Faculty Bibliography

BACKGROUND: Although large scale informatics studies on introns can be useful in making broad inferences concerning patterns of intron gain and loss, more specific questions about intron evolution at a finer scale can be addressed using a gene family where structure and function are well known. Genome wide surveys of tetraspanins from a broad array of organisms with fully sequenced genomes are an excellent means to understand specifics of intron evolution. Our approach incorporated several new fully sequenced genomes that cover the major lineages of the animal kingdom as well as plants, protists and fungi. The analysis of exon/intron gene structure in such an evolutionary broad set of genomes allowed us to identify ancestral intron structure in tetraspanins throughout the eukaryotic tree of life. METHODOLOGY/PRINCIPAL FINDINGS: We performed a phylogenomic analysis of the intron/exon structure of the tetraspanin protein family. In addition, to the already characterized tetraspanin introns numbered 1 through 6 found in animals, three additional ancient, phase 0 introns we call 4a, 4b and 4c were found. These three novel introns in combination with the ancestral introns 1 to 6, define three basic tetraspanin gene structures which have been conserved throughout the animal kingdom. Our phylogenomic approach also allows the estimation of the time at which the introns of the 33 human tetraspanin paralogs appeared, which in many cases coincides with the concomitant acquisition of new introns. On the other hand, we observed that new introns (introns other than 1-6, 4a, b and c) were not randomly inserted into the tetraspanin gene structure. The region of tetraspanin genes corresponding to the small extracellular loop (SEL) accounts for only 10.5% of the total sequence length but had 46% of the new animal intron insertions. CONCLUSIONS/SIGNIFICANCE: Our results indicate that tests of intron evolution are strengthened by the phylogenomic approach with specific gene families like tetraspanins. These tests add to our understanding of genomic innovation coupled to major evolutionary divergence events, functional constraints and the timing of the appearance of evolutionary novelty

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

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

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.

Reports an error in 'Cerebrospinal fluid monoaminergic metabolites in wild Papio anubis and P. hamadryas are concordant with taxon-specific behavioral ontogeny' by Clifford J. Jolly, Jane E. Phillips-Conroy, Jay R. Kaplan and J. John Mann (International Journal of Primatology, 2008[Dec], Vol 29[6], 1549-1566). Due to a production error, the author corrections were not incorporated into the published version of the manuscript. 1. Improved versions of Figs. 1 and 2 are printed with their captions: Fig. 1 Fig. 2 2. Table 5, missing from the published version, is given on this page Table 5 3. Reference citations in text are corrected by section, with the affected beginning portions of the paragraphs republished under the appropriate section heading, followed by a listing of references to be added to the original published paper. (The following abstract of the original article appeared in record 2008-18378-010). We used a cross-sectional sample to compare ontogenetic trajectories in the concentrations of monoamine neurotransmitter metabolites in cerebrospinal fluid of wild anubis (Papio anubis, n = 49) and hamadryas (P. hamadryas, n = 54) baboons to test the prediction that they would differ, especially in males, in association with their distinct behavioral ontogenies. Values of all 3 metabolites [3-methoxy-4- hydroxyphenylglycol (MHPG), the norepinephrine metabolite; 5-hydroxyindoleacetic acid (5-HIAA), the serotonin metabolite; and homovanillic acid (HVA), the dopamine metabolite] declined consistently with dentally-calibrated maturation, and few taxon-related differences were apparent among juveniles. Adult females were too few for adequate comparison, but a discriminant function suggested that they might differ by taxon. Adult males of the 2 species differed strikingly from juveniles and from each other. Contrary to our initial hypothesis, adult male anubis had significantly lower HVA and MHPG, and higher 5-HIAA levels, than predicted from the overall, age-related trend, and MHPG continued to decline with age among adults. As young adults, male hamadryas had low 5-HIAA and a high HVA/5-HIAA ratio, while older males [normatively one-male unit (OMU) leaders] showed a reversal in the trend, with 5-HIAA rising and the HVA/5-HIAA ratio tending to fall. We speculate that the results are related to the dispersing and philopatric ontogenies of anubis and hamadryas males, respectively. Adult male anubis, whose fitness depends on building social networks with nonkin, have high relative serotonin activity, commonly associated with greater social circumspection and skill. Young adult male hamadryas, living among agnatic kin and mating opportunistically, exhibit low 5-HIAA levels, generally associated with impulsivity and social irresponsibility. This reverses as a male approaches the age at which he is normatively the leader of a one-male unit (OMU), and his fitness depends on his maintaining stable relationships with other leaders and with females.

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

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