Biomedical subjects

P Pollesello

Publications and source records attributed to P Pollesello.

At least 19 recordsLinked to original sources

PubMed2003

Interaction of levosimendan with cardiac troponin C in the presence of cardiac troponin I peptides.

The interaction between troponin C (TnC) and troponin I (TnI) is essential for the regulation of muscle contraction. There are several binding sites for TnI on TnC that are differentially occupied depending on the phase of the contraction/relaxation cycle. TnI and TnC interact in an antiparallel fashion with each other. The C-domain of cTnC and the N-domain region of cTnI(residues 33-70) always interact under physiological conditions, whereas the interaction between regulatory regions of TnC and TnI (residues 128-166) is calcium dependent. Previously, it has been shown that levosimendan, a calcium sensitizer used as a treatment for acute heart failure, can interact with both domains of isolated cTnC. To understand which interaction is relevant for the mechanism of calcium sensitization, we used a more complete troponin model obtained by complexing cTnI(32-79) and cTnI(128-180) with calcium-saturated cTnC(CS). The cTnI peptides bound to cTnC(CS) to form a 1:1:1 complex. The interaction of levosimendan with this complex was followed by 1H-(15)N heteronuclear correlation spectroscopy. It was clear that based on chemical shift changes, cTnI(32-79) blocked the levosimendan interaction sites on the C-domain, whereas cTnI(128-180) did not compete with levosimendan for the binding site on the N-domain. Hence, the effective binding site of levosimendan on cTnC resulting in the calcium-sensitizing effect is located in the regulatory domain (N-domain).

Amino Acid Sequence↗

PubMed2001

Levosimendan is a mitochondrial K(ATP) channel opener.

Levosimendan, a new inodilator developed for the treatment of heart failure has been shown to have a vasodilatory effect via opening of K(ATP) channels in the plasma membrane of vascular smooth muscle cells. In this study, we investigated the effects of levosimendan on the mitochondrial K(ATP) channel. This compound did not influence mitochondrial transmembrane potential (DeltaPsi), and at up to 2.2 microM had no effect on the respiration rate of rat liver mitochondria, respiring on 5 mM succinate (+5 microM rotenone). A sensitive method was developed for assessing K(ATP) channel opening activity employing rat liver mitochondria, respiring only on endogenous substrates in the presence of 400 microM ATP and 1 microg oligomycin/mg mitochondrial protein. In this model, levosimendan (0.7-2.6 microM) decreased DeltaPsi by 6.5-40.4% (n=3, incubation time 15 min). This effect was dependent on the K+ concentration in the incubation medium and was abolished by the selective blocker of the mitochondrial K(ATP) channel-5-hydroxydecanoate (200 microM). Our results indicate that levosimendan opens mitochondrial K(ATP) channels.

Adenosine Triphosphate↗

PubMed2001

Improved survival with simendan after experimental myocardial infarction in rats.

This study compared the effects of simendan, a calcium sensitizer, with those of milrinone and enalapril on survival of rats with healed myocardial infarction. Seven days after ligation-induced myocardial infarction, the rats were randomized to control, milrinone, enalapril, or simendan groups. All compounds were administered via the drinking water for 312 days, at which time there was 80% mortality in the control group--the study's primary endpoint. The infarct sizes were similar across all groups. At endpoint, the mortality rates were: 63% (milrinone), 56% (enalapril) and 53% (simendan); the risk reductions were 25% (P = 0.04 vs. control) and 28% (P = 0.02 vs. control) with enalapril and simendan, respectively. Milrinone had no statistically significant effect on the survival rate. These findings suggest that, like enalapril, simendan improved survival in rats with healed myocardial infarction.

Animals↗

PubMed2001

Levosimendan increases diastolic coronary flow in isolated guinea-pig heart by opening ATP-sensitive potassium channels.

Levosimendan, a novel calcium sensitizer developed for the treatment of acute heart failure, is an inodilator that increases coronary flow. Because it was recently shown that levosimendan stimulates potassium current through K(ATP) channels in isolated rat arterial cells, our aim was to assess whether the levosimendan-induced increase in coronary flow is due to the opening of the K(ATP) channels in coronary smooth muscle. The effect of levosimendan on the diastolic coronary flow velocity (DCFV) was measured in the Langendorff perfused spontaneously beating guinea-pig heart in the absence and presence of glibenclamide. Pinacidil was used as a reference compound, and the protein kinase C inhibitor bisindolylmaleimide was used to study the dilatory effect of levosimendan when the K(ATP) channels in smooth muscle are not inhibited by PKC-dependent phosphorylation. Levosimendan (0.01-1 microM) increased DCFV concentration-dependently and was noncompetitively antagonized by 0.1 microM glibenclamide, whereas pinacidil was inhibited competitively by glibenclamide. In the presence of glibenclamide the positive inotropic and chronotropic effects of levosimendan were unaltered. The effect of bisindolylmaleimide and levosimendan on DCFV was additive. The results indicate that levosimendan induced coronary vasodilation through the opening of the K(ATP) channels. Levosimendan and pinacidil probably have different binding sites on the K(ATP) channels. The additive effect of bisindolylmaleimide and levosimendan on the increase of DCFV suggests that the latter binds to the unphosphorylated form of the channel.

Animals↗

PubMed2000

Binding of levosimendan, a calcium sensitizer, to cardiac troponin C.

Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca(2+)-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca(2+)-loaded regulatory domain of recombinant cTnC(C35S) was observed. The changes in the NMR spectra of the N-domain of full-length cTnC(C35S), due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnC(A-Cys), where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnC(C35S) and cTnC(A-Cys). The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca(2+)-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN(3)), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.

Calcium↗

PubMed2000

Further evidence for the cardiac troponin C mediated calcium sensitization by levosimendan: structure-response and binding analysis with analogs of levosimendan.

Levosimendan, an inodilatory drug discovered using troponin C as a target protein, has a cardiac effect deriving from the calcium sensitization of contractile proteins. The aim of this study was to give further evidence that levosimendan binds to cardiac troponin C and that the binding involves amino acid residues on helixepsilon of the N-terminal domain of this calcium-binding protein. Nine organic molecules, obtained by chemical modification of levosimendan, were tested both for their calcium-dependent binding to troponin C and troponin complex affinity HPLC columns, and for their ability to increase the calcium sensitivity of myofilaments in cardiac skinned fibers. A good correlation between the calcium sensitization and the calcium-dependent binding to troponin complex (r=0.90) and to cardiac troponin C (r=0.91) for the analogs of levosimendan was shown. In addition, the effect of levosimendan on the calcium-induced conformational changes in native and point-mutated cTnC was studied. Cys84-->Ser, Asp87-->Lys and Asp88-->Ala point-mutated cTnC were shown to maintain a high affinity to calcium, but their Ca(2+)titration curves were not influenced by levosimendan as for the native protein. Finally, it was demonstrated that the NMR chemical shifts of the terminal methyl groups of Met47, Met81, and Met85 on calcium-saturated cTnC were changed after addition of levosimendan in water solution at pH 7.4. This effect was not seen when adding an analog of levosimendan, which did not bind to the troponin C affinity HPLC column and did not increase the calcium-induced tension in cardiac skinned fibers.

Calcium↗

PubMed2000

Conformations of the regulatory domain of cardiac troponin C examined by residual dipolar couplings.

Conformations of the regulatory domain of cardiac troponin C (cNTnC) were studied by means of residual dipolar couplings measured from samples dissolved in dilute liquid crystals. Changes in the main chain HN residual dipolar couplings revealed a conformational change in cNTnC due to the complexation with the second binding region (amino acids 148-163) of cardiac troponin I (cTnI). Formation of the complex is accompanied with a molecular realignment in the liquid crystal. The residual dipolar couplings measured for apo-cNTnC and the complex with TnI were in agreement with the values computed from the corresponding closed and open solution structures, whereas for the calcium-loaded conformation the correlation and quality factor were only modest. Ca2+-cNTnC may be subject to conformational exchange. The data support the model that cardiac troponin C functions as a calcium-dependent open-closed switch, such as the skeletal troponin C.

Amino Acid Sequence↗

PubMed2000

Calcium sensitivity enhancers.

Patients suffering from acute decompensated heart failure, immediately after infarction, or late in the progression of heart failure, need short-term, positive inotropic support in their therapy. To date, drugs acting through cAMP are used to increase the contractile force of the heart of such patients, although it is well-known that these kind of drugs may trigger arrhythmias and as a result may worsen the long-term prognosis of the patients. Recently, levosimendan, a new drug acting through calcium sensitization of contractile proteins, has shed new light on inotropic therapy, and, importantly, has reduced mortality in acute heart failure patients. For this reason, this review focuses on calcium sensitizers and the mechanisms behind calcium sensitization. The compounds which have been selected for detailed consideration are limited only to positive inotropic compounds that produce calcium sensitization of contractile proteins. In addition, this review elucidates the differences between various calcium sensitizing mechanisms, and mainly focuses on the relaxation of cardiac muscle.

Journal Article↗

PubMed1999

Structural determination of the acidic exopolysaccharide produced by a Pseudomonas sp. strain 1.15.

Pseudomonas strain 1.15 was isolated from a freshwater biofilm and shown to produce considerable amounts of an acidic polysaccharide which was investigated by methylation analysis, NMR spectroscopy and ionspray mass spectrometry (ISMS). The polysaccharide was depolymerised by a bacteriophage-associated endoglucosidase and by autohydrolysis, and the resulting oligosaccharides were investigated by NMR spectroscopy and mass spectrometry. The resulting data showed that the parent repeating unit of the 1.15 exopolysaccharide (EPS) is a branched hexasaccharide. The main chain is constituted of the trisaccharide -->4)-alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->3)-beta-D-Glcp- (1--> and the side chain alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->3)-alpha-D-Galp-(1-->is linked to O-3 of the first Fuc residue. The terminal non-reducing Gal carries a 1-carboxyethylidene acetal in the R configuration at the positions 4 and 6. Of the four different O-acetyl groups present in non-stoichiometric amounts, two were established to be on O-2 of the 3-linked Gal and on O-2 of the 4-linked Fuc.

Carbohydrate Sequence↗

PubMed1999

Structure of the 1-36 amino-terminal fragment of human phospholamban by nuclear magnetic resonance and modeling of the phospholamban pentamer.

The structure of a 36-amino-acid-long amino-terminal fragment of phospholamban (phospholamban[1-36]) in aqueous solution containing 30% trifluoroethanol was determined by nuclear magnetic resonance. The peptide, which comprises the cytoplasmic domain and six residues of the transmembrane domain of phospholamban, assumes a conformation characterized by two alpha-helices connected by a turn. The residues of the turn are Ile18, Glu19, Met20, and Pro21, which are adjacent to the two phosphorylation sites Ser16 and Thr17. The proline is in a trans conformation. The helix comprising amino acids 22-36 is well determined (the root mean square deviation for the backbone atoms, calculated for a family of 18 nuclear magnetic resonance structures is 0.57 A). Recently, two molecular models of the transmembrane domain of phospholamban were proposed in which a symmetric homopentamer is composed of a left-handed coiled coil of alpha-helices. The two models differ by the relative orientation of the helices. The model proposed by,Simmerman et al. (H.K. Simmerman, Y.M. Kobayashi, J.M. Autry, and L.R. Jones, 1996, J. Biol. Chem. 271:5941-5946), in which the coiled coil is stabilized by a leucine-isoleucine zipper, is similar to the transmembrane pentamer structure of the cartilage oligomeric membrane protein determined recently by x-ray (V. Malashkevich, R. Kammerer, V Efimov, T. Schulthess, and J. Engel, 1996, Science 274:761-765). In the model proposed by Adams et al. (P.D. Adams, I.T. Arkin, D.M. Engelman, and A.T. Brunger, 1995, Nature Struct. Biol. 2:154-162), the helices in the coiled coil have a different relative orientation, i.e., are rotated clockwise by approximately 50 degrees. It was possible to overlap and connect the structure of phospholamban[1-36] derived in the present study to the two transmembrane pentamer models proposed. In this way two models of the whole phospholamban in its pentameric form were generated. When our structure was connected to the leucine-isoleucine zipper model, the inner side of the cytoplasmic domain of the pentamer (where the helices face one another) was lined by polar residues (Gln23, Gln26, and Asn30), whereas the five Arg25 side chains were on the outer side. On the contrary, when our structure was connected to the other transmembrane model, in the inner side of the cytoplasmic domain of the pentamer, the five Arg25 residues formed a highly charged cluster.

Amino Acid Sequence↗

PubMed1999

Regulation of total mitochondrial Ca2+ in perfused liver is independent of the permeability transition pore.

Triggering of the permeability transition pore (PTP) in isolated mitochondria causes release of matrix Ca2+, ions, and metabolites, and it has been proposed that the PTP mediates mitochondrial Ca2+ release in intact cells. To study the role of the PTP in mitochondrial energy metabolism, the mitochondrial content of Ca2+, Mg2+, ATP, and ADP was determined in hormonally stimulated rat livers perfused with cyclosporin A (CsA). Stimulation of livers perfused in the absence of CsA with glucagon and phenylephrine induced an extensive uptake of Ca2+, Mg2+, and ATP plus ADP by the mitochondria, followed by a release on omission of hormones. In the presence of CsA, the PTP was fully inhibited, but neither the hormone-induced uptake of Ca2+, ATP, or ADP by mitochondria nor their release after washout of hormones was significantly changed. We conclude that the regulation of sustained changes in mitochondrial Ca2+ content induced by hormonal stimulation is independent of the PTP.

Adenosine Diphosphate↗

PubMed1998

Sensitivity of chondrocytes of growing cartilage to reactive oxygen species.

Vascular invasion of calcified cartilage, during endochondral ossification, is initiated and sustained by invasive cells (endothelial cells and macrophages) which degrade the tissue by releasing lytic enzymes. Concurrently, reactive oxygen species (ROS) are also released by these cells and we hypothesize that ROS also contribute to the degradation of the tissue. As a preliminary approach to this problem, the antioxidant activities and the effect of ROS on hypertrophic cartilage and chondrocytes (HCs) were investigated. Compared to resting or articular chondrocytes, HCs exhibited higher catalase but lower SOD specific activities and lower PHGPx concentration, thus revealing a defence activity specific against H2O2. Moreover, dose-dependent depletion of ATP occurred after few minutes of exposure to ROS, and a long-term treatment (16 h incubation with ROS) promoted the release of LDH activity and a significant variation of the poly- to mono-unsaturated fatty acid ratio. Finally, the incubation of HCs with low ROS doses induced the release of sedimentable alkaline phosphatase activity (matrix vesicles). How the obtained results fit the in vivo occurring events is discussed.

Adenosine Triphosphate↗

PubMed1998

Solution structure and main chain dynamics of the regulatory domain (Residues 1-91) of human cardiac troponin C.

The three-dimensional structure of calcium-loaded regulatory, i.e. N-terminal, domain (1-91) of human cardiac troponin C (cNTnC) was determined by NMR in water/trifluoroethanol (91:9 v/v) solution. The single-calcium-loaded cardiac regulatory domain is in a "closed" conformation with comparatively little exposed hydrophobic surface. Difference distance matrices computed from the families of Ca2+-cNTnC, the apo and two-calcium forms of the skeletal TnC (sNTnC) structures reveal similar relative orientations for the N, A, and D helices. The B and C helices are closer to the NAD framework in Ca2+-cNTnC and in apo-sNTnC than in 2.Ca2+-sNTnC. However, there is an indication of a conformational exchange based on broad 15N resonances for several amino acids measured at several temperatures. A majority of the amides in the alpha-helices and in the calcium binding loop exhibit very fast motions with comparatively small amplitudes according to the Lipari-Szabo model. A few residues at the N and C termini are flexible. Data were recorded from nonlabeled and 15N-labeled samples, and backbone dynamics was investigated by 15N T1, T2, and heteronuclear nuclear Overhauser effect as well as by relaxation interference measurements.

Amino Acid Sequence↗

PubMed1998

Structural investigation of the exopolysaccharide produced by Pseudomonas flavescens strain B62--degradation by a fungal cellulase and isolation of the oligosaccharide repeating unit.

Pseudomonas flavescens strain B62 (NCPPB 3063) is a recently described bacterium isolated from walnut blight cankers. This strain has been designated as the type strain of a Pseudomonas rRNA group-I species. Strain B62 produced a mixture of two exopolysaccharides, differing in weight average relative molecular mass and composition. Only the most abundant exopolysaccharide (90% by mass), corresponding to the one with the lower molecular mass, was investigated by use of methylation analysis, partial acid hydrolysis, and NMR spectroscopy. The polysaccharide was depolymerised by the action of the cellulase produced by Penicillum funiculosum and the oligosaccharide obtained, corresponding to the repeating unit, was characterised by NMR spectroscopy and ion-spray mass spectrometry. The repeating unit of the B62 exopolysaccharide is [structure in text] where X is glucose (75%) or mannose (25%), and Lac is lactate. The O-acetyl groups are present only on 75% of the repeating units, and they are linked to the C6 of the hexose residues in non-stoichiometric amounts.

Carbohydrate Conformation↗

PubMed1997

Cellular UDP-glucose deficiency caused by a single point mutation in the UDP-glucose pyrophosphorylase gene.

We previously isolated a mutant cell that is the only mammalian cell reported to have a persistently low level of UDP-glucose. In this work we obtained a spontaneous revertant whose UDP-glucose level lies between those found in the wild type and the mutant cell. The activity of UDP-glucose pyrophosphorylase (UDPG:PP), the enzyme that catalyzes the formation of UDP-glucose, was in the mutant 4% and in the revertant 56% of the activity found in the wild type cell. Sequence analysis of UDPG: PP cDNAs from the mutant cell showed one missense mutation, which changes amino acid residue 115 from glycine to aspartic acid. The substituted glycine is located within the largest stretch of strictly conserved residues among eukaryotic UDPG:PPs. The analysis of the cDNAs from the revertant cell indicated the presence of an equimolar mixture of the wild type and the mutated mRNAs, suggesting that the mutation has reverted in only one of the alleles. In summary, we demonstrate that the G115D substitution in the Chinese hamster UDPG:PP dramatically impairs its enzymatic activity, thereby causing cellular UDP-glucose deficiency.

Amino Acid Sequence↗

PubMed1997

Organic solvent systems for 31P nuclear magnetic resonance analysis of lecithin phospholipids: applications to two-dimensional gradient-enhanced 1H-detected heteronuclear multiple quantum coherence experiments.

31P NMR of lipid extracts is a reproducible, rapid, and nondegradative method for qualitative and quantitative analyses of phospholipid mixtures. This analysis, however, is hampered by the instability of the solvent system commonly used for NMR spectroscopy (CHCl3/ CH3OH/H2O-EDTA). In this work we have investigated the effects of several monophasic solvent mixtures to overcome this disadvantage. Among these mixtures we have selected a solution of triethylamine, dimethylformamide, and guanidinium chloride (Et3N/DMF-GH+) as the most efficient system. In this solvent the chemical shift dispersion of the 31P signals is about four times the frequency range observed in the standard chloroform-methanol-water system. Moreover, the stability of this solvent, as a monophasic system, allows easy reproducibility of the analysis. The use of two-dimensional 1H-31P gradient-enhanced heteronuclear multiple quantum coherence experiments can further exploit the higher resolution of the signals obtained with this solvent system for the structure elucidation of known and unidentified phospholipids.

Acetamides↗

PubMed1997

Noninvasive in vivo quantitative assessment of fat content in human liver.

BACKGROUND/AIMS: Since the introduction of ultrasonography, liver steatosis has become an increasingly frequent diagnosis. Both ultrasonography (US) and computerized tomography (CT) provide qualitative rather than quantitative assessment of fatty infiltration. The objective of this study was to develop a noninvasive method for the quantification of the hepatic fat content in vivo. A test object containing solutions with CT scan density (CTD) similar to normal liver ("liver-equivalent") or "fat-equivalent material" in variable proportions was prepared to measure patients with variable degrees of steatosis in vivo. RESULTS: A linear correlation (r=0.99, p<0.001) linked CTD and the increasing percentage of fat-equivalent material. A CTD calibration curve was derived as a reference for the in vivo determinations. In 29 consecutive patients with steatosis diagnosed by histology, CTD was linearly correlated (r=0.83, p<0.001) with the hepatic fat content (HFC) expressed as percent of the whole liver, obtained by a computerized histomorphometric analysis. Based on the calibration curve obtained in 29 subjects who underwent liver biopsy, 38 additional consecutive steatotic patients were examined and the degree of hepatic fat content was calculated. The HFC was linearly correlated (r=-0.86, p<0.001) with the liver-to-spleen ratio. CONCLUSIONS: We conclude that the use of test objects allows an accurate and reproducible noninvasive quantitative assessment of hepatic fat infiltration in humans. This technique may prove useful in the evaluation of the natural course and treatment of hepatic steatosis as well as in the assessment of donor livers prior to transplantation.

Adult↗

PubMed1996

Analysis of total lipid extracts from human liver by 13C and 1H nuclear magnetic resonance spectroscopy.

Lipid extracts of liver tissue from normal donors and from patients with acute liver failure were analyzed by 13C NMR and 1H NMR at 9.4 T. The spectra allowed estimation of (i) the free fatty acid to total fatty acid chains ratio, (ii) the polyunsaturated to monounsaturated lipids ratio, (iii) the glycerophospholipids to triacylglycerols ratio, (iv) the total cholesterol to total fatty acids molar ratio, (v) the acylcholesterol to cholesterol ratio, (vi) the phosphatidylcholine to phosphatidylethanolamine molar ratio, and (vii) the unsaturation ratio of the fatty acid chains. The values obtained for normal livers agree with literature data on lipid composition obtained by other techniques. The two pathologic livers differ from the normal in the composition of some lipids and show a higher free fatty acid to total fatty acid chains ratio and a lower polyunsaturated to monounsaturated lipid ratio, a lower acylcholesterol to cholesterol ratio, and a lower phosphatidylcholine to phosphatidylethanolamine ratio, whereas the ratio of total cholesterol to fatty acids and the unsaturation ratio remained unchanged. The detailed assignment of the 13C NMR spectra of lipid extracts from normal and pathologic human liver may contribute to direct future research programs on 13C NMR spatially localized spectroscopy of the liver in vivo.

Cholesterol↗