Gene expression profiles and protein balance in skeletal muscle of burned children after {beta}-adrenergic blockade

David N. Herndon, Mohan R. K. Dasu, Robert R. Wolfe, and Robert E. Barrow

Department of Surgery, The University of Texas Medical Branch, and Shriners Hospitals for Children, Galveston, Texas 77550

Submitted 20 November 2002 ; accepted in final form 8 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Propranolol, a nonselective {beta}-blocker, has been shown effective in hypermetabolic burn patients by decreasing cardiac work, protein catabolism, and lipolysis. This study investigates the effect of propranolol on gene and protein expression changes in skeletal muscle of burned children by use of high-density oligonucleotide arrays to establish the genetic profiles and stable isotope technique to quantitate protein synthesis. Thirty-seven children (mean age 9.7 ± 1.1 yr) were randomized into groups to receive placebo (n = 23) or propranolol (n = 14) titrated to reduce heart rate by 15%. Children had >40% total body surface area burns (mean 43 ± 5.6%). Protein net balance was determined by stable-isotope infusion technique. Total RNA from muscle biopsies was isolated, labeled, and cRNA hybridized to the HG-U95Av2 Affymetrix array. Mean net balance of protein synthesis and breakdown was –14.3 ± 12.9 nmol · min1 · 100 ml leg volume1 for placebo and +69.3 ± 34.9 nmol · min1 · 100 ml leg volume1 in the propranolol-treated children (P = 0.012). Comparison of 12,000 genes in burned children receiving placebo showed increased expression of two genes with time, whereas children receiving propranolol showed increased expression of nine genes with a decrease in five genes. We conclude that burned children receiving propranolol showed a significant upregulation in genes involved in muscle metabolism and downregulation of an important enzyme involved in gluconeogenesis and insulin resistance compared with burned children receiving placebo. The upregulation of genes involved in muscle metabolism correlates well with the increase in net protein balance across the leg.

propranolol; microarray analysis; hypermetabolism; muscle catabolism


SEVERE BURNS ARE ASSOCIATED with a hypermetabolic response in which muscle protein is used to fuel the increase in energy expenditure. Typical features of the response include increased body temperature, recruitment of neutrophils, changes in lipid metabolism, increased gluconeogenesis, and stimulation of protective pathways such as coagulation and complement activation, hormonal changes, and increased muscle catabolism (32). These changes lead to increased energy expenditure and futile substrate cycling, with depletion of nutritional and functional fat and protein stores (5, 30). Protein catabolism is increased after a severe burn, leading to a breakdown of functional structural proteins that results in the loss of lean muscle mass (10, 21).

One of the primary mediators of the hypermetabolic response is thought to be endogenous catecholamines (19, 41). The increase in catecholamine plasma levels found early after burn trauma leads to a hyperdynamic circulation (2, 40), increased basal metabolic rate (34), and increased catabolism of structural protein in skeletal muscle (20, 21). {beta}-Adrenergic blockade has been shown to be effective in posttraumatic hypermetabolism by decreasing thermogenesis (22), decreasing increased heart rate (29) and cardiac work (3), lowering resting energy expenditure (8), and attenuating muscle catabolism (23).

We have used several anabolic agents to effectively diminish muscle catabolism in burn patients. Insulin has been shown beneficial in protein metabolism primarily by stimulating protein synthesis (14, 35). Recombinant human growth hormone and IGF-I-binding protein-3 have also shown efficacy in improving muscle protein kinetics and wound healing in severely burned children (12, 18, 24). Testosterone can increase protein synthesis; however, there are risks of virilism and hepatotoxicity (15). After burns, the elevations in basal energy expenditure and muscle protein catabolism have been found to be correlated (20). Antagonists to the increased catecholamine response to burn may also have utility in decreasing catabolism after injury. Propranolol, a nonselective {beta}-blocker, has been effective in decreasing cardiac work in catabolic burn patients with hyperdynamic circulation (22) and in decreasing lipolysis (31).

This study investigates the effect of propranolol on protein metabolism and gene expression changes in skeletal muscle of burned children by means of high-density oligonucleotide arrays and stable isotope infusion technique to establish the genetic and phenotypic events associated with {beta}-adrenergic blockade.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Patients. Thirty-seven children who were admitted to our hospital within 1 wk after injury were studied. Children ranged in age from 3 to <18 yr with flame or scald burns covering more than 40% of their total body surface area (TBSA). Those with other severe injuries not related to the burn or preexisting conditions, such as asthma or pneumonia, were excluded from this study.

Each patient underwent burn-wound excision and grafting with skin autografts and allografts within 72 h after admission. Sequential staged grafting procedures were performed until the wounds were closed. All patients were fed a commercial enteral formula (Vivonex T.E.N., Sandoz Nutritional, Minneapolis, MN) through a nasoduodenal tube. The daily caloric intake was calculated to deliver 1,500 kcal/m2 of body surface area burned plus another 1,500 kcal/m2 of TBSA. Enteral nutrition was started at admission and continued until the wounds healed. Patients remained in bed for 5 days after each excision and grafting procedure, and then they were allowed to walk daily. Nude weights were recorded twice weekly until discharge by use of a standard sling scale. All of the patients were routinely administered antianxiety medication after the 1st wk after burn. Fourteen received propranolol, and 23 served as controls. Randomization followed a random-number protocol. Immediately after the second operation, the children in the drug group began to receive propranolol by nasogastric tube at a dose ranging from 0.3 to 1.0 mg/kg body wt given every 4 or 6 h. The dose was adjusted to achieve a 10–15% decrease in heart rate compared with the 24-h average heart rate immediately before drug treatment. Heart rate and blood pressure were monitored continuously throughout the study. When the mean blood pressure fell below 65 mmHg, the dose of propranolol was withheld or decreased. The dose was then increased incrementally to meet the study goal.

All test subjects underwent metabolic studies (indirect calorimetry and net protein balance studies) on the 5th day after the first surgical procedure. Figure 1 depicts the schedule protocol. Beginning on the morning of the 5th day, children in the continuously fed state were infused with a primed constant infusion of L-[ring-2H5]phenylalanine (Cambridge Isotopes, Andover, MA). The initial priming dose was 2 µmol/kg and was followed by a dose of 0.08 µmol · kg1 · min1 given intravenously. Phenylalanine is not synthesized or degraded in the peripheral tissues; thus measurements across the leg reflect net balance of protein synthesis and breakdown. To determine blood flow in the leg, indocyanine green dye was infused into the femoral artery (23). Immediately after the second operation, the propranolol group received by nasogastric tube a dose ranging from 0.3 to 1.0 mg/kg body wt given every 4 or 6 h. Five days later, a second series of protein kinetic studies was performed. The blood levels of unlabeled phenylalanine and its isotope were determined by gas chromatography-mass spectrometry as previously described (6, 7). Biopsy of the vastus lateralis muscle was taken and snap-frozen before and after propranolol treatment (Fig. 1). Muscle biopsies for DNA microarrays, RT-PCR, and Western blot analysis were stored at –70°C.



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Fig. 1. Study protocol for burned children who were admitted within 1 wk after injury and nutritionally depleted. Initial baseline studies were conducted 5 days after the 1st operation and muscle biopsies taken. Five days after the second operation, protein kinetic studies and muscle biopsies were repeated. All patients were treated with placebo or propranolol until discharge.

 

RNA extraction. For microarray analysis, we used muscle biopsies from seven patients in the control group and seven patients in the treatment group. Total RNA was isolated from muscle biopsies by acid guanidinium thiocyanate-phenol-chloroform extraction using TRI Reagent (Molecular Research Center, Cincinnati, OH). This method was based on the single-step method of RNA isolation described by Chomczynski and Sacchi (11). Samples were homogenized in TRI Reagent on ice, and total RNA was extracted following the manufacturer's instructions. Purified RNA was quantified by UV absorbance at 260 and 280 nm and stored in 25-µg aliquots at –70°C for DNA microarray hybridization and analyses. The adequacy and integrity of the extracted RNA were determined by gel electrophoresis. Two propranolol-treated and two placebo muscle biopsies showed insufficient quantity of RNA for microarray analysis. These patients were omitted from any further analysis.

Microarray analysis. Probe labeling, hybridization, and image acquisition were done according to the standard Affymetrix protocol. Briefly, 25 µg of purified total RNA were transcribed into cRNA, purified, and used as templates for in vitro transcription of biotin-labeled antisense RNA. Biotinylated antisense RNA preparation was fragmented and placed in a hybridization mixture containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre). Ten samples from controls (5 chips for time period 1 and 5 chips for time period 2), and 10 samples from treatment (5 chips before treatment and 5 chips after treatment) were hybridized to an identical lot of 20 Affymetrix Gene Chip arrays (HG-U95 Av2) for 16 h. The arrays were washed and stained using the instrument's standard Eukaryotic GE Wash 2' protocol and antibody-mediated signal amplification. The images were scanned and analyzed with Affymetrix Gene Chip Analysis Suite 5.0. Images from each gene chip were scaled and adjusted to an average intensity value for all arrays of 1,500. Scaled average-difference values and absolute call data from each gene chip were exported to data files and used for statistical analysis. In vitro transcription and chip hybridization were performed in collaboration with the University of Texas Medical Branch Genomic Core Facility.

Data analysis. Microarray data analyses included chip validation, normalization, filtering, and generation of consensus gene lists by comparing burn or treatment groups. Invariant-set normalization and perfect match model-based expression values were generated using DNA chip analyzer (dChip) software (26). For microarray validation, data were clustered to detect gross discrepancies among different array data. Data of an array were discarded if only a small fraction of probes was present compared with the remaining arrays in the group. The degree of similarity or dissimilarity among transcription profiles was established using clustering methods (data not shown) (4, 26, 37). The next step was the elimination of genes that showed little variation across the samples or that were absent in the majority of the samples. The first criterion was that the ratio of standard deviation and the mean of a gene's expression values across all samples were greater than the threshold (of 0.85 and the upper limit of 8). Data were discarded if there was a large deviation in the number of present calls or if the correlation coefficient among samples within the group was <0.85. The second criterion required a gene to be called present in >80% of arrays at all times. We determined the "presence" (P) or "absence" (A) of each probe within the group (according to the Affymetrix algorithm). A probe is present if its absolute call was P for at least two members of the group containing three samples; otherwise, the gene was considered not expressed in the group. The primary goal was to identify genes with significant differences in expression between the test and control groups. The within-group average of expression was calculated, and comparisons were made between groups. Comparisons were done by computing the expression fold difference for each gene and listing differences that showed a >1.5-fold increase or decrease in activity. An entry was discarded as an outlier when its value was beyond three standard deviations. Only the statistically significant differences at P < 0.05 were retained (13). When the number of samples per group and its influence on the validity of the analysis are considered, the power of the t-test was computed. If the correlation coefficient was <0.85, results were discarded even when significant. The expression profiles of the skeletal muscle biopsies taken from propranolol- and placebo-treated burned children were analyzed. Samples taken at study 1 (baseline) were before propranolol treatment, and study 2 was after treatment began. By using HG-U95A Affymetrix arrays, ~4,000 genes of 12,000 genes present in the array were expressed. This was in agreement with Affymetrix DNA array analysis of mouse skeletal muscle (25). Table 1 indicates the number of probes present, those absent, and those marginally present with corresponding regression coefficient values.


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Table 1. Number of probes present, absent, and marginally present, with corresponding regression coefficient values of the 20 chips used in the study

 

RT-PCR. Total RNA extracted from the muscle was quantitated by UV spectrophotometer and stored at –80°C for RT-PCR analysis. The cDNA reaction as well as the PCR were performed with an optimized buffer and enzyme system (Titan One Tube RT-PCR System; Roche, Indianapolis, IN). This system is designed to use avian myeloblastosis virus (AMV) reverse transcriptase for first-strand synthesis and the Expand high-fidelity blend of thermostable DNA polymerases, which consists of Taq DNA polymerase and a proofreading polymerase, for the PCR part. The reaction was carried out in a 50-µl volume containing 50–100 ng of the total RNA, 10 pM of forward and reverse primers specific for heat shock protein (HSP)70 (GenBank acc. no. NM005923, sense 5'-CAGAAGGATCCATGGCCAAAGC-3' and antisense 5'-GGAATTCATCTACCTCCTCAATGG-3'); 1x PCR buffer with Mg2+, 0.2 mM dNTPs, 5 mM DTT solution, 5–10 U of RNAse inhibitor, and 0.05 U/µl reaction of the enzyme mix (High Fidelity enzyme mix, reverse transcriptase, AMV in storage buffer). An initial RT step was performed at 50°C for 30 min and at 94°C for 2 min for one cycle, followed by 35 cycles (denaturation at 94°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 1.5 min), and finally one cycle at 72°C for 5 min. In addition, a pair of primers was designed to amplify a portion of the {beta}-actin transcript that spans an exon-exon boundary (forward 5'-ACC CAC ACT GTG CCC ATC TA-3' and reverse 5'-CGG GAA CCG CTC ATT GCC-3'). {beta}-Actin was used as the housekeeping gene to provide an internal marker for mRNA integrity within the experiment. PCR products were separated on (1.5% wt/vol) agarose gels and visualized by ethidium bromide staining under UV light. Image capture and density analysis of bands was done with the SynGene gel documentation system (SynGene-Synoptics, Cambridge, UK), and the intensity of bands was expressed as the ratio of HSP70 to {beta}-actin (36).

Western blot analysis. Total protein from the muscle tissue was extracted, and 20 µg of the protein were separated on a 4–20% SDS-polyacrylamide gel under reducing conditions and transferred to nitrocellulose membranes (Hybond-C; Amersham Pharmacia Biotech) in a semidry blotting chamber. After blockage of nonspecific binding sites with 5% nonfat milk in TBS containing 0.1% Tween 20 (Sigma, St. Louis, MO), blots were incubated in 1:1,000 dilution of anti-vascular endothelial growth factor mouse monoclonal antibody, anti-growth arrest and DNA damage-inducible (GADD)45{gamma} goat polyclonal antibody (Santa Cruz Biotechnology), anti-dynein mouse monoclonal antibody (Chemicon International, Temecula, CA), anti-myosin goat polyclonal antibody (ventricular light chain, 1:2,500 dilution; Cortex Biochem, San Leandro, CA), and anti-actin rabbit polyclonal antibody (1:2,500 dilution; Sigma) as an internal control for 2 h at room temperature. After extensive washing, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit IgG, anti-mouse IgG, and anti-goat IgG, respectively (final concentration 1:2,000), for 90 min at room temperature. Bound antibodies were detected with ECL Western blotting detection reagents (Amersham Pharmacia Biotech) according to the manufacturer's instructions.

Statistical analysis. Data are presented as means ± SE. The Wilcoxon signed rank test was used for comparisons within groups, and an unpaired t-test was used between the groups. Statistical significance was accepted at P < 0.05. The statistical program used was Minitab.

This study was approved and conducted in compliance with the requirements for institutional review and informed consent at The University of Texas Medical Branch, Galveston, TX.


    RESULTS
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 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Patient characteristics, heart rates, and net protein balance. Age, gender distribution, weight, and burn size were not significantly different between placebo and drug groups (Table 2). Some children randomized to receive propranolol did not, in fact, receive continuous drug therapy, as administration orders are routinely canceled at surgery and the automatic recording for the blinded medication not reestablished in a timely manner. Initially, propranolol significantly decreased heart rate by 15% compared with the average for 3 days before the study (P < 0.01). An average lower heart rate of 8% was maintained in the drug groups compared with control until patients were 95% healed. Admission weight to 95% healed increased by 0.3 ± 4.9% in those receiving propranolol and decreased by 4.5 ± 3.7% in those not treated with propranolol (P = 0.44). The change in net protein balance for children receiving placebo was –14.3 ± 12.9 nmol phenylalanine · min1 · 100 ml leg volume1 (n = 23) compared with +69.3 ± 34.9 nmol phenylalanine · min1 · 100 ml leg volume1 (n = 14) in those receiving propranolol (P = 0.012).


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Table 2. Demographics of study patients

 

Blood pressure, temperature, and glucose levels did not differ significantly between groups. Neither the control group nor the propranolol group required mechanical ventilation except for brief periods perioperatively, and none had clinically identified pneumonia.

Gene expression patterns. Comparison of 12,000 genes in burned children receiving placebo showed increased expression of two genes with time compared with baseline, whereas children receiving propranolol showed increased expression of nine genes compared with placebo while the expression of five genes decreased. Thus 0.15% of the genes were altered in muscle from burned children treated with propranolol compared with placebo.

There were 14 genes pertinent to this study that were affected by propranolol treatment as determined by DNA microarray analysis (Table 3). Protein tyrosine phosphatase (acc. no. X68277 [GenBank] ) and diazepam-binding inhibitor (acc. no. AI557240 [GenBank] ) genes were increased two-fold when baseline was compared with placebo. Categories of genes affected were transcription factors, growth factors, stress response modulators, and muscle-associated proteins. We further described genes whose expression increased or decreased according to their function and involvement in metabolic pathways (9, 16, 17, 27, 28, 33, 38, 39, 44) (Table 4).


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Table 3. Muscle gene expression profile changed > 1.5 fold by propranolol treatment in burned children

 

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Table 4. List of genes altered in propranolol-treated burn children, indicating their function

 

Verification of mRNA. Changes observed in mRNA expression were verified using RT-PCR for HSP70 gene expression to show the corresponding stimulatory effect of propranolol. The ratio of HSP70 mRNA to {beta}-actin after propranolol treatment was 1.21 ± 0.06 compared with 0.54 ± 0.01 for placebo (P < 0.05). We further verified mRNA changes through the expression of related proteins by Western blot analyses. Dynein and GADD45{gamma}, whose transcription was upregulated by propranolol treatment, showed a significant increase in their protein expression (Fig. 2). The inhibitory effect of propranolol on myosin light chain was verified by a significant decrease in protein expression (Fig. 2).



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Fig. 2. Western blot using antibodies against vascular endothelial growth factor (VEGF), dynein, growth arrest and DNA damage-inducible (GADD){gamma}, and myosin light chain in muscle from burned children. {beta}-Actin is depicted to show any loading differences. Each lane represents total protein extracted from 1 muscle biopsy.

 


    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Hypermetabolism accompanying burn trauma has a significant effect on protein synthesis and metabolism. The bulk of muscle protein is made up of myofibrillar components, specifically actin and myosin. Animal studies have shown a decrease in net myofibrillar protein synthesis in response to burn and sepsis. It is the myofibrillar protein that constitutes the majority of protein undergoing breakdown and causing net protein loss. Adequate nutritional support with additional protein and amino acid intake several times higher than the normal requirement has failed to completely reverse the net protein catabolism. Increased activity of several protein breakdown pathways has shown the increase in expression and activity of the ubiquitin-proteosome pathways. {beta}-Adrenergic blockade has been used with success in several pathological conditions from excessive quantities of circulating thyroid hormone or catecholamines.

Increased heart rates, myocardial work, and lipolysis are responses to the high levels of catecholamines associated with thermal injury. Propranolol, given during acute hospitalization, has been proved effective in children for the control of cardiovascular derangements. Gene chip array analysis indicates that there are other possible attributes of propranolol in treating burned children. We had a therapeutic goal of decreasing the heart rate by 10–15%, a decrease that we had previously shown to be safe (1, 42, 43). The mean heart rate reduction was 8%, which was similar to that obtained by Baron et. al (3). They further showed a maximum average daily reduction of nearly 12% by increasing propranolol given orally up to 3 mg · kg1 · day1. Any pharmacotherapy carries risks. Given carelessly, propranolol could cause hypoperfusion as a result of decreased cardiac output, particularly in patients with sepsis. In patients with asthma, it could induce severe bronchospasm. The patients underwent continuous hemodynamic and respiratory monitoring, and there were no complications related to the therapy. We found no significant decrease in blood pressure with propranolol treatment in these doses. In our study, propranolol did not change the inward transport of protein but did increase the efficiency of protein synthesis in muscle. With an increase in protein synthesis while protein breakdown remained unchanged, the net balance of protein increased. This supports the hypothesis that the mechanism for improved net protein balance across the leg was the stimulation of muscle protein synthesis, which corresponds well with the increase in expression of genes involved in protein synthesis like HSP70 (GRP-78KD-NM005347), which plays a significant role in the recovery of mRNA translation during the stress response and eukaryotic initiation factor 2 cycling and ribosomal mRNA loading.

A comparison of the gene expression profile in muscle biopsies before and after propranolol treatment showed a general stimulatory effect on nine genes and an inhibitory effect on five genes whose functions vary (Table 4). The stability of the genetic profile in untreated individuals over this period of time is consistent with our previous observations that show a stability of the flow phase of the hypercatabolic response between 5 and 45 days after burn, during which time protein degradation and increased glucose inflow are predominant responses (20, 21). Two genes, protein tyrosine phosphatase (acc. no. X68277 [GenBank] ) and diazepam-binding inhibitor (acc. no. AI557240 [GenBank] ), were perturbed by our standard treatment, which involves administration of large doses of antianxiety medicine after the 1st wk after burn, and these changes simply reflect the effect of an additional drug (diazepam) that was given to all of the patient population. The effects of propranolol treatment on gene expression in burned children compared with untreated patients are perhaps more comprehensive in that a broad category of genes is suppressed by propranolol. For example, propranolol treatment stimulated 2.5 times the expression of GADD45{gamma}, which was found to have been suppressed in the untreated burned children, and HSP70 (GRP-78KD-NM005347), which plays a significant role in the recovery of mRNA translation during the stress response and eukaryotic initiation factor 2 cycling and ribosomal mRNA loading. The expression of fructose-1,6-biphosphatase 2 (Y12235 [GenBank] ) was suppressed nearly threefold after propranolol treatment. Fructose-1,6-bisphosphatase is a key regulatory enzyme of gluconeogenesis that catalyzes the hydrolysis of fructose 1,6-bisphosphate to generate fructose 6-phosphate and inorganic phosphate. Deficiency of fructose-1,6-bisphosphatase is associated with fasting hypoglycemia and metabolic acidosis because of impaired gluconeogenesis. Zinc finger protein-145 (AJ242778 [GenBank] ), which has been known to be regulated by activating protein (AP)-1 type transcription factor and involved in the negative feedback regulation of NF-{kappa}B activation, was suppressed twofold. The indirect involvement of AP-1/NF-{kappa}B signal transduction pathways is consistent with stress response pathways. Zinc finger protein-145 (A20) was first cloned as an immediate early response gene upregulated by TNF-{alpha} in endothelial cells and is also critical for terminating TNF-induced NF-{kappa}B responses. The nature of the changes may be more specific to severe trauma in light of the prolonged metabolic changes. Although the number of patients reported in this study is relatively small, the comparison of gene expression patterns in a paired study design provides evidence that the results are likely valid and not outliers associated with the large number of gene probes used (12,000, Affymetrix). It is possible that some of the important changes that might occur at a lower level of significance might be masked, as the degree of change is volume averaged over the entire muscle volume. This suggests that there are genes of low abundance that are highly regulated within small compartments of muscle that escape the level of detection possible with this assay.

The sequential muscle biopsies are one strength of our study, as they serve as a powerful control. A more thorough analysis with a larger data set is likely to provide further support of long-term changes in the levels of gene expression for signaling molecules, which are known to be part of the metabolic response to burns. The participation of a large number of genes critical to inflammation and other stress response signal transduction pathways in response to propranolol treatment suggests a beneficial mechanism for propranolol as consisting principally of the stimulation of gene expression resulting from thermal trauma. We believe that alterations in the expression of these selective genes may help identify molecular changes for specific therapeutic and diagnostic purposes.


    DISCLOSURES
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 ABSTRACT
 METHODS
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 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported by Shriners Hospitals for Children Grant no. 8660 and National Institute of General Medical Sciences Grants 1P50 [PDB] GM-60338-01 and 5R01 GM-57295-03.


    ACKNOWLEDGMENTS
 
We express our appreciation to Dr. Tom Wood (Genomics Core Facility, University of Texas Medical Branch) for help in processing the samples for DNA microarray hybridizations.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. E. Barrow, Shriners Hospitals for Children, 815 Market St., Galveston, TX 77550.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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