7,503 research outputs found
Binding of the heterogeneous ribonucleoprotein K (hnRNP K) to the Epstein-Barr virus nuclear antigen 2 (EBNA2) enhances viral LMP2A expression.
No full textThe Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties
Epstein-Barr virus latent proteins regulate expression of the anti-apoptotic cellular bfl-1 gene
No full textThe ubiquitous and oncogenic human herpes-virus Epstem-Barr virus (EB V) establishes a latent infection and promotes the long-term survival of the infected host cell by targeting the molecular machinery that controls cell fate decisions, including apoptosis, proliferation and differentiation. These host-virus interactions are likely to play a crucial role in the development of EBV-associated malignancies such, as Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma and tumours in lmmunosuppressed individuals. It has previously been shown in our laboratory that two EBV latent proteins, latent membrane protein 1 (LMP1) and EBV nuclear antigen 2 (EBNA2), which are major effectors of cellular phenotypic change, can independently regulate expression of the cellular bfl-1 gene Bfl-1 is an anti-apoptotic protein of the Bcl-2 family, whose preferential expression in hematopoietic and endothelial cells is controlled by inflammatory stimuli. In this study, it is reported that LMP1 and EBNA2 regulate bfl-1 activity through interactions with components of the NF-kB and Notch signalling pathways respectively NF-kB composed of p65 sub-units trans-activated the bfl-1 promoter m the EBV-negative cell line DG75, and an NF-icB-like binding site at position -52 to -43 relative to the transcription start site was essential for this effect. An RBP-Jk/CBF1 mutant blocked EBNA2-mediated trans-activation of bfl-1 in DG75 cells, indicating an important role for this DNA-binding protein in bfl-1 trans-activation by EBNA2. Although RBP-Jk/CBFI is also essential for signalling by the cellular equivalent of EBNA2, mtra-cellular Notch (NotchIC), this protein was not found to trans-activate the bfl-1 promoter. Both EBNA2 and LMP1 are expressed in EBVmfected cell lines, and EBNA2 is responsible for induction of LMP1 Blocking of either EBNA2- or LMP1-mediated signalling in EBV-mfected cell lines did not dramatically affect the level of bfl-1 promoter activity. However, when both EBNA2 and LMP1 signalling were blocked simultaneously, a significant decrease in the level of bfl-1 activity was observed. These data indicate a role for both EBNA2 and LMP1 in the regulation of the promoter for the bfl-1 gene m the context of the EBV-infected cell. These findings are relevant to our understanding of EBV persistence in the infected host, and its role in malignant disease
Barr, Edward B. (FA 813)
No full textFinding aid only for Folklife Archives Project 813. This collection “Hog Killing on the Farm”describes the slaughtering process on the Robert Hall farm in Crestwood, Oldham County, Kentucky. Narrative is in interview format and is illustrated with photographs. This information was collected by Western Kentucky University student Edward B. Barr for credit in a folklore class
Reduced sensitivity of fa/fa Zucker rats to adrenomedullin
No full textRat adrenomedullin is a peptide vasodepressor that may be of importance in the pathogenesis of hypertensive disease. Because of the known link between obesity and hypertension, we hypothesized that decreased responsiveness to adrenomedullin might be seen in an obese rodent model. In this study, the in vivo vasodilator actions of exogenous adrenomedullin were compared in anesthetized lean (n = 7) and obese (fa/fa) Zucker rats (n = 8). Adrenomedullin dose dependently lowered mean arterial pressure in both phenotypes, but the half-maximal dose (ID50) was 2-fold higher in fa/fa rats (1.7 +/- 0.22 vs. 0.83 +/- 0.06 nmol/kg). Moreover, the duration of effect was markedly reduced in the fa/fa rats, to 1-2 min from about 5 min in the lean animals. There was no evidence for an increased rate of degradation of adrenomedullin in the fa/fa rats. Although the rats used in this study were not hypertensive, adrenomedullin had reduced sensitivity and duration of action. The evidence suggests possible defects at the target receptor or altered metabolism of adrenomedullin in obesity.LR: 20061115; PUBM: Print; JID: 0372712; 0 (Peptides); 0 (Vasodilator Agents); 148498-78-6 (Adrenomedullin); ppublishSource type: Electronic(1
Identification of biochemical defects in pancreatic islets of fa/fa rats: a developmental study
No full textAdult obese (fa/fa) Zucker rats hypersecrete insulin in response to glucose and other secretagogues. Functional changes in islet alpha 2-adrenoceptors (8) and glycolytic regulation (9) have been reported. In this study, the development of these biochemical lesions in islets isolated from suckling (3 week old) and weanling (5 week old) lean and fa/fa rats was investigated and compared to results in adult animals. Glucose (15 mM)-induced insulin secretion was inhibited by mannoheptulose (MH) in lean (n = 8) but not fa/fa (n = 10) adult rats, indicating loss of sensitivity of glucokinase to competitive inhibition. Sensitivity to MH was somewhat reduced in the islets of 3- and 5-week-old fa/fa (n = 7 and 12) compared to lean (n = 15 and 9) rats, requiring 30-100 fold higher concentrations to achieve significant inhibition. At 3 weeks of age fa/fa rats did not differ from lean controls in either islet insulin content or body weight, but both parameters were increased in fa/fa rats by 5 weeks. The presence of altered alpha 2-adrenoceptor function in fa/fa rats could not be confirmed in this study. Unlike the previous report, prazosin did not antagonize alpha 2-agonist mediated inhibition of insulin secretion. The presence of defective regulation of the glycolytic pathway by mannoheptulose in suckling and weanling rats may contribute to development of hyperinsulinemia in fa/fa rats.LR: 20061115; PUBM: Print; JID: 9305691; 0 (Receptors, Adrenergic, alpha); 11061-68-0 (Insulin); 50-99-7 (Glucose); 654-29-5 (Mannoheptulose); EC 2.7.1.2 (Glucokinase); ppublishSource type: Electronic(1
Effect of adrenalectomy on the development of a pancreatic islet lesion in fa/fa rats
No full textAdrenalectomy prevents development of obesity and hyperinsulinaemia in obese (fa/fa) Zucker rats, thereby implicating the hypothalamo- pituitary-adrenal axis in the pathogenesis of obesity. In this study glucose-induced insulin secretion and glucokinase activity were investigated in isolated islets from adrenalectomized and control obese and lean female rats. Islets from control fa/fa rats were more sensitive to glucose with a half-maximal effective concentration (EC50) of 6.1 +/- 2.0 mmol. 1(-1) compared with 10.6 +/- 2.7 mmol. 1(-1) for adrenalectomized fa/fa rat islets. Adrenalectomy did not alter the islet sensitivity to glucose in the lean rats (EC50 of 9.4 +/- 1.5 mmol.1(-1) and 9.3 +/- 2.0 mmol. 1(-1) for adrenalectomized and control lean rats respectively). Mannoheptulose did not inhibit insulin secretion from control obese rats; however at concentrations of 1.0 mmol. 1(-1) or more it significantly inhibited glucose-induced insulin secretion in adrenalectomized obese and lean, and control lean rat islets (P < 0.05). In adrenalectomized fa/fa islets the glucokinase Km was increased twofold compared with the control fa/fa rats (9.5 +/- 1.5 mmol. 1(-1) vs 5.0 +/- 1.5 mmol. 1(-1), respectively), but there was no significant change in glucokinase Km in the lean rat islets after adrenalectomy. Mannoheptulose (10 mmol.1(-1) caused a significant reduction in glucose phosphorylation in disrupted islets of adrenalectomized fa/fa and lean, and of control lean rats, but not of control fa/fa rats. These data demonstrate that development of abnormal regulation of glycolysis in pancreatic islet beta cells of fa/fa rats, as indicated by the insulin response to manno-heptulose and glucokinase activity, is dependent on an intact hypothalamo-pituitary-adrenal axis.LR: 20061115; PUBM: Print; JID: 0006777; 0 (Blood Glucose); 11061-68-0 (Insulin); 50-22-6 (Corticosterone); 50-99-7 (Glucose); 654-29-5 (Mannoheptulose); EC 2.7.1.1 (Hexokinase); EC 2.7.1.2 (Glucokinase); ppublishSource type: Electronic(1
Ultrastructural and secretory heterogeneity of fa/fa (Zucker) rat islets
No full textMany previous studies of obese rodents documented biochemical changes in pancreatic islets that contribute to hyperinsulinemia in vivo. Those studies used heterogeneous populations of islets, although the size of islets from obese rats ranges from 500 microm. Here, functional and morphological changes in size-sorted ( 250 microm diameter) islets from obese Zucker (fa/fa) rats were correlated. Ultrastructural examination revealed that > 250 microm cultured islets had an increased number of immature secretory granules in the beta cells. The number of degranulated beta cells in > 250 and 250 microm, 250 microm islets compared with small islets. Studies of individual beta cells by reverse hemolytic plaque assay revealed 3-fold more cells from > 250 microm islets were stimulated by 1.4 mmol.l(-1) glucose than cells from < 125 microm islets. We conclude that functional defects in mixed size populations of islets from fa/fa rats are mainly due to alterations in the large islets, whereas smaller islets have relatively normal function. Exposure to high glucose exacerbates morphological and functional differences of large islets, which could have important implications in the transition to noninsulin-dependent diabetes when beta cell insulin production is unable to compensate for hyperglycemia.LR: 20061115; PUBM: Print; JID: 7500844; 11061-68-0 (Insulin); 50-99-7 (Glucose); 654-29-5 (Mannoheptulose); 7782-44-7 (Oxygen); ppublishSource type: Electronic(1
Evidence for defective glucose sensing by islets of fa/fa obese Zucker rats
No full textThe hypothesis that a defect in glucose sensing by islets of fa/fa Zucker rats contributes to hyperinsulinemia in these animals was tested. Islets from lean and fa/fa rats were isolated by collagenase digestion and step-density gradient purification and then cultured overnight in Dulbecco's modified Eagle's medium containing 12.5 mM glucose. Obese rat islets were more sensitive to hypoglycemic glucose levels with half-maximal effective concentration (EC50) of 5.6 mM compared with an EC50 of 8.2 mM for lean rat islets. In contrast, responsiveness of both phenotypes to alpha-ketoisocaproate and quinine was similar. Mannoheptulose did not inhibit insulin secretion from fa/fa islets, although inhibitors of later events in the stimulus-secretion coupling pathway were normally inhibited by iodoacetate and diazoxide. Finally, starvation in vivo and culture of islets in low glucose concentrations (5 mM) in vitro both decreased glucose-stimulated insulin secretion from lean but not fa/fa rat islets. We conclude that fa/fa rat islets have an exaggerated insulin response to hypoglycemic stimuli, possibly as a result of a defect in B-cell glucokinase function.LR: 20061115; PUBM: Print; JID: 0372712; 0 (Amino Acids); 0 (Blood Glucose); 0 (Iodoacetates); 11061-68-0 (Insulin); 130-95-0 (Quinine); 364-98-7 (Diazoxide); 50-99-7 (Glucose); 56-65-5 (Adenosine Triphosphate); 64-69-7 (Iodoacetic Acid); 654-29-5 (Mannoheptulose); EC 2.7.1.2 (Glucokinase); ppublishSource type: Electronic(1
Functional characterization of alpha-adrenoceptors on pancreatic islets of fa/fa Zucker rats
No full textRecently, a defect in pertussis toxin-independent actions of epinephrine on pancreatic B-cells of fa/fa Zucker rats was reported (Cawthorn and Chan (1991) Mol. Cell. Endocrinol. 75, 197-204). We now report studies of islet alpha 2-adrenoceptor function of fa/fa rats. Insulin and cAMP production by islets of obese rats were both inhibited by the alpha 2-adrenoceptor agonist clonidine. Calculated pD2 values for clonidine were 9.57 +/- 0.59 and 9.43 +/- 0.33 for lean and fa/fa rat islets, respectively. Yohimbine reversed clonidine effects equipotently in lean and obese rat islets (pA2 values of 7.48 +/- 0.57 vs 7.43 +/- 0.58). Unexpectedly, the alpha 1-antagonist prazosin stimulated insulin secretion from islets of obese but not lean rats. Functional characteristics of the alpha-adrenoceptors on fa/fa islets are thus similar to those recently designated alpha 2B. Altered expression of alpha-adrenoceptors on pancreatic islets of fa/fa rats may contribute to changes in the pertussis toxin-independent pathway of epinephrine action previously observed.LR: 20061115; PUBM: Print; JID: 7500844; 0 (Receptors, Adrenergic, alpha); 11061-68-0 (Insulin); 146-48-5 (Yohimbine); 19216-56-9 (Prazosin); 4205-90-7 (Clonidine); 50-99-7 (Glucose); 51-43-4 (Epinephrine); 60-92-4 (Cyclic AMP); 66428-89-5 (Forskolin); ppublishSource type: Electronic(1
Glucose refractoriness of beta-cells from fed fa/fa rats is ameliorated by nonesterified fatty acids
No full textThe aim of this study was to characterize the glucose responsiveness of individual beta-cells from fa/fa rats under ad libitum feeding conditions. Enlarged intact islets from fed fa/fa rats had a compressed insulin response curve to glucose compared with smaller islets. Size-sorted islets from obese rats yielded beta-cells whose glucose responsiveness was assessed by reverse hemolytic plaque assay to determine whether glucose refractoriness was caused by a decreased number of responsive cells or output per cell. In addition, the effects of palmitic acid on glucose-stimulated insulin secretion were assessed because of evidence that nonesterified fatty acids have acute beneficial effects. Two- to threefold more beta-cells from >250 microm diameter (large) islets than 10-fold increase in recruitment of active cells from small islets, compared with only a 2.6-fold increase in large islets. This refractoriness was partially reversed by preincubation of the cells in low glucose for 2 h. In addition, secretion per cell of the large islet beta-cell population was significantly reduced compared with lean beta-cells, so that the overall response capacity of large but not small islet beta-cells was significantly reduced at high glucose. Therefore, continued near-normal function of the beta-cells from small islets of fa/fa rats seems crucial for glucose responsiveness. Incubation of beta-cells from large islets with palmitic acid normalized the secretory capacity to glucose mainly by increasing recruitment and secondarily by increasing secretion per cell. In conclusion, these studies demonstrate refractoriness to glucose of beta-cells from large islets of fa/fa rats under ad libitum feeding conditions. When acutely exposed to nonesterified fatty acids, islets from fa/fa rats have a potentiated insulin response despite chronic elevation of plasma lipids in vivo.LR: 20061115; PUBM: Print; JID: 0372712; 0 (Fatty Acids, Nonesterified); 0 (Lipids); 11061-68-0 (Insulin); 50-99-7 (Glucose); 57-10-3 (Palmitic Acid); ppublishSource type: Electronic(1
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