Search PubMed⌕ Search

Biomedical subjects

T Conway

Publications and source records attributed to T Conway.

At least 37 records · Page 2Linked to original sources
PubMed1998

Sequence analysis of the GntII (subsidiary) system for gluconate metabolism reveals a novel pathway for L-idonic acid catabolism in Escherichia coli.

The presence of two systems in Escherichia coli for gluconate transport and phosphorylation is puzzling. The main system, GntI, is well characterized, while the subsidiary system, GntII, is poorly understood. Genomic sequence analysis of the region known to contain genes of the GntII system led to a hypothesis which was tested biochemically and confirmed: the GntII system encodes a pathway for catabolism of L-idonic acid in which D-gluconate is an intermediate. The genes have been named accordingly: the idnK gene, encoding a thermosensitive gluconate kinase, is monocistronic and transcribed divergently from the idnD-idnO-idnT-idnR operon, which encodes L-idonate 5-dehydrogenase, 5-keto-D-gluconate 5-reductase, an L-idonate transporter, and an L-idonate regulatory protein, respectively. The metabolic sequence is as follows: IdnT allows uptake of L-idonate; IdnD catalyzes a reversible oxidation of L-idonate to form 5-ketogluconate; IdnO catalyzes a reversible reduction of 5-ketogluconate to form D-gluconate; IdnK catalyzes an ATP-dependent phosphorylation of D-gluconate to form 6-phosphogluconate, which is metabolized further via the Entner-Doudoroff pathway; and IdnR appears to act as a positive regulator of the IdnR regulon, with L-idonate or 5-ketogluconate serving as the true inducer of the pathway. The L-idonate 5-dehydrogenase and 5-keto-D-gluconate 5-reductase reactions were characterized both chemically and biochemically by using crude cell extracts, and it was firmly established that these two enzymes allow for the redox-coupled interconversion of L-idonate and D-gluconate via the intermediate 5-ketogluconate. E. coli K-12 strains are able to utilize L-idonate as the sole carbon and energy source, and as predicted, the ability of idnD, idnK, idnR, and edd mutants to grow on L-idonate is altered.

Biological Transport↗
PubMed1998

Positive and negative transcriptional regulation of the Escherichia coli gluconate regulon gene gntT by GntR and the cyclic AMP (cAMP)-cAMP receptor protein complex.

The gntT gene of Escherichia coli is specifically induced by gluconate and repressed via catabolite repression. Thus, gluconate is both an inducer and a repressor of gntT expression since gluconate is a catabolite-repressing sugar. In a gntR deletion mutant, the expression of a chromosomal gntT::lacZ fusion is both high and constitutive, confirming that GntR is the negative regulator of gntT. Indeed, GntR binds to two consensus gnt operator sites; one overlaps the -10 region of the gntT promoter, and the other is centered at +120 with respect to the transcriptional start site. The binding of GntR to these sites was proven in vitro by gel redardation assays and in vivo by site-directed mutagenesis of the binding sites. Binding of GntR to the operators is eliminated by gluconate and also by 6-phosphogluconate at a 10-fold-higher concentration. Interestingly, when gntR deletion strains are grown in the presence of gluconate, there is a twofold decrease in gntT expression which is independent of catabolite repression and binding of GntR to the operator sites. This novel response of gntR mutants to the inducer is termed ultrarepression. Transcription of gntT is activated by binding of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex to a CRP binding site positioned at -71 upstream of the gntT transcription start site.

Bacterial Proteins↗
PubMed1997

Characterization of a novel transporter family that includes multiple Escherichia coli gluconate transporters and their homologues.

The nucleotide sequences of seven Escherichia coli genes that encode members of the gluconate permease (GntP) family have recently become available. These genes include gntP, gntU, gntW, ORf449, dsdX, and ORFo454. The deduced amino acid sequences of all seven E. coli genes are homologous to the gntP gene products from Bacillus subtilis and B. licheniformis as well as two additional gene products from Haemophilus influenzae. These 11 proteins are not demonstrably homologous to members of the major facilitator superfamily or other recognized permease families. Four of the E. coli gluconate transporter genes have been cloned and shown to encode gluconate transporters with apparent affinities ranging from 6 to 212 microM. These studies serve to characterize a novel family of bacterial permeases.

Bacillus↗
PubMed1997

Setting health priorities: community boards accurately reflect the preferences of the community's residents.

Setting priorities remains an important part of healthcare planning and program management. Local community input is often sought in government or publicly sponsored programs. Community policy/advisory boards are a common vehicle to represent the community's interests in program decisions and direction. Questions remain whether community boards accurately represent their communities' views. As part of a planning effort within Chicago and Cook County, Illinois, local District Health Councils (DHCs) have been created to provide assistance and leadership in systemization and improvement of the healthcare in communities with the poorest health status in the region. We sought to discover how closely the perceptions of health priorities of DHC members agreed with those of community members. A structured five-point Likert scale questionnaire of 22 of the most common diseases and conditions known to impact health were used for a random digit dialing telephone interview with a sample of 286 households from three under-served communities. The same interview was repeated with all DHC members (n = 80) representing those communities. Sociodemographic profiles and health-related behaviors were also collected. The results of this interview indicate a close and substantial agreement in priorities between community members and DHC members. Psychosocial conditions such as violence and substance abuse were ranked as the highest priorities by both groups. In contrast, sociodemographics and healthcare behavior differed significantly between DHC members and community's residents. This study demonstrates that these community policy/advisory boards can closely reflect the views of the communities they represent. Attention to their differences in sociodemographics and healthcare experiences with the community may strengthen their role even more.

Adult↗
PubMed1997

The role of expectations in the perception of health care quality: developing a conceptual model.

Proposes a conceptual model of quality which incorporates an expectation framework. The model was prompted by a related empirical study examining effectiveness in a health care setting which highlighted the importance of quality measures in a contracting environment. The model attempts to understand the relationships between perceived service quality and patient expectations, experience and satisfaction. Makes recommendations which are considered to be relevant to NHS policy makers.

Choice Behavior↗
PubMed1997

Kinetics of Sugar Transport and Phosphorylation Influence Glucose and Fructose Cometabolism by Zymomonas mobilis.

The competitive inhibition of fructokinase by glucose has been proposed as the mechanism by which Zymomonas mobilis preferentially consumes glucose from mixtures of glucose and fructose and accumulates fructose when growing on sucrose. In this study, incorporation of radioactive fructose into biomass was used as a measure of fructose catabolism. It was determined that the rate of fructose incorporation by Z. mobilis CP4 was somewhat lower in the presence of an equimolar concentration of glucose but that the inhibition of fructokinase by glucose was not nearly as severe in vivo as was predicted from in vitro studies. Interestingly, addition of glucose to a culture of Z. mobilis CP4-M2, a glucokinaseless mutant, resulted in an immediate and nearly complete inhibition of fructose incorporation. Furthermore, addition of nonmetabolizeable glucose analogs had a similar effect on fructose catabolism by the wild-type Z. mobilis CP4, and fructose uptake by Z. mobilis CP4-M2 was shown to be severely inhibited by equimolar amounts of glucose. These results suggest that competition for fructose transport plays an important role in preferential catabolism of glucose from sugar mixtures. Indeed, the apparent K(infm) values for sugar uptake by Z. mobilis CP4 were approximately 200 mM for fructose and 13 mM for glucose. Other experiments supported the conclusion that a single facilitated diffusion transport system, encoded by the glf gene, is solely responsible for the uptake of both glucose and fructose. The results are discussed with regard to the hypothesis that the kinetics of sugar transport and phosphorylation allow the preferential consumption of glucose and accumulation of fructose, making the fructose available for the enzyme glucose-fructose oxidoreductase, which forms sorbitol, an important osmoprotectant for Z. mobilis when growing in the presence of high sugar concentrations.

Journal Article↗
PubMed1997

Molecular genetic characterization of the Escherichia coli gntT gene of GntI, the main system for gluconate metabolism.

The Escherichia coli gntT gene was subcloned from the Kohara library, and its expression was characterized. The cloned gntT gene genetically complemented mutant E. coli strains with defects in gluconate transport and directed the formation of a high-affinity gluconate transporter with a measured apparent Km of 6 microM for gluconate. Primer extension analysis indicated two transcriptional start sites for gntT, which are separated by 66 bp and which give rise to what appears on a Northern blot to be a single, gluconate-inducible, 1.42-kb gntT transcript. Thus, it was concluded that gntT is monocistronic and is regulated by two promoters. Both of the promoters have - 10 and -35 sequence elements typical of sigma70 promoters and catabolite gene activator protein binding sites in appropriate locations to exert glucose catabolite repression. In addition, two putative gnt operator sites were identified in the gntT regulatory region. A search revealed the presence of nearly identical palindromic sequences in the regulatory regions of all known gluconate-inducible genes, and these seven putative gnt operators were used to derive a consensus gnt operator sequence. A gntT::lacZ operon fusion was constructed and used to examine gntT expression. The results indicated that gntT is maximally induced by 500 microM gluconate, modestly induced by very low levels of gluconate (4 microM), and partially catabolite repressed by glucose. The results also showed a pronounced peak of gntT expression very early in the logarithmic phase, a pattern of expression similar to that of the Fis protein. Thus, it is concluded that GntT is important for growth on low concentrations of gluconate, for entry into the logarithmic phase, and for cometabolism of gluconate and glucose.

Bacterial Proteins↗
PubMed1996

BRCA1 testing in families with hereditary breast-ovarian cancer. A prospective study of patient decision making and outcomes.

OBJECTIVES: To identify predictors of utilization of breast-ovarian cancer susceptibility (BRCA1 gene) testing and to evaluate outcomes of participation in a testing program. DESIGN: Prospective cohort study with baseline interview assessment of predictor variables (eg, sociodemographic factors, knowledge about hereditary cancer and genetic testing, perceptions of testing benefits, limitations, and risks). BRCA1 test results were offered after an education and counseling session in a research setting. Outcome variables (including depression, functional health status, and prophylactic surgery plans [follow-up only]) were assessed at baseline and 1-month follow-up interviews. PARTICIPANTS: Adult male and female members (n=279) of families with BRCA1-linked hereditary breast-ovarian cancer (HBOC). RESULTS: Of subjects who completed a baseline interview (n=192), 60% requested BRCA1 test results (43% of all study subjects requested results). Requests for results were more frequent for persons with health insurance (odds ration [OR], 3.74; 95% confidence interval [CI], 2.06-6.80); more first-degree relatives affected with breast cancer (OR, 1.59; 95% CI, 1.16-2.16); more knowledge about BRCA1 testing (OR, 1.85; 95% CI, 1.36-2.50); and indicating that test benefits are important (OR, 1.45; 95% CI, 1.13-1.86). At follow-up, noncarriers of BRCA1 mutations showed statistically significant reductions in depressive symptoms and functional impairment compared with carriers and nontested individuals. Individuals identified as mutation carriers did not exhibit increases in depression and functional impairment. Among unaffected women with no prior prophylactic surgery, 17% of carriers (2/12) intended to have mastectomies and 33% (4/12) to have oophorectomies. CONCLUSIONS: Only a subset of HBOC family members are likely to request BRCA1 testing when available. Rates of test use may be higher in persons of a higher socioeconomic status and those with more relatives affected with breast cancer. For some high-risk individuals who receive test results in a research setting that includes counseling, there may be psychological benefits. More research is needed to assess the generalizability of these results and evaluate the long-term consequences of BRCA1 testing.

Adult↗
PubMed1996

The internist's role in addressing violence. A review of current recommendations and a model for intervention.

Approximately 25,000 people die in the United States each year as the result of intentional homicide, and many millions more suffer the physical and emotional consequences of nonfatal interpersonal violence. Violence affects everybody's quality of life. The cost to the health care system is in the billions of dollars annually. Over the past 2 decades epidemiologic studies have defined risk factors for violence, and a number of physicians and medical groups have made recommendations for preventive action. As the largest physician group in the United States, internists have a clear role to play in carrying out the medical responsibility to help prevent violence. Internists should be aware of the signs and symptoms of violence in patients and be prepared to assist victims. To facilitate action against violence, a model for intervention is proposed that involves screening, counseling, and treatment for patients at greatest risk.

Adolescent↗
PubMed1996

Cloning and expression of the Zymomonas mobilis "production of ethanol" genes in Lactobacillus casei.

This study describes the expression of the Zymomonas mobilis genes coding for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) in Lactobacillus casei 686. To promote transcription, the promoter and ribosome binding site (RBS) from the Lactococcus lactis subsp. lactis-derived vector, pMGE36e, were inserted upstream of the pdc gene. The former sequences were positioned such that translation of pdc was coupled to translation of an 81-base pair open reading frame terminating within the pdc initiation site. The recombinant plasmid (pRSG02) was electroporated into L. casei, and transformants were obtained. Northern analysis confirmed the production of a 3. 1-kb transcript corresponding to the predicted size of the PET operon. Western blot analyses revealed that the recombinant strain expressed both enzymes. The recombinant produced more than twice the ethanol produced by the parental L. casei strain.

Alcohol Dehydrogenase↗
PubMed1996

Synthesis and preliminary evaluation of (S)-(2-[18F]fluoro-4,5-dihydroxyphenyl)-2-methyl L-alanine, (S)-[18F]-FMEDOPA, a potentially improved imaging agent for the presynaptic dopaminergic nervous system.

The synthesis and preliminary rat biodistribution studies of (S)-(2-[18F]fluoro-4,5-dihydroxyphenyl)-2-methyl L-alanine, (S)-[18F]-FMEDOPA, a potentially improved imaging agent for the presynaptic dopaminergic nervous system, are reported. (S)-[18F]-FMEDOPA produces a higher striatum-to-cerebellum (S/C) radioactivity ratio than the currently used PET imaging agent, (S)-[18F]-FDOPA, does at 180 min after administration.

Animals↗
PubMed1996

The gntP gene of Escherichia coli involved in gluconate uptake.

The gntP gene, located between the fim and uxu loci in Escherichia coli K-12, has been cloned and characterized. Nucleotide sequencing of a region encompassing the gntP gene revealed an open reading frame of 447 codons with significant homology to the Bacillus subtilis gluconate permease. Northern (RNA) blotting indicated that the gntP gene was monocistronic and was transcribed as an mRNA with an apparent molecular size of 1.54 kb. The transcriptional start point was determined by primer extension analysis. The gntP gene was found to be under catabolite repression and was not induced by gluconate. Also, expression seemed to be stringently controlled. Several observations indicated that the GntP protein is an inner membrane protein; it contains characteristic membrane-spanning regions and was isolated predominantly from the inner-membrane fraction of fractionated host cells. A topology analysis predicted a protein with 14 membrane-spanning segments. The inability of a mutant strain to grow on gluconate minimal medium could be relieved by introduction of a plasmid encoding the gntP gene. Finally, the kinetics of GntP-mediated gluconate uptake were investigated, indicating an apparent Km for gluconate of 25 microM.

Amino Acid Sequence↗
PubMed1996

Cloning and molecular genetic characterization of the Escherichia coli gntR, gntK, and gntU genes of GntI, the main system for gluconate metabolism.

Three genes involved in gluconate metabolism, gntR, gntK, and gntU, which code for a regulatory protein, a gluconate kinase, and a gluconate transporter, respectively, were cloned from Escherichia coli K-12 on the basis of their known locations on the genomic restriction map. The gene order is gntU, gntK, and gntR, which are immediately adjacent to asd at 77.0 min, and all three genes are transcribed in the counterclockwise direction. The gntR product is 331 amino acids long, with a helix-turn-helix motif typical of a regulatory protein. The gntK gene encodes a 175-amino-acid polypeptide that has an ATP-binding motif similar to those found in other sugar kinases. While GntK does not show significant sequence similarity to any known sugar kinases, it is 45% identical to a second putative gluconate kinase from E. coli,gntV. The 445-amino-acid sequence encoded by gntU has a secondary structure typical of membrane-spanning transport proteins and is 37% identical to the gntP product from Bacillus subtilis. Kinetic analysis of GntU indicates an apparent Km for gluconate of 212 microM, indicating that this is a low-affinity transporter. Studies demonstrate that the gntR gene is monocistronic, while the gntU and gntK genes, which are separated by only 3 bp, form an operon. Expression of gntR is essentially constitutive, while expression of gntKU is induced by gluconate and is subject to fourfold glucose catabolite repression. These results confirm that gntK and gntU, together with another gluconate transport gene, gntT, constitute the GntI system for gluconate utilization, under control of the gntR gene product, which is also responsible for induction of the edd and eda genes of the Entner-Doudoroff pathway.

Amino Acid Sequence↗
PubMed1996

Effects of dietary calcium and phosphorus on vitamin D metabolism and calcium absorption in hamster.

We studied the following responses to restriction of dietary calcium and phosphorus in the growing hamster: (i) serum concentrations of calcium, inorganic phosphorus, magnesium, and vitamin D metabolites; and (ii) calcium transport by ileum. Diets fed were normal calcium with normal or low phosphorus or low calcium with normal or low phosphorus. We found serum 1 alpha,25-dihydroxycalciferol (1,25-[OH]2D) concentration did not differ significantly among the diet groups. Calcium absorption, measured as serosal/mucosal calcium concentration ratio produced by everted ileal sac, was greater in the low calcium, normal phosphorous group than in all other groups. The other groups did not differ from one another in calcium absorption. Feeding the low calcium, normal phosphorus diet increased inorganic phosphorus and magnesium but decreased calcium concentration in serum in comparison with the three other diets. Both low phosphorus diets were without effect on serum calcium, but the low calcium, low phosphorus diet increased serum inorganic phosphorus and magnesium above that of the normal calcium, low phosphorus diet. Ileal calcium absorption in hamster (i) was independent of serum 1,25-(OH)2D concentration; (ii) increased in response to low dietary calcium if dietary phosphorus was normal; and (iii) was independent of dietary calcium, if dietary phosphorus was low. Despite increased calcium absorption, serum calcium was decreased in the low calcium-normal phosphorus group as compared with all other groups. Feeding low calcium diets increased serum inorganic phosphorus and magnesium as compared with feeding the corresponding normal calcium diets (i.e., independently of whether dietary phosphorus content was normal or low). These studies demonstrate that the interrelationships between calcium absorption and vitamin D and mineral metabolism in hamster differ from other mammals.

Absorption↗
PubMed1995

Single-run separation and detection of multiple metabolic intermediates by anion-exchange high-performance liquid chromatography and application to cell pool extracts prepared from Escherichia coli.

A method is described for analysis of the intracellular concentrations of metabolic intermediates of the Embden-Meyerhof-Parnas pathway, the Entner-Doudoroff pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle in cell pool extracts of Escherichia coli. A single anion-exchange HPLC run of 40 min allowed resolution of 27 anionic metabolite standards. Detection limits of 0.1 nmol per injection were achieved by use of a conductivity detector equipped with an anion self-regenerating suppressor and a uv detector. A boiling water extraction procedure was used to prepare cell pool extracts. Cochromatography of cell pool extracts and metabolite standards was used to confirm the identities of metabolites in the cell pool. As many as 16 metabolites could be detected and quantified in the cell pool extracts by using the described HPLC method. An analysis of metabolite concentrations in E. coli showed the dynamics of glucose metabolism during a 2-min transition from starvation to steady-state metabolism following addition of glucose. The ease and power of this method suggests general utility for in vivo metabolite analysis in a variety of experimental systems.

Carbohydrates↗
PubMed1995

Breast cancer and importance of zygosity determination in triplet sisters.

This study is the first to examine the genetic risk in a breast cancer-prone family wherein two of three triplet sisters and their mother manifested breast cancer. The unaffected triplet, the proband, was found to be monozygotic with her deceased sister through DNA testing of tumor blocks and dizygotic to her living affected sister. Genetic counseling implications are discussed.

Adult↗
PubMed1995

Characterization of the Zymomonas mobilis glucose facilitator gene product (glf) in recombinant Escherichia coli: examination of transport mechanism, kinetics and the role of glucokinase in glucose transport.

Zymomonas mobilis is known to transport glucose by a facilitated diffusion process. A putative glucose facilitator gene (glf), closely related to a large family of glucose transporters, is located in a cluster of genes that code for enzymes of glucose metabolism. The Z. mobilis glf gene is able to complement glucose transport in an Escherichia coli strain that is defective in native glucose transport and glucokinase. In this study, the recombinant E. coli was shown to be capable of influx counterflow when preloaded with glucose and had an apparent Km for glucose of approximately 1.1-2.9 mM, consistent with the function of Glf as a low-affinity glucose facilitator. The ability of glucokinase mutants expressing glf to transport glucose made it clear that glucokinase activity was not required for Glf-dependent glucose transport. The possibility that glucokinase can interact with Glf to improve the affinity for glucose was not supported since expression of the Z. mobilis glucokinase gene, in addition to glf, did not affect the Km of Glf for glucose in recombinant E. coli. The inability of various sugars to compete with glucose during glucose transport by recombinant E. coli expressing glf indicated that Glf is specific for glucose. While the results of fructose transport assays did not completely rule out the possibility of very low affinity for fructose, the apparent specificity of Glf for glucose makes it possible that Z. mobilis utilizes a different transporter(s) for fructose.

Bacterial Proteins↗