Biomedical subjects

A Pacini

Publications and source records attributed to A Pacini.

At least 19 recordsLinked to original sources

PubMed2005

Differential expression of tissue kallikrein in the skin of systemic sclerosis.

Systemic sclerosis (SSc) is characterised by ischemic damage, impaired angiogenesis and skin fibrosis. Tissue kallikrein (t-kallikrein) is involved through kinins in inflammation, vasorelaxation and angiogenesis. T-kallikrein is synthetised by endothelial, smooth muscle, and inflammatory cells and, in skin, also by dark cells of the sweat glands, where it is involved in sweat formation. Our aim was to analyse, by immunohistochemistry and RT-PCR, the expression of t-kallikrein in the skin of patients with different SSc subsets, limited (lSSc) and diffuse (dSSc), and phases, early and advanced. Skin biopsies were taken from 18 SSc patients and 10 controls. Immunohistochemistry was performed on paraffin sections with an antibody against human urinary t-kallikrein. For RT-PCR, cDNA from skin biopsies was amplified using primers specific for human t-kallikrein. In the control skin, dark cells of the secretory units of sweat glands showed immunopositivity for t-kallikrein as well as blood vessels. In the lSSc skin, immunoreactivity was observed only in some glands, with weak staining in the advanced phase. In early lSSc skin, immunoreactivity was observed in microvessel walls and in the inflammatory infiltrate. In dSSc skin, dark cells of the glandular fundus units, and the few remaining vessels showed scarcity (early phase) or lack (advanced phase) of immunoreactivity for t-kallikrein. RT-PCR confirmed a decrease of t-kallikrein mRNA levels from early to advanced phase in SSc subsets, reaching its lowest level in advanced dSSc. In conclusion, immunohistochemical and biomolecular results indicate that t-kallikrein is decreased in the skin of SSc patients and decreases progressively from the early to advanced phase of lSSc and dSSc. The decreased expression of t-kallikrein may be involved in the impairment of the sweating process, vessel functionality and angiogenesis.

Adult↗

PubMed2004

Multiple plasma proteins control atrial natriuretic peptide (ANP) aggregation.

We have recently demonstrated that human alpha-atrial natriuretic peptide (alpha-hANP), an amyloidogenic peptide responsible for isolated atrial amyloidosis, binds to a dimeric form of apo A-I belonging to small high-density lipoproteins (HDL). This binding phenomenon is considered a protective mechanism since it inhibits or strongly reduces the ANP aggregation process. The observation that plasma exhibits at least four times greater amyloid inhibitory activity than HDL prompted us to determine whether small HDL are the only ANP plasma-binding factors. After incubation of whole plasma with labelled ANP, the macromolecular complexes were subjected to two-dimensional gel electrophoresis followed by autoradiography. The results presented here provide novel evidence of additional binding proteins, in addition to apo A-I dimer, able to bind ANP in vitro and to prevent its aggregation. The mass spectrometry analysis of the radioactive spots identified them as albumin, alpha-1 antitrypsin, orosomucoid and apo A-IV-TTR complex. The putative impact of these findings in the amyloidogenic/antiamyloidogenic peptides network is discussed.

Amyloidosis↗

PubMed2003

Altered Cx43 expression during myocardial adaptation to acute and chronic volume overloading.

Gap-junctions are specialized regions of intercellular contacts allowing electrical impulse propagation among adjacent cardiomyocytes. Connexin43 (Cx43) is the predominant gap-junction protein in the working ventricular myocardium and its reduced expression has been extensively implicated in the genesis of conduction abnormalities and re-entry arrhythmia of chronically hypertrophied hearts. In contrast, data on the role played by this protein during cardiac remodeling and early phases of developing hypertrophy are lacking. Therefore, in the present study, we investigated this issue using an experimental model of pig left ventricle (LV) volume overloading consisting in the creation of an aorto-cava fistula. At scheduled times (6, 24, 48, 96, 168 h, and 2, 3 months after surgery) echocardiographic and haemodynamic measurements were performed and myocardial biopsies were taken for the morphological and biochemical analyses. When faced with the increased load, pig myocardium underwent an initial period (from 6 up to 48 h) of remarkable tissue remodeling consisting in the occurrence of cardiomyocyte damage and apoptosis. After that time, the tissue developed a hypertrophic response that was associated with early dynamic changes (up-regulation) in Cx43 protein expression, as demonstrated by Western blot and confocal immunofluorescence analyses. However, an initial transient increase of this protein was also found after 6 h from surgery. With the progression of LV hypertrophy (from 168 hr up to 3 months), a reduction in the myocardial Cx43 expression was, instead, observed. The increased expression of Cx43 protein during acute hypertrophic response was associated with a corresponding increase in the levels of its specific mRNA, as detected by RT-PCR. We concluded that up-regulation of Cx43 gap-junction protein could represent an immediate compensatory response to support the new working conditions in the early stages of ventricular overloading.

Adaptation, Physiological↗

PubMed2002

Biochemical changes and their relationship with morphological and functional findings in pig heart subjected to lasting volume overload: a possible role of acylphosphatase in the regulation of sarcoplasmic reticulum calcium pump.

We evaluated the changes in sarcoplasmic reticulum (SR) function and the parallel hemodynamic and morphological modifications in a heart subjected to volume overload. We also determined the levels of acylphosphatase, a cytosolic enzyme, that could play a regulatory effect on SR Ca(2+) pump by hydrolyzing the phosphorylated intermediate of this transport system. For this, swine hearts were subjected to volume overload by aorta-cava shunt for 1, 2, or 3 months. Changes in heart contractility reflected modifications of SR function, whose reduction after 1 month of overload was followed by a gradual recovery. A decrease in SERCA2a protein and mRNA content was shown from 1 month and remained for the following 2 months. Phospholamban content and its phosphorylation status were not modified. Acylphosphatase was unchanged at 1 month, but at 2 months this enzyme exhibited an increased activity, protein and mRNA expression. Morphological alterations consisting of the cytoskeletal architectures, intermyofibrillar oedema, swollen mitochondria and abnormality of the membrane system (T-tubule and SR cisternae) were particularly evident after 1 month but almost disappeared after 3 months. These results suggest that our overloaded hearts underwent a substantial recovery of their structural and biochemical properties at 3 months after surgery. A possible involvement of acylphosphatase in the modification of SR function is discussed.

Acid Anhydride Hydrolases↗

PubMed2001

Posttranscriptional regulation of gene expression in learning by the neuronal ELAV-like mRNA-stabilizing proteins.

The view that memory is encoded by variations in the strength of synapses implies that long-term biochemical changes take place within subcellular microdomains of neurons. These changes are thought ultimately to be an effect of transcriptional regulation of specific genes. Localized changes, however, cannot be fully explained by a purely transcriptional control of gene expression. The neuron-specific ELAV-like HuB, HuC, and HuD RNA-binding proteins act posttranscriptionally by binding to adenine- and uridine-rich elements (AREs) in the 3' untranslated region of a set of target mRNAs, and by increasing mRNA cytoplasmic stability and/or rate of translation. Here we show that neuronal ELAV-like genes undergo a sustained up-regulation in hippocampal pyramidal cells only of mice and rats that have learned a spatial discrimination paradigm. This learning-specific increase of ELAV-like proteins was localized within cytoplasmic compartments of the somata and proximal dendrites and was associated with the cytoskeleton. This increase was also accompanied by enhanced expression of the GAP-43 gene, known to be regulated mainly posttranscriptionally and whose mRNA is demonstrated here to be an in vivo ELAV-like target. Antisense-mediated knockdown of HuC impaired spatial learning performance in mice and induced a concomitant down-regulation of GAP-43 expression. Neuronal ELAV-like proteins could exert learning-induced posttranscriptional control of an array of target genes uniquely suited to subserve substrates of memory storage.

Animals↗

PubMed2001

Plasma factors controlling atrial natriuretic peptide (ANP) aggregation: role of lipoproteins.

We have previously shown that human plasma atrial alpha-natriuretic peptide (alpha-hANP) sequestering is a protective phenomenon against amyloid aggregation. In the present work, the possible role of lipoproteins as alpha-hANP binding factors has been investigated in vitro using an experimental model, developed in our laboratory, that allows to work at physiological concentrations. This approach consists of gel filtration on Sephacryl S-300 HR of big alpha-[(125)I]hANP generated in phosphate buffered saline or in human normal plasma supplemented or not with lipoproteins. The results of these experiments indicate that high density lipoproteins (HDL) are responsible for the ANP binding phenomenon observed in vitro, while low density lipoproteins and very low density lipoproteins do not directly interact with ANP. Moreover, the HDL remodeling process occurring in vitro has been analyzed during plasma incubation by monitoring the redistribution of lipids and apolipoproteins among the HDL subclasses. The changes in HDL size and composition observed in incubated plasma were compared with the redistribution of endogenous and labeled big ANP. The obtained results revealed that both tend to follow the molecular rearrangement in plasma of apolipoprotein A-I containing particles and suggested that, among HDL species, the small particles are mainly involved in the ANP binding phenomenon. This hypothesis was further demonstrated by ligand blotting experiments that confirmed the existence of differences in the ability of HDL particles to bind alpha-[(125)I]hANP.

Atrial Natriuretic Factor↗

PubMed2000

Molecular assembly of endogenous and synthetic big atrial natriuretic peptide (ANP) and its amyloidogenic implications.

The aggregation process of alpha-hANP has been investigated in vitro at physiological concentrations by gel chromatographic procedures using a radiolabeled tracer incubated in PBS and in plasma. In PBS big forms of ANP are organized as a peak eluting from both Sephacryl S-100 and S-300 HR in the void volume of the columns; in plasma, besides this major peak, a second radioactive peak is evident, eluting from Sephacryl S-100 HR around the HSA region. After gel chromatography on Sephacryl S-300 HR the major peak appears to consist of three components of different molecular size. Some information about the nature of these peak materials comes from the result of parallel incubations of partially aggregated (seed or nucleus) and aggregate depleted tracer. The comparison between the two time courses of big ANP formation indicates that: (a) ANP aggregation is a nucleation-dependent process, with a lag time longer than 8 days, at picogram peptide levels and (b) the aggregated forms of peptide are those eluting in the void volume, the other plasma peaks being probably expression of a binding, neither saturable or reversible, to some plasma components. The principle of seeded polymerization, used to detect ANP aggregates present in the plasma, indicates that: (a) the endogenous big ANP cannot act as a nucleus for polymerization and it likely consists of non-fibrillar ANP aggregates and/or bound ANP, and (b) this experimental approach can be suitable to evidence ANP binding plasma factors for further characterization studies.

Acrylic Resins↗

PubMed1999

Transcriptional down-regulation of poly(ADP-ribose) polymerase gene expression by E1A binding to pRb proteins protects murine keratinocytes from radiation-induced apoptosis.

Adenovirus E1A confers enhanced cell sensitivity to radiation and drug-induced DNA damage by a mechanism involving the binding to cellular proteins. Mutant analysis in E1A-transfected murine keratinocytes demonstrates that increased sensitivity to DNA damage requires at least E1A binding to the p300/CREB-binding protein (CBP) transcriptional coactivators and to pRb family members, indicating that this biological activity of E1A is the result of the concomitant perturbation of different cell pathways. Here we show that in the same cells E1A binding to members of the retinoblastoma protein family induces transcriptional down-regulation of the poly(ADP-ribose) polymerase (PARP) gene, coding for a NAD-dependent enzyme stimulated by DNA breaks. Inhibition of PARP expression is accompanied by a decrement of gamma-irradiation-induced apoptosis, which is overridden by reconstitution of wild type levels of PARP. Hence, E1A effects on PARP transcription are central determinant of the apoptotic sensitivity of E1A-expressing keratinocytes. Conversely, E1A binding to only p300/CBP results in an increase in PARP enzyme activity and consequently in cell death susceptibility to irradiation, which is effectively counteracted by the PARP chemical inhibitor 3-aminobenzamide. Therefore, our results identify in the E1A-mediated effects on PARP expression and activity a key molecular event involved in E1A-induced cell sensitization to genotoxic stress.

Adenovirus E1A Proteins↗

PubMed1999

A novel interaction mechanism accounting for different acylphosphatase effects on cardiac and fast twitch skeletal muscle sarcoplasmic reticulum calcium pumps.

In cardiac and skeletal muscle Ca2+ translocation from cytoplasm into sarcoplasmic reticulum (SR) is accomplished by different Ca2+-ATPases whose functioning involves the formation and decomposition of an acylphosphorylated phosphoenzyme intermediate (EP). In this study we found that acylphosphatase, an enzyme well represented in muscular tissues and which actively hydrolyzes EP, had different effects on heart (SERCA2a) and fast twitch skeletal muscle SR Ca2+-ATPase (SERCA1). With physiological acylphosphatase concentrations SERCA2a exhibited a parallel increase in the rates of both ATP hydrolysis and Ca2+ transport; in contrast, SERCA1 appeared to be uncoupled since the stimulation of ATP hydrolysis matched an inhibition of Ca2+ pump. These different effects probably depend on phospholamban, which is associated with SERCA2a but not SERCA1. Consistent with this view, the present study suggests that acylphosphatase-induced stimulation of SERCA2a, in addition to an enhanced EP hydrolysis, may be due to a displacement of phospholamban, thus to a removal of its inhibitory effect.

Acid Anhydride Hydrolases↗

PubMed1998

Oxidative stress and antioxidant defenses in renal patients receiving regular haemodialysis.

Patients with chronic renal failure, and particularly those receiving regular haemodialysis, have a high incidence of premature cardiovascular disease. Oxidative stress, which causes lipid peroxidation, may contribute to increase the risk of atherosclerosis. The results of the present study indicate that lipid peroxidation products (malonaldehyde and 4-hydroxyalkenals) are significantly increased in plasma of renal patients before dialysis and, although reduced, remained above the normal range after this treatment. Moreover, production of free radicals and reactive oxygen metabolites was increased in chronic renal failure patients, especially after dialysis. On the other hand, the antioxidant defenses of those patients were higher than those of normal subjects, as judged from the plasma levels of specific antioxidant molecules and from the plasma antioxidant capacity. We also found that triglycerides were significantly higher in renal patients, both before and after dialysis, than in the control group. These results suggest that patients on chronic haemodialysis are particularly prone to oxidative stress and that dialysis itself may worsen this condition. Rather than to a weakening of antioxidant defenses, the susceptibility of chronic renal failure patients to oxidative stress might be ascribed to an increased free radical and reactive oxygen metabolite production and to increased levels of oxidizable substrates, notably triglycerides with their unsaturated fatty acids.

Antioxidants↗

PubMed1997

Damage to the nasopharyngeal mucosa induced by current levels of urban air pollution: a field study in lambs.

This study concerns the effects of urban air pollution on the nasopharyngeal epithelium, with the aim of evaluating the possible harmful activity of levels of atmospheric pollution which are not currently considered to be dangerous. Over a 3 month period, 10 lambs kept in a zone characterized by numerous vehicles were sacrificed at regular intervals, and their nasopharyngeal mucosa was examined by scanning electron microscopy and image analysis. Two lambs kept in a rural area were used as controls. The local levels of some airborne contaminants (NO(x), NO2, NO, SO2, CO and particulate matter with aerodynamic diameter < or =10 microm (PM10)) were monitored throughout the experiment. The urban air had an irritating effect, inducing hypersecretion of mucus and morphological damage to the ciliated epithelium. These alterations increased with the duration of exposure to urban air and with increasing pollution levels, although the levels remained below current legal levels. We conclude that the harmful effects of airborne contaminants are probably underestimated. Moreover, physicochemical evaluation of pollution parameters should be complemented by morphological study of upper respiratory epithelium in exposed animals, since this mucosa is a sensitive target for irritating agents.

Air Pollutants↗

PubMed1996

Stimulation of cardiac sarcoplasmic reticulum calcium pump by acylphosphatase. Relationship to phospholamban phosphorylation.

Ca2+ transport by cardiac sarcoplasmic reticulum is tightly coupled with the enzymatic activity of Ca2+-dependent ATPase, which forms and decomposes an intermediate phosphoenzyme. Heart sarcoplasmic reticulum Ca2+ pump is regulated by cAMP-dependent protein kinase (PKA) phospholamban phosphorylation, which results in a stimulation of the initial rates of Ca2+ transport and Ca2+ ATPase activity. In the present studies we found that acylphosphatase from heart muscle, used at concentrations within the physiological range, actively hydrolyzes the phosphoenzyme of cardiac sarcoplasmic reticulum Ca2+ pump, with an apparent Km on the order of 10(-7) M, suggesting an high affinity of the enzyme for this special substrate. In unphosphorylated vesicles acylphosphatase enhanced the rate of ATP hydrolysis and Ca2+ uptake with a concomitant significant decrease in apparent Km for Ca2+ and ATP. In vesicles whose phospholamban was PKA-phosphorylated, acylphosphatase also stimulated the rate of Ca2+ uptake and ATP hydrolysis but to a lesser extent, and the Km values for Ca2+ and ATP were not significantly different with respect to those found in the absence of acylphosphatase. These findings suggest that acylphosphatase, owing to its hydrolytic effect, accelerates the turnover of the phosphoenzyme intermediate with the consequence of an enhanced activity of Ca2+ pump. It is known that phosphorylation of phospholamban results in an increase of the rate at which the phosphoenzyme is decomposed. Thus, as discussed, a competition between phospholamban and acylphosphatase effect on the phosphoenzyme might be proposed to explain why the stimulation induced by this enzyme is less marked in PKA-phosphorylated than in unphosphorylated heart vesicles.

Acid Anhydride Hydrolases↗

PubMed1996

In vitro effects of some differentiation inductors in metaplastic epithelium of the human nasal cavity.

The alterations in the mucociliary unit in the course of chronic inflammation of the upper respiratory tract correspond to morphologic anomalies of the respiratory epithelium and induce cuboidal and squamous metaplasia. While the squamous pattern is most probably irreversible, it is still not clear whether it is possible to restore ciliogenesis in cuboidal metaplasia. In the present study, the action of different inductors of differentiation was evaluated in vitro in isolated cells and explants from human nasal metaplastic epithelium. Polar/apolar compounds induced secretory activity, whereas retinoic acid was able to induce ciliogenesis in some cases. Therefore, the cuboidal metaplastic condition appears to be reversible, and two distinct pathways of differentiation, secretory and ciliogenetic, are identifiable.

Acetamides↗

PubMed1996

Acylphosphatase stimulates Ca2+ transport and Ca(2+)-dependent ATPase activity in cardiac sarcoplasmic reticulum.

Acylphosphatase purified from heart muscle actively hydrolyzes the phosphoenzyme intermediate of cardiac sarcoplasmic reticulum Ca(2+)-ATPase. This effect was evident with acylphosphatase concentrations (up to 100 units/mg sarcoplasmic reticulum protein) that fall within the physiological range, and the low value of the apparent Km, on the order of 10(-7)M, suggests a high affinity towards this special substrate. Moreover, acylphosphatase addition to sarcoplasmic reticulum vesicles significantly enhanced the rate of Ca(2+)-dependent ATP hydrolysis. Maximal stimulation, observed with 100 units/mg vesicular protein, resulted in an ATPase activity which was about two folds over basal value. The same acylphosphatase concentration increased at a similar extent the rate of ATP driven Ca2+ influx into sarcoplasmic reticulum vesicles. Taken together these findings lead to suppose that acylphosphatase, owing to its hydrolytic activity, induces an accelerated turnover of the phosphoenzyme intermediate, whence an overall stimulation of heart sarcoplasmic reticulum Ca2+ pump, affecting both ATP hydrolysis and Ca2+ influx.

Acid Anhydride Hydrolases↗

PubMed1993

Atrial natriuretic peptide and acute myocardial infarction: critical reappraisal.

The present investigation was designed to assess the correlation of atrial natriuretic peptide (ANP) with the ordinary markers of acute myocardial infarction (AMI). In 15 patients with uncomplicated AMI, no remarkable variation in ANP plasma levels was found. On the other hand, serum myoglobin and cardiac enzymes behaved as usual. Our results show that ANP is not correlated with serum myoglobin, creatine-phosphokinase and its isoenzyme MB, being completely independent from these markers of myocardial injury. These observations suggest that the plasma ANP variations observed in AMI by other investigators are probably dependent on hemodynamic dysfunction.

Aged↗

PubMed1991

An elastolytic proteinase from rabbit leukocytes: purification and partial characterization.

A proteinase with elastolytic activity was isolated from granules of rabbit bloodstream leukocytes, and purified to apparent homogeneity by a multi-step procedure consisting of ammonium sulfate precipitation, batch fractionation on DEAE-Sephadex A-50, and finally by preparative isoelectric focusing (IEF) on Sephadex G-75 Superfine. The molecule weight of the enzyme, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was 28,500. This enzyme shows an isoelectric point at pH 9.0. The proteinase is active against natural elastins as well as toward Suc-(Ala)3-NA, Methoxy-Suc-(Ala)2-Pro-Val-NA, and (to a lesser extent) against Suc-(Ala)2-Pro-Leu-NA and Boc-Ala-ONp. The inhibition profile of the isolated enzyme indicates that rabbit granulocyte elastase belongs to the group of serine proteinases. Inhibition by some natural proteinase inhibitors is also observed. Unlike other mammalian elastases, it is insensitive to elastatinal.

Amino Acid Sequence↗

PubMed1990

In vivo stimulation of lung collagen synthesis by collagen derived peptides.

In the present work we examined the "in vivo" effects of collagen breakdown products derived from elastase digestion on lung collagen synthesis in rabbits. It was found that i.v. injection of collagen peptides greatly enhances the collagen synthesis rate while does not affect the synthesis of non collagenous proteins. The increase of incorporation of 3H-proline in lung collagen parallels that of prolyl hydroxylase activity. The collagen synthesis, expressed as fractional rate (% day), amounted to 15% day in treated animals, resulting in a significant increase with respect to controls (11.7% day). The observations reported provide evidence that collagen breakdown products stimulate lung collagen synthesis and may play a role in collagen homeostasis.

Animals↗

PubMed1990

Development of interstitial lung fibrosis by long-term treatment with collagen breakdown products in rabbits.

We recently demonstrated that collagen breakdown products derived from elastase digestion (CDP) can stimulate "in vivo" lung collagen synthesis. The present work deals with the morphological and biochemical characteristics of an experimental model of lung fibrosis developed in rabbit by long-term treatment with CDP. Stimulation of collagen synthesis by CDP resulted in a significant thickening of alveolar septa due to accumulation of fibroblasts and a marked deposition of collagen fibrils as revealed by light as well as electron microscopy. Biochemical analysis confirmed the increase in lung collagen deposition. Total collagen content as determined by hydroxy-proline analysis was increased in CDP-treated animals of about 56% in respect to control animals. A relative increase of type I collagen in respect to type III was also observed. An additional interesting observation was a progressive hyperplasia of type II pneumocytes. Unlike other experimental models of lung fibrosis, the collagen deposition in our condition is not preceded or associated with inflammatory or degenerative processes. This fact renders this model very suitable to study matrix-cell interactions in pulmonary fibrogenesis.

Analysis of Variance↗