Different Effects of BORIS/CTCFL on Stemness Gene Expression, Sphere Formation and Cell Survival in Epithelial Cancer Stem Cells.

Cancer stem cells are cancer cells characterized by stem cell properties and represent a small population of tumor cells that drives tumor development, progression, metastasis and drug resistance. To date, the molecular mechanisms that generate and regulate cancer stem cells are not well defined. BORIS (Brother of Regulator of Imprinted Sites) or CTCFL (CTCF-like) is a DNA-binding protein that is expressed in normal tissues only in germ cells and is re-activated in tumors. Recent evidences have highlighted the correlation of BORIS/CTCFL expression with poor overall survival of different cancer patients. We have previously shown an association of BORIS-expressing cells with stemness gene expression in embryonic cancer cells. Here, we studied the role of BORIS in epithelial tumor cells. Using BORIS-molecular beacon that was already validated, we were able to show the presence of BORIS mRNA in cancer stem cell-enriched populations (side population and spheres) of cervical, colon and breast tumor cells. BORIS silencing studies showed a decrease of sphere formation capacity in breast and colon tumor cells. Importantly, BORIS-silencing led to down-regulation of hTERT, stem cell (NANOG, OCT4, SOX2 and BMI1) and cancer stem cell markers (ABCG2, CD44 and ALDH1) genes. Conversely, BORIS-induction led to up-regulation of the same genes. These phenotypes were observed in cervical, colon and invasive breast tumor cells. However, a completely different behavior was observed in the non-invasive breast tumor cells (MCF7). Indeed, these cells acquired an epithelial mesenchymal transition phenotype after BORIS silencing. Our results demonstrate that BORIS is associated with cancer stem cell-enriched populations of several epithelial tumor cells and the different phenotypes depend on the origin of tumor cells.

Birth Weight, Working Memory and Epigenetic Signatures in IGF2 and Related Genes: A MZ Twin Study.

Neurodevelopmental disruptions caused by obstetric complications play a role in the etiology of several phenotypes associated with neuropsychiatric diseases and cognitive dysfunctions. Importantly, it has been noticed that epigenetic processes occurring early in life may mediate these associations. Here, DNA methylation signatures at IGF2 (insulin-like growth factor 2) and IGF2BP1-3 (IGF2-binding proteins 1-3) were examined in a sample consisting of 34 adult monozygotic (MZ) twins informative for obstetric complications and cognitive performance. Multivariate linear regression analysis of twin data was implemented to test for associations between methylation levels and both birth weight (BW) and adult working memory (WM) performance. Familial and unique environmental factors underlying these potential relationships were evaluated. A link was detected between DNA methylation levels of two CpG sites in the IGF2BP1 gene and both BW and adult WM performance. The BW-IGF2BP1 methylation association seemed due to non-shared environmental factors influencing BW, whereas the WM-IGF2BP1 methylation relationship seemed mediated by both genes and environment. Our data is in agreement with previous evidence indicating that DNA methylation status may be related to prenatal stress and later neurocognitive phenotypes. While former reports independently detected associations between DNA methylation and either BW or WM, current results suggest that these relationships are not confounded by each other.

Dpp spreading is required for medial but not for lateral wing disc growth.

Drosophila Decapentaplegic (Dpp) has served as a paradigm to study morphogen-dependent growth control. However, the role of a Dpp gradient in tissue growth remains highly controversial. Two fundamentally different models have been proposed: the 'temporal rule' model suggests that all cells of the wing imaginal disc divide upon a 50% increase in Dpp signalling, whereas the 'growth equalization model' suggests that Dpp is only essential for proliferation control of the central cells. Here, to discriminate between these two models, we generated and used morphotrap, a membrane-tethered anti-green fluorescent protein (GFP) nanobody, which enables immobilization of enhanced (e)GFP::Dpp on the cell surface, thereby abolishing Dpp gradient formation. We find that in the absence of Dpp spreading, wing disc patterning is lost; however, lateral cells still divide at normal rates. These data are consistent with the growth equalization model, but do not fit a global temporal rule model in the wing imaginal disc.

Urine Fetuin-A is a biomarker of autosomal dominant polycystic kidney disease progression.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by numerous fluid-filled cysts that frequently result in end-stage renal disease. While promising treatment options are in advanced clinical development, early diagnosis and follow-up remain a major challenge. We therefore evaluated the diagnostic value of Fetuin-A as a new biomarker of ADPKD in human urine. RESULTS: We found that renal Fetuin-A levels are upregulated in both Pkd1 and Bicc1 mouse models of ADPKD. Measurement by ELISA revealed that urinary Fetuin-A levels were significantly higher in 66 ADPKD patients (17.5 ± 12.5 μg/mmol creatinine) compared to 17 healthy volunteers (8.5 ± 3.8 μg/mmol creatinine) or 50 control patients with renal diseases of other causes (6.2 ± 2.9 μg/mmol creatinine). Receiver operating characteristics (ROC) analysis of urinary Fetuin-A levels for ADPKD rendered an optimum cut-off value of 12.2 μg/mmol creatinine, corresponding to 94% of sensitivity and 60% of specificity (area under the curve 0.74 ; p = 0.0019). Furthermore, urinary Fetuin-A levels in ADPKD patients correlated with the degree of renal insufficiency and showed a significant increase in patients with preserved renal function followed for two years. CONCLUSIONS: Our findings establish urinary Fetuin-A as a sensitive biomarker of the progression of ADPKD. Further studies are required to examine the pathogenic mechanisms of elevated renal and urinary Fetuin-A in ADPKD.

Multilevel control of arabidopsis 3-hydroxy-3-methylglutaryl coenzyme A reductase by protein phosphatase 2A

Plants synthesize a myriad of isoprenoid products that are required both for essential constitutive processes and for adaptive responses to the environment. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes a key regulatory step of the mevalonate pathway for isoprenoid biosynthesis and is modulated by many endogenous and external stimuli. In spite of that, no protein factor interacting with and regulating plant HMGR in vivo has been described so far. Here, we report the identification of two B99 regulatory subunits of protein phosphatase 2A (PP2A), designated B99a and B99b, that interact with HMGR1S and HMGR1L, the major isoforms of Arabidopsis thaliana HMGR. B99a and B99b are Ca2+ binding proteins of the EF-hand type. We show that HMGR transcript, protein, and activity levels are modulated by PP2A in Arabidopsis. When seedlings are transferred to salt-containing medium, B99a and PP2A mediate the decrease and subsequent increase of HMGR activity, which results from a steady rise of HMGR1-encoding transcript levels and an initial sharper reduction of HMGR protein level. In unchallenged plants, PP2A is a posttranslational negative regulator of HMGR activity with the participation of B99b. Our data indicate that PP2A exerts multilevel control on HMGR through the fivemember B99 protein family during normal development and in response to a variety of stress conditions.

3,4-Methylenedioxymethamphetamine enhances kainic acid convulsive susceptibility

Abstract Kainic acid (KA) causes seizures and neuronal loss in the hippocampus. The present study investigated whether a recreational schedule of 3,4-methylenedioxymethamphetamine (MDMA) favours the development of a seizure state in a model of KA-induced epilepsy and potentiates the toxicity profile of KA (20 or 30 mg/kg). Adolescent male C57BL/6 mice received saline or MDMA t.i.d. (s.c. every 3 h), on 1 day a week, for 4 consecutive weeks. Twenty-four hours after the last MDMA exposure, the animals were injected with saline or KA (20 or 30 mg/kg). After this injection, we evaluated seizures, hippocampal neuronal cell death, microgliosis, astrogliosis, and calcium binding proteins. MDMA pretreatment, by itself, did not induce neuronal damage but increased seizure susceptibility in all KA treatments and potentiated the presence of Fluoro-Jade-positive cells in CA1. Furthermore, MDMA, like KA, significantly decreased parvalbumin levels in CA1 and dentate gyrus, where it potentiated the effects of KA. The amphetamine derivative also promoted a transient decrease in calbindin and calretinin levels, indicative of an abnormal neuronal discharge. In addition, treatment of cortical neurons with MDMA (1050 μM) for 6 or 48 h significantly increased basal Ca2 +, reduced basal Na+ levels and potentiated kainate response. These results indicate that MDMA potentiates KA-induced neurodegeneration and also increases KA seizure susceptibility. The mechanism proposed includes changes in Calcium Binding Proteins expression, probably due to the disruption of intracellular ionic homeostasis, or/and an indirect effect through glutamate release.

3,4-Methylenedioxymethamphetamine enhances kainic acid convulsive susceptibility

Abstract Kainic acid (KA) causes seizures and neuronal loss in the hippocampus. The present study investigated whether a recreational schedule of 3,4-methylenedioxymethamphetamine (MDMA) favours the development of a seizure state in a model of KA-induced epilepsy and potentiates the toxicity profile of KA (20 or 30 mg/kg). Adolescent male C57BL/6 mice received saline or MDMA t.i.d. (s.c. every 3 h), on 1 day a week, for 4 consecutive weeks. Twenty-four hours after the last MDMA exposure, the animals were injected with saline or KA (20 or 30 mg/kg). After this injection, we evaluated seizures, hippocampal neuronal cell death, microgliosis, astrogliosis, and calcium binding proteins. MDMA pretreatment, by itself, did not induce neuronal damage but increased seizure susceptibility in all KA treatments and potentiated the presence of Fluoro-Jade-positive cells in CA1. Furthermore, MDMA, like KA, significantly decreased parvalbumin levels in CA1 and dentate gyrus, where it potentiated the effects of KA. The amphetamine derivative also promoted a transient decrease in calbindin and calretinin levels, indicative of an abnormal neuronal discharge. In addition, treatment of cortical neurons with MDMA (1050 μM) for 6 or 48 h significantly increased basal Ca2 +, reduced basal Na+ levels and potentiated kainate response. These results indicate that MDMA potentiates KA-induced neurodegeneration and also increases KA seizure susceptibility. The mechanism proposed includes changes in Calcium Binding Proteins expression, probably due to the disruption of intracellular ionic homeostasis, or/and an indirect effect through glutamate release.

Evolution of Bioaffinity Reagents by Phage Display

Antibodies are natural binding proteins produced in vertebrates as a response to invading pathogens and foreign substances. Because of their capability for tight and specific binding, antibodies have found use as binding reagents in research and diagnostics. Properties of cloned recombinant antibodies can be further improved by means of in vitro evolution, combining mutagenesis with subsequent phage display selection. It is also possible to isolate entirely new antibodies from vast naïve or synthetic antibody libraries by phage display. In this study, library techniques and phage display selection were applied in order to optimise binding scaffolds and antigen recognition of antibodies, and to evolve new and improved bioaffinity reagents. Antibody libraries were generated by random and targeted mutagenesis. Expression and stability were mainly optimised by the random methods whereas targeted randomisation of the binding site residues was used for optimising the binding properties. Trinucleotide mutagenesis allowed design of defined randomisation patterns for a synthetic antibody library. Improved clones were selected by phage display. Capture by a specific anti- DHPS antibody was exploited in the selection of improved phage display of DHPS. Efficient selection for stability was established by combining phage display selection with denaturation under reducing conditions. Broad-specific binding of a generic anti-sulfonamide antibody was improved by selection with one of the weakest binding sulfonamides. In addition, p9 based phage display was studied in affinity selection from the synthetic library. A TIM barrel protein DHPS was engineered for efficient phage display by combining cysteinereplacement with random mutagenesis. The resulting clone allows use of phage display in further engineering of DHPS and possibly use as an alternative-binding scaffold. An anti-TSH scFv fragment, cloned from a monoclonal antibody, was engineered for improved stability to better suite an immunoassay. The improved scFv tolerates 8 – 9 °C higher temperature than the parental scFv and should have sufficient stability to be used in an immunoanalyser with incubation at 36 °C. The anti-TSH scFv fragment was compared with the corresponding Fab fragment and the parental monoclonal antibody as a capturing reagent in a rapid 5-min immunoassay for TSH. The scFv fragment provided some benefits over the conventionally used Mab in anayte-binding capacity and assay kinetics. However, the recombinant Fab fragment, which had similar kinetics to the scFv, provided a more sensitive and reliable assay than the scFv. Another cloned scFv fragment was engineered in order to improve broad-specific recognition of sulfonamides. The improved antibody detects different sulfonamides at concentrations below the maximum residue limit (100 μg/kg in EU and USA) and allows simultaneous screening of different sulfonamide drug residues. Finally, a synthetic antibody library was constructed and new antibodies were generated and affinity matured entirely in vitro. These results illuminate the possibilities of phage display and antibody engineering for generation and optimisation of binding reagents in vitro and indicate the potential of recombinant antibodies as affinity reagents in immunoassays.

DPS-Like Peroxide Resistance Protein: Structural and Functional Studies on a Versatile Nanocontainer

Oxidative stress is a constant threat to almost all organisms. It damages a number of biomolecules and leads to the disruption of many crucial cellular functions. It is caused by reactive oxygen species (ROS), such as hydrogen peroxide (H2O₂), superoxide (•O₂ -), and hydroxyl radical (•OH). The most harmful of these compounds is •OH, which is only formed in cells in the presence of redox-cycling transition metals, such as iron and copper. Bacteria have developed a number of mechanisms to cope with ROS. One of the most widespread means employed by bacteria is the DNA-binding proteins from starved cells (Dps). Dps proteins protect the cells by binding and oxidizing Fe2+, thus greatly reducing the production of •OH. The oxidized iron is stored inside the protein as an iron core. In addition, Dps proteins bind directly to DNA forming a protective coating that shields DNA from harmful agents. Moreover, Dps proteins have been found to elicit other protective functions in cells and to participate in bacterial virulence. Dps proteins are of special importance to Streptococci owing to the lack of catalase in this genus of bacteria.This study was focused on structural and functional characterization of streptococcal Dpslike peroxide resistance (Dpr) proteins. Initially, crystal structures of Streptococcus pyogenes Dpr were determined. The data confirmed the presence of a di-metal ferroxidase center (FOC) in Dpr proteins and revealed the presence of a novel N-terminal helix as well as a surface metal-binding site. The crystal structures of Streptococcus suis Dpr complexed with transition metals demonstrated the metal specificity of the FOC. Solution binding studies also indicated the presence of a di-metal FOC. These results suggested a possible role for Dpr in the detoxification of various metals. Iron was found to mineralize inside the protein as ferrihydrite based on X-ray absorption spectroscopy data. The iron core was found to exhibit clear superparamagnetic behaviour using magnetic and Mössbauer measurements. The results from this study are expected to further increase our understanding on the binding, oxidation, and mineralization of iron and other metals in Dpr proteins. In particular, the structural and magnetic properties of the iron core can form a basis for potential new applications in nanotechnology. From the streptococcal viewpoint, the results would help in understanding better the complicated picture of bacterial pathogenesis. Dpr proteins may also provide a novel target for drug design due to their tight involvement in bacterial virulence.

Identification of Epigenetic Targets in Prostate Cancer for Therapeutic Development

Recurrent castration resistant prostate cancer remains a challenge for cancer therapies and novel treatment options in addition to current anti-androgen and mitosis inhibitors are needed. Aberrations in epigenetic enzymes and chromatin binding proteins have been linked to prostate cancer and they may form a novel class of drug targets in the future. In this thesis we systematically evaluated the epigenenome as a prostate cancer drug target. We functionally silenced 615 known and putative epigenetically active protein coding genes in prostate cancer cell lines using high throughput RNAi screening and evaluated the effects on cell proliferation, androgen receptor (AR) expression and histone patterns. Histone deacetylases (HDACs) were found to regulate AR expression. Furthermore, HDAC inhibitors reduced AR signaling and inhibited synergistically with androgen deprivation prostate cancer cell proliferation. In particular, TMPRSS2- EGR fusion gene positive prostate cancer cell lines were sensitive to combined HDAC and AR inhibition, which may partly be related to the dependency of a fusion gene induced epigenetic pathway. Histone demethylases (HDMs) were identified to regulate prostate cancer cell line proliferation. We discovered a novel histone JmjC-domain histone demethylase PHF8 to be highly expressed in high grade prostate cancers and mediate cell proliferation, migration and invasion in in vitro models. Additionally, we explored novel HDM inhibitor chemical structures using virtual screening methods. The structures best fitting to the active pocket of KDM4A were tested for enzyme inhibition and prostate cancer cell proliferation activity in vitro. In conclusion, our results show that prostate cancer may efficiently be targeted with combined AR and HDAC inhibition which is also currently being tested in clinical trials. HDMs were identified as another feasible novel drug target class. Future studies in representative animal models and development of specific inhibitors may reveal HDMs full potential in prostate cancer therapy

Chromatoid body mediated RNA regulation in mouse male germline

Male germ cell differentiation, spermatogenesis is an exceptional developmental process that produces a massive amount of genetically unique spermatozoa. The complexity of this process along with the technical limitations in the germline research has left many aspects of spermatogenesis poorly understood. Post-meiotic haploid round spermatids possess the most complex transcriptomes of the whole body. Correspondingly, efficient and accurate control mechanisms are necessary to deal with the huge diversity of transcribed RNAs in these cells. The high transcriptional activity in round spermatids is accompanied by the presence of an uncommonly large cytoplasmic ribonucleoprotein granule, called the chromatoid body (CB) that is conjectured to participate in the RNA post-transcriptional regulation. However, very little is known about the possible mechanisms of the CB function. The development of a procedure to isolate CBs from mouse testes was this study’s objective. Anti-MVH immunoprecipitation of cross-linked CBs from a fractionated testicular cell lysate was optimized to yield considerable quantities of pure and intact CBs from mice testes. This protocol produced reliable and reproducible data from the subsequent analysis of CB’s protein and RNA components. We found that the majority of the CB’s proteome consists of RNA-binding proteins that associate functionally with different pathways. We also demonstrated notable localization patterns of one of the CB transient components, SAM68 and showed that its ablation does not change the general composition or structure of the CB. CB-associated RNA analysis revealed a strong accumulation of PIWI-interacting RNAs (piRNAs), mRNAs and long non-coding RNAs (lncRNAs) in the CB. When the CB transcriptome and proteome analysis results were combined, the most pronounced molecular functions in the CB were related to piRNA pathway, RNA post-transcriptional processing and CB structural scaffolding. In addition, we demonstrated that the CB is a target for the main RNA flux from the nucleus throughout all steps of round spermatid development. Moreover, we provided preliminary evidence that those isolated CBs slice target RNAs in vitro in an ATPdependent manner. Altogether, these results make a strong suggestion that the CB functions involve RNA-related and RNA-mediated mechanisms. All the existing data supports the hypothesis that the CB coordinates the highly complex haploid transcriptome during the preparation of the male gametes for fertilization. Thereby, this study provides a fundamental basis for the future functional analyses of ribonucleoprotein granules and offers also important insights into the mechanisms governing male fertility.

Pivotal role of leptin in insulin effects

The OB protein, also known as leptin, is secreted by adipose tissue, circulates in the blood, probably bound to a family of binding proteins, and acts on central neural networks regulating ingestive behavior and energy balance. The two forms of leptin receptors (long and short forms) have been identified in various peripheral tissues, a fact that makes them possible target sites for a direct action of leptin. It has been shown that the OB protein interferes with insulin secretion from pancreatic islets, reduces insulin-stimulated glucose transport in adipocytes, and increases glucose transport, glycogen synthesis and fatty acid oxidation in skeletal muscle. Under normoglycemic and normoinsulinemic conditions, leptin seems to shift the flux of metabolites from adipose tissue to skeletal muscle. This may function as a peripheral mechanism that helps control body weight and prevents obesity. Data that substantiate this hypothesis are presented in this review.

E. coli a-hemolysin: a membrane-active protein toxin

Alpha-Hemolysin is synthesized as a 1024-amino acid polypeptide, then intracellularly activated by specific fatty acylation. A second activation step takes place in the extracellular medium through binding of Ca2+ ions. Even in the absence of fatty acids and Ca2+ HlyA is an amphipathic protein, with a tendency to self-aggregation. However, Ca2+-binding appears to expose hydrophobic patches on the protein surface, facilitating both self-aggregation and irreversible insertion into membranes. The protein may somehow bind membranes in the absence of divalent cations, but only when Ca2+ (or Sr2+, or Ba2+) is bound to the toxin in aqueous suspensions, i.e., prior to its interaction with bilayers, can a-hemolysin bind irreversibly model or cell membranes in such a way that the integrity of the membrane barrier is lost, and cell or vesicle leakage ensues. Leakage is not due to the formation of proteinaceous pores, but rather to the transient disruption of the bilayer, due to the protein insertion into the outer membrane monolayer, and subsequent perturbations in the bilayer lateral tension. Protein or glycoprotein receptors for a-hemolysin may exist on the cell surface, but the toxin is also active on pure lipid bilayers.

Involvement of the actin cytoskeleton and p21rho-family GTPases in the pathogenesis of the human protozoan parasite Entamoeba histolytica

It has been estimated that infection with the enteric protozoan parasite Entamoeba histolytica kills more than 50,000 people a year. Central to the pathogenesis of this organism is its ability to directly lyse host cells and cause tissue destruction. Amebic lesions show evidence of cell lysis, tissue necrosis, and damage to the extracellular matrix. The specific molecular mechanisms by which these events are initiated, transmitted, and effected are just beginning to be uncovered. In this article we review what is known about host cell adherence and contact-dependent cytolysis. We cover the involvement of the actin cytoskeleton and small GTP-binding proteins of the p21rho-family in the process of cell killing and phagocytosis, and also look at how amebic interactions with molecules of the extracellular matrix contribute to its cytopathic effects.

Galectin-1, an alternative signal for T cell death, is increased in activated macrophages

Galectin-1 belongs to an evolutionarily conserved family of animal ß-galactoside-binding proteins, which exert their functions by crosslinking the oligosaccharides of specific glycoconjugate ligands. During the past decade, attempts to identify the functional role of galectin-1 suggested participation in the regulation of the immune response. Only in the last few years has the molecular mechanism involved in these properties been clearly elucidated, revealing a critical role for galectin-1 as an alternative signal in the generation of T cell death. In the present study we will discuss the latest advances in galectin research in the context of the regulation of the immune response, not only at the central level but also at the periphery. Moreover, we will review the purification, biochemical properties and functional significance of a novel galectin-1-like protein from activated rat macrophages, whose expression is differentially regulated according to the activation state of the cells. The novel role of a carbohydrate-binding protein in the regulation of apoptosis is providing a breakthrough in galectin research and extending the interface between immunology, glycobiology and clinical medicine.