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Biomedical subjects

R A Edwards

Publications and source records attributed to R A Edwards.

At least 19 recordsLinked to original sources
PubMed2002

Impaired apoptosis, extended duration of immune responses, and a lupus-like autoimmune disease in IEX-1-transgenic mice.

Susceptibility of activated T cells to apoptosis must be tightly regulated to ensure sufficient T cell progeny for an effective response, while allowing a rapid depletion of them at the end of the immune response. We show here that a previously isolated, NF-kappa B/rel target gene IEX-1 (Immediate Early response gene X-1) is highly expressed in T cells at early stages of activation, but declines with a prolonged period of activation time, coincident with an increased susceptibility of T cells to apoptosis during the late phases of an immune response. Transgenic expression of IEX-1 specifically in lymphocytes impaired apoptosis in activated T cells, extended a duration of an effector-phase of a specific immune response, and increased the accumulation of effector/memory-like T cells and the susceptibility to a lupus-like autoimmune disease. Our study demonstrated an antiapoptotic effect of IEX-1 on T cell apoptosis triggered by ligation of Fas and T cell receptor (TCR)/CD3 complex. The ability of extending life expectancy of T effectors, in line with a decrease in its expression following prolonged T cell activation, suggests a key role for IEX-1 in regulating T cell homeostasis during immune responses.

Animals↗
PubMed2001

Phenotypic consequences of lung-specific inducible expression of FGF-3.

Members of the fibroblast growth factor (FGF) family play a critical role in embryonic lung development and adult lung physiology. The in vivo investigation of the role FGFs play in the adult lung has been hampered because the constitutive pulmonary expression of these factors often has deleterious effects and frequently results in neonatal lethality. To circumvent these shortcomings, we expressed FGF-3 in the lungs under the control of the progesterone antagonist-responsive binary transgenic system. Four binary transgenic lines were obtained that showed ligand-dependent induction of FGF-3 with induced levels of FGF-3 expression dependent on the levels of expression of the GLp65 regulator as well as the dose of the progesterone antagonist, RU486, administered. FGF-3 expression in the adult mouse lung resulted in two phenotypes depending on the levels of induction of FGF-3. Low levels of FGF-3 expression resulted in massive free alveolar macrophage infiltration. High levels of FGF-3 expression resulted in diffuse alveolar type II cell hyperplasia. Both phenotypes were reversible after the withdrawal of RU486. This system will be a valuable means of investigating the diverse roles of FGFs in the adult lung.

Animals↗
PubMed2001

Removing a hydrogen bond in the dimer interface of Escherichia coli manganese superoxide dismutase alters structure and reactivity.

Among manganese superoxide dismutases, residues His30 and Tyr174 are highly conserved, forming part of the substrate access funnel in the active site. These two residues are structurally linked by a strong hydrogen bond between His30 NE2 from one subunit and Tyr174 OH from the other subunit of the dimer, forming an important element that bridges the dimer interface. Mutation of either His30 or Tyr174 in Escherichia coli MnSOD reduces the superoxide dismutase activity to 30--40% of that of the wt enzyme, which is surprising, since Y174 is quite remote from the active site metal center. The 2.2 A resolution X-ray structure of H30A-MnSOD shows that removing the Tyr174-->His30 hydrogen bond from the acceptor side results in a significant displacement of the main-chain segment containing the Y174 residue, with local rearrangement of the protein. The 1.35 A resolution structure of Y174F-MnSOD shows that disruption of the same hydrogen bond from the donor side has much greater consequences, with reorientation of F174 having a domino effect on the neighboring residues, resulting in a major rearrangement of the dimer interface and flipping of the His30 ring. Spectroscopic studies on H30A, H30N, and Y174F mutants show that (like the previously characterized Y34F mutant of E. coli MnSOD) all lack the high pH transition of the wt enzyme. This observation supports assignment of the pH sensitivity of MnSOD to coordination of hydroxide ion at high pH rather than to ionization of the phenolic group of Y34. Thus, mutations near the active site, as in the Y34F mutant, as well as at remote positions, as in Y174F, similarly affect the metal reactivity and alter the effective pK(a) for hydroxide ion binding. These results imply that hydrogen bonding of the H30 imidazole N--H group plays a key role in substrate binding and catalysis.

Amino Acid Substitution↗
PubMed2001

Outer sphere mutations perturb metal reactivity in manganese superoxide dismutase.

Tyrosine 34 and glutamine 146 are highly conserved outer sphere residues in the mononuclear manganese active site of Escherichia coli manganese superoxide dismutase. Biochemical and spectroscopic characterization of site-directed mutants has allowed functional characterization of these residues in the wild-type (wt) enzyme. X-ray crystallographic analysis of three mutants (Y34F, Q146L, and Q146H) reveal subtle changes in the protein structures. The Y34A mutant, as well as the previously reported Y34F mutant, retained essentially the full superoxide dismutase activity of the wild-type enzyme, and the X-ray crystal structure of Y34F manganese superoxide dismutase shows that mutation of this strictly conserved residue has only minor effects on the positions of active site residues and the organized water in the substrate access funnel. Mutation of the outer sphere solvent pocket residue Q146 has more dramatic effects. The Q146E mutant is isolated as an apoprotein lacking dismutase activity. Q146L and Q146H mutants retain only 5-10% of the dismutase activity of the wild-type enzyme. The absorption and circular dichroism spectra of the Q146H mutant resemble corresponding data for the superoxide dismutase from a hyperthermophilic archaeon, Pyrobaculum aerophilum, which is active in both Mn and Fe forms. Interestingly, the iron-substituted Q146H protein also exhibits low dismutase activity, which increases at lower pH. Mutation of glutamine 146 disrupts the hydrogen-bonding network in the active site and has a greater effect on protein structure than does the Y34F mutant, with rearrangement of the tyrosine 34 and tryptophan 128 side chains.

Binding Sites↗
PubMed2001

Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13.

The ubiquitin conjugating enzyme complex Mms2-Ubc13 plays a key role in post-replicative DNA repair in yeast and the NF-kappaB signal transduction pathway in humans. This complex assembles novel polyubiquitin chains onto yet uncharacterized protein targets. Here we report the crystal structure of a complex between hMms2 (Uev1) and hUbc13 at 1.85 A resolution and a structure of free hMms2 at 1.9 A resolution. These structures reveal that the hMms2 monomer undergoes a localized conformational change upon interaction with hUbc13. The nature of the interface provides a physical basis for the preference of Mms2 for Ubc13 as a partner over a variety of other structurally similar ubiquitin-conjugating enzymes. The structure of the hMms2-hUbc13 complex provides the conceptual foundation for understanding the mechanism of Lys 63 multiubiquitin chain assembly and for its interactions with the RING finger proteins Rad5 and Traf6.

Adenosine Triphosphatases↗
PubMed2001

Inside or outside: detecting the cellular location of bacterial pathogens.

Salmonella are intracellular pathogens that infect and multiply inside macrophages. Although Salmonella are some of the best-studied pathogens, it is difficult to determine quickly and reliably whether the bacteria are intracellular or extracellular. We have developed a novel method using differential fluorescence of two fluorescent proteins to determine the cellular location of pathogenic bacteria in macrophage infection assays. Using the differential expression of two unique fluorescent proteins that are expressed under specific conditions, we have developed a real-time assay for macrophage infections. The critical advantages of this system are that it does not alter the bacterial surface, it is not toxic to either the bacteria or the host cell, and it may be used in real-time quantitative assays. This assay can be readily applied to any other model pathogenic systems such as Listeria, Mycobacteria, and Legionella in which intracellular gene expression has been characterized.

Animals↗
PubMed2000

A role for Salmonella fimbriae in intraperitoneal infections.

Enteric bacteria possess multiple fimbriae, many of which play critical roles in attachment to epithelial cell surfaces. SEF14 fimbriae are only found in Salmonella enterica serovar Enteritidis (S. enteritidis) and closely related serovars, suggesting that SEF14 fimbriae may affect serovar-specific virulence traits. Despite evidence that SEF14 fimbriae are expressed by S. enteritidis in vivo, previous studies showed that SEF14 fimbriae do not mediate adhesion to the intestinal epithelium. Therefore, we tested whether SEF14 fimbriae are required for virulence at a stage in infection after the bacteria have passed the intestinal barrier. Polar mutations that disrupt the entire sef operon decreased virulence in mice more than 1,000-fold. Nonpolar mutations that disrupted sefA (encoding the major structural subunit) did not affect virulence, but mutations that disrupted sefD (encoding the putative adhesion subunit) resulted in a severe virulence defect. The results indicate that the putative SEF14 adhesion subunit is specifically required for a stage of the infection subsequent to transit across the intestinal barrier. Therefore, we tested whether SefD is required for uptake or survival in macrophages. The majority of wild-type bacteria were detected inside macrophages soon after i.p. infection, but the sefD mutants were not readily internalized by peritoneal macrophages. These results indicate that the potential SEF14 adhesion subunit is essential for efficient uptake or survival of S. enteritidis in macrophages. This report describes a role of fimbriae in intracellular infection, and indicates that fimbriae may be required for systemic infections at stages beyond the initial colonization of host epithelial surfaces.

Animals↗
PubMed2000

Unilocular retroperitoneal cyst of mesothelial origin presenting as a renal mass.

We report the first 2 cases, to our knowledge, of retroperitoneal cysts with features of mesothelial differentiation that clinically mimic renal masses. The first lesion occurred in a 71-year-old man who presented with flank pain. Ultrasound and magnetic resonance imaging studies showed a unilocular cystic structure arising from the upper pole of the left kidney. The second lesion was in a 44-year-old woman who presented with left flank pain. Imaging studies revealed an 8-cm hemorrhagic cyst at the lower pole of the left kidney. Histologic examination of the nephrectomy specimens in each case revealed a unilocular cyst with intracystic and pericystic hemorrhage. In each case, the cyst was lined by a single layer of cells with ample eosinophilic cytoplasm and benign nuclear features without mucinous or müllerian differentiation. Histochemical staining showed Alcian blue positivity on the cell surface, which was sensitive to hyaluronidase digestion. Intracytoplasmic mucin, however, was not detected. Immunostaining showed that the cyst lining cells were positive for keratin, vimentin, HBME-1, WT1, and thrombomodulin but negative for carcinoembryonic antigen, B72.3, Leu-M1, and BerEP4. The first case was positive for calretinin, whereas the second was negative. These findings support the mesothelial nature of the cysts.

Adult↗
PubMed1999

Increasing DNA transfer efficiency by temporary inactivation of host restriction.

E. coli and Salmonella typhimurium are widely used bacterial hosts for genetic manipulation of DNA from prokaryotes and eukaryotes. Introduction of foreign DNA by electroporation or transduction into E. coli and Salmonella is limited by host restriction of incoming DNA by the recipient cells. Here, we describe a simple method that temporarily inactivates host restriction, allowing high-frequency DNA transfer. This technique might be readily applied to a wide range of bacteria to increase DNA transfer between strains and species.

Bacteriophage P22↗
PubMed1998

Activation of calcineurin and smooth muscle myosin light chain kinase by Met-to-Leu mutants of calmodulin.

The effects of replacement of each of the individual Met in calmodulin (CaM) with Leu on the activation of two CaM target enzymes [smooth muscle myosin light chain kinase (smMLCK) and calcineurin (CN)] were investigated. The KD and Pmax (percentage maximal activation) values for activation of both enzymes by M76L-CaM were indistinguishable from wild-type (wt)-CaM, which is consistent with the location of Met-76 in the central linker that is not involved in target protein interaction. The other eight Met in CaM are exposed in the hydrophobic surfaces that are involved in target-enzymes binding, and in general equivalent effects are observed for substitutions of Leu for Met residues in homologous positions in the two CaM domains. However, the importance of the interaction of specific Met residues with the target enzyme depends on the particular enzyme. Leu substitution at Met-36 or Met-109 reduced the affinity of MLCK for the mutant and the maximal activation of CN. MLCK had a higher KD for M51L-CaM whereas M124L-CaM activated the kinase to only 68% of maximal activity induced by wt-CaM; these mutants were indistinguishable from wt-CaM in activation of CN. M71L- and M144L-CaMs behaved like wt-CaM in activation of MLCK, but activated the phosphatase to only about 80% of maximal activity induced by wt-CAM. M72L-CaM exhibited an increased affinity for MLCK compared to wt-CaM and slightly decreased maximal activation, whereas M145L-CaM exhibited maximal activation significantly greater than that due to wt-CaM; these mutants behaved like wt-CaM with respect to CN activation. Finally, a mutant CaM in which all four C-terminal Met were replaced by Leu (M4-CT-L4-CaM) had similar affinities for MLCK and CN as wt-CaM but maximal activation of these enzymes by this mutant was only 60-70% of that achieved with wt-CaM. These results imply that, in addition to removing the autoinhibitory domain from the active site of the target enzyme, CaM must induce a conformational change in the active site itself.

Animals↗
PubMed1998

Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression.

A series of vectors has been developed to provide improved positive and negative selection for allelic exchange. Based on homologous regions of DNA ranging in size from less than 200 bp to over 1 kb, we have successfully used these new plasmids to introduce or remove markers in chromosomal or plasmid DNA. Wild type fimbria genes were replaced both in Salmonella enteritidis (sefA, agfA and fimC) and Escherichia coli (fasA and fasH). Regulation of 987P fimbriation could be identified after replacement of fasA and fasH with allelic reporter fusions. The expression of fasA but not fasH is dependent upon the osmolarity of the growth medium in an HNS-dependent manner, but unlike some other fimbrial systems expression is not dependent on the exogenous iron concentration.

Adhesins, Escherichia coli↗
PubMed1998

Hospital admissions through the emergency department: does insurance status matter?

PURPOSE: To assess the effect of insurance status on the probability of admission and subsequent health status of patients presenting to emergency departments. SUBJECTS AND METHODS: We performed a prospective cohort study of patients with common medical problems at five urban, academic hospital emergency departments in Boston and Cambridge, Massachusetts. The outcome measure for the study was admission to the hospital from the emergency department and functional health status at baseline and follow-up. RESULTS: During a 1-month period, 2,562 patients younger than 65 years of age presented with either abdominal pain (52%), chest pain (19%) or shortness of breath (29%). Of the 1,368 patients eligible for questionnaire, 1,162 (85%) completed baseline questionnaires, and of these, 964 (83%) completed telephone follow-up interviews 10 days later. Fifteen percent of patients were uninsured and 34% were admitted to the hospital from the emergency department. Uninsured patients were significantly less likely than insured patients to be admitted, both when adjusting for urgency, chief complaint, age, gender and hospital (odds ratio = 0.5, 95% confidence interval 0.3 to 0.7), and when additionally adjusting for comorbid conditions, lack of a regular physician, income, employment status, education and race (odds ratio = 0.4, 95% confidence interval 0.2 to 0.8). However, there were no differences in adjusted functional health status between admitted and nonadmitted patients by insurance status, either at baseline or at 10-day follow-up. CONCLUSIONS: Uninsured patients with one of three common chief complaints appear to be less frequently admitted to the hospital than are insured patients, although health status does not appear to be affected. Whether these results reflect underutilization among uninsured patients or overutilization among insured patients remains to be determined.

Abdominal Pain↗
PubMed1998

Telemedicine in vascular surgery: feasibility of digital imaging for remote management of wounds.

PURPOSE: Telemedicine coupled with digital photography could potentially improve the quality of outpatient wound care and decrease medical cost by allowing home care nurses to electronically transmit images of patients' wounds to treating surgeons. To determine the feasibility of this technology, we compared bedside wound examination by onsite surgeons with viewing digital images of wounds by remote surgeons. METHODS: Over 6 weeks, 38 wounds in 24 inpatients were photographed with a Kodak DC50 digital camera (resolution 756 x 504 pixels/in2). Agreements regarding wound description (edema, erythema, cellulitis, necrosis, gangrene, ischemia, and granulation) and wound management (presence of healing problems, need for emergent evaluation, need for antibiotics, and need for hospitalization) were calculated among onsite surgeons and between onsite and remote surgeons. Sensitivity and specificity of remote wound diagnosis compared with bedside examination were calculated. Potential correlates of agreement, level of surgical training, certainty of diagnosis, and wound type were evaluated by multivariate analysis. RESULTS: Agreement between onsite and remote surgeons (66% to 95% for wound description and 64% to 95% for wound management) matched agreement among onsite surgeons (64% to 85% for wound description and 63% to 91% for wound management). Moreover, when onsite agreement was low (i.e., 64% for erythema) agreement between onsite and remote surgeons was similarly low (i.e., 66% for erythema). Sensitivity of remote diagnosis ranged from 78% (gangrene) to 98% (presence of wound healing problem), whereas specificity ranged from 27% (erythema) to 100% (ischemia). Agreement was influenced by wound type (p < 0.01) but not by certainty of diagnosis (p > 0.01) or level of surgical training (p > 0.01). CONCLUSIONS: Wound evaluation on the basis of viewing digital images is comparable with standard wound examination and renders similar diagnoses and treatment in the majority of cases. Digital imaging for remote wound management is feasible and holds significant promise for improving outpatient vascular wound care.

Amputation, Surgical↗
PubMed1998

Fimbrial expression in enteric bacteria: a critical step in intestinal pathogenesis.

The ability of species of enteric bacteria to recognize and colonize unique niches along the intestine is mainly based on receptor distribution and interpretation of a combination of environmental signals leading to the expression of specific adherence factors. Such elaborate orchestration of events is critical during the initial steps of pathogenesis.

Animals↗
PubMed1998

A method and apparatus for monitoring the level of tritiated water vapor in air using a solid scintillator.

Tritium is presently used in large quantities in laboratories for technological studies, as fuel for the process of nuclear fusion, and in the manufacture of radio-luminescent items. It is also produced in fission nuclear plants (with a yield of 10(-4) if the fuel is 235U and twice as much if the fuel is 239Pu), particularly in those which use D2O as moderator. The weak energy of beta particles (Emean=5.7 keV) produced by tritium implies that it is only harmful if internalized. In nature tritium in air is mainly present under two different chemical forms: elementary (HT, DT, T2) and oxidized (HTO, DTO, T2O). The latter is extremely more dangerous than the former as far as radioprotection is concerned; for this reason the derived air concentration limit (8 x 10(5) Bq m(-3)) for the gaseous form is 25,000 times higher than the value for the oxidized one. The purpose of the work presented here is to realize an area monitor that is able to discriminate in real time between the two chemical forms. Using the properties of zeolite as an absorber and scintillator, it was possible to construct such a detector. In 1 h the instrument can reveal HTO concentrations 40 times below the derived air concentration. A concentration equal to the derived air concentration can be revealed within the first minute of counting and the performance may be further improved. Moreover, the prototype realized is able to work automatically and continuously for 5 h. The capability of discriminating the oxidized chemical form, the sensitivity, and the possibility of obtaining real time information make this instrument a good monitor in those cases where there is a real risk of tritium air contamination.

Air Pollutants, Radioactive↗
PubMed1998

Alpha-periodicity analysis of small multidrug resistance (SMR) efflux transporters.

Proteins in the small multidrug resistance (SMR) family of transport proteins are about 110 amino acids in length and are predicted to have four transmembrane helices. This family is divided into a two groups, one of which we have referred to as small multidrug pumps (Smp) and confer resistance to a wide variety of quaternary ammonium compounds through a proton-drug efflux antiport mechanism. Members of the second group within this family have, as yet, not had their substrate profile characterized and are referred to as Sug proteins. Alpha-periodicity analysis was conducted on a set of six homologous proteins of the SMR family consisting of three established Smp and three Sug proteins. Several amino acid properties were used in the analysis including hydropathy, variability, and a substitution matrix for lipid exposed amino acids. The scanning window was varied between 8 and 14 residues and the alpha-periodicity was calculated from the peaks in the Fourier transform power spectra in the region between 3.0 and 4.3 residues/turn. This analysis adds to the hydropathy analysis to give a more confident prediction of which residues are within the lipid bilayer for each of the four transmembrane helices. Information was also obtained that allowed for the identification of zones within each transmembrane helix that face the interior of the helical bundle on one side and are lipid exposed on the other face.

Amino Acid Sequence↗
PubMed1997

Patient-specific predictors of ambulance use.

STUDY OBJECTIVE: To determine patient-specific socioeconomic and health status characteristics for patients arriving by ambulance at an emergency department. METHODS: Ambulance use among adult ED patients presenting with abdominal pain, chest pain, head trauma, or shortness of breath was studied at five urban teaching hospitals in the north-eastern United States. Cross-sectional analysis within a prospective cohort study of 4,979 consecutive patients was performed using an interval sequence subset of 2,315 patients (84% of those eligible) to whom questionnaires were administered. Ambulance use (21% of surveyed patients; 26% of all patients) was analyzed with logistic regression. RESULTS: Predictors of ambulance use included age greater than 65 years (odds ratio [OR], 1.95; 95% confidence interval [CI], 1.34 to 2.82); clinical severity (OR, 3.11; 95% CI, 2.27 to 4.25); poverty (OR, 1.40; 95% CI, 1.08 to 1.83); physical function (OR, 1.05; 95% CI, 1.02 to 1.09 for each point of worsening function on a 12-point physical function scale); and various types of health insurance coverage. Race, sex, education, Medicaid coverage, frequency of ED use, living arrangements, and primary physician availability were not predictive in multivariate analysis of surveyed patients. CONCLUSION: Ambulance use varies by age, clinical severity, income, patient-specific characteristics of physical function, and type of health insurance. Medicaid coverage and frequent ED use are not predictive of increased ambulance use.

Acute Disease↗