Biomedical subjects

T Bosma

Publications and source records attributed to T Bosma.

7 recordsLinked to original sources

PubMed2001

Characteristic hydrogen concentrations for various redox processes in batch study.

The dissolved hydrogen concentrations under various redox processes were investigated based on batch experiments. Chloroethenes including tetrachloroethene (PCE), cis-dichloroethene (cis-DCE) and vinylchloride (VC) were respectively used as culture substrates. For each chloroethene, a series of bottles were prepared with the additions of different electron acceptors or donors such as nitrate, manganese oxide, ferrous iron, sulfate, carbondioxide and volatile fatty acids. Hydrogen concentrations as well as redox species were measured over time to ensure the achievements of characteristic hydrogen levels in various enrichment batches. The results showed that redox processes with nitrate, manganese oxide and ferric iron as the electron acceptors exhibited hydrogen threshold values close to PCE/TCE dechlorination, whereas cis-DCE and VC dechlorinations exhibited hydrogen threshold values in the range of sulfate reduction and methanogenesis, respectively. Characteristic hydrogen concentrations for various redox processes were as follows (nM): denitrification, 0.1-0.4; manganese reduction, 0.1-2.0; iron reduction, 0.1-0.4; sulfate reduction, 1.5-4.5; methanogenesis, 2.5-24; PCE/TCE dechlorination, 0.6-0.9; eis-DCE dechlorination, 0.1-2.5; and VC dechlorination, 2-24.

Biodegradation, Environmental↗

PubMed2001

Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehydrogenases/reductases.

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity. The k(cat) and K(m) values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s(-1) and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK(a) of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.

Amino Acid Sequence↗

PubMed2000

Haloalkane-utilizing Rhodococcus strains isolated from geographically distinct locations possess a highly conserved gene cluster encoding haloalkane catabolism.

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.

Alkanes↗

PubMed1999

Utilization of trihalogenated propanes by Agrobacterium radiobacter AD1 through heterologous expression of the haloalkane dehalogenase from Rhodococcus sp. strain M15-3.

Trihalogenated propanes are toxic and recalcitrant organic compounds. Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful. Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp. strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better. Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp. strain m15-3 under the control of the heterologous promoters P(lac), P(dhlA), and P(trc). The resulting plasmids yielded functional expression in several gram-negative bacteria. A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth. The recombinant strain AD1(pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources. Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes.

Hydrolases↗

PubMed1996

Sequence variation in 5' termini of rubella virus genomes: changes affecting structure of the 5' proximal stem-loop.

Variation within a 523 nucleotide region proximal to the 5' terminus of seven rubella virus strains has been analysed. Compared to the Therien strain twenty sites of nucleotide variation have been identified, three of which are in the 5' untranslated region. Individual strains have between three and nine nucleotide differences, only three of which result in amino acid substitutions. TO-336 has a serine for threonine at amino acid (aa) 42 and CM arginine for histidine at aa 159. RA27/3 has arginine for lysine at aa 3 and serine for threonine at aa 42. Nucleotide differences which affect a stem-loop structure reported to be important for binding of host cell proteins have been identified.

Amino Acid Substitution↗

PubMed1995

Thermostability of respiratory terminal oxidases in the lipid environment.

The effect of the lipid environment on the thermostability of three respiratory terminal oxidases was determined. Cytochrome-c oxidase from beef heart and Bacillus stearothermophilus were used as representative proteins from mesophilic and thermophilic origin, respectively. Quinol oxidase from the archaeon Sulfolobus acidocaldarius represented the model for a extreme thermoacidophilic enzyme. All three integral membrane proteins were tested for their thermal inactivation in detergent and after reconstitution in liposomes composed of phospholipids of Escherichia coli or tetraether lipids from S. acidocaldarius. When preincubated at 0 degrees C, all three enzymes exhibited biphasic thermal inactivation curves. Data could be analysed according to a two-state model that defines two conformations of the enzyme, differing in their thermostability. Monophasic inactivation curves were observed when the enzymes were preincubated at higher temperatures prior to thermal inactivation. Lipids rendered the beef-heart cytochrome-c oxidase and S. acidocaldarius quinol oxidase more thermostable as compared to detergent solution. In contrast, the B. stearothermophilus oxidase, an intrinsically thermostable enzyme, was as thermostable in detergent as in the reconstituted state. These data suggest that the lipid environment can be an important factor in the thermostability of membrane proteins.

Animals↗

PubMed1987

Tumor necrosis factor-alpha (cachectin) stimulates bone resorption in mouse calvaria via a prostaglandin-mediated mechanism.

Recombinant human (h) and murine (m) tumor necrosis factor (TNF)-alpha stimulated bone resorption and the production of prostaglandin (PG) E2 in neonatal mouse calvaria in organ culture. In experiments of 72-h duration, the effect on bone resorption was of large magnitude (an average increase in medium calcium of 3.3 mg/dl above control values in 11 separate experiments) and occurred over a concentration range of 0.1-20 ng/ml mTNF and 0.5-50 ng/ml hTNF. Accompanying the TNF-enhanced release of bone calcium there was enhanced accumulation of PGE2 in the culture medium. The increases in medium calcium and PGE2 were both inhibited completely by nontoxic concentrations of 4 different PG cyclooxygenase inhibitors (indomethacin, piroxicam, ibuprofen, and acetylsalicylic acid) but not by the noncyclooxygenase inhibitor salicylic acid. The magnitude of the PGE2 response, but not the calcium release, was less for bones treated with TNF than for those treated with equipotent doses of epidermal growth factor or human transforming growth factors-alpha or -beta, suggesting that the local site of production of PGE2 in bone may be different for TNF than for the other factors. Repeated sc injections of hTNF to intact mice for a 48-h period produced a statistically significant elevation of the plasma calcium concentration. Because TNF is produced by cells of the monocyte/macrophage lineage in response to invasive stimuli such as the presence of tumor, our findings indicate that a host factor produced in response to malignant cells can cause enhanced bone resorption. Thus, the concept of the humoral hypercalcemias of malignancy must be expanded to include mediators not produced by the tumor cells themselves.

Animals↗