Genomic and proteomic profiling I: Leiomyomas in African Americans and Caucasians

<p>Abstract</p> <p>Background</p> <p>Clinical observations indicate that leiomyomas occur more frequently in African Americans compared to other ethnic groups with unknown etiology. To identify the molecular basis for the difference we compared leiomyomas form A. Americans with Caucasians using genomic and proteomic strategies.</p> <p>Methods</p> <p>Microarray, realtime PCR, 2D-PAGE, mass spectrometry, Western blotting and immunohistochemistry.</p> <p>Results</p> <p>Using Affymetrix U133A array and analysis based on P ranking (P < 0.01) 1470 genes were identified as differentially expressed in leiomyomas compared to myometrium regardless of ethnicity. Of these, 268 genes were either over-expressed (177 genes) or under-expressed (91 genes) based on P < 0.01 followed by 2-fold cutoff selection in leiomyomas of A. Americans as compared to Caucasians. Among them, the expression E2F1, RUNX3, EGR3, TBPIP, ECM2, ESM1, THBS1, GAS1, ADAM17, CST6, CST7, FBLN5, ICAM2, EDN1 and COL18 was validated using realtime PCR low-density arrays. 2D PAGE coupled with image analysis identified 332 protein spots of which the density/volume of 31 varied by greater than or equal to 1.5 fold in leiomyomas as compared to myometrium. The density/volume of 34 protein-spots varied by greater than or equal to 1.5 fold (26 increased and 8 decreased) in leiomyomas of A. Americans as compared to Caucasians. Tandem mass spectrometric analysis of 15 protein spots identified several proteins whose transcripts were also identified by microarray, including 14-3-3 beta and mimecan, whose expression was confirmed using western blotting and immunohistochemistry.</p> <p>Conclusion</p> <p>These findings imply that the level rather than the ethnic-specific expression of a number of genes and proteins may account for the difference between leiomyomas and possibly myometrium, in A. Americans and Caucasians. Further study using larger sample size is required to confirm these findings.</p

Gonadotropin releasing hormone analogue (GnRHa) alters the expression and activation of Smad in human endometrial epithelial and stromal cells

Gonadotropin releasing hormone analogues (GnRHa) are often used to regress endometriosis implants and prevent premature luteinizing hormone surges in women undergoing controlled ovarian stimulation. In addition to GnRH central action, the expression of GnRH and receptors in the endometrium implies an autocrine/paracrine role for GnRH and an additional site of action for GnRHa. To further examine the direct action of GnRH (Leuprolide acetate) in the endometrium, we determined the effect of GnRH on endometrial stromal (ESC) and endometrial surface epithelial (HES) cells expression and activation of Smads (Smad3, -4 and -7), intracellular signals activated by transforming growth factor beta (TGF-beta), a key cytokine expressed in the endometrium. The results show that GnRH (0.1 microM) increased the expression of inhibitory Smad7 mRNA in HES with a limited effect on ESC, while moderately increasing the common Smad4 and Smad7 protein levels in these cells (P < 0.05). GnRH in a dose- (0.01 to 10 microM) and time- (5 to 30 min) dependent manner decreased the rate of Smad3 activation (phospho-Smad3, pSmad3), and altered Smad3 cellular distribution in both cell types. Pretreatment with Antide (GnRH antagonist) resulted in further suppression of Smad3 induced by GnRH, with Antide inhibition of pSmad3 in ESC. Furthermore, co-treatment of the cells with GnRH + TGF-beta, or pretreatment with TGF-beta type II receptor antisense to block TGF-beta autocrine/paracrine action, in part inhibited TGF-beta activated Smad3. In conclusion, the results indicate that GnRH acts directly on the endometrial cells altering the expression and activation of Smads, a mechanism that could lead to interruption of TGF-beta receptor signaling mediated through this pathway in the endometrium

Genomic and proteomic profiling II: Comparative assessment of gene expression profiles in leiomyomas, keloids, and surgically-induced scars

<p>Abstract</p> <p>Background</p> <p>Leiomyoma have often been compared to keloids because of their fibrotic characteristic and higher rate of occurrence among African Americans as compared to other ethnic groups. To evaluate such a correlation at molecular level this study comparatively analyzed leiomyomas with keloids, surgical scars and peritoneal adhesions to identify genes that are either commonly and/or individually distinguish these fibrotic disorders despite differences in the nature of their development and growth.</p> <p>Methods</p> <p>Microarray gene expression profiling and realtime PCR.</p> <p>Results</p> <p>The analysis identified 3 to 12% of the genes on the arrays as differentially expressed among these tissues based on P ranking at greater than or equal to 0.005 followed by 2-fold cutoff change selection. Of these genes about 400 genes were identified as differentially expressed in leiomyomas as compared to keloids/incisional scars, and 85 genes as compared to peritoneal adhesions (greater than or equal to 0.01). Functional analysis indicated that the majority of these genes serve as regulators of cell growth (cell cycle/apoptosis), tissue turnover, transcription factors and signal transduction. Of these genes the expression of E2F1, RUNX3, EGR3, TBPIP, ECM-2, ESM1, THBS1, GAS1, ADAM17, CST6, FBLN5, and COL18A was confirmed in these tissues using quantitative realtime PCR based on low-density arrays.</p> <p>Conclusion</p> <p>the results indicated that the molecular feature of leiomyomas is comparable but may be under different tissue-specific regulatory control to those of keloids and differ at the levels rather than tissue-specific expression of selected number of genes functionally regulating cell growth and apoptosis, inflammation, angiogenesis and tissue turnover.</p

A Design Support System Using Analogy Based Reasoning

Abstract: This paper represents a procedure to support the designer in his/her process of mechanical system design, by inspiring the knowledge acquired from previous projects. To this end, the proposed method represents an appropriate means to capitalize the know-how of the professional experts. Based on this approach, an interactive programme is implemented, which assist designers in the specification of new products. The data structure of the implemented tool is based on the object oriented modelling. This structure allows several classifications of a same design, using different levels of abstraction. This approach enables designer to begin with a more general description of the product, and to refine the description by referring to similar data in the pattern bases

Adhesion Development and the Expression of Endothelial Nitric Oxide Synthase

Objective: This study was conducted to determine whether nitric oxide (NO), a potent vasodilator and inhibitor of thrombus formation, is involved in the formation and maintenance of adhesions. Methods: Skin, subcutaneous tissues, peritoneum and adhesions were collected from surgical patients and total RNA was isolated. Quantitative reverse transcription polymerase chain reaction (QRT-PCR) was performed to quantitate endothelial nitric oxide synthase (eNOS) and β-actin mRNA levels. Results: eNOS mRNA levels for skin, subcutaneous tissue, peritoneum and adhesions were ≤ 3.12 × 10(-4), ≤ 3.12 × 10(-4), 6.24 × 10(-4) and 2.5 × 10(-3) attomoles/μl, respectively. β-actin mRNA levels for all tissues were between 1.25 × 10(-1) and 6.25 × 10(-2) attomoles/μl. Conclusion: eNOS mRNA can be identified in tissue adhesions, and may therefore play a role in adhesion formation and maintenance

Epithelial Neutrophil-Activating Peptide (ENA-78), Acute Coronary Syndrome Prognosis, and Modulatory Effect of Statins

Endothelial inflammation with chemokine involvement contributes to acute coronary syndromes (ACS). We tested the hypothesis that variation in the chemokine gene CXCL5, which encodes epithelial neutrophil-activating peptide (ENA-78), is associated with ACS prognosis. We also investigated whether statin use, a potent modulator of inflammation, modifies CXCL5's association with outcomes and characterized the in vitro effect of atorvastatin on endothelial ENA-78 production. Using a prospective cohort of ACS patients (n = 704) the association of the CXCL5 −156 G>C polymorphism (rs352046) with 3-year all-cause mortality was estimated with hazard ratios (HR). Models were stratified by genotype and race. To characterize the influence of statins on this association, a statin*genotype interaction was tested. To validate ENA-78 as a statin target in inflammation typical of ACS, endothelial cells (HUVECs) were treated with IL-1β and atorvastatin with subsequent quantification of CXCL5 expression and ENA-78 protein concentrations. C/C genotype was associated with a 2.7-fold increase in 3-year all-cause mortality compared to G/G+G/C (95%CI 1.19–5.87; p = 0.017). Statins significantly reduced mortality in G/G individuals only (58% relative risk reduction; p = 0.0009). In HUVECs, atorvastatin dose-dependently decreased IL-1β-stimulated ENA-78 concentrations (p<0.0001). Drug effects persisted over 48 hours (p<0.01). CXCL5 genotype is associated with outcomes after ACS with potential statin modification of this effect. Atorvastatin lowered endothelial ENA-78 production during inflammation typical of ACS. These findings implicate CXCL5/ENA-78 in ACS and the statin response

Electron microscopic studies of erythroid cells and their isolated nuclei in Xenopus Laevius

Adult Xenovue laevis, rendered anaemic by phenylhydrazine injection have been studied during the recovery from such anaemia. Anaemia induced by phenylhydrazine resulted in stimulation of erythropoiesis and electron microscopic observation indicated that both the liver and spleen r e the sites of erythroid cell production. Electron microscopic observation suggests that there is a sudden release of a large number of immature erythroid cells (basophilic erythroblast) from liver and spleen, into the blood circulation. This is followed by mitosis of this new population in the circulation. No further release of erythroid cell from these organs is likely, until complete recovery from anaemia has occurred. Electron microscopy of liver and spleen also indicated that both organs are active in the phagocytosis and destruction of the old damaged red blood cells. These observations showed that liver, and to a lesser extent, spleen are the sites of red cello sequestration in the early days of anaemia. Finally, the spleen plays an important role in phagooytosis, until complete destruction of the damaged red blood cells has occured. This may account for spleen involvement as the site of red cell destruction in normal healthy Xenopus. Electron microscopic observation suggests that gross chromatin condensation within the isolated Xenopus erythrocyte nucleus, after the isolation, seems to be largely determined byothe ionic concentration of the isolating medium. The 200A beaded chromatin fibres observed in vivo are maintained in22edia containing MgCl and CaC12,'but not those containing &amp; as2$ the divalent cation. In the presence of divalent cation (Mg or Ca) decreasing ICC1 concentration causes peripheral condensation towards the nuclear membrane. Monovalent cation alone does not appear capable of inducing peripheral condensation. Ultrastructural studies of thin sections of isolated Xenopus erythrocyte nuclei under varying ionic conditions has revealed four different morphologies with characteristic chromatin conformations. An attempt has been made to study electron micrographs of thin nuclear sections using computer image analysis of their intensity of staining with uranyl acetate/lead citrate. This is explained by a model which suggests that there are only 'four conformation that chromatin can assume within the nucleus.</p