Altered Activity and Expression of Cytosolic Peptidases in Colorectal Cancer.

BACKGROUND AND OBJECTIVE: The role of peptidases in carcinogenic processes and their potential usefulness as tumor markers in colorectal cancer (CRC) have been classically attributed to cell-surface enzymes. The objective of the present study was to analyze the activity and mRNA expression of three cytosolic peptidases in the CRC and to correlate the obtained results with classic histopathological parameters for tumor prognosis and survival. METHODS: The activity and mRNA levels of puromycin-sensitive aminopeptidase (PSA), aminopeptidase B (APB) and pyroglutamyl-peptidase I (PGI) were measured by fluorimetric and quantitative RT-PCR methods in colorectal mucosa and tumor tissues and plasma samples from CRC patients (n=81). RESULTS: 1) PSA and APB activity was higher in adenomas and carcinomas than in the uninvolved mucosa. 2) mRNA levels of PSA and PGI was lower in tumors. 3) PGI activity in CRC tissue correlated negatively with histological grade, tumor size and 5-year overall survival of CRC patients. 4) Higher plasmatic APB activity was independently associated with better 5-year overall survival. CONCLUSIONS: Data suggest that cytosolic peptidases may be involved in colorectal carcinogenesis and point to the determination of this enzymes as a valuable method in the determination of CRC prognosis.

Dual-specific phosphatase-6 (Dusp6) and ERK mediate AMPA receptor-induced oligodendrocyte death.

Oligodendrocytes, the myelinating cells of the CNS, are highly vulnerable to glutamate excitotoxicity, a mechanism involved in tissue damage in multiple sclerosis. Thus, understanding oligodendrocyte death at the molecular level is important to develop new therapeutic approaches to treat the disease. Here, using microarray analysis and quantitative PCR, we observed that dual-specific phosphatase-6 (Dusp6), an extracellular regulated kinase-specific phosphatase, is up-regulated in oligodendrocyte cultures as well as in optic nerves after AMPA receptor activation. In turn, Dusp6 is overexpressed in optic nerves from multiple sclerosis patients before the appearance of evident damage in this structure. We further analyzed the role of Dusp6 and ERK signaling in excitotoxic oligodendrocyte death and observed that AMPA receptor activation induces a rapid increase in ERK1/2 phosphorylation. Blocking Dusp6 expression, which enhances ERK1/2 phosphorylation, significantly diminished AMPA receptor-induced oligodendrocyte death. In contrast, MAPK/ERK pathway inhibition with UO126 significantly potentiates excitotoxic oligodendrocyte death and increases cytochrome c release, mitochondrial depolarization, and mitochondrial calcium overload produced by AMPA receptor stimulation. Upstream analysis demonstrated that MAPK/ERK signaling alters AMPA receptor properties. Indeed, Dusp6 overexpression as well as incubation with UO126 produced an increase in AMPA receptor-induced inward currents and cytosolic calcium overload. Together, these data suggest that levels of phosphorylated ERK, controlled by Dusp6 phosphatase, regulate glutamate receptor permeability and oligodendroglial excitotoxicity. Therefore, targeting Dusp6 may be a useful strategy to prevent oligodendrocyte death in multiple sclerosis and other diseases involving CNS white matter.

Physical Activity at School Recess: A Key Element in Balancing Social Disparities.

BACKGROUND: Since children are generally not achieving desirable levels of physical activity (PA), schools can provide an opportunity to increase their active time. This study aimed to describe individual- and social-level variables associated with PA at school recess in a culturally diverse primary school. METHODS: This observational cross-sectional study included 131 children attending primary school. PA data were measured via accelerometry during school recess and analyzed in relation to data on sociodemographics, anthropometrics, and PA outside school. Data were analyzed using generalized linear modeling and logistic regression. FINDINGS: Girls were more sedentary and engaged in less moderate to vigorous physical activity (MVPA) than boys (p < .001), while older girls were less active than younger girls (p < .001). Also, non-Caucasian children were less sedentary (p < .001) and engaged in more MVPA (p = .007). Belonging to ethnic minorities was also associated with lower socioeconomic status, higher body mass index, and less PA outside of school and on weekends. CONCLUSIONS: Girls (especially older girls) and children from lower socioeconomic contexts should be prioritized when promoting PA. IMPLICATIONS FOR SCHOOL HEALTH POLICY: School recess can be used as an effective strategy aimed at increasing PA and reducing social inequalities.

Successful breastfeeding among women with intention to breastfeed: From physiology to socio-cultural factors.

BACKGROUND: Even if women have intention to breastfeed, they do not always achieve a successful breastfeeding. AIM: This study aims to analyse factors affecting breastfeeding prevalence among mothers that intended to breastfeed. METHODS: This is a prospective observational study involving 401 pregnant women that intended to breastfeed (asked at the 20th week). Breastfeeding prevalence was evaluated in reference to health-related, socio-cultural factors and healthcare professionals' interventions at 1 month, 6 months and 12 months after birth. Data were analysed using descriptive statistical methods, bivariate logistic regression and multivariate logistic regression modelling. RESULTS: Independent factors negatively affecting breastfeeding prevalence related to mothers' and newborns' health parameters and birth characteristics included smoking during pregnancy, anaemia and use of analgesia during labour. Regarding sociocultural parameters, being an immigrant, higher education level, intention to breastfeed before pregnancy, comfort with public breastfeeding and bedsharing were positively linked to breastfeeding, while teat or pacifier use in the first week was negatively linked. Regarding healthcare professionals' practices, mother and father/partner antenatal education course attendance and exclusive breastfeeding at the hospital were positively associated with breastfeeding. CONCLUSION: Breastfeeding is a very complex phenomenon affected by multiple and diverse variables. Physiological factors only affect the short term (1st month), while middle and long term BF affecting variables are mainly identical and include mostly socio-cultural factors and also BF related practices, especially in the first days after birth. These data should help to develop more effective breastfeeding promotion strategies.

Evidence for LKB1/AMP-activated protein kinase/ endothelial nitric oxide synthase cascade regulated by hepatocyte growth factor, S-adenosylmethionine, and nitric oxide in hepatocyte proliferation.

UNLABELLED: S-adenosylmethionine (SAMe) is involved in numerous complex hepatic processes such as hepatocyte proliferation, death, inflammatory responses, and antioxidant defense. One of the most relevant actions of SAMe is the inhibition of hepatocyte proliferation during liver regeneration. In hepatocytes, SAMe regulates the levels of cytoplasmic HuR, an RNA-binding protein that increases the half-life of target messenger RNAs such as cyclin D1 and A2 via inhibition of hepatocyte growth factor (HGF)-mediated adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. Because AMPK is activated by the tumor suppressor kinase LKB1, and AMPK activates endothelial nitric oxide (NO) synthase (eNOS), and NO synthesis is of great importance for hepatocyte proliferation, we hypothesized that in hepatocytes HGF may induce the phosphorylation of LKB1, AMPK, and eNOS through a process regulated by SAMe, and that this cascade might be crucial for hepatocyte growth. We demonstrate that the proliferative response of hepatocytes involves eNOS phosphorylation via HGF-mediated LKB1 and AMPK phosphorylation, and that this process is regulated by SAMe and NO. We also show that knockdown of LKB1, AMPK, or eNOS with specific interference RNA (iRNA) inhibits HGF-mediated hepatocyte proliferation. Finally, we found that the LKB1/AMPK/eNOS cascade is activated during liver regeneration after partial hepatectomy and that this process is impaired in mice treated with SAMe before hepatectomy, in knockout mice deficient in hepatic SAMe, and in eNOS knockout mice. CONCLUSION: We have identified an LKB1/AMPK/eNOS cascade regulated by HGF, SAMe, and NO that functions as a critical determinant of hepatocyte proliferation during liver regeneration after partial hepatectomy.

Loss of the glycine N-methyltransferase gene leads to steatosis and hepatocellular carcinoma in mice.

UNLABELLED: Glycine N-methyltransferase (GNMT) is the main enzyme responsible for catabolism of excess hepatic S-adenosylmethionine (SAMe). GNMT is absent in hepatocellular carcinoma (HCC), messenger RNA (mRNA) levels are significantly lower in livers of patients at risk of developing HCC, and GNMT has been proposed to be a tumor-susceptibility gene for liver cancer. The identification of several children with liver disease as having mutations of the GNMT gene further suggests that this enzyme plays an important role in liver function. In the current study we studied development of liver pathologies including HCC in GNMT-knockout (GNMT-KO) mice. GNMT-KO mice have elevated serum aminotransferase, methionine, and SAMe levels and develop liver steatosis, fibrosis, and HCC. We found that activation of the Ras and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways was increased in liver tumors from GNMT-KO mice coincidently with the suppression of the Ras inhibitors Ras-association domain family/tumor suppressor (RASSF) 1 and 4 and the JAK/STAT inhibitors suppressor of cytokine signaling (SOCS) 1-3 and cytokine-inducible SH2-protein. Finally, we found that methylation of RASSF1 and SOCS2 promoters and the binding of trimethylated lysine 27 in histone 3 to these 2 genes was increased in HCC from GNMT-KO mice. CONCLUSION: These data demonstrate that loss of GNMT induces aberrant methylation of DNA and histones, resulting in epigenetic modulation of critical carcinogenic pathways in mice.

Clinical impact of aspartyl aminopeptidase expression and activity in colorectal cancer.

Aspartyl aminopeptidase (ASP; EC 3.4.11.21) is a widely distributed and abundant cytosolic enzyme that regulates bioactive peptides such as angiotensin II. It has been demonstrated that the expression and activity of this enzyme is modified in tissue and serum of patients with several types of cancer. However, the involvement of ASP in the neoplastic development and survival of patients with colorectal cancer (CRC) has not been analyzed to date. The activity and messenger RNA expression of ASP in tumor tissue (n = 71) and plasma (n = 40) of patients with CRC was analyzed prospectively using fluorometric and quantitative real-time polymerase chain reaction methods. Data obtained from tumor tissue were compared with those from the surrounding normal mucosa. Classic pathologic parameters (grade, stage, nodal invasion, distant metastases and perineural, lymphatic, and vascular invasion) were stratified following ASP data and analyzed for 5-year survival. ASP was upregulated in CRC tissues, and greater activity correlated significantly with the absence of lymph node metastases and with better overall survival. Inversely, greater plasmatic ASP activity was associated with worse overall and disease-free survival. Data suggest that ASP is involved in colorectal neoplasia and point to this enzyme as a potential useful diagnostic tool in clinical practice.

Nonalcoholic steatohepatitis, animal models, and biomarkers: what is new?

Nonalcoholic fatty liver disease (NAFLD) is a clinicopathological term that encompasses a spectrum of abnormalities ranging from simple triglyceride accumulation in the hepatocytes (hepatic steatosis) to hepatic steatosis with inflammation (steatohepatitis, also known as nonalcoholic steatohepatitis or NASH). NASH can also progress to cirrhosis and hepatocellular carcinoma (HCC). Steatohepatitis has been estimated to affect around 5% of the total population and 20% of those who are overweight. The mechanisms leading to NASH and its progression to cirrhosis and HCC remain unclear, but it is a condition typically associated with obesity, insulin resistance, diabetes, and hypertriglyceridemia. This point corroborates the need for animal models and molecular markers that allow us to understand the mechanisms underlying this disease. Nowadays, there are numerous mice models to study abnormal liver function such as steatosis, NASH, and hepatocellular carcinoma. The study of the established animal models has provided many clues in the pathogenesis of steatosis and steatohepatitis, although these remain incompletely understood and no mice model completely fulfills the clinical features observed in humans. In addition, there is a lack of accurate sensitive diagnostic tests that do not involve invasive procedures. Current laboratory tests include some biochemical analysis, but their utility for diagnosing NASH is still poor. For that reason, a great effort is being made toward the identification and validation of novel biomarkers to assess NASH using high-throughput analysis based on genomics, proteomics, and metabolomics. The most recent discoveries and their validation will be discussed.

The N-terminal domain of the enzyme I is a monomeric well-folded protein with a low conformational stability and residual structure in the unfolded state.

The bacterial phosphoenolpyruvate-dependent sugar phosphotransferase system is a multiprotein complex that phosphorylates and, concomitantly, transports carbohydrates across the membrane into the cell. The first protein of the cascade is a multidomain protein so-called enzyme I (EI). The N-terminal domain of EI from Streptomyces coelicolor, EIN(sc), responsible for the binding to the second protein in the cascade (the histidine phosphocarrier, HPr), was cloned and successfully expressed and purified. We have previously shown that EI(sc) binds to HPr(sc) with smaller affinity than other members of the EI and HPr families [Hurtado-Gómez et al. (2008) Biophys. J., 95, 1336-1348]. We think that the study of the isolated binding HPr(sc) domain, that is EIN(sc), could shed light on the small affinity value measured. Therefore, in this work we present a detailed description of the structural features of the EIN domain, as a first step towards a complete characterization of the molecular recognition process between the two proteins. We show that EIN(sc) is a folded protein, with alpha-helix and beta-sheet structures and also random-coil conformations, as shown by circular dichroism (CD), FTIR and NMR spectroscopies. The acquisition of secondary and tertiary structures, and the burial of hydrophobic regions, occurred concomitantly at acidic pHs, but at very low pH, the domain acquired a molten-globule conformation. The EIN(sc) protein was not very stable, with an apparent conformational free energy change upon unfolding, DeltaG, of 4.1 +/- 0.4 kcal mol(-1), which was pH independent in the range explored (from pH 6.0 to 8.5). The thermal denaturation midpoint, which was also pH invariant, was similar to that measured in the isolated intact EI(sc). Although EIN(sc) shows thermal- and chemical denaturations that seems to follow a two-state mechanism, there is evidence of residual structure in the chemical and thermally unfolded states, as indicated by differential scanning calorimetry and CD measurements.

Non-alcoholic fatty liver disease proteomics.

PURPOSE: Non-alcoholic fatty liver disease (NAFLD) is an important cause of chronic liver injury that has gained concern in clinical hepatology. The principal aim of this study was to find differences in protein expression between patients with NAFLD and healthy controls. EXPERIMENTAL DESIGN: Changes in protein expression of liver samples from each of the three groups of subjects, controls, non-alcoholic steatosis, and non-alcoholic steatohepatitis (NASH), were analyzed by DIGE combined with MALDI TOF/TOF analysis, a proteomic approach that allows to compare hundreds of proteins simultaneously. RESULTS: Forty-three proteins exhibiting significant changes (ratio ≥1.5, p<0.05) were characterized, 22 comparing steatosis samples versus control samples and 21 comparing NASH versus control samples. Ten of these proteins were further analyzed by Western blot in tissue samples to confirm the observed changes of protein expression using DIGE. The proteins validated were further tested in serum samples of different cohorts of patients. CONCLUSIONS AND CLINICAL RELEVANCE: Following this approach we identified two candidate markers, carbamoyl phosphate synthase 1 and 78 kDa glucose-regulated protein, differentially expressed between control and NASH. This proteomics approach demonstrates that DIGE combined with MALDI TOF/TOF and Western blot analysis of tissue and serum samples is a useful approach to identify candidate markers associated with NAFLD, resulting in proteins whose level of expression can be correlated to a disease state.

Non-alcoholic steatohepatitis and animal models: understanding the human disease.

Non-alcoholic fatty liver disease includes a broad spectrum of liver abnormalities ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular carcinoma. Patients with primary NASH have the metabolic (or insulin resistance) syndrome, condition typically associated with obesity, diabetes, hyperlipidemia and hypertension. To understand the mechanisms implicated in development of NASH, animal models of non-alcoholic fatty liver disease have been generated. These have greatly improved our understanding of some of the aspects of this disease. The challenge now is to identify the common mechanisms between the animal models and humans, which could eventually lead to a better prognosis and development of novel therapeutic strategies.

S-adenosylmethionine and proliferation: new pathways, new targets.

SAMe (S-adenosylmethionine) is the main methyl donor group in the cell. MAT (methionine adenosyltransferase) is the unique enzyme responsible for the synthesis of SAMe from methionine and ATP, and SAMe is the common point between the three principal metabolic pathways: polyamines, transmethylation and transsulfuration that converge into the methionine cycle. SAMe is now also considered a key regulator of metabolism, proliferation, differentiation, apoptosis and cell death. Recent results show a new signalling pathway implicated in the proliferation of the hepatocyte, where AMPK (AMP-activated protein kinase) and HuR, modulated by SAMe, take place in HGF (hepatocyte growth factor)-mediated cell growth. Abnormalities in methionine metabolism occur in several animal models of alcoholic liver injury, and it is also altered in patients with liver disease. Both high and low levels of SAMe predispose to liver injury. In this regard, knockout mouse models have been developed for the enzymes responsible for SAMe synthesis and catabolism, MAT1A and GNMT (glycine N-methyltransferase) respectively. These knockout mice develop steatosis and HCC (hepatocellular carcinoma), and both models closely replicate the pathologies of human disease, which makes them extremely useful to elucidate the mechanism underlying liver disease. These new findings open a wide range of possibilities to discover novel targets for clinical applications.

Identification of a gene-pathway associated with non-alcoholic steatohepatitis.

BACKGROUND/AIMS: We have integrated gene expression profiling of liver biopsies of NASH patients with liver samples of a mouse model of steatohepatitis (MAT1A-KO) to identify a gene-pathway associated with NASH. METHODS: Affymetrix U133 Plus 2.0 microarrays were used to evaluate nine patients with NASH, six patients with steatosis, and six control subjects; Affymetrix MOE430A microarrays were used to evaluate wild-type and MAT1A-KO mice at 15 days, 1, 3, 5 and 8 months after birth. Transcriptional profiles of patients with NASH and MAT1A-KO mice were compared with those of their proficient controls. RESULTS: We identified a gene-pathway associated with NASH, that accurately distinguishes between patients with early-stage NASH and controls. Patients with steatosis have a gene expression pattern intermediate between that of NASH and controls. Promoter analysis revealed that 34 of the genes associated with NASH contained an Sp1 element. We found that Sp1 binding to these genes is increased in MAT1A-KO mice. Sp1 is also hyperphosphorylated in MAT1A-KO as well as in patients with NASH and steatosis. CONCLUSIONS: A gene-pathway associated with NASH has been identified. We speculate that hyperphosphorylation of Sp1 may be involved in the genesis of steatosis and that other factors, such as oxidative stress, may trigger its progression to NASH.