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J A Wright

Publications and source records attributed to J A Wright.

At least 91 records · Page 5Linked to original sources
PubMed1994

Comparison of glyceraldehyde-3-phosphate dehydrogenase and 28S-ribosomal RNA gene expression as RNA loading controls for northern blot analysis of cell lines of varying malignant potential.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been a gene of choice in Northern blot analyses as an internal RNA loading control. We have investigated the expression of GAPDH and 28S-ribosomal RNA (28S rRNA) genes in mouse 10T1/2 cells and in a variety of tumorigenic and highly malignant metastatic cell lines derived from the 10T1/2 cell line. We observed that GAPDH mRNA levels varied markedly among the tumorigenic and highly malignant cell lines and were elevated in these cell lines when compared to the normal mouse 10T1/2 cells. In contrast, the levels of 28S rRNA did not significantly vary among the tumorigenic and highly malignant cell lines and were approximately at the same level as that found in the normal parental mouse 10T1/2 cell line. These observations indicate that much caution should be taken when using GAPDH gene expression as an RNA loading control for Northern blots. Based upon these observations, we recommend the use of 28S rRNA gene expression as a preferred RNA loading control for Northern blot analysis in which total RNA is used.

Animals↗
PubMed1994

Aberrant expression of basic fibroblast growth factor in NIH-3T3 cells alters drug resistance and gene amplification potential.

We have investigated the genetic stability of NIH-3T3 cells transfected with sequences coding for basic fibroblast growth factor (bFGF) by determining drug resistance and gene amplification potential. Colony-forming experiments and fluctuation analyses showed that the frequency and rate of resistance to N-(phosphonacetyl)-L-aspartate (PALA) was dramatically elevated in cells transfected with either the normal bFGF coding sequence that lacks a known signal for secretion or a chimeric bFGF sequence that targets the growth factor to the secretory pathway. Basic FGF-transfected cells that grew in the presence of PALA were found to possess an amplification of the CAD gene, which codes for a multifunctional protein involved in pyrimidine biosynthesis and is the site of action for PALA. The observation that these alterations occur in cells transfected with a bFGF sequence, without a conventional signal sequence for secretion, suggests an intracrine as opposed to autocrine mechanism of action. The results describe a new function for this growth factor and suggest a novel role for aberrant expression of bFGF in mechanisms of tumor progression.

3T3 Cells↗
PubMed1994

Comparison of methods to categorize undernutrition in children.

We compared the results of three methods of categorizing undernutrition in a group of 258 children referred for evaluation of "failure to thrive." There was broad variation between the number of children classified as malnourished (75% to 98%) and the degree of undernutrition, depending on the method used. We recommend that categorization systems be used only to define a child as having a risk of possible adverse effects of undernutrition, not as being malnourished.

Failure to Thrive↗
PubMed1994

Randomized intervention to increase children's selection of low-fat foods in school lunches.

OBJECTIVE: The purpose of this study was to determine whether children would increase their selection of low-fat foods in school lunches if these foods were labeled on the menu and parents were notified of their availability. METHODS: The 16 elementary schools in the Bellevue (Washington) School District were randomly assigned to intervention and control groups. Participants were the students eating the school lunch--an average of 2445 students per day, of whom one third received free or reduced-price lunches. In all schools, one of the two daily lunch entrees was low in fat (< or = 30% of calories from fat). After a 5-month baseline period, eight schools received a 4-month intervention. The monthly menus carried home by students began to indicate the low-fat entree and to compare the fat content of both entrees. In the first month, parents in the intervention schools (2329 households) received a mailing with a copy of the menu, an informational pamphlet on dietary fat, and a letter that described the menu changes and asked the parents to encourage their children to select low-fat entrees. A follow-up telephone survey was performed in one school 1 month after the intervention mailing to assess the recall and impact of the intervention. The main outcome, based on 249,861 student meal selections, was the proportion of students who selected the low-fat entree, and the unit of analysis was the school. RESULTS: At baseline, there was little difference between intervention and control schools in the percentage of children choosing the low-fat entree (31.5% vs 30.8%). During the intervention, there was an increase in low-fat entree selection in the intervention schools compared with the control schools (35.5% vs 32.2%; p = 0.03). Of 221 parents surveyed, 71% recalled the mailing, 53% remembered that there were now low-fat entrees on the menu, and 10% reported that, after the mailing, they asked their child to choose a low-fat entree.

Child↗
PubMed1994

Fibroblast growth factor mediated alterations in drug resistance, and evidence of gene amplification.

We have investigated the drug resistance and gene amplification potential of NIH3T3 cells transfected with sequences coding for K-FGF, a known oncogene product, or bFGF, a non-oncogene member of the fibroblast growth factor family. Resistance to methotrexate, N-(phosphonacetyl)-L-aspartate and hydroxyurea was observed with K-fgf transfectants, due to amplification of dihydrofolate reductase, CAD or ribonucleotide reductase R2 genes, respectively. In keeping with the increase in gene amplification frequency, cells transfected with the K-fgf gene also exhibited a marked increase in CAD gene amplification rate, as determined by fluctuation analysis in the presence of N-(phosphonacetyl)-L-aspartate. Cells transfected with bFGF encoding cDNA also exhibited a significant elevation in N-(phosphonacetyl)-L-aspartate resistance, and CAD gene amplification. Treatment with suramin, which interferes with the interaction of fibroblast growth factors with their cell surface receptors, did not decrease the drug resistance properties of K-fgf transfected cells. These observations with suramin and the findings with bFGF, which lacks a conventional signal sequence for secretion, suggests that the growth factor-mediated effects on drug resistance and gene amplification occur through an intracellular as opposed to autocrine mode of action. The finding that aberrant growth factor expression regulates gene amplification opens up new possibilities for investigating intracellular mechanisms relevant to this process and also describes new functions for the altered expression of K-FGF and bFGF, which are relevant to mechanisms of malignant progression.

Animals↗
PubMed1993

Phosphorylation of ribonucleotide reductase R2 protein: in vivo and in vitro evidence of a role for p34cdc2 and CDK2 protein kinases.

Ribonucleotide reductase is responsible for supplying the deoxyribonucleotides required for DNA synthesis and repair. The active enzyme consists of two dissimilar protein components called R1 and R2. Immunoprecipitation of R1 and R2 proteins from [32P]orthophosphate-labeled exponentially growing mouse L cells showed that the R2 protein but not the R1 protein of ribonucleotide reductase could be phosphorylated in vivo. Two-dimensional phosphopeptide mapping experiments of trypsin-digested R2 protein showed a major spot containing more than 90% of the total radioactivity and a minor spot with the remaining radioactivity. Phosphoamino acid analysis of R2 phosphorylated protein indicated that phosphorylation occurred exclusively on serine. Protein kinase C, cAMP-dependent protein kinase, p34cdc2, and CDK2 were capable of phosphorylating the R2 protein in vitro, whereas casein kinase II was not. To determine whether any of these enzymes could phosphorylate peptides observed to be phosphorylated in actively growing cells, tryptic phosphopeptide maps of R2 that had been phosphorylated in vitro were compared with maps of R2 that had been isolated from [32P]-labeled cells. Only the phosphopeptide maps obtained with p34cdc2 and CDK2 matched the pattern found in [32P]-labeled cells. Experiments in which tryptic digests from different samples were mixed prior to two-dimensional separation demonstrated comigration of phosphopeptides obtained by in vivo phosphorylation with phosphopeptides derived from p34cdc2 or CDK2 obtained by in vitro phosphorylations. These studies indicate that protein R2 phosphorylation may play an important role in the regulation of ribonucleotide reduction, DNA synthesis, and cell cycle progression, and suggest a potentially important p34cdc2 and/or CDK2 regulation point in DNA replication.

Amino Acid Sequence↗
PubMed1993

A novel transforming growth factor-beta 1 responsive cytoplasmic trans-acting factor binds selectively to the 3'-untranslated region of mammalian ribonucleotide reductase R2 mRNA: role in message stability.

Ribonucleotide reductase is a highly regulated enzyme that provides the four deoxyribonucleotides required for DNA synthesis. Our studies showed that TGF-beta 1 treatment of BALB/c 3T3 mouse fibroblasts markedly elevated ribonucleotide reductase R2 mRNA levels, and also increased the half-life of R2 message by 4-fold from 1.5 h in untreated cells to 6 h in treated cells. We describe a novel 75 Kd sequence-specific cytoplasmic factor (p75) that binds selectively to a 83-nucleotide 3'-untranslated region of R2 mRNA and did not bind to the 5'UTR, the coding region of the R2 message or to the 3'UTRs of other mRNAs (from c-myc, GM-CSF and the iron responsive element from the transferrin receptor mRNA), or to the homopolymer poly(A) sequence. p75-RNA binding activity, which requires new protein synthesis, is not present in untreated cells, but is induced following TGF-beta 1 stimulation. The in vivo kinetics of appearance of p75 binding activity paralleled the accumulation of R2 mRNA. Insertion of the 3'-untranslated region into the chloramphenicol acetyltransferase (CAT) message confers TGF-beta 1 induced stability of RNA in stably transfected cells, while the same insert carrying a deletion of the 83-nucleotide fragment had little affect on RNA levels. Furthermore, in vitro decay reactions that contained the 83-nucleotide RNA or deletion of this fragment caused a significant decrease in TGF-beta 1 stabilization of R2 message. A model is presented of R2 message regulation in which TGF-beta 1 mediated stabilization of R2 message involves a specific interaction of a p75-trans-acting factor with a cis-element(s) stability determinant within the 83-nucleotide sequence which is linked to a reduction in the rate of R2 mRNA degradation.

3T3 Cells↗
PubMed1993

Studies on the effect of mevinolin (lovastatin) and mevastatin (compactin) on the fusion of L6 myoblasts.

The effect of mevastatin and mevinolin on the fusion of L6 myoblasts was studied. Both compounds were present inhibitors of myoblast fusion at concentrations as low as 0.25 microM, but fusion was restored when the inhibitors were removed. Both compounds resulted in decreased binding of conA and WGA to cell surface oligosaccharides showing they were causing a reduction in N-linked cell surface glycoproteins. There was a reduction in creatine phosphokinase activities in the presence of both compounds showing that they were affecting biochemical differentiation. The presence of both compounds inhibited the incorporation of labeled mannose from GDP-mannose into lipid-sugar and N-linked glycoprotein, but the inhibition was reversed by addition of exogenous dolichol phosphate to the incorporation mixture. The main conclusion from these studies is that mevinolin and mevastatin are inhibiting myoblast fusion by affecting the synthesis of fusogenic cell surface N-linked glycoproteins probably by affecting the synthesis of dolichol phosphate containing oligosaccharides that are required as intermediates in N-linked glycoprotein biosynthesis.

Animals↗
PubMed1993

Animal research.

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Animals↗
PubMed1993

Mammalian ribonucleotide reductase R1 mRNA stability under normal and phorbol ester stimulating conditions: involvement of a cis-trans interaction at the 3' untranslated region.

Ribonucleotide reductase R1 gene expression is elevated in BALB/c 3T3 fibroblasts treated with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). We show that TPA treatment increased the half-life of R1 mRNA by 5-fold, indicating that TPA regulates R1 gene expression by a post-transcriptional mechanism. We investigated the possibility that the 3' untranslated region (3'UTR) of R1 mRNA contains regulatory information for TPA-mediated message stability. Our studies demonstrated that a 49 nucleotide (nt) TPA-responsive region existed within the R1 mRNA 3'UTR. Deletion of the 49 nt region led to the abolishment of TPA-induced stability of R1 and hybrid CAT mRNAs. Further deletions of the 3'UTR did not significantly affect mRNA turnover rates. In addition, we detected by cross-linking a novel 52-57 kDa R1 mRNA-binding protein (R1BP) that bound selectively to the 49 nt region of the R1 mRNA 3'UTR and did not bind to the 5'UTR, the coding region or other mRNA 3'UTRs. The R1BP-RNA binding activity observed in unstimulated cells was rapidly and markedly down-regulated after TPA treatment, suggesting a role for R1BP in mRNA metabolism, and in the mechanism of action of TPA-induced R1 message stabilization. These results support a novel model of R1 gene regulation in which a cis-element(s) within the 49 nt region of the R1 mRNA 3'UTR interacts with R1BP in a mechanism that regulates R1 message stability. We propose that this model accounts for the TPA-mediated stability alteration of R1 message, through down-regulation of R1BP-RNA binding activity linked to a reduction in the rate of R1 mRNA degradation.

3T3 Cells↗
PubMed1993

Transforming growth factor beta 1 selectively regulates ornithine decarboxylase gene expression in malignant H-ras transformed fibrosarcoma cell lines.

Negative growth regulators such as the transforming growth factor beta (TGF-beta) family appear to be important inhibitors in most tissue types. However, inhibition of DNA synthesis and cell proliferation is frequently lost during malignant transformation, and in some cases, tumor cell proliferation is actually stimulated by TGF-beta. The present study demonstrates a novel link between alterations in TGF-beta regulation during malignant conversion, and the expression of ornithine decarboxylase, a key rate-limiting activity in the biosynthesis of polyamines, and an enzyme that plays an important role in cell growth and differentiation. A panel of radiation and H-ras transformed mouse 10T1/2 cell lines exhibiting increasing malignant potential was investigated for possible TGF-beta 1 mediated changes in ornithine decarboxylase gene expression. Selective induction of gene expression was observed since only H-ras transformed cell lines with malignant potential exhibited marked elevations in ornithine decarboxylase message levels. Ornithine decarboxylase gene expression in nontransformed 10T1/2 cells and cell lines capable of only benign tumor formation was unaffected by TGF-beta 1 treatment. H-ras transformed cells were transfected with a plasmid placing the TGF-beta 1 coding region under the control of a zinc sensitive metallothionein promoter. When these cells were cultured in the presence of zinc an elevation of TGF-beta 1 mRNA was observed within 30 min. This increase in TGF-beta 1 message closely coincided with an elevation in ornithine decarboxylase message, and preceded an induction of jun-B, an early response gene in cells sensitive to TGF-beta 1 stimulation. Evidence for regulation of ornithine decarboxylase gene expression by TGF-beta 1 at both transcription and posttranscription was found. Actinomycin D pretreatment of malignant cells prior to TGF-beta 1 exposure prevented the increase in ornithine decarboxylase message. Marked differences in the rates of ornithine decarboxylase message decay were observed when cells treated with TGF-beta 1 were compared to untreated controls, with the half-life of ornithine decarboxylase mRNA increasing from 2.5 h in untreated cells to 17.5 h in cells exposed to TGF-beta 1. In addition, evidence was obtained for a cycloheximide sensitive regulator of ornithine decarboxylase gene expression, since the presence of this protein synthesis inhibitor increased the levels of ornithine decarboxylase message, and this effect was synergistically augmented by exposure of cells to cycloheximide and induction of TGF-beta 1 gene expression together.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Cell motility, invasion, and malignancy induced by overexpression of K-FGF or bFGF.

We have tested the hypothesis that fibroblast growth factors contribute to cell locomotion and invasion, by investigating the properties of NIH-3T3 fibroblasts that secrete K-FGF following transfection with the K-fgf proto-oncogene and NIH-3T3 fibroblasts transfected with either the normal basic fibroblast growth factor (bFGF) coding sequence that lacks a known secretory signal sequence, or a chimeric bFGF sequence fused to an immunoglobulin signal sequence that targets the growth factor to the secretory pathway. The cell lines were tested for altered invasive characteristics on a physiologically relevant collagen substratum, and their motility rates were measured in cell locomotion assays using time-lapse video microscopy in the absence or presence of suramin, an inhibitor of growth factor-receptor interaction. The data were analyzed by comparing the motility rates of all K-fgf- and bFGF-transfected cell lines as a function of malignant potential. These studies show for the first time that K-fgf gene expression potently increases cell motility characteristics. Further, the bFGF transfectants generally exhibited increased rates of cell locomotion which were not dependent upon the presence of a signal sequence for secretion. These results support models of cell locomotion in which stimulation of this process can occur through both autocrine and intracrine pathways. In addition, a significant correlation was found to exist between cell locomotion and malignant potential, supporting the view that growth factor-induced motility, although not sufficient on its own to induce metastasis, is important in the promotion of tumor cell dissemination.

3T3 Cells↗
PubMed1993

School lunch: a comparison of the fat and cholesterol content with dietary guidelines.

OBJECTIVE: To compare the fat and cholesterol content of the foods offered and selected in an elementary school lunch program with current dietary guidelines. DESIGN: For 105 school days we recorded the food items selected by elementary school students in an entire school district (262,851 meals) who were given a choice between two entrees. The nutrient content of foods was assessed with a computerized nutrient data base supplemented by the food manufacturers' data. SETTING: Sixteen elementary schools in the Bellevue (Washington) School District. PARTICIPANTS: The number of students eating school lunch averaged 2500 per day, of whom 25% were from households with incomes less than 185% of poverty. INTERVENTION: None. MAIN OUTCOME MEASURES: We determined the nutritional content of the average meal selected; the proportion of days when one of the two offered entrees met fat and cholesterol guidelines; and the proportion of children selecting the entrees that met the guidelines. RESULTS: The average lunch selected had 35.9% of calories from total fat and 12.6% from saturated fat, exceeding the guidelines of 30% and 10%, respectively. Lunch contained an average of 57 mg cholesterol (106 mg/1000 kcal) and met guidelines. One of the two daily entree choices met guidelines for both total fat and saturated fat on 20% of days, and met both fat and cholesterol guidelines on 14% of days. When available, entrees meeting the fat guidelines were chosen by 37% of students, and entrees meeting both fat and cholesterol guidelines were chosen by 34% of students. CONCLUSIONS: In this school district the average lunch selected did not meet the current guidelines for dietary fat; when given the choice, more than one third of students selected the entrees that met these guidelines.

Child↗
PubMed1993

TGF-beta 1 stimulation of cell locomotion utilizes the hyaluronan receptor RHAMM and hyaluronan.

TGF-beta is a potent stimulator of motility in a variety of cell types. It has recently been shown that hyaluronan (HA) can directly promote locomotion of cells through interaction with the HA receptor RHAMM. We have investigated the role of RHAMM and HA in TGF-beta-stimulated locomotion and show that TGF-beta triggers the transcription, synthesis and membrane expression of the RHAMM receptor and the secretion of HA coincident with the induction of the locomotory response. This was demonstrated by both incubating cells with exogenous TGF-beta 1 and by stimulating the production of bioactive TGF-beta 1 in tumor cells transfected with TGF-beta 1 under the control of the metallothionein promoter. TGF-beta 1-induced locomotion was suppressed by antibodies that prevented HA/RHAMM interaction, using polyclonal antibodies to either RHAMM fusion protein or RHAMM peptides, or mAbs to purified RHAMM. Peptides corresponding to the HA-binding motif of RHAMM also suppressed TGF-beta 1-induced increases in motility rate. Spontaneous locomotion of fibrosarcoma cells was blocked by neutralizing secreted TGF-beta with panspecific TGF-beta antibodies and by inhibition of TGF-beta 1 secretion with antisense oligonucleotides. Polyclonal anti-RHAMM fusion protein antibodies and peptide from the RHAMM HA-binding motif also suppressed the spontaneous motility rate of fibrosarcoma cells. These data suggest that fibrosarcoma cell locomotion requires TGF-beta, and the pathway by which TGF-beta stimulates locomotion uses the HA receptor RHAMM and HA.

Amino Acid Sequence↗
PubMed1993

Neuropsychological, academic, and adaptive functioning in children who survive in-hospital cardiac arrest and resuscitation.

Children suffering cardiac arrest (CA) are not uncommon in certain pediatric populations. Due to the increasing survival rates of child CA patients, there is a growing interest in, and concern for, their long-term intellectual, academic, emotional, and adaptive functioning. This article describes the possible neurologic sequelae of CA in children and presents standardized assessment results on 25 children, 2 to 15 years of age, who survived a CA while in the hospital. A majority of these children exhibited low-average to deficient levels of performance on neuropsychologic, achievement, and adaptive behavior measures. Duration of cardiac arrest and a medical risk score were significantly correlated with decreased functioning in child CA patients. Children who suffer a cardiac arrest are at high risk for academic struggles, and many may need special education services.

Adolescent↗
PubMed1993

Attentional dysfunction in children with encopresis.

Encopresis and attentional dysfunction are common neurobehavioral disorders of childhood. The extent to which these disorders occur in association is unknown. The purpose of this study is to document the comorbidity of attentional dysfunction in a clinically identified population of encopresis patients. We used the Child Behavior Checklist (CBCL) to estimate the prevalence of disordered attention or hyperactivity in a group of children with encopresis seen at a tertiary care facility. Responses to CBCL questionnaires were analyzed to compare scores on the "hyperactive" behavior subscale with published normative data. The number of encopretic respondents with T scores above 70 (> 2 SD above the mean) on a hyperactivity subscale was ascertained for each age and gender cohort. From 347 eligible new clinic patients, responses from 167 were suitable for analysis. Overall, 23.4% of children with encopresis (95% confidence interval: 17.2%-30.5%) had T scores on the hyperactive subscale higher than 70. This prevalence (ten fold greater than expected in the normal population) was similar in both genders and across age groups. This association between attentional dysfunction and encopresis has significance for theories regarding etiology and for practical treatment strategies.

Adolescent↗
PubMed1993

Extracorporeal membrane oxygenation service at Egleston: two years' experience.

Extracorporeal membrane oxygenation (ECMO) is a perfusion support procedure that has been used to treat more than 7,000 patients with life threatening cardiac and/or respiratory failure. After 6 months of training and preparation, an ECMO service was opened on January 2, 1991, in Egleston Children's Hospital at Emory University. During the first 2 years, 96 neonatal, 31 pediatric, and 8 cardiac patients have been referred for possible ECMO. Of these 135 patients, 21 had disqualifying conditions. Sixty-four were considered candidates for ECMO but were able to be supported using less invasive therapies; only one of these died. Fifty patients were treated with ECMO of whom 39 survived (78%). Survival rates for neonatal, pediatric, and cardiac cases as separate groups as well as for each diagnostic category within these groups compare favorably with those reported by the international ELSO Registry. Notable in this series is the fact that 26/35 neonatal patients and 7/10 pediatric patients were successfully supported using venovenous (VV) rather than venoarterial (VA) perfusion, with the major indication for venoarterial ECMO being inability to introduce the 14F venovenous catheter into the patient's internal jugular vein. No patient initially managed with VV ECMO required conversion to VA. It is anticipated that avoidance of carotid ligation along with other innovations, such as the impending commercial availability of heparin-coated ECMO circuits, will make ECMO a highly attractive and appropriate therapy for an increasing number of high risk neonatal and pediatric patients in our state and region.

Adolescent↗