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Adult and neonatal immunocompetent cells exhibit important functional distinctions, including differences in cytokine production and susceptibility to tolerance induction. We have investigated the molecular features that characterize the... more
Adult and neonatal immunocompetent cells exhibit important functional distinctions, including differences in cytokine production and susceptibility to tolerance induction. We have investigated the molecular features that characterize the immune response of cord blood-derived T lymphocytes compared with that of adult T lymphocytes. Our findings demonstrate that phospholipase C (PLC) isozymes, which play a pivotal role in the control of protein kinase C activation and Ca2+ mobilization, are differently expressed in cord and adult T lymphocytes. PLCbeta1 and delta1 are expressed at higher levels in cord T cells, while PLCbeta2 and gamma1 expression is higher in adult T lymphocytes. PLCdelta2 and gamma2 appear to be equally expressed in both cell types. In addition, a functional defect in PLC activation via CD3 ligation or pervanadate treatment, stimuli that activate tyrosine kinases, was observed in cord blood T cells, whereas treatment with aluminum tetrafluoride (AlF4-), a G protein ...
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Molt-4 human leukemia cells were triggered to undergo apoptosis by various agents with different mechanisms of action. Staurosporine [a protein kinase C (PKC) inhibitor], camptothecin (a topoisomerase I blocking drug), and tiazofurin [an... more
Molt-4 human leukemia cells were triggered to undergo apoptosis by various agents with different mechanisms of action. Staurosporine [a protein kinase C (PKC) inhibitor], camptothecin (a topoisomerase I blocking drug), and tiazofurin [an inhibitor of inosine 5'-phosphate dehydrogenase (IMPDH)], were used. Ultrastructural analysis showed morphological changes characteristic for apoptosis that were very similar for all three agents. Nevertheless, DNA oligonucleosomic fragmentation was not detectable by agarose gel electrophoresis. However, a genomic DNA cleavage appeared after pulse-field gel electrophoresis (PFGE) in cells treated with these agents for 24 h. Furthermore, in situ nick translation (NT) showed a finely spotted nuclear labelling in all staurosporine-treated cells and a compact fluorescence after camptothecin incubation. In tiazofurin-treated cells an intermediate pattern was found. Therefore, apoptotis inducing agents with different mechanisms of action, induced the ...
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The data on the effects of aerobic training on plasma antioxidant vitamins are conflicting. Additionally, most studies focus on the oxidative profiles of professional athletes, but limited information is available for amateur athlete... more
The data on the effects of aerobic training on plasma antioxidant vitamins are conflicting. Additionally, most studies focus on the oxidative profiles of professional athletes, but limited information is available for amateur athlete populations. The aim of this study was to evaluate the effects of high-intensity exercise on antioxidant vitamins in non-professional runners with varying levels of aerobic power. Eighty-one male runners underwent an incremental test to exhaustion. The study population was then divided into the following tertiles according to VO2max: Group L (LowVO2max, less than 44.2 mLkg-1min-1), Group M (MediumVO2max, 44.2-49.7 mLkg-1min-1) and Group H (HighVO2max, >49.7). Comparative analyses were performed between Groups L and H. The total antioxidant capacity (TAC), Vitamin (Vit) E, Vitamin A, β-carotene, lycopene and thiobarbituric acid-reactive substances (TBARS) were determined before and 60 min after exercise testing. After the stress test, Vit A decreased ...
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Introduction: With menopause women face many changes that may lead to the loss of health related fitness, especially if sedentary. In particular, the estrogens deficiency affects the endothelial function thus increasing the incidence of... more
Introduction: With menopause women face many changes that may lead to the loss of health related fitness, especially if sedentary. In particular, the estrogens deficiency affects the endothelial function thus increasing the incidence of the cardiovascular diseases. The cardio-protective effects of physical exercise is at least partially due to its ability to improve the health of arterial walls by influencing the endothelial function. Nonetheless, a direct angiogenetic of the physical exercise cannot be ruled out. VEGF is an important modulator of vascular growth but there are contrasting results about its response to physical exercise. Aim of our study was to compare the effects of two aerobic training on the VEGF levels and on the angiogenesis in postmenopausal women. Material and Methods. 34 Postmenopausal women underwent a 13 weeks training. In order to analyse the angiogentic effects, plasmatic VEGF levels were analysed before (T0) and after the training (T1). Moreover, the abi...
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The characterization of Amniotic Fluid-derived multipotent Stem Cells (AFSCs) open new paths in stem cell research. hAFSCs have characteristics intermediate between pluripotent embryonic- (ESCs) and lineage-restricted adult stem cells,... more
The characterization of Amniotic Fluid-derived multipotent Stem Cells (AFSCs) open new paths in stem cell research. hAFSCs have characteristics intermediate between pluripotent embryonic- (ESCs) and lineage-restricted adult stem cells, and are non-tumorigenic and low immunogenic. Moreover, they are obtained without destroying human embryos, so that most of the ethical and social controversy could be prevented. We previously observed that human AFSCs express some genes specific of ESCs and primordial germ cells. We also shown hAFSCs ability to form in vitro three-dimensional aggregates of cells known as embryoid bodies (EBs), that express three germ layer markers. Recent studies reported the ability of hAFSCs to differentiate in vitro into adipocytes and osteocytes. Aim of our study was to analyse the cardiomyogenic potential of hAFSCs. EBs were obtained by modified hanging drops protocol from hAFSCs coltured in presence of ascorbic acid and 5-aza-2’-deoxycytidine (differentiation me...
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Ghrelin (Ghr) is a 28-amino acid peptide identified as endogenous, acylated ligand for the growth hormone (GH) secretagogue G-protein-coupled receptor (GHSR). Mainly synthesized from X/A like neuroendocrin cells of gastric fundus, Ghr... more
Ghrelin (Ghr) is a 28-amino acid peptide identified as endogenous, acylated ligand for the growth hormone (GH) secretagogue G-protein-coupled receptor (GHSR). Mainly synthesized from X/A like neuroendocrin cells of gastric fundus, Ghr acts directly on the pituitary gland inducing GH release; moreover Ghr regulates food intake resulting tightly associated with obesity. Several studies reported that Ghr is widely expressed in different tissues and, besides its orexigenic activity, effects on cardiovascular, pulmonary, reproductive and central nervous systems have been described. Recently, functional studies on rats and rabbits indicated Ghr as modulator of iris smooth muscles activity since induces relaxation of both sphincter and dilator muscles. Moreover Ghr mRNA has been found in the ciliary epithelium of ciliar body (CB). On the basis of these observations, we purposed to investigate Ghr and GHSR expression in human eye. The immunohistochemical analysis performed on iris and cilia...
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Despite the demonstrated exercise-induced increase in reactive oxygen species (ROS) production, growing epidemiological evidence indicates that habitual, moderate physical activity reduces the incidence of several oxidative stress-based... more
Despite the demonstrated exercise-induced increase in reactive oxygen species (ROS) production, growing epidemiological evidence indicates that habitual, moderate physical activity reduces the incidence of several oxidative stress-based diseases. This apparent paradox can be explained taking into account that ROS produced during repeated exercise bouts may act as mild stressors able to trigger physiological and biomolecular hormetic responses through a number of redox-sensitive transcription pathways. Unfortunately, much more limited information is available from general population-based research, which could better reflect the condition of common people interested in achieving and maintaining good fitness levels. The present work aimed at investigating whether and how exercise-related habits in non-professional regular runners (n=33) can affect the systemic anti-oxidative capacity, and the resting serum levels of typical lipid peroxidation-related by-products and oxidativelydamaged...
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PKCs are serine-threonine kinases that play an important role in many cellular functions. Almost all the 12 PKC isoforms described so far are expressed throughout erythroid differentiation of human CD34pos cells. Besides the... more
PKCs are serine-threonine kinases that play an important role in many cellular functions. Almost all the 12 PKC isoforms described so far are expressed throughout erythroid differentiation of human CD34pos cells. Besides the downmodulation of PKCε expression (Bassini et al, Blood93; 1178, 1999), little is known on the role exerted by each PKC on erythroid differentiation. PKCα and PKCδ are two isoforms expressed at comparable levels (as mRNA and protein) throughout differentiation of CD36highCD235alow pro-erythroblasts into orthochromatic erythroblasts. For both proteins, the ratio between phosphorylated and total form remains constant during the differentiation of adult cells. In contrast, the ratio P-PKCα/total PKCα (but not that of P-PKCδ/total PKCδ) increases by 3-fold (with slight donor-to-donor variability) during the differentiation of CD36highCD235alow cells from cord blood. Furthermore, the protein becomes prominently localized in the nucleus of these cells. Since the most ...
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GATA-1 is a transcription factor involved in the intrinsic control of erythroid, megakaryocytic and mast cell differentiation. Experimentally-induced deletion of the megakaryocytic (Mk)-specific regulatory sequences of GATA-1 (Gata1tm2Sho... more
GATA-1 is a transcription factor involved in the intrinsic control of erythroid, megakaryocytic and mast cell differentiation. Experimentally-induced deletion of the megakaryocytic (Mk)-specific regulatory sequences of GATA-1 (Gata1tm2Sho or GATA-1low mutation) results in severe thrombocytopenia, because of defective thrombocytopoiesis, and myelofibrosis. The relationship, if any, between defective megakaryocytopoiesis and development of the disease is still poorly understood. Electron microscopy studies, coupled with immuno-electron microscopy, have allowed us to determine that the GATA-1low mutation blocks Mk maturation between stage I and II, resulting in accumulation of defective Mk in the tissues of GATA-1low mice. The block in maturation includes failure to properly organize α-granules, since von Willebrand factor is barely detectable in mutant Mk while P-selectin, although expressed at normal level, is found mainly associated with the Demarcation Membrane System (DMS) rather ...
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Although much is known about the intracellular phospholipase C (PLC) specific for inositol phospholipids, few data are available about the presence of a less common PLC at the external side of the membrane bilayer of some cell types. This... more
Although much is known about the intracellular phospholipase C (PLC) specific for inositol phospholipids, few data are available about the presence of a less common PLC at the external side of the membrane bilayer of some cell types. This ectoenzyme seems to play particular roles in cellular function by hydrolyzing inositol lipids located on the outer leaflet of the plasma membrane. Here, we provide the first evidence that peripheral T lymphocytes express a discrete level of a PLCγ1 at the outer leaflet of the plasma membrane. Flow cytometry showed that the PLCγ1-positive (PLCγ1+) cells (∼37%) were CD8+ and CD45RA+. Biochemical evidence indicated that (1) this ectoenzyme displays a mass similar to the cytoplasmic form, (2) it is phosphorylated on tyrosine residues, and (3) its activity is Ca2+-dependent. In addition, this enzyme appeared to be correlated with the proliferative state of the cell, since stimulation with phytohemagglutinin (PHA) downregulated both its expression and ac...
Research Interests: Molecular Biology, Kinetics, Biology, Calcium, Medicine, and 15 moreCell Division, Humans, Blood, Membrane Lipids, Phosphorylation, Phosphotyrosine, Clinical Sciences, Adult, Isoenzymes, Hydrolysis, Enzyme Induction, Cell Membrane, Inositol Phosphates, Cardiovascular medicine and haematology, and Paediatrics and reproductive medicine
Previous results demonstrated that the occurrence of death in human peripheral B lymphocytes by TNF-a was paralleled by the activation of the cytoplasmic Jak1 and Tyk2 protein kinases, along with the recruitment of transcription factors... more
Previous results demonstrated that the occurrence of death in human peripheral B lymphocytes by TNF-a was paralleled by the activation of the cytoplasmic Jak1 and Tyk2 protein kinases, along with the recruitment of transcription factors Stat3 and Stat5b. In this study we demonstrate that the balance of survival signals in the presence of TNF-α was altered by the addition of a salicylate compound, the endonuclease inhibitor aurintricarboxylic acid (ATA). Apoptosis effected by TNF-α alone was suppressed by ATA and this event was paralleled by phosphorylation and nuclear translocation of Jak2, Stat2, Stat4 and NF-kB, along with inhibition of caspase activation. These results confirm that among the different cellular responses evoked by TNF-α in human B cells, recruitment of Jak/Stat proteins and possible related gene modulation represent contributing factors and address the issue of the development of potential therapeutic strategies aimed at the control of systemic or local effects pr...
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HIT Poster session 2P479Strain concordance in a real-world setting: experience in our laboratory after equipment upgradeP4803D echocardiography is a fast-learning and reliable method for the measurements of left atrial volumesP481Echocardiographic parameters associated with long-term appropriate ...more
HIT Poster session 2P486The effect of short term aerobic exercise and ACE polymorphism on cardiovascular remodeling in healthy sedentary postmenopausal womenP487Are there predictors of malignant progression of aortic stenosis severity?P488Quantitative und semiquantitative parameters in the classi...more
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B-cell receptor (BCR)-induced activation of phospholipase C-γ1 (PLCγ1) and PLCγ2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCγ activation, the mechanism coupling PLCγ to the BCR remains... more
B-cell receptor (BCR)-induced activation of phospholipase C-γ1 (PLCγ1) and PLCγ2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCγ activation, the mechanism coupling PLCγ to the BCR remains undefined. The role of PLCγ1 SH2 and SH3 domains at different steps of BCR-induced PLCγ1 activation was examined by reconstitution in a PLCγ-negative B-cell line. PLCγ1 membrane translocation required a functional SH2 N-terminal [SH2(N)] domain, was decreased by mutation of the SH3 domain, but was unaffected by mutation of the SH2(C) domain. Tyrosine phosphorylation did not require the SH2(C) or SH3 domains but depended exclusively on a functional SH2(N) domain, which mediated the association of PLCγ1 with the adapter protein, BLNK. Forcing PLCγ1 to the membrane via a myristoylation signal did not bypass the SH2(N) domain requirement for phosphorylation, indicating that the phosphorylation mediated by this domain is not due to membrane anchoring alone....
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ABSTRACT
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Adult and neonatal immunocompetent cells exhibit important functional distinctions, including differences in cytokine production and susceptibility to tolerance induction. We have investigated the molecular features that characterize the... more
Adult and neonatal immunocompetent cells exhibit important functional distinctions, including differences in cytokine production and susceptibility to tolerance induction. We have investigated the molecular features that characterize the immune response of cord blood-derived T lymphocytes compared with that of adult T lymphocytes. Our findings demonstrate that phospholipase C (PLC) isozymes, which play a pivotal role in the control of protein kinase C activation and Ca2+ mobilization, are differently expressed in cord and adult T lymphocytes. PLCbeta1 and delta1 are expressed at higher levels in cord T cells, while PLCbeta2 and gamma1 expression is higher in adult T lymphocytes. PLCdelta2 and gamma2 appear to be equally expressed in both cell types. In addition, a functional defect in PLC activation via CD3 ligation or pervanadate treatment, stimuli that activate tyrosine kinases, was observed in cord blood T cells, whereas treatment with aluminum tetrafluoride (AlF4-), a G protein ...
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The morphological features of cell undergoing programmed cell death is well known and has been widely described in a number of experimental models with a variety of apoptotic triggering agents. Despite the similar cell behaviour,... more
The morphological features of cell undergoing programmed cell death is well known and has been widely described in a number of experimental models with a variety of apoptotic triggering agents. Despite the similar cell behaviour, underlying molecular events seem variable and only partially understood. A multiple approach appears crucial to better clarify the phenomenon. The first technique, DNA gel electrophoresis, allows the identification of fragmented DNA and has been long considered the hallmark of apoptosis. Different patterns of DNA cleavage, which can be identified by conventional or "pulsed-field gel" electrophoresis, are presented and discussed. "In situ" labelling methods are also described both with terminal deoxynucleotidyl transferase and DNA polymerase I, aimed at the study of the distribution of DNA cleavage areas. Flow cytometry is also proposed and different technical approaches, based on different laser utilizations, are discussed. Ultrastructur...
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Chromogranin A (CgA (CHGA)) is the major soluble protein co-stored and co-released with catecholamines and can function as a pro-hormone by giving rise to several bioactive peptides. This review summarizes the physiological functions, the... more
Chromogranin A (CgA (CHGA)) is the major soluble protein co-stored and co-released with catecholamines and can function as a pro-hormone by giving rise to several bioactive peptides. This review summarizes the physiological functions, the pathogenic implications, and the recent use of these molecules as biomarkers in several pathological conditions. A thorough literature review of the electronic healthcare databases MEDLINE, from January 1985 to September 2013, was conducted to identify articles and studies concerned with CgA and its processing. The search strategies utilized keywords such as chromogranin A, vasostatins 1 and 2, chromofungin, chromacin, pancreastatin, catestatin, WE14, chromostatin, GE25, parastatin, and serpinin and was supplemented by the screening of references from included papers and review articles. A total of 209 English-language, peer-reviewed original articles or reviews were examined. The analysis of the retrospective literature suggested that CgA and its ...
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Research Interests: Mechanical Engineering, Sports Medicine, Oxidative Stress, Running, Sport, and 15 moreAntioxidants, Humans, Reactive Oxygen Species, Male, Exercise, Middle Aged, Adult, Physical Exercise, Exercise Test, Oxidation-Reduction, Malondialdehyde, Blood Proteins, Anaerobic Threshold, Protein carbonylation, and ergometry
Stromal derived factor-1 alpha (SDF-1 alpha), the high-affinity ligand of CXC-chemokine receptor 4 (CXCR4), was added to human CD34(+) hematopoietic progenitor cells that can be induced to differentiate along the monocytic or... more
Stromal derived factor-1 alpha (SDF-1 alpha), the high-affinity ligand of CXC-chemokine receptor 4 (CXCR4), was added to human CD34(+) hematopoietic progenitor cells that can be induced to differentiate along the monocytic or megakaryocytic lineages. In control liquid cell cultures supplemented with two different cytokine cocktails: stem cell factor (SCF), interleukin-3 (IL-3), macrophage-colony stimulating factor (M-CSF), and 10% fetal calf serum (FCS), or, SCF and thrombopoietin (TPO), the expression of surface CXCR4 progressively increased in both the CD14(+) monocytic and CD41(+) megakaryocytic lineages. While SDF-1 alpha caused only modest effects on cells of the monocytic lineage, it induced profound down-regulation of CXCR4 in megakaryocytic cells at all stages of differentiation. Moreover, while SDF-1 alpha initially up-regulated the early megakaryocytic antigen CD41, at later time points (days 12-16) it induced down-regulation of the late megakaryocytic antigen CD42b. Consistently, at day 16, the number of mature megakaryocytes was significantly decreased in cultures supplemented with SDF-1 alpha. These findings indicate that, besides its primary role in regulating the retention of precursor cells in hematopoietic tissues, the SDF-1 alpha/CXCR4 system participates in the regulation of megakaryocytic development by stimulating the formation of immature megakaryoblasts and inhibiting the formation of mature megakaryocytes.
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Molt-4 human leukemia cells were triggered to apoptosis by various agents with different mechanisms of action. Staurosporine, a protein kinase C (PKC) inhibitor; camptothecin, a topoisomerase I blocking drug; and tiazofurin, an inhibitor... more
Molt-4 human leukemia cells were triggered to apoptosis by various agents with different mechanisms of action. Staurosporine, a protein kinase C (PKC) inhibitor; camptothecin, a topoisomerase I blocking drug; and tiazofurin, an inhibitor of inosine 5'-phosphate dehydrogenase (IMPDH), were used. Ultrastructural analysis showed morphologic changes characteristic of apoptosis that were very similar for all three agents. Nevertheless, DNA oligonucleosomic fragmentation was not detectable by agarose gel electrophoresis. However, a genomic DNA cleavage appeared after pulse-field gel electrophoresis (PFGE) in cells treated with these agents for 24 h. Furthermore, in situ nick translation (NT) showed a finely spotted nuclear labelling in staurosporine-treated cells and a compact fluorescence after camptothecin incubation. In tiazofurin-treated cells an intermediate pattern was found. Therefore, apoptotic agents with different mechanisms of action induced the formation of large genomic DNA fragments and very similar ultrastructural changes. Therefore, both phenomena and the closely related apoptosis progression depend on target cell machinery and not on the triggering agent.
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The gastrointestinal peptide hormone ghrelin (Ghr) was discovered in 1999 as the endogenous ligand for the growth hormone secretagogue receptor (GHSR-1a). It is a pleiotropic peptide that modulates a wide spectrum of biological... more
The gastrointestinal peptide hormone ghrelin (Ghr) was discovered in 1999 as the endogenous ligand for the growth hormone secretagogue receptor (GHSR-1a). It is a pleiotropic peptide that modulates a wide spectrum of biological activities, such as growth hormone (GH) release, feeding stimulation, adiposity and cardiovascular actions. The presence of Ghr mRNA in the iris and ciliary body (CB) epithelium was recently demonstrated in animal models, where a possible myorelaxing effect on the iris muscles has been suggested. Based on these observations, the aim of our study was to investigate the Ghr and GHSR-1a expression and localization in the normal human eye. Five different ciliary body/iris samples from normal eyes were subjected to Western blot analysis. Immunohistochemical detection was performed on three enucleated eyes. Twenty aqueous humor (AqH) samples obtained from patients submitted to cataract surgery were analyzed with an ELISA for the presence of Ghr. Ghr and GHSR-1a were co-expressed by the pigmented epithelium (PE) of the CB, by the retinal pigmented epithelium (RPE) and by the anterior limiting layer (ALL) of the iris. No reaction was detected at the subepithelial level in the ciliary or pupillae smooth muscle cells. The AqH samples were positive for the presence of Ghr. This study provides the first evidence that Ghr and GHSR-1a are expressed in the human eye by specific cells. The understanding of the functional role of Ghr at the human eye level needs more efforts and investigation, but a hypothetical action on the GH retinal synthesis and/or on the circadian clock system could be suggested.
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This study aimed to evaluate the role of angiotensin type 1 receptor gene (AGTR1) polymorphism (A1166C) in left ventricular hypertrophy (LVH) mediated by the angiotensin-converting enzyme (ACE) in endurance athletes. A group of 74 white,... more
This study aimed to evaluate the role of angiotensin type 1 receptor gene (AGTR1) polymorphism (A1166C) in left ventricular hypertrophy (LVH) mediated by the angiotensin-converting enzyme (ACE) in endurance athletes. A group of 74 white, healthy male endurance athletes, aged between 25 and 40 yr, were enrolled in this study. All of them participated primarily in isotonic sports, training for at least >10 h x wk(-1), for at least 5 yr. The ACE genotype (insertion [I] or deletion [D] alleles) was ascertained by polymerase chain reaction (DD in 35, ID in 36, and II in 3). Group II was excluded from the analysis because of its small size. No difference was found between the two groups as regards age, blood pressure, HR, and echocardiographic data. The left ventricular mass index (LVMI) was significantly higher in group DD rather than in group ID (P = 0.029). The group DD showed a slightly higher prevalence of subjects with LVH (LVMI > 131 g x m(-2); 62.9%) than group ID (44.4%, P = 0.120). No association was found between ACE-DD and LVH (odds ratio (OR) = 2.12, 95% confidence interval = 0.82-5.46). Concerning the role of AGTR1 polymorphism, the highest LVMI was found in 15 athletes with ACE-DD and AGTR1-AC/CC genotypes (150 +/- 23 g x m(-2)); the lowest value of LVMI was found in the case of ACE-ID and AGTR1-AA (127 g x m(-2) +/- 18 g x m(-2)), whereas LVMI in subjects with ACE-DD + AGTR1-AA was similar to that in the ACE-ID + AGTR1-AC/CC group (134 +/- 18 g x m(-2) vs 133 +/- 20 g x m(-2), P = 0.880). The presence of ACE-DD + AGTR1 + AC/CC was strongly associated with LVH (OR = 4.6, P = 0.029). Moreover, subjects with LVH showed longer left ventricular isovolumetric relaxation time and higher end-systolic wall stress. The latter was strongly correlated to LVMI (r = 0.588), especially in the presence of ACE-DD + AGTR1 + AC/CC (r = 0.728). LVMI may be greater in the presence of ACE- DD and AGTR1-AC/CC polymorphisms.
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Evidence for a rapid and transient hyperphosphorylation of a 45 kD protein in isolated nuclei from interferon-alpha-treated Daudi lymphoma cells is presented. Extraction of nuclear matrices from these nuclei has provided further evidence... more
Evidence for a rapid and transient hyperphosphorylation of a 45 kD protein in isolated nuclei from interferon-alpha-treated Daudi lymphoma cells is presented. Extraction of nuclear matrices from these nuclei has provided further evidence for the association of such a protein in the nuclear matrix structure. Because phosphorylation assays performed directly on nuclear matrices from interferon-treated cells have revealed rapid and transient increase of gamma 32P-ATP incorporation into this 45 kD band, an early involvement of the nuclear matrix in the response of the nucleus to the interferon antiproliferative message is suggested.
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32D cells grown for 1 year in interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) generated the 32D Ro cell line which retained the parental mast cell phenotype but lost ability to generate erythroid cells in response... more
32D cells grown for 1 year in interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) generated the 32D Ro cell line which retained the parental mast cell phenotype but lost ability to generate erythroid cells in response to erythropoietin (EPO). In order to clarify the mechanisms underlying such restriction, we compared 32D and 32D Ro cells for their capacity to express erythroid-specific transcription factors (Gata1, Gata2, Scl, Nef2, Eklf, and Id) and the capacity of short exposure to 5-azacytidine (5-AzaC) to reactivate erythroid differentiation potential in 32D Ro cells. By Northern analysis, the two cell lines expressed similar levels of all these genes. However, after being treated with 5-AzaC, 32D Ro cells acquired the ability to generate EPO-dependent clones (1 clone/10(4) cells) which gave rise to EPO-dependent cell lines. All the 10 EPO-responsive cell lines independently isolated from 5-AzaC-treated 32D Ro cells had erythroid morphology and expressed high levels of alpha- and beta-globin. In contrast, none of the IL-3-dependent and granulocyte/macrophage colony-stimulating factor-dependent clones concurrently isolated, as a control, showed erythroid properties. Therefore, 5-AzaC treatment reactivates the potential of the myeloid-restricted 32D Ro cells to generate EPO-responsive erythroid clones suggesting that gene methylation played an important role in the G-CSF-mediated restriction/activation of the differentiation potential of these cells.
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Interferons exert their antiviral, antiproliferative and immunoregulatory activities by stimulating the expression of several genes. Such genes disclose a common element within their promoters, defined Interferon Stimulated Response... more
Interferons exert their antiviral, antiproliferative and immunoregulatory activities by stimulating the expression of several genes. Such genes disclose a common element within their promoters, defined Interferon Stimulated Response Element (ISRE), which binds a nuclear factor(s) translocated from the cytoplasm to the nucleus (ISGF3) after the binding of interferon (IFN) to the specific receptor. Here we report the induction of the synthesis and of the hydrolytic activity of phospholipase C gamma 1 (PLC gamma 1) in human T lymphocytes by IFN-beta. The increased level of PLC gamma 1 becomes evident after 90 min of IFN-beta treatment and is still detectable after 24 h. Neither the PLC gamma 1 overexpression induced by IFN nor the increased hydrolytic activity of the enzyme appear to be affected by pretreatment of the cells with the protein tyrosine kinase inhibitor genistein, which is known to prevent the association of ISGF3 components. These results suggest that in human T lymphocytes IFN-beta can activate other transcription factor(s) distinct from ISGF3 to regulate PLC gamma 1 expression. In addition, the ability of this enzyme to hydrolyse PIP2, also in the presence of genistein, implies the possibility that this enzyme can exert its hydrolytic activity independently of protein tyrosine kinase activation.
Research Interests: Immunology, Confocal Microscopy, Transcription Factors, Western blotting, Cell Division, and 13 moreHumans, Protein Tyrosine Kinase, Phosphorylation, Transcription Factor, Enzyme, T lymphocytes, Cytokine, Time Factors, Isoenzymes, Genistein, DNA binding proteins, Phospholipase C, and phytohemagglutinins
Here it is analyzed the expression of a mini locus dual reporter construct composed by a micro-LCR and by the promoters for Aγ-and β-globin gene, each one linked to a different Luciferase, in stably transfected GM979 cells for as long as... more
Here it is analyzed the expression of a mini locus dual reporter construct composed by a micro-LCR and by the promoters for Aγ-and β-globin gene, each one linked to a different Luciferase, in stably transfected GM979 cells for as long as 12 years from transfection. ...
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The expression of phosphoinositidase C (PIC) at nuclear and cytoplasmic level has been revealed in control and interferon treated Burkitt lymphoma cells by means of western blotting and immunocytochemical analysis employing specific... more
The expression of phosphoinositidase C (PIC) at nuclear and cytoplasmic level has been revealed in control and interferon treated Burkitt lymphoma cells by means of western blotting and immunocytochemical analysis employing specific monoclonal antibodies against beta 1, gamma 1 and delta 1 isozymes. Results have indicated that PIC isoform beta 1, mainly detectable in the nucleus, undergoes transient modifications early after interferon treatment. PIC delta 1 has been found only at cytoplasmic level, apparently insensitive to interferon treatment, while PIC gamma 1 was scarcely or not detected either in the cytoplasmic or in the nuclear compartment. These results suggest that interferon may exert its antiproliferative effect activating at least two distinct pathways of signal transduction, at cytoplasmic and nuclear level, involving inositol lipid cycle mainly in the nucleus by modulation of PIC beta 1 expression.
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The early molecular mechanisms activated by the treatment of human lymphocytes with human interferon beta have been studied. These identify an early increase with respect to control, in diacylglycerol (DG) levels as response to interferon... more
The early molecular mechanisms activated by the treatment of human lymphocytes with human interferon beta have been studied. These identify an early increase with respect to control, in diacylglycerol (DG) levels as response to interferon treatment. Such a DG production was derived from the rapid and sequential activation of phosphoinositide specific phospholipase C and phospholipase D pathway. This suggests that a synergistic involvement of phosphatidylinositol-bis-phosphate (PIP2) hydrolysis and phosphatidylcholine (PC) breakdown provide early molecular events upon the interaction between interferon beta and its cell surface receptors. This finally leads to the slowing down of cell growth.