Allergy in coronary in-stent restenosis.
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Coronary Disease↗
Biomedical subjects
T Ha
Publications and source records attributed to T Ha.
Physical activity-antidepressant treatment combination: impact on brain-derived neurotrophic factor and behavior in an animal model.
The mechanism of antidepressant action, at the cellular level, is not clearly understood. It has been reported that chronic antidepressant treatment leads to an up-regulation of brain-derived neurotrophic factor (BDNF) mRNA levels in the hippocampus, and that physical activity (voluntary running) enhances this effect. We wished to investigate whether BDNF expression brought about by these interventions may overcome deficits caused by acute stress, and might impact behavior in an animal model. In this report, we have tested the hypothesis that the combination of the antidepressant, tranylcypromine, and physical exercise could lead to decreased neurotrophin deficits and enhanced swimming time in animals that have been forced to swim in an inescapable water tank. Rats were either treated with tranylcypromine, engaged in voluntary running, or both for one week. After these treatments, the animals underwent a two-day forced swimming procedure. BDNF mRNA levels were significantly depressed in untreated animals subjected to forced swimming. Animals that either underwent prior activity or received antidepressant showed BDNF mRNA levels restored to baseline. Animals receiving the combined intervention showed an increase in hippocampal BDNF mRNA well above baseline. Swimming time during a five-minute test was significantly enhanced in animals receiving the combined intervention over untreated animals. Swimming time was not significantly enhanced over that of animals receiving antidepressant alone, however. Enhanced swimming time correlated with increased levels of BDNF mRNA in one hippocampal sub-region (CA4-hilus). These results suggest that the combination of exercise and antidepressant treatment may have significant neurochemical, and possibly behavioral, effects. In addition, these results support the possibility that the enhancement of BDNF expression may be an important element in the clinical response to antidepressant treatment. The induction of BDNF expression by activity/pharmacological treatment combinations could represent an important intervention for further study, to potentially improve depression treatment and management.
Animals↗
Single-molecule fluorescence methods for the study of nucleic acids.
Single-molecule fluorescence methods and biomechanical tools provide exciting new opportunities to probe biochemical processes in unprecedented detail. The detection and spectroscopy of single fluorophores have recently been used to observe conformational changes and biochemical events involving nucleic acids. A number of fluorescence observables, including localization, quenching, polarization response and fluorescence resonance energy transfer, have been utilized. An exciting new opportunity of combining fluorescence methods and biomechanical tools to study the structural changes and functions of enzymes that participate in nucleic acid metabolism has also arisen.
Biochemistry↗
Nucleotide excision repair gene XPD polymorphisms and genetic predisposition to melanoma.
The nucleotide excision repair pathway has evolved to deal with UV light-induced DNA damage. Individuals with the rare inherited nucleotide excision repair deficiency disease xeroderma pigmentosum have a 1000-fold increased incidence of skin cancer. We are interested in the possibility that more subtle changes in nucleotide excision repair genes, resulting in either a reduced capacity for repair or in altered interactions between repair proteins and components of the cell cycle control machinery, might constitute important genetic risk factors for the development of skin cancer in the general population. To investigate this hypothesis we have compared the frequency of polymorphisms in exons 6, 22 and 23 of the XPD gene in melanoma patients and a control group. For each of these two allele polymorphisms one of the alleles was over-represented in the melanoma group and there was a significant association with melanoma. Importantly, this association did not extend to markers immediately flanking the XPD gene, thus providing evidence that XPD gene polymorphisms might predispose to melanoma in the general population. There is a report that one of the polymorphic XPD alleles (exon 23 Lys), which is over-represented in the melanoma group, has reduced repair proficiency and we discuss the possibility that this is the causal change to the XPD gene that predisposes to melanoma.
Alleles↗
Early activation of IKKbeta during in vivo myocardial ischemia.
We have demonstrated that in vitro brief ischemia activates nuclear factor (NF)-kappaB in rat myocardium. We report in vivo ischemia-reperfusion (I/R)-induced NF-kappaB activation, IkappaB kinase -beta (IKKbeta) activity, and IkappaBalpha phosphorylation and degradation in rat myocardium. Rat hearts were subjected to occlusion of the coronary artery for up to 45 min or occlusion for 15 min followed by reperfusion for up to 3 h. Cytoplasmic and nuclear proteins were isolated from ischemic and nonischemic areas of each heart. NF-kappaB activation was increased in the ischemic area (680%) after 10 min of ischemia and in the nonischemic area (350%) after 15 min of ischemia and remained elevated during prolonged ischemia and reperfusion. IKKbeta activity was markedly increased in ischemic (1,800%) and nonischemic (860%) areas, and phosphorylated IkappaBalpha levels were significantly elevated in ischemic (180%) and nonischemic (280%) areas at 5 min of ischemia and further increased after reperfusion. IkappaBalpha levels were decreased in the ischemic (45%) and nonischemic (36%) areas after 10 min of ischemia and remained low in the ischemic area during prolonged ischemia and reperfusion. The results suggest that in vivo I/R rapidly induces IKKbeta activity and increases IkappaBalpha phosphorylation and degradation, resulting in NF-kappaB activation in the myocardium.
Animals↗
Fluorescence quenching: A tool for single-molecule protein-folding study.
By using titin as a model system, we have demonstrated that fluorescence quenching can be used to study protein folding at the single molecule level. The unfolded titin molecules with multiple dye molecules attached are able to fold to the native state. In the native folded state, the fluorescence from dye molecules is quenched due to the close proximity between the dye molecules. Unfolding of the titin leads to a dramatic increase in the fluorescence intensity. Such a change makes the folded and unfolded states of a single titin molecule clearly distinguishable and allows us to measure the folding dynamics of individual titin molecules in real time. We have also shown that fluorescence quenching can signal folding and unfolding of a small protein with only one immunoglobulin domain.
Animals↗
A single-molecule study of RNA catalysis and folding.
Using fluorescence microscopy, we studied the catalysis by and folding of individual Tetrahymena thermophila ribozyme molecules. The dye-labeled and surface-immobilized ribozymes used were shown to be functionally indistinguishable from the unmodified free ribozyme in solution. A reversible local folding step in which a duplex docks and undocks from the ribozyme core was observed directly in single-molecule time trajectories, allowing the determination of the rate constants and characterization of the transition state. A rarely populated docked state, not measurable by ensemble methods, was observed. In the overall folding process, intermediate folding states and multiple folding pathways were observed. In addition to observing previously established folding pathways, a pathway with an observed folding rate constant of 1 per second was discovered. These results establish single-molecule fluorescence as a powerful tool for examining RNA folding.
Animals↗
Adenosine prevents activation of transcription factor NF-kappa B and enhances activator protein-1 binding activity in ischemic rat heart.
BACKGROUND: Adenosine prevents myocardial TNF-alpha production induced by ischemia/reperfusion, but the mechanisms are poorly understood. Transcription factors NF-kappa B and AP-1 have been implicated in the regulation of a variety of inducible gene expressions in response to oxidative stress and cellular defense. The effects of adenosine on NF-kappa B and AP-1 activation have not been clearly defined. This study demonstrated differential effects of adenosine on NF-kappa B and AP-1 nuclear binding activity in ischemic myocardium. METHODS: Isolated working rat hearts were subjected to 0, 1, 2, 3, 4, 5, 7.5, 10, 15, and 30 minutes of ischemia, with 4 to 6 hearts for each time point with and without adenosine (100 mumol/L). NF-kappa B and AP-1 binding activity in the nucleus were analyzed by electrophoretic mobility shift assay (EMSA). I kappa B alpha levels in the cytoplasm were measured by Western blot analysis. TNF-alpha mRNA levels were determined by RT-PCR. RESULTS: NF-kappa B binding activity in the nucleus significantly increased after 4 minutes of ischemia and remained to 30 minutes. The levels of I kappa B alpha protein in the cytoplasm markedly decreased after 4, 5, 7.5, and 10 minutes of ischemia. TNF-alpha mRNA levels peaked after 10 minutes of ischemia. AP-1 DNA binding activity was induced and persisted during all ischemic periods. Adenosine significantly inhibited NK-kappa B binding activity in the nucleus, markedly prevented the loss of I kappa B alpha proteins from the cytoplasm, and concomitantly down-regulated TNF-alpha mRNA expression, but enhanced AP-1 binding activity in the nucleus of ischemic myocardium. CONCLUSIONS: Adenosine modulation of NF-kappa B activation may be the cellular molecular mechanism of down-regulation of TNF-alpha mRNA expression. The cardioprotective properties of adenosine may be involved in the differential modulation of NF-kappa B and AP-1 activation during myocardial ischemia.
Adenosine↗
Inhibition of LPS-induced NFkappaB activation by a glucan ligand involves down-regulation of IKKbeta kinase activity and altered phosphorylation and degradation of IkappaBalpha.
Growing evidence supports the role of transcription factor activation in the pathophysiology of inflammatory disorders, sepsis, ARDS, SIRS, and shock. Kinase mediated phosphorylation of IkappaBalpha is a crucial step in the NFkappaB activation pathway. We investigated IKBalpha phosphorylation in murine liver and lung extracts after cecal ligation and puncture (CLP) in the presence and absence of a glucan ligand. ICR mice were subjected to CLP. Unoperated and sham-operated mice served as the controls. Glucan phosphate (50 mg/kg) was administered 1 h before or 15 min after CLP. CLP increased hepatic and pulmonary levels of phospho-IkappaBalpha by 48-192%. Pre- or post-treatment with glucan phosphate decreased (P < 0.05) tissue phospho-IkappaBalpha levels in CLP mice. Phospho-IkappaBalpha in the glucan-CLP group were not significantly different from the unoperated controls. To investigate mechanisms we examined IKKbeta kinase activity, IkappaBalpha phosphorylation and degradation, and NFkappaB activity in a murine macrophage cell line, J774a.1, treated with LPS (1 microg/mL) and/or glucan phosphate (1 microg/mL) for up to 120 min. The glucan ligand blunted LPS-induced IKKbeta kinase activity, phosphorylation and degradation of IkappaBalpha, and NFkappaB nuclear binding activity. The data indicate that one mechanism by which (1-->3)-beta-D-glucan may alter the response to endotoxin or polymicrobial sepsis involves modulation of IKK3 kinase activity with subsequent decreases in IkappaBalpha phosphorylation and NFkappaB activation.
Animals↗
Nuclear factor kappaB activation in acute appendicitis: a molecular marker for extent of disease?
Nuclear factor-kappaB (NF-kappaB) has been demonstrated to regulate the transcription of target genes and stimulate inflammatory cytokine responses in a variety of inflammatory diseases. Preliminary studies have demonstrated that NF-kappaB is activated early in acute inflammation and sepsis and may serve as an indicator of clinical severity. The present study was designed to evaluate the degree of activation of NF-kappaB in patients with acute appendicitis and correlate activation with clinical extent of disease. Ten patients with acute appendicitis and five control patients (elective inguinal hernia repair) were evaluated by assaying NF-kappaB activity preoperatively and 12 to 18 hours postoperatively. Assaying of NF-kappaB was determined by binding activity for consensus probes in nuclear extracts from peripheral mixed white blood cells obtained by venous puncture. The bands of NF-kappaB activity from gel electrophoresis were quantified with a phosphor imager and reported as units of integrated intensity. The preoperative NF-kappaB activity was increased in all patients with appendicitis versus the controls [mean 151 (range 97-189) vs mean 50.3 (range 13.7-77); P < 0.0001]. The increased NF-kappaB activity also correlated with length of time of symptoms before operation. The patients who were symptomatic for less than 24 hours had an average NF-kappaB value of 103 (range 97-105) versus 171.4 (range 152-189) (P < 0.0001) in those who were symptomatic 24 or more hours. The NF-kappaB activity did not correlate with the white blood cell count. Postoperative NF-kappaB binding activity in the appendicitis patients dropped to minimal levels (mean 50.3), even lower than the control patients' baseline values (mean 55.6). Control patients demonstrated low baseline values preoperatively and a slight rise postoperatively [mean 50.3 (range 13.7-77) vs mean 100 (range 45-186)]. We conclude the following: (1) NF-kappaB binding activity is elevated in patients with acute appendicitis and correlates with symptoms longer than 24 hours. (2) This increased activity returns to baseline values within 18 hours after appendectomy. (3) Molecular indicators of inflammation may have a role in both staging surgical inflammatory conditions and predicting ultimate outcome.
Acute Disease↗
Physical activity and cardiovascular disease risk in middle-aged and older women.
The authors investigated the relation between physical activity and cardiovascular disease (CVD) in women by following 1,564 University of Pennsylvania alumnae (mean age, 45.5 years), initially free of CVD, from 1962 until 1993. Energy expenditure was estimated from the daily number of flights of stairs climbed and blocks walked as well as the sports played and was categorized into approximate thirds (<500, 500-999, > or = 1,000 kcal/week). During 35,021 person-years, 181 CVD cases were identified. After adjustment for coronary risk factors, the relative risks of CVD were 0.99 (95% confidence interval (CI): 0.69, 1.41) and 0.88 (95% CI: 0.62, 1.25) for women who expended 500-999 and > or = 1,000, respectively, compared with <500 kcal/week (p for trend = 0.45). Only walking was found to be inversely related to CVD risk (p for trend = 0.054). Compared with women who walked <4 blocks/day, the relative risks of CVD were 0.84 (95% CI: 0.59, 1.19) and 0.67 (95% CI: 0.45, 1.01) for women who walked 4-9 and > or = 10 blocks/day, respectively. Finally, an interaction (p = 0.023) between body mass index and physical activity on CVD risk was observed, with an inverse association only for leaner (<23 kg/m2) women. These data showed no overall association of physical activity with CVD risk in women. However, walking > or = 10 blocks/day (approximately 6 miles (9.7 km)/week) was associated with a 33% decreased risk. One explanation for this finding may be that walking was reported more precisely than other kinds of activities.
Aged↗
Ligand-induced conformational changes observed in single RNA molecules.
We present the first demonstration that fluorescence resonance energy transfer can be used to track the motion of a single molecule undergoing conformational changes. As a model system, the conformational changes of individual three-helix junction RNA molecules induced by the binding of ribosomal protein S15 or Mg(2+) ions were studied by changes in single-molecule fluorescence. The transition from an open to a folded configuration was monitored by the change of fluorescence resonance energy transfer between two different dye molecules attached to the ends of two helices in the RNA junction. Averaged behavior of RNA molecules closely resembles that of unlabeled molecules in solution determined by other bulk assays, proving that this approach is viable and suggesting new opportunities for studying protein-nucleic acids interactions. Surprisingly, we observed an anomalously broad distribution of RNA conformations at intermediate ion concentrations that may be attributed to foldability differences among RNA molecules. In addition, an experimental scheme was developed where the real-time response of single molecules can be followed under changing environments. As a demonstration, we repeatedly changed Mg(2+) concentration in the buffer while monitoring single RNA molecules and showed that individual RNA molecules can measure the instantaneous Mg(2+) concentration with 20-ms time resolution, making it the world's smallest Mg(2+) meter.
Base Sequence↗
Identification and characterization of a serine hydroxymethyltransferase isoform in Caenorhabditis briggsae.
In the nematode Caenorhabditis elegans, the maternal effect lethal gene mel-32 encodes a serine hydroxymethyltransferase isoform. Since interspecies DNA comparison is a valuable tool for identifying sequences that have been conserved because of their functional importance or role in regulating gene activity, mel-32(SHMT) genomic DNA from C. elegans was used to screen a genomic library from the closely related nematode Caenorhabditis briggsae. The C. briggsae genomic clone identified fully rescues the Mel-32 phenotype in C. elegans, indicating functional and regulatory conservation. Computer analysis reveals that CbMEL-32(SHMT) is 92% identical (97% similar) to CeMEL-32(SHMT) at the amino acid level over the entire length of the protein (484 amino acids), whereas the coding DNA is 82.5% identical (over 1455 nucleotides). Several highly conserved non-coding regions upstream and downstream of the mel-32(SHMT) gene reveal potential regulatory sites that may bind trans-acting protein factors.
Amino Acid Sequence↗
Single-pair fluorescence resonance energy transfer on freely diffusing molecules: observation of Förster distance dependence and subpopulations.
Photon bursts from single diffusing donor-acceptor labeled macromolecules were used to measure intramolecular distances and identify subpopulations of freely diffusing macromolecules in a heterogeneous ensemble. By using DNA as a rigid spacer, a series of constructs with varying intramolecular donor-acceptor spacings were used to measure the mean and distribution width of fluorescence resonance energy transfer (FRET) efficiencies as a function of distance. The mean single-pair FRET efficiencies qualitatively follow the distance dependence predicted by Förster theory. Possible contributions to the widths of the FRET efficiency distributions are discussed, and potential applications in the study of biopolymer conformational dynamics are suggested. The ability to measure intramolecular (and intermolecular) distances for single molecules implies the ability to distinguish and monitor subpopulations of molecules in a mixture with different distances or conformational states. This is demonstrated by monitoring substrate and product subpopulations before and after a restriction endonuclease cleavage reaction. Distance measurements at single-molecule resolution also should facilitate the study of complex reactions such as biopolymer folding. To this end, the denaturation of a DNA hairpin was examined by using single-pair FRET.
DNA↗
Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism.
Fluorescence resonance energy transfer and fluorescence polarization anisotropy are used to investigate single molecules of the enzyme staphylococcal nuclease. Intramolecular fluorescence resonance energy transfer and fluorescence polarization anisotropy measurements of fluorescently labeled staphylococcal nuclease molecules reveal distinct patterns of fluctuations that may be attributed to protein conformational dynamics on the millisecond time scale. Intermolecular fluorescence resonance energy transfer measurements provide information about the dynamic interactions of staphylococcal nuclease with single substrate molecules. The experimental methods demonstrated here should prove generally useful in studies of protein folding and enzyme catalysis at single-molecule resolution.
Base Sequence↗
Intracellular Ca2+ and adrenergic responsiveness of cardiac myocytes in streptozotocin-induced diabetes.
1. The contractile function of diabetic hearts is impaired. In addition, the responsiveness of diabetic cardiac muscle to sympathetic stimulation is altered. Previous studies have revealed a depressed response to beta-adrenoceptor stimulation; however, the response to alpha-adrenoceptor activation remains controversial. Because alpha- and beta-adrenoceptor agonists increase cardiac contractility, largely through increased mobilization of intracellular Ca2+, the aim of the present study was to investigate the effects of alpha- and beta-adrenoceptor stimulation on intracellular Ca2+ handling in cardiac myocytes from streptozotocin-induced diabetic rats. 2. Intracellular Ca2+ was measured using fura-2. Under basal conditions (27 degrees C, 2.5 mmol/L extracellular [Ca2+], 0.3 Hz stimulation), there was no significant difference in resting or peak Ca2+ levels between control and diabetic cardiomyocytes. However, the time course of the intracellular Ca2+ transient was significantly prolonged in cells from diabetic hearts. 3. The beta-adrenoceptor agonist orciprenaline (at 10(-7) and 10(-6) mol/L) increased the amplitude of the Ca2+ transient in both groups; however, the extent of potentiation was less in diabetic compared with control cardiomyocytes. Orciprenaline decreased the duration of the transient to the same extent in both groups. 4. The alpha-adrenoceptor agonist phenylephrine (at 10(-7) and 10(-6) mol/L) had no effect on the Ca2+ transient in control myocytes but caused a significant concentration-dependent increase in its amplitude in diabetic cardiomyocytes. Phenylephrine had no effect on the time course of the transient in either group. 5. These results demonstrate differential effects of insulin-dependent diabetes on the responsiveness of cardiomyocytes to alpha- and beta-adrenoceptor stimulation. The heightened response to alpha-adrenoceptor stimulation observed in diabetic cardiomyocytes may partly compensate for the diminished myocardial beta-adrenoceptor response.
Adrenergic alpha-Agonists↗
Inhibiting early activation of tissue nuclear factor-kappa B and nuclear factor interleukin 6 with (1-->3)-beta-D-glucan increases long-term survival in polymicrobial sepsis.
BACKGROUND: Recent data implicate the activation of nuclear factor-kappa B (NF-kappa B) and nuclear factor interleukin 6 (NF-IL6) as important steps in the pathophysiologic mechanisms of adult respiratory distress syndrome and systemic inflammatory response syndrome. METHODS: This study evaluated the effect of immunomodulating polysaccharides on transcription factor activation, cytokine expression, and mortality in a murine cecal ligation and puncture (CLP) model. ICR/HSD mice were treated with glucan (50 mg/kg) 1 hour before or 15 minutes after CLP. Liver and lung tissue were harvested at 3 hours and mortality trends were observed for 20 days. RESULTS: CLP increased liver and lung NF-kappa B and NF-IL6 nuclear binding activity as well as tumor necrosis factor-alpha and interleukin 6 messenger RNA levels at 3 hours. Pretreatment or posttreatment with glucans inhibited tissue NF-kappa B and NF-IL6 nuclear binding activity and tissue cytokine messenger RNA levels. Prophylaxis with glucan phosphate or scleroglucan increased (P < .001) long-term survival (20% CLP vs 65% glucan phosphate, 75% scleroglucan). Posttreatment with glucan phosphate also increased (P < .05) long-term survival (20% vs 65%). CONCLUSIONS: Pretreatment or posttreatment with biologic response modifiers decreased tissue transcription factor nuclear binding activity and cytokine message in liver and lung of septic mice. Inhibiting early transcription factor activation and cytokine message expression correlates with improved outcome in polymicrobial sepsis as denoted by increased long-term survival.
Animals↗
Early activation of hepatic NFkappaB and NF-IL6 in polymicrobial sepsis correlates with bacteremia, cytokine expression, and mortality.
BACKGROUND: The role of transcription factor activation in the pathophysiology of sepsis syndrome has not been established. This study investigated the relation between tissue nuclear factor kappaB (NFkappaB) and nuclear factor interleukin 6 (NF-IL6 or C/EBP) activation and bacteremia, inflammatory cytokine expression, and mortality in a murine model of cecal ligation and puncture (CLP). METHODS: Transcription factor activation was assessed by the electrophoretic mobility shift assay. Cytokine mRNA levels were established by reverse transcription-polymerase chain reaction and quantified by scanning densitometry. Bacteremia was evaluated by standard aerobic and anaerobic microbiologic methods. RESULTS: CLP stimulated hepatic NFkappaB activation at 2, 3, 4, 5, 6, and 8 hours compared with control and sham-operated mice. Hepatic NFkappaB activation during CLP peaked at 4 hours (1114% vs. no surgery, 609% vs. sham). Hepatic NF-IL6 activation was observed at 3, 4, and 6 hours after CLP. Hepatic and splenic levels of tumor necrosis factor-alpha and IL-6 mRNA were also elevated after CLP. Bacteremia in CLP mice consisted of Bacteroides species and to a lesser extent facultative gram-negative bacilli and group D Enterococcus. CONCLUSIONS: Early activation of hepatic and splenic NFkappaB and NF-IL6 positively correlates with tissue cytokine mRNA expression and mortality in a surgical model of polymicrobial sepsis. The data suggest that transcription factor activation is an early event in the pathophysiology of sepsis.
Animals↗