Beta-adrenergic receptor regulation of pigment epithelial-derived factor expression in rat retina

In the present study, we have examined a potential mechanism by which sympathetic nerves regulate PEDF and whether its down regulation may be responsible for increased capillary density observed after sympathectomy. Six weeks post-sympathectomy, eyes were removed from female Sprague-Dawley rats for Western blot analysis, RNA isolation, real-time PCR, and immunohistochemistry for measurement of PEDF expression. The contralateral or left eye was used as an intra-animal control. In addition, retinal pigment epithelial cells were grown in culture and treated with norepinephrine and propranolol. An ELISA assay was used to determine the amount of PEDF secreted into the RPE media. Quantitative results of Western blot analysis and real-time PCR confirm that both steady-state gene expression and protein levels of PEDF are significantly decreased in the sympathectomized retina (P<0.05) when compared to the contralateral retina. Qualitative results of immunohistochemistry verify that PEDF is located predominantly in the RPE cell layer of the retina, and levels are decreased in the sympathectomized retina. ELISA results illustrate that norepinephrine significantly increases PEDF secretion by RPE cells and propranolol slightly decreases PEDF secretion into RPE cell medium. In conclusion, down regulation of PEDF may contribute to the increased capillary density of the outer plexiform layer in the retina noted after sympathectomy. Furthermore, expression of PEDF was significantly increased after treatment of norepinephrine in RPE medium demonstrating a role of beta-adrenergic regulation of PEDF. Since sympathetic nerves are damaged in diabetes and PEDF appears to be regulated by beta-adrenergic receptors, these results suggest a role for sympathetic nerves in diabetic retinopathy. This knowledge, in turn, may be used for future treatment and prevention of diabetic retinopathy and other ocular diseases.     

Cervical sympathectomy causes photoreceptor-specific cell death in the rat retina

Changes in the regulation of the vasculature of the eye may be related to some age-related ocular diseases. We have previously shown that loss of sympathetic innervation, as can normally occur with age, resulted in substantial vascular growth of the choroid. The current study was designed to determine whether changes induced by sympathetic denervation causes significant loss of photoreceptors and increased glial cell reactivity in the retina. Sympathetic denervation was performed followed by immunohistochemistry, TUNEL staining, and protein expression analysis to investigate photoreceptor loss. There was a significant reduction (30%) in photoreceptor numbers in the sympathectomized eye. This loss was due to apoptosis, as there was over a doubling in apoptotic cell numbers after sympathectomy. This loss of photoreceptors in the sympathectomized eye resulted in a significantly reduced width of the outer nuclear layer of the retina when compared to the contralateral eye. Increased glial fibrillary acidic protein (GFAP) staining was also noted after sympathectomy in the ganglion cell layer with streaking toward the bipolar cell layer. These results suggest that loss of sympathetic innervation may cause significant changes to the physiology of the choroid.     

Eating behavior in the Old Order Amish: heritability analysis and a genome-wide linkage analysis

BACKGROUND: Eating behavior and thus dietary intake affect the development of obesity-related diseases such as diabetes, hypertension, and hyperlipidemia. OBJECTIVE: We investigated the genetic underpinnings of eating behavior. DESIGN: We administered a standardized eating behavior inventory to 624 adults from 28 families participating in the Amish Family Diabetes Study. Three quantifiable components of eating behavior were measured: restraint, disinhibition, and hunger. Associations between eating behavior scores and physical characteristics were evaluated. Heritability analysis and a genome-wide multipoint linkage analysis were performed. RESULTS: Eating behavior scores were associated with obesity and obesity-related phenotypes. Heritability estimates were 0.28 +/- 0.09 for restraint, 0.40 +/- 0.10 for disinhibition, and 0.23 +/- 0.09 for hunger (P < 0.001). The linkage analysis showed 4 regions of suggestive linkage. We observed suggestive evidence for linkage of restraint scores to 2 chromosomal regions, near markers D3S1304 [LOD (log of odds) = 2.5, P = 0.0003] and D6S276 (LOD = 2.3, P = 0.0006). We previously reported that D3S1304 is linked to a locus influencing percentage body fat in this same population (LOD = 1.6), suggesting that this behavioral phenotype may be secondary to obesity. The maximum LOD scores for disinhibition were 1.6 (P = 0.003) near marker D7S657 and 1.4 (P = 0.005) near marker D16S752. The maximum LOD score for hunger was 1.4 (P = 0.005) near marker D3S1278. CONCLUSION: Significant familial effects on eating behavior and suggestive genetic linkage were found in Amish adults.     

Increased ocular blood vessel numbers and sizes following chronic sympathectomy in rat

Disease states characterized by ocular vascular pathology are often associated with impaired sympathetic function. This study examined the effect of sympathetic denervation on ocular vasculature of the adult rat. Uveal perfusion and choroidal and retinal blood vessel sizes and numbers were assessed in rats with intact innervation and after short- (2 days) or long-term (6 weeks) sympathetic denervation induced by ipsilateral superior cervical ganglion excision. In rats with intact innervation and after short-term sympathectomy, blood flow in both eyes was comparable. However, after long-term sympathectomy, blood flow was four-fold greater in the denervated than in the innervated eye, but was unaltered in lacrimal gland, cerebral cortex, and masseter muscle. Choroid surface area was not affected by long-term sympathectomy, but choroidal thickness was increased and choroidal cross-sectional area occupied by vascular lumina was greater. Arteriolar number per unit cross-sectional area of choroid was not altered although arteriolar diameters were enlarged. Choroidal venules were larger and more abundant. Choroidal capillary numbers were unchanged, but retinal capillaries of the outer plexiform layer were increased. To determine if these changes result from loss of sympathetic activity, sympathetic preganglionic innervation was excised chronically. This produced significant increases in choroidal thickness and vascular luminal area, and in numbers of arterioles, small venules, and capillaries in the outer plexiform layer. These findings show that sympathetic innervation is critical in regulating choroidal and retinal vascularity, and that chronic loss of sympathetic activity may contribute to abnormal vascular proliferation in diseases such as age-related macular degeneration and diabetic retinopathy.     

ZAP-70 expression in B-cell hematologic malignancy is not limited to CLL/SLL

ZAP-70 is a tyrosine kinase expressed in normal T cells and NK cells. Expression of ZAP-70 has been associated with poor prognosis in B-cell chronic lymphocytic leukemia and might be a surrogate marker for immunoglobin heavy chain (IGH) mutational status in that disease. Little is known about ZAP-70 expression in other hematologic malignant neoplasms. We examined 446 specimens representing a range of hematopoietic malignant neoplasms for ZAP-70 expression by immunohistochemical analysis. As expected, most T cell-lineage disorders and a subset of small lymphocytic lymphomas were positive. IGH mutational status corresponded to ZAP-70 expression in a small subset of small lymphocytic lymphoma cases subjected to sequence analysis. Of note, however, ZAP-70 was expressed in a minority of other types of B-cell lymphomas, including precursor B-cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma, mantle cell lymphoma, and very rare cases of classic Hodgkin lymphoma. Immunohistochemical analysis represents an alternative method for assessing ZAP-70 expression and reveals that other B-cell malignant neoplasms express ZAP-70.     

Gene expression measurements in the context of epidemiological studies

Background: Gene expression measurements became an attractive tool to assess biological responses in epidemiological studies. However, collection of blood samples poses various technical problems. We used gene expression data from two epidemiological studies to evaluate differences between sampling methods, comparability of two methods for measuring RNA levels and stability of RNA samples over time. Methods: For the PARSIFAL study, PBLC of 1155 children were collected using EDTA tubes in two countries. In the PASTURE study, tubes containing RNA‐stabilizing solutions (PAXgene^® Blood RNA Tubes; PreAnalytiX) were used to collect cord blood leucocytes of 982 children in five countries. Real‐time PCR (conventional single tube assay and high‐throughput low density arrays) was used to quantify expression of various innate immunity genes. In 77 PARSIFAL samples, gene expression was measured repeatedly during prolonged storage. Results: In PARSIFAL (EDTA tubes) the median RNA yield after extraction significantly differed between the two centres (70 and 34 ng/μl). Collecting blood into an RNA‐stabilizing solution markedly reduced differences in RNA yield in PASTURE (range of medians 91–107 ng/μl). The agreement [Spearman rank correlation (r)] between repeated measurements of gene expression decreased with increasing storage time [e.g., for CD14: r (first/second measurement) = 0.35; r (first/third measurement) = 0.03]. RNA levels measured with either the conventional method or low‐density arrays were comparable (r > 0.9). Conclusion: Collecting blood samples into tubes containing an RNA‐stabilizing solution increases RNA yield and reduces its variability. Long‐term storage of samples may lead to RNA degradation, requiring special attention in longitudinal studies.     

Complex structure of the activating immunoreceptor NKG2D and its MHC class I-like ligand MICA

The major histocompatibility complex (MHC) class I homolog, MICA, is a stress-inducible ligand for NKG2D, a C-type lectin-like activating immunoreceptor. The crystal structure of this ligand-receptor complex that we report here reveals an NKG2D homodimer bound to a MICA monomer in an interaction that is analogous to that seen in T cell receptor-MHC class I protein complexes. Similar surfaces on each NKG2D monomer interact with different surfaces on either the alpha1 or alpha2 domains of MICA. The binding interactions are large in area and highly complementary. The central section of the alpha2-domain helix, disordered in the structure of MICA alone, is ordered in the complex and forms part of the NKG2D interface. The extensive flexibility of the interdomain linker of MICA is shown by its altered conformation when crystallized alone or in complex with NKG2D.     

(+)-Z-Bisdehydrodoisynolic acid ameliorates obesity and the metabolic syndrome in female ZDF rats

OBJECTIVE: The putative selective estrogen receptor modulator (+)-Z-bisdehydrodoisynolic acid (Z-BDDA) has been found to improve cardiovascular risk in rodents. The objective of this study was to investigate the effectiveness of (+)-Z-BDDA compared with the antidiabetic drug, rosiglitazone, in treating obesity and risk factors associated with the metabolic syndrome. RESEARCH METHODS AND PROCEDURES: Female Zucker Diabetic Fatty rats were randomly assigned to three treatment groups for 29 weeks: control (C), 1.8 mg (+)-Z-BDDA/kg diet [control diet + (+)-Z-BDDA (CB)], or 100 mg rosiglitazone/kg diet [control diet + rosiglitazone (CR)]. At sacrifice, physiological, biochemical, and molecular parameters were examined. RESULTS: CB animals gained less weight and exhibited a decrease in total body lipids (p < 0.05) as compared with C or CR rats. Body weight and total body lipids were the highest in CR rats (p < 0.05). Liver weights in CB and CR rats were lower (p < 0.05) than in C rats, whereas kidney weights were lower in CB (p < 0.05) than in C and CR animals. Fasting plasma glucose was lower (p < 0.05) in the CB and CR animals when compared with C animals. C rats exhibited the highest concentration of total plasma cholesterol, and CR-treated rats exhibited the lowest concentration. Plasma triglycerides followed the same pattern as plasma cholesterol. Histomorphometry of heart vasculature revealed that CB and CR treatments produced a significant shift from small to large venules and arterioles compared with C (p < 0.05). Liver expression profiles of peroxisome proliferator-activated receptor (PPAR) alpha, PPARgamma, and PPAR-regulated genes revealed encouraging CB-induced effects. DISCUSSION: These results suggest that (+)-Z-BDDA may have applications in treating obesity and complications associated with the metabolic syndrome.     

Presynaptic muscarinic facilitation of parasympathetic neurotransmission after sympathectomy in the rat choroid

The effect of sympathectomy on parasympathetic regulation of ocular perfusion was investigated. Uveal blood flow through the vortex veins was measured by laser Doppler flowmetry during electrical stimulation of the superior salivatory nucleus, which activates ocular parasympathetic nerves, in adult rats with intact innervation and 2 days or 6 weeks after excision of the ipsilateral superior cervical ganglion. In all groups, parasympathetic stimulation produced comparable increases in flux, which were abolished by the selective neuronal nitric-oxide synthetase inhibitor, 1-(2-trifluoromethylphenyl) imidazole. Atropine had no effect in control and acutely sympathectomized rats but abolished the flux increase in four of six chronically sympathectomized animals, and 1-(2-trifluoromethylphenyl) imidazole eliminated the residual response. The muscarinic receptor agonist bethanechol did not affect basal flow in control or sympathectomized rats. However, bethanechol enhanced parasympathetically mediated vasodilation, but only in rats studied at 6 weeks after sympathectomy, a finding consistent with the appearance of muscarinic prejunctional facilitation of nitrergic transmission. In chronically sympathectomized rats, the M(2) and M(4) receptor antagonists methoctramine and tropicamide did not affect choroidal flow during parasympathetic activation. However, pirenzepine increased flux, implying the presence of M(1) inhibitory autoreceptors on these nerves. Parasympathetically mediated increased flux was partially blocked by the M(3) antagonist 4-diphenylacetoxy-N-methylpiperdine, and the remaining vasodilation was blocked by atropine. We conclude that parasympathetic prejunctional facilitatory M(3) and probably M(5) receptors adopt a crucial role after chronic sympathectomy in maintaining nitrergic vasodilatory ocular neurotransmission in the face of down-regulated nitric oxide transmitter mechanisms.