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J A Wright

Publications and source records attributed to J A Wright.

At least 55 records · Page 3Linked to original sources
PubMed1997

Meeting the challenge of emerging pathogens: the role of the United States Air Force in global influenza surveillance.

Influenza virus is one of the most ubiquitous organisms on the planet, causing illness in much of the population each year. The dynamic nature of the influenza virus requires similarly dynamic surveillance and prevention initiatives. The efforts of national surveillance programs, overseen by the World Health Organization and administered by institutions such as the U.S. Centers for Disease Control and Prevention, the U.S. armed forces, and 60 to 70 collaborating laboratories, annually culminate in the development of effective influenza vaccines. The U.S. Air Force's contribution is via Project Gargle, through which bases in various locations worldwide conduct active surveillance and submit throat swab specimens for virus isolation and characterization; the results of these laboratory analyses help determine the composition of the following year's influenza vaccine. These collaborative efforts have resulted in an identical or close antigenic match between vaccine and epidemic strains in 8 of the last 9 influenza seasons.

Centers for Disease Control and Prevention, U.S.↗
PubMed1996

Relative levels of inhibition of p24 gene expression by different 20-mer antisense oligonucleotide sequences targeting nucleotides + 1129 to + 1268 of the HIV-1 gag genome: an analysis of mechanism.

GPI2A is a 20-mer antisense oligonucleotide sequence that is complementary to a region of the HIV-1 gag gene. An analysis of viral core antigen p24 protein synthesis inhibition was performed with cells expressing HIV-1 proteins, following treatment with GPI2A or eight other unique antisense constructs designed to bind to regions of the gag gene, at positions that 5' or 3' flank the GPI2A target site. GPI2A was found to be the most effective construct, indicating that the GPI2A target region is a particularly sensitive site for antisense activity. An analysis of energy-related parameters important in complementary duplex formation was performed for each antisense construct. Also, the potential of each antisense sequence to exhibit self-complementarity or to self-dimerize was assessed. The results from these analyses provided an explanation for the high specificity and the superior inhibitory characteristics of GPIA when compared to the eight other antisense oligonucleotides. GPI2A exhibited the second most favorable energy-related characteristics for hybridization reactions, and most importantly, unlike the other eight antisense sequences, it did not show the potential to self-complement or to dimerize. The results of this study and a previous investigation of sequence specificity requirements for GPI2A inhibition of HIV-1 gene expression provide strong evidence for an antisense mode of action for this oligonucleotide construct, a useful tool for analysis of viral gene expression and perhaps a potential therapeutic agent.

Animals↗
PubMed1996

Ribonucleotide reductase R2 component is a novel malignancy determinant that cooperates with activated oncogenes to determine transformation and malignant potential.

Ribonucleotide reductase is a highly regulated cell cycle-controlled activity that is essential for DNA synthesis and repair. A retroviral vector for the R2 component of mammalian ribonucleotide reductase, the rate-limiting protein for enzyme activity and DNA synthesis in proliferating cells, was constructed and introduced into mammalian cells. Expression of Myc epitope-tagged R2 protein in benign BALB/c 3T3 and NIH 3T3 cells leads to a greatly increased frequency of focus formation in cooperation with H-ras transformation. Four lines of H-ras-transformed mouse 10T1/2 fibroblasts showed increased growth efficiency in soft agar after infection with the recombinant R2 expression virus vector. Furthermore, cells with altered R2 expression also exhibited significantly reduced subcutaneous tumor latency and increased tumor growth rates in syngeneic mice, and showed markedly elevated metastatic potential in lung metastasis assays. The results indicate that altered R2 gene expression cooperates with ras in mechanisms of malignant progression. A major Ras pathway involves the Raf-1 protein, which is recruited to the plasma membrane for activation. We show that recombinant R2 expression leads to significant increases in membrane-associated Raf-1 protein and mitogenactivating protein kinase-2 activity suggesting a mechanism for the observed Ras/R2 synergism. In support of this finding, we observed that activated Rac-1, which operates parallel to Raf-1 and cooperates with Raf-1 in Ras activated pathways, also cooperates with R2 in cellular transformation. These studies demonstrate that the R2 protein can participate in other critical cellular functions in addition to ribonucleotide reduction, and that deregulated R2 is a novel tumor progressor determinant that cooperates in oncogene-mediated mechanisms, which control malignant potential.

3T3 Cells↗
PubMed1996

A cis-trans interaction at the 3'-untranslated region of ribonucleotide reductase mRNA is regulated by TGF-beta 1, TGF-beta 2, and TGF-beta 3.

The R2 component of ribonucleotide reductase is rate-limiting for DNA synthesis in proliferating cells, and recently, it has been shown that aberrant expression of R2 directly alters the malignant potential of tumor cells. We show that R2 gene expression is elevated in BALB/c 3T3 cells treated with transforming growth factor (TGF)-beta 1, TGF-beta 2, or TGF-beta 3, as determined by Northern blot analysis. Gel shift assays and UV crosslinking studies demonstrated similar post-transcriptional regulation at the 3'-untranslated region (3'-UTR) of the R2 mRNA, by TGF-beta 1, TGF-beta 2, and TGF-beta 3. The three growth factors induced a common 75 kDa RNA-protein complex. A 9 nucleotide sequence, GAGUUUGAG, previously shown to be responsive to TGF-beta 1-mediated R2 message stability changes, effectively competed out the formation of the R2 3'-UTR complex. We propose that these three different members of the TGF-beta family work through a common mechanism to control an important component of cell proliferation and a potential determinant of malignant progression.

3T3 Cells↗
PubMed1996

Suppression of malignancy by the 3' untranslated regions of ribonucleotide reductase R1 and R2 messenger RNAs.

Mammalian ribonucleotide reductase is rate limiting for the synthesis of DNA. The active enzyme is composed of two dissimilar components called R1 and R2, encoded by different genes. The 3' untranslated regions (3' UTRs) of R1 and R2 messages contain sequences that are important in regulating gene expression through changes in message stability. We have constructed expression plasmids containing the R1 or R2 mRNA 3' UTRs, and we show that transfection of these plasmids into highly malignant mouse 10 T1/2 cells significantly suppresses the tumorigenic properties of these cells in syngeneic mice when compared with cells transfected with the same plasmid lacking R1 or R2 3' UTR sequences or when compared with cells transfected with the same plasmid expressing a heterologous sequence as a control. Furthermore, cells expressing the R2 3' UTR exhibit significantly reduced potential to disseminate to the lungs of syngeneic animals in experimental metastasis assays. The tumor-suppressive effects of the mouse R1 and R2 3' UTRs were not confined to mouse cells, because human HeLa cells transfected with expression plasmids containing either RI or R2 3' UTRs were also significantly less tumorigenic in assays using BALB/c nu/nu mice. These studies demonstrate that the untranslated regions of ribonucleotide reductase mRNAs can function as modifiers of tumor cell development and for the more complex process of tumor dissemination. We propose that these malignancy-suppressive effects are mediated through RNA interactions with cellular components involved in growth regulation through mechanisms of posttranscriptional control of gene expression. In addition, these observations emphasize the enormous potential of untranslated RNA to act directly as modifiers of biological characteristics relevant to mechanisms of malignancy.

Animals↗
PubMed1996

Defining a novel cis-element in the 3'-untranslated region of mammalian ribonucleotide reductase component R2 mRNA. cis-trans-interactions and message stability.

Mammalian ribonucleotide reductase is a highly regulated activity essential for DNA synthesis and repair. The 3'-untranslated region (3'-UTR) of mammalian ribonucleotide reductase R2 mRNA has been implicated in the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate-mediated stabilization of mouse BALB/c 3T3 R2 message. We investigated the possibility that the 3'-UTR contains regulatory information for R2 mRNA turnover. Using 3'-end-labeled RNA in gel shift and UV cross-linking analyses, we detected in the 3'-UTR a novel 9-nucleotide cis-element, 5'-UCGUGUGCU-3', which interacted with a widely distributed cellular cytosolic protease-sensitive factor(s) in a sequence-specific manner to form a 45-kDa R2 binding protein complex. The binding activity was redox-sensitive and down-regulated by 12-O-tetradecanoylphorbol-13-acetate and okadaic acid in a dose-dependent manner. Insertion of a 154-base pair fragment containing the cis-element led to markedly reduced accumulation of chloramphenicol acetyltransferase hybrid mRNA relative to the same insert carrying a series of G --> A mutations within this element that eliminated binding. We suggest that the 9-nucleotide region functions as a destabilizing element. These results provide a model for ribonucleotide reductase gene expression through a novel and specific mRNA cis-trans-interaction involving a phosphorylation signal pathway that leads to changes in the stability of R2 message.

3T3 Cells↗
PubMed1996

Transforming growth factor-beta1 stimulates multiple protein interactions at a unique cis-element in the 3'-untranslated region of the hyaluronan receptor RHAMM mRNA.

The receptor for hyaluronan mediated motility (RHAMM) gene expression is markedly elevated in fibrosarcomas exposed to transforming growth factor-beta1 (TGF-beta1). The half-life of RHAMM mRNA was increased by 3 fold in cells treated with TGF-beta1, indicating that growth factor regulation of RHAMM gene expression at least in part involves a posttranscriptional mechanism. Our studies demonstrated that a unique 30-nucleotide (nt) region that has three copies of the sequence, GCUUGC, was the TGF-beta1-responsive region in the 3'-untranslated region (3'-UTR) that mediated message stability. This region interacted specifically with cytoplasmic trans-factors to form multiple protein complexes of approximately 175, 97, 63, 26, and 17 kDa post-TGF-beta1 treatment, suggesting a role for these complexes in the mechanism of action of TGF-beta1-induced message stabilization. Insertion of the 3'-UTR into the chloramphenicol acetyltransferase gene conferred TGF-beta1 induced stability of chloramphenicol acetyltransferase-hybrid RNA in stably transfected cells, while the same insert carrying a deletion containing the 30-nt region had no significant effect on mRNA stability. These results provide a model of RHAMM message regulation in which TGF-beta1-mediated alteration of RHAMM message stability involves the up-regulation of multiple protein interactions with a 30-nt cis-element stability determinant in the 3'-UTR. This model also suggests that this 30-nt base region functions in cis to destabilize RHAMM mRNA in resting normal cells.

Animals↗
PubMed1996

Soluble hyaluronan receptor RHAMM induces mitotic arrest by suppressing Cdc2 and cyclin B1 expression.

The hyaluronan (HA) receptor RHAMM is an important regulator of cell growth. Overexpression of RHAMM is transforming and is required for H-ras transformation. The molecular mechanism underlying growth control by RHAMM and other extracellular matrix receptors remains largely unknown. We report that soluble RHAMM induces G2/M arrest by suppressing the expression of Cdc2/Cyclin B1, a protein kinase complex essential for mitosis. Down-regulation of RHAMM by use of dominant negative mutants or antisense of mRNA also decreases Cdc2 protein levels. Suppression of Cdc2 occurs as a result of an increased rate of cdc2 mRNA degradation. Moreover, tumor cells treated with soluble RHAMM are unable to form lung metastases. Thus, we show that mitosis is directly linked to RHAMM through control of Cdc2 and Cyclin B1 expression. Failure to sustain levels of Cdc2 and Cyclin B1 proteins leads to cell cycle arrest.

Animals↗
PubMed1996

Basic fibroblast growth factor selectively regulates ornithine decarboxylase gene expression in malignant H-ras transformed cells.

Cell growth regulation by fibroblast growth factors (FGFs) is highly complex. The present study demonstrates a novel link between alterations in bFGF regulation during malignant conversion and the expression of ornithine decarboxylase, a key rate-limiting and regulatory activity in the biosynthesis of polyamines. H-ras transformed mouse 10T 1/2 cell lines exhibiting increasing malignant potential were investigated for possible bFGF-mediated changes in ornithine decarboxylase gene expression. Selective induction of ornithine decarboxylase gene expression was observed, since, in contrast to nontransformed 10T 1/2 cells and cells capable of only benign tumor formation, H-ras transformed metastatic cells exhibited marked elevations in ornithine decarboxylase message levels. Evidence for regulation of ornithine decarboxylase gene expression by bFGF at both transcription and posttranscription was found. Actinomycin D pretreatment of malignant cells prior to bFGF exposure inhibited the increase in ornithine decarboxylase message. Furthermore, striking differences in the rates of ornithine decarboxylase message decay were observed when cells treated with bFGF were compared to untreated control cells, with the half-life of ornithine decarboxylase mRNA increasing from 2.4 h in untreated cells to 12.5 h in cells exposed to bFGF. Evidence was also obtained for a cycloheximide-sensitive regulator of ornithine decarboxylase gene expression whose effect, in combination with bFGF, resulted in a further augmentation of ornithine decarboxylase gene expression. Furthermore, evidence is presented to suggest a possible role for G-protein-coupled events in the bFGF-mediated regulation of ornithine decarboxylase gene expression. The bFGF regulation of ornithine decarboxylase expression in H-ras transformed malignant cells appeared to occur independent of protein kinase C-mediated events. These results show that bFGF can modulate ornithine decarboxylase gene expression in malignant H-ras transformed cells and further suggests a mechanism of growth factor stimulation of malignant cells wherein early alterations in the regulatory control of ornithine decarboxylase gene expression are critical.

Animals↗
PubMed1996

A link between ferritin gene expression and ribonucleotide reductase R2 protein, as demonstrated by retroviral vector mediated stable expression of R2 cDNA.

We have constructed a retroviral expression vector for the mammalian ribonucleotide reductase R2 component. Stable infectants, which express a myc epitope tagged R2 protein from the vector cDNA were obtained and described for the first time. Cells containing the recombinant protein exhibited increased ribonucleotide reductase activity, and were resistant to the antitumour agent hydroxyurea, which targets the R2 component of ribonucleotide reductase. Furthermore, a direct link between ferritin gene expression and R2 protein was observed, since cells containing vector expressed recombinant R2 protein exhibited increased H-chain and L-chain ferritin gene expression.

3T3 Cells↗
PubMed1996

Neutrophil and cytokine activation with neonatal extracorporeal membrane oxygenation.

OBJECTIVE: To determine whether extracorporeal membrane oxygenation (ECMO), like cardiopulmonary bypass, produces systemic inflammatory responses that could potentiate organ injury in infants with respiratory failure. STUDY DESIGN: We evaluated the effects of neonatal ECMO on neutrophil surface adherence proteins, elastase release, and cytokine levels in blood samples from 15 patients before and during ECMO, and from banked blood and ECMO circuit blood before cannulation. Neutrophil elastase, tumor necrosis factor alpha, and interleukin types 1 beta, 6, and 8 were measured. Chest radiographs were evaluated by a radiologist using a lung injury score in blinded fashion. RESULTS: Primed ECMO circuit blood, in comparison with patient pre-ECMO blood, demonstrated marked up-regulation of CD11b (mean fluorescence intensity 1660 +/- 109 vs 361 +/- 81; p < 0.001 (mean +/- SEM)), shedding of L-selectin (mean fluorescence intensity 10 +/- 2 vs 89 +/- 38; p < 0.01), and elevated elastase levels (349 +/- 76 vs 154 ng/ml +/- 38; p < 0.001), consistent with neutrophil activation. During ECMO, neutrophil CD11b levels increased but L-selectin was not significantly shed. Concentrations of circulating neutrophil elastase increase significantly during ECMO. Corrected circulating quantities of interleukin-8 also rose significantly, but the responses of tumor necrosis factor alpha and interleukin-1 beta were minimal. Radiographic lung injury scores worsened with the initiation of ECMO (median score: 6 before ECMO vs 11 in first hour of ECMO; p = 0.012), in conjunction with indicators of neutrophil activation. CONCLUSION: Neonates with respiratory failure have activation of the inflammatory cascade. ECMO incites additional neutrophil and cytokine activation in association with early pulmonary deterioration. Routine leukodepletion of blood for circuit priming to remove activated neutrophils may be beneficial.

Cytokines↗
PubMed1996

Pediatric emergency physician interpretation of plain radiographs: Is routine review by a radiologist necessary and cost-effective?

STUDY OBJECTIVE: To determine the concordance rate of plain radiograph interpretations by pediatric emergency physicians and pediatric radiologists, to evaluate the effect of incorrect radiologic diagnosis on patient management, and to evaluate the necessity and cost-effectiveness of routine follow-up review of all plain radiographs by a radiologist. METHODS: We assembled a prospective series of all patients who presented to the emergency department of an urban tertiary care children's hospital and underwent plain radiography between October 1 and October 31, 1994. Pediatric emergency physicians documented their interpretations. Within 24 hours, films were reviewed by a pediatric radiologist. The two interpretations were classified as concordant or discordant and were further assessed for medical significance and subsequent change in management. RESULTS: During the study period, 707 radiographic examinations were performed: chest, 56%; skeletal excluding spine, 20.1%; abdomen, 11.9%; sinus, 4.2%; spine, 3.6%; and other, 4%. The accuracy or concordance rate was 90.2% (638 of 707) for pediatric emergency physician interpretations; clinical management was unchanged in 96.9% (685 of 707) of the cases. Of the 69 discordant interpretations, 48 were clinically significant, with 22 requiring changes in management. They included 9 false-negative interpretations by pediatric emergency physicians: (5 fractures, 2 cases of pneumonia, 1 case of sinusitis, 1 case of cardiomegaly); 10 false-positive interpretations by pediatric emergency physicians (5 fractures, 4 cases of pneumonia, 1 case of sinusitis), and 3 false-positive interpretations by radiologists (1 case of C-2 spine subluxation, 1 retropharyngeal abscess, and 1 case of necrotizing enterocolitis). No adverse outcomes resulted from these misinterpretations. Routine review of all plain radiographs by a radiologist represents an estimated $210,000 annual cost to the patients and payers. CONCLUSION: Radiograph interpretations by pediatric emergency physicians were generally accurate, and no adverse outcomes occurred as a result of misinterpretation. Clinical assessment probably assisted these physicians in interpreting the radiographs of high-risk patients. Judicious consultation with a radiologist during the initial presentation of a high-risk patient, when deemed warranted by the pediatric emergency physician, will help the emergency physician deliver high-quality, cost-effective health care. Given the overall clinical accuracy rate of radiograph interpretations by the pediatric emergency physicians and the cost of routine review of all plain radiographs in the ED by a radiologist, routine review versus selective specialty consultation must be further evaluated.

Child↗
PubMed1996

Outcome of respiratory failure: a case-control study.

Despite the availability of ECMO (extracorporeal membrane oxygenation) services for nearly a decade, the criteria for the institution of ECMO for pediatric respiratory failure are still not clearly defined. Therefore, a chart review was performed on children who were mechanically ventilated more than 48 hours in 1989-1990 in order to evaluate possible predictors of death from pediatric respiratory failure. Twenty-three children died as a consequence of respiratory failure. Nonsurvivors in both years were compared with the 78 survivors in 1990, and potential predictors were subjected to multivariate analysis. After 4 days of mechanical ventilation, an alveolar-arterial oxygen gradient (AaDO2) greater than 400 torr (53.3 kPa) was a weak predictor of death due to respiratory failure, and yet an AaDO2 less than 400 torr (53.3 kPa) was a stronger predictor of survivability. Combination of variables did not yield a better predictor than any single variable. Early prediction of mortality from respiratory failure in this population was not found.

Acute Disease↗
PubMed1996

Growth factors in mechanisms of malignancy: roles for TGF-beta and FGF.

Malignant progression is a complex process involving the accumulation of multiple genetic alterations leading to changes in many specialized cell functions. Important in this process is the loss of growth control which is frequently associated with modifications in growth factor production, and growth factor response pathways. Indeed, oncogenes have been characterized that code for polypeptide growth factors or their receptors, and many tumor cell populations release potently mitogenic growth factors which contribute to the malignant properties of tumor cells. In this review, the importance of growth factors in mechanisms of malignant progression is emphasized, using as examples the transforming growth factor-beta (TGF-beta) and fibroblast growth factor (FGF) families. We describe many of the properties and biological activities of these two families of growth factors, focusing on mechanisms of autocrine and intracrine mitogenic stimulation of tumor cell proliferation and malignant progression. The discussion includes evidence for altered growth factor expression in tumor cells, and the relationship between these changes in growth factors and alterations in the regulation of DNA synthesis, cell proliferation, protease production and cell motility required for invasion and metastasis. Recent studies are described that show that aberrant expression of TGF-beta1, bFGF or K-FGF results in dramatic changes in the genetic stability of cells, leading to increased rates of spontaneous gene amplification and the generation of drug resistant variants. These findings describe new malignancy relevant functions for altered growth factor expression.

Animals↗
PubMed1995

Antiviral activity and protection of cells against human immunodeficiency virus type-1 using an antisense oligodeoxyribonucleotide phosphorothioate complementary to the 5'-LTR region of the viral genome.

A COS-like monkey kidney cell line stably transfected with the plasmids pCMVgagpol-rre-r with the gag and pol genes, and pCMV rev with the rev gene of HIV-1 derived from the cDNA clone BH10, was used as a model for assessing the effectiveness of antisense (AS) constructs, A 20-mer oligodeoxyribonucleotide (oligo) phosphorothioate sequence (5'-CCG CCC CTC GCC TCT TGC CG) complementary to a portion of the 5'-long terminal repeat (5'-LTR) of the HIV-1 genome was tested for its inhibitory effects on the biologically important processes of HIV-1 replication and proliferation. We observed a concentration-dependent inhibition of HIV protein synthesis. Desitometric analysis of data from Western blot analysis showed sequence-specific and concentration-dependent oligo inhibition of p24 viral core antigen formation in the low-microM range. When lipofectin was used as a delivery vehicle, a markedly increased potentiation of the AS activity of the sequence was observed at a lower concentration (0.1 microM), following a 24-h preincubation. The AS construct specifically inhibited intracellular p24 production in chronically HIV-1-infected cells of lymphoid origin (H9/IIIB cells) by 95%, resulting in a 15-fold inhibitory effect relative to a similar sequence thiolated at only seven single-base positions. A concentration-dependent attenuation in the reverse transcriptase activity and a reduction in viral p24 level was observed in the culture supernatant of AS-pretreated HIV-1-infected phytohemagglutinin A-stimulated human cord blood mononuclear cells. Incubation of a HIV-1-infected lymphoid cell line with AS sequence resulted in a marked reduction in syncytium formation, and therefore protected cells from the cytopathic effects of the virus. Furthermore, the AS oligo did not appear to be cytotoxic in cell growth rate and colony-forming ability assays. The AS oligo described in this report is a useful new tool for the molecular analysis of HIV-1 gene expression and proliferation, and may have potential as a therapeutic agent.

Animals↗
PubMed1995

Fibroblasts transformed by combinations of ras, myc and mutant p53 exhibit increased phosphorylation of histone H1 that is independent of metastatic potential.

H1 histones play an important role in regulating higher order structure of chromatin and are potential regulators of gene expression. H1s are phosphorylated, a modification which alters their interaction with DNA. We measured the abundance of three phosphorylated H1 subtypes in mouse fibroblasts transformed by combinations of ras, myc and mutant p53 which differ in metastatic potential. We found that there is an increase in phosphorylation of H1 subtypes in fibroblasts transformed with ras, myc and mutant p53. This increase was found to correlate with cellular transformation but not with induction of the metastatic phenotype.

Animals↗
PubMed1995

Altered regulation of message stability and tumor promoter-responsive cis-trans interactions of ribonucleotide reductase R1 and R2 messenger RNAs in hydroxyurea-resistant cells.

Mammalian ribonucleotide reductase is a highly regulated activity essential for DNA synthesis and repair. The activity and message levels of the enzyme are elevated in cells treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, and this appears to be mediated through specific cis elements in the 3'-untranslated region of the R1 and R2 mRNAs that interact with R1 and R2 binding proteins called R1BP and R2BP, respectively. Hydroxyurea-resistant cells with increased R1 and R2 message levels were observed to have increased R1 and R2 message half-lives. This was accompanied by alterations in R1 and R2 3'-untranslated region cis-trans interactions, as judged by band shift and UV cross-linking assays, in which R1BP and R2BP binding was markedly reduced. This first description of mutant mammalian cells altered in message stability regulatory determinants indicates another mechanism for acquiring resistance to an antitumor agent. Furthermore, the present study strongly supports the concept that R1BP and R2BP are important general regulators of ribonucleotide reductase message stability and act as message destabilizing factors.

Animals↗