Search PubMed⌕ Search

Biomedical subjects

W Ross

Publications and source records attributed to W Ross.

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

Transcription activation at Class II CRP-dependent promoters: identification of determinants in the C-terminal domain of the RNA polymerase alpha subunit.

Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase. At Class II CRP-dependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase alpha subunit C-terminal domain (alphaCTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase alpha subunit N-terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within alphaCTD for transcription activation at a Class II CRP-dependent promoter. Our results indicate that Class II CRP-dependent transcription requires the side chains of residues 265, 271, 285-288 and 317. Residues 285-288 and 317 comprise a discrete 20x10 A surface on alphaCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between alpha subunits and CRP, but do not affect DNA binding by alpha subunits. We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in alphaCTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for alphaCTD-CRP interaction in Class I CRP-dependent transcription.

Alanine↗
PubMed1998

RNA polymerase mutants that destabilize RNA polymerase-promoter complexes alter NTP-sensing by rrn P1 promoters.

Mutations in Escherichia coli rpoB or rpoC, selected for the ability to confer prototrophy on relA spoT strains, were found to affect transcription from rrn P1 promoters. Two mutant strains (beta RH454 and beta' delta 215-220) reduced transcription of rrn P1 core promoter-lacZ fusions but not of control promoter-lacZ fusions. Purified mutant RNAPs formed complexes with rrn P1 promoters that were much less stable than those formed by wild-type RNAP and required high concentrations of the initiating NTP for efficient rrn P1 transcription. The instability of the rrn P1 core promoter complexes with the mutant RNAPs and their altered regulatory properties support a recently proposed model for the control of rRNA transcription by changing concentrations of the initiating NTPs. We further suggest that destabilization of promoter complexes by the mutant RNAPs mimics effects of ppGpp, decreasing or increasing transcription depending on the kinetic properties of the specific promoter.

Adenosine Triphosphate↗
PubMed1998

Quantitative risk assessment for Escherichia coli O157:H7 in ground beef hamburgers.

Quantitative Risk Assessment (QRA) is a methodology used to organize and analyze scientific information to estimate the probability and severity of an adverse event. Applied to microbial food safety, the methodology can also help to identify those stages in the manufacture, distribution, handling, and consumption of foods that contribute to an increased risk of foodborne illness, and help focus resources and efforts to most effectively reduce the risk of foodborne pathogens. The term Process Risk Model (PRM) is introduced in this paper to describe the integration and application of QRA methodology with scenario analysis and predictive microbiology to provide an objective assessment of the hygienic characteristics of a manufacturing process. The methodology was applied to model the human health risk associated with Escherichia coli O157:H7 in ground beef hamburgers. The PRM incorporated two mathematical submodels; the first was intended to described the behaviour of the pathogen from the production of the food through processing, handling, and consumption to predict human exposure. The exposure estimate was then used as input to a dose-response model to estimate the health risk associated with consuming food from the process. Monte Carlo simulation was used to assess the effect of the uncertainty and variability in the model parameters on the predicted human health risk. The model predicted a probability of Hemolytic Uremic Syndrome of 3.7 x 10(-6) and a probability of mortality of 1.9 x 10(-7) per meal for the very young. These estimates are likely high for all hamburger meals, but may be reasonable for the home-prepared hamburgers described by this model. The efficacy of three risk mitigation strategies were evaluated by modifying the values of the predictive factors and comparing the new predicted risk. The average probability of illness was predicted to be reduced by 80% under a hypothetical mitigation strategy directed at reducing microbial growth during retail storage through a reduction in storage temperature. This strategy was predicted to be more effective than a hypothetical intervention which estimated a plausible reduction in the concentration of E. coli O157:H7 in the feces of cattle shedding the pathogen and one aimed at convincing consumers to cook hamburgers more thoroughly. The conclusions of this approach are only accurate to the extent that the model accurately represents the process. Currently, uncertainty and ignorance about the hygienic effects of the individual operations during production, processing, and handling limit the applicability of a PRM to specify HACCP criteria in a quantitative manner. However, with continuous improvement through stimulated research, a PRM should encompass all available information about the process, food, and pathogen and should be the most appropriate decision-support tool since it represents current knowledge.

Animals↗
PubMed1998

Escherichia coli promoters with UP elements of different strengths: modular structure of bacterial promoters.

The alpha subunit of Escherichia coli RNA polymerase (RNAP) participates in promoter recognition through specific interactions with UP element DNA, a region upstream of the recognition hexamers for the sigma subunit (the -10 and -35 hexamers). UP elements have been described in only a small number of promoters, including the rRNA promoter rrnB P1, where the sequence has a very large (30- to 70-fold) effect on promoter activity. Here, we analyzed the effects of upstream sequences from several additional E. coli promoters (rrnD P1, rrnB P2, lambda pR, lac, merT, and RNA II). The relative effects of different upstream sequences were compared in the context of their own core promoters or as hybrids to the lac core promoter. Different upstream sequences had different effects, increasing transcription from 1.5- to approximately 90-fold, and several had the properties of UP elements: they increased transcription in vitro in the absence of accessory protein factors, and transcription stimulation required the C-terminal domain of the RNAP alpha subunit. The effects of the upstream sequences correlated generally with their degree of similarity to an UP element consensus sequence derived previously. Protection of upstream sequences by RNAP in footprinting experiments occurred in all cases and was thus not a reliable indicator of UP element strength. These data support a modular view of bacterial promoters in which activity reflects the composite effects of RNAP interactions with appropriately spaced recognition elements (-10, -35, and UP elements), each of which contributes to activity depending on its similarity to the consensus.

Bacterial Proteins↗
PubMed1998

Activation of Escherichia coli rRNA transcription by FIS during a growth cycle.

rRNA transcription in Escherichia coli is activated by the FIS protein, which binds upstream of rrnp1 promoters and interacts directly with RNA polymerase. Analysis of the contribution of FIS to rrn transcription under changing physiological conditions is complicated by several factors: the wide variation in cellular FIS concentrations with growth conditions, the contributions of several other regulatory systems to rRNA synthesis, and the pleiotropy of fis mutations. In this report, we show by in vivo footprinting and Western blot analysis that occupancy of the rrnBp1 FIS sites correlates with cellular levels of FIS. We find, using two methods of measurement (pulse induction of a FIS-activated hybrid promoter and primer extension from an unstable transcript made from rrnBp1), that the extent of transcription activation by FIS parallels the degree of FIS site occupancy and therefore cellular FIS levels. FIS activates transcription throughout exponential growth at low culture density, but rrnp1 transcription increases independently of FIS immediately following upshift, before FIS accumulates. These results support the model that FIS is one of a set of overlapping signals that together contribute to transcription from rrnp1 promoters during steady-state growth.

Base Sequence↗
PubMed1997

Transcription regulation by initiating NTP concentration: rRNA synthesis in bacteria.

The sequence of a promoter determines not only the efficiency with which it forms a complex with RNA polymerase, but also the concentration of nucleoside triphosphate (NTP) required for initiating transcription. Escherichia coli ribosomal RNA (rrn P1) promoters require high initiating NTP concentrations for efficient transcription because they form unusually short-lived complexes with RNA polymerase; high initiating NTP concentrations [adenosine or guanosine triphosphate (ATP or GTP), depending on the rrn P1 promoter] are needed to bind to and stabilize the open complex. ATP and GTP concentrations, and therefore rrn P1 promoter activity, increase with growth rate. Because ribosomal RNA transcription determines the rate of ribosome synthesis, the control of ribosomal RNA transcription by NTP concentration provides a molecular explanation for the growth rate-dependent control and homeostatic regulation of ribosome synthesis.

Adenosine Triphosphate↗
PubMed1997

The RNA polymerase alpha subunit carboxyl-terminal domain is required for both basal and activated transcription from the alkA promoter.

Expression of the Escherichia coli adaptive response genes (ada, aidB, and alkA) is regulated by the transcriptional activator, Ada. However, the interactions of RNA polymerase and Ada with these promoters differ. In this report we characterize the interactions of Ada, methylated Ada (meAda), and RNA polymerase at the alkA promoter and contrast these interactions with those characterized previously for the ada and aidB promoters. At the alkA promoter, we do not detect the RNA polymerase alpha subunit-mediated binary complex detected at the ada and aidB promoters. In the presence of either of these two activators, RNA polymerase protects the alkA core promoter, including the elements at -35 and -10, and is more efficient in transcription initiation in vitro. RNA polymerase holoenzyme containing the alpha subunit mutation R265A is severely impaired in Ada-independent basal alkA transcription, shows no activation by Ada or meAda, and fails to bind the alkA promoter in vitro. Binding of the purified wild type alpha subunit to alkA was not detected, but a complex of promoter DNA, Ada or meAda, and alpha was observed in gel shift assays. These observations suggest that both forms of Ada protein activate alkA transcription by enhancing RNA polymerase holoenzyme and alpha subunit binding.

Bacterial Proteins↗
PubMed1997

Molecular anatomy of a transcription activation patch: FIS-RNA polymerase interactions at the Escherichia coli rrnB P1 promoter.

FIS, a site-specific DNA binding and bending protein, is a global regulator of gene expression in Escherichia coli. The ribosomal RNA promoter rrnB P1 is activated 3- to 7-fold in vivo by a FIS dimer that binds a DNA site immediately upstream of the DNA binding site for the C-terminal domain (CTD) of the alpha subunit of RNA polymerase (RNAP). In this report, we identify several FIS side chains important specifically for activation of transcription at rrnB P1. These side chains map to positions 68, 71 and 74, in and flanking a surface-exposed loop adjacent to the helix-turn-helix DNA binding motif of the protein. We also present evidence suggesting that FIS activates transcription at rrnB P1 by interacting with the RNAP alphaCTD. Our results suggest a model for FIS-mediated activation of transcription at rrnB P1 that involves interactions between FIS and the RNAP alphaCTD near the DNA surface. Although FIS and the transcription activator protein CAP have little structural similarity, they both bend DNA, use a similarly disposed activation loop and target the same region of the RNAP alphaCTD, suggesting that this is a common architecture at bacterial promoters.

Arginine↗
PubMed1997

Patterns of health maintenance on Michigan equine operations.

Data from two 12 month prospective monitoring programs that followed management, economics and animal health from randomly-sampled equine operations in Michigan were used to determine patterns of health maintenance. Health maintenance measures were grouped, and average uses per year were computed for the most-common measures reported: respiratory, Potomac Horse Fever, neurological and multiple-system vaccinations, deworming, and general farrier work. Factors examined for potential association with health maintenance measure use were numbers of equids and horse-days on the operation, average age of equids, disease prevention management procedures, and operation and individual equine activities. The most-common vaccinations reported were respiratory vaccines (administered an average of 0.9 times per equid per year, of which 61% were for rhinopneumonitis). Multiple-system, Potomac Horse Fever and neurological vaccines were given on average 0.9, 0.9 and 0.7 times per equid per year, respectively. Deworming treatments were administered approximately 2.7 times per equid per year, of which 64% were ivermectin. Overall farrier work was reported 3.3 times per equid per year, trimming was done 2.9 times and shoeing was done 1.5 times per equid per year. In general, operations with active equids engaged in training, breeding or showing showed increased use of health maintenance measures. Overall use of health maintenance measures in the Michigan equine operations monitored did not appear to reach recommended levels, because many operations reported no use of these measures. However, on operations where health maintenance measures were reported, operators were using these measures at or near recommended levels.

Aging↗
PubMed1997

Does repetitive physical loading inhibit radial growth in female gymnasts?

OBJECTIVE: Stress-related injuries to the distal radius have been noted in female gymnasts with potential for resultant premature closure and abnormal growth at this site. The purpose of this study was comprehensively to review and critically to appraise the available literature to examine the evidence related to this question: does repetitive physical loading inhibit growth of the radius in female gymnasts? DATA SOURCES: MEDLINE and SPORT Discuss were searched from 1975 to the present by using "gymnast" in combination with injury, growth plate, epiphyseal, and ulnar variance. Additional references were retrieved from the bibliographies of the retrieved articles. STUDY SELECTION: All descriptive and analytic studies that included data related to stress-related injuries affecting the distal radius of competitive female gymnasts were included. Conclusions regarding the effects of gymnastics training on radial growth of female gymnasts were limited to data from case reports, clinical series, cross-sectional studies, and descriptive cohort studies. Data from relevant experimental animal studies also were included. DATA EXTRACTION AND SYNTHESIS: In reviewing the literature, particular attention was paid to the relative strengths of the different study designs. From these data, information associated with growth inhibition at the distal radius was examined. MAIN RESULTS: The descriptive research reviewed included clinical, cross-sectional, and cohort studies that establish the existence of stress-related injuries affecting one or more constituent parts of the epiphyseal-physeal-metaphyseal (EPM) complex of the distal radius, symptomatic ulna-radial-length difference (URLD), and distal radius physeal arrest among female gymnasts. Five cross-sectional studies showed radiographic abnormalities consistent with distal radius physeal-stress reaction in 10-85% of gymnasts studied. Two cross-sectional studies indicated "abnormal" positive URLD in 8-20% of wrists radiographed. Four cross-sectional studies showed significant correlations between training intensity and URLD, suggesting a dose-response relation. Three cross-sectional studies indicate greater URLD in gymnasts compared with nongymnasts. Radiographic evidence of distal radius physeal arrest involving physically immature female gymnasts is presented in four studies (two clinical series, one cross-sectional, and one descriptive cohort). In animal studies, prolonged physical training has also been shown to inhibit or stop growth in weight-bearing long bones. However, there were no rigorous studies (i.e., randomized control trials or analytic cohorts) examining the question. CONCLUSION: The results of this critical review of the scientific literature support the plausibility of stress-related distal radius physeal arrest with secondary URLD. However, the strength of evidence is inadequate to be conclusive.

Adolescent↗
PubMed1996

DNA-binding determinants of the alpha subunit of RNA polymerase: novel DNA-binding domain architecture.

The Escherichia coli RNA polymerase alpha-subunit binds through its carboxy-terminal domain (alpha CTD) to a recognition element, the upstream (UP) element, in certain promoters. We used genetic and biochemical techniques to identify the residues in alpha CTD important for UP-element-dependent transcription and DNA binding. These residues occur in two regions of alpha CTD, close to but distinct from, residues important for interactions with certain transcription activators. We used NMR spectroscopy to determine the secondary structure of alpha CTD, alpha CTD contains a nonstandard helix followed by four alpha-helices. The two regions of alpha CTD important for DNA binding correspond to the first alpha-helix and the loop between the third and fourth alpha-helices. The alpha CTD DNA-binding domain architecture is unlike any DNA-binding architecture identified to date, and we propose that alpha CTD has a novel mode of interaction with DNA. Our results suggest models for alpha CTD-DNA and alpha CTD-DNA-activator interactions during transcription initiation.

Amino Acid Sequence↗
PubMed1996

rRNA transcription and growth rate-dependent regulation of ribosome synthesis in Escherichia coli.

The synthesis of ribosomal RNA is the rate-limiting step in ribosome synthesis in bacteria. There are multiple mechanisms that determine the rate of rRNA synthesis. Ribosomal RNA promoter sequences have evolved for exceptional strength and for regulation in response to nutritional conditions and amino acid availability. Strength derives in part from an extended RNA polymerase (RNAP) recognition region involving at least two RNAP subunits, in part from activation by a transcription factor and in part from modification of the transcript by a system that prevents premature termination. Regulation derives from at least two mechanistically distinct systems, growth rate-dependent control and stringent control. The mechanisms contributing to rRNA transcription work together and compensate for one another when individual systems are rendered inoperative.

DNA, Bacterial↗
PubMed1995

The transcriptional activator protein FIS: DNA interactions and cooperative interactions with RNA polymerase at the Escherichia coli rrnB P1 promoter.

The E. coli rrnB P1 promoter owes its strength, in part, to the transcriptional activator protein FIS. FIS binds to three sites upstream of the RNA polymerase (RNAP) binding site and increases transcription in vivo four to ten-fold. In this report, hydroxyl radical and DMS footprinting analyses show that FIS binds to its three sites along one side of the DNA helix, and that FIS bound at the promoter-proximal site (site I) and RNAP bound at the promoter are in close proximity. The binding of FIS at site I and RNAP at the promoter are mutually cooperative. These observations support a model for direct interaction between the FIS protein bound at site I and RNAP in transcription activation at rrnB P1. We also find that FIS does not bind cooperatively to its three sites upstream of rrnB P1, and that the relatively small activation associated with FIS bound at sites II and III does not result indirectly by facilitation of binding of FIS to site I.

Base Sequence↗
PubMed1995

Depressive symptoms in Alzheimer disease: assessment and determinants.

Depression is difficult to assess in Alzheimer disease (AD) and controversy surrounds the prevalence, etiology, and characteristics of mood alterations in patients with this disorder. We used a variety of standardized instruments to assess mood changes in 33 patients with AD. The frequency of depression ranged from 6 to 30%, depending on the diagnostic criteria employed. No relationship was found between patient depression and dementia severity, self-awareness of cognitive deficits (as measured by a memory self-rating scale), or mood of the caregiver. Delusional patients had higher scores on mood rating scales than nondelusional patients. The results suggest that depression in AD is not severe and is unrelated to patient self-awareness of illness. We hypothesize that the cholinergic deficit of AD may ameliorate depressive symptoms.

Activities of Daily Living↗
PubMed1995

Obtaining consent for organ donation in nine NSW metropolitan hospitals.

Organ donation rates vary markedly around the world. In an attempt to analyse why some patients' families are not asked about organ donation, the case notes of 6080 patients who died over a twelve-month period from April 1991 to March 1992 in nine hospitals in Sydney, NSW, were studied. Irreversible coma occurred in 515 patients. Of these, 177 were considered to be potentially brain dead donors, 126 of whom had a formal diagnosis of brain death. The clinicians caring for the patients at the time of death were asked at follow-up about the reasons for not considering donation, or the reasons for family refusal. Consent to proceed to organ donation was requested in 112 cases (49 donated and 63 refused) and not requested in 65. Analysis of the proportions asked and consenting in each hospital revealed no correlation. Half of the families refusing gave no reason (24/50) while eleven gave religious or cultural reasons, and six expressed a desire to prevent mutilation of the body as their primary reason for not consenting. There was universal failure to gain consent from families when the patient was not in an Intensive Care Unit. Analysis of those patients of whom consent was not sought showed that they died more quickly after admission, were older and died from causes other than trauma or intracranial haemorrhage. The odds of the family being asked dropped by about half as the age increased from one group to the next.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗