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Qun S Zang,
Hesham Sadek,
David L Maass,
Bobbie Martinez,
Lisha Ma,
Jessica A Kilgore,
Noelle S Williams,
Doug E Frantz,
Jane G Wigginton, Fiemu E Nwariaku,
Steven E Wolf,
Joseph P Minei
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ABSTRACT: Using a mitochondria-targeted vitamin E (Mito-Vit-E) in a rat pneumonia-related sepsis model, we examined the role of mitochondrial reactive oxygen species in sepsis-mediated myocardial inflammation and subsequent cardiac contractile dysfunction. Sepsis was produced in adult male Sprague-Dawley rats via intratracheal injection of S. pneumonia (4 × 10(6) colony formation units per rat). A single dose of Mito-Vit-E, vitamin E, or control vehicle, at 21.5 μmol/kg, was administered 30 min postinoculation. Blood was collected, and heart tissue was harvested at various time points. Mito-Vit-E in vivo distribution was confirmed by mass spectrometry. In cardiac mitochondria, Mito-Vit-E improved total antioxidant capacity and suppressed H(2)O(2) generation, whereas vitamin E offered little effect. In cytosol, both antioxidants decreased H(2)O(2) levels, but only vitamin E strengthened antioxidant capacity. Mito-Vit-E protected mitochondrial structure and function in the heart during sepsis, demonstrated by reduction in lipid and protein oxidation, preservation of mitochondrial membrane integrity, and recovery of respiratory function. While both Mito-Vit-E and vitamin E suppressed sepsis-induced peripheral and myocardial production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and interleukin-6), Mito-Vit-E exhibited significantly higher efficacy (P < 0.05). Stronger anti-inflammatory action of Mito-Vit-E was further shown by its near-complete inhibition of sepsis-induced myeloperoxidase accumulation in myocardium, suggesting its effect on neutrophil infiltration. Echocardiography analysis indicated that Mito-Vit-E ameliorated cardiac contractility of sepsis animals, shown by improved fractional shortening and ejection fraction. Together, our data suggest that targeted scavenging of mitochondrial reactive oxygen species protects mitochondrial function, attenuates tissue-level inflammation, and improves whole organ activities in the heart during sepsis.
AJP Heart and Circulatory Physiology 03/2012; 302(9):H1847-59. · 3.71 Impact Factor
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Youxue Wang,
Qun S Zang,
Zijuan Liu,
Qian Wu,
David Maass,
Genevieve Dulan,
Philip W Shaul,
Lisa Melito,
Doug E Frantz,
Jessica A Kilgore,
Noelle S Williams,
Lance S Terada, Fiemu E Nwariaku
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ABSTRACT: Endothelial migration is a crucial aspect of a variety of physiologic and pathologic conditions including atherosclerosis and vascular repair. Reactive oxygen species (ROS) function as second messengers during endothelial migration. Multiple intracellular sources of ROS are regulated by cellular context, external stimulus, and the microenvironment. However, the predominant source of ROS during endothelial cell (EC) migration and the mechanisms by which ROS regulate cell migration are incompletely understood. In this study, we tested the hypothesis that mitochondria-derived ROS (mtROS) regulate EC migration. In cultured human umbilical vein endothelial cells, VEGF increased mitochondrial metabolism, promoted mtROS production, and induced cell migration. Either the targeted mitochondrial delivery of the antioxidant, vitamin E (Mito-Vit-E), or the depletion of mitochondrial DNA abrogated VEGF-mediated mtROS production. Overexpression of mitochondrial catalase also inhibited VEGF-induced mitochondrial metabolism, Rac activation, and cell migration. Furthermore, these interventions suppressed VEGF-stimulated EC migration and blocked Rac1 activation in endothelial cells. Constitutively active Rac1 reversed Mito-Vit-E-induced inhibition of EC migration. Mito-Vit-E also attenuated carotid artery reendothelialization in vivo. These results provide strong evidence that mtROS regulate EC migration through Rac-1.
AJP Cell Physiology 06/2011; 301(3):C695-704. · 3.54 Impact Factor
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ABSTRACT: Reactive oxygen species (ROS) mediate various cell fate decisions in normal and transformed cells. In this issue of Cancer Cell, Zhu et al. demonstrate the ability of ANGPTL4 to engage integrin-dependent survival signals by activation of the NADPH oxidase Nox1, thus mimicking anchorage conditions and bypassing anoikis by controlling ROS.
Cancer cell 03/2011; 19(3):297-9. · 25.29 Impact Factor
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ABSTRACT: Cytologically indeterminate thyroid nodules represent a diagnostic and therapeutic challenge. In 2007, the National Cancer Institute recommended The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) as a means of improving the accuracy of thyroid cytopathology. Our objective was to determine the effect of TBSRTC on thyroidectomy rates and malignancy risk in cytologically indeterminate lesions.
We compared thyroidectomy rates and malignancy risk in patients with indeterminate thyroid cytopathology across 2 time periods, spanning January 2000 and November 2009; pre-TBSRTC (January 2000 to September 2003) and post-TBSRTC (June 2008 to November 2009). Statistical comparisons were performed using the Fisher's exact test and chi-square analysis (P = .05 significant).
We performed 938 fine-needle aspirations in the first period, 765 in the second. We identified 78 (8.3%) cytologically indeterminate lesions in the pre-TBSRTC group and 91 (11.9%) lesions in the post-TBSRTC group. We found no difference in thyroidectomy rates between the groups (37/78 [47%] pre-Bethesda versus 32/91 [35%] post-Bethesda; P = .12). However, the malignancy rate was significantly lower in the post-TBSRTC group (13/37 [35%] pre-Bethesda versus 4/32 [13%] post-Bethesda; P = .02).
Application of TBSRTC is associated with lower malignancy risk in indeterminate thyroid nodules, despite similar thyroidectomy rates. These findings imply that standardization of cytologic classification improves diagnostic accuracy.
Surgery 12/2010; 148(6):1267-72; discussion 1272-3. · 3.10 Impact Factor
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ABSTRACT: Macronodular adrenocortical hyperplasia classically presents with progressive hypercortisolemia and Cushing syndrome. We describe a 29-yr-old man with massive macronodular adrenocortical hyperplasia without hypercortisolemia but rather markedly elevated and nonsuppressible production of dehydroepiandrosterone (DHEA) and its sulfate (DHEAS).
To characterize the clinical and molecular features of this case and to determine whether the tissue biochemically resembles the zona reticularis or fetal adrenal.
University clinic, hospital, and laboratories.
Static and dynamic blood and urine testing were performed preoperatively. Tissue was studied by light microscopy, immunoblot, RNA microarray, and enzyme assay.
A 29-yr-old man with incidentally discovered bilateral adrenal enlargement.
Bilateral adrenalectomy.
Molecular studies compared with control samples.
Hypercortisolism and 21-hydroxylase deficiency were excluded. DHEA, DHEAS, and 17-hydroxypregnenolone were markedly elevated and did not suppress with dexamethasone 2 mg/d for 4 d. Homogenates of the adrenals demonstrated high 17-hydroxylase, good 17,20-lyase, and low or absent 21-hydroxylase and 3β-hydroxysteroid dehydrogenase activities. Immunoblots confirmed robust expression of cytochrome P450c17 and AKR1C3 but not P450c21. Microarray analysis demonstrated high CYP11A1 and CYP17A1 expression but low or absent HSD3B1, HSD3B2, and CYP21A2 expression. Expression of mRNA for cytochrome b(5) (CYB5A) and AKR1C3, markers of the zona reticularis, were markedly elevated.
This is the first case of macronodular hyperplasia of the adrenal zona reticularis confirmed with studies of enzyme activity, mRNA expression, and protein identification. We speculate that this condition can be clinically silent in men but might cause severe hyperandrogenemia in women.
The Journal of clinical endocrinology and metabolism 11/2010; 96(2):E243-50. · 6.50 Impact Factor
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ABSTRACT: Studies from animal models suggest that myocardial mitochondrial damage contributes to cardiac dysfunction after burn injury. In this report, we used an ex vivo model of primary cardiomyocyte culture to investigate the mechanisms of burn-induced mitochondrial impairment. Briefly, blood serum was collected from Sprague-Dawley (SD) rats subjected to 40% total body surface area burn and added (10% vol/vol) to primary cardiomyocytes prepared from SD rats. The effect of the burn serum on mitochondrial function and membrane integrity in the myocytes was analyzed. Exposure of myocytes to burn serum doubled the mitochondrial membrane damage measured by two independent assays. This treatment also significantly elevated mitochondrial oxidative stress, indicated by a more than 30% increase in lipid oxidation. Downregulation of mitochondrial antioxidant defense was also evident since the activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase were reduced by about 30% and 50%, respectively. Burn serum also induced deficiency of mitochondrial metabolism, indicated by a 30% decrease in the activity of cytochrome c oxidase. These mitochondrial dysfunctions appear to be generated by oxidative stress because burn serum induced a significant increase of mitochondrial oxygen species (mtROS) in cardiomyocytes, and pretreatment of cardiomyocytes with the antioxidant N-acetyl-cysteine prevented the mitochondrial damages induced by burn serum. Remarkably, the increase in mtROS was abolished by an antibody-mediated blockade of CD14. Furthermore, burn injury-induced mitochondrial damage in cardiomyocytes was prevented in CD14 knockout mice. Taken together, these data suggested that burn injury produces CD14-dependent mitochondrial damage via oxidative stress in myocardium.
AJP Heart and Circulatory Physiology 03/2010; 298(6):H1951-8. · 3.71 Impact Factor
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ABSTRACT: Vascular endothelial cadherin (VE-cad) is essential for endothelial barrier integrity and vascular sprouting. However, the role of this important protein in cardiovascular development is only recently becoming apparent.
To characterize the role of VE-cadherin in cardiovascular development, we analyzed cardiovascular development in a zebrafish VE-cad knockdown model. Embryos deficient in VE-cad show profoundly impaired cardiac development despite having apparently normal peripheral vasculature. Initial formation of the heart proceeds normally in knockdown embryos, but subsequent looping morphogenesis is impaired. Consistent with these results, VE-cad knockdown embryos demonstrate impaired cardiac function and early circulatory arrest. Histologic examination of knockdown embryos shows persistent, abnormal separation of the endocardial and myocardial layers. Using transmission electron microscopy, we demonstrate that endocardial junctions form poorly in VE-cad knockdown embryos, with resulting leak across the endothelial layer and reduction in the density of the cardiac jelly.
Our results demonstrate a significant role for VE-cadherin in cardiac development independent of its effects on the formation of the peripheral vasculature.
PLoS ONE 01/2010; 5(1):e8807. · 4.09 Impact Factor
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ABSTRACT: We hypothesized that an adrenal vein sampling (AVS) algorithm incorporating rapid cortisol assays, which enables resampling of the adrenal veins, would improve the success rate by a team of radiologists.
AVS is the most accurate means to localize aldosterone production in primary aldosteronism (PA). However, cannulation of the right adrenal vein (RAV) is difficult, and success is assumed from venography without the support of steroid assays. Furthermore, few institutions can assign all studies to 1 dedicated and experienced AVS interventional radiologist.
Retrospective chart review of patients with PA at our university hospitals who underwent AVS. We compared results for 30 AVS studies incorporating rapid cortisol assays with 30 conventional AVS studies.
The success rate for the control period was 73% (22/30 studies). For the first 30 studies after incorporating rapid cortisol assay, the success rate increased to 97% (29/30 studies). Resampling the RAV was required for 2 studies, and prolonged sheath insertion did not cause any complications.
High AVS success rates may be achieved by a team of interventional radiologists at 1 center using defined AVS protocols. Rapid cortisol assay allows for resampling of the RAV and improves AVS success rates.
Annals of surgery 03/2009; 249(2):318-21. · 7.90 Impact Factor
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Fiemu E Nwariaku
Journal of Surgical Research 07/2008; 154(2):304-11. · 2.25 Impact Factor
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ABSTRACT: The incidence of thyroid cancer is increasing. Our objective was to characterize the demographic pattern of this increase and to examine trends in surgical therapy for thyroid cancer.
Analysis of the SEER and NHDS databases was performed from 1974 to 2000 and from 1979 to 2004, respectively. Thyroid-related diagnoses were extracted, and thyroid cancer (ICD 193.X) were analyzed using the SAS statistical package. We compared the population-adjusted incidence of thyroid cancer and examined regional variations in the operative therapy for thyroid cancer.
The incidence of thyroid cancer has increased during the past 26 years. This increase occurred predominantly in women and in the Northeastern and Southern United States, whereas there has been a decrease in thyroid cancers in the Midwest. Papillary cancer accounts for most of this increase. Total thyroidectomy (TT) is now the most common operation for thyroid cancer. No differences in the use of TT were observed based on hospital size or insurance status.
The increasing incidence of thyroid cancer in the United States is predominantly in women. These results suggest that women are a high-risk group for developing thyroid cancer although men have higher stage disease.
Surgery 01/2008; 142(6):823-8; discussion 828.e1. · 3.10 Impact Factor
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ABSTRACT: A subgroup of patients with adrenal cortisol hypersecretion fails to meet the biochemical criteria for Cushing's syndrome. Appropriate therapy for this entity, subclinical Cushing's syndrome (subclinical CS), is unclear. We examined outcomes for patients who underwent unilateral adrenalectomy for subclinical CS.
Between 2003 and 2006, all patients who underwent adrenalectomy for cortisol hypersecretion caused by an adrenal mass were examined. We analyzed biochemical, metabolic, and clinical outcomes.
Overall, 24 patients underwent adrenalectomy for adrenal cortisol hypersecretion, of which 9 were found to have subclinical CS. Median serum cortisol was 2.0 microg/dL (range, 1.1-6.1) after 1-mg overnight dexamethasone suppression testing. Suspicious clinical findings on preoperative examination included skin bruising, unexplained weight gain, proximal muscle weakness, abnormal fat pads, skin thinning, fatigue, and facial plethora. During a median follow-up period of 5 months (range, 1-30 months), all 8 patients with easy bruising noted resolution postoperatively. Fatigue improved in 4 of 5 patients, muscle weakness in 6 of 8 patients, and weight in 7 of 9 patients, with a median body mass index change of -2.0 kg/m(2) (range, -7.1 to +0.5 kg/m(2)).
Adrenalectomy improves clinical and metabolic parameters for many patients with subclinical CS.
Surgery 01/2008; 142(6):900-5; discussion 905.e1. · 3.10 Impact Factor
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ABSTRACT: Primary aldosteronism is one of the few potentially curable forms of hypertension. This article highlights recent advances in the pathophysiology, diagnosis, and treatment of this relatively common secondary form of hypertension. These topics include the recognition that this disorder is more prevalent than previously assumed, the identification of high-risk populations that benefit most from screening, and the improved approaches to screening, diagnosis, localization, and treatment. This review uses illustrative examples to describe our approach to these patients.
Current Cardiology Reports 12/2007; 9(6):447-52.
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ABSTRACT: Detachment of parenchymal cells from a solid matrix switches contextual cues from survival to death during anoikis. Marked shape changes accompany detachment and are thought to trigger cell death, although a working model to explain the coordination of attachment sensation, shape change, and cell fate is elusive. The constitutive form of the adapter Shc, p52Shc, confers survival properties, whereas the longer p66Shc signals death through association with cytochrome c. We find that cells that lack p66Shc display poorly formed focal adhesions and escape anoikis. However, reexpression of p66Shc restores anoikis through a mechanism requiring focal adhesion targeting and RhoA activation but not an intact cytochrome c-binding motif. This pathway stimulates the formation of focal adhesions and stress fibers in attached cells and tension-dependent cell death upon detachment. p66Shc may thus report attachment status to the cell by imposing a tension test across candidate anchorage points, with load failure indicating detachment.
The Journal of Cell Biology 11/2007; 179(1):23-31. · 10.26 Impact Factor
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Richard J Auchus,
Donald W Chandler,
Sarita Singeetham,
Neema Chokshi, Fiemu E Nwariaku,
Bart L Dolmatch,
Shelby A Holt,
Frank H Wians,
Shellie C Josephs,
Clayton K Trimmer,
Jorge Lopera,
Wanpen Vongpatanasin,
Shawna D Nesbitt,
David Leonard,
Ronald G Victor
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ABSTRACT: In primary aldosteronism, elevated serum 18-hydroxycorticosterone (18OHB) suggests aldosterone-producing adenoma (APA) rather than bilateral, idiopathic hyperaldosteronism (IHA), but little is known about the relative production of 18OHB and aldosterone (A) in APAs compared with IHA.
We measured 18OHB, A, and cortisol (F) in blood from adrenal vein sampling (AVS) studies. We compared the discriminatory power of gradients in 18OHB/A and 18OHB/F ratios with A/F ratio gradients for distinguishing APA from IHA.
We measured 18OHB and A in excess serum from 23 AVS studies performed at our university hospitals.
We calculated the ratios 18OHB/A, 18OHB/F, and A/F for all specimens, and determined the adrenal vein gradients for these ratios.
The 18OHB/A ratios were much lower in blood draining APAs (2.17 +/- 0.62) than in blood draining the contralateral adrenals (12.96 +/- 12.76; P < 0.001) but similar to blood draining IHA adrenals (4.69 +/- 4.32; P = 0.02). In contrast, the 18OHB/F ratios were elevated in specimens from APAs (26.03 +/- 11.51) compared with IHA adrenals (9.22 +/- 5.18; P < 0.001) or the contralateral adrenals (6.23 +/- 2.97; P < 0.001). Using 18OHB/F gradient greater than two or 18OHB/A gradient less than 0.5 as criteria for lateralization, interpretations agreed with lateralizations based on A/F gradients in 21 of 23 cases.
High serum 18OHB in APA reflects augmented production of both 18OHB and A, not disproportionate 18OHB secretion relative to A. The 18OHB/A and 18OHB/F gradients are useful adjuncts but not as reliable as A/F gradients for A lateralization during AVS.
Journal of Clinical Endocrinology & Metabolism 08/2007; 92(7):2648-51. · 6.50 Impact Factor
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ABSTRACT: Anaplastic thyroid carcinoma is an aggressive form of cancer with no treatment. Angiogenesis inhibitors, such as TNP-470, a synthetic derivative of fumagillin, have been shown to reduce tumor size and increase survival in heterotopic animal models of thyroid cancer. Our goals were to determine the effect of TNP-470 on anaplastic thyroid cancer using an orthotopic murine model, to identify the molecular pathways of TNP-470 actions on endothelial cells, and to determine the non-endothelial tumor effects of TNP-470. We injected human anaplastic thyroid carcinoma cells (DRO'90) into the thyroid glands of nude mice. Mice received TNP-470 (30 mg/kg) s.c. for 6 weeks. TNP-470 prolonged survival and reduced liver metastases. TNP-470 had direct cytotoxic effects on anaplastic thyroid carcinoma cells in vitro and in vivo. Paradoxically, TNP-470 increased vascular endothelial growth factor secretion from tumor cells in vitro and in vivo. However, there was no associated increase in tumor microvessel density. In endothelial cells, TNP-470 prevented vascular endothelial growth factor-induced endothelial permeability, intercellular gap formation, and ruffle formation by preventing Rac1 activation.
Molecular Cancer Therapeutics 05/2007; 6(4):1329-37. · 5.23 Impact Factor
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ABSTRACT: An increasing number of patients with a history of solid organ malignancy now undergo surveillance imaging as part of their follow-up or for evaluation of other conditions. This imaging has led to both greater identification of asymptomatic adrenal masses and subsequent confusion among clinicians regarding the evaluation and treatment. Although established algorithms exist for treating such "incidentalomas" in otherwise healthy patients, the most effective way to do so in patients with known prior or concurrent malignancies is unclear. In this review, we explore methods of biochemical testing in such patients and discuss the role of imaging techniques in their ability to differentiate benign versus malignant lesions. In this population, we examine the increasing use of biopsy and discuss current data on both surveillance and resection of lesions based on their identity. Finally, we propose an algorithm to aid the clinician in evaluating and treating these complex patients efficiently.
The Oncologist 03/2007; 12(2):168-74. · 3.91 Impact Factor
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Fiemu E Nwariaku,
Barbra S Miller,
Richard Auchus,
Shelby Holt,
Lori Watumull,
Bart Dolmatch,
Shawna Nesbitt,
Wanpen Vongpatanasin,
Ronald Victor,
Frank Wians,
Edward Livingston,
William H Snyder
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ABSTRACT: Adrenal vein sampling is superior to computed tomography for subtype differentiation of primary hyperaldosteronism.
Retrospective review.
University medical center.
Forty-eight patients (32 men and 16 women) with biochemically confirmed primary hyperaldosteronism.
We compared demographic factors, results of biochemical and imaging studies (computed tomography and adrenal vein sampling), therapy, and patient outcomes.
Mean +/- SEM adrenal nodule size was 1.54 +/- 0.2 cm. Adrenal vein sampling was performed in 41 (85%) of 48 patients, and it was successful in 39 (95%) of those 41 patients. Concordance between computed tomography and adrenal vein sampling was observed in 22 (54%) of the 41 patients. Thirty-two patients underwent successful laparoscopic adrenalectomy. There was 1 complication and no deaths. All 32 patients were cured of hypokalemia.
Adrenal vein sampling is superior to image-based techniques for subtype differentiation of primary hyperaldosteronism.
Archives of Surgery 06/2006; 141(5):497-502; discussion 502-3. · 4.24 Impact Factor
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ABSTRACT: Endogenous oxidants participate in endothelial cell migration, suggesting that the enzymatic source of oxidants, like other proteins controlling cell migration, requires precise subcellular localization for spatial confinement of signaling effects. We found that the nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase adaptor p47(phox) and its binding partner TRAF4 were sequestered within nascent, focal complexlike structures in the lamellae of motile endothelial cells. TRAF4 directly associated with the focal contact scaffold Hic-5, and the knockdown of either protein, disruption of the complex, or oxidant scavenging blocked cell migration. An active mutant of TRAF4 activated the NADPH oxidase downstream of the Rho GTPases and p21-activated kinase 1 (PAK1) and oxidatively modified the focal contact phosphatase PTP-PEST. The oxidase also functioned upstream of Rac1 activation, suggesting its participation in a positive feedback loop. Active TRAF4 initiated robust membrane ruffling through Rac1, PAK1, and the oxidase, whereas the knockdown of PTP-PEST increased ruffling independent of oxidase activation. Our data suggest that TRAF4 specifies a molecular address within focal complexes that is targeted for oxidative modification during cell migration.
The Journal of Cell Biology 01/2006; 171(5):893-904. · 10.26 Impact Factor
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ABSTRACT: The frequency of surgery for peptic ulcer disease (PUD) has decreased dramatically during the last 3 decades. The purpose of this study was to characterize the Veteran patients undergoing surgery for peptic ulcer disease in a modern series and to examine the effect of H. pylori status on surgical outcome and recurrence of PUD.
An Institutional Review Board-approved retrospective review of all patients undergoing operations for peptic ulcer disease during a 66-month period at a single Veterans Administration medical center was performed. Patient records were examined for demographics, medication use, Helicobacter pylori status, operative details, and surgical outcomes.
From January 1999 to July 2004, 43 of 128 upper gastrointestinal operations were performed for PUD. Thirty-five operations (81%) were performed for bleeding or perforated ulcers, and 26 (60%) patients had no history of PUD. The mean age was 60 years, and 66% of patients were American Society of Anesthesiologists (ASA) class 3 or 4; 47% were Helicobacter pylori positive, and 54% used nonsteroidal anti-inflammatory (NSAID) medication. Hospital mortality was 23%. By univariate analysis, emergent surgery, higher ASA status, H. pylori status, and absence of a history of ulcer disease were risk factors for mortality (P <.05). Only 36% underwent definitive ulcer surgery. With a median follow-up of 18 months, there has been only 1 single recurrence (3%).
PUD still accounts for 33% of all gastroduodenal surgery performed in a Veterans Administration medical center. The majority of these operations are emergent operations in high-risk patients. In this era of effective acid suppression and H. pylori treatment, definitive ulcer surgery in the emergent setting may not be necessary.
The American Journal of Surgery 12/2005; 190(5):775-9. · 2.78 Impact Factor
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ABSTRACT: Gain-of-function mutations in the RET tyrosine kinase receptor cause the multiple endocrine neoplasia syndromes type 2a and 2b, and medullary thyroid cancer. We have previously shown that RET signals through focal adhesion kinase (FAK) in medullary thyroid cancer cells and that extracellular signal-regulated kinase (ERK) activity can be blocked by pp2, an inhibitor of both Src and RET. We hypothesized that RET could directly phosphorylate FAK and ERK.
RET and ERK kinase activity were measured with the use of an in vitro kinase assay. The relative contribution of RET in phosphorylation of ERK was tested by treating cells with PD98059, an inhibitor of MEK, and the RET inhibitor PP2, then measuring ERK activity.
Immunoprecipitated, mutant RET from cells or the recombinant RET kinase domain was able to directly phosphorylate tyrosine residues on FAK. Specifically Y576/577, Y861, and Y925, but not the autophosphorylation site Y397 of FAK, were phosphorylated by RET. Similarly ERK 2 could be phosphorylated at Y187 (Y204 in ERK1). Inhibition of both MEK (upstream of ERK) and RET was more potent than inhibition of either alone in decreasing ERK activity. Furthermore, tyrosine residues in DOK1, the p85 subunit of phosphatidylinositol 3' kinase, JNK 1 and 2, P-38, and phospholipase-gamma were directly phosphorylated by RET.
RET directly phosphorylates tyrosine residues on FAK, ERK 1/2, DOK1, the p85 subunit of of phosphatidylinositol 3' kinase, JNK 1 and 2, P-38, and phospholipase-gamma. These data indicate a direct interaction between RET and a broad range of effector molecules that may contribute to tumor pathogenesis.
Surgery 09/2005; 138(2):269-74. · 3.10 Impact Factor
