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S Stefanini

Publications and source records attributed to S Stefanini.

At least 37 records · Page 2Linked to original sources
PubMed1996

Thermal stability of horse spleen apoferritin and human recombinant H apoferritin.

The thermal stability of horse spleen apoferritin, a heteropolymer composed of 90% L and 10% H chains, has been studied by differential scanning calorimetry and compared with that of the human recombinant H homopolymer. The denaturation temperatures (Tm) are significantly higher for the horse spleen polymer than for the recombinant protein under all experimental conditions (e.g., at pH 7, Tm values are > or = 93 and 77 degrees C, respectively). The thermal denaturation process displays substantial reversibility for both polymers up to a few degrees below Tm, as indicated by CD measurements in the far and near uv regions. At temperatures higher than Tm the thermograms are influenced by the exothermic contribution of aggregation and/or precipitation. The H homopolymer thermogram, which is not distorted by the exotherm, is consistent with a multistate denaturation process. Acid dissociation of apoferritin produces stable dimeric subunits. The thermal unfolding of both dimeric subunits is reversible at least up to Tm and is characterized by an inversion of stability relative to the polymers (at pH 3.5, Tm is 42 degrees C for the horse spleen and 50 degrees C for the H subunit). These results indicate that the stabilization of the polymeric structure arises mainly from interactions between dimers, in accordance with the crystallographic evidence that the dimers are the building blocks of the polymeric molecule.

Animals↗
PubMed1996

Insulin binding and fluid-phase endocytosis stimulation in the mouse neuroblastoma cell line 41A3.

As well as many other hormones and growth factors, insulin is known to influence several processes in the CNS; its specific effects, however, are still poorly understood. Neuroblastoma cell lines represent a useful experimental system for the analysis of the insulin-specific effect on neurons, in the absence of possible regulatory mechanisms elicited by other neuronal/glial cells and/or soluble factors. The expression and the binding properties of insulin receptors, as well as the insulin effects on both membrane fluidity and cell surface architecture, have been investigated in 41A3 mouse neuroblastoma cells, by radioligand-binding fluorescence spectroscopy and scanning electron microscopy, respectively the same cells, insulin-induced modifications on cytoskeletal organisation also have been studied. Binding studies were performed using 125I-insulin, while the cationic fluorescent probe trimethylammonium 1,6-diphenyl-1,3,5-hexatriene was used for biophysical investigations. The results presented in this paper provide evidence that insulin interacts with 41A3 neuroblastoma cells through a receptor-mediated mechanism and that, in these cells, insulin binding modifies the cell surface morphology and stimulates endocytosis.

Animals↗
PubMed1996

Thallium-201 reverse redistribution at reinjection imaging correlated with coronary lesion, wall motion abnormality and tissue viability.

UNLABELLED: Previous studies based on standard stress-redistribution 201TI scintigraphy provided conflicting results about the clinical significance 201Tl reverse redistribution. Recent observations indicate that the majority of these defects normalize following reinjection reflecting viable myocardium. METHODS: In this study, the meaning of reverse redistribution occurring at reinjection imaging, its relation to standard 4-hr redistribution, coronary lesion, abnormal wall motion and tissue viability were assessed. A region with normal activity in the stress image was considered as having reverse redistribution if 201Tl activity at reinjection imaging was definitely abnormal with a decrease in relative tracer uptake >15% of the peak. From a series of 270 patients, 29 showed reverse redistribution. Of these 29 patients, 27 had evidence of previous myocardial infarction. Coronary lesions were detected in all but 1 patient. Average ejection fraction was 0.38 +/- 0.11. RESULTS: On a segmental basis, 50/377 regions showed the pattern of reverse redistribution. A significant coronary lesion (> or = 50%) was found in 78% of these regions; occlusion rate was 50%, and collateral circulation was found in 35% of occluded vessels. Hypokinesis or akinesis was present in 72% of segments. Tissue viability, defined as an uptake >55% of the peak, was found in 44% of these segments. The 50 segments showing reverse redistribution were divided into two groups according to an abnormal uptake also at 4-hr redistribution (group 1, 25 segments) or appearing only following reinjection (group 2,25 segments). Despite segments of group 1 showing a higher degree of coronary stenosis (80 +/- 32 versus 59 +/- 43%, p < 0.01), a similar rate of coronary occlusion, ventricular dysfunction and maintained viability was found in the two groups. CONCLUSION: Reverse redistribution in chronic coronary artery disease is frequently associated with significant coronary lesion, collateral-dependent dysfunctioning myocardium and preserved tissue viability. The occurrence of reverse redistribution following reinjection expands the indication for viability imaging to all patients with known coronary artery disease and regional wall motion abnormalities who undergo diagnostic and prognostic 201TI scintigraphy.

Coronary Angiography↗
PubMed1995

Morphometric analysis of liver and kidney peroxisomes in lactating rats and their pups after treatment with the peroxisomal proliferator di-(2-ethylhexyl)phthalate.

Di-(2-ethylexyl)phthalate (DEHP) administered to adult lactating rats from delivery to weaning induces age- and organ-specific modifications of the peroxisomal morphometric parameters (VV, NA and D) in the liver and kidney of both rats and their pups. In both tissues, peroxisomal relative volume and catalase biochemical activity show a similar pattern during the development, as well as under DEHP treatment. Morphometric results suggest that two modalities of peroxisomal proliferation exist, involving: a) increases in both number and mean diameter of the organelles; b) a purely numerical increase of the organelles, accompanied by a remarkable decrement in their mean diameter. A peroxisomal population proliferated through the latter model appears unable to return to normal conditions, following treatment withdrawal. These two proliferation systems, the first implying a swelling and the latter a fragmentation of pre-existing peroxisomal profiles, are supposed to be tissue-specific in the adult animal. In particular, in the liver the 'swelling' model appears more suitable to explain peroxisome proliferation, while the kidney this process would follow the 'fragmentation' model. Immature animals might instead show in both organs intermediate features of peroxisomal proliferation modalities.

Animals↗
PubMed1994

Differentiation of kidney cortex peroxisomes in fetal and newborn rats.

Peroxisomal enzyme assays as well as cytochemical detection of catalase were carried out on fetal and newborn rat kidney cortex throughout the last 3 days of prenatal life and the first month of postnatal development. Concerning the patterns of peroxisomal enzymes, catalase activity, hardly detectable in the fetus, shows the strongest increment after the second week of postnatal life; beta-oxidation system and D-amino acid oxidase increase soon after birth; urate oxidase activity, detected in fetal life, rapidly decreases after birth; dihydroxyacetone phosphate-acyltransferase activity doubles at birth, remaining constant thereafter. Since by cytochemistry no catalase particles were detected in fetal kidneys, morphometric parameters were studied only postnatally. The numerical density shows only minor variations, mainly at day 3; the mean diameter remains practically unchanged between birth and day 14 but strongly increases later. The volume density pattern correlates in the early phase with the numerical density and later with the profile mean diameter. The results suggest that enzymes are asynchronously incorporated into pre-existing peroxisomes; that this import is faster in smaller organelles than in the larger, adult ones; that catalase increases after the H2O2-producing oxidases; and that the abrupt rise of beta-oxidation capacity and DH-APAT is related to the increased renal work immediately after birth.

Animals↗
PubMed1994

Effects of Di-(2-ethylhexyl)phthalate on peroxisomes of liver, kidney and brain of lactating rats and their pups.

Di-(2-ethylhexyl)phthalate administered to adult lactating rats, from delivery to weaning, induces modifications of the peroxisomal enzymatic pattern in the liver, kidney and brain of both dams and pups. These modifications are age- and organ-dependent. Biochemical analysis shows that: 1) catalase specific activity is two-fold increased in the liver of both adult and newborn animals, in the kidney of newborns and in the brain of adults. 2)D-amino acid oxidase doubles in all newborn organs and in adult brain; it increases, although to a lesser extent, also in adult kidney, while it is half-reduced in adult liver. 3) Dihydroxyacetone phosphate acyl transferase only doubles in newborn liver, remaining fairly unchanged in all the other tested tissues. 4) Palmitoyl-CoA oxidase is greatly induced in the liver of both dams and litters, doubled in the kidneys and slightly increased or not at all in the brain of pups and mothers, respectively. The effect of the drug on enzyme activities is reversible, with different time courses depending on the considered enzyme and organ. Western blottings confirm the biochemical data. Electron microscopy shows proliferated peroxisomes in the liver and kidney of treated animals but not in the brain, where high catalase-like immunoreactivity is observed in the cytosol of neurons. Taken together, our data demonstrate that the response of peroxisomal enzymes to DEHP treatment is age- as well as tissue-dependent and specific for each enzyme studied.

Acyltransferases↗
PubMed1993

Nucleation of the iron core occurs at the three-fold channels of horse spleen apoferritin: an EXAFS study on the native and chemically-modified protein.

Extended X-ray absorbance fine structure measurements have been carried out on the initial Fe(III)-apoferritin complex at a Fe/subunit ratio of 2 in native and modified horse spleen apoferritin. Analysis of the data indicates that in the native protein the iron forms a protein-bound polynuclear cluster (Fe-Fe distance 3.4 A) with a first coordination sphere constituted by 5-6 low-Z atoms, e.g., nitrogen atoms, carboxylate-like ligands or oxo bridges between the iron atoms. Modification of Cys-126, a residue localized on the outer surface of the hydrophilic three-fold channels, with p-chloromercuribenzoate (PMB) or phenylmercuric acetate (PMA) brings about distinctive differences. In particular, in the PMB-reacted protein the feature assigned to the iron-iron interaction disappears from the spectrum, whilst in the PMA-reacted protein the main differences with respect to the native protein are observed at the level of the first coordination sphere. These results confirm the formation of protein-Fe(III)-clusters and localize these sites at the hydrophilic three-fold channels of horse spleen apoferritin.

Animals↗
PubMed1992

Liver peroxisomes in newborns from clofibrate-treated rats. I. A morphometric study of the recovery period.

Morphological and morphometric parameters (volume density (Vv), numerical density (NA) and mean diameter (D)) of newborn liver peroxisomes were measured throughout the first week of life in rats born to mothers treated with clofibrate (ethyl 2 p-chlorophenoxy isobutyrate) during the last five days of pregnancy. In control studies the same analyses were carried out in newborns from untreated rats. At birth (day 0), treated animals exhibited a proliferated, pleiomorphic peroxisomal population (higher Vv, NA and D, and a spread distribution of profile diameter with respect to the controls). In the subsequent two days, many peroxisomes disappeared (decrease of Vv and NA to values even lower than controls), with a persisting high pleiomorphism (no change of D and diameter distribution) in residual ones. Starting from day 3, and up to day 6, larger peroxisomes were no longer detectable in test animals, and a significant, not pleiomorphic proliferation took place (D and diameter distributions strictly comparable to the controls and progressively increasing Vv and NA). The correlation analysis validated these morphological results, from which it can be surmised that the postnatal peroxisome recovery period consists of a destructive phase followed by a proliferative one. The possible mechanism(s) of disposal of the excess of drug-induced peroxisomes are discussed.

Animals↗
PubMed1992

Liver peroxisomes in newborns from clofibrate-treated rats. II. A biochemical study of the recovery period.

The fatty-acyl-CoA beta-oxidation (FAO) and catalase activities, as well as membrane fluidity of liver peroxisomes of newborns from normal and clofibrate-treated rats were studied during the recovery period, ie, throughout the first week of postnatal life. In the test animals the enzyme activities, which are significantly higher than controls at birth return to normal levels showing a somewhat different time course with FAO rapidly decreasing to control values within three days but with catalase still higher than controls at day 6. The half-life and degradation rate (Kd) of FAO are identical to those calculated by us for the whole organelles and to those reported by others for total catalase in normal or clofibrate-treated adult animals in the presence of catalase inhibitors. Soluble catalase shows turnover values which are similar though not identical to those of FAO, while total catalase has a very long half-life and a low Kd. Peroxisomal membrane fluidity, as determined by fluorescence anisotropy of 1-anilinonaphthalene-8-sulfonate (ANS) bound to purified peroxisomal fractions is higher in tests than in controls, recovering normal values within 6 days. Our results demonstrate that liver peroxisomes of rats prenatally exposed to clofibrate return to control conditions within about 1 week. The turnover parameters of enzymes and the membrane fluidity values are discussed in terms of disposal mechanism(s) for the excess of induced peroxisomes.

Acyl-CoA Oxidase↗
PubMed1992

Insulin receptor in mouse neuroblastoma cell line N18TG2: binding properties and visualization with colloidal gold.

Insulin function in the nervous system is still poorly understood. Possible roles as a neuromodulator and as a growth factor have been proposed (Baskin et al., 1987, Ann. Rev. Physiol. 49, 335-347). Stable cell lines may provide an appropriate experimental system for the analysis of insulin action on the various cellular components of the central nervous system. We report here a study to investigate the presence and the properties of insulin specific binding sites in the murine neuroblastoma line, N18TG2, together with insulin action on cell growth and metabolism. Also, receptor internalization has been studied. Binding experiments, carried out in standard conditions at 20 degrees C, enabled us to demonstrate that these cells bind insulin in a specific manner, thus confirming previous findings on other cell lines. Saturation curves showed the presence of two binding sites with Kd 0.3 and 9.7 nM. Competition experiments with porcine and bovine insulin showed an IC50 of 1 and 10 nM, respectively. Competition did not occur in the presence of the unrelated hormones ACTH and FSH. Dissociation experiments indicated the existence of an internalization process of the ligand-receptor complex; this was confirmed by an ultrastructural study using gold conjugated insulin. As far as the insulin action in N18TG2 cells is concerned, physiological concentrations stimulate cell proliferation, whereas no stimulation of glucose uptake was observed, indicating that insulin action in these cells is not mediated by general metabolic effects. On the basis of these data, N18TG2 line appears to be a very suitable model for further studies of the neuronal type insulin receptors, and possibly insulin specific action on the nervous system.

Animals↗
PubMed1991

Iron entry route in horse spleen apoferritin. Involvement of the three-fold channels as probed by selective reaction of cysteine-126 with the spin label 4-maleimido-tempo.

Apoferritin has been selectively labeled with a maleimide nitroxide derivative at Cys-126, located in the hydrophilic 3-fold channels. Titration of this derivative with Fe(II), which gives rise to the initial Fe(III)-apoferritin complex, produces, at low metal-to-protein ratios, a decrease of the intensity of the label EPR signal due to the occurrence of a magnetic dipolar interaction. A label-metal distance ranging between 8-12 A can be estimated from titrations performed with VO(IV), which is known to bind in the 3-fold channels, and likewise produces a decrease in the label EPR signal. The present findings indicate that iron binds in the hydrophilic channels in its higher oxidation state and that these channels represent the metal entry route at least at low metal-to-protein ratios.

Animals↗
PubMed1991

The interaction of hydroxypyridinones with human serum transferrin and ovotransferrin.

The interaction of hydroxypyridinones with human serum transferrin and ovotransferrin has been studied by analyzing the distribution of iron between the chelator and the proteins as a function of both ligand concentration and transferrin saturation. The kinetics of iron removal by 3-hydroxypyridin-4-ones from both transferrins is slow; in ovotransferrin it appears to be monophasic, in contrast to that observed for serum transferrin. After 24 hours incubation at a 40:1 chelator:protein molar ratio, the percentage of iron removed from Fe(III)-ovotransferrin is 50%-60%, and is somewhat higher in the case of serum transferrin, in line with the respective affinity constants for the metal. The 3-hydroxypyridin-2-ones and the 3-hydroxypyran-4-ones, both of which have lower affinities for Fe(III), remove smaller proportions of the metal. The percentage of desaturation obtained with bidentate and hexadentate pyridinones appears to be similar for both transferrin classes at chelator:protein molar ratios from 40:1. The degree of transferrin saturation influences the extent of chelator mediated iron mobilization in the case of serum transferrin, but not of ovotransferrin. 59Fe competition studies demonstrate that bidentate pyridin-4-ones are capable of donating iron to serum apotransferrin; the relative concentrations of ligand and protein influence the distribution of iron because their effective binding constants (at pH 7.4) for Fe(III) are similar.

Apoproteins↗
PubMed1989

Identification of the iron entry channels in apoferritin. Chemical modification and spectroscopic studies.

The knowledge of the route through which iron can enter and leave the apoferritin shell is a prerequisite for the understanding of ferritin's function. The involvement of the hydrophilic 3-fold channels in the iron uptake process has been studied by taking advantage of the reactivity of specific residues that line such channels, i.e., glutamic acid-127 and aspartic acid-130, the major Cd(II) binding sites, and cysteine-126. 113Cd NMR experiments have provided direct evidence for the competition between Fe(II) and Cd(II) binding to major Cd(II) binding sites on the protein and or a higher affinity of Fe(II) for these sites, in line with the well-known inhibitory effect of Cd(II) on iron uptake. Further evidence for the use of the 3-fold channels in the iron entry process has been obtained by means of chemical modification of Cys-126 with different mercurials. In particular, the introduction of the additional carboxylate carried by p-(chloromercuri)benzoate near Asp-127 and Glu-130 increases the initial rate of iron uptake and affects the coordination geometry of the metal in the Fe(III)-apoferritin complex as indicated by optical absorption and EPR data. The assignment of these effects to the carboxylate moiety of p-(chloromercuri)benzoate is brought out by the observation that the introduction in the 3-fold channel of the benzene ring only by means of phenylmercuric acetate has no effect on the initial iron uptake kinetics and on the spectroscopic properties of the Fe(III)-apoferritin complex.

Animals↗
PubMed1989

Proline-incorporating cells in chronic active liver diseases.

The incorporation of 3H-proline in cells of liver biopsy specimens from patients with chronic active liver diseases has been studied by light and electron microscopic autoradiography. The labeled proline is incorporated by hepatocytes of the external rows of the residual liver lobule, by the cells of the proliferating bile ductule and very actively by the plasma cells localized at the boundary between the inflammatory infiltrate and the liver lobule. These plasma cells, which are often in close contact with the hepatocytes at the edge of the liver lobule, appear to be either negative or positive after the immunohistochemical tests for the k and lambda chains of immunoglobulins. Results are discussed in relation to both the synthesis of collagen and the role of the immunocompetent cells during the process of the piecemeal necrosis.

Autoradiography↗
PubMed1989

Proliferative response of foetal liver peroxisomes to clofibrate treatment of pregnant rats. A quantitative evaluation.

Liver peroxisomes during prenatal development were studied by means of morphological and morphometric analysis in foetuses growing both in untreated and in clofibrate-treated rats. Pregnant rats were fed a standard diet (25 g/d) containing or not 0.8% clofibrate during the week preceding sacrifice and livers were excised from 13, 15, 17, 19 and 21-day old foetuses and newborn rats. The morphometric analysis of hepatocyte peroxisomes, performed by a semiautomatic method in specimens incubated with 3,3' diaminobenzidine, shows that the peroxisome volume density and average diameter in test animals were significantly increased over the control values. While the increase in the average diameter was roughly the same (X 1.4) at all examined stages, the volume density increased over the control values particularly in foetuses over 19d-old and in newborn rats; over the same period the peroxisome numerical density also significantly increased over the control values. Finally, the average diameters of peroxisome profiles in test rats showed a more dispersed distribution (SD 40%) than in control animals (SD 30%). These results demonstrate that clofibrate, given to rats during pregnancy, induces peroxisomal proliferation in the livers of their foetuses. Data are discussed in view of the models proposed for peroxisomal biogenesis.

Animals↗
PubMed1987

Time-resolved fluorescence of apoferritin and its subunits.

The decay of the intrinsic fluorescence of the apoferritin polymer and its subunits has been studied by pulse and phase shift techniques. Both techniques show that the fluorescence decay of all the samples tested cannot be described by a single exponential function. The fluorescence decay data of the apoferritin subunits obtained with either technique can be fitted satisfactorily with a function resulting from the sum of two exponential components. However, the polymer data obtained with the high resolution phase shift technique operated either by synchrotron radiation or by a mode-locked argon ion laser can be fitted better using a bimodal gaussian continuous distribution of lifetime components. The molecular basis for this distribution of lifetime values may lie in the heterogeneity of the tryptophan environment generated by the assembly of the subunits into the polymer. The binding of the first 100 irons to apoferritin quenches the intrinsic fluorescence without affecting the lifetimes in a proportional way. This finding may be taken as an indication that the quenching of the tryptophan fluorescence induced by the binding of iron has both static and dynamic components.

Apoferritins↗
PubMed1987

On the mechanism of horse spleen apoferritin assembly: a sedimentation velocity and circular dichroism study.

The apoferritin shell is known to assemble spontaneously from its subunits obtained at acid pH upon neutralization. The reassembly of apoferritin from horse spleen has been followed by means of sedimentation velocity and circular dichroism experiments as a function of the pH and the nature of the assembly buffer in order to obtain information on the assembly pathway. In all the buffer systems tested the subunits sediment as a single peak of varying sedimentation and diffusion coefficients, and shell assembly starts at pH values around 3.5. In dilute glycine-acetate buffers the subunits are essentially dimeric up to this pH value. Therefore, the dimeric building blocks of the apoferritin shell that are apparent in the X-ray structure represent the first assembly intermediates. When the pH is increased to 4.0-4.3, the weight-average sedimentation velocity of the subunits increases to 3.6-4.7 S, respectively, and the subunit population becomes heterogeneous. Concomitantly, significant changes in the circular dichroism properties of the aromatic residues take place. On the basis of the X-ray structure, where aromatic residues appear to be located at or near the fourfold symmetry axes, these data suggest that assembly proceeds from dimers through tetramers and octamers. In the pH range 4.5-6.5 the reassembly process cannot be followed due to reversible precipitation of the subunits near their isoelectric point; at neutral pH values essentially quantitative reassembly is obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗