Search PubMed⌕ Search

Biomedical subjects

F Palmieri

Publications and source records attributed to F Palmieri.

At least 73 records · Page 4Linked to original sources
PubMed1998

[Blood lead levels in the population of La Spezia].

The Istituto Superiore di Sanità, according to the Presidential Decree 496 of 1982 (known as DPR 486/82), has promoted, starting from 1992, the second Italian campaign of biological monitoring of the general population against the risk of lead intoxication. Within the framework of this campaign, a total of 927 subjects residing in the city of La Spezia have been examined in 1992. Biological monitoring of lead exposure has been carried out by means of the determination of lead in blood. Both non exposed subjects and all the traffic wardens (vigili urbani) of La Spezia--exposed to the combustion of gasoline containing organic lead as antiknock additive--have been examined. Among non occupationally exposed adult subjects, median values for males and females were 102.25 micrograms/l and 69 micrograms/l, respectively. In a group of 74 children (45 males and 29 females) median values were 46.5 micrograms/l in male subjects and 38 micrograms/l in female subjects. The results obtained for the same group of traffic wardens in 1990 and 1996 are also reported in this paper. Among 55 subjects observed in 1990, 1993 and 1996 median blood lead levels were 140 micrograms/l, 85 micrograms/l and 72 micrograms/l, respectively. The comparison of data obtained starting from 1990 indicate a significant stepwise decline in both environmental lead pollution and blood lead levels of subjects residing in the city of La Spezia. These time trends can be attributed to the decreased concentration of lead in gasoline.

Adult↗
PubMed1997

Identification of the yeast ACR1 gene product as a succinate-fumarate transporter essential for growth on ethanol or acetate.

The protein encoded by the ACR1 gene in Saccharomyces cerevisiae belongs to a family of 35 related membrane proteins that are encoded in the fungal genome. Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 28 members of the family are unknown. The yeast ACR1 gene was introduced into Escherichia coli on an expression plasmid. The protein was over-produced as inclusion bodies, which were purified and solubilised in the presence of sarkosyl. The solubilised protein was reconstituted into liposomes and shown to transport fumarate and succinate. Its physiological role in S. cerevisiae is probably to transport cytoplasmic succinate, derived from isocitrate by the action of isocitrate lyase in the cytosol, into the mitochondrial matrix in exchange for fumarate. This exchange activity and the subsequent conversion of fumarate to oxaloacetate in the cytosol would be essential for the growth of S. cerevisiae on ethanol or acetate as the sole carbon source.

Acetates↗
PubMed1997

The purified and reconstituted ornithine/citrulline carrier from rat liver mitochondria: electrical nature and coupling of the exchange reaction with H+ translocation.

The mechanism and the electrical nature of ornithine/citrulline exchange has been investigated in proteoliposomes reconstituted with the ornithine/citrulline carrier purified from rat liver mitochondria. The stoichiometry of the exchanging substrates was close to 1:1. The exchange was not affected by inducing electrogenic flux of K+ with valinomycin. In contrast, the pH gradient generated by the K+/H+ exchanger nigericin in the presence of an outwardly directed K+ gradient stimulated the ornithineout/citrullinein exchange, but not the ornithine/ornithine homoexchange. Experiments in which either the internal or the external pH was varied, while keeping constant the pH in the other compartment, indicated that maximal exchange rates are found at pH 6 in the compartment containing citrulline and at pH 8 in the compartment containing ornithine. Changes in fluorescence of the pH indicator pyranine, included inside the proteoliposomes, showed that the exchanges ornithineout/citrullinein and citrullineout/ornithinein are accompanied by translocation of H+ in the same direction as citrulline. It is concluded that the mitochondrial ornithine/citrulline carrier catalyses an electroneutral exchange of ornithine+ for citrulline plus an H+. A reasonable model is one in which ornithine binds to a deprotonated carrier and citrulline to a protonated carrier and both substrate-carrier complexes are neutral. The physiological implications of this transport process are discussed.

Amino Acid Transport Systems, Acidic↗
PubMed1997

Identification of the yeast ARG-11 gene as a mitochondrial ornithine carrier involved in arginine biosynthesis.

The ARG-11 gene in Saccharomyces cerevisiae encodes a protein with the characteristic features of a family of 35 related membrane proteins that are encoded in the fungal genome. Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 29 members of the family are unknown. The yeast ARG-11 protein has been over-produced as inclusion bodies in Escherichia coli. It has been solubilized in the presence of sarkosyl, re-constituted into liposomes and shown to transport ornithine in exchange for protons. Its main physiological role is probably to take ornithine synthesized from glutamate in the mitochondrial matrix to the cytosol where it is converted to arginine.

Amino Acid Transport Systems, Basic↗
PubMed1997

The mitochondrial carnitine carrier protein: cDNA cloning, primary structure and comparison with other mitochondrial transport proteins.

GENBANK/o acid sequence of the rat carnitine carrier protein, a component of the inner membranes of mitochondria, has been deduced from the sequences of overlapping cDNA clones. These clones were generated in polymerase chain reactions with primers and probes based on amino acid sequence information, obtained from the direct sequencing of internal peptides of the purified carnitine carrier protein from rat. The protein sequence of the carrier, including the initiator methionine, has a length of 301 amino acids. The mature protein has a modified alpha-amino group, although the nature of this modification and the precise position of the N-terminal residue have not been ascertained. Analysis of the carnitine carrier sequence shows that the protein contains a 3-fold repeated sequence about 100 amino acids in length. Dot plot comparisons and sequence alignment demonstrate that these repeated domains are related to each other and also to the repeats of similar length that are present in the other mitochondrial carrier proteins sequenced so far. The hydropathy analysis of the carnitine carrier supports the view that the domains are folded into similar structural motifs, consisting of two transmembrane alpha-helices joined by an extensive extramembranous hydrophilic region. Southern blotting experiments suggest that both the human and the rat genomes contain single genes for the carnitine carrier. These studies provide the primary structure of the mitochondrial carnitine carrier protein and allow us to identify this metabolically important transporter as a member of the mitochondrial carrier family, and the sixth of the members whose biochemical function has already been identified.

Amino Acid Sequence↗
PubMed1997

Cloning of the human carnitine-acylcarnitine carrier cDNA and identification of the molecular defect in a patient.

The carnitine-acylcarnitine carrier (CAC) catalyzes the translocation of long-chain fatty acids across the inner mitochondrial membrane. We cloned and sequenced the human CAC cDNA, which has an open reading frame of 903 nucleotides. Northern blot studies revealed different expression levels of CAC in various human tissues. Furthermore, mutation analysis was performed for a CAC-deficient infant. Direct sequencing of the patient's cDNA revealed a homozygous cytosine nucleotide insertion. This insertion provokes a frameshift and an extension of the open reading frame with 23 novel codons. This is the first report documenting a mutation, in the CAC cDNA, responsible for mitochondrial beta-oxidation impairment.

Amino Acid Sequence↗
PubMed1997

Organization and sequence of the human gene for the mitochondrial citrate transport protein.

The citrate (tricarboxylate) carrier transports citrate (or other tricarboxylates) across the inner membranes of mitochondria in an electroneutral exchange for malate (or other dicarboxylic acids). We have determined the sequence of the human citrate transporter gene from overlapping genomic clones generated by polymerase chain reactions by use of primers and probes based on the rat cDNA sequence and on emerging sequences. The gene is spread over 2.8 kb of human DNA and is divided into eight exons. All the introns are located at the level of the sequences coding for the extramembranous loops (and not for the transmembrane segments) of the mature protein. The open reading frame of the human gene encodes the mature protein consisting of 298 amino acids, preceded by a presequence of 13 amino acids to help to target it into mitochondria. 84 identities and 106 highly conservative substitutions are present in CTPs from man to yeast. In addition, we have determined the sequences of two human pseudogenes related to the citrate carrier gene encompassing the coding sequence of the gene between nucleotides 260 and 720.

Amino Acid Sequence↗
PubMed1997

Lichens and moss as bioindicators and bioaccumulators in air pollution monitoring.

In this study, we review research conducted in the La Spezia district during 1989, 1992, and 1994, using lichens and moss as indicators of air pollution. SO2 pollution was examined by means of an Index of Atmospheric Purity (IAP) based on the frequency of epiphytic lichen within a sampling grid. Metal deposits were estimated using the lichen Parmelia caperata and the moss Hypnum cupressiforme as bioaccumulators. IAP maps show progressive air quality improvement from 1989 to 1994. This trend correlates to a decrease in SO2 emissions during recent years that is attributed to the use of methane for residential heating and the closing of a coal-fired power plant. Metal contamination maps show that the most polluted area is now in the southeastern part of the gulf. The pattern of pollution coincides with the location of the chief pollution sources in the area. From 1989 to 1994, the metal concentrations in lichens decreased, but metal deposits in the southeastern area were cause for concern. High concentrations of lead in the area are related to emissions from a waste incinerator and a plant that produces lead oxide. Epidemiological investigations reveal that the area population has the highest levels of lead in their blood. The use of bioindicators and bioaccumulators permits long-term and large-scale monitoring of environmental pollutant levels in full agreement with traditional methods.

Air Pollution↗
PubMed1996

Identification by bacterial expression and functional reconstitution of the yeast genomic sequence encoding the mitochondrial dicarboxylate carrier protein.

The inner membranes of mitochondria contain a family of transport proteins of related sequence and structure. The DNA sequence of the genome of Saccharomyces cerevisiae encodes at least 35 members of this family. Three of them can be recognised as known isoforms of the ADP-ATP translocase and two others as the phosphate and citrate carriers. The transport functions of the remainder cannot be identified with certainty. One of them, encoded on yeast chromosome xii, shows a fairly close sequence relationship to the known sequence of the bovine mitochondrial oxoglutarate-malate carrier. The yeast protein has been obtained by over-expression in Escherichia coli, reconstituted into phospholipid vesicles and shown to have transport properties characteristic of the mitochondrial carrier for dicarboxylate ions, such as malate, and also phosphate, previously biochemically characterised, but not sequenced, from both mammalian and yeast mitochondria. This is the first example of the biochemical identification of an unknown membrane protein encoded in the yeast genome since the completion of the genomic sequence.

Carrier Proteins↗
PubMed1996

Tissue-specific expression of the two isoforms of the mitochondrial phosphate carrier in bovine tissues.

Comparison of the sequence of the human mitochondrial phosphate carrier (PiC) gene with cDNA clones characterised from a human heart cDNA library suggested the existence of two isoforms of the PiC, which were generated by alternative splicing of exon IIIA or exon IIIB and which differed in 13 amino acids [Dolce et al. (1994) J. Biol. Chem. 269, 10451]. In this work the expression of isoforms A and B of the PiC was investigated in different bovine tissues by Northern blot analysis using two probes that are specific for bovine exon IIIA and exon IIIB, respectively. Isoform A is highly expressed in heart and skeletal muscle. Isoform B is ubiquitously expressed in all tissues that were examined, although at different levels. The tissue-specific expression pattern of the two PiC isoforms is similar to that reported for the isoforms of several mitochondrial proteins required for energy production.

Alternative Splicing↗
PubMed1996

Cloning and sequencing of the bovine cDNA encoding the mitochondrial tricarboxylate carrier protein.

The tricarboxylate or citrate transporter protein (CTP) catalyzes the transport of citrate across the inner mitochondrial membrane by an exchange for malate or some other anionic metabolite. Using primers based on the rat liver cDNA sequence, overlapping cDNA clones encoding the bovine CTP were isolated from bovine liver poly(A+) cDNA. The entire bovine cDNA is 1151 bp in length with 5' and 3' untranslated regions of 7 and 204 bp, respectively. The open reading frame encodes the mature protein consisting of 298 amino acids, preceded by a presequence of 13 amino acids. The amino acid sequence of the mature bovine CTP is 95.6, 94.9, 32.2% identical to that of the citrate carrier from man, rat and yeast, respectively.

Amino Acid Sequence↗
PubMed1996

The mitochondrial oxoglutarate carrier protein contains a disulfide bridge between intramembranous cysteines 221 and 224.

The oxoglutarate carrier (OGC) purified from bovine heart mitochondria was treated, both in its active and in its SDS-denatured state, with the fluorescent N-(1-pyrenyl)maleimide and other SH reagents before and after reduction with dithioerythritol or beta-mercaptoethanol. The number of SH groups per OGC polypeptide chain was found to be about 1 for the oxidized carrier and 3 for the reduced carrier. The bovine oxoglutarate carrier contains three cysteines: Cys-184, Cys-221 and Cys-224. Sequencing of BrCN cleavage products of oxoglutarate carrier showed that N-(1-pyrenyl)maleimide binds to only Cys-184 of the oxidized protein and also to Cys-221 and Cys-224 after reduction of the protein. These results show the presence of a disulfide bridge between the latter two cysteines of the purified carrier. The oxidized and the reduced forms of the oxoglutarate carrier exhibited different Vmax but virtually the same K(m) values for oxoglutarate.

Amino Acid Sequence↗
PubMed1996

The mitochondrial oxoglutarate carrier: sulfhydryl reagents bind to cysteine-184, and this interaction is enhanced by substrate binding.

The interaction of sulfhydryl reagents with the oxoglutarate carrier (OGC) of bovine heart mitochondria was investigated in proteoliposomes reconstituted from purified carrier and lipids. Incubation of the proteoliposomes with maleimides or mercurials led to inhibition of the oxoglutarate carrier protein. The inhibition of oxoglutarate transport by mercurials was removed by dithioerythritol (DTE), whereas inhibition by maleimides was not. Preincubation of the proteoliposomes with mercurials protected the carrier protein against inactivation by the fluorescent sulfhydryl reagent N-(1-pyrenyl)maleimide (PM) and decreased the fluorescence associated with the carrier, indicating that mercurials bind to the same cysteine which is modified by PM. The presence of the substrates oxoglutarate and malate increased the binding of PM to the reconstituted carrier as well as the degree of inhibition of the reconstituted transport activity caused by PM, other maleimides, and mercurials. This result is consistent with the assumption that substrate binding causes a change in the tertiary structure of the carrier protein. The primary sequence of the oxoglutarate carrier contains three cysteines (Cys-184, Cys-221, and Cys-224). We provide evidence that PM labels only Cys-184, whereas Cys-221 and Cys-224 are linked by a disulfide bridge.

Amino Acid Sequence↗
PubMed1996

Inhibition of the reconstituted mitochondrial oxoglutarate carrier by arginine-specific reagents.

The effect of arginine-specific reagents on the function of the purified and reconstituted oxoglutarate carrier protein of the inner mitochondrial membrane has been investigated. The alpha-dicarbonyl reagents 2,3-butanedione, 2,3-pentanedione, 2,3- and 3,4-hexanedione, 1-phenyl-1,2-propanedione, phenylglyoxal, and phenylglyoxal derivatives caused a concentration-dependent inhibition of oxoglutarate transport with an IC50 of 0.05 mM for 2,3-hexanedione, 0.08 mM for 4-hydroxy-3-nitrophenylglyoxal, and 0.17 mM for 2,3-pentanedione. The inhibition increased with pH from 6.0 to 8.0, indicating that the pK of the reacting group(s) is rather high. Mersalyl and pyridoxal 5'-phosphate (or 4,4'-dinitrostilbene-2,2'-disulfonate), which are known to react specifically and reversibly with cysteine residues and lysine residues, respectively, were unable to protect the oxoglutarate carrier against inhibition by alpha-dicarbonyl reagents. Other diketone compounds, which do not react with arginine residues, had no significant effect on the oxoglutarate transport activity. Oxoglutarate and L-malate effectively protected the oxoglutarate carrier against inactivation caused by arginine-specific reagents; other dicarboxylates, which are not substrates of the carrier, had no protective effect. A 50% substrate protection was observed at half-saturation of the external binding site. These results indicate that the arginine-specific reagents used in this investigation interact with the oxoglutarate carrier at the level of an arginine residue(s), which is essential for binding and/or translocation of substrates and which may be localized in, or near, the substrate-binding site.

Animals↗
PubMed1996

Purification and characterization of the tricarboxylate carrier from eel liver mitochondria.

The tricarboxylate carrier from eel (Anguilla anguilla) liver mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and Matrex Gel Blue B. On SDS-polyacrylamide gel electrophoresis, the purified fraction showed a single polypeptide band with an apparent molecular mass of 30.4 kDa. When reconstituted into liposomes, the tricarboxylate transport protein catalyzed a very active 1,2,3-benzenetricarboxylate-sensitive citrate/citrate exchange. It was purified 641-fold with a recovery of 13.3% and a protein yield of 0.02% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., requirement for a counteranion, substrate specificity and inhibitor sensitivity, were similar to those of the tricarboxylate carrier purified from rat liver mitochondria. These studies provide the first information on the mitochondrial tricarboxylate transport protein of a fish.

Anguilla↗
PubMed1996

The formation of a disulfide cross-link between the two subunits demonstrates the dimeric structure of the mitochondrial oxoglutarate carrier.

Isolated oxoglutarate carrier (OGC) can be cross-linked to dimers by disulfide-forming reagents such as Cu2+-phenanthroline and diamide. Acetone and other solvents increase the extent of Cu2+ -phenanthroline-induced cross-linking of OGC. Cross-linked OGC re-incorporated in proteoliposomes fully retains the oxoglutarate transport activity. The amount of cross-linked OGC calculated by densitometry of scanned gels depends on the method of staining, since cross-linked OGC exhibits a higher sensitivity to Coomassie brilliant blue as compared to silver nitrate. Under optimal conditions the formation of cross-linked OGC dimer (stained with Coomassie brilliant blue) amounts to 75% of the total protein. Approximately the same cross-linking efficiency was evaluated from Western blots. Cross-linking of OGC is prevented by SH reagents and reversed by SH-reducing reagents, which shows that it is mediated by disulfide bridge(s). The formation of S-S bridge(s) requires the native state of the protein, since it is suppressed by SDS and by heating. Furthermore, the extent of cross-linking is independent of OGC concentration indicating that disulfide bridge(s) must be formed between the two subunits of native dimers. The number and localization of disulfide bridge(s) in the cross-linked OGC were examined by peptide fragmentation and subsequent cleavage of disulfide bond(s) by beta-mercaptoethanol. Our experimental results show that cross-linking of OGC is accomplished by a single disulfide bond between the cysteines 184 of the two subunits and suggest that these residues in the putative transmembrane helix four are fairly close to the twofold axis of the native dimer structure.

1-Propanol↗