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Biomedical subjects

G Cuda

Publications and source records attributed to G Cuda.

At least 19 recordsLinked to original sources
PubMed2005

Modeling and designing a proteomics application on PROTEUS.

OBJECTIVES: Biomedical applications, such as analysis and management of mass spectrometry proteomics experiments, involve heterogeneous platforms and knowledge, massive data sets, and complex algorithms. Main requirements of such applications are semantic modeling of the experiments and data analysis, as well as high performance computational platforms. In this paper we propose a software platform allowing to model and execute biomedical applications on the Grid. METHODS: Computational Grids offer the required computational power, whereas ontologies and workflow help to face the heterogeneity of biomedical applications. In this paper we propose the use of domain ontologies and workflow techniques for modeling biomedical applications, whereas Grid middleware is responsible for high performance execution. As a case study, the modeling of a proteomics experiment is discussed. RESULTS: The main result is the design and first use of PROTEUS, a Grid-based problem-solving environment for biomedical and bioinformatics applications. CONCLUSION: To manage the complexity of biomedical experiments, ontologies help to model applications and to identify appropriate data and algorithms, workflow techniques allow to combine the elements of such applications in a systematic way. Finally, translation of workflow into execution plans allows the exploitation of the computational power of Grids. Along this direction, in this paper we present PROTEUS discussing a real case study in the proteomics domain.

Algorithms↗
PubMed2003

Molecular mechanisms of corticosteroid actions in chronic inflammatory airway diseases.

Although corticosteroids have been used for a long time as a very effective therapy of airway inflammatory diseases such as asthma, only recently the molecular basis of their mechanism of action has begun to be elucidated. These hormones exert their biological and pharmacological actions by binding to cytoplasmic receptors that, upon activation, translocate to the nucleus where they interact with specific genomic sequences thus modulating gene expression. However, many glucocorticoid effects responsible for their anti-inflammatory and anti-asthmatic activity take place irrespectively of receptor binding to DNA. In particular, ligand-bound glucocorticoid receptors can repress several different pro-inflammatory genes by physically associating, via protein-protein interactions, with various transcription factors and with the macromolecular complexes implicated in regulation of chromatin structure and function. In this regard, an important role is played by the influences of corticosteroids on the intrinsic histone acetyltransferase and deacetylase functions of coactivators and corepressors, respectively. Furthermore, the signal transduction pathways mediated by mitogen-activated protein kinases are newly recognized, key targets of glucocorticoids. Indeed, these enzymatic cascades are crucially involved in the regulation of gene expression in that they are essential for the activity of a high number of transcription factors. Therefore, the recent advances made in such a rapidly growing research field are providing new insights into the mode of action of corticosteroids, thereby also unveiling novel promising therapeutic strategies directly targeted to the molecular events underlying the inflammatory, immune, and apoptotic processes implicated in the pathogenesis of asthma and other airway diseases.

Asthma↗
PubMed2001

Effects of glucocorticoids on activation of c-jun N-terminal, extracellular signal-regulated, and p38 MAP kinases in human pulmonary endothelial cells.

Mitogen-activated protein kinases (MAPK) play a central role in signal transduction by regulating many nuclear transcription factors involved in inflammatory, immune, and proliferative responses. The aim of this study was to investigate, in human pulmonary endothelial cells, the effects of synthetic glucocorticosteroids on activation of c-jun N-terminal kinases, extracellular signal-regulated kinases, and p38 subgroups of the MAPK family. Human microvascular endothelial cells from lung were stimulated for 2 h with either H(2)O(2) (2 mM), IL-1beta (10 ng/mL), or tumour necrosis factor-alpha (10 ng/mL). Under these conditions, a remarkable increase in the phosphorylation pattern of c-jun N-terminal kinases, extracellular signal-regulated kinases 1/2, and p38 was detected. Pretreatment for 12 h with dexamethasone (100 nM) was able to prevent phosphorylation-dependent MAPK activation in stimulated cells, without substantially affecting the expression levels of these enzymes. Our results suggest that inhibition of MAPK signaling pathways in human pulmonary endothelial cells may significantly contribute, by interfering with activation of several different transcription factors, to the antiinflammatory and immunosuppressive effects of glucocorticosteroids.

Cells, Cultured↗
PubMed2001

Transcriptional regulation of the mismatch repair gene hMLH1.

We have characterized the promoter region of the human gene coding for the MLH1 mismatch repair protein. The total transcriptional activity of the hMLH1 promoter is driven by two positive cis-elements included between nucleotides -300 and -220. The upstream element is a canonical CCAAT box, and it is recognized by the heterotrimeric transcription factor NF-Y. On the other hand, the downstream element is recognized by a nuclear factor of about 120 kDa. Variations in hMLH1 intracellular levels may influence the surveillance of the genome integrity. The identification of the two elements may shad some light on the regulation of the transcriptional regulation of hMLH1 expression.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Opposing functions of Ki- and Ha-Ras genes in the regulation of redox signals.

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1-2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.

3T3 Cells↗
PubMed2001

Evidence of a founder mutation of BRCA1 in a highly homogeneous population from southern Italy with breast/ovarian cancer.

Several genes have been involved in the pathogenesis of hereditary breast/ovarian cancer (BOC), but mutations in the BRCA1 gene are by far the most recurrent. In this study, we report the identification of a founder mutation in a geographically and historically homogeneous population from Calabria, a south Italian region. A screening performed on 24 patients from unrelated families highlighted the high prevalence of a 5083del19 alteration in the BRCA1 gene, which accounts for 33% of the overall gene mutations. The same mutation was also detected in 4 patients, all of Calabrian origin, referred to us by research centres from the north of Italy. Allelotype analysis, performed on probands and unaffected family members revealed the presence a common allele, therefore suggesting a founder effect due to a common ancestor. Our findings underscore the importance of ethnic background homogeneity in patients' selection and highlight the usefulness of founder mutations as a potential tool for optimisation of preclinical diagnosis in gene carriers and therapeutic approaches in affected individuals.

Adult↗
PubMed2000

Changes in myocardial cytoskeletal intermediate filaments and myocyte contractile dysfunction in dilated cardiomyopathy: an in vivo study in humans.

AIM: To investigate in vivo the intermediate cytoskeletal filaments desmin and vimentin in myocardial tissues from patients with dilated cardiomyopathy, and to determine whether alterations in these proteins are associated with impaired contractility. METHODS: Endomyocardial biopsies were performed in 12 patients with dilated cardiomyopathy and in 12 controls (six women with breast cancer before anthracycline chemotherapy and six male donors for heart transplantation). Biopsy specimens were analysed by light microscopy and immunochemistry (desmin, vimentin). Myocyte contractile protein function was evaluated by the actin-myosin in vitro motility assay. Left ventricular ejection fraction was assessed by echocardiography and radionuclide ventriculography. RESULTS: Patients with dilated cardiomyopathy had a greater cardiomyocyte diameter than controls (p < 0.01). The increase in cell size was associated with a reduction in contractile function, as assessed by actin-myosin motility (r = -0.643; p < 0.01). Quantitative immunochemistry showed increased desmin and vimentin contents (p < 0.01), and the desmin distribution was disturbed in cardiomyopathy. There was a linear relation between desmin distribution and actin-myosin sliding in vitro (r = 0.853; p < 0.01) and an inverse correlation between desmin content and ejection fraction (r = -0.773; p < 0.02). Negative correlations were also found between myocardial vimentin content and the actin-myosin sliding rate (r = -0.74; p < 0.02) and left ventricular ejection fraction (r = -0.68; p < 0.01). CONCLUSIONS: Compared with normal individuals, the myocardial tissue of patients with dilated cardiomyopathy shows alterations of cytoskeletal intermediate filament distribution and content associated with reduced myocyte contraction.

Actins↗
PubMed1999

Calcium antagonist isradipine improves abnormal endothelium-dependent vasodilation in never treated hypertensive patients.

OBJECTIVE: To examine whether middle (two months) and long-term (six months) isradipine sustained-release treatment improves endothelium-dependent vasodilation in never treated hypertensive patients. METHODS: The responses of the forearm vasculature to acetylcholine (7.5, 15 and 30 micrograms/min) and sodium nitroprusside (0.8, 1.6, 3.2 micrograms/min) were evaluated in 12 normotensive controls (seven men and five women, aged 25 to 49 years), and in 12 hypertensives (eight men and four women, aged 20 to 47 years) at baseline and after two and six months of isradipine sustained-release treatment. Drugs were infused into the brachial artery, and forearm blood flow was measured by strain-gauge plethysmography. RESULTS: At baseline, the response to acetylcholine was significantly lower in hypertensives vs controls: at the highest dose (30 micrograms/min), forearm blood flow was 28.6 +/- 2.4 ml/100 ml of tissue per min in the controls vs 8.9 +/- 1.0 ml/100 ml of tissue per min in hypertensive (p < 0.0001). Similarly, vascular resistance was significantly (p < 0.0001) higher in hypertensives: 4.8 +/- 0.5 units (controls) vs 15.1 +/- 1.7 units (hypertensives). After isradipine treatment, the forearm blood flow in hypertensive patients changed from 8.9 +/- 1.0 ml/100 ml of tissue per min to 16.0 +/- 1.2 ml/100 ml of tissue per min (two months; p < 0.0001) and 15.2 +/- 1.4 ml/100 ml of tissue per min (six months; p < 0.0001). Isradipine treatment did not modify the vasodilating effect of sodium nitroprusside. CONCLUSIONS: Our data demonstrate for the first time that the calcium antagonist isradipine improves acetylcholine-induced vasodilation in hypertensives.

Acetylcholine↗
PubMed1998

Familial hypertrophic cardiomiopathy: molecular basis and genotype-phenotype correlations.

The impact of molecular genetics in the diagnosis and management of various forms of heritable cardiac or vascular disorders is continuously increasing thanks to the newly available laboratory tools. Familial hypertrophic cardiomyopathy (FHC), an autosomal dominant inherited disease characterized by unexplained left ventricular hypertrophy and a wide range of clinical symptoms, is the first cardiac disorder whose genetic bases have been elucidated. Linkage analysis studies have shown a statistically significant association between the disease status and at least seven genetic loci, all coding for sarcomeric proteins, in unrelated kindreds. A major challenge for physicians is to make an accurate and early diagnosis, not only on the basis of the traditional tools (i.e. physical examination and electro-echocardiography) but also to focus on the impact of genotype on clinical manifestations of FHC. In this review we present the more recent findings on the genetic basis of FHC and analyze the genotype-phenotype correlations of this disorder, whose expression may be modulated by additional factors (modifier genes, genetic background, environmental factors) other than mutations in any of the sarcometric proteins.

Animals↗
PubMed1997

Isometric tension and mutant myosin heavy chain content in single skeletal myofibers from hypertrophic cardiomyopathy patients.

Several mutations in the beta-myosin heavy chain (beta-MHC) gene have been linked to hypertrophic cardiomyopathy (HCM). Because this gene is also expressed in slow-twitch fibers of skeletal muscle, we have been able to study the mutant beta-myosin content and mechanical properties associated with these myosin mutations in single skinned skeletal muscle fibers obtained from HCM patients. We found that in patients carrying the 403Arg-->Gln mutation, the mutant beta-MHC comprises 47.3 +/- 9.1% of the total beta-MHC present in single slow-twitch fibers. Therefore, both alleles of the beta-MHC gene are on average equally expressed. Isometric tension was decreased by 18% in slow fibers from HCM patients with the 403Arg-->Gln mutation, but was unchanged in slow fibers from patients with two other beta-MHC gene mutations. Taken together with the previous demonstration of reduced velocities generated by these myosins in an in vitro assay, our results suggest that the mutant beta-myosins are functional molecular motors that are able to generate tension and movement, but with abnormal kinetics.

Calcium↗
PubMed1997

In vitro actin filament sliding velocities produced by mixtures of different types of myosin.

Using in vitro motility assays, we examined the sliding velocity of actin filaments generated by pairwise mixings of six different types of actively cycling myosins. In isolation, the six myosins translocated actin filaments at differing velocities. We found that only small proportions of a more slowly translating myosin type could significantly inhibit the sliding velocity generated by a myosin type that translocated filaments rapidly. In other experiments, the addition of noncycling, unphosphorylated smooth and nonmuscle myosin to actively translating myosin also inhibited the rapid sliding velocity, but to a significantly reduced extent. The data were analyzed in terms of a model derived from the original working cross-bridge model of A.F. Huxley. We found that the inhibition of rapidly translating myosins by slowly cycling was primarily dependent upon only a single parameter, the cross-bridge detachment rate at the end of the working powerstroke. In contrast, the inhibition induced by the presence of noncycling, unphosphorylated myosins required a change in another parameter, the transition rate from the weakly attached actomyosin state to the strongly attached state at the beginning of the cross-bridge power stroke.

Actin Cytoskeleton↗
PubMed1997

The in vitro motility activity of beta-cardiac myosin depends on the nature of the beta-myosin heavy chain gene mutation in hypertrophic cardiomyopathy.

Several mutations in the beta-myosin heavy chain gene cause hypertrophic cardiomyopathy. This study investigates (1) the in vitro velocities of translocation of fluorescently-labelled actin by beta-myosin purified from soleus muscle of 30 hypertrophic cardiomyopathy patients with seven distinct beta-myosin heavy chain gene mutations: Thr124Ile, Tyr162Cys, Gly256Glu, Arg403Gln, Val606Met, Arg870His, and Leu908Val mutations; and (2) motility activity of beta-myosin purified from cardiac and soleus muscle biopsies in the same patients. The velocity of translocation of actin by beta-myosin purified from soleus or cardiac muscle of 22 normal controls was 0.48 +/- 0.09 micron s-1. By comparison, the motility activity was reduced in all 30 patients with beta-myosin heavy chain gene mutations (range, 0.112 +/- 0.041 to 0.292 +/- 0.066 micron s-1. Notably, the Tyr162Cys and Arg403Gln mutations demonstrated significantly lower actin sliding velocities: 0.123 +/- 0.044, and 0.112 +/- 0.041 micron s-1, respectively. beta-myosin purified from soleus muscle from four patients with the Arg403Gln mutation had a similar actomyosin motility activity compared to beta-myosin purified from their cardiac biopsies (0.127 +/- 0.045 micron s-1 versus 0.119 +/- 0.068 micron s-1, respectively). Since these seven mutations lie in several distinct functional domains, it is likely that the mechanisms of their inhibitions of motility are different.

Actins↗
PubMed1997

Inhibition of myocardial crossbridge cycling by hypoxic endothelial cells: a potential mechanism for matching oxygen supply and demand?

Previous studies have shown that cardiac endothelial cells release substances that influence myocardial contraction. Since PO2 is an important stimulus that modulates endothelial function, we investigated the effects of acute moderate hypoxia and reoxygenation on the release of cardioactive factors by endothelial cells. Endothelial cells cultured from several vascular beds were superfused with normoxic (equilibrated with room air; PO2, approximately 160 mm Hg) or hypoxic (PO2, 40 to 50 mm Hg) physiological buffer solution, and the superfusates were reequilibrated to a PO2 of approximately 160 mm Hg and then tested for their effects on various myocardial assays. Endothelial cell viability and buffer ionic composition were unaltered after the superfusion procedures. The superfusates of hypoxic endothelial cells induced rapid, potent, reversible inhibition of isolated cardiac myocyte contraction without reducing cytosolic Ca2+ transients. This activity was not lost after heating (95 degrees C) and was present in low molecular weight (Mr, <500) superfusate fractions. Hypoxic endothelial superfusate reduced unloaded shortening velocity of human skinned soleus muscle fibers. It markedly depressed in vitro actin motility over cardiac myosin and reduced the rate of actin-activated cardiac myosin ATPase activity but had no effect on corresponding smooth muscle myosin assays. Reoxygenation of hypoxic endothelial cells resulted in loss of this inhibitory activity. These data indicate that cultured endothelial cells respond to acute moderate hypoxia by releasing an unidentified substance(s) that inhibits myocardial crossbridge cycling, independent of Ca2+ or other second messenger signaling pathways. Such a mechanism could have important implications for the regulation of oxygen supply-demand balance in the heart and be relevant to conditions such as myocardial hibernation.

Actin Cytoskeleton↗
PubMed1997

Embryonic gene expression in nonoverloaded ventricles of hereditary hypertrophic cardiomyopathic hamsters.

Current information regarding the molecular and biochemical mechanisms of myocardial hypertrophy, as obtained from isolated cardiomyocytes and/or healthy animals with aortic banding, does not permit dissection of the hierarchical relationship among different steps and triggers of the pathogenic process in vivo. The aim of the present study was to depict the temporal relationship among myocardial structural and functional characteristics, the embryonic gene program, and transforming growth factor (TGF) beta 1 expression in euthyroid hereditary hypertrophic cardiomyopathic hamsters (CMPH). This investigation was performed using Western and Northern blot and in situ hybridization techniques. The results show that in CMPH, the severity of the hemodynamic overload is not related to any modification in structural myocardial characteristics (cardiac mass, cardiomyocyte dimensions, total RNA, and protein content), whereas an early activation of the embryonic gene program occurs in not yet overloaded 90-day-old CMPH (left ventricular end diastolic pressure < 15 mm Hg). In these animals, a 30% to 90% decrease in the alpha myosin heavy chain (alpha MHC) relative content was found in ventricles, whereas beta MHC increased 5-fold. In addition, the alpha skeletal actin expression was enhanced 2-fold versus age-matched controls. No modifications were observed in myosin function evaluated by in vitro motility assay, whereas the administration of L-thyroxine (100 micrograms/kg intraperitoneally daily) to CMPH was able to reinduce the ventricular expression of the alpha MHC isoform (5-fold increase). Conversely, no changes were found in alpha cardiac actin and myosin light chain 2 (MLC2) expression. A close temporal relationship occurred in CMPH ventricles between the re-expression of the embryonic gene program and a 3-fold enhancement of the expression of TGF beta 1. These results indicate that the CMPH provides a useful model for investigating the expression of embryonic genes in hypertrophic ventricles in the absence of mechanical and hormonal stimuli, and that TGF beta 1 is involved in regulating in vivo the "embryonic step" of myocardial hypertrophy. Furthermore, the study offers new insights into the pathophysiologic mechanisms leading to heart failure.

Actins↗
PubMed1996

A previously undescribed de novo insertion-deletion mutation in the beta myosin heavy chain gene in a kindred with familial hypertrophic cardiomyopathy.

A previously undescribed de novo insertion-deletion mutation in the beta cardiac myosin heavy chain gene was found in a kindred with familial hypertrophic cardiomyopathy. In the mutated allele there is an inserted-deleted guanine at nucleotides 8823 and 8850 of the beta myosin heavy chain gene, resulting in a dramatic change of the amino acid sequence (AA 395-404). such a mutation, detected in the proband and in his son but not in the proband's parents, is likely to produce major impairment of myosin function.

Adult↗
PubMed1994

Angiotensin II maintains, but does not mediate, isoproterenol-induced cardiac hypertrophy in rats.

The role of angiotensin II (ANG II) in the development of isoproterenol (Iso)-induced cardiac hypertrophy was examined in rats. Iso increased cardiac mass, left ventricular RNA-to-DNA ratio, and the cardiac content of both myosin heavy chain and hydroxyproline in a dose-dependent manner, indicating that Iso-induced cardiac hypertrophy involves growth of both muscle and connective tissue. Cardiac hypertrophy reverted within 11-14 days after cessation of Iso. Propranolol prevented development of Iso-induced cardiac hypertrophy but did not affect the rate of its reversal. The ANG II receptor blocker losartan (Los) did not significantly decrease the hypertrophic response to Iso. Los injected after cessation of Iso dramatically enhanced the reversal of cardiac hypertrophy, even in rats that received Los with Iso during the induction of Iso-induced cardiac hypertrophy. ANG II, injected continuously at a subpressor dose that did not affect heart weight when given alone, inhibited reversal of cardiac hypertrophy when given after cessation of Iso. Los did not significantly affect the induction of the protooncogene c-fos by Iso. We conclude that endogenous ANG II has a major function in maintaining Iso-induced cardiac hypertrophy but does not mediate its induction. This suggests that different interactive stimuli may be required for development of cardiac hypertrophy, i.e., for initiation and for maintenance.

Actins↗