Bovine prion (PrP) and Doppel (Dpl) proteins expression after in vitro leukocyte activation or Dpl/PrP blocking

It has been postulated that Doppel (Dpl) and Prion (PrP) proteins have yet undetermined interactions, since Dpl is overexpressed in transgenic PrP-deficient mice. In this study we investigated the expression levels of Dpl and PrP on lymphocytes, monocytes and neutrophils (PMNs) isolated from bovine blood and incubated (2 and 18 h) with TNFalpha, IL-1, IL-2, IL-8, C5a, IFNgamma, anti-PrP, and anti-Dpl antibodies by flow cytometry. The isolation procedures yielded cell populations with high purity, viability and recovery rates. After 2 h incubation, expression of PrP or Dpl was altered only in PMNs. These cells overexpressed PrP when incubated with TNFalpha and IFNgamma, and both PrP and Dpl when incubated with C5a; incubation with TNFalpha, IL-8 and IFNgamma led to down-regulation of Dpl. Lymphocytes incubated for 18 h with IL-2 and with IFNgamma overexpressed Dpl. Incubation with the anti-PrP antibody induced down-regulation of Dpl in PMNs after 2 h and overexpression of Dpl in lymphocytes after 18 h. The differences recorded after 2 h were likely due to redistribution of pre-existing PrP or Dpl molecules, while those seen at 18 h were most probably due to increased protein synthesis. The variations seen using the different activators depend on different receptors and/or signaling pathways. These results demonstrate that is possible to alter the expression of Dpl and PrP in blood cells in vitro by incubation with either cytokines or anti-PrP antibodies. This opens an interesting opportunity to study the biology of these proteins using in vitro systems.     

Lac repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion

The tethered particle motion (TPM) allows the direct detection of activity of a variety of biomolecules at the single molecule level. First pioneered for RNA polymerase, it has recently been applied also to other enzymes. In this work we employ TPM for a systematic investigation of the kinetics of DNA looping by wild-type Lac repressor (wt-LacI) and by hinge mutants Q60G and Q60 + 1. We implement a novel method for TPM data analysis to reliably measure the kinetics of loop formation and disruption and to quantify the effects of the protein hinge flexibility and of DNA loop strain on such kinetics. We demonstrate that the flexibility of the protein hinge has a profound effect on the lifetime of the looped state. Our measurements also show that the DNA bending energy plays a minor role on loop disruption kinetics, while a strong effect is seen on the kinetics of loop formation. These observations substantiate the growing number of theoretical studies aimed at characterizing the effects of DNA flexibility, tension and torsion on the kinetics of protein binding and dissociation, strengthening the idea that these mechanical factors in vivo may play an important role in the modulation of gene expression regulation.     

Searching for a needle in the haystack: comparing six methods to evaluate heteroplasmy in difficult sequence context

Mitochondrial DNA (mtDNA) mutations have been involved in disease, aging and cancer and furthermore exploited for evolutionary and forensic investigation. When investigating mtDNA mutations the peculiar aspects of mitochondrial genetics, such as heteroplasmy and threshold effect, require suitable approaches which must be sensitive enough to detect low-level heteroplasmy and, precise enough to quantify the exact mutational load. In order to establish the optimal approach for the evaluation of heteroplasmy, six methods were experimentally compared for their capacity to reveal and quantify mtDNA variants. Drawbacks and advantages of cloning, Fluorescent PCR (F-PCR), denaturing High Performance Liquid Chromatography (dHPLC), quantitative Real-Time PCR (qRTPCR), High Resolution Melting (HRM) and 454 pyrosequencing were determined. In particular, detection and quantification of a mutation in a difficult sequence context were investigated, through analysis of an insertion in a homopolymeric stretch (m.3571insC).     

mda-7/IL-24 differentially regulates soluble and nuclear clusterin in prostate cancer

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), a unique member of the IL-10 gene family, displays a broad range of antitumor properties including cancer-specific induction of apoptosis, inhibition of tumor angiogenesis, and modulation of anti-tumor immune responses. Here, we identify clusterin (CLU) as a MDA-7/IL-24 interacting protein in DU-145 cells and investigate the role of MDA-7/IL-24 in regulating CLU expression and mediating the antitumor properties of mda-7/IL-24 in prostate cancer. Ad.mda-7 decreased expression of soluble CLU (sCLU) and increased expression of nuclear CLU (nCLU). In the initial phase of Ad.mda-7 infection sCLU expression increased and CLU interacted with MDA-7/IL-24 producing a cytoprotective effect. Infection of stable clones of DU-145 prostate cancer cells expressing sCLU with Ad.mda-7 resulted in generation of nCLU that correlated with decreased cell viability and increased apoptosis. In the presence of mda-7/IL-24, sCLU-DU-145 cells displayed G(2)/M phase arrest followed by apoptosis. Similarly, Ad.mda-7 infection decreased cell migration by altering cytoskeleton in sCLU-DU-145 cells. Ad.mda-7-treated sCLU-DU-145 cells displayed a significant reduction in tumor growth in mouse xenograft models and reduced angiogenesis when compared to the vector control group. Tumor tissue lysates demonstrated enhanced nCLU generated from sCLU with increased apoptosis in the presence of MDA-7/IL-24. Our findings reveal novel aspects relative to the role of sCLU/nCLU in regulating the anticancer properties of MDA-7/IL-24 that may be exploited for developing enhanced therapies for prostate cancer.     

Environmental exposure to benzene, micronucleus formation and polymorphisms in DNA-repair genes: a pilot study

This report is part of a biomarker study conducted in an Italian population with exposure to environmental benzene ranging from 1.43 to 31.41 μg/m3 (values from personal sampling). DNA damage induced by benzene is the crucial mechanism of its genotoxicity, which leads to chronic benzene poisoning, haematotoxicity and leukaemia. Therefore, genetic variation in DNA-repair genes may modulate susceptibility to benzene-induced DNA damage. In light of this, the effects of polymorphisms in DNA-repair genes (APEX1, hOGG1, NBS1, XPD, XRCC1, and XRCC3) on micronucleus (MN) formation as a biomarker of early biological effects were evaluated. A significantly higher median MN frequency was recorded in traffic wardens than in controls. However, none of the analysed polymorphisms was significantly associated with the median MN frequency. A gene-gender interaction was observed for the APEX1 genotype. The APEX1 variant genotype was associated with significantly lower median MN frequency in men, not in women. Statistical analysis did not reveal any association between the score of the protective alleles - hypothetically pushing the pathway towards optimal DNA-damage repair - and MN. Even though there are some limitations in the study, our results indicate that the general population may be exposed to benzene concentrations higher than the threshold level for air-quality standards in the European Union of 10 μg/m3. Furthermore, urban traffic wardens are exposed to significantly higher levels of benzene than individuals spending most of the time indoors. This higher exposure may contribute to DNA damage, suggesting that benzene might be implicated both as an environmental and occupational risk factor in leukaemia and other haematological diseases. In conclusion, this study suggest the need for (i) regular monitoring of traffic wardens for possible exposure to benzene, as a precautionary step to reduce the associated health risks, and (ii) more comprehensive studies in order to better elucidate the involvement of APEX1 genotypes in benzene genotoxicity.     

mTrop1/Epcam Knockout Mice Develop Congenital Tufting Enteropathy through Dysregulation of Intestinal E-cadherin/β-catenin

Congenital tufting enteropathy (CTE) is a life-threatening hereditary disease that is characterized by enteric mucosa tufting degeneration and early onset, severe diarrhea. Loss-of-function mutations of the human EPCAM gene (TROP1, TACSTD1) have been indicated as the cause of CTE. However, loss of mTrop1/Epcam in mice appeared to lead to death in utero, due to placental malformation. This and indications of residual Trop-1/EpCAM expression in cases of CTE cast doubt on the role of mTrop1/Epcam in this disease. The aim of this study was to determine the role of TROP1/EPCAM in CTE and to generate an animal model of this disease for molecular investigation and therapy development. Using a rigorous gene-trapping approach, we obtained mTrop1/Epcam -null (knockout) mice. These were born alive, but failed to thrive, and died soon after birth because of hemorrhagic diarrhea. The intestine from the mTrop1/Epcam knockout mice showed intestinal tufts, villous atrophy and colon crypt hyperplasia, as in human CTE. No structural defects were detected in other organs. These results are consistent with TROP1/EPCAM loss being the cause of CTE, thus providing a viable animal model for this disease, and a benchmark for its pathogenetic course. In the affected enteric mucosa, E-cadherin and β-catenin were shown to be dysregulated, leading to disorganized transition from crypts to villi, with progressive loss of membrane localization and increasing intracellular accumulation, thus unraveling an essential role for Trop-1/EpCAM in the maintenance of intestinal architecture and functionality.Supporting information is available for this article.     

2-arachidonoylglycerol signaling in forebrain regulates systemic energy metabolism

The endocannabinoid system plays a critical role in the control of energy homeostasis, but the identity and localization of the endocannabinoid signal involved remain unknown. In the present study, we developed transgenic mice that overexpress in forebrain neurons the presynaptic hydrolase, monoacylglycerol lipase (MGL), which deactivates the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). MGL-overexpressing mice show a 50% decrease in forebrain 2-AG levels but no overt compensation in other endocannabinoid components. This biochemical abnormality is accompanied by a series of metabolic changes that include leanness, elevated energy cost of activity, and hypersensitivity to β(3)-adrenergic-stimulated thermogenesis, which is corrected by reinstating 2-AG activity at CB(1)-cannabinoid receptors. Additionally, the mutant mice are resistant to diet-induced obesity and express high levels of thermogenic proteins, such as uncoupling protein 1, in their brown adipose tissue. The results suggest that 2-AG signaling through CB(1) regulates the activity of forebrain neural circuits involved in the control of energy dissipation.     

Additional contribution of emerging risk factors to the prediction of the risk of type 2 diabetes: evidence from the Western New York Study

Objective: To examine whether several biomarkers of endothelial function and inflammation improve prediction of type 2 diabetes over 5.9 years of follow‐up, independent of traditional risk factors. Methods and Procedures: A total of 1,455 participants from the Western New York Study, free of type 2 diabetes at baseline, were selected. Incident type 2 diabetes was defined as fasting glucose exceeding 125 mg/dl or on antidiabetic medication at the follow‐up visit. Sixty‐one people who met the case definition (8/1,000 person years) were identified and individually matched with up to three controls on gender, race, year of study enrollment, and baseline fasting glucose (<110 or 110–125 mg/dl). Biomarkers were measured from frozen baseline samples. Results: In conditional logistic regression analyses accounting for traditional risk factors (age, family history of diabetes, smoking, drinking status, and BMI), E‐selectin was positively related (3rd vs. 1st tertile: odds ratio 2.77, 95% confidence interval (CI) 1.13–6.79, P for linear trend = 0.023) and serum albumin was inversely related (3rd vs. 1st tertile: odds ratio 0.36, 95% CI 0.14–0.93, P for linear trend = 0.032) to type 2 diabetes incidence. The addition of E‐selectin, serum albumin, and leukocyte count to a basic risk factor model including only traditional risk factors significantly increased the area under the receiver operating characteristic curve (AUC) (from 0.646 to 0.726, P value = 0.04). Discussion: These results support the role of endothelial dysfunction and subclinical inflammation as important mechanisms in the etiopathogenesis of type 2 diabetes; moreover, they indicate that novel biomarkers may improve the prediction of type 2 diabetes beyond the use of traditional risk factors alone.     

Differential screening of phage-ab libraries by oligonucleotide microarray technology

A novel and efficient tagArray technology was developed that allows rapid identification of antibodies which bind to receptors with a specific expression profile, in the absence of biological information. This method is based on the cloning of a specific, short nucleotide sequence (tag) in the phagemid coding for each phage-displayed antibody fragment (phage-Ab) present in a library. In order to set up and validate the method we identified about 10,000 different phage-Abs binding to receptors expressed in their native form on the cell surface (10 k Membranome collection) and tagged each individual phage-Ab. The frequency of each phage-Ab in a given population can at this point be inferred by measuring the frequency of its associated tag sequence through standard DNA hybridization methods. Using tiny amounts of biological samples we identified phage-Abs binding to receptors preferentially expressed on primary tumor cells rather than on cells obtained from matched normal tissues. These antibodies inhibited cell proliferation in vitro and tumor development in vivo, thus representing therapeutic lead candidates.