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Biomedical subjects

S Scacco

Publications and source records attributed to S Scacco.

At least 19 recordsLinked to original sources
PubMed2005

Glatiramer acetate induces pro-apoptotic mechanisms involving Bcl-2, Bax and Cyt-c in peripheral lymphocytes from multiple sclerosis patients.

Apoptotic deletion of autoreactive T-cells is defective in patients with multiple sclerosis (MS). Glatiramer acetate (GA) treatment seems to restore apoptosis of detrimental T-cells. We analyzed the mitochondria membrane pro- (Bax) and anti-apoptotic (Bcl- 2) and cytosolic pro-apoptotic (Cyt-c, APAF-1) proteins expression in peripheral lymphocytes from relapsing-remitting (RR) MS patients during GA treatment. Blood samples were collected from 8 healthy controls (HCs) and from 8 RR MS patients prior to and every three months during the 9 months of GA treatment. Peripheral blood mononuclear cells (PBMNCs) Bcl-2, Bax, Cyt-c and APAF-1 were quantified by western blot followed by densitometric scanning and Bax/Bcl-2, cytosolic Cyt-c/Bcl-2 and APAF-1/Bcl-2 ratios were calculated. T-cells were in vitro tested for oxygen consumption by a respirometric analysis. Bax/Bcl-2, cytosolic Cyt-c/Bcl-2 and APAF-1/Bcl-2 ratios in untreated MS patients were significantly (p < 0.05) lower than in HCs. Bax/Bcl-2 ratio increased (p = 0.03) and Cyt-c/Bcl-2 ratio showed a trend to increase during the 9 months of GA treatment in MS patients. A reduction of 58% and 59% in oxygen consumption by PBMNCs was evident after GA treatment in vitro or when GA treated patients' cells were compared with those from HCs, respectively. Our findings suggest that GA exerts a regulatory effect on peripheral T lymphocytes through pro-apoptosis mechanisms involving mitochondria and cytosolic proteins.

Adult↗
PubMed2003

Characterization of plasma membrane respiratory chain and ATPase in the actinomycete Nonomuraea sp. ATCC 39727.

We have characterized the respiratory system of the aerobic actinomycete Nonomuraea sp. ATCC 39727. The plasma membrane of the microorganism is shown to contain a protonmotive respiratory chain and H+-ATPase. The respiratory chain is made up of a rotenone-sensitive NADH-quinone oxidoreductase, a four subunits aa3-type cytochrome c oxidase and a bc1 complex. The H+-ATPase is characterized as an F0F1-type on the basis of its sensitivity to specific inhibitors; the enzyme is also inhibited by mM concentrations of Ca2+. The activity of the respiratory chain increases during the exponential growth phase, but is depressed in the stationary phase. The H+-ATPase activity reaches, as the respiratory chain, a maximal activity at the end of the exponential growth phase and then remains constant in the stationary phase.

Actinomycetales↗
PubMed2003

Expression and subcellular distribution of Bcl-2 and BAX proteins in serum-starved human keratinocytes and mouth carcinoma epidermoid cultures.

Squamous cell carcinoma (SSC) is the most frequent malignant tumor of the oral cavity. Aberration of programmed cell death is thought to participate in cancer. Using specific antibodies a study of the expression and subcellular distribution of Bcl-2, BAX, caspase-3 and cytochrome c in normal human keratinocytes and mouth carcinoma slowly (HN) and rapidly growing (KB) cells has been carried out. In carcinoma cells depressed expression of BAX, presence in the cytosol of procaspase-3 and absence in this fraction of cytochrome c have been found. PGE2 treatment prevented cell growth depression induced by pro-apoptotic serum starvation both in control and carcinoma cell cultures. It is also shown that PGE2 promoted both in keratinocytes and KB cells expression of Bcl-2, which was accompanied in the first case by increase in its mitochondrial level. These results indicate that in carcinoma cells there is an apparent down regulation of the apoptotic cascade as compared to normal keratinocytes. Thus the possibility that down regulation of apoptosis is associated with promotion of tumor development in the oral mucosa cells seems to be supported by these observations.

Carcinoma↗
PubMed2002

The NADH: ubiquinone oxidoreductase (complex I) of the mammalian respiratory chain and the cAMP cascade.

Recent work has revealed cAMP-dependent phosphorylation of the 18-kDa IP subunit of the mammalian complex I of the respiratory chain, encoded by the nuclear NDUFS4 gene (chromosome 5). Phosphorylation of this protein has been shown to take place in fibroblast cultures in vivo, as well as in isolated mitochondria, which in addition to the cytosol also contain, in the inner-membrane matrix fraction, a cAMP-dependent protein kinase. Mitochondria appear to have a Ca2+-inhibited phosphatase, which dephosphorylates the 18-kDa phosphoprotein. In fibroblast and myoblast cultures cAMP-dependent phosphorylation of the 18-kDa protein is associated with potent stimulation of complex I and overall respiratory activity with NAD-linked substrates. Mutations in the human NDUFS4 gene have been found, which in the homozygous state are associated with deficiency of complex I and fatal neurological syndrome. In one case consisting of a 5 bp duplication, which destroyed the phosphorylation site, cAMP-dependent activation of complex I was abolished in the patient's fibroblast cultures. In another case consisting of a nonsense mutation, leading to termination of the protein after only 14 residues of the putative mitochondria targeting peptide, a defect in the assembly of complex I was found in fibroblast cultures.

Animals↗
PubMed2002

Complex I and the cAMP cascade in human physiopathology.

A cAMP-dependent protein kinase (PKA) is localized in mammalian mitochondria with the catalytic site at the matrix side of the membrane where it phosphorylates a number of proteins. One of these is the 18 kDa(IP) subunit of the mammalian complex I of the respiratory chain, encoded by the nuclear NDUFS4 gene. Mitochondria have a Ca(2+)-inhibited phosphatase, which dephosphorylates the 18 kDa phosphoprotein of complex I. In fibroblast and myoblast cultures cAMP-dependent phosphorylation of the 18 kDa protein is associated with stimulation of complex I and overall respiratory activity with NAD-linked substrates. Mutations in the human NDUFS4 gene have been found, which in the homozygous state are associated with deficiency of complex I and fatal neurological syndrome.

Base Sequence↗
PubMed2001

Cyclic adenosine monophosphate-dependent phosphorylation of mammalian mitochondrial proteins: enzyme and substrate characterization and functional role.

A study is presented on cyclic adenosine monophosphate- (cAMP-) dependent phosphorylation of mammalian mitochondrial proteins. Immunodetection with specific antibodies reveals the presence of the catalytic and the regulatory subunits of cAMP-dependent protein kinase (PKA) in the inner membrane and matrix of bovine heart mitochondria. The mitochondrial cAMP-dependent protein kinase phosphorylates mitochondrial proteins of 29, 18, and 6.5 kDa. With added histone as substrate, PKA exhibits affinities for ATP and cAMP and pH optimum comparable to those of the cytosolic PKA. Among the mitochondrial proteins phosphorylated by PKA, one is the nuclear-encoded (NDUFS4 gene) 18 kDa subunit of complex I, which has phosphorylation consensus sites in the C terminus and in the presequence. cAMP promotes phosphorylation of the 18 kDa subunit of complex I in myoblasts in culture and in their isolated mitoplast fraction. In both cases cAMP-dependent phosphorylation of the 18 kDa subunit of complex I is accompanied by enhancement of the activity of the complex. These results, and the finding of mutations in the NDUFS4 gene in patients with complex I deficiency, provide evidence showing that cAMP-dependent phosphorylation of the 18 kDa subunit of complex I plays a major role in the control of the mitochondrial respiratory activity.

Animals↗
PubMed2001

Mutation in the NDUFS4 gene of complex I abolishes cAMP-dependent activation of the complex in a child with fatal neurological syndrome.

Evidence is presented showing that in a patient with fatal neurological syndrome, the homozygous 5 bp duplication in the cDNA of the NDUFS4 18 kDa subunit of complex I abolishes cAMP-dependent phosphorylation of this protein and activation of the complex. These findings show for the first time that human complex I is regulated via phosphorylation of the subunit encoded by the NDUFS4 gene.

Amino Acid Sequence↗
PubMed2001

Morphological and biochemical characterization of mitochondria in Torpedo red blood cells.

A study is presented on the morphology and respiratory functions of mitochondria from Torpedo marmorata red blood cells. In vivo staining of red blood cells and transmission electron microscopy showed the existence of a considerable number of vital and orthodox mitochondria which decreased from young erythroblasts to mature erythrocytes from 60-50 to 30-20 per cell. In erythrocytes mitochondria exhibited a canonical, functional respiratory chain. The content and activity of cytochromes in erythrocytes were, however, significantly lower as compared to mammalian tissues.

Animals↗
PubMed2000

cAMP-dependent phosphorylation of the nuclear encoded 18-kDa (IP) subunit of respiratory complex I and activation of the complex in serum-starved mouse fibroblast cultures.

A study is presented on the in vivo effect of elevated cAMP levels induced by cholera toxin on the phosphorylation of subunits of the mitochondrial respiratory complexes and their activities in Balb/c 3T3 mouse fibroblast cultures. Treatment of serum-starved fibroblasts with cholera toxin promoted serine phosphorylation in the 18-kDa subunit of complex I. Phosphorylation of the 18-kDa subunit, in response to cholera toxin treatment of fibroblasts, was accompanied by a 2-3-fold enhancement of the rotenone-sensitive endogenous respiration of fibroblasts, of the rotenone-sensitive NADH oxidase, and of the NADH:ubiquinone oxidoreductase activity of complex I. Direct exposure of fibroblasts to dibutyryl cAMP resulted in an equally potent stimulation of the NADH:ubiquinone oxidoreductase activity. Stimulation of complex I activity and respiration with NAD-linked substrates were also observed upon short incubation of isolated fibroblast mitoplasts with dibutyryl cAMP and ATP, which also promoted phosphorylation of the 18-kDa subunit. These observations document an extension of cAMP-mediated intracellular signal transduction to the regulation of cellular respiration.

3T3 Cells↗
PubMed2000

Sequential serum-dependent activation of CREB and NRF-1 leads to enhanced mitochondrial respiration through the induction of cytochrome c.

Progression through the cell cycle requires ATP for protein synthesis, cytoskeletal rearrangement, chromatin remodeling, and protein degradation. The mechanisms by which mammalian cells increase respiratory capacity and ATP production in preparation for cell division are largely unexplored. Here, we demonstrate that serum induction of cytochrome c mRNA and processed protein in quiescent BALB/3T3 fibroblasts is associated with a marked increase in mitochondrial respiration. Cytochrome c was induced in the absence of any increase in citrate synthase activity or in subunit IV of the cytochrome c oxidase complex mRNA or protein, indicating that the enhanced respiratory rate did not require a general increase in mitochondrial biogenesis or respiratory chain expression. Transfections with a series of cytochrome c promoter mutants showed that both nuclear respiratory factor 1 (NRF-1) and cAMP-response element-binding protein (CREB) binding sites contributed equally to induced expression by serum. Moreover, CREB and NRF-1 were phosphorylated sequentially in response to serum, and the NRF-1 phosphorylation was accompanied by an increase in its ability to trans-activate target gene expression. The results demonstrate that the differential transcriptional expression of cytochrome c, through sequential transcription factor phosphorylations, leads to enhanced mitochondrial respiratory capacity upon serum-induced entry to the cell cycle.

3T3 Cells↗
PubMed1999

cAMP-dependent protein kinase and phosphoproteins in mammalian mitochondria. An extension of the cAMP-mediated intracellular signal transduction.

Evidence has been obtained for the occurrence of a cAMP-dependent serine protein kinase associated with the inner membrane/matrix of mammalian mitochondria. The catalytic site of this kinase is localized at the inner side of the inner membrane, where it phosphorylates a number of mitochondrial proteins. One of these has been identified as the AQDQ subunit of complex I. cAMP-dependent phosphorylation of this protein promotes the activity of complex I and mitochondrial respiration. A 5 bp duplication in the nuclear gene encoding this protein has been found in a human patient, which eliminates the phosphorylation site. PKA anchoring proteins have recently been identified in the outer membrane of mammalian mitochondria, which could direct phosphorylation of proteins at contact sites with other cell structures.

Binding Sites↗
PubMed1998

Comparison of oxidative phosphorylation in the anastomosis of the small and large bowel. An experimental study in the rabbit.

Dehiscence of anastomosis in the large bowel (LB) is more frequent compared with that in the small bowel (SB). The pathophysiological mechanisms underlying the phenomenon are well known. The goal of this study is to demonstrate the different oxidative phosphorylation roles in the response of the anastomosed SB and LB. In 45 male rabbits under general anaesthesia, the ileum and colon were transected always on the same side and an end-to-end anastomosis was constructed using single-layer inverting interrupted 5-0 Ethilon sutures. Tracts containing bowel anastomoses were collected at 3, 12 and 24 h and 3, 7 and 21 days after the operation and compared with non-anastomosed bowel. Respiratory rates with different substrates, enzymatic activities, respiratory complex structures and cytochrome contents were measured in smooth muscle purified mitochondria. Mitochondrial respiration showed significant differences in the LB versus anastomosed SB: respiratory rates were markedly reduced in the LB (residual activity: 25 vs. 95% of the SB). Detailed analysis of other mitochondrial parameters confirmed a better tolerance of the ileum to anastomosis. From these preliminary studies we can consider two different aerobic thresholds for either the large or the small rabbit intestine, the latter being oxygen dependent. This can explain the mitochondrial sufferance of anastomosis in the LB whose prevalent aerobic metabolism can be more easily affected by different noxae if compared with the tendency to glycolytic metabolism of the small intestine.

Anastomosis, Surgical↗
PubMed1998

Respiratory activity and growth of human skin derma fibroblasts.

A study has been made on the speed of growth and respiratory activity of fibroblast cultures from control derma, cheloid (hypertrophic) scar and stabilized scar taken from human skin. The speed of growth and the efficiency of plaque formation of fibroblasts from cheloid scar were greater in comparison with those of fibroblasts from stabilized scar and were stimulated by the addition to the culture medium of the exudate from post-traumatic ulcer. Measurement of the contents of cytochromes showed a decrease in the content of cytochromes b562 and c + c1 in the fibroblast culture from both cheloid and stabilized scar as compared to the fibroblast culture from control derma. Cytochrome aa3 content did not show significant difference among the three types of fibroblast cultures. The respiratory activities supported by pyruvate plus malate, succinate or ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine did not show, however, significant difference among the three fibroblast cultures. These observations show that the speed of growth of skin fibroblasts does not depend on the overall respiratory capacity. The exudate stimulated the activity of cytochrome c oxidase in fibroblasts from control derma, and cheloid scar. This effect and the accompanying stimulation of fibroblast growth might be correlated with the balance of oxygen free radicals.

Adult↗
PubMed1996

Topology of the mitochondrial cAMP-dependent protein kinase and its substrates.

In intact bovine heart mitochondria, cAMP-dependent phosphorylation of 42, 29, 18 and 6.5 kDa proteins was inhibited by carboxyatractyloside. This shows that both mitochondrial cAMP-dependent protein kinase (mtPKA) and its protein substrates are localized at the matrix side of the inner mitochondrial membrane. Proteins of 42, 29, 18, and 6.5 kDa were also bound at the outer surface of mitochondria where they were phosphorylated by the added purified catalytic subunit of PKA. In the cytosol from bovine heart proteins of the above molecular weights were phosphorylated by the cytosolic PKA.

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

Role of nuclear-encoded subunits of mitochondrial cytochrome c oxidase in proton pumping revealed by limited enzymatic proteolysis.

The role of supernumerary subunits of bovine heart cytochrome c oxidase has been investigated by examining the effect on the enzymatic activity of limited proteolysis by chymotrypsin, thermolysin, and trypsin. All three proteases, when added to the soluble oxidase, digested subunits III, VIa, and VIb and caused inhibition of electron flow in the oxidase. In addition, trypsin and thermolysin also digested subunit IV. Trypsin cleaved off an N-terminal segment of seven residues; thermolysin cleaved only the first four residues at the N-terminus of subunit IV. Digestion of the soluble oxidase by trypsin but not by thermolysin caused decoupling of redox-linked proton pumping in the oxidase. It is concluded that the sequence V5-V6-K7 of the hydrophilic N-terminus of subunit IV, which protrudes out of the matrix side of the mitochondrial membrane, mediates the access of protons into the transmembrane proton translocating pathway in the oxidase.

Amino Acid Sequence↗