Mass spectrometry-based protein profiling strategies for biomarker discovery in liver and inflammatory bowel diseases

The study of protein expression profiles for biomarker discovery in serum and in mammalian cell populations needs the continuous improvement and combination of proteins/peptides separation techniques, mass spectrometry, statistical and bioinformatic approaches. In this thesis work two different mass spectrometry-based protein profiling strategies have been developed and applied to liver and inflammatory bowel diseases (IBDs) for the discovery of new biomarkers. The first of them, based on bulk solid-phase extraction combined with matrix-assisted laser desorption/ionization - Time of Flight mass spectrometry (MALDI-TOF MS) and chemometric analysis of serum samples, was applied to the study of serum protein expression profiles both in IBDs (Crohn’s disease and ulcerative colitis) and in liver diseases (cirrhosis, hepatocellular carcinoma, viral hepatitis). The approach allowed the enrichment of serum proteins/peptides due to the high interaction surface between analytes and solid phase and the high recovery due to the elution step performed directly on the MALDI-target plate. Furthermore the use of chemometric algorithm for the selection of the variables with higher discriminant power permitted to evaluate patterns of 20-30 proteins involved in the differentiation and classification of serum samples from healthy donors and diseased patients. These proteins profiles permit to discriminate among the pathologies with an optimum classification and prediction abilities. In particular in the study of inflammatory bowel diseases, after the analysis using C18 of 129 serum samples from healthy donors and Crohn’s disease, ulcerative colitis and inflammatory controls patients, a 90.7% of classification ability and a 72.9% prediction ability were obtained. In the study of liver diseases (hepatocellular carcinoma, viral hepatitis and cirrhosis) a 80.6% of prediction ability was achieved using IDA-Cu(II) as extraction procedure. The identification of the selected proteins by MALDITOF/ TOF MS analysis or by their selective enrichment followed by enzymatic digestion and MS/MS analysis may give useful information in order to identify new biomarkers involved in the diseases. The second mass spectrometry-based protein profiling strategy developed was based on a label-free liquid chromatography electrospray ionization quadrupole - time of flight differential analysis approach (LC ESI-QTOF MS), combined with targeted MS/MS analysis of only identified differences. The strategy was used for biomarker discovery in IBDs, and in particular of Crohn’s disease. The enriched serum peptidome and the subcellular fractions of intestinal epithelial cells (IECs) from healthy donors and Crohn’s disease patients were analysed. The combining of the low molecular weight serum proteins enrichment step and the LCMS approach allowed to evaluate a pattern of peptides derived from specific exoprotease activity in the coagulation and complement activation pathways. Among these peptides, particularly interesting was the discovery of clusters of peptides from fibrinopeptide A, Apolipoprotein E and A4, and complement C3 and C4. Further studies need to be performed to evaluate the specificity of these clusters and validate the results, in order to develop a rapid serum diagnostic test. The analysis by label-free LC ESI-QTOF MS differential analysis of the subcellular fractions of IECs from Crohn’s disease patients and healthy donors permitted to find many proteins that could be involved in the inflammation process. Among them heat shock protein 70, tryptase alpha-1 precursor and proteins whose upregulation can be explained by the increased activity of IECs in Crohn’s disease were identified. Follow-up studies for the validation of the results and the in-depth investigation of the inflammation pathways involved in the disease will be performed. Both the developed mass spectrometry-based protein profiling strategies have been proved to be useful tools for the discovery of disease biomarkers that need to be validated in further studies.

Sindrome di Down e fattori di rischio nel declino neurocognitivo

Down syndrome (DS) or Trisomy 21, occurring in 1/700 and 1/1000 livebirths, is the most common genetic disorder, characterized by a third copy of the human chromosome 21 (Hsa21). DS is associated with various defects, including congenital heart diseases, craniofacial abnormalities, immune system dysfunction, mental retardation (MR), learning and memory deficiency. The phenotypic features in DS are a direct consequence of overexpression of genes located within the triplicated region on Hsa21. In addition to developmental brain abnormalities and disabilities, people with DS by the age of 30-40 have a greatly increased risk of early-onset of Alzheimer’s disease (AD) and an apparent tendency toward premature aging. Many of the immunological anomalies in DS can be enclosed in the spectrum of multiple signs of early senescence. People with DS have an increased vulnerability to oxidative damage and many factors, including amyloid beta protein (Abeta), genotype ApoE4, oxidative stress, mutations in mitochondrial DNA (mtDNA), impairment of antioxidant enzymes, accelerated neuronal cell apoptosis, are related to neuronal degeneration and early aging in DS. SUBJECTS and METHODS: Since 2007 a population of 50 adolescents and adults with DS, 26 males and 24 females (sex-ratio: M/F = 1.08), has been evaluated for the presence of neurological features, biomarkers and genetic factors correlated with neuronal degeneration and premature aging. The control group was determined by the mother and the siblings of the patients. A neuropsychiatric evaluation was obtained from all patients. The levels of thyroid antibodies (antiTg and antiTPO) and of some biochemical markers of oxidative stress, including homocysteine (tHcy), uric acid, cobalamin, folate were measured. All patients, the mother and the siblings were genotyped for ApoE gene. RESULTS: 40% of patients, with a mild prevalence of females aged between 19 and 30 years, showed increased levels of antiTg and antiTPO. The levels of tHcy were normal in 52% patients and mildly increased in 40%; hyperomocysteinemia was associated with normal levels of thyroid antibodies in 75% of patients (p<0.005). The levels of uric acid were elevated in 26%. Our study showed a prevalence of severe MR in patients aged between 1-18 years and over 30 years. Only 3 patients, 2 females and one male, over 30 years of age, showed dementia. According to the literature, the rate of Down left-handers was high (25%) compared to the rest of population and the laterality was associated with increased levels of thyroid antibodies (70%). 21.5% of patients were ApoE4 positive (ApoE4+) with a mean/severe MR. CONCLUSIONS: Until now no biochemical evidence of oxidative damage and no deficiency or alteration of antioxidant function in our patients with DS were found. mtDNA sequencing could show some mutations age-related and associated with oxidative damage and neurocognitive decline in the early aging of DS. The final aim is found predictive markers of early-onset dementia and a target strategy for the prevention and the treatment of diseases caused by oxidative stress. REFERENCES: 1) Rachidi M, Lopes C: “Mental retardation and associated neurological dysfunctions in Down syndrome: a consequence of dysregulation in critical chromosome 21 genes and associated molecular pathways.” - Eur J Paediatr Neurol. May;12(3):168-82 (2008). 2) Lott IT, Head E: “Down syndrome and Alzheimer's disease: a link between development and aging.” - Ment Retard Dev Disabil Res Rev, 7(3):172-8 (2001). 3) Lee HC, Wei YH: “Oxidative Stress, Mitochondrial DNA Mutation, and Apoptosis in Aging.” - Exp Biol Med (Maywood), May;232(5):592-606 (2007).

Down Syndrome: Neuropsychological phenotype and mitochondrial DNA

Introduction. Down Syndrome (DS) is the most known autosomal trisomy, due to the presence in three copies of chromosome 21. Many studies were designed to identify phenotypic and clinical consequences related to the triple gene dosage. However, the general conclusion is a senescent phenotype; in particular, the most features of physiological aging, such as skin and hair changes, vision and hearing impairments, thyroid dysfunction, Alzheimer-like dementia, congenital heart defects, gastrointestinal malformations, immune system changes, appear in DS earlier than in normal age-matched subjects. The only established risk factor for the DS is advanced maternal age, responsible for changes in the meiosis of oocytes, in particular the meiotic nondisjunction of chromosome 21. In this process mitochondria play an important role since mitochondrial dysfunction, due to a variety of extrinsic and intrinsic influences, can profoundly influence the level of ATP generation in oocytes, required for a correct chromosomal segregation. Aim. The aim of this study is to investigate an integrated set of molecular genetic parameters (sequencing of complete mtDNA, heteroplasmy of the mtDNA control region, genotypes of APOE gene) in order to identify a possible association with the early neurocognitive decline observed in DS. Results. MtDNA point mutations do not accumulate with age in our study sample and do not correlate with early neurocognitive decline of DS subjects. It seems that D-loop heteroplasmy is largely not inherited and tends to accumulate somatically. Furthermore, in our study sample no association of cognitive impairment and ApoE genotype is found. Conclusions. Overall, our data cast some doubts on the involvement of these mutations in the decline of cognitive functions observed in DS.

Regulation der Selenoproteinsynthese durch den Mevalonatweg

Im Rahmen dieser Arbeit sollte der Einfluss des Mevalonatpfads auf die Expression von Selenoproteinen untersucht werden. Im Mevalonatpfad, einem universellen Stoffwechselweg eukaryontischer Zellen, entstehen neben Cholesterol auch verschiedene Isoprenoide, die z.B. für die post-transkriptionelle Modifikation der Selenocystein-tRNA herangezogen werden. Selenocystein ist funktioneller Bestandteil von Selenoproteinen, welche häufig in den Abbau von oxidativem Stress involviert sind. rnDer Mevalonatpfad wird hauptsächlich durch die HMG-CoA-Reduktase (HMGCR) reguliert. Pharmaka vom „Statin“-Typ gelten als wirkungsvolle kompetitive Inhibitoren dieses Enzyms und finden ihren Einsatz bei Patienten zur Behandlung von Hypercholesterolämie, welche eine Grundlage für vaskuläre Krankheiten bildet. Trotz der allgemein guten Verträglichkeit der Statine treten jedoch auch unerwünschte Nebeneffekte, wie Erhöhung der Leberenzyme oder Myopathien auf, deren biochemischer Hintergrund bislang noch im Dunkeln liegt. rnDie in dieser Arbeit durchgeführten Experimente belegen, dass Atorvastatin, Cerivastatin und Lovastatin in klinisch relevanten Dosen die Synthese bestimmter Selenoproteine, wie der Glutathionperoxidase (GPx), in klonalen humanen Hepatocyten post-transkriptionell unterdrücken, wodurch die Zellen anfälliger für oxidativen Stress in Form von Peroxiden werden. Dieser Mechanismus könnte eine Erklärung für die häufig beobachteten abnormen Leberwerte von Statin-behandelten Patienten darstellen.rnEndogenes Cholesterol gilt ebenfalls als potenter Inhibitor der HMGCR. Die in dieser Arbeit erzielten Ergebnisse zeigen, dass Cholesterol in verschiedenen Formen, als Low-Density-Lipoprotein (LDL), als 25-Hydroxycholesterol, und als Methylcyclodextrin-Komplex in unterschiedlichen humanen Zelltypen die Selenoproteinsynthese ebenfalls unterdrücken. Der negative Zusammenhang zwischen Cholesterol und bestimmten Selenoproteinen konnte auch in vivo beobachtet werden. In juvenilen Mäusen konnte gezeigt werden, dass ein Knockout des LDL-Rezeptors sowie auch ein Knockout von Apolipoprotein E zu einer Senkung des Lebercholesterols führte, was in einer Zunahme der GPx in der Leber resultierte.rnDie vorliegenden Daten belegen erstmals einen direkten und funktionellen Zusammenhang zwischen dem Mevalonatpfad und der Selenoproteinsynthese. Unterdrückung dieses Pfades, entweder durch exogene Substanzen wie Statine, oder durch endogene Substanzen wie Cholesterol, hat offenbar zur Folge, dass essentielle Zwischenprodukte für die Modifizierung der Selenocystein-tRNA fehlen, was in einer post-transkriptionellen Verminderung der induzierbaren Selenoproteine resultiert. Dies könnte die biochemische Grundlage für einen Teil der vielfältigen gesundheitlich negativen Auswirkungen schon geringfügig erhöhter Cholesterolspiegel sein.

Characterization of the Caspr2 and NLGN2 ligands: a proteomic and biochemical approach

Autism Spectrum Disorder (ASD) is a range of early-onset conditions classified as neurodevelopmental disorders, characterized by deficits in social interactions and communication, as well as by restricted interest and repetitive behaviors. Among the proteins associated with this spectrum of disease there are Caspr2, α-NRXN1, NLGN1-4. Caspr2 is involved in the clustering of K+ channels at the juxtaparanodes, where it is proposed to bind TAG-1. Recent works reported a synaptic localization of Caspr2, but little is know on its role in this compartment. NRXNs and their ligand NLGNs, instead, have a well-defined role in the formation and maintenance of synapses. Among the neuroligins, NLGN2 binds NRXNs with the lowest affinity, suggesting that it could have other not yet characterized ligands. The aim of this work was to better characterize the binding of Caspr2 to TAG-1 and to identify new potential binding partner for Caspr2 and NLGN2. Unexpectedly, using Isothermal Titration Calorimetry and co-immunoprecipitation experiments the direct association of the first two proteins could not be verified and the results indicate that the first evidences reporting it were biased by false-positive artifacts. These findings, together with the uncharacterized synaptic localization of Caspr2, made the identification of new potential binding partners for this protein necessary. To find new proteins that associate with Caspr2 and NLGN2, affinity chromatography in tandem with mass spectrometry experiments were performed. Interestingly, about 25 new potential partners were found for these two proteins and NLGN1, that was originally included as a control: 5 of those, namely SFRP1, CLU, APOE, CNTN1 and TNR, were selected for further investigations. Only the association of CLU to NLGN2 was confirmed. In the future, screenings of the remaining candidates have to be carried out and the functional role for the proposed NLGN2-CLU complex has to be studied.

Rheumatoide Arthritis und Arteriosklerose : Bedeutung der zu Grunde liegenden chronisch entzündlichen Prozesse für die endotheliale Dysfunktion ; Wirkung der antiinflammatorischen Substanz Galiellalacton in murinen Arteriosklerosemodellen

Erhöhte arteriosklerotische und thrombotische Vorfälle sind ein Hauptgrund für die gesteigerten Zahlen kardiovaskulärer Todesfälle von Patienten mit chronisch entzündlichen Erkrankungen wie der rheumatoiden Arthritis (RA). Diese erhöhte Mortalität ist nicht auf die traditionellen Risikofaktoren, wie Alter, Geschlecht, Bluthochdruck oder Diabetes zurückzuführen. Man nimmt an, dass die systemische Entzündung einen nicht-traditionellen Risikofaktor für die erhöhten kardiovaskulären Todesfälle von RA-Patienten darstellt. Da die derzeitige Behandlung der RA zum Teil schwere Nebenwirkungen zur Folge haben kann, war es das Ziel dieser Doktorarbeit, die Zusammenhänge zwischen RA und Arteriosklerose (AS) näher zu untersuchen, sowie die neue antiinflammatorische Substanz Galiellalacton (Gal) für die Behandlung der AS zu charakterisieren.rnIn dem chronisch inflammatorischen Tiermodell der TTP-defizienten Mäuse, dessenrnPhänotyp dem einer humanen RA-Erkrankung ähnelt, konnte eine verschlechterternEndothelfunktion, die als ein erstes Symptom einer erworbenen AS gilt, nachgewiesen werden. Dies konnte auf eine erhöhte Stabilität der Nox2-mRNA zurückgeführt werden, die unabhängig von der erhöhten Expression des Entzündungsmarkers TNFα war. Diese gesteigerte Nox2-Menge führte wiederum zu einer erhöhten Bildung von reaktiven Sauerstoff- und Stickstoffspezies und somit zu einer verringerten Menge an bioaktivem Stickstoffmonoxid, welches die endotheliale Dysfunktion (eDF) bedingte.rnAls ein traditioneller Risikofaktor für das Auftreten von kardiovaskulären Ereignissen gilt unter anderem eine Diabeteserkrankung. Durch die Ausbildung einer Nitrattoleranz bei der Therapie mit organischen Nitraten wie NTG, ISMN oder ISDN kommt es zu der Entwicklung einer eDF. PETN, ein weiteres organisches Nitrat zeigt diese Nebenwirkung nicht. PETN, vermittelt seinen antioxidativen Effekt über die Nrf2-abhängige Induktion der HO-1-Promotoraktivität.rnDie Behandlung von arteriosklerotischen Mäusen (ApoE-/-- und ApoE-/-TFPI+/--Mäuse) mit dem antiinflammatorischen Pilzsekundärmetaboliten Gal zeigte eine verringerte mRNA-Expression von arteriosklerotischen und inflammatorischen Mediatoren, sowie eine reduzierte Thrombenbildung durch eine verringerte Plättchenadhäsion.rnZusammenfassend konnte gezeigt werden, dass inflammationsabhängiger oxidativerrnStress ein Hauptgrund für die entzündungsgetriebene Artheriogenese ist und Galrneine neue Leitsubstanz für die Behandlung dieser Erkrankung ist.

Type 2 diabetes is associated with reduced ATP-binding cassette transporter A1 gene expression, protein and function

Objective Increasing plasma glucose levels are associated with increasing risk of vascular disease. We tested the hypothesis that there is a glycaemia-mediated impairment of reverse cholesterol transport (RCT). We studied the influence of plasma glucose on expression and function of a key mediator in RCT, the ATP binding cassette transporter-A1 (ABCA1) and expression of its regulators, liver X receptor-α (LXRα) and peroxisome proliferator-activated receptor–γ (PPARγ). Methods and Results Leukocyte ABCA1, LXRα and PPARγ expression was measured by polymerase chain reaction in 63 men with varying degrees of glucose homeostasis. ABCA1 protein concentrations were measured in leukocytes. In a sub-group of 25 men, ABCA1 function was quantified as apolipoprotein-A1-mediated cholesterol efflux from 2–3 week cultured skin fibroblasts. Leukocyte ABCA1 expression correlated negatively with circulating HbA1c and glucose (rho = −0.41, p<0.001; rho = −0.34, p = 0.006 respectively) and was reduced in Type 2 diabetes (T2DM) (p = 0.03). Leukocyte ABCA1 protein was lower in T2DM (p = 0.03) and positively associated with plasma HDL cholesterol (HDL-C) (rho = 0.34, p = 0.02). Apolipoprotein-A1-mediated cholesterol efflux correlated negatively with fasting glucose (rho = −0.50, p = 0.01) and positively with HDL-C (rho = 0.41, p = 0.02). It was reduced in T2DM compared with controls (p = 0.04). These relationships were independent of LXRα and PPARγ expression. Conclusions ABCA1 expression and protein concentrations in leukocytes, as well as function in cultured skin fibroblasts, are reduced in T2DM. ABCA1 protein concentration and function are associated with HDL-C levels. These findings indicate a glycaemia- related, persistent disruption of a key component of RCT.

Role of retroviral restriction factors in the interferon-α-mediated suppression of HIV-1 in vivo

The antiviral potency of the cytokine IFN-α has been long appreciated but remains poorly understood. A number of studies have suggested that induction of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 (APOBEC3) and bone marrow stromal cell antigen 2 (BST-2/tetherin/CD317) retroviral restriction factors underlies the IFN-α-mediated suppression of HIV-1 replication in vitro. We sought to characterize the as-yet-undefined relationship between IFN-α treatment, retroviral restriction factors, and HIV-1 in vivo. APOBEC3G, APOBEC3F, and BST-2 expression levels were measured in HIV/hepatitis C virus (HCV)-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated IFN-α/ribavirin (IFN-α/riba) combination therapy. IFN-α/riba therapy decreased HIV-1 viral load by -0.921 (±0.858) log(10) copies/mL in HIV/HCV-coinfected patients. APOBEC3G/3F and BST-2 mRNA expression was significantly elevated during IFN-α/riba treatment in patient-derived CD4+ T cells (P < 0.04 and P < 0.008, paired Wilcoxon), and extent of BST-2 induction was correlated with reduction in HIV-1 viral load during treatment (P < 0.05, Pearson's r). APOBEC3 induction during treatment was correlated with degree of viral hypermutation (P < 0.03, Spearman's ρ), and evolution of the HIV-1 accessory protein viral protein U (Vpu) during IFN-α/riba treatment was suggestive of increased BST-2-mediated selection pressure. These data suggest that host restriction factors play a critical role in the antiretroviral capacity of IFN-α in vivo, and warrant investigation into therapeutic strategies that specifically enhance the expression of these intrinsic immune factors in HIV-1-infected individuals.

A proteome signature for intrauterine growth restriction derived from multifactorial analysis of mass spectrometry-based cord blood serum profiling

Intrauterine growth restriction (IUGR) is defined as a condition in which the fetus does not reach its genetically given growth potential, resulting in low birth weight. IUGR is an important cause of perinatal morbidity and mortality, thus contributing substantially to medically indicated preterm birth in order to prevent fetal death. We subjected umbilical cord blood serum samples either belonging to the IUGR group (n = 15) or to the control group (n = 15) to fractionation by affinity chromatography using a bead system with hydrophobic interaction capabilities. So prepared protein mixtures were analyzed by MALDI-TOF mass spectrometric profiling. The six best differentiating ion signals at m/z 8205, m/z 8766, m/z 13 945, m/z 15 129, m/z 15 308, and m/z 16 001 were collectively assigned as IUGR proteome signature. Separation confidence of our IUGR proteome signature reached a sensitivity of 0.87 and a specificity of 0.93. Assignment of ion signals in the mass spectra to specific proteins was substantiated by SDS-PAGE in conjunction with peptide mass fingerprint analysis of cord blood serum proteins. One constituent of this proteome signature, apolipoprotein C-III(0) , a derivative lacking glycosylation, has been found more abundant in the IUGR cord blood serum samples, irrespective of gestational age. Hence, we suggest apolipoprotein C-III(0) as potential key-marker of the here proposed IUGR proteome signature, as it is a very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) member and as such involved in triglyceride metabolism that itself is discussed as being of importance in IUGR pathogenesis. Our results indicate that subtle alterations in protein glycosylation need to be considered for improving our understanding of the pathomechanisms in IUGR.

The cytidine deaminases AID and APOBEC-1 exhibit distinct functional properties in a novel yeast selectable system

Activation-induced cytidine deaminase (AID) is indispensable for immunoglobulin maturation by somatic hypermutations and class switch recombination and is supposed to deaminate cytidines in DNA, while its homolog APOBEC-1 edits apolipoprotein (apo) B mRNA by cytidine deamination. We studied the editing activity of APOBEC-1 and AID in yeast using the selectable marker Gal4 linked to its specific inhibitor protein Gal80 via an apo B cassette (Gal4-C) or via the variable region of a mouse immunoglobulin heavy chain gene (Gal4-VH). Expression of APOBEC-1 induced C to U editing in up to 15% of the Gal4-C transcripts, while AID was inactive in this reaction even in the presence of the APOBEC-1 complementation factor. After expression of APOBEC-1 as well as AID approximately 10(-3) of yeast cells survived low stringency selection and expressed beta-galactosidase. Neither AID nor APOBEC-1 mutated the VH sequence of Gal4-VH, and consequently the yeast colonies did not escape high stringent selection. AID, however, induced frequent plasmid recombinations that were only rarely observed with APOBEC-1. In conclusion, AID cannot substitute APOBEC-1 to edit the apo B mRNA, and the expression of AID in yeast is not sufficient for the generation of point mutations in a highly transcribed Gal4-VH sequence. Cofactors for AID induced somatic hypermutations of immunoglobulin variable regions, that are present in B cells and a variety of non-B cells, appear to be missing in yeast. In contrast to APOBEC-1, AID alone does not exhibit an intrinsic specificity for its target sequences.

MRI follow-up of TNF-dependent differential progression of atherosclerotic wall-thickening in mouse aortic arch from early to advanced stages

OBJECTIVES: An optimized, longitudinal in vivo magnetic resonance vessel wall-imaging protocol was evaluated regarding its capability of detecting differences in the time-dependent atherosclerotic lesion progression in the aortic arch between ApoE(-/-) and double-deficient ApoE(-/-)/TNF(-/-) mice at comparatively early plaque development stages. MATERIALS AND METHODS: Seven ApoE(-/-) and seven ApoE(-/-)/TNF(-/-) female mice underwent MRI at 11.75 teslas at four stages up to 26 weeks of age. A double-gated spin-echo MRI sequence was used with careful perpendicular slice positioning to visualize the vessel wall of the ascending aortic arch. RESULTS: Wall-thickness progression measured with MRI was significant at 11 weeks of age in ApoE(-/-) mice, but only at 26 weeks in ApoE(-/-)/TNF(-/-) mice. A significant correlation was found between MRI wall-thickness and lesion area determined on histology. CONCLUSION: MRI was shown to be sensitive enough to reveal subtle genetically-induced differences in lesion progression at ages earlier than 25 weeks.

Contribution of 20 single nucleotide polymorphisms of 13 genes to dyslipidemia associated with antiretroviral therapy

BACKGROUND: HIV-1 infected individuals have an increased cardiovascular risk which is partially mediated by dyslipidemia. Single nucleotide polymorphisms in multiple genes involved in lipid transport and metabolism are presumed to modulate the risk of dyslipidemia in response to antiretroviral therapy. METHODS: The contribution to dyslipidemia of 20 selected single nucleotide polymorphisms of 13 genes reported in the literature to be associated with plasma lipid levels (ABCA1, ADRB2, APOA5, APOC3, APOE, CETP, LIPC, LIPG, LPL, MDR1, MTP, SCARB1, and TNF) was assessed by longitudinally modeling more than 4400 plasma lipid determinations in 438 antiretroviral therapy-treated participants during a median period of 4.8 years. An exploratory genetic score was tested that takes into account the cumulative contribution of multiple gene variants to plasma lipids. RESULTS: Variants of ABCA1, APOA5, APOC3, APOE, and CETP contributed to plasma triglyceride levels, particularly in the setting of ritonavir-containing antiretroviral therapy. Variants of APOA5 and CETP contributed to high-density lipoprotein-cholesterol levels. Variants of CETP and LIPG contributed to non-high-density lipoprotein-cholesterol levels, a finding not reported previously. Sustained hypertriglyceridemia and low high-density lipoprotein-cholesterol during the study period was significantly associated with the genetic score. CONCLUSIONS: Single nucleotide polymorphisms of ABCA1, APOA5, APOC3, APOE, and CETP contribute to plasma triglyceride and high-density lipoprotein-cholesterol levels during antiretroviral therapy exposure. Genetic profiling may contribute to the identification of patients at risk for antiretroviral therapy-related dyslipidemia.

Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer

G protein-coupled receptors (GPCRs) are seven transmembrane domain proteins that transduce extracellular signals across the plasma membrane and couple to the heterotrimeric family of G proteins. Like most intrinsic membrane proteins, GPCRs are capable of oligomerization, the function of which has only been established for a few different receptor systems. One challenge in understanding the function of oligomers relates to the inability to separate monomeric and oligomeric receptor complexes in membrane environments. Here we report the reconstitution of bovine rhodopsin, a GPCR expressed in the retina, into an apolipoprotein A-I phospholipid particle, derived from high density lipoprotein (HDL). We demonstrate that rhodopsin, when incorporated into these 10 nm reconstituted HDL (rHDL) particles, is monomeric and functional. Rhodopsin.rHDL maintains the appropriate spectral properties with respect to photoactivation and formation of the active form, metarhodopsin II. Additionally, the kinetics of metarhodopsin II decay is similar between rhodopsin in native membranes and rhodopsin in rHDL particles. Photoactivation of monomeric rhodopsin.rHDL also results in the rapid activation of transducin, at a rate that is comparable with that found in native rod outer segments and 20-fold faster than rhodopsin in detergent micelles. These data suggest that monomeric rhodopsin is the minimal functional unit in G protein activation and that oligomerization is not absolutely required for this process.

Olfactory bulb interneurons releasing NO exhibit the Reelin receptor ApoEr2 and part of those targeted by NO express Reelin

Nitric oxide (NO) and Reelin both modulate neuronal plasticity in developing and mature synaptic networks. We recently showed a loss of neuronal nitric oxide synthase (nNOS) protein in the olfactory bulb of reeler mutants and advanced the hypothesis that the Reelin and NO signalling pathways may influence each other. We now studied the distribution of NO sensitive guanylyl cyclase (NOsGC), Reelin and its receptor Apolipoprotein E2 (ApoEr2) in the olfactory bulb by multiple fluorescence labelling and tested whether nNOS and ApoEr2 colocalize in this area. We also essayed the protein content of NOsGC in the reeler olfactory bulb and tested whether there are any changes in nNOS and NOsGC protein in other reeler brain areas. Olfactory bulb interneurons expressing ApoEr2 and nNOS are only few in the glomerular layer but represent the large majority of granule cell layer interneurons. Conversely, NOsGC interneurons are rare in the granule cell layer and abundant as periglomerular cells. Reelin containing periglomerular cells almost entirely belong to the NOsGC subset. These data further support the hypothesis of a reciprocal signalling between Reelin/NOsGC and ApoEr2/nNOS expressing neurons to affect olfactory bulb activity. We also show that a significant rise in NOsGC content accompanies the decrease of nNOS protein in the reeler olfactory bulb. The same reciprocal changes present in the cortex/striatum and the hippocampus of reeler mice. Thus, the influence that the deficit of extracellular Reelin seems to exert on nNOS and its receptor is not limited to the olfactory bulb but is a general feature of the reeler brain.

Microsomal triglyceride transfer protein polymorphism (-493G/T) is associated with hepatic steatosis in patients with chronic hepatitis C

BACKGROUND: Hepatic steatosis may promote progression of chronic hepatitis C (CHC). Microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of ApoB lipoprotein and is implicated in hepatitis C virus (HCV)-related steatosis. The MTP -493G/T polymorphism may promote liver fat accumulation, but its role in HCV-related steatosis is still unclear. METHODS: Two hundred ninety-eight CHC patients were studied and genotyped for MTP -493G/T variants. Hepatic MTP mRNA expression and activity were determined in a subgroup. RESULTS: Patients with grades 2/3 steatosis were older, had a higher body mass index (BMI), more advanced fibrosis and lower MTP mRNA expression and carried more often HCV genotype 3 and the MTP T allele. Age, BMI, HCV-3 and MTP T allele [odds ratio (OR) 2.05; 95% confidence interval (CI) 1.2-3.53; P=0.009] were independent risk factors for steatosis grades 2/3, and in HCV genotype non-3 patients, the MTP T allele was the strongest predictor for steatosis grade 2/3 (OR 2.17; 95% CI 1.22-3.86; P=0.008). Moreover, TT carriers had higher high-density lipoprotein (65.6+/-14.6 vs 56.1+/-16.2 mg/dl; P=0.003) and apolipoprotein AI (1.80+/-0.3 vs 1.60+/-0.3 g/L; P=0.005) levels than G allele carriers. CONCLUSIONS: Chronic hepatitis C patients with the MTP -493T allele reveal higher grades of steatosis, indicating a relevant contribution to liver fat accumulation, particularly in HCV non-3 patients.