The Mutyh Base Excision Repair Gene Influences the Inflammatory Response in a Mouse Model of Ulcerative Colitis

Background

The Mutyh DNA glycosylase is involved in the repair of oxidized DNA bases. Mutations in the human MUTYH gene are responsible for colorectal cancer in familial adenomatous polyposis. Since defective DNA repair genes might contribute to the increased cancer risk associated with inflammatory bowel diseases, we compared the inflammatory response of wild-type and Mutyh^−/− mice to oxidative stress.

Methodology/Principal Findings

The severity of colitis, changes in expression of genes involved in DNA repair and inflammation, DNA 8-oxoguanine levels and microsatellite instability were analysed in colon of mice treated with dextran sulfate sodium (DSS). The Mutyh^−/− phenotpe was associated with a significant accumulation of 8-oxoguanine in colon DNA of treated mice. A single DSS cycle induced severe acute ulcerative colitis in wild-type mice, whereas lesions were modest in Mutyh^−/− mice, and this was associated with moderate variations in the expression of several cytokines. Eight DSS cycles caused chronic colitis in both wild-type and Mutyh^−/− mice. Lymphoid hyperplasia and a significant reduction in Foxp3⁺ regulatory T cells were observed only in Mutyh^−/− mice.

Conclusions

The findings indicate that, in this model of ulcerative colitis, Mutyh plays a major role in maintaining intestinal integrity by affecting the inflammatory response.     

Pre-analytical factors in clinical proteomics investigations: Impact of ex vivo protein modifications for multiple sclerosis biomarker discovery

Serum proteome investigations have raised an incredible interest in the research of novel molecular biomarker, nevertheless few of the proposed evidences have been translated to the clinical practice. One of the limiting factors has been the lack of generally accepted guidelines for clinical proteomics studies and the lack of a robust analytical and pre-analytical ground for the proposed classification models. Pre-analytical issues may results in a deep impact for biomarker discovery campaign. In this study we present a systematic evaluation of sample storage and sampling conditions for clinical proteomics investigations.We have developed and validated a linear MALDI-TOF-MS protein profiling method to explore the low protein molecular weight region (5–20 kDa) of serum samples. Data normalization and processing was performed using optimise peak detection routine (LIMPIC) able to describe each group under investigation. Data were acquired either from healthy volunteers and from multiple sclerosis patients in order to highlight ex vivo protein profile alteration related to different physio-pathological conditions. Our data showed critical conditions for serum protein profiles depending on storage times and temperatures: 23 °C, 4 °C, ? 20 °C and ? 80 °C. We demonstrated that upon a ? 20 °C short term storage, characteristic degradation profiles are associated with different clinical groups. Protein signals were further identified after preparative HPLC separation by peptide sequencing on a nanoLC-Q-TOF TANDEM mass spectrometer. Apolipoprotein A-IV and complement C3 protein fragments, transthyretin and the oxidized isoforms in different apolipoprotein species represent the major molecular features of such a degradation pattern.     

Residual Antimalarials in Malaria Patients from Tanzania – Implications on Drug Efficacy Assessment and Spread of Parasite Resistance

Background

Repeated antimalarial treatment for febrile episodes and self-treatment are common in malaria-endemic areas. The intake of antimalarials prior to participating in an in vivo study may alter treatment outcome and affect the interpretation of both efficacy and safety outcomes. We report the findings from baseline plasma sampling of malaria patients prior to inclusion into an in vivo study in Tanzania and discuss the implications of residual concentrations of antimalarials in this setting.

Methods and Findings

In an in vivo study conducted in a rural area of Tanzania in 2008, baseline plasma samples from patients reporting no antimalarial intake within the last 28 days were screened for the presence of 14 antimalarials (parent drugs or metabolites) using liquid chromatography-tandem mass spectrometry. Among the 148 patients enrolled, 110 (74.3%) had at least one antimalarial in their plasma: 80 (54.1%) had lumefantrine above the lower limit of calibration (LLC = 4 ng/mL), 7 (4.7%) desbutyl-lumefantrine (4 ng/mL), 77 (52.0%) sulfadoxine (0.5 ng/mL), 15 (10.1%) pyrimethamine (0.5 ng/mL), 16 (10.8%) quinine (2.5 ng/mL) and none chloroquine (2.5 ng/mL).

Conclusions

The proportion of patients with detectable antimalarial drug levels prior to enrollment into the study is worrying. Indeed artemether–lumefantrine was supposed to be available only at government health facilities. Although sulfadoxine–pyrimethamine is only recommended for intermittent preventive treatment in pregnancy (IPTp), it was still widely used in public and private health facilities and sold in drug shops. Self-reporting of previous drug intake is unreliable and thus screening for the presence of antimalarial drug levels should be considered in future in vivo studies to allow for accurate assessment of treatment outcome. Furthermore, persisting sub-therapeutic drug levels of antimalarials in a population could promote the spread of drug resistance. The knowledge on drug pressure in a given population is important to monitor standard treatment policy implementation.     

Characterization of oxidation end product of plasma albumin 'in vivo'

Anti-oxidants are paradoxically much lower in plasma than inside cells even blood is comparably exposed to the oxidative stress. 'In vitro' models suggest a critical role of albumin as substitutive anti-oxidant in plasma but no proof for this role is available 'in vivo.' Herein, we demonstrate by LC/MS/MS that plasma albumin undergoes massive oxidation in primary nephrotic syndrome, involving stable sulphonation SO3- of the free SH of Cys 34 with +48Da increase in exact mass of the protein (ESI-MS) and formation of a fast moving isoform in the pH range between 5 and 7. Physical-chemical experiments with DSC and fluorescence spectra indicate a thermal stabilization of the structure upon oxidation. This is the first demonstration of massive oxidation of albumin 'in vivo' that reflects a functional role of the protein. Free radicals should be implicated in the pathogenesis of proteinuria in human FSGS.     

PED interacts with Rac1 and regulates cell migration/invasion processes in human non‐small cell lung cancer cells

PED (phosphoprotein enriched in diabetes) is a 15 kDa protein involved in many cellular pathways and human diseases including type II diabetes and cancer. We recently reported that PED is overexpressed in human cancers and mediates resistance to induced apoptosis. To better understand its role in cancer, we investigated on PED interactome in non‐small cell lung cancer (NSCLC). By the Tandem Affinity Purification (TAP), we identified and characterized among others, Rac1, a member of mammalian Rho GTPase protein family, as PED‐interacting protein. In this study we show that PED coadiuvates Rac1 activation by regulating AKT mediated Rac1‐Ser⁷¹ phosphorylation. Furthermore, we show that the expression of a constitutively active Rac, affected PED‐Ser¹⁰⁴ phosphorylation, which is important for PED‐regulated ERK 1/2 nuclear localization. Through specific Rac1‐siRNA or its pharmacological inhibition, we demonstrate that PED augments migration and invasion in a Rac1‐dependent manner in NSCLC. In conclusion, we show for the first time that PED and Rac1 interact and that this interaction modulates cell migration/invasion processes in cancer cells through ERK1/2 pathway. J. Cell. Physiol. 225: 63–72, 2010. © 2010 Wiley‐Liss, Inc.     

Hop2-Mnd1 condenses DNA to stimulate the synapsis phase of DNA strand exchange

Hop2-Mnd1 is a meiotic recombination mediator that stimulates DNA strand invasion by both Dmc1 and Rad51. To understand the biochemical mechanism of this stimulation, we directly visualized the heterodimer acting on single molecules of duplex DNA using optical tweezers and video fluorescence microscopy. The results show that the Hop2-Mnd1 heterodimer efficiently condenses double-stranded DNA via formation of a bright spot or DNA condensate. The condensation of DNA is Hop2-Mnd1 concentration-dependent, reversible, and specific to the heterodimer, as neither Hop2 nor Mnd1 acting alone can facilitate this reaction. The results also show that the rate-limiting nucleation step of DNA condensation is overcome in the presence of divalent metal ions, with the following order of preference: Mn²⁺>Mg²⁺>Ca²⁺. Hop2-Mnd1/Dmc1/single-stranded DNA nucleoprotein filaments also condense double-stranded DNA in a heterodimer concentration-dependent manner. Of importance, the concentration dependence parallels that seen in DNA strand exchange. We propose that rapid DNA condensation is a key factor in stimulating synapsis, whereas decondensation may facilitate the invasion step and/or the ensuing branch migration process.     

Structural investigation of bilayers formed by 1-palmitoyl-2-oleoylphosphatidylnucleosides

Bilayers of palmitoyl-oleoylphosphatidylnucleoside derivatives (1-palmitoyl-2-oleoyl-phosphatidyl-adenosine and 1-palmitoyl-2-oleoyl-phosphatidyl-uridine) were synthesized and investigated in the low-water content regime by a combination of neutron diffraction and Fourier transform infrared linear dichroism (LD-FTIR). Attention was focused on the modulation of structural properties operated by the presence of nucleic acid bases (either adenosine or uridine, a purine and a pyrimidine that are complementary in RNA). Base substitution causes major differences in phase behavior of the phospholipids, i.e., water sorption from a controlled humidity atmosphere and smectic periodicity. The profile of scattering length density can be inferred from five diffraction orders for 1-palmitoyl-2-oleoyl-phosphatidyl-uridine lamellar phase. 1-Palmitoyl-2-oleoyl-phosphatidyl-adenosine is characterized by lower and less ready hydration, giving rise to a powder-like sample. A linear dichroism FTIR investigation on the same lamellar phases was undertaken with the purpose of gathering details at the submolecular level on different portions of the molecule. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers were also investigated with the same technique for the sake of comparison. Besides a confirmation of the diffraction data interpretation, FTIR has provided evidence that the same chemical groups at the bilayer interface (namely the sugar-phosphate) have a different orientation depending on whether the base is a purine or a pyrimidine. A very simple geometrical optimization agrees with this observation. This indicates that a different pattern of base interaction is operating in the two cases and that base substitution acts as a modulator of the phase properties.