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Biomedical subjects

C Fiore

Publications and source records attributed to C Fiore.

At least 19 recordsLinked to original sources
PubMed2001

Fluorometric detection of ADP/ATP carrier deficiency in human muscle.

Defects in mitochondrial energy metabolism lead to severe disorders in humans referred to as mitochondriocytopathies. Most of them have been reported to result from deficiencies of one or more complexes of the respiratory chain and, more rarely, from mitochondrial transmembrane metabolite carrier defects. Dysfunctioning of the ADP/ATP carrier, which catalyses the export of matrix ATP in exchange for cytosolic ADP, has been demonstrated to induce myopathies in mouse and in humans. To screen for ADP/ATP carrier deficiency in patients suffering from mitochondriocytopathy with no defined etiology, we have set up a fluorometric assay to quantify the ADP/ATP carrier in small muscle homogenates, without preliminary isolation of mitochondria. The assay is based on the use of a fluorescent derivative of atractyloside, namely naphthoyl-atractyloside, a highly specific inhibitor of ADP/ATP transport. Here, we describe analysis of healthy and pathological muscle samples, and characterization of ADP/ATP carrier deficiencies in two patients, one displaying an absence of the carrier and the second one containing a limited amount of the carrier with altered binding properties.

Biopsy↗
PubMed2001

Purification and preliminary characterization of the zonula occludens toxin receptor from human (CaCo2) and murine (IEC6) intestinal cell lines.

In the present study, we report the preliminary characterization of the epithelial cell receptor for Vibrio cholerae zonula occludens toxin (Zot). Zot receptor was purified by ligand-affinity chromatography. Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis revealed a protein of ca. 66 kDa. Partial N-terminal sequence obtained from purified murine and human Zot receptor revealed homology between the two proteins and with human alpha-1-chimaerin. Zot protein domain(s) involved in receptor binding were also analyzed by constructing several in frame deletion derivatives of a recombinant fusion Zot protein tagged with maltose binding protein. Our results suggest that Zot binding to its cellular membrane receptor requires a sequence that spans between amino acids 118 and 299.

Amino Acid Sequence↗
PubMed2001

Mitochondrial adaptation to in vivo polyunsaturated fatty acid deficiency: increase in phosphorylation efficiency.

Polyunsaturated fatty acid (PUFA) deficiency affects respiratory rate both in isolated mitochondria and in hepatocytes, an effect that is normally ascribed to major changes in membrane composition causing, in turn, protonophoriclike effects. In this study, we have compared the properties of hepatocytes isolated from PUFA-deficient rats with those from control animals treated with concentrations of the protonophoric uncoupler 2,4-dinitrophenol (DNP). Despite identical respiratory rate and in situ mitochondrial membrane potential (delta psi), mitochondrial and cytosolic ATP/ADP-Pi ratios were significantly higher in PUFA-deficient cells than in control cells treated with DNP. We show that PUFA-deficient cells display an increase of phosphorylation efficiency, a higher mitochondrial ATP/ADP-Pi ratio being maintained despite the lower delta psi. This is achieved by (1) decreasing mitochondrial Pi accumulation, (2) increasing ATP synthase activity, and (3) by increasing the flux control coefficient of adenine nucleotide translocation. As a consequence, oxidative phosphorylation efficiency was only slightly affected in PUFA-deficient animals as compared to protonophoric uncoupling (DNP). Thus, the energy waste induced by PUFA deficiency on the processes that generate the proton motive force (pmf) is compensated in vivo by powerful adaptive mechanisms that act on the processes that use the pmf to synthesize ATP.

2,4-Dinitrophenol↗
PubMed2000

Two distinct regions of the yeast mitochondrial ADP/ATP carrier are photolabeled by a new ADP analogue: 2-azido-3'-O-naphthoyl-[beta-32P]ADP. Identification of the binding segments by mass spectrometry.

A novel photoactivatable radioactive ADP derivative, namely, 2-azido-3'-O-naphthoyl-[beta-(32)P]ADP (2-azido-N-[(32)P]ADP), was synthesized with the aim at mapping the substrate binding site(s) of the yeast mitochondrial ADP/ATP carrier. It was used with mitochondria isolated from genetically modified strains of Saccharomyces cerevisiae, producing the native or the His-tagged Anc2p isoform of the carrier. In darkness, 2-azido-N-[(32)P]ADP was reversibly bound to the carrier in mitochondria, without being transported. Upon photoirradiation, only the ADP/ATP carrier was covalently radiolabeled among all mitochondrial proteins. Specificity of labeling was demonstrated since carboxyatractyloside (CATR), a potent inhibitor of ADP/ATP transport, totally prevented the incorporation of the photoprobe. To localize the radioactive region(s), the purified photolabeled carrier was submitted to CNBr or hydroxylamine cleavage. The resulting fragments were characterized and identified by SDS-PAGE, Western blotting, amino acid sequencing, and MALDI-MS and ESI-MS analyses. Two short photolabeled distinct segments, eight and nine residues long, were identified: S183-R191, located in the central part of the ADP/ATP carrier; and I311-K318, belonging to its C-terminal end. Plausible models of organization of the nucleotide binding site(s) of the carrier involving the two regions specifically labeled by 2-azido-N-[(32)P]ADP are proposed.

Adenosine Diphosphate↗
PubMed2000

A covalent tandem dimer of the mitochondrial ADP/ATP carrier is functional in vivo.

The adenine nucleotide carrier, or Ancp, is an integral protein of the inner mitochondrial membrane. It is established that the inactive Ancp bound to one of its inhibitors (CATR or BA) is a dimer, but different contradictory models were proposed over the past years to describe the organization of the active Ancp. In order to decide in favor of a single model, it is necessary to establish the orientations of the N- and C-termini and thus the parity of the Ancp transmembrane segments (TMS). According to this, we have constructed a gene encoding a covalent tandem dimer of the Saccharomyces cerevisiae Anc2p and we demonstrate that it is stable and active in vivo as well as in vitro. The properties of the isolated dimer are strongly similar to those of the native Anc2p, as seen from nucleotide exchange and inhibitor binding experiments. We can therefore conclude that the native Anc2p has an even number of TMS and that the N- and C-terminal regions are exposed to the same cellular compartment. Furthermore, our results support the idea of a minimal dimeric functional organization of the Ancp in the mitochondrial membrane and we can suggest that TMS 1 of one monomer and TMS 6 of the other monomer in the native dimer are very close to each other.

Amino Acid Sequence↗
PubMed2000

Purification of histidine-tagged mitochondrial ADP/ATP carrier: influence of the conformational states of the C-terminal region.

A functional recombinant mitochondrial ADP/ATP carrier from the yeast Saccharomyces cerevisiae that bears a six-histidine tag at the C-terminus, Anc2(His(6))p, has been engineered to allow its purification by immobilized metal-ion affinity chromatography (IMAC). The tagged carrier was expressed at a level similar to that of unmodified Anc2p as determined by immunodetection and titration of the specific atractyloside binding sites. Anc2(His(6))p, enriched by chromatography on hydroxyapatite of detergent extracts of mitochondria, was still contaminated by mitochondrial proteins and a large amount of ergosterol. It was highly purified after adsorption on Ni-NTA resin and elution by imidazole buffer, with a 90-95% overall yield. Anc2(His(6))p interacted differently with immobilized ions depending on whether it was unliganded or bound to carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors of the ADP/ATP transport, thus indicating that accessibility of the C-terminus is markedly influenced by the conformational state of the carrier. Fluorometric assays demonstrated that purified unliganded Anc2(His(6))p was in a functional state since it underwent CATR- and BA-sensitive and ADP (or ATP)-induced conformational changes. Large-scale purification of Anc2(His(6))p-CATR and Anc2(His(6))p-BA complexes by IMAC will be of major interest for structural analysis of the ADP/ATP carrier.

Anti-Bacterial Agents↗
PubMed1998

The mitochondrial ADP/ATP carrier: structural, physiological and pathological aspects.

Under the conditions of oxidative phosphorylation, the mitochondrial ADP/ATP carrier catalyses the one to one exchange of cytosolic ADP against matrix ATP across the inner mitochondrial membrane. The ADP/ATP transport system can be blocked very specifically by two families of inhibitors: atractyloside (ATR) and carboxyatractyloside (CATR) on one hand, and bongkrekic acid (BA) and isobongkrekic acid (isoBA) on the other hand. It is well established that these inhibitors recognise two different conformations of the carrier protein, the CATR- and BA-conformations, which exhibit different chemical, immunochemical and enzymatic reactivities. The reversible transition of the ADP/ATP carrier between the two conformations was studied by fluorometric techniques. This transconversion, which is only triggered by transportable nucleotides, is probably the same as that which occurs during the functioning of ADP/ATP transport system. The fluorometric approach, using the tryptophanyl residues of the yeast carrier as intrinsic fluorescence probes, was combined to a mutagenesis approach to elucidate the ADP/ATP transport mechanism at the molecular level. Finally, recent reports that myopathies might result from defect in ADP/ATP transport led us to develop a method to quantify the carrier protein in muscular biopsies.

Adenosine Diphosphate↗
PubMed1998

Surface replacement hemiarthroplasty for the treatment of osteonecrosis of the femoral head.

We reviewed the results of thirty-three femoral resurfacing procedures in twenty-five patients who had stage-III or early stage-IV osteonecrosis of the femoral head according to the classification system of Ficat and Arlet. There were no perioperative complications. Thirty hip prostheses (91 percent) survived for a minimum of five years. At a mean of 10.5 years (range, four to fourteen years) postoperatively, sixteen (62 percent) of the twenty-six hips with stage-III disease had a good or excellent Harris hip score. Four of the seven hips with stage-IV disease did not have or need a total hip arthroplasty. Overall, twenty hips (61 percent) had a good or excellent result according to the scoring system of Harris, and thirteen (39 percent) had a fair or poor result and subsequently had or needed a total hip arthroplasty. The mean interval between the hemiarthroplasty and the total hip arthroplasty was sixty months (range, thirty-six to 136 months). These thirteen hips all had a successful clinical result (a Harris hip score of at least 80 points) at a mean of thirty months (range, twenty-four to seventy-two months) after the total hip arthroplasty. The results of the present study suggest that resurfacing of the femoral head can be a successful interim procedure for the management of patients who have Ficat and Arlet stage-III or early stage-IV disease with a large lesion that is not amenable to other treatment options except total hip arthroplasty.

Adult↗
PubMed1997

The enterotoxic effect of zonula occludens toxin on rabbit small intestine involves the paracellular pathway.

BACKGROUND & AIMS: Zonula occludens toxin is a novel toxin elaborated by Vibrio cholerae that modulates intestinal tight junctions. The aim of this study was to establish whether the permeabilizing effect of the toxin leads to intestinal secretion. METHODS: Rabbit intestine was mounted in Ussing chambers and exposed to increasing concentrations of purified toxin. The tissues were also fixed, exposed to zonula occludens toxin, and processed for fluorescence microscopy to determine the distribution of the toxin receptor within the intestine. Then purified toxin was simultaneously perfused in three distinct rabbit intestinal segments in vivo, and water and electrolyte absorption were measured. RESULTS: Zonula occludens toxin induced a time- and dose-dependent decrease of tissue resistance starting at a toxin concentration of 1.1 x 10(-13) mol/L. When tested in vivo, the toxin induced a secretion of water and chloride and the passage of polyethylene glycol 4000 in the bloodstream. Both the in vitro and in vivo effects of the toxin were observed only in the small intestine but not in the colon and paralleled the distribution of the toxin receptor within the intestine. CONCLUSIONS: The intestinal secretion induced by zonula occludens toxin follows the opening of tight junctions caused by the toxin, possibly representing a novel mechanism of intestinal secretion.

Animals↗
PubMed1996

Conformational changes of the yeast mitochondrial adenosine diphosphate/adenosine triphosphate carrier studied through its intrinsic fluorescence. 1. Tryptophanyl residues of the carrier can be mutated without impairing protein activity.

During the transport process the mitochondrial adenine nucleotide carrier (Ancp) undergoes conformational changes which result in modifications of the intrinsic fluorescence of the carrier. To further study these changes by a fluorometric approach, the three tryptophanyl residues (Trp87, Trp126, and Trp235) of the Saccharomyces cerevisiae Anc2p were individually mutated to their tyrosine counterparts. The resulting mutated genes (two-Trp, one-Trp or Trp-less variants) were integrated at the ANC2 locus. A prerequisite for such studies is that all the engineered carrier molecules are still able to catalyze ADP/ATP exchange. The cellular characteristics of the strains expressing the mutated Anc2p and the biochemical properties of the variant Anc2p in mitochondria were examined. Although Trp87 is absolutely conserved in all 30 available Ancp sequences, none of the tryptophanyl residues is essential to the carrier protein folding and the transport activity. The mutated and wild-type Anc2p were expressed to the same level, as evidenced by both ligand binding and immunochemical analyses. When isolated in the presence of detergent, all the variant Anc2p preparations contained ergosterol in similar amounts (9 mol/mol of 35 kDa Anc2p) but no specific interaction was revealed. Our results show that the tryptophanmutated Anc2p are suitable for fluorescence studies, which are reported in the accompanying paper by Roux et al. [(1996) Biochemistry 35, 16125-16131].

Atractyloside↗
PubMed1996

Conformational changes of the yeast mitochondrial adenosine diphosphate/adenosine triphosphate carrier studied through its intrinsic fluorescence. 2. Assignment of tryptophanyl residues of the carrier to the responses to specific ligands.

Tryptophanyl substitution of the Saccharomyces cerevisiae adenine nucleotide carrier (Anc2p isoform) was not deleterious for the transport activity or the folding of the carrier [preceding paper by Le Saux et al. (1996) Biochemistry 35, 16116-16124]. Conformational changes of the isolated wild-type and Trp-substituted Anc2p variants, induced upon binding of specific substrates [adenosine triphosphate (ATP) or diphosphate (ADP)] or inhibitors [carboxyatractyloside (CATR) or bongkrekic acid (BA)], were studied by measurement of intrinsic fluorescence. Titration of CATR and BA binding sites ended in the same number of sites, namely, 6-7 nmol/mg of wild-type and variant Anc2p. Isolated Anc2p in detergent presented similar emission spectra, suggesting that all tryptophanyl residues were in environments of similar hydrophobicity. Trp87 and Trp126 contributed largely and to a similar extent to the fluorescence enhancement observed in response to ATP binding, while Trp235 contributed negatively and to a small extent to the fluorescence change. Both Trp126 and Trp235, and to a lower extent Trp87, participate in the CATR-induced fluorescence decrease of Anc2p. Responses to BA binding were observed only in the presence of ATP; they consisted of a further fluorescence increase of the Anc2p.ATP complex, which was mainly due to Trp87 and Trp126, Trp235 being much less responsive. The different fluorescence responses of the three Trp residues of Anc2 variants to ATP, CATR, and BA are in agreement with distinct binding sites for these ligands and distinct conformations of the carrier protein recognizing specifically CATR or BA. A mechanistic model is proposed to interpret the transitions between the different conformational states of Anc2p.

Adenosine Diphosphate↗
PubMed1996

Fluorometric titration of the mitochondrial ADP/ATP carrier protein in muscle homogenate with atractyloside derivatives.

We describe here the chemical synthesis of the novel methylanthraniloyl (Mant-) derivative of atractyloside (ATR), which is a specific inhibitor of the mitochondrial ADP/ATP carrier. The spectral properties of Mant-ATR and naphthoyl-ATR (N-ATR) are analyzed. Both derivatives bind to the membrane-bound ADP/ATP carrier at the same sites as ATR and carboxyatractyloside (CATR). When Mant-ATR and N-ATR are displaced by CATR, their fluorescence emissions are decreased and increased, respectively. These fluorescence changes allow the titration of the CATR binding sites and therefore the quantitation of the amount of ADP/ATP carrier protein in a biological preparation. The validity of the fluorometric titration was tested with beef heart mitochondria and confirmed by binding assays using radioactive ATR. The fluorometric method was applied to rabbit skeletal muscle homogenate and the results of titration were confirmed by binding assays of radioactive ATR. The reliability of the fluorometric method was assessed by comparing the amounts of CATR binding sites and the content of heme aa3 in muscle homogenates and in isolated mitochondria from the same homogenates. Because of its high sensitivity, the fluorometric titration of the ADP/ATP carrier requires small amounts of tissue. Mant-ATR and N-ATR can therefore be considered as convenient, reliable, and sensitive probes to quantify the amount of ADP/ATP carrier and detect a putative carrier protein deficiency in biopsy samples from human patients suffering from myopathies with no clear identified etiology.

Animals↗
PubMed1995

Evaluation of functionality of cochlear outer hair cells in patients with retinitis pigmentosa and in their relatives.

Hearing functionality was studied in 36 patients with retinitis pigmentosa (R P) and 29 of their relatives. These patients were defined as having bilateral normal hearing on the basis of tonal-threshold audiometric and acoustic-immittance tests. The transiently evoked otoacoustic emissions (T E O A Es) were studied in these normal hearing patients. T E O A Es represent an extremely sensitive method to study the functionality of the outer hair cells of the organ of Corti. When the values of T E O A E amplitude, intensity, and frequency in R P patients and in their relatives were compared with those in control subjects, they were found to be significantly reduced. The T E O A Es were clearly pathological in 52.8% of patients with R P and in 24.1% of their relatives. During embryologic development, there is one transitory axoneme in the outer hair cells of the organ of Corti; this transitory axoneme is important for the organization of the stereocilia. Axonemes are found in mature hair cells, including photoreceptors. The alteration of cochlear outer hair cells in a high percentage of patients with R P and in some of their relatives corroborates the hypothesis that, in some instances, retinitis pigmentosa may be due to a structural anomaly of the ciliated cells.

Adolescent↗
PubMed1994

Evoked otoacoustic emissions behaviour in retinitis pigmentosa.

The hearing function was studied in 26 patients affected by retinitis pigmentosa (RP) and in their relatives. Sixteen patients showed bilateral normal hearing when examined with traditional audiometric methods. In these normoacusic patients evoked otoacoustic emissions (EOE) have been studied. The EOE offer a unique opportunity to measure objectively the function of outer hair cells: they record the amplitude of the energy produced by the outer hair cells of the coclea following an acoustic stimulation. The data have been statistically compared, using the Student's t-test, with those obtained in a homogeneous control-group of normal subjects. In normoacusic subjects with RP the average values of EOE intensity are statistically lower than those of normal subjects in 64 of the 127 frequency bands examined. Moreover, the distribution of the EOE in patients with retinitis pigmentosa proved to be more discontinous than that observed in the normal subjects. The EOE recorded in 14 normoacusic relatives show in some cases small anomalies but the data, on account of the limited sample group, cannot be statistically evaluated. Therefore a subclinical alteration of the Organ of Corti is found in 100% of the patients affected by RP, although they appear to be normoacusic to usual audiometric tests.

Acoustic Stimulation↗
PubMed1994

Stages of change and decisional balance for 12 problem behaviors.

This integrative study investigated the generalization of the transtheoretical model across 12 problem behaviors. The cross-sectional comparisons involved relationships between two key constructs of the model, the stages of change and decisional balance. The behaviors studied were smoking cessation, quitting cocaine, weight control, high-fat diets, adolescent delinquent behaviors, safer sex, condom use, sunscreen use, radon gas exposure, exercise acquisition, mammography screening, and physicians' preventive practices with smokers. Clear commonalities were observed across the 12 areas, including both the internal structure of the measures and the pattern of changes in decisional balance across stages.

Adult↗
PubMed1993

Pleural empyema: early pulmonary decortication and lung biopsy.

Presented herein is a case of a patient with infectious acute pneumopathy who developed exudative pleural effusions. A diagnostic and therapeutic thoracoscopy was carried out. The usefulness of this method in early pulmonary decortication is emphasized.

Biopsy↗
PubMed1991

Activating tasks for the study of visual-spatial attention in ADHD children: a cognitive anatomic approach.

The clinical label attention deficit hyperactivity disorder (ADHD) suggests that this syndrome is a disorder of attention. However, the presumed attentional deficits have not been linked either to specific cognitive operations or to specific neural systems. To provide this link, theories of the cognitive anatomy of attention were used to generate hypotheses about specific visual-spatial attentional deficits in children with ADHD. A cued reaction-time test was used to assess covert and overt shifts of attention theoretically linked to two neuroanatomically defined attentional systems in the posterior and anterior parts of the human brain. The early, posterior-based covert shift of attention was found to be normal in ADHD children, but a later, anterior-based overt shift of attention was abnormal as reflected by a significant lateral difference in reaction time. This was interpreted as a failure to sustain focused attention.

Analysis of Variance↗