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Biomedical subjects

E W Holmes

Publications and source records attributed to E W Holmes.

At least 19 recordsLinked to original sources
PubMed2002

Hepatic expression of polymerase beta, Ref-1, PCNA, and Bax in WY 14,643-exposed rats and hamsters.

The hepatic levels of three protein markers of oxidative stress, polymerase beta, Ref-1, and PCNA, and of the pro-apoptotic protein, Bax, were quantitated after exposure to WY 14,643 (500 ppm in the feed) for 6 or 34 days in a rodent that is susceptible peroxisome proliferator (PP)-induced liver tumors (the Sprague Dawley rat) and in a rodent that is relatively resistant PP-induced liver tumors (the Syrian hamster). The analysis of detergent-extracted whole liver homogenates by immunoblotting showed a marked increase in the abundance of a 45-kDa variant of polymerase beta immunoreactivity and significant increases in the expression of Ref-1 and PCNA in WY 14,643-exposed rats. In contrast. WY 14,643-exposed hamsters expressed only trace levels of the polymerase beta variant and showed significant decreases in the expression of Ref-1 and PCNA. Long-term WY 14,643 exposure was associated with marked decreases in Bax expression in both species. Dose-response studies in the rat showed that the hepatic expression of the polymerase beta and Ref-1 were significantly increased after 6 days of exposure to WY 14,643 at levels of 5 and 50 ppm, respectively. The analysis of subcellular fractions of rat liver showed that the pathological increases in the levels of polymerase beta, Ref-1, and PCNA were especially prominent in mitochondria-enriched particulate liver subfractions. These results indicate that WY 14,643 exposure is associated with an increase in oxidative stress to the liver and that liver mitochondria are a major target of WY 14,643-associated liver damage. Our data are consistent with the hypothesis that the chronic overexpression of mutagenic or oncogenic effectors like polymerase beta and Ref-1 in a setting of increased hepatocyte proliferation and decreased apoptosis may facilitate peroxisome proliferator-induced hepatocellular carcinoma in the rat.

Animals↗
PubMed2001

A novel bipartite intronic splicing enhancer promotes the inclusion of a mini-exon in the AMP deaminase 1 gene.

Alternative splicing of the 12-base exon 2 of the adenosine monophosphate deaminase (AMPD) gene is subject to regulation by both cis- and trans-regulatory signals. The extent of exon 2 inclusion is stage- and cell type-specific and is subject to the physiological state of the cell. In adult skeletal muscle, a cell type that regulates the activity of this allosteric enzyme at several levels, the exon 2-plus form of AMPD, predominates. We have performed a systematic analysis of the cis-acting regulatory sequences that reside in the intron immediately downstream of this mini-exon. A complex element comprising sequences that enhance exon 2 inclusion and sequences that counteract this effect resides in the middle of this intron. We demonstrate that the enhancing component is bipartite, with more than a kilobase of sequence separating the two functional sites. The presence of even minimal levels the mini-exon in the fully processed AMPD mRNA requires both of these sites, neither of which appears in any other published splicing enhancer. An RNA binding activity derived from a muscle cell line requires both of the enhancing sites. Mutations in either of the sites that eliminate exon 2 inclusion abrogate this binding activity.

3T3 Cells↗
PubMed2001

Preventing gut leakiness by oats supplementation ameliorates alcohol-induced liver damage in rats.

Only 30% of alcoholics develop liver disease (ALD) suggesting that additional factors are needed. Endotoxin is one such factor, but its etiology is unclear. Since the gut is the main source of endotoxin, we sought to determine whether an increase in intestinal permeability (leaky gut) is required for alcohol-induced endotoxemia and liver injury and whether the gut leakiness is preventable. For 10 weeks, rats received by gavage increasing alcohol doses (to 8 g/kg/day) and either oats (10 g/kg) or chow b.i.d. Intestinal permeability was then assessed by urinary excretion of lactulose and mannitol. Liver injury was evaluated histologically, biochemically (liver fat content), and by serum aminotransferase. Alcohol caused gut leakiness that was associated with both endotoxemia and liver injury. Oats prevented these changes. We conclude that chronic gavage of alcohol in rats is a simple experimental model that mimics key aspects of ALD, including endotoxemia and liver injury, and can be useful to study possible mechanisms of endotoxemia in ALD. Since preventing the gut leakiness by oats also prevented the endotoxemia and ameliorated liver damage in rat, our results suggest that alcohol-induced gut leakiness 1) may cause alcohol-induced endotoxemia and liver injury and 2) may be the critical cofactor in the 30% of alcoholics who develop ALD. Further studies are needed to determine whether ALD in humans can be prevented by preventing alcohol-induced gut leakiness, studies that should lead to the development of useful therapeutic agents for the prevention of ALD.

Animals↗
PubMed2000

Circadian rhythm of serum total homocysteine in men.

Serum homocysteine levels were examined in a 24-hour study of 7 healthy and 5 diabetic men, revealing a statistically significant circadian rhythm (p = 0.030), normal concentrations of 11.83 +/- 1.2 vs 12.99 +/- 1.2 micromol/L, with peak values occurring during the evening (10:37 P.M.) and lowest levels occurring during the morning. These findings imply that increased atherosclerotic risk in insulin-resistant diabetics during morning hours does not appear to be explained by differences in homocysteine levels in the normal population.

Adult↗
PubMed2000

Positive and negative elements mediate control of alternative splicing in the AMPD1 gene.

The second exon of the AMP deaminase (AMPD) 1 gene is alternatively spliced in response to stage-specific signals elaborated during myocyte differentiation. Since inheritance of the mutation in exon 2 of the AMPD1 gene has been recently shown to be associated with a better prognosis of congestive heart failure and the alternative splicing of exon 2 modulates the residual activity of AMPD1 in individuals with this mutant allele, the regulatory mechanism of alternative splicing in the AMPD1 gene is clinically intriguing. Retention or exclusion of exon 2 results from the interplay between negative and positive elements in the primary transcript. Exon 2 is intrinsically defective and difficult to recognize. Herein, we show that this property of exon 2 is the consequence of three defects; a suboptimal 3' splice acceptor site, a suboptimal 5' splice donor site and the small size of the exon. An improvement in any one of these defects relieves the masking of this exon. Further, this defective exon can only be identified in the presence of the adjacent downstream intron.

3T3 Cells↗
PubMed2000

Exacerbation of alcoholic liver injury by enteral endotoxin in rats.

Increased gut permeability (leaky gut) and endotoxin-mediated Kupffer cell activation are proposed as the mechanisms of alcoholic liver injury. Although ethanol feeding is shown to sensitize the liver for injury induced by parental administration of lipopolysaccharide (LPS), how enteral LPS loading affects alcoholic liver injury is yet to be tested. The present study provides direct evidence for enhanced entrance to portal circulation of LPS enterally administered to the intragastric ethanol infusion model. Portal and systemic blood endotoxin levels increased to 43.0 +/- 4.1 and 6.2 +/- 4.3 pg/mL at 2 hours following enteral LPS administration (5 mg/kg) in alcohol-fed animals, while no such increases were observed in pair-fed controls. However, endotoxin levels in systemic blood of alcohol-fed rats were reduced to 0 to 1. 5 pg/mL 16 hours after LPS administration. Weekly enteral administration of LPS to the model for 9 weeks exacerbated an increase in plasma alanine transaminase (ALT) levels (227 +/- 75 vs. 140 +/- 70; P <.01), mononuclear infiltration (25 +/- 22 vs. 6.4 +/- 4.4/10 mm(2); P =.02), sinusoidal congestion, and spotty necrosis, and induced diffuse coagulative necrosis and centrilobular fibrosis in some animals. Reverse-transcription polymerase chain reaction (RT-PCR) analysis confirmed the LPS effect at the tissue level by demonstrating accentuated induction of tumor necrosis factor alpha (TNF-alpha) and Cox-2 mRNA. In conclusion, enteral LPS administration potentiates alcoholic liver necrosis, inflammation, and fibrosis despite efficient endotoxin clearance by the liver and mild systemic endotoxemia that occurs episodically following enteral LPS challenge.

Animals↗
PubMed2000

Measuring contributions to the research mission of medical schools.

The authors of this article, who were the members and staff of a research panel formed by the AAMC as part of its mission-based management initiative, reflect on the growing interest in quantitative information in the management of the research mission of medical schools. They note the serious limitations of any such system of measures for research, particularly its inability to represent directly the quality of the research effort. Despite these concerns, the authors acknowledge that leaders in academic medicine have always used quantitative measures in one form or another to compare performance or assess progress. Two factors appear to be driving increases in this practice: (1) the need to demonstrate to institutional stakeholders that resources are being used wisely and that the school's performance justifies continued investment in the research mission; and (2) the need to fashion an economic strategy to manage precious institutional resources, particularly research space. Given these realities, the authors offer guidelines for the proper development and use of measures to assess contributions by faculty, departments, and institutions to the research mission. They also comment on the measures most commonly used in four areas: grants and other revenue-generating activities; publications; faculty members' research reputation and contributions to the national research enterprise; and support to the general research mission of the school. The authors conclude that quantitative information can help institutional leaders in important management decisions. However, the potential for misuse is great. The key is always to regard this information as an aid to judgment, not a substitute for it.

Research↗
PubMed1999

Common variant in AMPD1 gene predicts improved clinical outcome in patients with heart failure.

BACKGROUND: This study was undertaken to identify gene(s) that may be associated with improved clinical outcome in patients with congestive heart failure (CHF). The adenosine monophosphate deaminase locus (AMPD1) was selected for study. We hypothesized that inheritance of the mutant AMPD1 allele is associated with increased probability of survival without cardiac transplantation in patients with CHF. METHODS AND RESULTS: AMPD1 genotype was determined in 132 patients with advanced CHF and 91 control reference subjects by use of a polymerase chain reaction-based, allele-specific oligonucleotide detection assay. In patients with CHF, those heterozygous (n=20) or homozygous (n=1) for the mutant AMPD1 allele (AMPD1 +/- or -/-, respectively) experienced a significantly longer duration of heart failure symptoms before referral for transplantation evaluation than CHF patients homozygous for the wild-type allele (AMPD1 +/+; n=111; 7.6+/-6.5 versus 3.2+/-3.6 years; P<0.001). The OR of surviving without cardiac transplantation >/=5 years after initial hospitalization for CHF symptoms was 8.6 times greater (95% CI: 3.05, 23.87) in those patients carrying >/=1 mutant AMPD1 allele than in those carrying 2 wild-type AMPD1 +/+ alleles. CONCLUSIONS: After the onset of CHF symptoms, the mutant AMPD1 allele is associated with prolonged probability of survival without cardiac transplantation. The mechanism by which the presence of the mutant AMPD1 allele may modify the clinical phenotype of heart failure remains to be determined.

AMP Deaminase↗
PubMed1999

Determination of protein carbonyl groups by immunoblotting.

Free radical-mediated oxidation of proteins results in the formation of carbonyl groups in quantities that reflect the intensity of the oxidative stress. We have developed an immunochemical technique for the quantification of carbonyl groups in protein samples prepared from small tissue samples and cell cultures. Protein samples were slot-blotted onto a polyvinylidene difluoride membrane, which was sequentially treated with 2,4-dinitrophenylhydrazine (DNPH), a primary antibody specific for the 2,4-dinitrophenol group, and a peroxidase-labeled second antibody. After the blots were developed with a chemiluminescent substrate and exposed to X-ray film, the level of immunostaining was quantitated by densitometry. Using oxidized bovine serum albumin as a standard and loading 5 microg of protein per slot, the minimum detectable carbonyl content was approximately 60 pmol carbonyl/mg protein. When necessary, nonspecific staining by noncarbonyl constituents in complex sample matrices was accounted for by using sodium borohydride-treated blanks. Results by the new method were highly correlated (r = 0.932, P < 0.0001) with those of the standard DNPH-based spectrophotometric technique. The coefficient of variation at a carbonyl level of 1.5 nmol/mg protein was 9.7%. The utility of this new method was demonstrated by measuring protein oxidation in cultured human colon cells (SW620) that were briefly exposed to H2O2.

Cells, Cultured↗
PubMed1999

Altered purine and glycogen metabolism in skeletal muscle during exercise in patients with heart failure.

Plasma levels of ammonia and hypoxanthine (HX) can be indices of purine nucleotide degradation. The present study determined if patients with heart failure (HF) have altered exercise plasma ammonia and HX levels relative to the peak work rate performed. Blood lactate, plasma ammonia, and plasma HX levels were measured in 59 patients with HF (New York Heart Association [NYHA] classes I:20, II:21, and III:18) and 21 controls at rest and after a maximal cardiopulmonary exercise test. The peak work rate (normal and NYHA I, II, and III, 163+/-11, 152+/-9, 94+/-5, and 69+/-5 W) and peak oxygen uptake ([VO2] 32.3+/-1.7, 25.1+/-0.9, 18.6+/-0.5, and 14.1+/-0.6 mL/min/kg) decreased as the NYHA functional class increased. The increment from rest to peak exercise (delta) for lactate ([(delta)lactate] 6.1+/-0.3, 4.8+/-0.4, 4.6+/-0.3, and 2.9+/-0.3 mmol/L), (delta)ammonia (132+/-14, 119+/-20, 94+/-13, and 32+/-6 microg/dL), and (delta)HX (33.5+/-3.4, 24.9+/-4.7, 20.6+/-3.0, and 9.9+/-1.2 micromol/L) was progressively smaller as HF worsened. The ratio for (delta)lactate to peak work rate (0.037+/-0.003, 0.032+/-0.004, 0.049+/-0.003, and 0.042+/-0.005) was higher in classes II to III HF, while the ratio for (delta)ammonia to peak work rate (0.81+/-0.14, 0.78+/-0.16, 0.99+/-0.11, and 0.47+/-0.11) was significantly lower in class III HF. In summary, patients with HF exhibited a smaller ammonia response with a higher lactate response to exercise when normalized with the peak work rate. These results suggest there may be an altered purine and glycogen metabolism during exercise in skeletal muscle in patients with HF.

Ammonia↗
PubMed1999

Leaky gut in alcoholic cirrhosis: a possible mechanism for alcohol-induced liver damage.

OBJECTIVE: Only 30% of alcoholics develop cirrhosis, suggesting that the development of alcohol-induced liver injury requires one or more additional factors. Animal studies have shown that gut-derived endotoxin is one such factor. Because increased intestinal permeability has been shown to cause endotoxemia, we hypothesized that increased gastrointestinal permeability contributes to the pathogenesis of alcoholic liver disease. This study aimed to measure gastroduodenal and intestinal permeability in alcoholics with and without chronic liver disease and in nonalcoholic subjects with chronic liver disease. METHODS: Gastroduodenal permeability was assessed by measurement of urinary excretion of sucrose after oral administration. Intestinal permeability was assessed by measurement of urinary lactulose and mannitol after oral administration of these sugars. RESULTS: Alcoholics with no liver disease showed a small but significant increase in sucrose excretion. Alcoholics with chronic liver disease demonstrated a marked and highly significant increase in urinary sucrose excretion relative to the controls, to the alcoholics with no liver disease, and to the nonalcoholics with liver disease. Alcoholics with chronic liver disease demonstrated a marked and highly significant increase in both lactulose absorption and in the urinary lactulose/mannitol ratio (alcoholics 0.703 vs controls 0.019, p = 0.01). In contrast, alcoholics with no liver disease and nonalcoholics with liver disease showed normal lactulose absorption and normal lactulose/mannitol ratio. CONCLUSION: Because only the alcoholics with chronic liver disease had increased intestinal permeability, we conclude that a "leaky" gut may be a necessary cofactor for the development of chronic liver injury in heavy drinkers.

Aged↗
PubMed1999

Analysis of risk factors for the development of bronchiolitis obliterans syndrome.

Chronic rejection after lung transplantation, manifesting as bronchiolitis obliterans syndrome (BOS), has become the dominant challenge to long-term patient and graft survival. In order to elucidate risk factors for development of BOS we utilized the 1995 revision of the working formulation for the classification of lung allograft rejection (), and devised a quantitative method to retrospectively study lung transplant biopsies from all patients who survived at least 90 d. All transbronchial biopsies were regraded 0 to 4 for acute perivascular rejection and lymphocytic bronchitis/bronchiolitis (LBB), and the grades were totaled over a period of time to give two scores, respectively, for each patient. Also examined were timing of acute rejection and LBB episodes and decreased immunosuppression defined as two or more cyclosporine A levels < 200 ng/ml. Sixty-six patients with BOS and 68 with no BOS (NBOS) satisfied our criteria for inclusion in the study. Demographics including age, sex, and primary diagnoses were similar. The mean perivascular score for BOS was 6.2 over a mean follow-up of 822 d (range, 113 to 2,146) compared with 3.2 for NBOS over 550 d (range, 97 to 1,734) mean follow-up. Airway scores were 5.3 and 1.7, respectively, for the same follow-up periods. There was no correlation between length of follow-up and rejection or LBB scores, although mean length of follow-up for the two groups was significantly different. Late acute rejection and LBB were significantly associated with BOS as was decreased immunosuppression. In addition to perivascular rejection, LBB, late acute rejection, and decreased immunosuppression are significant risk factors for the development of BOS. Analysis of the current data leads us to believe that LBB, in the absence of infection, is in fact a manifestation of acute rejection, with similar implications for graft function as acute perivascular rejection.

Adolescent↗
PubMed1998

Glutathione content of colonic mucosa: evidence for oxidative damage in active ulcerative colitis.

Oxidative stress appears to play a role in the tissue damage of active ulcerative colitis, and it has been suggested that a defect in mucosal antioxidant defenses is a etiological factor in the disease. This study was undertaken to investigate the mucosal content and oxidation state of glutathione in ulcerative colitis in the active and inactive states and to examine the relationship between glutathione content and disease activity in this patient population. Endoscopic biopsies of colon mucosa were collected from normal subjects, from macroscopically normal tissue of patients with inactive and active ulcerative colitis, and from inflamed tissue of patients with active ulcerative colitis. The mucosal contents of GSH and GSSG were determined by liquid chromatography. We found no significant differences in tissue contents of reduced glutathione among the four groups. The median tissue level of oxidized glutathione in inflamed mucosa from patients with active ulcerative colitis was increased 1.7-fold (P = 0.017) over that of patients with inactive disease. The oxidized glutathione content of the mucosa also showed significant positive correlations with clinical and histological indices of disease severity among ulcerative colitis patients. In conclusion, a change in the redox status of mucosal glutathione is associated with inflammation and disease activity in ulcerative colitis. This change appears to be a consequence of inflammation rather than a pathogenic factor for the disease.

Colitis, Ulcerative↗
PubMed1998

Expression and regulation of interferon-gamma-induced tryptophan catabolism in cultured skin fibroblasts.

Interferon-gamma (IFN-gamma)-induced, indoleamine dioxygenase-catalyzed tryptophan catabolism was studied in cultured human foreskin fibroblasts using the increase in cellular kynurenine synthesis as an index of gene expression. The time courses of the inhibition of IFN-gamma-induced kynurenine synthesis by actinomycin D and cycloheximide showed that the indoleamine dioxygenase gene was transcribed as early as 2 h and translated as early as 5 h after initiation of IFN treatment. Expression was completely inhibited by the Ser/Thr kinase inhibitor, H-7 (66 microM), during the first 2 h after IFN-gamma treatment. Prolonged pretreatment of cells with high concentrations of staurosporine (380 nM) or genestein (610 microM) inhibited expression by 38% and 53%, respectively. Genestein also inhibited expression when it was added to cultures between 8 and 24 h after IFN-gamma treatment. The expression of kynurenine synthesis was inhibited by A23817 during the first 4 h after IFN treatment by mechanisms that were independent of cyclooxygenase, calmodulin, and calcineurin. Exogenous gangliosides (bovine brain gangliosides and purified GM1) inhibited IDO expression throughout the first 24 h after IFN-gamma treatment by mechanisms that did not involve effects on Ca2+ channels. Other biologic response modifiers, including phorbol myristic acetate, arachidonic acid, lipopolysaccharide, analogs of cAMP and cGMP, W-7, and sphingosine, did not induce IDO in the absence of IFN-gamma, nor did they modulate IFN-gamma-induced expression. These results indicate that the expression of kynurenine synthesis is modulated at the transcriptional and posttranscriptional levels by protein tyrosine kinase and by a Ser/Thr kinase with properties distinctly different from those of conventional protein kinase C. The capacity for attenuation of this IFN-gamma-induced response over its entire time course by many effectors and through multiple cellular signaling pathways may represent a mechanism for fine-tuning the level of oxidative tryptophan metabolism to meet the needs of a particular cytostatic or antiproliferative response.

Calcimycin↗
PubMed1998

Control of AMP deaminase 1 binding to myosin heavy chain.

AMP deaminase (AMPD) plays a central role in preserving the adenylate energy charge in myocytes following exercise and in producing intermediates for the citric acid cycle in muscle. Prior studies have demonstrated that AMPD1 binds to myosin heavy chain (MHC) in vitro; binding to the myofibril varies with the state of muscle contraction in vivo, and binding of AMPD1 to MHC is required for activation of this enzyme in myocytes. The present study has identified three domains in AMPD1 that influence binding of this enzyme to MHC using a cotransfection model that permits assessment of mutations introduced into the AMPD1 peptide. One domain that encompasses residues 178-333 of this 727-amino acid peptide is essential for binding of AMPD1 to MHC. This region of AMPD1 shares sequence similarity with several regions of titin, another MHC binding protein. Two additional domains regulate binding of this peptide to MHC in response to intracellular and extracellular signals. A nucleotide binding site, which is located at residues 660-674, controls binding of AMPD1 to MHC in response to changes in intracellular ATP concentration. Deletion analyses demonstrate that the amino-terminal 65 residues of AMPD1 play a critical role in modulating the sensitivity to ATP-induced inhibition of MHC binding. Alternative splicing of the AMPD1 gene product, which alters the sequence of residues 8-12, produces two AMPD1 isoforms that exhibit different MHC binding properties in the presence of ATP. These findings are discussed in the context of the various roles proposed for AMPD in energy production in the myocyte.

AMP Deaminase↗
PubMed1997

Coupled enzymatic assay for the determination of sucrose.

An enzymatic spectrophotometric assay for the determination of sucrose in unextracted samples of serum and urine was developed. The method entailed the coupling of invertase-catalyzed sucrose hydrolysis with a fructose dehydrogenase-catalyzed oxidation of the liberated fructose. The latter reaction generated reducing equivalents that were transferred to a tetrazolium salt with a concomitant increase in absorbance at 570 nm. The assay, which was carried out in microtiter plates, had a minimum detectable sucrose concentration of 0.03 mmol/liter and run-to-run and within-run coefficients of variation of 7.5 and 6.7%, respectively, and showed a good correlation with urine sucrose determination by GLC (r = 0.92). The assay range of 0.03-2.10 mmol/liter is suitable for the quantitation of serum sucrose following iv administration and for the quantitation of urine sucrose at basal levels and following the consumption of an oral test dose of sucrose. This method was used to analyze urine samples from a group of human subjects who consumed 20 g of sucrose for the assessment of gastroduodenal permeability. This convenient assay provides for the rapid and specific estimation of sucrose and has the potential to be used in a variety of manual, semiautomated, or automated formats.

Carbohydrate Dehydrogenases↗