Faculty Bibliography

Gap junction redistribution and reduced expression, a phenomenon termed gap junction remodeling (GJR), is often seen in diseased hearts and may predispose towards arrhythmias. We have recently shown that short-term pacing in the mouse is associated with changes in connexin43 (Cx43) expression and localization, but not with increased inducibility into sustained arrhythmias. We hypothesized that short-term pacing, if imposed on murine hearts with decreased Cx43 abundance, could serve as a model for evaluating the electrophysiologic effects of GJR. We paced wildtype (normal Cx43 abundance) and heterozygous Cx43 knockout mice (Cx43(+/-), 66% mean reduction in Cx43) for six hours at 10-15% above their average sinus rate. We investigated the electrophysiologic effects of pacing on the whole animal using programmed electrical stimulation, and in isolated ventricular myocytes with patch clamp studies. Cx43(+/-) myocytes had significantly shorter action potential durations (APD) and increased steady state and inward rectifier potassium currents (Iss and IK1, respectively) compared to wildtype littermate cells. In Cx43(+/-) hearts, pacing resulted in significant prolongation of ventricular effective refractory period and action potential duration, and significant diminution of Iss compared to unpaced Cx43(+/-) hearts. However, these changes were not seen in paced wildtype mice. These data suggest that Cx43 abundance plays a critical role in regulating currents involved in myocardial repolarization and their response to pacing. Our study may aid in understanding how dyssynchronous activation of diseased, Cx43-deficient myocardial tissue can lead to electrophysiologic changes which may contribute to the worsened prognosis often associated with pacing in the failing heart. Key words: Connexin43, ventricular myocytes, mouse, gap junction

Magnetic resonance (MR) imaging is becoming a pivotal diagnostic method to identify and characterize vulnerable atherosclerotic plaques. We previously reported a reconstituted high-density lipoprotein (rHDL) nanoparticle platform enriched with Gd-based amphiphiles as a plaque-specific MR imaging contrast agent. Further modification can be accomplished by inserting targeting moieties into this platform to potentially allow for improved intraplaque macrophage uptake. Since studies have indicated that intraplaque macrophage density is directly correlated to plaque vulnerability, modification of the rHDL platform may allow for better detection of vulnerable plaques. In the current study we incorporated a carboxyfluoresceine-labeled apolipoprotein E-derived lipopeptide, P2fA2, into rHDL. The in vitro macrophage uptake and in vivo MR efficacy were demonstrated using murine J774A.1 macrophages and the apolipoprotein E knock-out (apoE(-/-)) mouse model of atherosclerosis. The in vitro studies indicated enhanced association of murine macrophages to P2fA2 enriched rHDL (rHDL-P2A2) nanoparticles, relative to rHDL, using optical techniques and MR imaging. The in vivo studies showed a more pronounced and significantly higher signal enhancement of the atherosclerotic wall 24 h after the 50 micromol Gd/kg injection of rHDL-P2A2 relative to administration of rHDL. The normalized enhancement ratio for atherosclerotic wall of rHDL-P2A2 contrast agent injection was 90%, while that of rHDL was 53% 24 h post-injection. Confocal laser scanning microscopy revealed that rHDL-P2A2 nanoparticles co-localized primarily with intraplaque macrophages. The results of the current study confirm the hypothesis that intraplaque macrophage uptake of rHDL may be enhanced by the incorporation of the P2fA2 peptide into the modified HDL particle

High density lipoprotein (HDL) is an important natural nanoparticle that may be modified for biomedical imaging purposes. Here we developed a novel technique to create unique multimodality HDL mimicking nanoparticles by incorporation of gold, iron oxide, or quantum dot nanocrystals for computed tomography, magnetic resonance, and fluorescence imaging, respectively. By including additional labels in the corona of the particles, they were made multifunctional. The characteristics of these nanoparticles, as well as their in vitro and in vivo behavior, revealed that they closely mimic native HDL.

Erythropoietin-producing hepatocellular carcinoma (Eph) receptors play important roles in development, neural plasticity, and cancer. We used an Orbitrap mass spectrometer and stable isotope labeling by amino acids in cell culture (SILAC) to identify and quantify 204 proteins with significantly changed abundance in antiphosphotyrosine immunoprecipitates after ephrinB1-Fc stimulation. More than half of all known effectors downstream of EphB receptors were identified in this study, as well as numerous novel candidates for EphB signaling

0507BS3 virus was isolated from a mixed pool of Culex sp. and Anopheles sp. collected in Kashi, Xinjiang, China. 0507BS3 virus could cause cytopathic effects on C6/36 cells but not on Vero and BHK-21 cells. Viral particles had no envelope and appeared round with diameter of about 60 nm (n = 20). Viral capsid was composed of a single layer and a central core. Capsomeres on the surface of capsid were clearly visible. Electrophoresis of viral genome showed a profile of 10 double stranded RNA (dsRNA) segments. Sequencing of the tenth segment revealed the length of 964bp (GenBank ID: FJ150869). A single open reading frame (ORF) was found and encoded a protein of 275 amino acids with a molecular mass of 30.8kDa. The nucleotide sequence had no similarity with any other viral genomic sequences, but the deduced amino acid sequence significantly matched the polyhedrin genes of cytoplasmic polyhedrosis virus (CPV) in some sections. A phylogenetic tree was constructed to compare the polyhedrin gene sequences of all CPV types in GenBank. The tree demonstrated that 0507BS3 virus was only distantly related to the other CPV types and belonged to a new CPV type.

Meiosis is coupled to gamete development and must be well regulated to prevent aneuploidy. During meiotic maturation, Drosophila oocytes progress from prophase I to metaphase I. The molecular factors controlling meiotic maturation timing, however, are poorly understood. We show that Drosophila alpha-endosulfine (endos) plays a key role in this process. endos mutant oocytes have a prolonged prophase I and fail to progress to metaphase I. This phenotype is similar to that of mutants of cdc2 (synonymous with cdk1) and of twine, the meiotic homolog of cdc25, which is required for Cdk1 activation. We found that Twine and Polo kinase levels are reduced in endos mutants, and identified Early girl (Elgi), a predicted E3 ubiquitin ligase, as a strong Endos-binding protein. In elgi mutant oocytes, the transition into metaphase I occurs prematurely, but Polo and Twine levels are unaffected. These results suggest that Endos controls meiotic maturation by regulating Twine and Polo levels, and, independently, by antagonizing Elgi. Finally, germline-specific expression of the human alpha-endosulfine ENSA rescues the endos mutant meiotic defects and infertility, and alpha-endosulfine is expressed in mouse oocytes, suggesting potential conservation of its meiotic function.

p202, an interferon-inducible protein that belongs to an interferon-inducible p200 family, was highly induced in the course of osteogenesis of pluripotent C2C12 cells and the chondrogenesis of C3H10T1/2 cells. Differential expression of p202 is probably due, at least in part, to the transactivation of the p202 gene by Smad transcription factors. Overexpressing p202 inhibited, whereas lowering p202 via a siRNA approach enhanced, chondrocyte differentiation. In contrast, overexpression of p202 enhanced, whereas knockdown of p202 inhibited, osteoblast differentiation. Molecular mechanism studies revealed that p202 and parathyroid hormone-related peptide (PTHrP) formed a positive feedback loop, since (1) overexpressing p202 markedly enhanced whereas knocking down p202 suppressed the expression of PTHrP; and (2) p202 expression was increased in growth plate chondrocytes of PTHrP receptor transgenic mouse embryos; however, its expression was reduced in PTHrP knockout mouse embryos. Taken together, our findings demonstrate that p202 protein is a novel, important mediator of chondrogenic and osteogenic differentiation

The hedgehog family of morphogens (sonic [Shh], Indian, and desert hedgehog) are central regulators of embryologic growth and tissue patterning. Although recent work implicates Shh in postnatal tissue repair and development, conclusive evidence is lacking. Here, we demonstrated the importance of Shh in wound repair, by examining the effects of cyclopamine, a specific inhibitor of the Shh signaling cascade, on tissue repair. Using a murine-splinted excisional wound model, which attenuates wound contraction in this loose-skinned rodent, we established that, by all measures (wound closure, epithelialization, granulation formation, vascularity, and proliferation), wound healing was profoundly impaired when Shh signaling was disrupted. Because embryonic disruption of Shh is associated with distinct phenotypic defects, our findings invite investigation of the potential role of Shh signaling under postnatal conditions associated with disregulated wound healing

0507JS60 virus was isolated from a pool of Culex sp. collected in Kashi, Xinjiang, which could be propagated stably on C6/36 cells and caused cytopathic effects continuously. Viral particles had no envelope and appeared round with diameter of about 55nm (n = 10). Capsomeres on the surface of capsid were clearly visible. Electrophoresis of viral genome showed a profile of 12 double stranded RNA (dsRNA) segments. Sequencing of the twelfth segment revealed the length of 760bp (GenBank ID: FJ157354). A single open reading frame (ORF) was found and encoded a protein of 174 amino acids with a molecular mass of 18.9kD. The nucleotide sequence had similarity over 89% with that of LNV, but the deduced amino acid sequence had similarity over 91% with that of LNV. A phylogenetic tree was constructed to compare the corresponding genetic sequences in Seadornavirus. The tree demonstrated that 0507JS60 virus lied in the same branch with LNV and more closely related to LNV-NE9712. 0507JS60 virus was identified as LNV, which was firstly isolated outside the Northeast of China.