Faculty Bibliography

Protein misfolding in the endoplasmic reticulum (ER) activates a set of intracellular signaling pathways, collectively termed the Unfolded Protein Response (UPR). UPR signaling promotes cell survival by reducing misfolded protein levels. If homeostasis cannot be restored, UPR signaling promotes cell death. The molecular basis for the switch between prosurvival and proapoptotic UPR function is poorly understood. The ER-resident proteins, PERK and IRE1, control two key UPR signaling pathways. Protein misfolding concomitantly activates PERK and IRE1 and has clouded insight into their contributions toward life or death cell fates. Here, we employed chemical-genetic strategies to activate individually PERK or IRE1 uncoupled from protein misfolding. We found that sustained PERK signaling impaired cell proliferation and promoted apoptosis. By contrast, equivalent durations of IRE1 signaling enhanced cell proliferation without promoting cell death. These results demonstrate that extended PERK and IRE1 signaling have opposite effects on cell viability. Differential activation of PERK and IRE1 may determine life or death decisions after ER protein misfolding

Introduction: Connexin43 (Cx43) gap junction channels close during myocardial infarction (MI). To assess the importance of Cx43 regulation on arrhythmia susceptibility, mice in which the coding region of Cx43 was replaced with a mutation lacking most of the carboxyl-terminal domain (K258stop) were subjected to MI. This mutation has been shown to prevent chemical regulation of Cx43 channels by low intracellular pH in vitro. Due to reduced viability of homozygous K258stop mice, studies were carried out in animals harboring one Cx43 knockout allele and one K258stop or Cx43 allele, respectively (i.e., K258stop/KO; Cx43/KO). Methods: Langendorff-perfused hearts (n=12 per group) were subjected to 1 hour of ischemia and 4 hours of reperfusion by reversibly occluding the left anterior descending (LAD) coronary artery. Hearts were monitored for spontaneous ventricular tachyarrhythmias (VT) and for inducibility of VT by endocardial burst pacing near the apex of the left ventricle (3 x 18 S1 stimuli; 80, 60, 40 and 20ms cycle length; 2.5 times threshold) 15, 30, 45 and 60 minutes after the onset of LAD occlusion. Results: 45 minutes after the onset of LAD occlusion, VT could be induced by at least one pacing frequency in 81.8% of K258stop/KO hearts. The number increased to 100% at 60 minutes. For Cx43/KO hearts only 41.7% and 75% of the hearts developed VT at 45 min and 60 min, respectively. The average number of VT events elicited (regardless of burst pacing frequency) was significantly higher in the K258stop/KO hearts (2.64 +/- 0.56 v. 0.83 +/- 0.39 at 45min; p: 0.014; 3.18 +/- 0.55 v.1.67 +/- 0.47 at 60min; p= 0.047). VT episodes were also of longer duration in the K258stop/KO group. During reperfusion, K258stop/KO hearts showed a higher incidence of spontaneous VT (85.7% v. 42.9% of hearts) and increased numbers of episodes (7.14 +/- 2.27 v. 1.57 +/- 0.95; p=0.043). Conclusions: Loss of the regulatory domain of Cx43 leads to an increased susceptibility to arrhythmias following acute coronary occlusion. Whether similar results would be obtained when Cx43 channels remain open but structurally intact remains to be determined

Invasive aspergillosis remains difficult to diagnose despite advances in imaging and antigen-based serological testing. To overcome this problem, nucleic acid (NA)-based amplification assays were introduced to identify infecting pathogens. Unfortunately, the reliability of such assays to detect Aspergillus spp. has met with mixed success. A new generation of NA platforms are emerging, which greatly improve our ability to detect Aspergillus-specific DNA and RNA from respiratory and blood samples. These platforms can accurately detect a single genome, and the emergence of pan-fungal and pan-Aspergillus probes offer promise for broader detection. PCR remains the most important platform, especially when coupled with real-time probes. It is multiplex friendly and can distinguish between closely related target sequences. Nucleic acid sequence-based amplification (NASBA) is an RNA-directed isothermal transcription-based amplification platform, which is more robust than PCR resulting in a 10(14)-fold amplification. RNA-based detection facilitates more target options and can be used to assess cell viability. Both DNA and RNA amplification platforms take advantage of allele-specific properties of probes, which are valuable for assessing drug resistance markers. Finally, as new molecular diagnostic platforms mature, their role may expand to include early monitoring of therapy.

AIMS: The effects of deleting genes encoding uroplakins II (UPII) and III (UPIIIa) on mouse bladder physiology/dysfunction were studied in male and female wild type and knockout (KO) mice. METHODS: UPII, UPIIIa, and WT mice were catheterized using previously described techniques. Continuous cystometry was conducted in conscious, freely moving animals. Bladder strips were harvested after animal sacrifice and pharmacological studies and EFS were conducted in an organ chamber. Histological studies were also carried on with H&E staining to identify differences among the three mouse types. RESULTS: These studies have revealed numerous alterations, some of which were apparently gender-specific. Nonvoiding contractions were common in both UPII and UPIIIa KO mice, although more severe in the former. In particular, the increased bladder capacity, micturition pressure and demonstrable nonvoiding contractions observed in the male UPII KO's, were reminiscent of an obstruction-like syndrome accompanied by evidence of emerging bladder decompensation, as reflected by an increased residual volume. Pharmacological studies revealed a modest, gender-specific reduction in sensitivity of isolated detrusor strips from UPII KO female mice to carbachol-induced contractions. A similar reduction was observed in UPIIIa KO female mice. Histological investigation showed urothelial hyperplasia in both UPII KO and UPIIIa KO mice, although again, apparently more severe in the former. CONCLUSIONS: These results confirm and extend previous work to indicate that urothelial defects due to uroplakin deficiency are associated with significant alterations in bladder function and further highlight the importance of the urothelium to bladder physiology/dysfunction

Purpose: Unlike other DMARDs methotrexate diminishes the risk of Atherosclerotic Cardiovascular Disease (ASCVD) in patients with Rheumatoid Arthritis and adenosine, acting at adenosine A2A receptors, has been shown to mediate the anti-inflammatory effects of methotrexate. Adenosine inhibits the first step in formation of atherosclerotic plaque, foam cell formation in macrophages and this effect appears to be mediated by enhanced expression of cholesterol 27-hydroxylase, an enzyme involved in reverse cholesterol transport. We therefore asked whether the effect of adenosine A2A receptors on foam cell formation in vitro are mediated by apoE or 27-hydroxylase (27OH'ase), proteins involved in reverse cholesterol transport. Method: THP-1 cells, a human monocytoid cell line, were infected with lentiviral vectors expressing siRNA for either apoE or 27OH'ase or scrambled RNA and infected cell lines were selected by incubation with puromycin. Foam cell formation was induced in THP-1 cells by incubation with interferon- (500U/ml) and % foam cells enumerated in 5 high power fields. 3H-Cholesterol efflux was measured after loading with label. Results: Specific lentiviral siRNA infection markedly reduces apoE (p< 0.0001, apoE siRNA vs. control, n=3) or 27OH'ase mRNA (p< 0.0001, 27-hydroxylase siRNA vs. control, n=3) and protein (p< 0.0107, 27-hydroxylase siRNA vs. control n= 3) in THP-1 cells. Despite diminished apoE expression CGS-21680 (1muM), an adenosine A2A receptor agonist, inhibits IFN-induced foam cell formation (p< 0.0002, IFN CGS vs. IFN alone, n= 4) but has no effect on foam cell formation in 27OH'ase KD cells. CGS21680 increases cholesterol efflux in wild type and apoE1 KD cells (from 9.5% to 17.5+2.5% and from 10.0+2% to 17.5 +2%, respectively) but not 27OH'ase KD cells. Conclusion: Adenosine A2A receptor-mediated increases in reverse cholesterol transport leading to diminished foam cell formation explains the anti-atherosclerotic effects of methotrexate

Introduction: A critical event in the development of cardiac fibrosis is the transformation of fibroblasts into myofibroblasts. Fibroblasts isolated from healthy hearts and grown under standard tissue culture conditions express alpha-SMA and have been referred to as myofibroblasts. However, recent data suggest the in vitro transformation does not fully replicate the in vivo activation process. The purpose of this study was to investigate the potential of activated fibroblasts to contribute to an arrhythmogenic substrate through paracrine and direct coupling effects. Methods: Confluent neonatal rat myocyte monolayers were treated with media (CM) conditioned by cardiac fibroblasts isolated from ventricles of healthy (Fb) and infarcted (MI-Fb) hearts and optically mapped 16-20 hours later. To study the combined paracrine and direct coupling effect, Fb and MI-Fb were plated on top of myocyte monolayers. Results: Treatment with both Fb CM (16.6+/-0.4 cm/s) and MIFb CM (15.8+/-0.4 cm/s) significantly decreased conduction velocity (CV) compared to homocellular myocyte monolayers (Myo; 19.7+/-0.7 cm/s). Action potential duration (APD70) was significantly reduced by MI-Fb CM (143.6+/-1.7 ms) treatment compared to Myo (159.4+/-4.0 ms) and Fb CM (153.4+/-2.7 ms). In heterocellular cultures, Fb significantly decreased (17.0+/-0.5 cm/s) and MI-Fb increased (22.0+/-0.6 cm/s) average CV compared to Myo. In addition, CV was significantly faster with MI-Fb compared to Fb (p=1.95E-8). Fb (145.0+/-3.9 ms) and MIFb (131.1+/-3.7 ms) significantly reduced APD70 compared to Myo (159.4+/-4.0 ms), and APD70 was significantly shorter with MI-Fb compared to Fb (p=0.01). Analysis of Cx43 levels showed a significant upregulation of Cx43 in MI-Fb compared to Fb. Conclusions: These data demonstrate Fb exert predominantly paracrine effects while MI-Fb affect myocyte electrophysiology through a combination of paracrine and direct coupling mechanisms. Moreover, APD shortening and increased Cx43 levels in MI-Fb could contribute to the greater incidence of arrhythmias observed in fibrotic hearts. These findings may lead to the development of new anti-arrhythmic therapeutic approaches targeting the fibroblast activation process

Stress triggers crucial responses, including elevated blood pressure and heart rate (HR), to handle the emergency and restore homeostasis. However, continuation of these effects following cessation of the stress is implicated with many stress-related disorders. Here, we examine the kinetics and persistence of cardiovascular and locomotor responses to single and repeated immobilization stress (IMO), with and without prior treatment with adrenocorticotropic hormone (ACTH). Radiotelemetry probes were implanted into male Sprague-Dawley rats to continually monitor mean arterial pressure (MAP), HR and locomotor activity. Rats were subjected to IMO for 2 h daily (10 a.m. to noon, 6 consecutive days). The first IMO induced the greatest change in MAP (about 30 mm Hg) and HR (about 200 bpm). Following each IMO, MAP and HR were elevated during the remaining light phase and in the subsequent dark phase, HR was lower than prior to IMO. We further examined whether elevation of ACTH to a level similar to IMO will elicit similar effects, and if it will alter subsequent responses to IMO. Injection of ACTH (13 IU/kg, s.c.) triggered a short-lived rise in MAP, and decreased HR. After six daily injections of ACTH and recovery time (8 days), rats were immobilized as above. The cardiovascular responses were similar during the IMO, but the ACTH-pretreated group displayed differences following cessation of the IMO. In addition, IMO led to a large reduction of locomotor activity during the dark (normally active) phase to levels similar to the light phase. Following the IMOs, locomotor activity recovered more slowly in the ACTH-pretreated group. The study revealed that IMO-triggered cardiovascular and locomotor responses are evident after termination of the stress. In addition, prior exposure to ACTH delayed recovery in cardiovascular and locomotor functions following cessation of stress.

Neuronal ELAV (nELAV) proteins are RNA-binding proteins which play a physiological role in controlling gene expression in memory formation, and their alteration may contribute to cognitive impairment associated with neurodegenerative pathologies such as Alzheimer's disease (AD). Indeed, we found that the content of nELAV proteins is significantly decreased along with clinical dementia progression in the hippocampi of AD brains, where it inversely correlates with the amount of amyloid-beta (Abeta). To check the direct influence of Abeta on nELAV, we performed in vitro experiments using human SH-SY5Y cells, finding that Abeta(1-42) specifically determines nELAV proteins reduction. Since ADAM10 mRNA has the predicted sequences targeted by nELAV, we investigated whether Abeta, through nELAV proteins, could originate a vicious circle affecting amyloid-beta protein precursor (AbetaPP) processing. Immunoprecipitation experiments showed that indeed nELAV proteins bind to ADAM10 mRNA and that this binding is disrupted by Abeta(1-42) exposure, resulting in a decreased ADAM10 protein expression. ADAM10 protein diminution was also found in AD hippocampi. These data show for the first time the involvement of nELAV in AD pathology and suggest that their alteration may affect genes implicated in AbetaPP processing

The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding.