1,721,005 research outputs found
Supplementation of anti-oxidants in leucofiltered erythrocyte concentrates: Assessment of morphological changes through scanning electron microscopy
No full textThe cardioprotective effect of sildenafil is mediated by the activation of malate dehydrogenase and an increase in the malate-aspartate shuttle in cardiomyocytes
No full textRecent evidence has shown the cardioprotective effect of PDE5 inhibition in myocardial ischemia/reperfusion injury, heart failure and cardiac hypertrophy. To investigate the biochemical changes that occur during PDE5 inhibition in cardiac cells, this study assessed the metabolic profile of the HL1 cell line, a murine atrial cell line with adult cardiomyocyte properties. After one hour of treatment with sildenafil, glycolysis was moderately but selectively stimulated, unlike the pentose phosphate pathway and the Krebs cycle. Moreover, malate and a-Ketoglutarate accumulated, paralleled by a decrease in aspartate and glutamate. Interestingly, increased activity of malate dehydrogenase (MDH) was also detected in these cells after sildenafil treatment. Thus, we hypothesized that sildenafil stimulates the malate-aspartate shuttle (MAS) with the final effect of transferring electrons and protons from glycolysis-derived cytosolic NADH into the matrix for use by the electron transport chain, using malate as an electron carrier. Through this metabolic modification, sildenafil may counteract what is often observed in ischemia, i.e. reduced MAS flux as well as a dramatic acceleration of glycolysis, which switches to lactate production. Additionally, the results observed in HL1 cells were also found in isolated mouse hearts. The documented metabolic alteration in cardiomyocytes upon treatment with sildenafil occurred by stimulating cGMP production, which did not activate PKG (cGMP-PKG signaling), since the addition of DT-2, a PKG inhibitor, did not block malate accumulation and increased MDH activity. Conversely, the addition of chelerythrine, a PKC inhibitor, counteracted both malate accumulation and MAS activation, supporting previous evidence that, upon the addition of sildenafil, some PKC isoforms may be implicated in cardioprotection (cGMP-PKC signaling). Interestingly, an increase in cGMP, driven by sildenafil, another cGMP stimulator such as nitroprusside (SNP), or a C-type natriuretic peptide (CNP) which does not inhibit PDE5, led to MAS stimulation and increased MDH activity
Effects of arg8-vasopressin on developing skeletal muscle
No full textArg8-vasopressin (AVP), a nonapeptide produced by neuroendocrine cells of the hypothalamus and released into the blood stream at the level of the·neurohypophysis, exerts i!s multiple physiological actions through two types of receptors: the V1 receptor (liver, smooth muscle cells) coupled to phosphoinositidase c, and the V 2 receptor (kidney), coupled to adenylate cydase. Very little information is available about biologic::al actions of AVP on skeletal muscle. We have recently reported that AVP induces significant responses in skeletal myogenic cells in culture, consisting in stimulation of phosp4oinositide (PI) turnover, with rapid (3-5 s") production of inositol 1•4-5 trisphosphate, and modifications of intracellular calcium concentration and pH. The response to AVP seems to be regulated by protein kinase C (PKC), a key enzyme in myogenic differentiation, as shown by the modifications induced by TPA, and their reversion by the PKC inhibitor staurosporine
Changes in morphology, cell wall composition and soluble proteome in Rhodobacter sphaeroides cells exposed to chromate
No full textThe response of the carotenoidless Rhodobacter sphaeroides mutant R26 to chromate stress under photosynthetic conditions is investigated by biochemical and spectroscopic measurements, proteomic analysis and cell imaging. Cell cultures were found able to reduce chromate within 3-4 days. Chromate induces marked changes in the cellular dimension and morphology, as revealed by atomic force microscopy, along with compositional changes in the cell wall revealed by infrared spectroscopy. These effects are accompanied by significant changes in the level of several proteins: 15 proteins were found up-regulated and 15 down-regulated. The protein content found in chromate exposed cells is in good agreement with the biochemical, spectroscopic and microscopic results. Moreover at the present stage no specific chromate-reductase could be found in the soluble proteome, indicating that detoxification of the pollutant proceeds via aspecific reductants
Additional file 3: Figure S1. of Urinary metabolomics of young Italian autistic children supports abnormal tryptophan and purine metabolism
Full text linkAccurate mass and MS/MS fragmentation data for (a) kynurenine, (b) melatonin, and (c) tryptophan. (TIF 191 kb
CDK1 phosphorylates WRN at collapsed replication forks
No full textRegulation of end-processing is critical for accurate repair and to switch between homologous recombination (HR) and non-homologous end joining (NHEJ). End resection is a two-stage process but very little is known about regulation of the long-range resection, especially in humans. WRN participates in one of the two alternative long-range resection pathways mediated by DNA2 or EXO1. Here we demonstrate that phosphorylation of WRN by CDK1 is essential to perform DNA2-dependent end resection at replication-related DSBs, promoting HR, replication recovery and chromosome stability. Mechanistically, S1133 phosphorylation of WRN is dispensable for relocalization in foci but is involved in the interaction with the MRE11 complex. Loss of WRN phosphorylation negatively affects MRE11 foci formation and acts in a dominant negative manner to prevent long-range resection altogether, thereby licensing NHEJ at collapsed forks. Collectively, we unveil a CDK1-dependent regulation of the WRN-DNA2-mediated resection and identify an undescribed function of WRN as a DSB repair pathway switch
Different methods for two-dimensional electrophoresis of membrane proteins
No full textDottorato di ricerca in Genetica e biologia cellulareIl progredire delle metodiche scientifiche e delle tecnologie nelle ultime decadi, ha
catalizzato un’estensione degli scopi connessi agli studi in campo biologico
spostando l’attenzione dalle riduttive analisi biochimiche a carico delle singole
proteine ad un esame attento ed approfondito esteso all’intero proteoma. La
proteomica nasce come naturale evoluzione della chimica delle proteine che
rappresentava uno dei capisaldi nello studio della biologia durante gli anni ottanta.
L’analisi proteomica implica la necessità di separare le proteine prima della loro
caratterizzazione. Così tutta la qualità dell’intera analisi molto dipende dalle
performance dei metodi di separazione adottati. Questa lavoro di tesi nasce dal
condensamento di diverse pubblicazioni e vuole proporsi come un saggio delle
diverse metodiche di elettroforesi bidimensionale applicabili allo studio di proteine
di membrana. Utilizzando differenti tecniche elettroforetiche bidimensionali e di
rivelazione sono state affrontate due tematiche di ricerca diverse su differenti
sistemi biologici: nel capitolo 2 con una proteomica differenziale di tipo “classico”
(2D IEF-SDS-PAGE) è stata studiata le degradazione delle proteine della membrana
eritrocitaria durante la conservazione del sangue ad uso trasfusionale, nel capitolo 3
invece, prendendo come modello le proteine delle membrane fotosintetiche
tilacoidali, è stato proposto un nuovo sistema elettroforetico bidimensionale nativo,
sia in prima che in seconda dimensione (2D N-LP-IEF-SDS-PAGE), accoppiabile
con una terza dimensione denaturante.Developments in methods and technologies have resulted in an expansion of the
purpose of biological studies from the reductionist biochemical analysis of single
proteins to proteome-wide measurements. Proteomics and other complementary
analysis methods are essential components of the emerging 'systems biology'.
Proteome analysis implies the ability to separate proteins as a first step prior the
characterization. Thus, the overall performance of the analysis strongly depends on
the performance of the separation tool, usually two dimensional electrophoresis.
This thesis gives an introduction to the proteomic technique and shows how twodimensional
electrophoresis works with membrane proteins. Different subjects and
biological systems were investigated using different separative two-dimensional
electrophoresis techniques. In chapter one, two-dimensional gel electrophoresis (2D
IEF-SDS-PAGE) and mass spectrometry were used to identify protein profile
changes in red blood cell membranes stored over time under atmospheric oxygen, in
the presence or absence of protease inhibitors. In chapter two, a new 3D native
electrophoretic protocol is proposed for an exhaustive separation and identification
of membrane proteins. This method is based on native liquid phase
isoelectrofocusing (N-LP-IEF) of protein complexes in the first dimension, followed
by blue native polyacrylamide gel electrophoresis (BN-PAGE) in the second
dimension, where both the pI and the molecular masses of protein complexes (2D
N-LP-IEF-BN) were used to separate them in their native form
Proteomic analysis of 4-hydroxynonenal and nitrotyrosine modified proteins in RTT fibroblasts
No full textRett syndrome (RTT) is a pervasive developmental disorder, primarily affecting girls with a prevalence of 1 in every 10,000 births. A clear etiological factor present in more than 90% of classical RTT cases is the mutation of the gene encoding methyl-CpG-binding protein 2 (MECP2). Recent work from our group was able to shown a systemic oxidative stress (OxS) in these patients that correlates with the gravity of the clinical features. Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have performed a two-dimensional gel electrophoresis in order to evidence the oxidative modifications of proteins with special focus on the formation of protein adducts with 4-hydroxynonenal (4-HNE PAs)—a major secondary product of lipid peroxidation— and Nitrotyrosine, a marker derived from the biochemical interaction of nitric oxide (NO) or nitric oxide-derived secondary products with reactive oxygen species (ROS). Then, oxidatively modified spots were identified by mass spectrometry, LC-ESI-CID-MS/MS. Our results showed that 15 protein spots presented 4-HNE PAs and/or nitrotyrosine adducts in fibroblasts proteome from RTT patients compared to healthy control cells. Post-translationally modified proteins were related to several functional categories, in particular to cytoskeleton structure and protein folding. In addition, clear upregulated expression of the inducible NO synthase (iNOS) with high nitrite levels were observed in RTT fibroblasts, justifying the increased nitrotyrosine protein modifications. The present work describes not only the proteomic profile in RTT fibroblasts, but also identifies the modified proteins by 4-HNE and nitrotyrosine. Of note, for the first time, it appears that a dysregulation of NO pathway can be associated to RTT pathophysiology. In conclusion, the evidence of a wide range of proteins able to forms adducts with 4-HNE, Nitrotyrosine or with both confirms the possible alteration of several aspects of cellular functions that well correlates to the complex clinical features of RTT patients
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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