Biomedical subjects

D Boffoli

Publications and source records attributed to D Boffoli.

At least 19 recordsLinked to original sources

PubMed2006

Mitochondrial dysfunction in hepatitis C virus infection.

The mechanisms of liver injury in chronic hepatitis C virus (HCV) infection are poorly understood though HCV induces a state of hepatic oxidative stress that is more pronounced than that present in many other inflammatory diseases. This mini-review will focus on recent findings revealing an unexpected role of mitochondria in providing a central role in the innate immunity and in addition will illustrate the application of stably transfected human-derived cell lines, inducibly expressing the entire HCV open reading frame for in vitro studies on mitochondria. Results obtained by a comparative analysis of the respiratory chain complexes activities along with mitochondrial morpho-functional confocal microscopy imaging show a detrimental effect of HCV proteins on the cell oxidative metabolism with specific inhibition of complex I activity, decrease of mtDeltaPsi, increased production of reactive oxygen species. A possible de-regulation of calcium recycling between the endoplasmic reticulum and the mitochondrial network is discussed to provide new insights in the pathogenesis of hepatitis C.

Gene Expression Regulation, Viral↗

PubMed2006

Influence of human menopausal gonadotrophin treatment on testicular blood flow and on seminal plasma nitric oxide levels in infertile males.

Gonadotrophin treatment is capable of increasing intratesticular blood flow and improving sperm fertilization potential. A nitric oxide (NO)-mediated mechanism may play a role. This study aimed at evaluating whether the increase in intratesticular blood flow, in response to human menopausal gonadotrophin (hMG) treatment, is accompanied by an increase in seminal plasma NO levels in 20 normogonadotropic males affected by severe oligoasthenoteratozoospermia. Patients received 150 IU of hMG, three times a week for 3 months. Before starting treatment and at month 3 of therapy, levels of NO in the seminal plasma were determined, followed by ultrasound scanning of testis, Doppler evaluation of intratesticular vascular resistance, serum hormone and conventional semen evaluation. Statistical analysis was performed by using Wilcoxon test; the limit of statistical significance was p<0.05. At the end of treatment, seminal plasma concentration of NO, conventional semen parameters and testicular volume did not increase significantly. Follicle-stimulating hormone serum level significantly increased (p=0.014) after treatment, whereas luteinizing hormone, testosterone and oestradiol values did not change. The resistance index of both intratesticular arteries significantly decreased (p=0.012 and 0.005 for right and left testes respectively). The results of this study confirmed the positive effect of exogenous hMG on testicular blood flow, but failed to demonstrate any effect of hMG treatment on NO levels in the seminal plasma in infertile males.

Adult↗

PubMed2001

A nonsense mutation in the NDUFS4 gene encoding the 18 kDa (AQDQ) subunit of complex I abolishes assembly and activity of the complex in a patient with Leigh-like syndrome.

Sequence analysis of mitochondrial and nuclear candidate genes of complex I in children with deficiency of this complex and exhibiting Leigh-like syndrome has revealed, in one of them, a novel mutation in the NDUFS4 gene encoding the 18 kDa subunit. Phosphorylation of this subunit by cAMP-dependent protein kinase has previously been found to activate the complex. The present mutation consists of a homozygous G-->A transition at nucleotide position +44 of the coding sequence of the gene, resulting in the change of a tryptophan codon to a stop codon. Such mutation causes premature termination of the protein after only 14 amino acids of the putative mitochondrial targeting peptide. Fibroblast cultures from the patient exhibited severe reduction of the rotenone-sensitive NADH-->UQ oxidoreductase activity of complex I, which was insensitive to cAMP stimulation. Two-dimensional electrophoresis showed the absence of detectable normally assembled complex I in the inner mitochondrial membrane. These findings show that the expression of the NDUFS4 gene is essential for the assembly of a functional complex I.

Amino Acid Sequence↗

PubMed2000

The proton/electron coupling ratio at heme a and Cu(A) in bovine heart cytochrome c oxidase.

Measurements of the H(+)/heme a, Cu(A) ratios for proton-electron coupling at these centers (redox Bohr effect) in CO-inhibited cytochrome c oxidase purified from bovine heart mitochondria, both in the soluble state and reconstituted in liposomes, are presented. In the soluble oxidase, the H(+)/heme a, Cu(A) ratios were experimentally determined upon oxidation by ferricyanide of these centers as well as upon their reduction by hexammineruthenium(II). These measurements showed that in order to obtain H(+)/heme a, Cu(A) ratios approaching 1, one-step full oxidation of both metal centers by ferricyanide had to be induced by a stoicheiometric amount of the oxidant. Partial stepwise oxidation or reduction of heme a and Cu(A) did produce H(+)/heme a, Cu(A) ratios significantly lower or higher than 1, respectively. The experimental H(+)/heme a, Cu(A) ratios measured upon stepwise reduction/oxidation of the metals were reproduced by mathematical simulation based on the coupling of oxido-reduction of both heme a and Cu(A) to pK shifts of common acid-base groups. The vectorial nature of the proton-electron coupling at heme a/Cu(A) was analyzed by measuring pH changes in the external bulk phase associated with oxido-reduction of these redox centers in the CO-inhibited oxidase reconstituted in liposomes. The results show that the proton release associated with the oxidation of heme a and Cu(A) takes place in the external aqueous phase. Protons taken up by the oxidase upon rereduction of the centers derive, on the other hand, from the inner space. These results provide evidence supporting the view that cooperative proton-electron coupling at heme a/Cu(A) is involved in the proton pump of the oxidase.

Animals↗

PubMed2000

Tyrosinase gene expression in the Kupffer cells of Rana esculenta L.

The liver of Amphibia and Reptilia shows a dark-brown pigmentation due to the presence of particular melanin-containing cells that are different from melanocytes and derive from cells of macrophage lineage (Kupffer Cells), which have been shown to have an autonomous capacity to synthesize melanins. To date, as far as we know, there are no reports in the literature about the genetic system of tyrosinase as regards these melanin-synthesizing cells; we carried out the present study to analyze how the tyrosinase gene may function. We showed that the Kupffer cells of Rana esculenta L. do indeed have a transcriptionally active tyrosinase gene. Evidence of this was obtained by reverse transcription polymerase chain reaction analysis carried out on both the liver tissue and the Kupffer cells in culture. Moreover, analysis of the cells in culture enabled us to observe that, by increasing the culture time from 0 to 72 hr, an appreciable increase occurred in the amplification products of the tyrosinase gene, as well as in the level of dopa oxidase activity and in the quantity of melanin in the cells. The results of the present study demonstrate that frog Kupffer cells possess an active tyrosinase gene and that the increase of the tyrosinase mRNA accumulation closely correlates with phenotypic differentiation, in terms of increased dopa oxidase activity and melanosome content. This provides further strong support of the hypothesis that amphibian Kupffer cells possess an endogenous ability to synthesize melanin and suggests the involvement of the transcriptional level of control in the modulation of their melanogenic activity.

Animals↗

PubMed1998

Ultrastructural and functional study of the liver pigment cells from Rana esculenta L.

A study of the liver pigment cells of Rana esculenta L. has been performed on both liver in toto and cells in culture. Ultrastructural and cytochemical analyses showed a close relationship between this visceral pigment cell system and the cells of hepatic macrophage lineage. Like the latter, the liver pigment cells present phagocytic activity, in the sinusoids and in vitro, and give a positive response to tests for peroxidase and lipase. The liver pigment cells are isolated, together with the Kupffer cells, from the sinusoidal cell fraction of the liver. In culture, they maintain their melanogenetic ability, demonstrated by the presence of dopaoxidase activity in the soluble, membranous, and melanosome fractions. Analysis of the cultures showed that as culture time increased, so did melanosome dopaoxidase activity, the number of pigmented fields, and the level of pigmentation of the cells. The values of dopaoxidase activity of the pigment cells in culture show the same seasonal oscillations as the system in toto, indicating that the cells maintain an internal clock, at least in the first 72 h of culture. There is evidence that the pigment cells are macrophages which can express a melanogenetic function. Our results and other experimental data provide a basis for hypothesizing that the pigment cells in Rana esculenta L. liver may derive from, or have a common origin with, the Kupffer cells.

Animals↗

PubMed1996

Ageing is associated in females with a decline in the content and activity on the b-c1 complex in skeletal muscle mitochondria.

The activity of cytochrome-c oxidase [E.C. 1.9.3.1] and b-c1 complex [E.C. 1.10.2.2] and the content of cytochromes b, c + c1 and a + a3 in human skeletal muscle mitochondria from orthopaedic patients (108 women and 68 males), of age ranging between 10 and 50 years, have been analyzed. The activity of cytochrome c-oxidase declines with age both in females and males. The activity of b-c1 complex, which in young females is significantly higher than in young males, declines sharply in females, but not in males, with ageing. These results reveal that the content of active b-c1 complex in muscle mitochondria is specifically controlled by female sex hormones.

Adolescent↗

PubMed1996

Fast-to-slow muscle conversion by chronic electrostimulation: effects on mitochondrial respiratory chain function with possible implications for the gracilis neosphincter procedure.

The effects of chronic, around the clock, low-frequency electrostimulation on the respiratory chain activity and cytochrome content of freshly isolated mitochondria were evaluated in rabbit skeletal muscle before and after 30 days of continuous or cyclical electrostimulation using a totally implantable system and a training programme now used in humans. The respiratory activity measured in state III increased strongly after electrostimulation. The efficiency of the respiratory chain increased significantly after electrostimulation but the activity of complex [(reduced nicotinamide adenine dinucleotide dehydrogenase) did not increase. The amount of cytochromes a and a3, b562, and c and c1 increased clearly after electrostimulation. The respiratory activity rate of mitochondria obtained after continuous electrostimulation was apparently higher than after cyclical electrostimulation. Chronic uninterrupted low-frequency electrostimulation, using a clinical training programme, induces an increase in mitochondrial respiratory chain activity in purified mitochondria of skeletal muscle. These changes are the basis of induced resistance to fatigue in fast-to-slow muscle conversion by chronic electrostimulation.

Animals↗

PubMed1994

Correlation between mitochondrial DNA 4977-bp deletion and respiratory chain enzyme activities in aging human skeletal muscles.

The content of the mitochondrial DNA 4977-bp deletion and the respiratory chain enzyme activities were determined in the same human skeletal muscle specimens. A direct correlation between damage to mtDNA and bioenergetic deficiency was observed. The time-course of the appearance of the mtDNA deletion was followed. The highest percentage of mtDNA-deleted molecules was 0.26% and it was found in the eighties which corresponds to the age of the major reduction in the respiratory chain enzyme activities. Two samples with very low mitochondrial respiratory enzyme activities exhibited much higher levels of deletion compared to the similar age counterparts. Given, however, the low absolute level of the deletion also in these samples, we suggest that damage to the respiratory chain complexes, especially complex IV, might be the cause more than the effect of the increased number of mtDNA molecules bearing deletions in aged human skeletal muscle.

Adult↗

PubMed1994

Decline with age of the respiratory chain activity in human skeletal muscle.

Mitochondrial respiratory systems have been screened in 63 orthopaedic patients of age ranging between 17 and 91 years. The results show a statistically significant definite decrease with ageing of mitochondrial respiratory activity with pyruvate plus malate, succinate and ascorbate plus TMPD. This pattern is associated with an equally significant decrease with age of the enzymatic activity of complex I, II and IV. No significant decrease with age is, on the contrary, observed in the mitochondrial content of cytochromes a+a3, and c+c1. Preliminary Western blot analysis indicates an altered polypeptide pattern in cytochrome c oxidase. This study provides evidence for a decline with age of mitochondrial respiratory activity in human skeletal muscle, affecting complex I, II and IV.

Adolescent↗

PubMed1990

Morpho-functional characterization of cultured pigment cells from Rana esculenta L. liver.

A simple method to isolate and culture liver pigment cells from Rana esculenta L. is described which utilizes a pronase digestion of perfused liver, followed by sedimentation on a Ficoll gradient. A first characterization of isolated and cultured cells is also reported. They show both positivity for nonspecific esterases, and phagocytosis ability, like the cells of phagocytic lineage. Furthermore, after stimulation with a phorbol ester, these cells generate superoxide anions. At phase contrast microscope, liver pigment cells present variability in size, morphology, and in their content of dark-brown granules. Inasmuch as a cell extract obtained from cultured cells exhibits a specific protein band with dopa-oxidase activity, when run on nondenaturing polyacrylamide gel electrophoresis, liver pigment cells from Rana esculenta L. should not be considered as melanophages, but as cells that can actively synthesize melanin. The method presented here seems to be useful to more directly investigate this extra-cutaneous melanin-containing cell system and to clarify its physiologic relevance.

Animals↗

PubMed1989

Effect of papain digestion on polypeptide subunits and electron-transfer pathways in mitochondrial b-c1 complex.

Papain digestion of subunits of mitochondrial b-c1 complex (ubiquinol-cytochrome-c reductase) isolated from bovine heart and its impact on redox and proton-motive activity of the whole complex were investigated. A 5-min incubation of the oxidized enzyme with papain resulted in digestion of core protein II and the 14-kDa subunit, and limited digestion of the iron-sulfur protein. This was accompanied by a small inhibition of the rate of electron flow and a marked inhibition of proton translocation with decrease of the H+/e- ratio for proton pumping. When papain treatment was performed on the b-c1 complex pre-reduced with ascorbate, partial proteolysis of the iron-sulfur protein and the 14-kDa subunit was greatly accelerated and the electron transfer activity was more markedly inhibited. In all the conditions tested, digestion of the Rieske iron-sulfur protein paralleled the inhibition of reductase activity. Under ascorbate-reduced conditions, papain digestion of the complex gave rise to an alteration of the EPR line shape of the iron-sulfur cluster, namely a broadening and shift of the gx negative peak and destabilization of the protein-bound antimycin-sensitive semiquinone. The latter paralleled the decrease in electron transfer activity and inhibition of antimycin-sensitive cytochrome-b reduction. The results obtained indicate the following. 1. Core protein II and the 14-kDa protein may contribute to the proton-conducting pathway(s) from the matrix aqueous phase to the primary protolytic redox center (protein-bound semiquinone/quinone couple). 2. The iron-sulfur protein contributes, together with other protein(s) (the 14-kDa subunit), to the stabilization of the protein-bound antimycin-sensitive semiquinone species in a protein pocket in the complex. 3. Reduction of the high-potential redox centers induces a change in the quaternary structure of the complex which results in an enhanced surface exposure of segments of the 14-kDa protein and the iron-sulfur protein.

Animals↗

PubMed1987

Chemical modification studies of beef-heart mitochondrial b-c1 complex. Effect of modification by ethoxyformic anhydride.

The effect of the histidine-modifier ethoxyformic anhydride (EFA) on the enzymatic properties of the mitochondrial b-c1 complex (ubiquinol-cytochrome c reductase) has been investigated. Chemical modification by EFA inhibited to the same extent the reductase and the proton translocating activity of the complex. In particular EFA modification of the complex resulted in: strong inhibition of the antimycin-insensitive reduction of b cytochromes; inhibition of the antimycin-promoted oxidant-induced reduction of b cytochromes and inhibition of oxidation of pre-reduced b cytochromes. Analysis of the absorbance at 238 nm, indicative of N-(ethoxyformyl)histidine derivative, of the various polypeptide subunits separated by high-pressure liquid chromatography procedure, showed that EFA modified residues in core proteins and in the low-molecular-mass proteins. Both the inhibition of the redox and the protonmotive activity of the complex and the absorbance increase at 238 nm of the core protein fraction were readily reversed by hydroxylamine, indicating that modification of histidine residue(s) in core protein(s) is critical for the activity of the complex. This was supported by the finding that modification of the reductase with EFA prevented binding of fluorescein isothiocyanate to histidine residue(s) in core protein II. EFA modification of the reductase was without effect on the binding of N-(7-dimethylamino-4-methylcoumarinyl)maleimide to the various polypeptides of the complex except for the binding to the Fe-S protein which was greatly potentiated. Thus primary chemical modification of histidine residue(s) in core protein (II) appears to cause, in turn, a conformational change in the Rieske Fe-S protein.

Animals↗

PubMed1983

Redox-linked proton translocation in the b-c1 complex from beef-heart mitochondria reconstituted into phospholipid vesicles. General characteristics and control of electron flow by delta micro H+.

A study is presented of the characteristics of redox-linked proton translocation in the b-c1 complex isolated from beef-heart mitochondria and reconstituted into phospholipid vesicles. Measurements of the H+/e- stoichiometry, with three different methods, show that four protons are released from the vesicles per 2e- flowing from quinols to cytochrome c, two of these protons formally deriving from scalar oxidation of quinols by cytochrome c. This H+/e- stoicheiometry is independent of the initial redox state of the b-c1 complex (fully reduced or oxidized) and the rate of electron flow through the complex. It does not change in the pH range 6.0 - 7.2, but declines to 1.5 going with pH from 7.2 - 8.3. This decrease is accompanied by enhancement of the rate of electron flow in the coupled state. Collapse of delta psi effected by valinomycin addition to turning-over b-c1 vesicles resulted in substantial oxidation of cytochrome b-566 and comparable reduction of cytochrome c1, with little oxidation of cytochrome b-562. Nigericin alone had no effect on the steady-state redox levels of b and c cytochromes. Its addition in the presence of valinomycin caused oxidation of b cytochromes but no change in the redox state of cytochrome c1. Valinomycin alone caused a marked enhancement of the rate of electron flow through the complex. Nigericin alone was ineffective, but caused further stimulation of electron flow when added in the presence of valinomycin. The data presented are discussed in terms of two mechanisms: the Q cycle and a model based on combination of protonmotive catalysis by special bound quinone and proton conduction along pathways in the apoproteins.

Animals↗

PubMed1983

Redox-linked proton translocation in the b-c1 complex from beef-heart mitochondria reconstituted into phospholipid vesicles. Studies with chemical modifiers of amino acid residues.

Possible involvement of polypeptides of b-c1 complex of beef-heart mitochondria in its redox and protonmotive activity has been investigated, by means of chemical modification of amino acid residues in the soluble as well as in the phospholipid-reconstituted b-c1 complex. Treatment of the enzyme with tetranitromethane (C(NO2)4) or with ethoxyformic anhydride (EFA), that modify reversibly tyrosyl and hystidyl residues respectively, resulted in a marked inhibition of electron transport from reduced quinols to cytochrome c. This was accompanied, in b-c1 reconstituted into phospholipid vesicles, by a parallel inhibition of respiratory-linked proton translocation; the H+/e- stoichiometry remained unchanged. Treatment of b-c1 complex with DCCD, that specifically modifies carboxylic groups of glutammic or aspartic residues caused a marked depression of proton translocation in b-c1 vesicles, under conditions where the rate of electron flow in the coupled state, was enhanced. As a consequence the H+/e- stoichiometry was lowered. SDS gel electrophoresis and [14C]DCCD-labelling of the polypeptides of the b-c1 complex showed a major binding of 14C-DCCD to the 8-kDa subunit of the complex and possible cross-linking, induced by DCCD treatment, of polypeptide(s) in the 8-kDa band and the 12-kDa band, with the Fe-s protein of the complex, with the appearance of a new polypeptide band with an apparent molecular mass of about 40 kDa. Involvement of polypeptides of low molecular mass, for which no functional role was so far described, and possibly of the Fe-S protein in the redox-linked proton translocation in b-c1 complex is suggested.

Amino Acids↗

PubMed1983

Mechanism of proton translocation associated to oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine in rat liver mitochondria.

A kinetic analysis is presented of proton translocation, TMPD+ formation and oxidation of endogenous respiratory carriers during oxygen pulses of TMPD supplemented rat-liver mitochondria. The results show that antimycin-insensitive proton ejection observed under coupled conditions derives from oxidation of endogenous respiratory carriers and re-reduction of TMPD+ by hydrogenated donors and not from proton pumping by cytochrome oxidase as claimed by other investigators. The observations presented provide an example of certain interpretative difficulties in the use of redox mediators and of the methodological approaches that can be used to avoid these.

Animals↗

PubMed1980

The H+/e- stoicheiometry of respiration-linked proton translocation in the cytochrome system of mitochondria.

1. The -->H(+)/e(-) quotients for proton release from mitochondria associated with electron flow from succinate and duroquinol to O(2), ferricyanide or ferricytochrome c, and from NNN'N'-tetramethyl-p-phenylenediamine+ascorbate to O(2), were determined from rate measurements of electron flow and proton translocation. 2. Care was taken to avoid, or to take into account, unrelated electron flow and proton translocation, which might take place in addition to the oxido-reductions that were the subject of our analysis. Spectrophotometric techniques were chosen to provide accurate measurement of the rate of consumption of oxidants and reductants. The rate of proton translocation was measured with fast pH meters with a precision of 10(-3) pH unit. 3. The -->H(+)/O quotient for succinate or duroquinol oxidation was, at neutral pH, 4, when computed on the basis of spectrophotometric determinations of the rate of O(2) consumption or duroquinol oxidation. Higher -->H(+)/O quotients for succinate oxidation, obtained from polarographic measurements of O(2) consumption, resulted from underestimation of the respiratory rate. 4. The -->H(+)/2e(-) quotient for electron flow from succinate and duroquinol to ferricyanide or ferricytochrome c ranged from 3.9 to 3.6. 5. Respiration elicited by NNN'N'-tetramethyl-p-phenylenediamine+ascorbate by antimycin-inhibited mitochondria resulted in extra proton release in addition to that produced for oxidation of ascorbate to dehydroascorbate. Accurate spectrophotometric measurement of respiration showed that the -->H(+)/e(-) ratio was only 0.25 and not 0.7-1.0 as obtained with the inadequate polarographic assay of respiration. Proton release was practically suppressed when mitochondria were preincubated aerobically in the absence of antimycin. Furthermore, the rate of scalar proton consumption for water production was lower than that expected from the stoicheiometry. Thus the extra proton release observed during respiration elicited by NNN'N'-tetramethyl-p-phenylenediamine+ascorbate is caused by oxidation of endogenous hydrogenated reductants. 6. It is concluded that (i) the -->H(+)/O quotient for the cytochrome system is, at neutral pH, 4 and not 6 or 8 as reported by others; (ii) all the four protons are released during electron flow from quinol to cytochrome c; (iii) the oxidase transfers electrons from cytochrome c to protons from the matrix aqueous phase and does not pump protons from the matrix to the outer aqueous phase.

Animals↗

PubMed1979

The mechanism of transmembrane delta muH+ generation in mitochondria by cytochrome c oxidase.

In rat liver mitochondria treated with rotenone, N-ethylmaleimide or oligomycin the expected alkalinization caused by proton consumption for aerobic oxidation of ferrocyanide was delayed with respect to ferrocyanide oxidation, unless carbonyl cyanide p-trifluoromethoxyphenylhydrazone was present. 2. When valinomycin or valinomycin plus antimycin were also present, ferricyanide, produced by oxidation of ferrocyanide, was re-reduced by hydrogenated endogenous reductants. Under these circumstances the expected net proton consumption caused by ferrocyanide oxidation was preceded by transient acidification. It is shown that re-reduction of formed ferricyanide and proton release derive from rotenone- and antimycin-resistant oxidation of endogenous reductants through the proton-translocating segments of the respiratory chain on the substrate side of cytochrome c. The number of protons released per electron flowing to ferricyanide varied, depending on the experimental conditions, from 3.6 to 1.5. 3. The antimycin-insensitive re-reduction of ferricyanide and proton release from mitochondria were strongly depressed by 2-n-heptyl-4-hydroxyquinoline N-oxide. This shows that the ferricyanide formed accepts electrons passing through the protonmotive segments of the respiratory chain at the level of cytochrome c and/or redox components of the cytochrome b-c1 complex situated on the oxygen side of the antimycin-inhibition site. Dibromothymoquinone depressed and duroquinol enhanced, in the presence of antimycin, the proton-release process induced by ferrocyanide respiration. Both quinones enhanced the rate of scalar proton production associated with ferrocyanide respiration, but lowered the number of protons released per electron flowing to the ferricyanide formed. 4. Net proton consumption caused by aerobic oxidation of exogenous ferrocytochrome c by antimycin-supplemented bovine heart mitochondria was preceded by scalar proton release, which was included in the stoicheiometry of 1 proton consumed per mol of ferrocytochrome c oxidized. This scalar proton production was associated with transition of cytochrome c from the reduced to the oxidized form and not to electron flow along cytochrome c oxidase. 5. It is concluded that cytochrome c oxidase only mediates vectorial electron flow from cytochrome c at the outer side to protons that enter the oxidase from the matrix side of the membrane. In addition to this consumption of protons the oxidase does not mediate vectorial proton translocation.

Animals↗