The regulatory subunit of PKA-I remains partially structured and undergoes β-aggregation upon thermal denaturation

Background: The regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B. Methodology/Principal Findings: As revealed by circular dichroism (CD) and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212-216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers. Conclusions/Significance: Thermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation. © 2011 Dao et al

Coexpression of VEGF-C and COX-2 and its association with lymphangiogenesis in human breast cancer

<p>Abstract</p> <p>Background</p> <p>Lymphangiogenesis has become a new research frontier in tumor metastasis since the discovery of reliable lymphatic markers that have allowed observation and isolation of lymphatic endothelium. Cyclooxygenase-2 (COX-2) has been reported to be involved in the critical steps in carcinogenesis. However, possible role of COX-2 in lymphangiogenesis and lymphatic metastasis is still poorly understood. In present study, we aimed to investigate the relationship between vascular endothelial growth factor-C (VEGF-C) and COX-2 in human breast cancer, and correlations with lymphangiogenesis and prognosis.</p> <p>Methods</p> <p>Tissue samples of primary tumors from 70 patients undergoing intentionally curative surgical resections for breast cancer were immunohistochemically examined for VEGF-C, COX-2, and D2-40 expressions. The association between COX-2 and VEGF-C expressions and clinicopathological parameters as well as prognosis were analysised. To demonstrate the presence of proliferating lymphatic endothelial cells, 10 random cases with high LVD counts were selected for D2-40/Ki-67 double immunostaining.</p> <p>Results</p> <p>A significant correlation was found between the expression of VEGF-C and COX-2 (<it>r </it>= 0.529, <it>P </it>< 0.001), and both elevated VEGF-C expression and elevated COX-2 expression were associated with higher lymph vessel density (LVD), lymph node metastasis and D2-40 positive lymphatic invasion (LVI) as well as worse disease free survival (DFS) and overall survival (OS) in a univariate analysis. In the double immunostain for the lymph vessel marker D2-40 and the proliferation marker Ki-67, the results confirmed Ki-67-positive nuclei in a proportion of lymph vessel endothelial cells.</p> <p>Conclusion</p> <p>There is indeed lymphangiogenesis in breast cancer, the most compelling evidence being the presence of proliferating lymphatic endothelial cells. VEGF-C and COX-2 are coexpressed and significantly associated with lymphangiogenesis and prognosis in invasive breast cancer. Suggesting COX-2 may up-regulate VEGF-C expression and thus promote lymph node metastasis via lymphangiogenesis pathway in human breast cancer.</p

Narrow-band imaging does not improve detection of colorectal polyps when compared to conventional colonoscopy: a randomized controlled trial and meta-analysis of published studies

<p>Abstract</p> <p>Background</p> <p>A colonoscopy may frequently miss polyps and cancers. A number of techniques have emerged to improve visualization and to reduce the rate of adenoma miss.</p> <p>Methods</p> <p>We conducted a randomized controlled trial (RCT) in two clinics of the Gastrointestinal Department of the Sanitas University Foundation in Bogota, Colombia. Eligible adult patients presenting for screening or diagnostic elective colonoscopy were randomlsy allocated to undergo conventional colonoscopy or narrow-band imaging (NBI) during instrument withdrawal by three experienced endoscopists. For the systematic review, studies were identified from the Cochrane Library, PUBMED and LILACS and assessed using the Cochrane risk of bias tool.</p> <p>Results</p> <p>We enrolled a total of 482 patients (62.5% female), with a mean age of 58.33 years (SD 12.91); 241 into the intervention (NBI) colonoscopy and 241 into the conventional colonoscopy group. Most patients presented for diagnostic colonoscopy (75.3%). The overall rate of polyp detection was significantly higher in the conventional group compared to the NBI group (RR 0.75, 95%CI 0.60 to 0.96). However, no significant differences were found in the mean number of polyps (MD -0.1; 95%CI -0.25 to 0.05), and the mean number of adenomas (MD 0.04 95%CI -0.09 to 0.17). Meta-analysis of studies (regardless of indication) did not find any significant differences in the mean number of polyps (5 RCT, 2479 participants; WMD -0.07 95% CI -0.21 to 0.07; I2 68%), the mean number of adenomas (8 RCT, 3517 participants; WMD -0.08 95% CI -0.17; 0.01 to I2 62%) and the rate of patients with at least one adenoma (8 RCT, 3512 participants, RR 0.96 95% CI 0.88 to 1,04;I2 0%).</p> <p>Conclusion</p> <p>NBI does not improve detection of colorectal polyps when compared to conventional colonoscopy (Australian New Zealand Clinical Trials Registry <a href="http://www.anzctr.org.au/ACTRN12610000456055.aspx">ACTRN12610000456055</a>).</p

Curcumin Promotes A-beta Fibrillation and Reduces Neurotoxicity in Transgenic Drosophila

The pathology of Alzheimer's disease (AD) is characterized by the presence of extracellular deposits of misfolded and aggregated amyloid-β (Aβ) peptide and intraneuronal accumulation of tangles comprised of hyperphosphorylated Tau protein. For several years, the natural compound curcumin has been proposed to be a candidate for enhanced clearance of toxic Aβ amyloid. In this study we have studied the potency of feeding curcumin as a drug candidate to alleviate Aβ toxicity in transgenic Drosophila. The longevity as well as the locomotor activity of five different AD model genotypes, measured relative to a control line, showed up to 75% improved lifespan and activity for curcumin fed flies. In contrast to the majority of studies of curcumin effects on amyloid we did not observe any decrease in the amount of Aβ deposition following curcumin treatment. Conformation-dependent spectra from p-FTAA, a luminescent conjugated oligothiophene bound to Aβ deposits in different Drosophila genotypes over time, indicated accelerated pre-fibrillar to fibril conversion of Aβ1–42 in curcumin treated flies. This finding was supported by in vitro fibrillation assays of recombinant Aβ1–42. Our study shows that curcumin promotes amyloid fibril conversion by reducing the pre-fibrillar/oligomeric species of Aβ, resulting in a reduced neurotoxicity in Drosophila

COX-2-mediated stimulation of the lymphangiogenic factor VEGF-C in human breast cancer

Increased expression of COX-2 or VEGF-C has been correlated with progressive disease in certain cancers. Present study utilized several human breast cancer cell lines (MCF-7, T-47D, Hs578T and MDA-MB-231, varying in COX-2 expression) as well as 10 human breast cancer specimens to examine the roles of COX-2 and prostaglandin E (EP) receptors in VEGF-C expression or secretion, and the relationship of COX-2 or VEGF-C expression to lymphangiogenesis. We found a strong correlation between COX-2 mRNA expression and VEGF-C expression or secretion levels in breast cancer cell lines and VEGF-C expression in breast cancer tissues. Expression of LYVE-1, a selective marker for lymphatic endothelium, was also positively correlated with COX-2 or VEGF-C expression in breast cancer tissues. Inhibition of VEGF-C expression and secretion in the presence of COX-1/2 or COX-2 inhibitors or following downregulation of COX-2 with COX-2 siRNA established a stimulatory role COX-2 in VEGF-C synthesis by breast cancer cells. EP1 as well as EP4 receptor antagonists inhibited VEGF-C production indicating the roles of EP1 and EP4 in VEGF-C upregulation by endogenous PGE2. Finally, VEGF-C secretion by MDA-MB-231 cells was inhibited in the presence of kinase inhibitors for Her-2/neu, Src and p38 MAPK, indicating a requirement of these kinases for VEGF-C synthesis. These results, for the first time, demonstrate a regulatory role of COX-2 in VEGF-C synthesis (and thereby lymphangiogenesis) in human breast cancer, which is mediated at least in part by EP1/EP4 receptors