High-Resolution Magic-Angle-Spinning NMR in Revealing Hepatoblastoma Hallmarks

Cancer is one of the leading causes of death in children and adolescents worldwide; among the types of liver cancer, hepatoblastoma (HBL) is the most common in childhood. Although it affects only two to three individuals in a million, it is mostly asymptomatic at diagnosis, so by the time it is detected it has already advanced. There are specific recommendations regarding HBL treatment, and ongoing studies to stratify the risks of HBL, understand the pathology, and predict prognostics and survival rates. Although magnetic resonance imaging spectroscopy is frequently used in diagnostics of HBL, high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy of HBL tissues is scarce. Using this technique, we studied the alterations among tissue metabolites of ex vivo samples from (a) HBL and non-cancer liver tissues (NCL), (b) HBL and adjacent non-tumor samples, and (c) two regions of the same HBL samples, one more centralized and the other at the edge of the tumor. It was possible to identify metabolites in HBL, then metabolites from the HBL center and the border samples, and link them to altered metabolisms in tumor tissues, highlighting their potential as biochemical markers. Metabolites closely related to liver metabolisms such as some phospholipids, triacylglycerides, fatty acids, glucose, and amino acids showed differences between the tissues

HOXB7 mRNA is overexpressed in pancreatic ductal adenocarcinomas and its knockdown induces cell cycle arrest and apoptosis

Background\ud Human homeobox genes encode nuclear proteins that act as transcription factors involved in the control of differentiation and proliferation. Currently, the role of these genes in development and tumor progression has been extensively studied. Recently, increased expression of HOXB7 homeobox gene (HOXB7) in pancreatic ductal adenocarcinomas (PDAC) was shown to correlate with an invasive phenotype, lymph node metastasis and worse survival outcomes, but no influence on cell proliferation or viability was detected. In the present study, the effects arising from the knockdown of HOXB7 in PDAC cell lines was investigated.\ud \ud Methods\ud Real time quantitative PCR (qRT-PCR) (Taqman) was employed to assess HOXB7 mRNA expression in 29 PDAC, 6 metastatic tissues, 24 peritumoral tissues and two PDAC cell lines. siRNA was used to knockdown HOXB7 mRNA in the cell lines and its consequences on apoptosis rate and cell proliferation were measured by flow cytometry and MTT assay respectively.\ud \ud Results\ud Overexpression of HOXB7 mRNA was observed in the tumoral tissues and in the cell lines MIA PaCa-2 and Capan-1. HOXB7 knockdown elicited (1) an increase in the expression of the pro-apoptotic proteins BAX and BAD in both cell lines; (2) a decrease in the expression of the anti-apoptotic protein BCL-2 and in cyclin D1 and an increase in the number of apoptotic cells in the MIA PaCa-2 cell line; (3) accumulation of cell in sub-G1 phase in both cell lines; (4) the modulation of several biological processes, especially in MIA PaCa-2, such as proteasomal ubiquitin-dependent catabolic process and cell cycle.\ud \ud Conclusion\ud The present study confirms the overexpression of HOXB7 mRNA expression in PDAC and demonstrates that decreasing its protein level by siRNA could significantly increase apoptosis and modulate several biological processes. HOXB7 might be a promising target for future therapies.This study was supported in part by a FAPESP grant 2010/01421-1

Differential expression of genes encoding proteins of the HGF/MET system in insulinomas

Abstract\ud \ud Background\ud Insulinomas are the most common functional pancreatic neuroendocrine tumors, whereas histopathological features do not predict their biological behaviour. In an attempt to better understand the molecular processes involved in the tumorigenesis of islet beta cells, the present study evaluated the expression of genes belonging to the hepatocyte growth factor and its receptor (HGF/MET) system, namely, MET, HGF; HGFAC and ST14 (encode HGF activator and matriptase, respectively, two serine proteases that catalyze conversion of pro-HGF to active HGF); and SPINT1 and SPINT2 (encode serine peptidase inhibitors Kunitz type 1 and type 2, respectively, two inhibitors of HGF activator and of matriptase).\ud \ud \ud Methods\ud Quantitative real-time reverse transcriptase polymerase chain reaction was employed to assess RNA expression of the target genes in 24 sporadic insulinomas: 15 grade 1 (G1), six grade 2 (G2) and three hepatic metastases. Somatic mutations of MET gene were searched by direct sequencing of exons 2, 10, 14, 16, 17 and 19.\ud \ud \ud Results\ud Overexpression of MET was observed in the three hepatic metastases concomitantly with upregulation of the genes encoding HGF and matriptase and downregulation of SPINT1. A positive correlation was observed between MET RNA expression and Ki-67 proliferation index while a negative correlation was detected between SPINT1 expression and the mitotic index. No somatic mutations were found in MET gene.\ud \ud \ud Conclusion\ud The final effect of the increased expression of HGF, its activator (matriptase) and its specific receptor (MET) together with a decreased expression of one potent inhibitor of matriptase (SPINT1) is probably a contribution to tumoral progression and metastatization in insulinomas

Isolamento e caracterização dos simbiontes do tripanossomatídeo Crithidia deanei : 1 - caracterização da síntese de proteínas e ácido nucléico; 2 - clonagem molecular de genes

Orientador: Dr. José Franco da Silveira FilhoDissertação (mestrado) - Universidade Federal do Paraná, Curso de Pós-Graduação em BioquímicaInclui referências: p. 104-112Resumo: Neste trabalho nós descrevemos um método de isolamento de simbionte de Crithidia deanei em gradiente de Percoll. Este método permite o isolamento de simbiontes intactos em quantidade suficiente para se efetuar estudos bioquímicos. O flagelado Crithidia deanei foi rompido por sonicação e os simbiontes liberados foram separados por gradiente descontínuo de Percoll dos restos celulares e de células de Crithidia deanei que não se romperam pelo processo de ultrassom. Os simbiontes isolados pelo método citado acima conservam as suas características morfológicas e apresentam ainda uma atividade metabólica intensa. A enzima ornitina carbamil transferase (OCT), foi utilizada como marcador bioquímico dos simbiontes. Esta enzima encontra-se com atividade enzimática três vezes maior na fração do gradiente de Percoll aonde se encontram os simbiontes isolado do que no extrato bruto, e aproximadamente 6 % da atividade total da OCT do extrato bruto foi recuperada na fração rica em simbiontes do gradiente de Percoll. Nós estimamos que 20 % dos simbiontes presentes no homogenato foram recuperados nesta fração. Os simbiontes isolados pelo gradiente de Percoll incorporaram L-(4-5-H) leucina e L-(S) metionina ativamente nos primeiros 45 e 60 minutos respectivamente. O mecanismo de síntese proteica do simbionte foi analisada através do efeito dos antibióticos cloranfenicol e rifampicina na incorporação com L-(4-5-H) leucina nas suas proteínas Os antibióticos cloranfenicol e rifampicina em concentração de 20 (jg/ml diminuem a incorporação entorno de 50 % e 70%, respectivamente. Estes antibióticos em concentração de 50 ug/ml inibem a incorporação quase completamente. A inibição da síntese proteica pelos antibióticos cloranfenicol e rifampicina produzem evidências díiretas da existência de um mecanismo de síntese proteica procariótica. Os polipeptídeos sintetizados pelo simbiontes foram marcados com L-( S) metionina e submetidos a eletroforese em gel de poliacrilamida-SDS. Os polipeptídeos fortemente marcados apresentaram pesos moleculares aparentes de 90, 88, 78, 60 e 58 x 10.0 perfil dos polipeptídeos sintetizados pelo simbionte difere daqueles polipeptídeos encontrados na cepa de Crithidia deanei com simbionte. Vários polipeptídeos estão ausentes ou fracamente marcados no flagelado, por exemplo 88,78,60 e 58 kDa .Este resultado sugere que a síntese destes polipeptídeos está inibida no flagelado intacto. A incorporação de metil ( H )-timidina na fração ácido insolúvel dos simbiontes foi linear até 35 minutos, demonstrando que o simbionte isolado sintetiza DNA. Este resultado sugere a presença de uma via alternativa de síntese de DNA e a depencia de tiraidina quinase nesta via. A integridade do DNA do simbionte isolado foi analisada em eletroforose de gel de agarose. Este comigra com o DNA de E.coli em uma simples banda, cujo o peso molecular está acima do marcador de massa molecular 23 Kb. O DNA do simbionte foi digerido com enzimas de restrição Mbo I, obtendo-se fragmentos de 2,3-1 Kb que foram clonados em plasmídio pUC 13 . O banco construído em pUC 13 com DNA do simbionte de Crithidia deanei é representativo. Os Clones contendo sequências específicas de endosimbiontes foram isolados por hibridização diferencial.Abstract: The method described here allows the isolation intact symbionts in sufficient amounts for biochemical studies. The flagellates are disrupted by sonication and the symbionts separated from unbroken cells and cellular debris by centrifugation through a discontinous Percoll gradient. The isolated symbionts retain their characteristic morphological features and were reasonably free of subcellular debris. Ornithine carbaomoyltransferase (OCT) a symbiont enzymatic marker is present in the symbiont fraction with a specific activity 3 fold higher than in crude homogenates. We estimate that about 20 % of symbionts present in the homogenate are recovered in this fraction. The isolated symbionts actively incorporate L-(4-5-H)-leucine and L-(S) methionine into acid-insoluble material for the first 45 and 60min, respectively. In order to characterize the protein-synthesis machinery operanting in the symbionts, have studied the effects of chloramphenicol and rifampicin on the incorporation of leucine into proteins. Chloramphenicol and rifampicin at 20 ug/ml reduce the incorporation by about 70 and 50%, respectively. Both antibiotics at 50 ug/ml almost completely inhibited the incorporation L-(4-5-H) leucine. The inhibition of protein synthesis by chloramphenicol and rifampicin provides direct evidence for the existence of a prokaryotic protein-synthesizing system in this unusual subcellular structure. The labeled polypeptides synthesized by the isolated endosymbionts range between 200,000-18,000, as determined by SDS-polyacrilamide gel electrophoresis. The most heavily-labeled polypeptides are those with apparent molecular weight of 90, 88, 78, 60 and 58 x 10. The profile of symbiont labeled polypeptides differs from that obtained with the symbiont strain C. deanei. Several symbiont polypeptides are absent or poorly represented in the flagellate, for example, the 88, 78, 60, and 58 kDa polypeptides, suggesting that synthesis of these polypeptides is inhibited in the intact flagellate. The incorporation metil-(H) thymidine into acid insoluble symbiont fraction was linear up to 45 min showing that isolated symbionts can synthesize DNA. This result suggests the presence in the symbionts of a thymidine kinase dependent salvage pathway for DNA synthesis. The integrity of the DNA extracted from isolated symbiont was analyzed by agarose gel electrophoresis. lt comigrates with intact E. coli DNA as a single heavy band. The symbiont DNA has been purified and fragment with restriction endonuclease Mbo I. The fragments (2,3-1,0 Kb) have been incorporated in vitro into pUC 13 to make libraries which most of the endosymbiont DNA is represented. Specific endosymbiont sequences can be isolated in this library by differential screening

Study of activity transcription factors C/EBPalpha in region - 53 to - 33 of promoter apolipoprotein B gene

A apolipoproteina B (apoB) tem um importante papel na regulação na homeostasia celular, do colesterol e na patogênese da aterosclerose. Esta proteína age como ligante para o reconhecimento e catabolismo lipoproteínas de baixa densidade (LBD) através do receptor de LDL. Estudos anteriores mostraram que a expressão do gene da apolipoproteína B (APOB) em células hepáticas é regulada pela interação de fatores ligados ao elemento enhancer no intron 2, e em 3 elementos denominados de III, IV e V localizados nas regiões -86 a -62, -72 a -53 e -53 a -33 , respectivamente, do promotor do gene da APOB. Neste trabalho, nós sugerimos que o fator de transcrição C/EBPalfa ligado a região -53 a -33 da APOB interage com o complexo HNF-4 e C/EBPalfa localizado dentro da região -86 a -53 do APO B e contribui para aumentar a transcrição do gene APOB.Apolipoprotein B (ApoB) plays a major role in the regulation of cellular cholesterol homeostasis and pathogenesis of atherosclerosis. This protein acts as a ligand for the cellular recognition and catabolism of low density lipoprotein (LDL) by the LDL receptor. Previous studies have shown that the expression of apoB in hepatic cells is regulated by the interaction of factors binding to enhancer elements in intron 2 and three elements designated III, IV and V. These elements lie within regions respectively -86 to -62, -72 to -53 and -53 to -33 from the ApoB promoter. In this study, we have suggested that transcription factor C/EBPalpha, which binds to the -53 to -33 region of the apoB, interacts with the HNF-4 synergistic complex and C/EBPalpha factors within -86 to -53 and may contribute to increase transcription of the ApoB gene

Uncommon allele in APO AI-CIII-AIV gene cluster in a family with congenital generalized lipodystrophy

Congenital generalized lipodystrophy is a rare inherited disease. One of its features is a disturbance in lipid metabolism characterized by hypercholesterolemia and hypertriglyceridemia. A brother and a sister with congenital generalized lipodystrophy, an 8-year old male and a 12-year old female were studied. The mother and a 6-year old brother were healthy. The genetic analysis of Sstl RFLP of the apo Al-CIII-AIV gene cluster showed the presence of the rare Sstl allele (S2) in the patients but not in the healthy mother and brother. As this uncommon allele has been reported to be related to high plasma triglyceride levels, this association could be relevant in explaining in part the hypertriglyceridemia observed in these patients