Biomedical subjects

K Breese

Publications and source records attributed to K Breese.

At least 19 recordsLinked to original sources

PubMed2001

Anaerobic metabolism of 3-hydroxybenzoate by the denitrifying bacterium Thauera aromatica.

The anaerobic metabolism of 3-hydroxybenzoate was studied in the denitrifying bacterium Thauera aromatica. Cells grown with this substrate were adapted to grow with benzoate but not with 4-hydroxybenzoate. Vice versa, 4-hydroxybenzoate-grown cells did not utilize 3-hydroxybenzoate. The first step in 3-hydroxybenzoate metabolism is a coenzyme A (CoA) thioester formation, which is catalyzed by an inducible 3-hydroxybenzoate-CoA ligase. The enzyme was purified and characterized. Further metabolism of 3-hydroxybenzoyl-CoA by cell extract required MgATP and was coupled to the oxidation of 2 mol of reduced viologen dyes per mol of substrate added. Purification of the 3-hydroxybenzoyl-CoA reducing enzyme revealed that this activity was due to benzoyl-CoA reductase, which reduced the 3-hydroxy analogue almost as efficiently as benzoyl-CoA. The further metabolism of the alicyclic dienoyl-CoA product containing the hydroxyl substitution obviously required additional specific enzymes. Comparison of the protein pattern of 3-hydroxybenzoate-grown cells with benzoate-grown cells revealed several 3-hydroxybenzoate-induced proteins; the N-terminal amino acid sequences of four induced proteins were determined and the corresponding genes were identified and sequenced. A cluster of six adjacent genes contained the genes for substrate-induced proteins 1 to 3; this cluster may not yet be complete. Protein 1 is a short-chain alcohol dehydrogenase. Protein 2 is a member of enoyl-CoA hydratase enzymes. Protein 3 was identified as 3-hydroxybenzoate-CoA ligase. Protein 4 is another member of the enoyl-CoA hydratases. In addition, three genes coding for enzymes of beta-oxidation were present. The anaerobic 3-hydroxybenzoate metabolism here obviously combines an enzyme (benzoyl-CoA reductase) and electron carrier (ferredoxin) of the general benzoyl-CoA pathway with enzymes specific for the 3-hydroxybenzoate pathway. This raises some questions concerning the regulation of both pathways.

Acyl Coenzyme A↗

PubMed1998

Genes coding for the benzoyl-CoA pathway of anaerobic aromatic metabolism in the bacterium Thauera aromatica.

Many aromatic compounds are anaerobically oxidized to CO2 via benzoyl-CoA as the common aromatic intermediate. In Thauera aromatica, the central benzoyl-CoA pathway comprises the ATP-driven two-electron reduction of the benzene ring; this reaction uses a ferredoxin as electron donor and is catalyzed by benzoyl-CoA reductase. The first intermediate, cyclohex-1,5-diene-1-carboxyl-CoA, is subsequently hydrated by dienoyl-CoA hydratase to 6-hydroxycyclohex-1-ene-1-carboxyl-CoA. Formation of the main product produced by cell extracts, 3-hydroxypimelyl-CoA, requires at least two further steps; the oxidation of a hydroxyl group and the hydrolytic carbon ring cleavage of a CoA-activated beta-oxoacid. In addition, enoyl-CoA hydratase may come into play. A cluster of eight adjacent genes, which are transcribed in the same direction and may form an operon, was found in this bacterium. The cluster codes for proven and postulated enzymes of the benzoyl-CoA pathway. The genes for the enzymes code for ferredoxin, four subunits of benzoyl-CoA reductase, dienoyl-CoA hydratase, 6-hydroxycyclohex-1-ene-1-carboxyl-CoA dehydrogenase (NAD+), and the ring hydrolyzing enzyme. The deduced amino acid sequences of these proteins were 35-86% similar to the corresponding sequences found in Rhodopseudomonas palustris. Benzoyl-CoA reductase subunits exhibit distinct similarities with 2-hydroxyglutaryl-CoA dehydratase and its ATP-hydrolysing activase protein of Acidaminococcus fermentans as well as with open reading frames of unknown function in other bacteria. Conversion of benzoyl-CoA to 3-hydroxypimelyl-CoA can be explained by a minimal model of the benzoyl-CoA pathway assuming the four enzymes whose genes were characterized and an additional enoyl-CoA hydratase. In R. palustris the dienoyl-CoA hydratase gene is lacking suggesting the operation of a modified benzoyl-CoA pathway with cyclohex-1-ene-1-carboxyl-CoA as intermediate.

Acyl Coenzyme A↗

PubMed1998

4-Hydroxybenzoyl-CoA reductase (dehydroxylating) from the denitrifying bacterium Thauera aromatica--prosthetic groups, electron donor, and genes of a member of the molybdenum-flavin-iron-sulfur proteins.

4-Hydroxybenzoyl-CoA reductase catalyzes an important reaction in the anaerobic metabolism of phenolic compounds, i.e. the reductive removal of an aromatic hydroxyl group. The prosthetic groups and the natural electron donor of the enzyme were investigated and the genes were cloned and sequenced. The enzyme is a molybdenum-flavin-iron-sulfur protein of subunit composition of alpha2beta2gamma2. It contains approximately 1.3 flavin nucleotide, probably FAD, 1.9 Mo, 15 Fe, and 12.5 acid-labile sulfur. Sequence interpretation suggests that the native enzyme contains two [4Fe-4S] and four [2Fe-2S] clusters. A 9.8-kDa ferredoxin with two [4Fe-4S] clusters functions as the natural electron donor. The genes coding for the three subunits, hcrABC, show high similarities to other molybdenum-flavin-iron-sulfur proteins of the xanthine oxidase family, notably to the three putative 4-hydroxybenzoyl-CoA reductase genes in Rhodopseudomonas palustris. In addition, there are close similarities to three open reading frames (orf) in E. coli. A major difference is the presence of an additional domain in the beta-subunit (HcrB, 35 kDa) probably carrying an additional iron-sulfur cluster. The 82-kDa alpha-subunit (HcrA) contains a Mo-cofactor-binding site. The 17-kDa gamma-subunit (HcrC) harbors two [2Fe-2S] clusters. Upstream of the hcrCAB region, an ORF was found coding for a regulatory protein of the MarR family. Downstream of the hcrCAB region lies an ORF presumably coding for a hydrophobic permease.

Amino Acid Sequence↗

PubMed1998

Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica.

Differential induction of enzymes involved in anaerobic metabolism of aromatic substrates was studied in the denitrifying bacterium Thauera aromatica. This metabolism is divided into (1) peripheral reactions transforming the aromatic growth substrates to the common intermediate benzoyl-CoA, (2) the central benzoyl-CoA pathway comprising ring-reduction of benzoyl-CoA and subsequent beta-oxidation to 3-hydroxypimelyl-CoA, and (3) the pathway of beta-oxidation of 3-hydroxypimelyl-CoA to three acetyl-CoA and CO2. Regulation was studied by three methods. 1. Determination of protein patterns of cells grown on different substrates. This revealed several strongly substrate-induced polypeptides that were missing in cells grown on benzoate or other intermediates of the respective metabolic pathways. 2. Measurement of activities of known enzymes involved in this metabolism in cells grown on different substrates. The enzyme pattern found is consistent with the regulatory pattern deduced from simultaneous adaptation of cells to utilisation of other aromatic substrates. 3. Immunological detection of catabolic enzymes in cells grown on different substrates. Benzoate-CoA ligase and 4-hydroxybenzoate-CoA ligase were detected only in cells yielding the respective enzyme activity. However, presence of the subunits of benzoyl-CoA reductase and 4-hydroxybenzoyl-CoA reductase was also recorded in some cell batches lacking enzyme activity. This possibly indicates an additional level of regulation on protein level for these two reductases.

Acyl Coenzyme A↗

PubMed1997

Structure of the puf operon of the obligately aerobic, bacteriochlorophyll alpha-containing bacterium Roseobacter denitrificans OCh114 and its expression in a Rhodobacter capsulatus puf puc deletion mutant.

Roseobacter denitrificans (Erythrobacter species strain OCh114) synthesizes bacteriochlorophyll a (BChl) and the photosynthetic apparatus only in the presence of oxygen and is unable to carry out primary photosynthetic reactions and to grow photosynthetically under anoxic conditions. The puf operon of R. denitrificans has the same five genes in the same order as in many photosynthetic bacteria, i.e., pufBALMC. PufC, the tetraheme subunit of the reaction center (RC), consists of 352 amino acids (Mr, 39,043); 20 and 34% of the total amino acids are identical to those of PufC of Chloroflexus aurantiacus and Rubrivivax gelatinosus, respectively. The N-terminal hydrophobic domain is probably responsible for anchoring the subunit in the membrane. Four heme-binding domains are homologous to those of PufC in several purple bacteria. Sequences similar to pufQ and pufX of Rhodobacter capsulatus were not detected on the chromosome of R. denitrificans. The puf operon of R. denitrificans was expressed in trans in Escherichia coli, and all gene products were synthesized. The Roseobacter puf operon was also expressed in R. capsulatus CK11, a puf puc double-deletion mutant. For the first time, an RC/light-harvesting complex I core complex was heterologously synthesized. The strongest expression of the R. denitrificans puf operon was observed under the control of the R. capsulatus puf promoter, in the presence of pufQ and pufX and in the absence of pufC. Charge recombination between the primary donor P+ and the primary ubiquinone Q(A)- was observed in the transconjugant, showing that the M and L subunits of the RC were correctly assembled. The transconjugants did not grow photosynthetically under anoxic conditions.

Amino Acid Sequence↗

PubMed1996

Predicted and trifluoroethanol-induced alpha-helicity of polypeptides.

The alpha-helix stabilizing solvent 2,2,2-trifluoroethanol (TFE) is frequently used as a medium for determining the average alpha-helicity of polypeptides by CD spectroscopy. CD spectra measured in solutions containing 10, 15, 20, 50, and 90% (vol/vol) TFE are presented for 5 peptides that were selected to demonstrate possible variations in the effect of TFE concentration on the secondary structure. The analysis is extended to 6 further peptides whose CD spectra as measured in TFE are documented in the literature. The observed alpha-helicity at a high TFE concentration is compared with the alpha-helicity determined by a structure prediction method that combines conformational filtering [S. Vajda, (1993) Journal of Molecular Biology, Vol. 229, pp. 125-145], and a Monte Carlo simulation [J. Figge et al. (1993) Protein Science, Vol. 2, pp. 155-164]. For the set of 11 peptides we find a correlation of 0.84 between the predicted [theta]222 values and the corresponding values observed by CD spectroscopy in a high concentration of TFE (p < 0.01). Although we generally find a good correlation at high TFE concentration between observed and predicted alpha-helicity, there are several peptides that do not follow the predicted behavior. An analysis of the TFE titration curves in one such case revealed that TFE can induce a sharp transition from a partial beta-sheet conformation to an alpha-helical conformation as the TFE concentration is increased above a critical value.

Amino Acid Sequence↗

PubMed1995

Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by the E. coli lac repressor.

Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that the Escherichia coli ldc operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two synthetic lac operators. Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-beta-D-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of the E. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.

Animals↗

PubMed1995

p53 gene status in endometrial carcinomas showing diffuse positivity for p53 protein by immunohistochemical analysis.

Although detection of p53 protein by immunohistochemical testing was originally thought to indicate p53 gene mutation, recent analyses of human malignancies have shown that high expression of p53 protein may occur without detectable gene mutation. Several explanations have been proposed for this phenomenon, including mutation out of "hot spot" regions, overexpression of wild-type protein, sampling error in molecular analyses, and conformational changes of wild-type p53 protein. As discussed, it is unlikely that the first two possibilities contribute significantly to the occurrence of this phenomenon, and the current study examined the possibility that sampling error in molecular analyses might account for a lack of concordance between immunohistochemical and molecular analyses. Such a possibility exists because immunohistochemical studies frequently report high expression when staining is only focal or regional and molecular analyses are based on the polymerase chain reaction, which is highly exponential in nature and may not detect mutation if the target gene segment is not amplified early in the chain reaction. In the current report, p53 protein expression was examined by immunohistochemical testing in 45 cases of endometrioid carcinoma, and all cases showing diffuse positivity were then examined by polymerase chain reaction in combination with single-strand conformational analysis for exons 4 to 9 with the use of a microdissection technique to separate malignant from benign cells. Of the 45 cases, diffuse staining was found in four cases, and only two of the four were found to show evidence of gene mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1994

Alteration of the p53 locus in benign hyperplastic prostatic epithelium associated with high-grade prostatic adenocarcinoma.

Recent evidence suggests that the tumor suppressor protein, p53, protects somatic cells against the accumulation of genomic mutations. The genomes of cells lacking normal p53 function may become hypermutable, a condition that might result in the accumulation of multiple genetic alterations as the affected cells proliferate. Such cells may then become more susceptible to malignant transformation. We hypothesized that some high-grade prostate cancers might arise from foci of morphologically benign cells that had previously sustained p53 lesions. As an initial test of this hypothesis, we employed a microdissection technique to isolate morphologically benign cells within hyperplastic glands located near foci of high-grade adenocarcinoma. Genomic DNA from these cells was subjected to polymerase chain reaction amplification and single-stranded conformational polymorphism analysis for detecting alterations in the p53 locus. With use of this approach, gross alterations in the p53 locus were demonstrated in benign cells in 1 of 20 (5%) specimens harboring high-grade malignancy (Gleason grade 7 or higher). Thus, in some cases, hyperplastic prostatic epithelium harbors preneoplastic genetic alterations that could possibly give rise to high-grade malignancies.

Adenocarcinoma↗

PubMed1993

The binding domain structure of retinoblastoma-binding proteins.

The retinoblastoma gene product (Rb), a cellular growth suppressor, complexes with viral and cellular proteins that contain a specific binding domain incorporating three invariant residues: Leu-X-Cys-X-Glu, where X denotes a nonconserved residue. Hydrophobic and electrostatic properties are strongly conserved in this segment even though the nonconserved amino acids vary considerably from one Rb-binding protein to another. In this report, we present a diagnostic computer pattern for a high-affinity Rb-binding domain featuring the three conserved residues as well as the conserved physico-chemical properties. Although the pattern encompasses only 10 residues (with only 4 of these explicitly defined), it exhibits 100% sensitivity and 99.95% specificity in database searches. This implies that a certain pattern of structural and physico-chemical properties encoded by this short sequence is sufficient to govern specific Rb binding. We also present evidence that the secondary structural conformation through this region is important for effective Rb binding.

Amino Acid Sequence↗

PubMed1993

A competitive inhibitor assay for SV40 T/pRB complex formation employing extracts of SV40-transformed human lung cells.

Simian virus 40 (SV40) large T antigen (T), a potent dominant oncogene product, forms a specific complex with the human retinoblastoma protein (pRb), a cellular growth suppressor. We have used a recombinant pRb fusion protein (GST-Rb) in combination with extracts from a line of SV40-transformed human lung cells (WI-26 VA4) to develop a simple, non-radioactive assay to rapidly screen for competitive inhibitors of T/pRb binding. We illustrate the use of the assay by demonstrating that several short peptides containing the signature sequence, Leu-X-Cys-X-Glu, can inhibit T/pRb complex formation. In contrast, peptides containing the related motif, Leu-X-Glu-X-Glu, including two peptides derived from the transcription factor E2F, are inactive in this assay. These results show that Glu cannot substitute for Cys in the Leu-X-Cys-X-Glu motif. This assay will facilitate the identification of agents that are inhibitors of T/pRb complex formation and that might exert effects on cellular growth regulation.

Amino Acid Sequence↗

PubMed1991

Effect of atherosclerosis on cerebral vascular responses to activation of leukocytes and platelets in monkeys.

The goal of this study was to test the hypothesis that atherosclerosis alters responses of cerebral arteries and the ocular circulation to the activation in vivo of leukocytes and platelets. We measured blood flow to the brain and eye using microspheres and pressure in the cerebral microvessels of normal and atherosclerotic monkeys. The intracarotid injection of 10(-7) M N-formyl-L-methionyl-L-leucyl-L-phenylalanine to activate leukocytes did not alter cerebral blood flow in 11 normal or 10 atherosclerotic monkeys but increased the resistance of large cerebral arteries by 46 +/- 11% (mean +/- SEM) in the atherosclerotic animals. The injection of N-formyl-L-methionyl-L-leucyl-L-phenylalanine did not alter blood flow to the eye in 10 normal monkeys but decreased blood flow to the choroid by 38 +/- 9% in 11 atherosclerotic monkeys. The intracarotid injection of 3 x 10(-9) M prostaglandin E2, a leukocyte product, produced an increase in the resistance of large cerebral arteries in five atherosclerotic but not in six normal monkeys. Prostaglandin E2 reduced blood flow to the retina and choroid in the atherosclerotic monkeys by 62 +/- 22% and 65 +/- 17%, respectively. The intracarotid infusion of 25 micrograms/min collagen to activate platelets increased cerebral blood flow by 21 +/- 5% in 10 normal monkeys but did not alter it in 11 atherosclerotic monkeys. Collagen did not alter blood flow to the choroid in 10 normal monkeys but decreased it by 29 +/- 8% in 11 atherosclerotic monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1980