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R Roberti

Publications and source records attributed to R Roberti.

At least 19 recordsLinked to original sources
PubMed2004

Binding and release of cytochrome c in brain mitochondria is influenced by membrane potential and hydrophobic interactions with cardiolipin.

Factors influencing the release and anchorage of cytochrome c to the inner membrane of brain mitochondria have been investigated. Metabolic activity of mitochondria caused a decrease in the membrane potential delta psi(m), accompanied by detachment of the protein from the inner membrane. In a model system of cytochrome c reconstituted in cardiolipin (CL) liposomes, phosphate was used to breach the hydrophilic lipid-protein interactions. About 44% cytochrome c was removable when heart CL (80% 18:2n-6) was employed, whereas the remaining protein accounted for the tightly bound conformation characterized by hydrophobic lipid-protein interactions. Cytochrome c release from brain CL liposomes was higher compared to heart CL, consistent with lower polyunsaturated fatty acid content. The release was even higher with CL extracted from metabolically stressed mitochondria, exhibiting more saturated fatty acid profile compared to control (30% vs. 17%). Therefore, weakening of the hydrophobic interactions due to saturation of CL may account for the observed cytochrome c release from mitochondria following metabolic stress. Moreover, mitochondria enriched with polyunsaturated CL exhibited higher delta psi(m), compared to less unsaturated species, suggesting that CL fatty acid composition influences delta psi(m). Mitochondria incorporated exogenous cytochrome c without protease-sensitive factors or delta psi(m). The internalized protein anchored to the inner membrane without producing swelling, as monitored by forward and side light scattering, but produced delta psi(m) consumption, suggesting recovery of respiratory activity. The delta psi(m) decrease is ascribed to a selected mitochondrial population containing the incorporated cytochrome c.

Animals↗
PubMed2001

Acidic pH generated by H+-ATPase pumps triggers the activity of a fusogenic protein associated with rat liver endoplasmic reticulum.

Fusogenic protein (FP) is a glycoprotein ( approximately 50 kDa), previously purified by us from rat liver endoplasmic reticulum, which explicates fusogenic activity at acidic pH in vitro. To suggest a possible role of FP in membrane fusion, the topology of the protein in the membrane and the conditions in which FP is operating in microsomes have been investigated. Anti-FP polyclonal antibodies inhibited pure FP activity, but not the protein activity in microsomes, suggesting interaction of antibodies with a part of FP concealed in intact membranes. FP activity in microsomes was lost after treatment with Pronase. Western blot analysis of Pronase-treated microsomes showed that the proteolysis removed a fragment ( approximately 5 kDa). This fragment is exposed on the outer surface of microsomes and involved in fusogenic activity, whereas the largest part of FP is embedded in microsomal vesicles. Therefore, FP can be affected by modifications on the cytosolic and luminal sides of microsomal membranes. Indeed, when microsomal lumen was acidified by H+-ATPase activity, binding and fusion of fluorescent labelled liposomes to microsomes occurred. Direct involvement of FP in the fusogenic event was observed by reconstituting pure FP in liposomes with a preformed H+ gradient. FP triggered a fusion process in response to the acidic interior of liposomes, despite an exterior 7.4 pH unable to promote fusogenic protein activity. As intracellular membrane fusion occurs at neutral pH involving the cytosolic sides of membranes, FP may participate in this event by exploiting the acidic pH formed in the lumen of endoplasmic reticulum through H+-translocating ATPase activity.

Adenosine Triphosphate↗
PubMed2000

Respiratory state and phosphatidylserine import in brain mitochondria in vitro.

The mechanism of phosphatidylserine (PS) movement from donor membranes into rat brain mitochondria was investigated. Mitochondria were incubated with liposomes and subjected to density gradient centrifugation. The energized state was monitored by flow cytometry measuring the fluorescence of membrane-potential-sensitive rhodamine-123 dye. Mitochondria density decreased upon increase of the respiratory rate, as a consequence of their association with liposomes. After interaction of mitochondria with (14)C-PS containing liposomes, (14)C-PS became a substrate of PS decarboxylase, as monitored by the formation of (14)C-phosphatidylethanolamine (PE), indicating translocation of (14)C-PS to the inner membrane. The kinetics of (14)C-PE formation showed a high rate upon addition of ADP, malate and pyruvate (state 3) compared to control (state 1). In state 3, (14)C-PE formation decreased in the presence of NaN(3). Mitochondria-associated membranes (MAM) are the major site of PS synthesis. However, their role in the translocation of PS to mitochondria has not been completely elucidated. A crude mitochondrial fraction (P(2)) containing MAM, synaptosomes and myelin was prelabeled with (14)C-PS and incubated in different respiratory states. At a high respiratory rate, low-density labeled mitochondria, whose band overlaps that of synaptosomes, were obtained by centrifugation. A parallel decrease of both radioactivity and protein in MAM fraction was observed, indicating that the association of MAM and mitochondria had occurred. Synthesis and translocation of (14)C-PS in P(2) membranes were also studied by incubating P(2) with (14)C-serine. In the resting state (14)C-PS accumulated in MAM, indicating that the transfer to mitochondria was a limiting step. In state 3 both the transfer rate of (14)C-PS and its conversion to (14)C-PE increased. Respiratory mitochondrial activity modulated the association of MAM and mitochondria, triggering a mechanism that allowed the transport of PS across the outer mitochondrial membrane.

Adenosine Diphosphate↗
PubMed1999

Purification of ethanolaminephosphotransferase from bovine liver microsomes.

CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (EC 2. 7.8.1) has been purified to electrophoretic homogeneity and in a catalytically active form from bovine liver microsomes. The purification method is based on the high hydrophobicity of the protein whose charged sites appear to be masked from the interaction with the chromatographic stationary phases when membranes are solubilized with an excess of non-ionic detergent. The isolated protein has a molecular mass of about 38 kDa, as estimated by SDS-PAGE mobility, and exhibits both ethanolaminephosphotransferase and cholinephosphotransferase activities. Evidence is given that both activities are Mn2+-dependent and that the same catalytic site is involved in cholinephosphotransferase and ethanolaminephosphotransferase reactions. Mg2+-dependent CDP-choline:diacylglycerol cholinephosphotransferase (EC 2.7.8.2) is completely inactivated during the solubilization and purification steps.

Animals↗
PubMed1999

Determinants of psychiatric inpatient admission to general hospital psychiatric wards: an epidemiological study in a region of central Italy.

OBJECTIVE: To determine which factors contribute to the decision to admit individuals to psychiatric wards in general hospitals. METHOD: Data on 1,379 individuals undergoing psychiatric evaluation in eight emergency rooms in a region of central Italy were collected. A logistic regression analysis was used to evaluate the likelihood of psychiatric admission considering the independent effects of demographic, social, and clinical factors and of the history of psychiatric treatment. RESULTS: The adjusted odds ratio for psychiatric admission significantly increased with the following variables: severity of symptoms; presence of paranoid states and schizophrenic psychoses, affective psychoses and acute psychotic conditions (with neurotic disorders used as reference); a history of outpatient treatment; the presence of a staff member of a community mental health facility upon presentation at the emergency room; and the availability of beds in the psychiatric ward. CONCLUSION: The independent effect played by the presence of a staff member of a community mental health facility is of particular interest, suggesting the existence of a collaborative relationship between inpatient and outpatient services.

Adult↗
PubMed1998

A glycoprotein from rat liver endoplasmic reticulum promotes both aggregation and fusion of liposomes at acidic pH.

Low-pH-induced fusion of liposomes with rat liver endoplasmic reticulum was evidenced. Fusion was inactivated by treatment of microsomes with trypsin or EEDQ (N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline), indicating the involvement of a protein. The protein was purified 555-fold by chromatographic steps. The identification and purification to homogeneity was obtained by electroelution from a slab gel, which gave a still active protein of about 50 kDa. The protein promoted the fusion of liposomes; laser light scattering showed an increase of mean radius of vesicles from 60 up to about 340 nm. Fusion was studied as mass action kinetics, describing the overall fusion as a two-step sequence of a second order aggregation followed by a first order fusion of liposomes. For phosphatidylcholine containing liposomes aggregation was not rate-limiting at pH 5.0 and fusion followed first order kinetics with a rate constant of 13 . 10(-3) sec-1. For phosphatidylethanolamine/phosphatidic acid liposomes aggregation was rate-limiting; however, the overall fusion was first order process, suggesting that fusogenic protein influences both aggregation and fusion of liposomes. The protein binds to the lipid bilayer of liposomes, independently of pH, probably by a hydrophobic segment. Exposed carboxylic groups might be able to trigger pH-dependent aggregation and fusion. It is proposed that the protein inserted in the lipid bilayer bridges with an adjacent liposome forming a fused doublet. Since at endoplasmic reticulum level proton pumps are operating to generate a low-pH environment, the membrane bound fusogenic protein may be responsible for both aggregation and fusion of neighboring membranes and therefore could operate in the exchange of lipidic material between intracellular membranes.

Animals↗
PubMed1997

Detection of coronary collaterals using dipyridamole PET myocardial perfusion imaging with rubidium-82.

UNLABELLED: This study evaluated the ability of dipyridamole PET myocardial perfusion imaging to detect coronary collaterals. A previous study showed an association between dipyridamole-induced coronary steal on PET imaging and the presence of coronary collaterals on angiography. METHODS: Dipyridamole PET myocardial perfusion imaging using 82Rb was performed in 45 patients who had recent coronary angiography. The stress/rest count ratio (rubidium activity with stress divided by activity at rest)-was used to express the change in regional tracer uptake with dipyridamole and was calculated manually and automatically. The accuracy of the stress/rest count ratio for detecting coronary collaterals was determined. RESULTS: A manual stress/rest count ratio < or = 0.80 identified coronary collaterals with 81% sensitivity, 92% specificity and 90% accuracy (p < 0.0001). An automated ratio < or = 0.80 had 90% sensitivity, 88% specificity and 90% accuracy (p < 0.0001). Vascular beds incorrectly identified by PET as having collaterals had an increased frequency of severe stenoses and abnormal wall motion. CONCLUSION: PET perfusion imaging using the stress/rest count ratio can serve as a unique imaging method to identify coronary collaterals noninvasively.

Collateral Circulation↗
PubMed1996

Quantitation of glycerophosphorylcholine by flow injection analysis using immobilized enzymes.

A method for quantitating glycerophosphorylcholine by flow injection analysis is reported in the present paper. Glycerophosphorylcholine phosphodiesterase and choline oxidase, immobilized on controlled porosity glass beads, are packed in a small reactor inserted in a flow injection manifold. When samples containing glycerophosphorylcholine are injected, glycerophosphorylcholine is hydrolyzed into choline and sn-glycerol-3-phosphate. The free choline produced in this reaction is oxidized to betain and hydrogen peroxide. Hydrogen peroxide is detected amperometrically. Quantitation of glycerophosphorylcholine in samples containing choline and phosphorylcholine is obtained inserting ahead of the reactor a small column packed with a mixed bed ion exchange resin. The time needed for each determination does not exceed one minute. The present method, applied to quantitate glycerophosphorylcholine in samples of seminal plasma, gave results comparable with those obtained using the standard enzymatic-spectrophotometric procedure. An alternative procedure, making use of co-immobilized glycerophosphorylcholine phosphodiesterase and glycerol-3-phosphate oxidase for quantitating glycerophosphorylcholine, glycerophosphorylethanolamine and glycerophosphorylserine is also described.

Choline↗
PubMed1996

Isolated guinea pig gastric chief cells express tumour necrosis factor receptors coupled with the sphingomyelin pathway.

The tumour necrosis factor alpha (TNF), has been implicated in the pathogenesis of non-steroidal anti-inflammatory drug (NSAID) induced gastropathy and Helicobacter pylori induced gastritis. Both conditions are characterised by high plasma pepsinogen concentrations, which are thought to reflect an increased rate of enzyme release by the pepsinogen secreting (chief) cells. The mechanisms responsible for this cell dysfunction are unknown. This study investigates whether chief cells express TNF receptors and, if so, whether their activation results in cell death. Immunohistochemical studies conducted with monoclonal antibodies (mAbs) directed against two TNF receptor associated proteins of 55 kDa (TNF-R1) and 75 kDa (TNF-R2) showed that TNF binding sites were expressed in approximately 100% gastric chief cells. Western blot analysis of whole chief cell lysates probed with the TNF-R1 and TNF-R2 mAbs gave two distinct bands of 55 and 75 kDa in the immunoprecipitate. Incubating chief cells with TNF caused concentration and time dependent cell death, which was prevented by pretreating the cells with anti-TNF receptor mAbs. Exposing the cells to TNF reduced sphingomyelin content by 25%. Sphingomyelinase (10(-6) to 10(-2) IU/ml) mimicked the effect of TNF in that it provoked a concentration and time dependent reduction in chief cell viability and increased pepsinogen release. In conclusion, gastric chief cells express two TNF receptors partially linked to the sphingomyelin pathway. TNF induced chief cell dysfunction might be responsible for the high plasma pepsinogen concentrations seen in patients with NSAID gastropathy or H pylori induced gastritis.

Animals↗
PubMed1995

Serial myocardial perfusion imaging with dipyridamole and rubidium-82 to assess restenosis after angioplasty.

UNLABELLED: The purpose of this study was to determine whether patients at high risk for clinical restenosis, following coronary angioplasty, could be identified by myocardial perfusion imaging performed with dipyridamole- 82Rb PET. METHODS: Forty-five patients (34 men, 11 women; mean age 58.5 yr) who had successful single-vessel angioplasty and were asymptomatic had dipyridamole-82Rb PET at 1 and 3 mo after the procedure. Abnormal flow reserve in the distribution of the angioplasty artery on PET was considered to be a decrease of > or = 1 perfusion grade in response to dipyridamole (assessed qualitatively from tomographic images and polar coordinate maps). Follow-up was performed for 6 mo postangioplasty. Clinical restenosis was defined as recurrent angina similar to that occurring before angioplasty and/or > or = 50% stenosis at the angioplasty site documented angiographically. We analyzed abnormal flow reserve in the distribution of the angioplasty vessel to identify which patients were at high risk for clinical restenosis. RESULTS: Fourteen patients developed clinical restenosis between 1 and 6 mo postangioplasty. Abnormal relative flow reserve in the distribution of the angioplasty vessel was present prior to the development of symptoms in 13 of 14 patients with clinical restenosis and in 8 of 31 patients without clinical restenosis (sensitivity 93%, specificity 74%, p < 0.0001). PET imaging successfully separated postangioplasty patients into groups with high (62%) and low (4%) risk of clinical restenosis. CONCLUSION: Abnormal relative flow reserve in the distribution of the angioplasty vessel on dipyridamole PET identifies asymptomatic postangioplasty patients at risk for clinical restenosis.

Angioplasty, Balloon, Coronary↗
PubMed1994

Exercise echocardiographic correlates of transient dilatation of the left ventricular cavity on exercise thallium-201 SPECT imaging.

Transient dilatation of the left ventricular cavity on exercise thallium perfusion imaging is recognized as a marker of significant coronary disease, but the mechanisms that produce this finding are not fully understood. We studied 32 patients who underwent exercise thallium imaging and exercise echocardiography to determine the changes in left ventricular cavity size that underlie transient dilatation. Left ventricular area from the apical four-chamber view was used to approximate left ventricular cavity size. There were 24 patients who did not have transient dilatation (group 1) and 8 patients who did have transient dilatation (group 2) on thallium imaging. Systolic area decreased from rest to exercise in group 1 patients but not in group 2 patients. There was no significant change in diastolic area from rest to exercise in either group 1 or group 2 patients. Thus, exercise-induced systolic dysfunction, manifested as a failure to decrease left ventricular systolic cavity size in exercise, may be an important mechanism in producing scintigraphic transient dilatation.

Echocardiography↗
PubMed1993

Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C.

Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P-labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

Blood Platelets↗
PubMed1992

Reversibility of the reactions catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase solubilized from rat-brain microsomes.

The incorporation of CMP into CDP-ethanolamine and CDP-choline, catalyzed by ethanolaminephosphotransferase (EC 2.7.8.1) and cholinephosphotransferase (EC 2.7.8.2), respectively, has been studied in solubilized preparations of rat-brain microsomes. Mn2+ ions were required for the maximal activity of both enzymes. The CMP concentration needed to reach the half-maximal reaction rate was 1.6 microM for both activities. The rate of incorporation of CMP into CDP-choline and CDP-ethanolamine was increased by increasing the concentration of phosphatidylcholine and phosphatidylethanolamine, respectively, in detergent-phospholipid micellar systems. The rate of the reaction at pH 6.5 was comparable with that measured at pH 8.5, whereas the rate of synthesis of phosphatidylcholine and phosphatidylethanolamine, catalyzed by the same enzymes, increased with pH. Ethanolaminephosphotransferase, which catalyzes the synthesis of phosphatidylethanolamine from CDP-ethanolamine and diacylglycerol, was co-eluted with the enzyme activity catalyzing the reverse reaction, when solubilized microsomes were submitted to anion exchange chromatography on DEAE Bio-Gel A. Cholinephosphotransferase was inactivated during the chromatographic procedure.

Animals↗
PubMed1990

In vitro synthesis and transbilayer movement of phosphatidylethanolamine molecules labelled with different fatty acids in chick brain microsomes.

The transbilayer fatty acid distribution of diacylglycerophosphoethanolamine and the translocation of newly synthesized phosphatidylethanolamine molecules labelled with different fatty acids has been investigated in chick brain microsomes using trinitrobenzensulfonic acid. The determination of the fatty acid composition of diacylglycerophosphoethanolamine in both the outer and the inner leaflet of the microsomal vesicles revealed a similar distribution indicating that both leaflets share the same molecular species. The in vitro incorporation of radioactive fatty acids (16:0, 18:1 and 20:4(n-6] into ethanolamine phospholipids, known to be catalyzed by the lyosphosphatidylethanolamine acyl transferase, showed that the radioactive diacylglycerophosphoethanolamine molecules appeared first in the outer leaflet and were thereafter transferred to the inner leaflet. The apparent rate of translocation of the newly synthesized ethanolamine phospholipid molecules was the highest for those labelled with 16:0 and the lowest for those labelled with 20:4(n-6). The results indicate that the active site of the acyl-CoA:lysophosphatidylethanolamine acyltransferases is located on the outer leaflet of the microsomal vesicles and that the different newly synthesized molecular species of diacylglycerophosphoethanolamine may be translocated from the outer to the inner leaflet at different rates.

Animals↗
PubMed1990

Determination of phosphatidylcholine in a flow injection system using immobilized enzyme reactors.

Two alternative procedures are described for the quantitative determination of phosphatidylcholine in a flow-injection system utilizing immobilized enzymes. Phospholipase C from Bacillus cereus and phospholipase D from cabbage were covalently bound to the surface of controlled-pore glass beads and the enzyme-derivatized beads were packed in small columns. In the first procedure, the phospholipase C column was connected with a second column containing coimmobilized alkaline phosphatase and choline oxidase. In the alternative procedure, the column packed with immobilized phospholipase D was connected with a column packed with immobilized choline oxidase. The hydrogen peroxide produced through the action of choline oxidase in both flow-injection systems was detected amperometrically. Both procedures are suitable for an accurate and rapid quantitation of phosphatidylcholine. The sensitivity of the method based on phospholipase C and alkaline phosphatase is higher than that using phospholipase D. Quantitation of phosphatidylcholine at the nanomole level can be easily obtained using the first method.

Alcohol Oxidoreductases↗
PubMed1990

[Functional changes in the biliary system following extrahepatic biliary obstruction].

To assess the potential structural changes of the biliary tree and liver in patients with extrahepatic biliary obstruction, the resected specimens of 20 patients operated for benign biliary stricture were evaluated by means of immunocytochemical and histological methods. Furthermore, liver biopsies were taken for the same purposes. The results showed that in the dilated segment of the hepatic duct proximal to the stricture, innervation was greatly reduced or completely absent with associated advanced morphological and histological changes and high intrabiliary pressure levels. Similar findings were observed in the liver biopsies, too. These biopsies showed advanced morphological and histological changes associated with reduced innervation. By contrast, the nondilated segment of the hepatic duct, distal to the obstruction, showed normal innervation, normal morphology and histology and normal levels of intrabiliary pressure. The present study provides evidence that in cases of extrahepatic biliary obstruction, there are advanced pathological changes in the biliary tree associated with innervation impairment. These structural changes are associated with functional changes in both the liver and the biliary tree. Such functional changes represent a threat to the patient, particularly if major surgery is required. Increased biliary pressure appears to be a major cause of the development of these changes. Biliary drainage, either surgical or endoscopic, is indicated as the only alternative to reduce intrabiliary pressure and to contribute to a reversal of these structural and functional changes.

Bile↗