Phospholipase A(2) - A structural review

Phospholipases A(2) (PLA(2)) are widely distributed in nature and are well characterized proteins with respect to their catalytic and pharmacological activities, A wealth of structural information has recently become available both from X-ray diffraction and NMR studies, and although a detailed model of the catalytic mechanism of PLA(2) has been proposed, the structural bases of other aspects of PLA(2) function, such as interfacial activation and venom PLA(2) pharmacological activities, are still under debate. An appreciation of the PLA(2) protein structure will yield new insights with regard to these activities, the salient structural features of the class I, II and III PLA(2) are discussed with respect to their functional roles. Copyright (C) 1996 Published by Elsevier B.V. Ltd

Immunohistochemical expression of PCNA, p53, bax and bcl-2 in oral lichen planus and epithelial dysplasia.

The potential for malignant transformation of oral lichen planus is still controversial. The expression of proteins related to cell proliferation and apoptosis in oral lichen planus and epithelial dysplasia was analyzed to evaluate the true potential for malignant transformation of this disease. Twenty-four cases of each lesion were subjected to the streptoavidin-biotin technique for identifying the immunohistochemical expression of PCNA, p53, bax, and bcl-2 proteins. Of the 24 cases of oral lichen planus, 14 (58.33%) were positive for PCNA, 10 (41.67%) for p53, 4 (16.67%) for bcl-2 and 12 (50%) for bax, whereas of the 24 cases of epithelial dysplasia, 20 (83.33%) were positive for PCNA, 10 (41.67%) for p53, 6 (25%) for bcl-2, and 20 (83.33%) for bax. Chi-squared test showed no statistically significant differences between the expression of p53 and bcl-2 in oral lichen planus and epithelial dysplasia, regardless of the grade (P > 0.05). However, the expression of PCNA and bax was significantly increased in epithelial dysplasia (P < 0.05). The results of this study showed that alterations in expression of these proteins are observed in oral lichen planus and epithelial dysplasia, suggesting the potential for malignant transformation in both lesions.

Hepatitis C Virus NS2 Protein Inhibits DNA Damage Pathway by Sequestering p53 to the Cytoplasm

Chronic hepatitis C virus (HCV) infection is an important cause of morbidity and mortality globally, and often leads to end-stage liver disease. The DNA damage checkpoint pathway induces cell cycle arrest for repairing DNA in response to DNA damage. HCV infection has been involved in this pathway. In this study, we assess the effects of HCV NS2 on DNA damage checkpoint pathway. We have observed that HCV NS2 induces ataxia-telangiectasia mutated checkpoint pathway by inducing Chk2, however, fails to activate the subsequent downstream pathway. Further study suggested that p53 is retained in the cytoplasm of HCV NS2 expressing cells, and p21 expression is not enhanced. We further observed that HCV NS2 expressing cells induce cyclin E expression and promote cell growth. Together these results suggested that HCV NS2 inhibits DNA damage response by altering the localization of p53, and may play a role in the pathogenesis of HCV infection. © 2013 Bitter et al.

Estudo da expressão da proteína BCL-2 em mulheres com carcinoma ductal invasivo de mama. Correlação anatomopatólogica e da sobrevida

Pós-graduação em Ginecologia, Obstetrícia e Mastologia - FMB

Mecanismo de transdução de sinal na próstata de cão: avaliação nas vias da p-ERK1/2 e da CYR61

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cellular and molecular studies of the effects of a selective COX-2 inhibitor celecoxib in the cardiac cell line H9c2 and their correlation with death mechanisms

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Role of cyclooxygenase-2 in Trypanosoma cruzi survival in the early stages of parasite host-cell interaction

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Caracterização funcional e estrutural das proteínas RUV-1 e RUV-2 do fungo Neurospora crassa

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Avaliação da teneurina-2 (Ten-2) nos astrócitos do córtex cerebral de ratos adultos após lesão mecânica. Estudo imuno-histoquímico e de expressão gênica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação da teneurina-2 (Ten-2) nos astrócitos do córtex cerebral de ratos adultos após lesão mecânica. Estudo imuno-histoquímico e de expressão gênica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Synthesis, anti-inflammatory activity and molecular docking studies of 2,5-diarylfuran amino acid derivatives

A series of 2,5-diaryl substituted furans functionalized with several amino acids were synthesized and evaluated as the cyclooxygenases COX-1 and COX-2 enzymes inhibitors. The proline-substituted compound inhibited PGE(2) secretion by LPS-stimulated neutrophils, suggesting selectivity for COX-2. Molecular docking studies in the binding site of COX-2 were performed. (C) 2011 Elsevier Masson SAS. All rights reserved.

Identification of 2 Novel ANTXR2 Mutations in Patients With Hyaline Fibromatosis Syndrome and Proposal of a Modified Grading System

Juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) are rare, autosomal recessive disorders of the connective tissue caused by mutations in the gene encoding the anthrax toxin receptor 2 protein (ANTXR2) located on chromosome 4q21. Characteristically, these conditions present with overlapping clinical features, such as nodules and/or pearly papules, gingival hyperplasia, flexion contractures of the joints, and osteolytic bone defects. The present report describes a pair of sibs and three other JHF/ISH patients whose diagnoses were based on typical clinical manifestations and confirmed by histopathologic analyses and/or molecular analysis. A comparison of ISH and JHF, additional thoughts about new terminology (hyaline fibromatosis syndrome) and a modified grading system are also included. (C) 2012 Wiley Periodicals, Inc.

Influence of low-level laser associated with osteogenic proteins recombinant human BMP-2 and Hevea brasiliensis on bone repair in Wistar rats

This study analyzed the newly formed bone tissue after application of recombinant human BMP-2 (rhBMP-2) and P-1 (extracted from Hevea brasiliensis) proteins, 2 weeks after the creation of a critical bone defect in male Wistar rats treated or not with a low-intensity laser (GaAlAs 780 nm, 60 mW of power, and energy density dose of 30 J/cm2). The animals were divided into two major groups: (1) bone defect plus low-intensity laser treatment and (2) bone defect without laser irradiation. The following subgroups were also analyzed: (a) 5 mu g of pure rhBMP-2; (b) 5 mu g of pure P-1 fraction; (c) 5 mu g of rhBMP-2/monoolein gel; (d) 5 mu g of P-1 fraction/monoolein gel; (e) pure monoolein gel. Comparisons of the groups receiving laser treatment with those that did not receive laser irradiation show differences in the areas of new bone tissue. The group treated with 5 mu g of rhBMP-2 and laser irradiation was not significantly different (P >0.05) than the nonirradiated group that received the same treatment. The irradiated, rhBMP-2/monoolein gel treatment group showed a lower area of bone formation than the nonirradiated, rhBMP-2/gel monoolein treatment group (P < 0.001). The area of new bone tissue in the other nonirradiated and irradiated groups was not significantly different (P > 0.05). Furthermore, the group that received the 5 mu g of rhBMP-2 application showed the greatest bone formation. We conclude that the laser treatment did not interfere with the area of new bone tissue growth and that the greatest stimulus for bone formation involved application of the rhBMP-2 protein. Microsc. Res. Tech. 2011. (c) 2011 Wiley Periodicals, Inc.

HER-2, p53, p21 and hormonal receptors proteins expression as predictive factors of response and prognosis in locally advanced breast cancer treated with neoadjuvant docetaxel plus epirubicin combination

Abstract Background Neoadjuvant chemotherapy has been considered the standard care in locally advanced breast cancer. However, about 20% of the patients do not benefit from this clinical treatment and, predictive factors of response were not defined yet. This study was designed to evaluate the importance of biological markers to predict response and prognosis in stage II and III breast cancer patients treated with taxane and anthracycline combination as neoadjuvant setting. Methods Sixty patients received preoperative docetaxel (75 mg/m2) in combination with epirubicin (50 mg/m2) in i.v. infusion in D1 every 3 weeks after incisional biopsy. They received adjuvant chemotherapy with CMF or FEC, attaining axillary status following definitive breast surgery. Clinical and pathologic response rates were measured after preoperative therapy. We evaluated the response rate to neoadjuvant chemotherapy and the prognostic significance of clinicopathological and immunohistochemical parameters (ER, PR, p51, p21 and HER-2 protein expression). The median patient age was 50.5 years with a median follow up time 48 months after the time of diagnosis. Results Preoperative treatment achieved clinical response in 76.6% of patients and complete pathologic response in 5%. The clinical, pathological and immunohistochemical parameters were not able to predict response to therapy and, only HER2 protein overexpression was associated with a decrease in disease free and overall survival (P = 0.0007 and P = 0.003) as shown by multivariate analysis. Conclusion Immunohistochemical phenotypes were not able to predict response to neoadjuvant chemotherapy. Clinical response is inversely correlated with a risk of death in patients submitted to neoadjuvant chemotherapy and HER2 overexpression is the major prognostic factor in stage II and III breast cancer patients treated with a neoadjuvant docetaxel and epirubicin combination.

RNA Interference and cyclooxygenase-2 (COX-2) regulation in colon cancer cells

Despite new methods and combined strategies, conventional cancer chemotherapy still lacks specificity and induces drug resistance. Gene therapy can offer the potential to obtain the success in the clinical treatment of cancer and this can be achieved by replacing mutated tumour suppressor genes, inhibiting gene transcription, introducing new genes encoding for therapeutic products, or specifically silencing any given target gene. Concerning gene silencing, attention has recently shifted onto the RNA interference (RNAi) phenomenon. Gene silencing mediated by RNAi machinery is based on short RNA molecules, small interfering RNAs (siRNAs) and microRNAs (miRNAs), that are fully o partially homologous to the mRNA of the genes being silenced, respectively. On one hand, synthetic siRNAs appear as an important research tool to understand the function of a gene and the prospect of using siRNAs as potent and specific inhibitors of any target gene provides a new therapeutical approach for many untreatable diseases, particularly cancer. On the other hand, the discovery of the gene regulatory pathways mediated by miRNAs, offered to the research community new important perspectives for the comprehension of the physiological and, above all, the pathological mechanisms underlying the gene regulation. Indeed, changes in miRNAs expression have been identified in several types of neoplasia and it has also been proposed that the overexpression of genes in cancer cells may be due to the disruption of a control network in which relevant miRNA are implicated. For these reasons, I focused my research on a possible link between RNAi and the enzyme cyclooxygenase-2 (COX-2) in the field of colorectal cancer (CRC), since it has been established that the transition adenoma-adenocarcinoma and the progression of CRC depend on aberrant constitutive expression of COX-2 gene. In fact, overexpressed COX-2 is involved in the block of apoptosis, the stimulation of tumor-angiogenesis and promotes cell invasion, tumour growth and metastatization. On the basis of data reported in the literature, the first aim of my research was to develop an innovative and effective tool, based on the RNAi mechanism, able to silence strongly and specifically COX-2 expression in human colorectal cancer cell lines. In this study, I firstly show that an siRNA sequence directed against COX-2 mRNA (siCOX-2), potently downregulated COX-2 gene expression in human umbilical vein endothelial cells (HUVEC) and inhibited PMA-induced angiogenesis in vitro in a specific, non-toxic manner. Moreover, I found that the insertion of a specific cassette carrying anti-COX-2 shRNA sequence (shCOX-2, the precursor of siCOX-2 previously tested) into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT-29) without activating any interferon response. Phenotypically, COX-2 deficient HT-29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, results reported here indicate an easy-to-use, powerful and high selective virus-based method to knockdown COX-2 gene in a stable and long-lasting manner, in colon cancer cells. Furthermore, they open up the possibility of an in vivo application of this anti-COX-2 retroviral vector, as therapeutic agent for human cancers overexpressing COX-2. In order to improve the tumour selectivity, pSUPER.retro vector was modified for the shCOX-2 expression cassette. The aim was to obtain a strong, specific transcription of shCOX-2 followed by COX-2 silencing mediated by siCOX-2 only in cancer cells. For this reason, H1 promoter in basic pSUPER.retro vector [pS(H1)] was substituted with the human Cox-2 promoter [pS(COX2)] and with a promoter containing repeated copies of the TCF binding element (TBE) [pS(TBE)]. These promoters were choosen because they are partculary activated in colon cancer cells. COX-2 was effectively silenced in HT-29 and HCA-7 colon cancer cells by using enhanced pS(COX2) and pS(TBE) vectors. In particular, an higher siCOX-2 production followed by a stronger inhibition of Cox-2 gene were achieved by using pS(TBE) vector, that represents not only the most effective, but also the most specific system to downregulate COX-2 in colon cancer cells. Because of the many limits that a retroviral therapy could have in a possible in vivo treatment of CRC, the next goal was to render the enhanced RNAi-mediate COX-2 silencing more suitable for this kind of application. Xiang and et al. (2006) demonstrated that it is possible to induce RNAi in mammalian cells after infection with engineered E. Coli strains expressing Inv and HlyA genes, which encode for two bacterial factors needed for successful transfer of shRNA in mammalian cells. This system, called “trans-kingdom” RNAi (tkRNAi) could represent an optimal approach for the treatment of colorectal cancer, since E. Coli in normally resident in human intestinal flora and could easily vehicled to the tumor tissue. For this reason, I tested the improved COX-2 silencing mediated by pS(COX2) and pS(TBE) vectors by using tkRNAi system. Results obtained in HT-29 and HCA-7 cell lines were in high agreement with data previously collected after the transfection of pS(COX2) and pS(TBE) vectors in the same cell lines. These findings suggest that tkRNAi system for COX-2 silencing, in particular mediated by pS(TBE) vector, could represent a promising tool for the treatment of colorectal cancer. Flanking the studies addressed to the setting-up of a RNAi-mediated therapeutical strategy, I proposed to get ahead with the comprehension of new molecular basis of human colorectal cancer. In particular, it is known that components of the miRNA/RNAi pathway may be altered during the progressive development of colorectal cancer (CRC), and it has been already demonstrated that some miRNAs work as tumor suppressors or oncomiRs in colon cancer. Thus, my hypothesis was that overexpressed COX-2 protein in colon cancer could be the result of decreased levels of one or more tumor suppressor miRNAs. In this thesis, I clearly show an inverse correlation between COX-2 expression and the human miR- 101(1) levels in colon cancer cell lines, tissues and metastases. I also demonstrate that the in vitro modulating of miR-101(1) expression in colon cancer cell lines leads to significant variations in COX-2 expression, and this phenomenon is based on a direct interaction between miR-101(1) and COX-2 mRNA. Moreover, I started to investigate miR-101(1) regulation in the hypoxic environment since adaptation to hypoxia is critical for tumor cell growth and survival and it is known that COX-2 can be induced directly by hypoxia-inducible factor 1 (HIF-1). Surprisingly, I observed that COX-2 overexpression induced by hypoxia is always coupled to a significant decrease of miR-101(1) levels in colon cancer cell lines, suggesting that miR-101(1) regulation could be involved in the adaption of cancer cells to the hypoxic environment that strongly characterize CRC tissues.