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The enzyme carnitine-acylcarnitine translocase (CACT) is involved in the transport of long-chain fatty acids into mitochondria. CACT deficiency is a life-threatening, recessively inherited disorder of lipid beta-oxidation which manifests in early infancy with hypoketotic hypoglycemia, cardiomyopathy, liver failure, and muscle weakness. We report here the clinical, biochemical, and molecular features of six CACT-deficient patients from Italy, Spain, and North America who exhibited significant clinical heterogeneity. In five patients (Patients 1, 2, 4, 5, and 6) the disease manifested in the neonatal period, while the remaining patient (Patient 3), the younger sibling of an infant who had died with clinical suspicion of fatty acid oxidation defect, has been treated since birth and was clinically asymptomatic at 4.5 years of age. Patients 1 and 4 were deceased within 6 months from the onset of this study, while the remaining four are still alive at 8, 4.5, 3.5, and 2 years, respectively. Sequence analysis of the CACT gene (SLC25A20) disclosed five novel mutations and three previously reported mutations. Three patients were homozygous for the identified mutations. Two of the novel mutations (c.718+1G>C and c.843+4_843+50del) altered the donor splice site of introns 7 and 8, respectively. The 47-nt deletion in intron 8 caused both skipping of exon 8 only and skipping of exons 6-8. Four mutations [[c.159dupT;c.163delA] ([p.Gly54Trp;p.Thr55Ala]) c.397C>T (p.Arg133Trp), c.691G>C (p.Asp231His), and c.842C>T (p.Ala281Val)] resulted in amino acid substitutions affecting evolutionarily conserved regions of the protein. Interestingly, one of these exonic mutations (p.Ala281Val) was associated with a splicing defect also characterized by skipping of exons 6-8. The deleterious effect of the p.Arg133Trp substitution was demonstrated by measuring CACT activity upon expression of the normal and the mutant protein in E. coli and functional reconstitution into liposomes. Combined analysis of clinical, biochemical, and molecular data failed to indicate a correlation between the phenotype and the genotype., (Copyright 2004 Wiley-Liss, Inc.)

Objectives: Lower Urinary Tract Symptoms (LUTS) associated to Benign Prostatic Hyperplasia (BPH) are very common in middle aged and older men. Since BPH, and the related therapies have a significant impact on a patient's Quality of Life (QoL), the health related quality of life (HQoL) evaluation is becoming an important aspect to be considered. The present study deals with the development and validation of a BPH disease specific questionnaire (Bononian Satisfaction Profile - BSP-BPH), considering HQoL in patients seeking medical help for BPH. The innovation of this questionnaire is that the patients' scores achieved are related to their subjective satisfaction., Materials and Methods: Questionnaire development: On the base of our previous experience with BSP-PC (a disease specific questionnaire assessing HQoL in prostate Cancer patients), of a careful review of other available instruments (SAT-P, SF-36, ICS-QoL, BPH-HqoL) and of three subsequent meetings with experts (10) and patients (20), we defined the ten life aspects mostly impaired by BPH. We created a first 72-item version of the BSP-BPH. Ten patients cross matched for age end education to our study population were asked to fill in the questionnaire. A 31 item questionnaire version, together with EuroQoL, an already generic validated instrument, was administered to 121 patients as well. Further meetings and statistical analysis defined the 18 items of the BSP- BPH. Questionnaire validation: the BSP-BPH was filled in by 435 patients enrolled in a campaign for detection of prostatic diseases held among the male population of Bologna., Results: Questionnaire development: the participants' mean age was 63 years (N = 121); 47.1% had BPH. The 18 items were selected on the basis of the following criteria: a) r > 0.50, p < 0.05 (correlation test re-test); b) p < 0.05 (ANOVA presence vs absence of BPH); c) r > 0.50, p < 0.05 (correlation with EuroQol). Questionnaire validation: the participants' mean age was 63 years (N = 436). 16 patients with a history of neoplasia were excluded from our study. 21 were missing data. 45% of the sample had BPH. Principal component analysis identified 5 components: 1) satisfaction about sexual functionality (Cronbach alpha = 0.94); 2) satisfaction about social functionality (alpha = 0.80); 3) satisfaction about cognitive/emotional functionality (alpha = 0.82); 4) satisfaction about urinary functionality (alpha = 0.87); 5) satisfaction about physical functionality (alpha = 0.66); total Cronbach alpha was 0.88., Conclusions: The 18 items version of BSP-BPH questionnaire can be used as an instrument for HQoL evaluation in patients with BPH focusing on patients' subjective satisfaction. At the moment only the Italian version is available.

The structures of the first and the second transmembrane segment of the bovine mitochondrial oxoglutarate carrier (OGC) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. Peptides 21-46 and 78-108 of its primary sequence were synthesized and structurally characterized in membrane-mimetic environments. CD data showed that at high concentrations of TFE (>50%) and SDS (>2%) both peptides assume alpha-helical structures, whereas in more hydrophilic environments only peptide 78-108 has a helical structure. (1)H-NMR spectra of the two peptides in TFE/water and SDS were fully assigned, and the secondary structures of the peptides were obtained from nuclear Overhauser effects, (3)J(alphaH-NH) coupling constants and alphaH chemical shifts. The three-dimensional solution structures of the peptides in TFE/water were generated by distance geometry calculations. A well-defined alpha-helix was found in the region K24-V39 of peptide 21-46 and in the region A86-F106 of peptide 78-108. We cannot exclude that in intact OGC the extension of these helices is longer. The helix of peptide 21-46 is essentially hydrophobic, whereas that of peptide 78-108 is predominantly hydrophilic.

The yeast peroxisomal adenine nucleotide carrier, Ant1p, was shown to catalyse unidirectional transport in addition to exchange of substrates. In both transport modes, proton movement occurs. Nucleotide hetero-exchange is H+-compensated and electroneutral. Furthermore, microscopic fluorescence imaging of a pH-sensitive green fluorescent protein targeted to peroxisomes shows that Ant1p is involved in the formation of a DeltapH across the peroxisomal membrane, acidic inside.

The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites, nucleotides, and cofactors through this membrane and thereby connect and/or regulate cytoplasm and matrix functions. ATP-Mg is transported in exchange for phosphate, but no protein has ever been associated with this activity. We have isolated three human cDNAs that encode proteins of 458, 468, and 489 amino acids with 66-75% similarity and with the characteristic features of the mitochondrial carrier family in their C-terminal domains and three EF-hand Ca(2+)-binding motifs in their N-terminal domains. These proteins have been overexpressed in Escherichia coli and reconstituted into phospholipid vesicles. Their transport properties and their targeting to mitochondria demonstrate that they are isoforms of the ATP-Mg/Pi carrier described in the past in whole mitochondria. The tissue specificity of the three isoforms shows that at least one isoform was present in all of the tissues investigated. Because phosphate recycles via the phosphate carrier in mitochondria, the three isoforms of the ATP-Mg/Pi carrier are most likely responsible for the net uptake or efflux of adenine nucleotides into or from the mitochondria and hence for the variation in the matrix adenine nucleotide content, which has been found to change in many physiopathological situations.

Highly active antiretroviral therapy (HAART) greatly reduces the risk of developing tuberculosis for HIV-infected persons. Nonetheless, HIV-associated tuberculosis continues to occur in countries where HAART is widely used. To identify the characteristics of HIV-infected persons who develop tuberculosis in the context of the availability of HAART, the current authors analysed data taken from 271 patients diagnosed, in Italy, during 1999-2000. These patients represent 0.7% of the 40,413 HIV-infected patients cared for in the clinical units participating in this current study. From the data it was observed that 20 patients (7.4%) had a previous episode of tuberculosis whose treatment was not completed. Eighty-one patients (29.9%) were diagnosed with HIV at tuberculosis diagnosis, 108 (39.8%) were aware of their HIV status but were not on antiretroviral treatment and 82 (30.3%) were on antiretroviral treatment. Patients on antiretroviral treatment were significantly less immunosuppressed than patients with HIV diagnosed concurrently with tuberculosis, or other patients not on antiretrovirals (median CD4 lymphocytes count: 220 cells x mm(-3) versus 100 cells x mm(-3), and 109 cells x mm(-3), respectively). No significant differences in clinical presentation of tuberculosis according to antiretroviral therapy status were recorded. Failure of tuberculosis control interventions (e.g. noncompletion of treatment) and of HIV care (delayed diagnosis of HIV infection and suboptimal uptake of therapy) may contribute to continuing occurrence of HIV-associated tuberculosis in a country where highly active antiretroviral therapy is largely available. However, a significant proportion of cases occur in patients who are on antiretroviral treatment.

Intravascular catheters have become essential devices for the management of critically and chronically ill patients. However, their use is often associated with serious infectious complications, mostly catheter-related bloodstream infection (CRBSI), resulting in significant morbidity, increased duration of hospitalization, and additional medical costs. The majority of CRBSIs are associated with central venous catheters (CVCs), and the relative risk for CRBSI is significantly greater with CVCs than with peripheral venous catheters. However, most CVC-related infections are preventable, and different measures have been implemented to reduce the risk for CRBSI, including maximal barrier precautions during catheter insertion, catheter site maintenance, and hub handling. The focus of the present review is on new technologies for preventing infections that are directed at CVCs. New preventive strategies that have been shown to be effective in reducing risk for CRBSI, including the use of catheters and dressings impregnated with antiseptics or antibiotics, the use of new hub models, and the use of antibiotic lock solutions, are briefly described.

Screening of a cDNA library prepared from liver of the freeze-tolerant wood frog (Rana sylvatica) identified a freeze-responsive clone containing a 1370-nt sequence with an open reading frame of 360 amino acids. Sequence analysis revealed 84-86% identity with the mammalian inorganic phosphate carrier (PiC) that spans the inner mitochondrial membrane. Northern blot analysis showed that pic transcript levels increased over a time course of freezing, reaching 60-fold upregulation after 24-h frozen. Transcript levels were also assessed under freezing-related stresses with results showing a strong increase in pic transcript levels in response to dehydration (elevated 9.0-fold in 40% dehydrated frogs) but not under anoxia. Western blotting revealed elevated PiC protein over a time course of freeze-thaw whereas other mitochondrial carriers (dicarboxylate carrier, oxoglutarate transporter) of the same family were not affected by freezing. This modulation of PiC protein levels may play a role in mitochondrial ionic and/or osmotic balance during freeze-induced cell volume reduction.

The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 16 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates, and cofactors across the inner membrane. Here the identification and functional characterization of the mitochondrial GTP/GDP carrier (Ggc1p) is described. The ggc1 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. It transported GTP and GDP and, to a lesser extent, the corresponding deoxynucleotides and the structurally related ITP and IDP by a counter-exchange mechanism. Transport was saturable with an apparent K(m) of 1 microm for GTP and 5 microm for GDP. It was strongly inhibited by pyridoxal 5'-phosphate, bathophenanthroline, tannic acid, and bromcresol purple but little affected by the inhibitors of the ADP/ATP carrier carboxyatractyloside and bongkrekate. Furthermore, in contrast to the ADP/ATP carrier, the Ggc1p-mediated GTP/GDP heteroexchange is H(+)-compensated and thus electroneutral. Cells lacking the ggc1 gene had reduced levels of GTP and increased levels of GDP in their mitochondria. Furthermore, the knock-out of ggc1 results in lack of growth on nonfermentable carbon sources and complete loss of mitochondrial DNA. The physiological role of Ggc1p in S. cerevisiae is probably to transport GTP into mitochondria, where it is required for important processes such as nucleic acid and protein synthesis, in exchange for intramitochondrially generated GDP.

Deficiency of carnitine/acylcarnitine translocase (CACT) is an autosomal recessive disorder of the carnitine cycle resulting in the inability to transfer fatty acids across the inner mitochondrial membrane. Only a limited number of affected patients have been reported and the effect of therapy on this condition is still not well defined. Here, we report a new patient with this disorder and follow the response to therapy. Our patient was the product of a consanguineous marriage. He presented shortly after birth with cardiac myopathy and arrhythmia coupled with severe non-ketotic hypoglycemia. Initial metabolic studies indicated severe non-ketotic C6-C10 dicarboxylic aciduria, plasma carnitine deficiency, and a characteristic elevation of plasma C:16:0, C18:1, and C18:2 acylcarnitine species. Enzyme assay confirmed deficiency of CACT activity. Molecular studies indicated that this child was homozygous, and both parents heterozygous, for a single bp change converting glutamine 238 to arginine (Q238R). Therapy with a formula providing most of the fat via medium chain triglycerides (MCT) and carnitine supplementation reduced the concentration of long-chain acylcarnitines and reversed cardiac symptoms and the hypoglycemia. These results suggest that carnitine and MCT may be effective in treating this defect of long-chain fatty acid oxidation., (Copyright 2003 Wiley-Liss, Inc.)

The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites and cofactors through this membrane, and connect cytoplasmic functions with others in the matrix. SAM (S-adenosylmethionine) has to be transported into the mitochondria where it is converted into S-adenosylhomocysteine in methylation reactions of DNA, RNA and proteins. The transport of SAM has been investigated in rat liver mitochondria, but no protein has ever been associated with this activity. By using information derived from the phylogenetically distant yeast mitochondrial carrier for SAM and from related human expressed sequence tags, a human cDNA sequence was completed. This sequence was overexpressed in bacteria, and its product was purified, reconstituted into phospholipid vesicles and identified from its transport properties as the human mitochondrial SAM carrier (SAMC). Unlike the yeast orthologue, SAMC catalysed virtually only countertransport, exhibited a higher transport affinity for SAM and was strongly inhibited by tannic acid and Bromocresol Purple. SAMC was found to be expressed in all human tissues examined and was localized to the mitochondria. The physiological role of SAMC is probably to exchange cytosolic SAM for mitochondrial S-adenosylhomocysteine. This is the first report describing the identification and characterization of the human SAMC and its gene.

The South-eastern Area (SA) of the Municipality of La Spezia (Liguria Region, Italy) is characterised by a heavy environmental lead (Pb) contamination, chiefly due to the emissions of a Pb-processing plant in operation since 1930. In order to assess the risk of Pb poisoning of residents of SA, and to estimate the degree of association between the blood Pb level (BLL) and haematocrit % (HCT), intended as a biomarker of early haematological dysfunction, we reanalysed data of 785 individuals collected in 1992 as a part of a larger national biological monitoring project. Multiple normal regression modelling was applied to estimate the role of residence on log-transformed BLL, and Median Ratio (MR) was used as an index of effect. The same statistical modelling was also applied to reveal the relationship between HCT and BLL. Allowing for several confounders (including occupational exposure to Pb), residents of SA showed a 14% increase (MR=1.14, 95% IC=1.06-1.23%) in the median BLL value compared to people living outside SA. The excess reached 27% (MR=1.27, 95% IC=1.14-1.41%) after 30 years of residence. Parallel results were also obtained in a subgroup composed only of pupils (<18 years, non-smokers, non-drinkers). Finally, regression analysis highlighted a statistically significant parabolic trend in HCT in relation to BLL. The non-linear dose-response relationship, which attests to an adverse effect on the erythrocytic function of BLLs at least over 17.00 microg/dl, is in agreement with the findings of other authors and consistent with the results of an excess occurrence of self-reported anaemia obtained from a previous comparative survey carried out on the same population.

Objectives: Radical prostatectomy is actually the gold-standard treatment for organ-confined prostate cancer. Since Schuessler et al. performed the first laparoscopical radical prostatectomy (LRP) in 1992 this surgical approach for prostate cancer treatment has been widely diffused among european urologists. In this study we report our initial experience with laparoscopic surgery focusing on the oncological assessments and comparing these results to those of the retropubic approach., Material and Methods: Between March 2002 and November 2003, 50 laparoscopic radical prostatectomy were performed at our Institute. We reviewed the operative times, intraoperative complications, mean catheterization and postoperative hospital stay of these first 50 cases. Moreover during the same period a group of 50 consecutive patients underwent retropubic radical prostatectomy (RRP) and data were analyzed and compared to laparoscopic issues. The laparoscopic approach was performed according to the Montsouris technique. Patient age, Gleason score at biopsy, PSA and clinical stage of the two groups were compared. Positive margins of the two groups were compared in relation to their location and pathological stage., Results: No significative statistical differences of age, preoperatory PSA, Gleason score at biopsy and clinical stage were observed between the two groups (p > 0.05). Also in post-operative data no significative statistical differences regarding the pathological stage (p = 0.54) and the Gleason score (p = 0.714) were noted between the two groups. In RRP group the pathological stage was pT2 in 28 patients and pT3 in 22 patients; in LRP group was pT2 in 31 patients and pT3 in 19 patients. The mean Gleason score resulted 6.16 in RRP group and 6.10 in LRP group. The number of positive surgical margins was low in both groups and the location was similar in retropubic and laparoscopic specimens. We reported 13 positive surgical margins in RRP group and 12 in LRP (p = 0.8)., Conclusions: Basing on our initial experience with 50 patients we can affirm that laparoscopic radical prostatectomy can be performed with a lower morbidity and oncological results similar to the retropubic approach even in the early phase of the learning curve. Our experience could be useful to encourage all the urologists approaching laparoscopy.

It is crucial to ensure an optimal clinical management of HCV infection in HIV-co-infected persons. The reasons for the development of guidelines on HCV-infection treatment in HIV-infected persons arise from the need for a standardised management of HIV/HCV coinfection in our Institute. The aim of these guidelines are: to clarify principles of clinical management of HCV infection in HIV-infected patients to care-providers; to improve the awareness of HIV-infected patients cared for our Institute on current management of HCV infection; to improve the quality of care on this topic. These guidelines, based on Evidence based Medicine principles, have been developed by a panel of experts, who conducted a systematic review of the literature, mainly taking into account current international recommendations. In the present document, the most frequent clinical presentation occurring in the management of HIV/HCV co-infected patients at our Institution are discussed. The adherence to present guidelines and their effectiveness at our Institution, outcome indicators will be evaluated. The present guidelines cannot entirely substitute the judgement of an expert clinician. However, adherence to these guidelines will contribute to the improvement of the standard of care of HIV/HCV-co-infected persons.

The mitochondrial carriers (MCs) shuttle a variety of metabolites across the inner mitochondrial membrane (i.m.m.). In man they are encoded by the SLC25 genes. Some MCs have isoforms encoded by different SLC25 genes, whereas the phosphate carrier has two variants arising from an alternative splicing of SLC25A3. Six MCs have been sequenced after purification, and many more have been identified from their transport and kinetic properties following heterologous over-expression and reconstitution into liposomes. All MCs of known function belong to the same protein family, since their polypeptide chains consist of three tandemly related sequences of about 100 amino acids, and the repeats of the different carriers are homologous. They probably function as homodimers, each monomer being folded in the membrane into six transmembrane segments. The functional information obtained in studies with mitochondria and/or the reconstituted system has helped to gain an insight into the physiological role of the MCs in cell metabolism, as have tissue distribution, the use of knock-out mice (and/or yeast) and over-expression in human cell lines (or yeast) of individual carriers and isoforms. At the same time, the cloning and functional identification of many SLC25 genes has made it possible (i) to identify the genes (and their defects) responsible for some diseases, e.g. Stanley syndrome and Amish microcephaly, and (ii) where the genes were already known, to characterize the function of the gene products and hence understand the molecular basis and the symptoms of the diseases, e.g. hyperornithinaemia, hyperammonaemia and homocitrullinuria (HHH) syndrome and type II citrullinemia. It is likely that further extension and functional characterization of the SLC25 gene family will elucidate other diseases caused by MC deficiency.

A 34-year-old woman presented with a 10-year history of recurrent oral and genital ulcerations and recurrent episodes of bilateral conjunctivitis associated with HIV infection. A diagnosis of Behçet's disease (BD) in association with keratoconjunctivitis sicca (KCS) was made after exclusion of other viral and autoimmune diseases according to the international criteria for BD. This is the first reported case of a combination of BD and KCS in a patient with HIV infection in which a complete resolution was observed as a result of successful highly active antiretroviral therapy. The likelihood that a direct viral effect or HIV-induced autoimmune mechanisms act in the pathogenesis of both BD and KCS in HIV-infected patients is discussed.

This work is the result of two surveys carried out to obtain an overview of the specific types of activities conducted by public institutions and non-governmental organisations (NGO) with regard to HIV infection and AIDS among migrant populations in Italy. The results reveal that the types of services offered to migrant populations differ when comparing public institutions to NGOs. Public institutions mainly provide services related to medical-health issues, whereas NGOs focus on social assistance and acceptance. Both types of organisations report problems in communicating to migrant populations information on HIV infection, sexually transmitted diseases, prevention, and therapy, which is a critical issue in the relationship between the psycho-social-health operator and the foreigner.

The genome of Saccharomyces cerevisiae contains 35 members of the mitochondrial carrier protein family, most of which have not yet been functionally identified. Here the identification of the mitochondrial carrier for S-adenosylmethionine (SAM) Sam5p is described. The corresponding gene has been overexpressed in bacteria and the protein has been reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, (i) the Sam5p-GFP protein was found to be targeted to mitochondria; (ii) the cells lacking the gene for this carrier showed auxotrophy for biotin (which is synthesized in the mitochondria by the SAM-requiring Bio2p) on fermentable carbon sources and a petite phenotype on non-fermentable substrates; and (iii) both phenotypes of the knock-out mutant were overcome by expressing the cytosolic SAM synthetase (Sam1p) inside the mitochondria.

The malate-aspartate NADH shuttle in mammalian cells requires the activity of the mitochondrial aspartate-glutamate carrier (AGC). Recently, we identified in man two AGC isoforms, aralar1 and citrin, which are regulated by calcium on the external face of the inner mitochondrial membrane. We have now identified Agc1p as the yeast counterpart of the human AGC. The corresponding gene was overexpressed in bacteria and yeast mitochondria, and the protein was reconstituted in liposomes where it was identified as an aspartate-glutamate transporter from its transport properties. Furthermore, yeast cells lacking Agc1p were unable to grow on acetate and oleic acid, and had reduced levels of valine, ornithine and citrulline; in contrast they grew on ethanol. Expression of the human AGC isoforms can replace the function of Agc1p. However, unlike its human orthologues, yeast Agc1p catalyses both aspartate-glutamate exchange and substrate uniport activities. We conclude that Agc1p performs two metabolic roles in Saccharomyces cerevisiae. On the one hand, it functions as a uniporter to supply the mitochondria with glutamate for nitrogen metabolism and ornithine synthesis. On the other, the Agc1p, as an aspartate-glutamate exchanger, plays a role within the malate-aspartate NADH shuttle which is critical for the growth of yeast on acetate and fatty acids as carbon sources. These results provide strong evidence of the existence of a malate-aspartate NADH shuttle in yeast.

Deficiency of citrin encoded by SLC25A13 causes adult-onset type II citrullinemia (CTLN2) and idiopathic neonatal hepatitis (NICCD). So far we have diagnosed 126 (3) CTLN2 and 103 (4) NICCD patients in Japan (and other countries). From preliminary population analysis of the known nine SLC25A13 mutations, we found that the carrier frequency is high in China (1/79), Taiwan (1/98), and Korea (1/50) as well as Japan (1/69), suggesting that many patients with citrin deficiency exist in East Asia.

The Ca(2+)-sensitive dehydrogenases of the mitochondrial matrix are, so far, the only known effectors to allow Ca2+ signals to couple the activation of plasma membrane receptors to the stimulation of aerobic metabolism. In this study, we demonstrate a novel mechanism, based on Ca(2+)-sensitive metabolite carriers of the inner membrane. We expressed in Chinese hamster ovary cells aralar1 and citrin, aspartate/glutamate exchangers that have Ca(2+)-binding sites in their sequence, and measured mitochondrial Ca2+ and ATP levels as well as cytosolic Ca2+ concentration with targeted recombinant probes. The increase in mitochondrial ATP levels caused by cell stimulation with Ca(2+)-mobilizing agonists was markedly larger in cells expressing aralar and citrin (but not truncated mutants lacking the Ca(2+)-binding site) than in control cells. Conversely, the cytosolic and the mitochondrial Ca2+ signals were the same in control cells and cells expressing the different aralar1 and citrin variants, thus ruling out an indirect effect through the Ca(2+)-sensitive dehydrogenases. Together, these data show that the decoding of Ca2+ signals in mitochondria depends on the coordinate activity of mitochondrial enzymes and carriers, which may thus represent useful pharmacological targets in this process of major pathophysiological interest.

Two isoforms of the human ornithine carrier, ORC1 and ORC2, have been identified by overexpression of the proteins in bacteria and by study of the transport properties of the purified proteins reconstituted into liposomes. Both transport L-isomers of ornithine, lysine, arginine, and citrulline by exchange and by unidirectional mechanisms, and they are inactivated by the same inhibitors. ORC2 has a broader specificity than ORC1, and L- and D-histidine, L-homoarginine, and D-isomers of ornithine, lysine, and ornithine are all substrates. Both proteins are expressed in a wide range of human tissues, but ORC1 is the predominant form. The highest levels of expression of both isoforms are in the liver. Five mutant forms of ORC1 associated with the human disease hyperornithinemia-hyperammonemia-homocitrullinuria were also made. The mutations abolish the transport properties of the protein. In patients with hyperornithinemia-hyperammonemia-homocitrullinuria, isoform ORC2 is unmodified, and its presence compensates partially for defective ORC1.

Deficiency of the carnitine/acylcarnitine translocase (CACT), the most severe disorder of fatty acid beta-oxidation, is usually lethal in both humans and animals, precluding the development of animal models of the disease. In contrast, CACT deficiency is conditionally lethal in the fungus Aspergillus nidulans, since loss-of-function mutations in acuH, the translocase structural gene, do not prevent growth on carbon sources other than ketogenic compounds, such as fatty acids. Here, we describe the molecular characterization of extant acuH alleles and the development of a fungal model for CACT deficiency based on the ability of human CACT to fully complement, when expressed at physiological levels, the growth defect of an A. nidulans DeltaacuH strain on acetate and long-chain fatty acids. By using growth tests and in vitro assays this model enabled us to carry out a functional characterization of human CACT mutations showing that it may be useful for distinguishing potentially pathogenic human CACT missense mutations from neutral, single residue substitution-causing polymorphisms.

Aim: To evaluate whether highly active antiretroviral therapy (HAART) modifies radiographic appearances of pulmonary tuberculosis (TB), in terms of patterns and their relative frequencies, among patients with human immunodeficiency virus (HIV) infection., Materials and Methods: Chest radiographs were obtained in 209 HIV-infected patients with culture confirmed pulmonary TB. Computed tomography (CT) images were also reviewed for 42 patients whose chest radiographs were normal or showed questionable abnormalities. Imaging was evaluated for the presence and distribution of consolidation, cavitation, interstitial changes, pleural disease, adenopathy, and were classified as a primary or post-primary pattern., Results: A post-primary pattern was more frequent after 1996 when HAART came into clinical use. Forty-four percent (77/176) of patients not on HAART had a post-primary pattern in comparison with 82% (27/33) of patients receiving HAART (p<0.001). A primary pattern was significantly more frequent (p<0.001), in patients with more severe immunosuppression (CD4 lymphocyte less than 200/mm(3))., Conclusion: HIV patients receiving HAART with pulmonary TB, had a post-primary pattern more frequently than those not receiving this treatment. This observation is consistent with the partial restoration of cell-mediated immunity that can be induced by HAART.

The structural and dynamic features of the fourth transmembrane segment of the mitochondrial oxoglutarate carrier were investigated using site-directed spin labeling and electron paramagnetic resonance (EPR). Using a functional carrier protein with native cysteines replaced with serines, the 18 consecutive residues from S184 to S201 which are believed to form the transmembrane segment IV were substituted individually with cysteine and labeled with a thiol-selective nitroxide reagent. Most of the labeled mutants exhibited significant oxoglutarate transport in reconstituted liposomes, where they were examined by EPR as a function of the incident microwave power in the presence and absence of two paramagnetic perturbants, i.e., the hydrophobic molecular oxygen or the hydrophilic chromium oxalate complex. The periodicity of the sequence-specific variation in the spin-label mobility and the O(2) accessibility parameters unambiguously identifies the fourth transmembrane segment of the mitochondrial oxoglutarate carrier as an alpha-helix. The accessibility to chromium oxalate is out of phase with oxygen accessibility, indicating that the helix is amphipatic, with the hydrophilic face containing the residues found to be important for transport activity by site-directed mutagenesis and chemical modification. The helix is strongly packed, as indicated by the values of normalized mobility, which also suggest that the conformational changes occurring during transport probably involve the N-terminal region of the helix.

The early secretory antigenic target (ESAT)-6 purified protein and peptides from Mycobacterium tuberculosis were evaluated as antigens for the immunodiagnosis of tuberculosis (TB). Because the control of TB requires improved diagnostic procedures, efforts have increased to identify Mycobacterium tuberculosis-specific epitopes for the immunodiagnosis of active TB and to discriminate between active and latent states of infection. Two multiepitopic peptides from ESAT-6 protein were selected by computational analysis. Patients with active TB (7 HIV(+) and 20 HIV(-)) and control patients (17 HIV(+) and 28 HIV(-)) were enrolled. Enzyme-linked immunospot assay analysis for interferon-g expression by peripheral blood mononuclear cells was quantified after stimulation with selected ESAT-6 peptides, purified protein derivative, or the intact ESAT-6 protein. During active TB, 20 of 27 patients responded in vitro to ESAT-6 peptides and 23 of 27 patients to purified protein derivative. None of the controls without active TB, including individuals with latent TB infection, recognized ESAT-6 peptides. By contrast, latently infected individuals did respond in vitro to both intact ESAT-6 protein and purified protein derivative. Thus, high T-cell response frequencies to ESAT-6 peptides are present only during active TB and can be used to discriminate between active and latent forms of infection.

We describe the identification and functional characterization of two Arabidopsis mitochondrial basic amino acid carriers (BAC), AtmBAC1 and AtmBAC2, which are related to the yeast ornithine (Orn) carrier Ort1p, also known as Arg11p. The arg11 mutant requires arginine (Arg) supplementation because it fails to export sufficient ornithine from the mitochondrion to the cytosol where it is converted to arginine. AtmBAC1 and, to a lesser extent, AtmBAC2 partially replaced the function of Ort1p in yeast arg11. The more efficient putative carrier, AtmBAC1, was expressed in E. coli, purified, and reconstituted into phospholipid vesicles, where it transported the basic l-amino acids arginine, lysine, ornithine and histidine (in order of decreasing affinity). AtmBAC1 recognized l-histidine whereas both yeast Ort1p and the mammalian ortholog ORNT1p do not. Also different from ORNT1p, AtmBAC1 did not transport citrulline. AtmBAC1 appeared to be more stereospecific than the yeast and mammalian ornithine carriers, exhibiting greater preference for the l-forms of arginine, lysine and ornithine. By RT-PCR, both AtmBAC1 and AtmBAC2 transcripts were detected in stems, leaves, flowers, siliques, and seedlings. Expression of AtmBAC1 in seedlings is consistent with its involvement in Arg breakdown in early seedling development, i.e. delivery of Arg to mitochondrial arginase. The Km (0.19 mm) for Arg uptake by AtmBAC1 was close to the value we previously determined for the saturable component of Arg uptake into intact mitochondria from soybean seedling cotyledons.

Most mitochondrial preproteins carry a cleavable N-terminal presequence that mediates targeting to mitochondria and translocation across the mitochondrial membranes. In this study, we characterized the presequence of the citrate carrier (CIC, tricarboxylate carrier) of rat liver mitochondria. The CIC presequence was found to be dispensable both for targeting to mitochondria and insertion into the inner membrane. Unlike the presequence of the related phosphate carrier, fusion of the CIC presequence to the cytosolic enzyme dihydrofolate reductase did not confer mitochondrial targeting, indicating that the CIC presequence does not act as a targeting signal. However, the presequence was required to keep the CIC in a soluble state. Mature CIC lacking the presequence was prone to aggregation. We conclude that mitochondrial presequences do not necessarily act as mediators of targeting. In the case of the CIC, the presequence appears to determine the folding state of the preprotein.

During 1998-1999 a survey assessed the health impact of living in an area (Zona D) of the city of La Spezia (Liguria Region, Italy) characterised by industrial plants, urban waste disposal sites and incinerators, a coal-fired power-station and a heavily trafficked thoroughfare. One of the main results was an excess prevalence and incidence of self-reported anaemia among residents in area D compared to those of the control area (Zone C). Given the environmental lead pollution in Zone D due to the emissions of a lead processing plant active since 1930, and in order to confirm the validity of previous findings, was investieable the association between residence and blood lead levels, as well as the relationship between the latter and an anaemia-specific biomarker. We re-analysed personal, occupational and haematological data of 785 residents of La Spezia Province collected by the Liguria Environmental Protection Agency (ARPAL) during 1992 as part of a biological monitoring survey of the general population about the risk of lead intoxication. Subjects were geo-referenced by the 1992 residence address, and the haematocrit value was used as the anaemia-specific biomarker. Multiple regression analysis estimated the effect of residence on log-transformed lead blood levels, and to assess the relationship between the latter and haematocrit values. After adjusting for several potential confounding factors, residents from Zone D showed a 14% increase (95% IC = 6%-23%) in the blood lead median value estimated via the log-normal regression model compared to people living elsewhere. The excess became 27% (95% IC = 14%-41%) after 30 years of residence in the study area. Similar results were obtained by focusing analyses on students (n = 89, < 18 years, non-smokers, non-drinkers). The same modelling highlighted a significant non-linear (parabolic) dose-response relationship between lead blood and haematocrit values. This trend is consistent with the findings of other Authors and might reveal an adverse haematological effect of lead values over 170, 0 microgram/l. In conclusion, The results of this investigation strengthen the epidemiological evidence of the 1998-1999 survey.

Starvation has been associated with a reduced citrate carrier (CTP) activity in rat liver mitochondria. In the present study the molecular mechanism responsible for this reduction was investigated. Northern blot analysis performed with hepatic total RNA showed a decrease of about 40% in the CTP mRNA abundance in starved rats, when compared to fed animals. Nuclear run-on assay did not reveal any appreciable difference in the rate of CTP mRNA synthesis between the two groups of animals, while the apparent half-life of CTP mRNA in hepatocytes from fed and starved rats was 11 and 6h, respectively. Therefore, these results suggest that in starved rats the regulation of hepatic CTP expression occurs at posttranscriptional level. Moreover, the reduced CTP activity in starved animals gradually increased by refeeding. The carrier activity reached fed rat values 6-9h following refeeding. Interestingly, the accumulation of CTP mRNA raised in parallel with the transport activity.

The adenine nucleotide carrier from Jerusalem artichoke (Helianthus tuberosus L.) tubers mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxapatite and Matrex Gel Blue B in the presence of cardiolipin and asolectin. SDS gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 33 kDa. When reconstituted in liposomes, the adenine nucleotide carrier catalyzed a pyridoxal 5'-phosphate-sensitive ATP/ATP exchange. It was purified 75-fold with a recovery of 15% and a protein yield of 0.18% with respect to the mitochondrial extract. Among the various substrates and inhibitors tested, the reconstituted protein transported only ATP, ADP, and GTP and was inhibited by bongkrekate, phenylisothiocyanate, pyridoxal 5'-phosphate, mersalyl and p-hydroxymercuribenzoate (but not N-ethylmaleimide). Atractyloside and carboxyatractyloside (at concentrations normally inhibitory in animal and plant mitochondria) were without effect in Jerusalem artichoke tubers mitochondria. Vmax of the reconstituted ATP/ATP exchange was determined to be 0.53 micromol/min per mg protein at 25 degrees C. The half-saturation constant Km and the corresponding inhibition constant Ki were 20.4 microM for ATP and 45 microM for ADP. The activation energy of the ATP/ATP exchange was 28 KJ/mol between 5 and 30 degrees C. The N-terminal amino acid partial sequence of the purified protein showed a partial homology with the ANT protein purified from mitochondria of maize shoots.

The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 15 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates and cofactors across the inner membrane. Here the identification of the mitochondrial carrier for the essential cofactor thiamine pyrophosphate (ThPP) is described. The protein has been overexpressed in bacteria, reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, cells lacking the gene for this carrier had reduced levels of ThPP in their mitochondria, and decreased activity of acetolactate synthase, a ThPP-requiring enzyme found in the organellar matrix. They also required thiamine for growth on fermentative carbon sources.

The disorder Amish microcephaly (MCPHA) is characterized by severe congenital microcephaly, elevated levels of alpha-ketoglutarate in the urine and premature death. The disorder is inherited in an autosomal recessive pattern and has been observed only in Old Order Amish families whose ancestors lived in Lancaster County, Pennsylvania. Here we show, by using a genealogy database and automated pedigree software, that 23 nuclear families affected with MCPHA are connected to a single ancestral couple. Through a whole-genome scan, fine mapping and haplotype analysis, we localized the gene affected in MCPHA to a region of 3 cM, or 2 Mb, on chromosome 17q25. We constructed a map of contiguous genomic clones spanning this region. One of the genes in this region, SLC25A19, which encodes a nuclear mitochondrial deoxynucleotide carrier (DNC), contains a substitution that segregates with the disease in affected individuals and alters an amino acid that is highly conserved in similar proteins. Functional analysis shows that the mutant DNC protein lacks the normal transport activity, implying that failed deoxynucleotide transport across the inner mitochondrial membrane causes MCPHA. Our data indicate that mitochondrial deoxynucleotide transport may be essential for prenatal brain growth.

By use of site-directed mutagenesis in combination with chemical modification of mutated proteins, the role of the six Cys residues in the transport function of the rat mitochondrial carnitine carrier (CAC) was studied. Several CAC mutants, in which one or more Cys residues had been replaced with Ser, were overexpressed in Escherichia coli, purified, and reconstituted in liposomes. The efficiency of incorporation into liposomes of the reconstituted proteins was lower for all constructs lacking Cys-23. Single, double, and quadruple replacement mutants showed V(max) comparable to that of the wild type. On the basis of the values of internal and external transport affinities (K(m)) for carnitine and of their comparison with those measured in mitochondria, the recombinant CAC is oriented unidirectionally in the liposomes, right side out compared to mitochondria. Substitution of Cys-136 with Ser caused a nearly complete loss of sensitivity of the CAC to N-ethylmaleimide, (2-aminoethyl)methanethiosulfonate hydrobromide (MTSES), and other hydrophilic SH reagents but not to the very hydrophobic N-phenylmaleimide. The wild-type CAC and the mutants containing Cys-136 showed substrate protection against NEM and MTSES inhibition and against NEM labeling. The data show that none of the native cysteines is essential for the transport mechanism and that Cys-136 is the major target of SH reagents and raise the hypothesis that Cys-136 is accessible from the external medium and is located at, or near, the substrate binding site. A model of the CAC is proposed in which the matrix hydrophilic loop containing Cys-136 protrudes into the membrane between the transmembrane domains of the protein.

A cDNA from Arabidopsis thaliana and four related cDNAs from Nicotiana tabacum that we have isolated encode hitherto unidentified members of the mitochondrial carrier family. These proteins have been overexpressed in bacteria and reconstituted into phospholipid vesicles. Their transport properties demonstrate that they are orthologs/isoforms of a novel mitochondrial carrier capable of transporting both dicarboxylates (such as malate, oxaloacetate, oxoglutarate, and maleate) and tricarboxylates (such as citrate, isocitrate, cis-aconitate, and trans-aconitate). The newly identified dicarboxylate-tricarboxylate carrier accepts only the single protonated form of citrate (H-citrate2-) and the unprotonated form of malate (malate2-) and catalyzes obligatory, electroneutral exchanges. Oxoglutarate, citrate, and malate are mutually competitive inhibitors, showing K(i) close to the respective K(m). The carrier is expressed in all plant tissues examined and is largely spread in the plant kingdom. Furthermore, nitrate supply to nitrogen-starved tobacco plants leads to an increase in its mRNA in roots and leaves. The dicarboxylate-tricarboxylate carrier may play a role in important plant metabolic functions requiring organic acid flux to or from the mitochondria, such as nitrogen assimilation, export of reducing equivalents from the mitochondria, and fatty acid elongation.

Sengers syndrome is characterized by congenital cataracts, hypertrophic cardiomyopathy, mitochondrial myopathy, and lactic acidosis, but no abnormalities have been found with routine mitochondrial biochemical diagnostics (the determination of pyruvate oxidation rates and enzyme measurements). In immunoblot analysis, the protein content of the mitochondrial adenine nucleotide translocator 1 (ANT1) was found to be strongly reduced in the muscle tissues of two unrelated patients with Sengers syndrome. In addition, low residual adenine nucleotide translocator activity was detected upon the reconstitution of detergent-solubilized mitochondrial extracts from the patients' skeletal or heart muscle into liposomes. Sequence analysis and linkage analysis showed that ANT1 was not the primary genetic cause of Sengers syndrome. We propose that transcriptional, translational, or posttranslational events are responsible for the ANT1 deficiency associated with the syndrome.

The use of central vascular catheters (CVC) is associated with a substantial number of complications, amongst which infections predominate. A diagnosis of CVC-related infection usually requires catheter removal for culture. Semiquantitative (roll-plate method) and quantitative methods (flush, vortex, centrifugation or sonication methods) are the most reliable diagnostic methodologies requiring catheter removal, because of their greater specificity. The roll-plate method is the simplest and most commonly used technique. This method only samples the external surface of the catheter, and is particularly indicated for recently inserted catheters in which extraluminal colonisation is the primary mechanism of infection. Luminal culture techniques, such as the quantitative methods, may be more relevant for catheters that have been in place for a long period of time. However, in up to 85% of removed CVC the culture is negative, and other diagnostic techniques that do not require catheter removal have been proposed, including paired quantitative blood cultures, endoluminal brushing, and differential time to positivity (DTP) of paired blood cultures. DTP, that compares the time to positivity for qualitative cultures of blood samples simultaneously drawn from the CVC and a peripheral vein, appears to be the most reliable in the routine clinical practice since many hospitals use automatic devices for qualitative blood culture positivity detection. More recently catheter-sparing direct diagnostic methods, which include Gram stain and acridin-orange leucocyte cytospin (AOLC) test, appeared to be especially useful because of the rapidity of results and the ability to distinguish different microorganisms, allowing early targeted antimicrobial therapy.

A fully self-contained discrete framework with discrete equivalents of Stokes's, Gauss's, and Green's theorems is presented. The formulation is analogous to that of continuous operators, but totally discrete in nature, and the exact relationships derived are shown to hold provided that a set of predefined rules is followed in building discrete contours and domains. The method allows for an analytical rigor that is not guaranteed if one translates the classical continuous formulations onto a discretized approximated framework. We clarify several issues related to the use of discrete operators, which may play a crucial role in specific applications such as the two-dimensional phase-unwrapping problem, chosen as our main application example, and we show that reconstruction on irregular domains and/or in the presence of undersampling and noise is better formulated in the discrete framework than in the continuous domain.

The mitochondrial carriers are a family of transport proteins in the inner membranes of mitochondria. They shuttle substrates, metabolites, and cofactors through this membrane and connect cytoplasm functions with others in the matrix. Glutamate is co-transported with H(+) (or exchanged for OH(-)), but no protein has ever been associated with this activity. Two human expressed sequence tags encode proteins of 323 and 315 amino acids with 63% identity that are related to the aspartate-glutamate carrier, a member of the carrier family. They have been overexpressed in Escherichia coli and reconstituted into phospholipid vesicles. Their transport properties demonstrate that the two proteins are isoforms of the glutamate/H(+) symporter described in the past in whole mitochondria. Isoform 1 is expressed at higher levels than isoform 2 in all the tissues except in brain, where the two isoforms are expressed at comparable levels. The differences in expression levels and kinetic parameters of the two isoforms suggest that isoform 2 matches the basic requirement of all tissues especially with respect to amino acid degradation, and isoform 1 becomes operative to accommodate higher demands associated with specific metabolic functions such as ureogenesis.

Insulin resistance (IR) and its health consequences (diabetes, hypertension, cardiovascular disease, obesity etc.) affect between 25 and 35% of Westernized populations. Decreased fatty acid (FA) oxidation in skeletal muscle is implicated in obesity-related IR. Carnitine-acylcarnitine translocase (CACT) transports long-chain FAs both into mitochondria (as carnitine esters for energy-generating processes) and out of mitochondria. To determine whether CACT activity correlates with decreased FA oxidation we measured CACT concentrations in cellular and mitochondrial extracts from the skeletal muscle of 19 obese IR individuals and of 19 lean controls. We also evaluated carnitine transport in skeletal muscle mitochondria in both groups. Mitochondrial CACT was decreased at translational and transductional level, and carnitine-carnitine and acylcarnitine-carnitine exchange rates were significantly lower in IR subjects. Aberrant acylcarnitine flux into mitochondria was not correlated with decreased activity of other components of the mitochondrial carnitine system (i.e., carnitine palmitoyl transferase-I and II). Our data suggest that by restraining entry of FA-coenzyme A into mitochondria, low CACT levels increase cytosolic FA levels and their incorporation into glycerolipids. The low level of CACT in IR muscle may contribute to the elevated muscle concentrations of triglycerides, diacylglycerol, and FA-coenzyme A characteristic of IR muscle.

Background: HIV-infected patients with pulmonary tuberculosis exhibit atypical radiological presentation and negative sputum smear more frequently than their HIV-negative counterparts., Patients and Methods: We performed a retrospective study based on a chart review of 146 HIV-infected patients with pulmonary symptoms and culture-proven pulmonary tuberculosis. We compared clinical characteristics and the outcome in 71 patients (49%) with positive sputum smear (SS+), 62 patients (42%) with negative sputum smear/abnormal chest X-ray (SS-/CXR+) and 13 patients (9%) with negative sputum smear/normal chest X-ray (SS-/CXR-). Patients were enrolled from January 1987 to December 1998, and were followed up until December 1999., Results: On hospital admission the three groups of patients examined did not differ significantly in demographic characteristics, degree of immunosuppression or Mycobacterium tuberculosis drug-susceptibility pattern. SS-/CXR- patients were significantly Less LikeLy to present with prolonged fever and dyspnea. Median survival was shorter for SS-/CXR- patients (6.4 months vs 20.2 and 18.8 months in the other two groups). In multivariate analysis, SS-/CXR-patients had a significantly increased risk of death (hazard ratio 3.0, 95% confidence interval, 1.4 to 6.4, p = 0.004) compared to SS+ patients. This increase in risk was no longer statistically significant when initiation of antituberculous therapy within 8 weeks from the collection date of the first specimen yielding M. tuberculosis was included in the multivariate model., Conclusion: Decreased survival was observed in HIV-infected patients with pulmonary tuberculosis and with both negative sputum smear and normaL chest X-ray presentation. This may primarily be a resuLt of delayed tuberculosis diagnosis and initiation of antituberculous therapy. The latter delay may also lead to a faster progression of HIV infection in SS-/CXR patients, in whom diagnostic oversight may be common.

Using a functional mitochondrial oxoglutarate carrier mutant devoid of Cys residues (C-less carrier), each amino acid residue in transmembrane domain IV and flanking hydrophilic loops (from T179 to S205) was replaced individually with Cys. The great majority of the 27 mutants exhibited significant oxoglutarate transport in reconstituted liposomes as compared to the activity of the C-less carrier. In contrast, Cys substitution for G183, R190, Q198, and Y202, in either C-less or wild-type carriers, yielded molecules with complete loss of oxoglutarate transport activity. G183 and R190 could be partially replaced only by Ala and Lys, respectively, whereas Q198 and Y202 were irreplaceable with respect to oxoglutarate transport. Of the single-Cys mutants tested, only T187C, A191C, V194C, and N195C were strongly inactivated by N-ethylmaleimide and by low concentrations of methanethiosulfonate derivatives. Oxoglutarate protects Cys residues at positions 187, 191, and 194 against reaction with N-ethylmaleimide. These positions as well as the residues found to be essential for the carrier activity, except Y202 which is located in the extramembrane loop IV-V, reside on the same face of transmembrane helix IV, probably lining part of a water-accessible crevice or channel between helices of the oxoglutarate carrier.

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.

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.

Aspergillus endocarditis is a rare event; a very few cases have been reported in injection drug users (IDUs) and patients infected with human immunodeficiency virus (HIV). We report 2 proven cases and 1 highly suggestive case of Aspergillus endocarditis in IDUs, 2 of whom were infected with HIV, and discuss some related clinical and pathogenic aspects.

The requirement for small molecule transport systems across the peroxisomal membrane has previously been postulated, but not directly proven. Here we report the identification and functional reconstitution of Ant1p (Ypr128cp), a peroxisomal transporter in the yeast Saccharomyces cerevisiae, which has the characteristic sequence features of the mitochondrial carrier family. Ant1p was found to be an integral protein of the peroxisomal membrane and expression of ANT1 was oleic acid inducible. Targeting of Ant1p to peroxisomes was dependent on Pex3p and Pex19p, two peroxins specifically required for peroxisomal membrane protein insertion. Ant1p was essential for growth on medium-chain fatty acids as the sole carbon source. Upon reconstitution of the overexpressed and purified protein into liposomes, specific transport of adenine nucleotides could be demonstrated. Remarkably, both the substrate and inhibitor specificity differed from those of the mitochondrial ADP/ATP transporter. The physiological role of Ant1p in S.cerevisiae is probably to transport cytoplasmic ATP into the peroxisomal lumen in exchange for AMP generated in the activation of fatty acids.

The mitochondrial aspartate/glutamate carrier catalyzes an important step in both the urea cycle and the aspartate/malate NADH shuttle. Citrin and aralar1 are homologous proteins belonging to the mitochondrial carrier family with EF-hand Ca(2+)-binding motifs in their N-terminal domains. Both proteins and their C-terminal domains were overexpressed in Escherichia coli, reconstituted into liposomes and shown to catalyze the electrogenic exchange of aspartate for glutamate and a H(+). Overexpression of the carriers in transfected human cells increased the activity of the malate/aspartate NADH shuttle. These results demonstrate that citrin and aralar1 are isoforms of the hitherto unidentified aspartate/glutamate carrier and explain why mutations in citrin cause type II citrullinemia in humans. The activity of citrin and aralar1 as aspartate/glutamate exchangers was stimulated by Ca(2+) on the external side of the inner mitochondrial membrane, where the Ca(2+)-binding domains of these proteins are localized. These results show that the aspartate/glutamate carrier is regulated by Ca(2+) through a mechanism independent of Ca(2+) entry into mitochondria, and suggest a novel mechanism of Ca(2+) regulation of the aspartate/malate shuttle.