl-CoA thiolase gene of Saccharomyces cerevisiae. Similar sequences to UAS(ALK) were also found on several peroxisomal enzyme-encoding genes of C. tropicalis. (+info)
Haloalkane-utilizing Rhodococcus strains isolated from geographically distinct locations possess a highly conserved gene cluster encoding haloalkane catabolism.
(34/908)
The sequences of the 16S rRNA and haloalkane dehalogenase (dhaA) genes of five gram-positive haloalkane-utilizing bacteria isolated from contaminated sites in Europe, Japan, and the United States and of the archetypal haloalkane-degrading bacterium Rhodococcus sp. strain NCIMB13064 were compared. The 16S rRNA gene sequences showed less than 1% sequence divergence, and all haloalkane degraders clearly belonged to the genus Rhodococcus. All strains shared a completely conserved dhaA gene, suggesting that the dhaA genes were recently derived from a common ancestor. The genetic organization of the dhaA gene region in each of the haloalkane degraders was examined by hybridization analysis and DNA sequencing. Three different groups could be defined on the basis of the extent of the conserved dhaA segment. The minimal structure present in all strains consisted of a conserved region of 12.5 kb, which included the haloalkane-degradative gene cluster that was previously found in strain NCIMB13064. Plasmids of different sizes were found in all strains. Southern hybridization analysis with a dhaA gene probe suggested that all haloalkane degraders carry the dhaA gene region both on the chromosome and on a plasmid (70 to 100 kb). This suggests that an ancestral plasmid was transferred between these Rhodococcus strains and subsequently has undergone insertions or deletions. In addition, transposition events and/or plasmid integration may be responsible for positioning the dhaA gene region on the chromosome. The data suggest that the haloalkane dehalogenase gene regions of these gram-positive haloalkane-utilizing bacteria are composed of a single catabolic gene cluster that was recently distributed worldwide. (+info)
Taxol and discodermolide represent a synergistic drug combination in human carcinoma cell lines.
(35/908)
Recently, three natural products have been identified, the epothilones, eleutherobin, and discodermolide, whose mechanism of action is similar to that of Taxol in that they stabilize microtubules and block cells in the mitotic phase of the cell cycle. In this report, we have compared and contrasted the effects of these new agents in Taxol-sensitive and -resistant cell lines. We also have taken advantage of a human lung carcinoma cell line, A549-T12, that was isolated as a Taxol-resistant cell line and found to require low concentrations of Taxol (2-6 nM) for normal cell division. This study then examined the ability of these new compounds to substitute for Taxol in sustaining the growth of A549-T12 cells. Immunofluorescence and flow cytometry have both indicated that the epothilones and eleutherobin, but not discodermolide, can substitute for Taxol in this Taxol-dependent cell line. In A549-T12 cells, the presence of Taxol significantly amplified the cytotoxicity of discodermolide, and this phenomenon was not observed in combinations of Taxol with either the epothilones or eleutherobin. Median effect analysis using the combination index method revealed a schedule-independent synergistic interaction between Taxol and discodermolide in four human carcinoma cell lines, an effect that was not observed between Taxol and epothilone B. Flow cytometry revealed that concurrent exposure of A549 cells to Taxol and discodermolide at doses that do not induce mitotic arrest caused an increase in the hypodiploid population, thereby indicating that a possible mechanism for the observed synergy is the potentiation of apoptosis. Our results suggest that Taxol and discodermolide may constitute a promising chemotherapeutic combination. (+info)
Trichosporon veenhuisii sp. nov., an alkane-assimilating anamorphic basidiomycetous yeast.
(36/908)
A morphological and physiological description of an alkane-assimilating anamorphic basidiomycetous yeast species, named Trichosporon veenhuisii, is presented. The ability to assimilate several aliphatic and aromatic compounds as sole source of carbon and energy is reported. The phylogenetic position within the genus, based on nuclear base sequencing of the D1/D2 region of the large subunit of rDNA, is discussed. The type strain is CBS 7136T. (+info)
Block of voltage-dependent calcium channels by aliphatic monoamines.
(37/908)
We have recently identified farnesol, an intermediate in the mevalonate pathway, as a potent endogenous modulator and blocker of N-type calcium channels (Roullet, J. B., R. L. Spaetgens, T. Burlingame, and G. W. Zamponi. 1999. J. Biol. Chem. 274:25439-25446). Here, we investigate the action of structurally related compounds on various types of voltage-dependent Ca(2+) channels transiently expressed in human embryonic kidney cells. 1-Dodecanol, despite sharing the 12-carbon backbone and headgroup of farnesol, exhibited a significantly lower blocking affinity for N-type Ca(2+) channels. Among several additional 12-carbon compounds tested, dodecylamine (DDA) mediated the highest affinity inhibition of N-type channels, indicating that the functional headgroup is a critical determinant of blocking affinity. This inhibition was concentration-dependent and relatively non-discriminatory among N-, L-, P/Q-, and R-Ca(2+) channel subtypes. However, whereas L-type channels exhibited predominantly resting channel block, the non-L-type isoforms showed substantial rapid open channel block manifested by a speeding of the apparent time course of current decay and block of the inactivated state. Consistent with these findings, we observed significant frequency-dependence of block and dependence on external Ba(2+) concentration for N-type, but not L-type, channels. We also systematically investigated the drug structural requirements for N-type channel inhibition. Blocking affinity varied with carbon chain length and showed a clear maximum at C12 and C13, with shorter and longer molecules producing progressively weaker peak current block. Overall, our data indicate that aliphatic monoamines may constitute a novel class of potent inhibitors of voltage-dependent Ca(2+) channels, with block being governed by rigid structural requirements and channel-specific state dependencies. (+info)
Molecular analysis of surfactant-driven microbial population shifts in hydrocarbon-contaminated soil.
(38/908)
We analyzed the impact of surfactant addition on hydrocarbon mineralization kinetics and the associated population shifts of hydrocarbon-degrading microorganisms in soil. A mixture of radiolabeled hexadecane and phenanthrene was added to batch soil vessels. Witconol SN70 (a nonionic, alcohol ethoxylate) was added in concentrations that bracketed the critical micelle concentration (CMC) in soil (CMC') (determined to be 13 mg g(-1)). Addition of the surfactant at a concentration below the CMC' (2 mg g(-1)) did not affect the mineralization rates of either hydrocarbon. However, when surfactant was added at a concentration approaching the CMC' (10 mg g(-1)), hexadecane mineralization was delayed and phenanthrene mineralization was completely inhibited. Addition of surfactant at concentrations above the CMC' (40 mg g(-1)) completely inhibited mineralization of both phenanthrene and hexadecane. Denaturing gradient gel electrophoresis of 16S rRNA gene segments showed that hydrocarbon amendment stimulated Rhodococcus and Nocardia populations that were displaced by Pseudomonas and Alcaligenes populations at elevated surfactant levels. Parallel cultivation studies revealed that the Rhodococcus population can utilize hexadecane and that the Pseudomonas and Alcaligenes populations can utilize both Witconol SN70 and hexadecane for growth. The results suggest that surfactant applications necessary to achieve the CMC alter the microbial populations responsible for hydrocarbon mineralization. (+info)
Long-chain aldehyde dehydrogenase that participates in n-alkane utilization and wax ester synthesis in Acinetobacter sp. strain M-1.
(39/908)
A long-chain aldehyde dehydrogenase, Ald1, was found in a soluble fraction of Acinetobacter sp. strain M-1 cells grown on n-hexadecane as a sole carbon source. The gene (ald1) was cloned from the chromosomal DNA of the bacterium. The open reading frame of ald1 was 1,512 bp long, corresponding to a protein of 503 amino acid residues (molecular mass, 55,496 Da), and the deduced amino acid sequence showed high similarity to those of various aldehyde dehydrogenases. The ald1 gene was stably expressed in Escherichia coli, and the gene product (recombinant Ald1 [rAld1]) was purified to apparent homogeneity by gel electrophoresis. rAld1 showed enzyme activity toward n-alkanals (C(4) to C(14)), with a preference for longer carbon chains within the tested range; the highest activity was obtained with tetradecanal. The ald1 gene was disrupted by homologous recombination on the Acinetobacter genome. Although the ald1 disruptant (ald1Delta) strain still had the ability to grow on n-hexadecane to some extent, its aldehyde dehydrogenase activity toward n-tetradecanal was reduced to half the level of the wild-type strain. Under nitrogen-limiting conditions, the accumulation of intracellular wax esters in the ald1Delta strain became much lower than that in the wild-type strain. These and other results imply that a soluble long-chain aldehyde dehydrogenase indeed plays important roles both in growth on n-alkane and in wax ester formation in Acinetobacter sp. strain M-1. (+info)
Entry into and release of solvents by Escherichia coli in an organic-aqueous two-liquid-phase system and substrate specificity of the AcrAB-TolC solvent-extruding pump.
(40/908)
Growth of Escherichia coli is inhibited upon exposure to a large volume of a harmful solvent, and there is an inverse correlation between the degree of inhibition and the log P(OW) of the solvent, where P(OW) is the partition coefficient measured for the partition equilibrium established between the n-octanol and water phases. The AcrAB-TolC efflux pump system is involved in maintaining intrinsic solvent resistance. We inspected the solvent resistance of delta acrAB and/or delta tolC mutants in the presence of a large volume of solvent. Both mutants were hypersensitive to weakly harmful solvents, such as nonane (log P(OW) = 5.5). The delta tolC mutant was more sensitive to nonane than the delta acrAB mutant. The solvent entered the E. coli cells rapidly. Entry of solvents with a log P(OW) higher than 4.4 was retarded in the parent cells, and the intracellular levels of these solvents were maintained at low levels. The delta tolC mutant accumulated n-nonane or decane (log P(OW) = 6. 0) more abundantly than the parent or the delta acrAB mutant. The AcrAB-TolC complex likely extrudes solvents with a log P(OW) in the range of 3.4 to 6.0 through a first-order reaction. The most favorable substrates for the efflux system were considered to be octane, heptane, and n-hexane. (+info)