Faculty Bibliography

Aqueous humor is formed by fluid transfer from the ciliary stroma sequentially across the pigmented ciliary epithelial (PE) cells, gap junctions, and nonpigmented ciliary epithelial (NPE) cells. Which connexins (Cx) contribute to PE-NPE gap junctional formation appears species specific. We tested whether small interfering RNA (siRNA) against Cx43 (siCx43) affects bovine PE-NPE communication and whether cAMP affects communication. Native bovine ciliary epithelial cells were studied by dual-cell patch clamping, Lucifer Yellow (LY) transfer, quantitative polymerase chain reaction with reverse transcription (qRT-PCR), and Western immunoblot. qRT-PCR revealed at least 100-fold greater expression for Cx43 than Cx40. siCx43 knocked down target mRNA expression by 55 +/- 7% after 24 h, compared with nontargeting control siRNA (NTC1) transfection. After 48 h, siCx43 reduced Cx43 protein expression and LY transfer. The ratio of fluorescence intensity (R(f)) in recipient to donor cell was 0.47 +/- 0.09 (n = 11) 10 min after whole cell patch formation in couplets transfected with NTC1. siCx43 decreased R(f) by approximately 60% to 0.20 +/- 0.07 (n = 13, P < 0.02). Dibutyryl-cAMP (500 microM) also reduced LY dye transfer by approximately 60%, reducing R(f) from 0.41 +/- 0.05 (n = 15) to 0.17 +/- 0.05 (n = 20) after 10 min. Junctional currents were lowered by approximately 50% (n = 6) after 10-min perfusion with 500 microM dibutyryl-cAMP (n = 6); thereafter, heptanol abolished the currents (n = 5). Preincubation with the PKA inhibitor H-89 (2 microM) prevented cAMP-triggered current reduction (n = 6). We conclude that 1) Cx43, but not Cx40, is a major functional component of bovine PE-NPE gap junctions; and 2) under certain conditions, cAMP may act through PKA to inhibit bovine PE-NPE gap junctional communication.

Recent studies suggest that Stat3, a transcription factor that mediates cytokine signaling, plays a critical role in the pathogenesis of diabetic nephropathy. Complete Stat3 gene knockout is embryonic lethal; therefore, we crossed Stat3+/- mice with Stat3 mutant mice (SA/SA) that lack full Stat3 activity. This strategy generated Stat3SA/- mice (25% activity) and Stat3SA/+ mice (75% activity), which were made diabetic using streptozotocin in order to define the role of Stat3 in diabetic kidney disease. While the glomerular number was not different between these two groups of mice, the diabetic SA/- mice had significantly less proteinuria, mesangial expansion, glomerular cell proliferation, and macrophage infiltration than the diabetic SA/+ mice. The reduction in Stat3 activity did not affect glomerular hyperfiltration seen after the induction of diabetes, as it was increased to the same degree in both groups of mice. Phosphorylation of Stat3 was markedly increased in the glomeruli of diabetic SA/+ mice compared to diabetic SA/- mice. The expression of inflammatory markers, IL-6, MCP-1, and activated NF-kappaB; type IV collagen, TGF-beta, and ICAM-1 mRNA; or type IV collagen and TGF-beta protein, were all found to be significantly less in glomeruli isolated from diabetic SA/- mice, as compared with diabetic SA/+ mice. Our study shows that Stat3 plays a critical role in the regulation of inflammation and abnormal matrix synthesis at an early stage of DN

Tay-Sachs disease (TSD) is a progressive neurodegenerative disorder due to an autosomal recessively inherited deficiency of beta-hexosaminidase A (Hex A). Deficiency of Hex A in TSD is caused by a defect of the alpha-subunit resulting from mutations of the HEXA gene. To date, there is no effective treatment for TSD. Animal models of genetic diseases, similar to those known to exist in humans, are valuable and essential research tools for the study of potentially effective therapies. However, there is no ideal animal model of TSD available for use in therapeutic trials. In the present study, we report an animal model (American flamingo; Phoenicopterus ruber) of TSD with Hex A deficiency occurring spontaneously in nature, with accumulation of G(M2)-ganglioside, deficiency of Hex A enzymatic activity, and a homozygous P469L mutation in exon 12 of the hexa gene. In addition, we have isolated the full-length cDNA sequence of the flamingo, which consists of 1581 nucleotides encoding a protein of 527 amino acids. Its coding sequence indicates approximately 71% identity at the nucleotide level and about 72.5% identity at the amino acid level with the encoding region of the human HEXA gene. This animal model, with many of the same features as TSD in humans, could represent a valuable resource for investigating therapy of TSD

Canavan disease (CD), an autosomal recessive neurodegenerative disorder, is caused by mutations in the aspartoacylase (ASPA) gene. In the present study, the ASPA gene was analyzed in 24 non-Jewish patients with CD from 23 unrelated families. Within this cohort, we found three large novel deletions of approximate 92, 56, and 12.13 kb in length, using both self-ligation of restriction endonuclease-digested DNA fragments with long-distance inverse PCR and multiplex dosage quantitative PCR analysis of genomic DNA. The 92 kb large deletion results in complete absence of the ASPA gene in one homozygous and one compound heterozygous patient, respectively. The 56 kb large deletion causes absence of the majority of the ASPA gene except for exon 1 alone in a compound heterozygous patient. The 12.13 kb deletion involves deletion of the ASPA gene from intron 3 to intron 5 including exons 4 and 5 (I3 to E4E5I5) in a compound heterozygous patient. Patients with the three large deletions clinically manifested severe symptoms at birth, including seizures. Our study showed that the combined use of long-distance inverse PCR and multiplex dosage quantitative PCR analysis of genomic DNA is a helpful and rapid technique to search for large deletions, particularly for detection of large deletions in compound heterozygous patients

AIM: To explore the prevention of atherosclerosis by PPARy agonist rosiglitazone. METHODS: 24 male New Zealand white rabbits weighing 1.8 to 2.2 kg were randomly divided into 3 groups: control group, normal rabbit chow; cholesterol group, 1% cholesterol diet; rosiglitazone group, 1% cholesterol diet supplemented with rosiglitazone 0.5 mg x kg(-1) x d(-1) for 6 weeks. Rabbits in cholesterol group and rosiglitazone group were sequentially fed 1% cholesterol-containing diet for 16 weeks. At the end of the experiment, blood glucose, serum lipids levels, ratio of plaque area to aorta area and ratio of intima to media were determined. RESULTS: Hypercholesterolemia was successfully reproduced in rabbits. Adnimistration of rosiglitazone significantly decreased serum TC and LDL-C. The ratio of intima to media and ratio of plaque area to aorta area were also reduced. CONCLUSION: Rosiglitazone could prevent atherosclerosis by decreasing levels of TC and LDL-C

Atriofascicular accessory bundles with AV-node like conduction properties can sustain atrioventricular (AV) re-entrant tachycardia (Mahaim tachycardia). During early embryogenesis, the AV canal is situated above the primitive left ventricle (LV), and a right AV connection has not been achieved yet. We studied the formation of the right ventricular (RV) inflow tract in relation to the developing cardiac conduction system and hypothesized a morphological explanation for functional atriofascicular bypass tracts. Analysis of lacZ-expression during sequential stages of cardiogenesis was performed in CCS-lacZ transgenic mice (E9.5 to 15.5). Embryos were stained for beta-galactosidase activity and the myocardial marker HHF35. At early stages CCS-lacZ expression was observed in a ring surrounding the AV canal, which connected at the inner curvature to the primary fold. The first sign of formation of the (CCS-lacZ negative) RV inlet component was a groove in the CCS-lacZ positive tissue of the primary fold. Outgrowth of the RV inlet tract resulted in division of the primary fold in a septal part, the trabecula septomarginalis and a lateral part, the moderator band, which extended laterally up to the right AV ring. Electrophysiological measurements in embryonic hearts (E15.5) in which the right atrium (RA) and RV were isolated from the left atrium (LA) and LV supported the functionality of this AV-connection via the moderator band, by demonstrating sequential atrial and ventricular activation in both RA/RV and LA/LV preparations. In conclusion, our observations may provide a possible morphological and functional explanation for atriofascicular accessory pathways via the moderator band, underlying Mahaim tachycardia.