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Biomedical subjects

J A Wright

Publications and source records attributed to J A Wright.

At least 19 recordsLinked to original sources
PubMed2001

Statistical models to predict the need for postoperative intensive care and hospitalization in pediatric surgical patients.

OBJECTIVE: To develop statistical models for predicting postoperative hospital and ICU stay in pediatric surgical patients based on preoperative clinical characteristics and operative factors related to the degree of surgical stress. We hypothesized that preoperative and operative factors will predict the need for ICU admission and may be used to forecast the length of ICU stay or postoperative hospital stay. DESIGN: Prospective data collection from 1,763 patients. SETTING: Tertiary care children's hospital. PATIENTS AND PARTICIPANTS: All pediatric surgical patients, including those undergoing day surgery. Patients undergoing dental or ophthalmologic surgical procedures were excluded. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: A logistic regression model predicting ICU admission was developed from all patients. Poissonregression models were developed from 1,161 randomly selected patients and validated from the remaining 602 patients. The logistic regression model for ICU admission was highlypredictive (area under the receiver operating characteristics (ROC) curve = 0.981). In the data set used for development of Poisson regression models, significant correlations occurred between the observed and predicted ICU stay (Pearson r = 0.468, p < 0.0001, n = 131) and between the observed and predicted hospital stay for patients undergoing general (r = 0.695, p < 0.0001), orthopedic (r = 0.717, p < 0.0001), cardiothoracic (r = 0.746, p < 0.0001), urologic (r = 0.458, p < 0.0001), otorhinolaryngologic (r = 0.962, p < 0.0001), neurosurgical (r = 0.7084, p < 0.0001) and plastic surgical (r = 0.854, p < 0.0001) procedures. In the validation data set, correlations between predicted and observed hospital stay were significant for general (p < 0.0001), orthopedic (p < 0.0001), cardiothoracic (p = 0.0321) and urologic surgery (p = 0.0383). The Poisson models for length of ICU stay, otorhinolaryngology, neurosurgery or plastic surgery could not be validated because of small numbers of patients. CONCLUSIONS: Preoperative and operative factors may be used to develop statistical models predicting the need for ICU admission in pediatric surgical patients, and hospital stay following general surgical, orthopedic, cardiothoracic and urologic procedures. These statistical models need to be refined and validatedfurther, perhaps using data collection from multiple institutions.

Arkansas↗
PubMed2001

Standardized method of measuring acanthamoeba antibodies in sera from healthy human subjects.

Acanthamoeba species can cause serious, debilitating, and sometimes life-threatening infections. Three groups have been identified using morphological and immunological comparisons. Previous serological studies have utilized a variety of antigen preparations and assay methods and reported disparate (3 to 100%) results. This study was designed to (i) optimize an enzyme-linked immunosorbent assay for detecting serum antibodies to each of the Acanthamoeba serogroups and (ii) test 55 healthy individuals for specific immunoglobulin G reactivity. The highest signal-to-background ratio was found when 3,000 fixed, intact trophozoites per well were used with a 1:10 serum dilution. Sera yielding optical densities of <0.25 against all three Acanthamoeba serogroups were used to define the cutoff for positive results. The highest background reactivity with these sera was seen with Acanthamoeba polyphaga (serogroup 2), followed by Acanthamoeba culbertsoni (serogroup 3) and Acanthamoeba astronyxis (serogroup 1). Of 55 subjects tested, the highest number of positive results was seen with A. polyphaga (81.8%), followed by A. astronyxis (52.8%) and A. culbertsoni (40%). Seven serum samples (12.7%) were negative for all three Acanthamoeba serogroups, 16 (29.1%) were positive for one serogroup only, 16 were positive for two serogroups, and 16 reacted to all three serogroups. Further analysis showed no significant associations between serogroup reactivity and age or gender. However, some ethnic differences were noted, especially with A. polyphaga antigens. In that case, serum samples from Hispanic subjects were 14.5 times less likely to be positive (P = 0.0025) and had lower mean absorbance values (P = 0.047) than those from Caucasian subjects. Overall, these data suggest that Acanthamoeba colonization or infection is more common than previously thought. Mild or asymptomatic infections may contribute to the observed serum reactivities.

Acanthamoeba↗
PubMed2001

A randomized controlled trial of point-of-care evidence to improve the antibiotic prescribing practices for otitis media in children.

CONTEXT: Prescribing practices for otitis media are not consistent with current evidence-based recommendations. OBJECTIVE: To determine whether point-of-care evidence delivery regarding the use and duration of antibiotics for otitis media decreases the duration of therapy from 10 days and decreases the frequency of prescriptions written. DESIGN: Randomized, controlled trial. SETTING: Primary care pediatric clinic affiliated with university training program. Intervention. A point-of-care evidence-based message system presenting real time evidence to providers based on their prescribing practice for otitis media. MAIN OUTCOME MEASURES: Proportion of prescriptions for otitis media that were for <10 days and frequency with which antibiotics were prescribed. RESULTS: Intervention providers had a 34% greater reduction in the proportion of time they prescribed antibiotics for <10 days. Intervention providers were less likely to prescribe antibiotics than were control providers. CONCLUSIONS: A point-of-care information system integrated into outpatient pediatric care can significantly influence provider behavior for a common condition.

Adult↗
PubMed2001

Isolation of extrachromosomal elements by histone immunoprecipitation.

Here, we describe a gentle and effective method for the rapid and reproducible isolation of histone-bound extrachromosomal DNA molecules called extrachromosomal elements (EEs). This method facilitates the harvest of a specific population of EEs following their isolation from cultured cells, primary tissues, and tumor cells. Active EEs are bound to histone proteins, and these histone-bound EEs carry actively transcribing genes such as c-myc. Our method exploits the presence of histones on EEs and serves as a first-step purification procedure, allowing for the cloning or multivariant analysis of an immunopurified sample of EEs. We isolated EEs from 4-hydroxytamoxifen (4-HT)-activated Myc-ER-regulatable Pre-B ABM cells. Following one round of immunoprecipitation, we demonstrate the purification of histone-bound EEs. We confirmed that our purification enriched for EEs that carry genes by fluorescent in situ hybridization of EEs (FISH-EEs), and we probed non-enriched and immunopurified EEs with a dihydrofolate reductase (DHFR) cDNA probe that is known to detect extrachromosomal amplification in Myc-activated cells. We demonstrate the enrichment of immunoprecipitated DHFR-containing extrachromosomal DNA molecules.

Animals↗
PubMed2000

Acute animal studies of the STARFlex system: a new self-centering cardioSEAL septal occluder.

The STARFlex system is a modified CardioSEAL device with a flexible self-centering mechanism comprised of nitinol springs strung between opposing arms, a connecting ball (sleeve joint that allows the device to pivot prerelease), and a front-loading delivery system. It was designed to allow a smaller device/defect sizing ratio and delivery profile, provide centering capability, and improve closure rates. To test this system, 13 devices (23, 28, and 33 mm) were deployed in six sheep within created atrial septal defects (12- to 22-mm diameter; n = 10), in the left atrium (n = 2), and in inferior vena cava (n = 1). All implantations in atrial septal defects were successful, with device/defect ratio ranging from 1.3 to 1.9 (median, 1.3), with no residual leak by angiography or echocardiography in seven (3/10 had </= small immediate leaks). The STARFlex system was effective in closing created atrial septal defects using a 10 Fr delivery sheath and low device/defect sizing ratios, comparing favorably with the standard CardioSEAL. Cathet. Cardiovasc. Intervent. 49:225-233, 2000.

Animals↗
PubMed2000

Effects of 5-aminoisoquinolinone, a water-soluble, potent inhibitor of the activity of poly (ADP-ribose) polymerase on the organ injury and dysfunction caused by haemorrhagic shock.

Poly (ADP-ribose) synthetase (PARP) is a nuclear enzyme activated by strand breaks in DNA, which are caused inter alia by reactive oxygen species (ROS). Here we report on (i) a new synthesis of a water-soluble and potent PARP inhibitor, 5-aminoisoquinolinone (5-AIQ) and (ii) investigate the effects of 5-AIQ on the circulatory failure and the organ injury/dysfunction caused by haemorrhage and resuscitation in the anaesthetized rat. Exposure of human cardiac myoblasts (Girardi cells) to hydrogen peroxide (H(2)O(2), 3 mM for 1 h, n=9) caused a substantial increase in PARP activity. Pre-treatment of these cells with 5-AIQ (1 microM - 1 mM, 10 min prior to H(2)O(2)) caused a concentration-dependent inhibition of PARP activity (IC(50): approximately 0.01 mM, n=6). Haemorrhage and resuscitation resulted (within 4 h after resuscitation) in a delayed fall in blood pressure (circulatory failure) as well as in rises in the serum levels of (i) urea and creatinine (renal dysfunction), (ii) aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyl-transferase (gamma-GT) (liver injury and dysfunction), (iii) lipase (pancreatic injury) and (iv) creatine kinase (CK) (neuromuscular injury) (n=10). Administration (5 min prior to resuscitation of 5-AIQ) (0.03 mg kg(-1) i.v., n=8, or 0.3 mg kg(-1) i.v., n=10) reduced (in a dose-related fashion) the multiple organ injury and dysfunction, but did not affect the circulatory failure, associated with haemorrhagic shock. Thus, 5-AIQ abolishes the multiple organ injury caused by severe haemorrhage and resuscitation.

Alanine Transaminase↗
PubMed2000

The association between greater continuity of care and timely measles-mumps-rubella vaccination.

OBJECTIVES: This study assessed whether greater continuity of care is associated with timely administration of measles-mumps-rubella (MMR) vaccination. METHODS: We studied 11,233 patients continuously enrolled in Group Health Cooperative (GHC) from birth to 15 months. We used a preestablished index to quantify continuity of care based on the number of primary care providers in relation to the number of clinic visits. MMR vaccination status at 15 months was assessed with automated immunization data systems at GHC. RESULTS: In a logistic regression model, both medium continuity (odds ratio [OR] = 1.20, 95% confidence interval [CI] = 1.08, 1.33) and high continuity (OR = 1.36, 95% CI = 1.22, 1.52) were associated with increased likelihood of being immunized by 15 months compared with patients in the lowest tercile of continuity of care. CONCLUSION: Greater continuity of care is associated with more timely immunization.

Child Health Services↗
PubMed2000

A trade-off analysis of routine newborn circumcision.

BACKGROUND: The risks associated with newborn circumcision have not been as extensively evaluated as the benefits. OBJECTIVES: The goals of this study were threefold: 1) to derive a population-based complication rate for newborn circumcision; 2) to calculate the number needed to harm for newborn circumcision based on this rate; and 3) to establish trade-offs based on our complication rates and published estimates of the benefits of circumcision including the prevention of urinary tract infections and penile cancer. METHODS: Using the Comprehensive Hospital Abstract Reporting System for Washington State, we retrospectively examined routine newborn circumcisions performed over 9 years (1987-1996). We used International Classification of Diseases, Ninth Revision codes to identify both circumcisions and complications and limited our analyses to children without other surgical procedures performed during their initial birth hospitalization. RESULTS: Of 354, 297 male infants born during the study period, 130,475 (37%) were circumcised during their newborn stay. Overall 287 (.2%) of circumcised children and 33 (.01%) of uncircumcised children had complications potentially associated with circumcision coded as a discharge diagnosis. Based on our findings, a complication can be expected in 1 out every 476 circumcisions. Six urinary tract infections can be prevented for every complication endured and almost 2 complications can be expected for every case of penile cancer prevented. CONCLUSIONS: Circumcision remains a relatively safe procedure. However, for some parents, the risks we report may outweigh the potential benefits. This information may help parents seeking guidance to make an informed decision.

Circumcision, Male↗
PubMed1999

The ribonucleotide reductase R2 gene is a non-transcribed target of c-Myc-induced genomic instability.

The c-Myc oncoprotein is highly expressed in malignant cells of many cell types, but the mechanism by which it contributes to the transformation process is not fully understood. Here, we show for the first time that constitutive or activated overexpression of the c-myc gene in cultured mouse B lymphocytes is followed by chromosomal and extrachromosomal amplification as well as rearrangement of the ribonucleotide reductase R2 gene locus. Electron micrographs and fluorescent in situ hybridization (FISH) demonstrate the c-Myc-dependent generation of extrachromosomal elements, some of which contain R2 sequences. However, unlike other genes that have been shown to be targets of c-Myc-dependent genomic instability, amplification of the R2 gene is not associated with alterations in R2 mRNA or protein expression. These data suggest that c-Myc-dependent genomic instability involves a greater number of genes than previously anticipated, but not all of the genes that are amplified in this system are transcriptionally upregulated.

Animals↗
PubMed1999

Increased Ser-10 phosphorylation of histone H3 in mitogen-stimulated and oncogene-transformed mouse fibroblasts.

When the Ras mitogen-activated protein kinase (MAPK) signaling pathway of quiescent cells is stimulated with growth factors or phorbol esters, the early response genes c-fos and c-myc are rapidly induced, and concurrently there is a rapid phosphorylation of histone H3. Using an antibody specific for phosphorylated Ser-10 of H3, we show that Ser-10 of H3 is phosphorylated, and we provide direct evidence that phosphorylated H3 is associated with c-fos and c-myc genes in stimulated cells. H3 phosphorylation may contribute to proto-oncogene induction by modulating chromatin structure and releasing blocks in elongation. Previously we reported that persistent stimulation of the Ras-MAPK signaling pathway in oncogene-transformed cells resulted in increased amounts of phosphorylated histone H1. Here we show that phosphorylated H3 is elevated in the oncogene-transformed mouse fibroblasts. Further we show that induction of ras expression results in a rapid increase in H3 phosphorylation. H3 phosphatase, identified as PP1, activities in ras-transformed and parental fibroblast cells were similar, suggesting that elevated H3 kinase activity was responsible for the increased level of phosphorylated H3 in the oncogene-transformed cells. Elevated levels of phosphorylated H1 and H3 may be responsible for the less condensed chromatin structure and aberrant gene expression observed in the oncogene-transformed cells.

3T3 Cells↗
PubMed1999

Ribonucleotide reductase R2 protein is phosphorylated at serine-20 by P34cdc2 kinase.

Ribonucleotide reductase is a rate-limiting enzyme in DNA synthesis and is composed of two different proteins, R1 and R2. The R2 protein appears to be rate-limiting for enzyme activity in proliferating cells, and it is phosphorylated by p34cdc2 and CDK2, mediators of cell cycle transition events. A sequence in the R2 protein at serine-20 matches a consensus sequence for p34cdc2 and CDK2 kinases. We tested the hypothesis that the serine-20 residue was the major p34cdc2 kinase site of phosphorylation. Three peptides were synthesized (from Asp-13 to Ala-28) that contained either the wild type amino acid sequence (Asp-Gln-Gln-Gln-Leu-Gln-Leu-Ser-Pro-Leu-Lys-Arg-Leu-Thr-Leu-Ala, serine peptide) or a mutation, in which the serine residue was replaced with an alanine residue (alanine peptide) or a threonine residue (threonine peptide). Only the serine peptide and threonine peptide were phosphorylated by p34cdc2 kinase. In two-dimensional phosphopeptide mapping experiments of serine peptide and Asp-N endoproteinase digested R2 protein, peptide co-migration patterns suggested that the synthetic phosphopeptide containing serine-20 was identical to the major Asp-N digested R2 phosphopeptide. To further test the hypothesis that serine-20 is the primary phosphorylated residue on R2 protein, three recombinant R2 proteins (R2-Thr, R2-Asp and R2-Ala) were generated by site-directed mutagenesis, in which the serine-20 residue was replaced with threonine, aspartic acid or alanine residues. Wild type R2 and threonine-substituted R2 proteins (R2-Thr) were phosphorylated by p34cdc2 kinase, whereas under the same experimental conditions, R2-Asp and R2-Ala phosphorylation was not detected. Furthermore, the phosphorylated amino acid residue in the R2-Thr protein was determined to be phosphothreonine. Therefore, by replacing a serine-20 residue with a threonine, the phosphorylated amino acid in R2 protein was changed to a phosphothreonine. In total, these results firmly establish that a major p34cdc2 phosphorylation site on the ribonucleotide reductase R2 protein occurs near the N-terminal end at serine-20, which is found within the sequence Ser-Pro-Leu-Lys-Arg-Leu. Comparison of ribonucleotide reductase activities between wild type and mutated forms of the R2 proteins suggested that mutation at serine-20 did not significantly affect enzyme activity.

Amino Acid Sequence↗
PubMed1999

Microstructures for studies of cultured neural networks.

A description is given of a functional silicon micromachined device that permits non-invasive, bidirectional, highly specific communication with cultured mammalian neurons. The heart of the system is a well structure that holds the cell in close proximity to a metal extracellular electrode while permitting normal outgrowth of axons and dendrites. An iterative approach is used to create a design that allows normal growth of the neurons while preventing their escape. An array of 16 such neurowells makes it possible to perform studies of biological neural network development and function with unprecedented detail.

Animals↗
PubMed1999

fas-mediated lysis of chronic lymphocytic leukaemia cells: role of type I versus type II cytokines and autologous fasL-expressing T cells.

Given the known role of the fas cytolytic pathway in B-cell regulation, we evaluated whether fas-fasL interactions might induce chronic lymphocytic leukaemia (CLL) cell death. De novo CLL cells expressed a low level of surface fas, and were not lysed by fasL-bearing cells. CLL cells cultured in media containing the type I cytokines interleukin (IL)-12 or interferon (IFN)-alpha had increased fas expression, and were readily lysed by fasL-bearing cells. In contrast, the type II cytokine IL-4 did not increase CLL cell fas, and abrogated type I cytokine-induced fas up-regulation. With prolonged culture, IL-4 exposed CLL cells expressed an intermediate level of fas; however, such CLL cells were resistant to fas-mediated lysis. These results indicate that IL-4 inhibits fas-mediated killing of CLL cells at the level of both fas receptor expression and post-receptor events. Additionally, we have defined in vitro culture conditions which generate fasL-bearing T cells from CLL patients; such T cells efficiently mediated fas-based lysis of autologous fas-positive CLL cells. We therefore conclude that type I and type II cytokines differentially regulate the fas pathway in CLL cells, and that a combination of type I cytokines and fasL-expressing T cells may represent a new approach to the immunotherapy of CLL.

Apoptosis↗
PubMed1999

Is greater continuity of care associated with less emergency department utilization?

BACKGROUND: The benefits of continuity of care (COC) have not been firmly established for pediatric patients. OBJECTIVE: To assess whether greater COC is associated with lower emergency department (ED) utilization. SETTING: Outpatient teaching clinic at Children's Hospital and Regional Medical Center, Seattle, WA. PATIENTS: All 785 Medicaid managed care children ages 0 to 19 years followed at Children's Hospital and Regional Medical Center between 1993 to 1997 who had at least four outpatient visits. METHODS: Retrospective claims-based analysis. COC was quantified based on the number of different care providers in relation to the number of clinic visits. RESULTS: Attending COC was significantly greater than resident COC. In a multiple event survival analysis, compared with those patients in the lowest tertile of attending COC, those in the middle tertile had 30% lower ED utilization (hazard ratio 0.70 [0.53-0.93]) and those in the highest tertile had 35% lower ED use (hazard ratio 0.65 [0.50-0.80]). Resident COC was not significantly associated with ED use. CONCLUSION: Greater COC with attending physicians in outpatient teaching clinics is associated with lower ED utilization.

Adolescent↗
PubMed1999

Organization of chromatin in cancer cells: role of signalling pathways.

The role of mechanical and chemical signalling pathways in the organization and function of chromatin is the subject of this review. The mechanical signalling pathway consists of the tissue matrix system that links together the three-dimensional skeletal networks, the extracellular matrix, cytoskeleton, and nuclear matrix. Intermediate filament proteins are associated with nuclear DNA, suggesting that intermediate filaments may have a role in the organization of chromatin. In human hormone-dependent breast cancer cells, the interaction between cytokeratins and chromatin is regulated by estrogens. Transcription factors, histone acetyltransferases, and histone deacetylases, which are associated with the nuclear matrix, are components of the mechanical signalling pathway. Recently, we reported that nuclear matrix-bound human and chicken histone deacetylase 1 is associated with nuclear DNA in situ, suggesting that histone deacetylase has a role in the organization of nuclear DNA. Chemical signalling pathways such as the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway stimulate the activity of kinases that modify transcription factors, nonhistone chromosomal proteins, and histones. The levels of phosphorylated histones are increased in mouse fibroblasts transformed with oncogenes, the products of which stimulate the Ras/MAPK pathway. Histone phosphorylation may lead to decondensation of chromatin, resulting in aberrant gene expression.

Amino Acid Sequence↗
PubMed1998

Ras-associated nuclear structural change appears functionally significant and independent of the mitotic signaling pathway.

An altered nuclear morphology has been previously noted in association with Ras activation, but little is known about the structural basis, functional significance, signaling pathway, or reproducibility of any such change. We first tested the reproducibility of Ras-associated nuclear change in a series of rodent fibroblast cell lines. After independently developing criteria for recognizing Ras-associated nuclear change in a Papanicolaou stained test cell line with an inducible H(T24)-Ras oncogene, two cytopathologists blindly and independently assessed 17 other cell lines. If the cell lines showed Ras-associated nuclear change, a rank order of increasing nuclear change was independently scored. Ras-associated nuclear changes were identified in v-Fes, v-Src, v-Mos, v-Raf, and five of five H(T24)-Ras transfectants consisting of a change from a flattened, occasionally undulating nuclear shape to a more rigid spherical shape and a change from a finely textured to a coarse heterochromatic appearance. Absent or minimal changes were scored in six control cell lines. The two cytopathologists' independent morphologic rank orders were similar (P < .0002). The mitogen signaling pathway per se does not appear to transduce the change since no morphologic alterations were identified in cell lines with activations of downstream components of this pathway--MAPKK or c-Myc--and the rank orders did not correlate with markers of mitotic rate (P > .11). The rank order correlated closely with metastatic potential (P < .0014 and P < .0003) but not with histone H1 composition or global nuclease sensitivity. Based on published studies of five of the cell lines, there may be a correlation between increases in certain nuclear matrix proteins and the Ras-associated nuclear change.

3T3 Cells↗
PubMed1998

Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control.

In fission yeast, the rad3 gene product plays a critical role in sensing DNA structure defects and activating damage response pathways. A structural homologue of rad3 in humans (ATR) has been identified based on sequence similarity in the protein kinase domain. General information regarding ATR expression, protein kinase activity, and cellular localization is known, but its function in human cells remains undetermined. In the current study, the ATR protein was examined by gel filtration of protein extracts and was found to exist predominantly as part of a large protein complex. A kinase-inactivated form of the ATR gene was prepared by site-directed mutagenesis and was used in transfection experiments to probe the function of this complex. Introduction of this kinase-dead ATR into a normal fibroblast cell line, an ATM-deficient fibroblast line derived from a patient with ataxia-telangiectasia, or a p53 mutant cell line all resulted in significant losses in cell viability. Clones expressing the kinase-dead ATR displayed increased sensitivity to x-rays and UV and a loss of checkpoint control. We conclude that ATR functions as a critical part of a protein complex that mediates responses to ionizing and UV radiation in human cells. These responses include effects on cell viability and cell cycle checkpoint control.

Ataxia Telangiectasia Mutated Proteins↗