Biomedical subjects

F Palmieri

Publications and source records attributed to F Palmieri.

At least 37 records · Page 2Linked to original sources

PubMed2001

The human mitochondrial deoxynucleotide carrier and its role in the toxicity of nucleoside antivirals.

The synthesis of DNA in mitochondria requires the uptake of deoxynucleotides into the matrix of the organelle. We have characterized a human cDNA encoding a member of the family of mitochondrial carriers. The protein has been overexpressed in bacteria and reconstituted into phospholipid vesicles where it catalyzed the transport of all four deoxy (d) NDPs, and, less efficiently, the corresponding dNTPs, in exchange for dNDPs, ADP, or ATP. It did not transport dNMPs, NMPs, deoxynucleosides, nucleosides, purines, or pyrimidines. The physiological role of this deoxynucleotide carrier is probably to supply deoxynucleotides to the mitochondrial matrix for conversion to triphosphates and incorporation into mitochondrial DNA. The protein is expressed in all human tissues that were examined except for placenta, in accord with such a central role. The deoxynucleotide carrier also transports dideoxynucleotides efficiently. It is likely to be medically important by providing the means of uptake into mitochondria of nucleoside analogs, leading to the mitochondrial impairment that underlies the toxic side effects of such drugs in the treatment of viral illnesses, including AIDS, and in cancer therapy.

Amino Acid Sequence↗

PubMed2001

Functional analysis of mutant human carnitine acylcarnitine translocases in yeast.

Long chain fatty acids are translocated as carnitine esters across the mitochondrial inner membrane by carnitine acylcarnitine translocase (CACT). We report functional studies on the mutant CACT proteins from a severe and a mild patient with CACT deficiency. CACT activities in fibroblasts of both patients were markedly deficient with some residual activity (<1%) in the milder patient. Palmitate oxidation activity in cells from the severe patient was less than 5% but in the milder patient approximately 27% residual activity was found. Sequencing of the CACT cDNAs revealed a c.241G>A (G81R) in the severe and a c.955insC mutation (C-terminal extension of 21 amino acids (CACT(+21aa)) in the milder patient. The effect of both mutations on the protein was studied in a sensitive expression system based on the ability of human CACT to functionally complement a CACT-deletion strain of yeast. Expression in this strain revealed significant residual activity for CACT(+21aa), while the CACT(G81R) was inactive.

Amino Acid Sequence↗

PubMed2001

Kinetic mechanism of antiports catalyzed by reconstituted ornithine/citrulline carrier from rat liver mitochondria.

The transport mechanism of the reconstituted ornithine/citrulline carrier purified from rat liver mitochondria was investigated kinetically. A complete set of half-saturation constants (K(m)) was established for ornithine, citrulline and H(+) on both the external and internal side of the liposomal membrane. The internal affinity for ornithine was much lower than that determined on the external surface. The exclusive presence of a single transport affinity for ornithine on each side of the membrane indicated a unidirectional insertion of the ornithine/citrulline carrier into liposomes, probably right-side-out with respect to mitochondria. Two-reactant initial velocity studies of the homologous (ornithine/ornithine) and heterologous (ornithine/citrulline) exchange reactions resulted in a kinetic pattern which is characteristic of a simultaneous antiport mechanism. This type of mechanism implies that the carrier forms a ternary complex with the substrates before the transport reaction occurs. A quantitative analysis of substrate interaction revealed that rapid-equilibrium random conditions were fulfilled, characterized by a fast and independent binding of internal and external substrates.

Amino Acid Transport Systems, Basic↗

PubMed2001

Mass spectrometric identification of a candidate biomarker peptide from the in vitro interaction of epichlorohydrin with red blood cells.

The reaction products of epichlorohydrin with human alpha- and beta- globins, obtained through in vitro incubation of these compounds and red blood cells, were determined by using reversed-phase high-performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization tandem mass spectrometry. The alpha-globin was much more reactive than the beta-globin. At low incubation ratios, approximating the order of magnitude of epichlorohydrin concentration as found in workplaces, the only modified peptide still detectable was the 62-90 belonging to the alpha-chain and carrying an incremental mass of 92 u on either His72 or His89. Given that the two peptides co-eluted in a single chromatographic peak during RP-HPLC separation, they could be chosen as suitable biomarkers for quantification in the setting up of a new methodology for the biological monitoring of persons occupationally exposed, replacing currently known procedures.

Amino Acids↗

PubMed2001

Covariance of tricarboxylate carrier activity and lipogenesis in liver of polyunsaturated fatty acid (n-6) fed rats.

The mitochondrial tricarboxylate (citrate) carrier plays an important role in hepatic intermediary metabolism because, among other functions, it supplies the cytosol with acetyl units for fatty-acid synthesis. In this study, the effect of polyunsaturated fatty acids (PUFA, n-6) on the function of this mitochondrial transporter and on lipogenic enzyme activities was investigated by feeding rats for 4 weeks with a 15%-fat diet composed of high linoleic safflower oil. Citrate transport was strongly reduced in liver mitochondria isolated from PUFA-treated rats. A reduced transport activity was also observed when solubilized mitochondrial citrate carrier from PUFA-treated rats was reconstituted into liposomes. In the same animals, a decrease of cytosolic lipogenic enzyme activities was observed. These results indicate a coordinated modulation of citrate carrier and of lipogenic enzyme activities by PUFA feeding. Kinetic analysis of the carrier activity showed that only V(max) decreased, whereas K(m) was almost virtually unaffected. The PUFA-mediated effect is most likely due to the reduced mRNA level and lower content of the citrate carrier protein observed in the safflower oil-fed rats.

Animals↗

PubMed2001

ACE gene polymorphism and insulin action in older subjects and healthy centenarians.

OBJECTIVES: To evaluate the possible relationship between angiotensin-converting enzyme (ACE) insertion-deletion (ID) genotype and insulin resistance in a population of healthy older Italian subjects. DESIGN: Prospective recruitment of a convenience sample. PARTICIPANTS: One hundred twenty-five subjects age 62 to 105 in good health and not taking any drug known to interfere with glucose metabolism. RESULTS: In the sample population, the relative frequencies of the ACE genotypes deletion-deletion (DD) (0.424), ID (0.400), and insertion-insertion (II) (0.176) were not significantly different from values predicted by Hardy-Weinberg equilibrium. The genotype distribution was similar in men and women. Subjects carrying the II genotype had a higher FPG (P <.001) and FPI (P <.001) than did subjects with DD or ID genotype. Subjects with II genotype also had a significantly higher HOMA index than did subjects with DD or ID genotype (P for trend <.002). In a multivariate stepwise regression analysis, the ACE ID polymorphism was significantly and independently associated with the HOMA index (P <.001). The same result was confirmed performing multivariate analysis in the younger group and centenarians separately. CONCLUSIONS: In an older population, the presence of II ACE genotype is associated with a high degree of insulin resistance independent of other anthropometric variables known to interfere with insulin action; this association is significant in both the younger subjects and the centenarians.

Age Distribution↗

PubMed2001

Pathogenesis of HIV-related pulmonary hypertension.

Human immunodeficiency virus (HIV)-related pulmonary hypertension (HRPR) is a cardiovascular complication of HIV infection that has been recognized in the last years with increasing frequency. The etiology of HRPH is unknown. All the attempts to isolate HIV on pulmonary vessels in HRPH patients failed, and an indirect role for HIV in this disease has been hypothesized. Current theories on the pathogenesis focus on abnormalities of endothelial and smooth muscle cells of pulmonary vasculature. Endothelial and smooth muscle cell injury could be due to a high production or to a reduced clearance of cytokines in these patients. In fact, in several studies high levels of ET-1, IL-1alpha, IL-6 and PDGF in primary pulmonary hypertension (PPH) and in HRPH have been found. HIV gp 120 could induce the production of these cytokines by a stimulation of monocytes/macrophages. A high alpha1-adrenoreceptors stimulation of pulmonary vessels could be also implicated in the pathogenesis of HRPH. Chronic hypoxia is observed with increased frequency in HIV patients, and this could induce a chronic stimulation of alpha1-receptors of pulmonary vasculature with typical pathological changes. However, only a small percentage of HIV- patients develop HRPH. This observation suggests the existence of an idiosyncratic susceptibility to the development of vascular disease. This susceptibility could have a genetic basis, and might be determined by particular major histocompatibility complex alleles.

Autoimmune Diseases↗

PubMed2001

Genomic organization and mapping of the gene (SLC25A19) encoding the human mitochondrial deoxynucleotide carrier (DNC).

The deoxynucleotide carrier (DNC) transports deoxynucleotides into mitochondria and is therefore essential for mtDNA synthesis. The human DNC gene (SLC25A19) spans about 16.5 kb and consists of nine exons with the translation start site in exon 4. It is located on chromosome 17q25.3. Three transcripts, which differ in their 5' ends and are generated by alternative splicing, have been identified.

Alternative Splicing↗

PubMed2001

Primary pulmonary hypertension in HIV patients: a systematic review.

The relationship between grade of pulmonary hypertension and factors associated with human immunodeficiency virus among patients with HIV infection is poorly documented. This report documents the most extensive attempt made thus far to determine whether a relationship exists between degree of pulmonary hypertension and the following conditions: HIV risk factor, degree of immunosuppression, presence or absence of AIDS, and presence or absence of liver cirrhosis. A retrospective study involving a search of the published literature on primary pulmonary hypertension among HIV cases from 1987 to 1998, using the Medline and Aidsline databases was conducted. Patients for whom secondary causes of pulmonary hypertension could be excluded were selected, and the following information for each was recorded: age, gender, risk factors for HIV infection, HIV disease stage according to the Centers for Disease Control, previous opportunistic and neoplastic diseases, CD4+ cell count (cells/L), presence or absence of liver cirrhosis, pulmonary systolic artery pressure level, and lung pathology specimens. Information about the patient's survival time was also recorded. Seventy-six patients were judged to have primary pulmonary hypertension and were included in the study. While no correlation was found between pulmonary systolic artery pressure level and CD4+ cell counts, a statistically significant difference was found between HIV-positive patients with and without AIDS as determined by the Centers for Disease Control criteria with regard to the degree of pulmonary hypertension, expressed as pulmonary systolic artery pressure level (85.4 +/- 17 mm Hg vs 71.8 +/- 15 mm Hg, p < 0.013). Although a higher PAPS was present in HIV cirrhotic patients, a statistically significant difference was not found between degree of pulmonary hypertension and evidence of hepatic cirrhosis (85 +/- 21 mm Hg vs 73.1 +/- 15 mm Hg, p < 0.062). Patients with AIDS and primary pulmonary hypertension present a higher degree of pulmonary hypertension than non-AIDS patients. Pulmonary hypertension associated with HIV seems to be related to a cytokine-related stimulation and proliferation of endothelium. High levels of cytokines present in AIDS patients can favor pulmonary hypertension, but the role of a host response to HIV--determined by one or more HLA subtypes--is suspected to enhance high cytokine production levels.

Adult↗

PubMed2000

Identification of the human mitochondrial oxodicarboxylate carrier. Bacterial expression, reconstitution, functional characterization, tissue distribution, and chromosomal location.

In Saccharomyces cerevisiae, the genes ODC1 and ODC2 encode isoforms of the oxodicarboxylate carrier. They both transport C5-C7 oxodicarboxylates across the inner membranes of mitochondria and are members of the family of mitochondrial carrier proteins. Orthologs are encoded in the genomes of Caenorhabditis elegans and Drosophila melanogaster, and a human expressed sequence tag (EST) encodes part of a closely related protein. Information from the EST has been used to complete the human cDNA sequence. This sequence has been used to map the gene to chromosome 14q11.2 and to show that the gene is expressed in all tissues that were examined. The human protein was produced by overexpression in Escherichia coli, purified, and reconstituted into phospholipid vesicles. It has similar transport characteristics to the yeast oxodicarboxylate carrier proteins (ODCs). Both the human and yeast ODCs catalyzed the transport of the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a counter-exchange mechanism. Adipate, glutarate, and to a lesser extent, pimelate, 2-oxopimelate, 2-aminoadipate, oxaloacetate, and citrate were also transported by the human ODC. The main differences between the human and yeast ODCs are that 2-aminoadipate is transported by the former but not by the latter, whereas malate is transported by the yeast ODCs but not by the human ortholog. In mammals, 2-oxoadipate is a common intermediate in the catabolism of lysine, tryptophan, and hydroxylysine. It is transported from the cytoplasm into mitochondria where it is converted into acetyl-CoA. Defects in human ODC are likely to be a cause of 2-oxoadipate acidemia, an inborn error of metabolism of lysine, tryptophan, and hydroxylysine.

Adipates↗

PubMed2000

Identification in Saccharomyces cerevisiae of two isoforms of a novel mitochondrial transporter for 2-oxoadipate and 2-oxoglutarate.

The nuclear genome of Saccharomyces cerevisiae encodes 35 members of a family of membrane proteins. Known members transport substrates and products across the inner membranes of mitochondria. We have localized two hitherto unidentified family members, Odc1p and Odc2p, to the inner membranes of mitochondria. They are isoforms with 61% sequence identity, and we have shown in reconstituted liposomes that they transport the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a strict counter exchange mechanism. Intraliposomal adipate and glutarate and to a lesser extent malate and citrate supported [14C]oxoglutarate uptake. The expression of Odc1p, the more abundant isoform, made in the presence of nonfermentable carbon sources, is repressed by glucose. The main physiological roles of Odc1p and Odc2p are probably to supply 2-oxoadipate and 2-oxoglutarate from the mitochondrial matrix to the cytosol where they are used in the biosynthesis of lysine and glutamate, respectively, and in lysine catabolism.

Adipates↗

PubMed2000

Identification and functions of new transporters in yeast mitochondria.

The genome of Saccharomyces cerevisiae encodes 35 putative members of the mitochondrial carrier family. Known members of this family transport substrates and products across the inner membranes of mitochondria. We are attempting to identify the functions of the yeast mitochondrial transporters via high-yield expression in Escherichia coli and/or S. cerevisiae, purification and reconstitution of their protein products into liposomes, where their transport properties are investigated. With this strategy, we have already identified the functions of seven S. cerevisiae gene products, whose structural and functional properties assigned them to the mitochondrial carrier family. The functional information obtained in the reconstituted system and the use of knock-out yeast strains can be usefully exploited for the investigation of the physiological role of individual transporters. Furthermore, the yeast carrier sequences can be used to identify the orthologous proteins in other organisms, including man.

Amino Acid Transport Systems, Basic↗

PubMed2000

Monitoring of heavy metal deposition in Northern Italy by moss analysis.

A survey of heavy metal deposition in the mountainous territories of Northern Italy was carried out in 1995-96. Moss samples (mainly Hylocomium splendens) were collected in a dense network of sites (about 3.2 sites/1000 km(2)) and the data of metal concentrations in moss tissues were statistically correlated with environmental and climatic factors, as well as with bulk deposition of elements and elemental concentrations in the soil. Three main geographic patterns of metal concentration in mosses could be defined: (1) Fe, Ni, and Cr, all derived both by soil particulates and anthropogenic emissions connected with ferrous metal manufacturing, were mostly concentrated in Northwestern Italy; (2) Cu and Zn, as typical multi-source elements, showed rather high concentrations with little ranges of variation over the whole area and small peaks reflecting local source points; (3) Cd and Pb reflected long-distance transport and showed highest concentrations in the regions with highest precipitation, especially in the Eastern Alps.

Journal Article↗

PubMed2000

Yeast mitochondrial carriers: bacterial expression, biochemical identification and metabolic significance.

The genome of Saccharomyces cerevisiae encodes 35 members of a family proteins that transport metabolites and substrates across the inner membranes of mitochondria. They include three isoforms of the ADP/ATP translocase and the phosphate and citrate carriers. At the start of our work, the functions of the remaining 30 members of the family were unknown. We are attempting to identify these 30 proteins by overexpression of the proteins in specially selected host strains of Escherichia coli that allow the carriers to accumulate at high levels in the form of inclusion bodies. The purified proteins are then reconstituted into proteoliposomes where their transport properties are studied. Thus far, we have identified the dicarboxylate, succinate-fumarate and ornithine carriers. Bacterial overexpression and functional identification, together with characterization of yeast knockout strains, has brought insight into the physiological significance of these transporters. The yeast dicarboxylate carrier sequence has been used to identify the orthologous protein in Caenorhabditis elegans and, in turn, this latter sequence has been used to establish the sequence of the human ortholog.

Animals↗

PubMed2000

[Vascular trauma of the lower limbs].

BACKGROUND: This study was conducted to evaluate the results of treatment of vascular trauma of the lower extremities and those factors associated with limb loss. DESIGN: a retrospective evaluation of a series with lower extremities vascular trauma. SETTING: University Hospital. METHODS: Thirty-one patients accounting for 45 vascular lesions of the lower extremities (27 arterial and 18 venous injuries), over a 15 years period ending December 1998. Age, sex, modality of the trauma, site of the lesion and associated skeletal injuries, diagnostic procedures, ischemic time, arterial and venous repair performed were analyzed. RESULTS: Perioperative mortality was 7.4%. For arterial injuries, limb salvage was obtained in 22 patients (81.5%). Five amputations (18.5%), 1 primary and 4 secondary have been performed. Amputation rate was 26.7% for popliteal lesions versus 8.3% for other locations, 40% when a skeletal lesion was associated versus 5.9% for those without such injuries, 37.5% for reverse saphenous vein interpositions versus 5.6% for arterial repair without interposition. CONCLUSIONS: In this study, the factors influencing limb loss in vascular trauma of the lower extremities are popliteal location, the association with skeletal injuries, the need of saphenous vein interposition for arterial repair.

Accidents, Occupational↗

PubMed1999

Organization and sequence of the gene for the human mitochondrial dicarboxylate carrier: evolution of the carrier family.

The dicarboxylate carrier (DIC) is a nuclear-encoded protein located in the mitochondrial inner membrane. It catalyses the transport of dicarboxylates such as malate and succinate across the mitochondrial membrane in exchange for phosphate, sulphate and thiosulphate. We have determined the sequences of the human cDNA and gene for the DIC. The gene sequence was established from overlapping genomic clones generated by PCRs by use of primers and probes based upon the human cDNA sequence. It is spread over 8.6 kb of human DNA and is divided into 11 exons. Five short interspersed repetitive Alu sequences are found in intron I. The protein encoded by the gene is 287 amino acids long. In common with the rat protein, it does not have a processed presequence to help to target it into mitochondria. It has been demonstrated by Northern- and Western-blot analyses that the DIC is present in high amounts in liver and kidney, and at lower levels in all the other tissues analysed. The positions of introns contribute towards an understanding of the processes involved in the evolution of human genes for carrier proteins.

Alu Elements↗

PubMed1999

Identification of the mitochondrial carnitine carrier in Saccharomyces cerevisiae.

The mitochondrial carrier protein for carnitine has been identified in Saccharomyces cerevisiae. It is encoded by the gene CRC1 and is a member of the family of mitochondrial transport proteins. The protein has been over-expressed with a C-terminal His-tag in S. cerevisiae and isolated from mitochondria by nickel affinity chromatography. The purified protein has been reconstituted into proteoliposomes and its transport characteristics established. It transports carnitine, acetylcarnitine, propionylcarnitine and to a much lower extent medium- and long-chain acylcarnitines.

Acetylcarnitine↗

PubMed1999

Identification of the yeast mitochondrial transporter for oxaloacetate and sulfate.

Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family, including the OAC protein. The transport specificities of some family members are known, but most are not. The function of the OAC has been revealed by overproduction in Escherichia coli, reconstitution into liposomes, and demonstration that the proteoliposomes transport malonate, oxaloacetate, sulfate, and thiosulfate. Reconstituted OAC catalyzes both unidirectional transport and exchange of substrates. In S. cerevisiae, OAC is in inner mitochondrial membranes, and deletion of its gene greatly reduces transport of oxaloacetate sulfate, thiosulfate, and malonate. Mitochondria from wild-type cells swelled in isoosmotic solutions of ammonium salts of oxaloacetate, sulfate, thiosulfate, and malonate, indicating that these anions are cotransported with protons. Overexpression of OAC in the deletion strain increased greatly the [(35)S]sulfate/sulfate and [(35)S]sulfate/oxaloacetate exchanges in proteoliposomes reconstituted with digitonin extracts of mitochondria. The main physiological role of OAC appears to be to use the proton-motive force to take up into mitochondria oxaloacetate produced from pyruvate by cytoplasmic pyruvate carboxylase.

Anion Transport Proteins↗