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Biomedical subjects

S Papa

Publications and source records attributed to S Papa.

At least 55 records · Page 3Linked to original sources
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↗
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↗
PubMed2001

[Lung base disease after orthotopic liver transplantation. Functional ultrasonography of the diaphragm and correlated alterations].

PURPOSE: To demonstrate the greater accuracy of B- and M-probe ultrasound (US) compared to traditional examinations in the evaluation of diseases of the lung-base, a frequent localisation of pathology in patients undergoing orthotopic liver transplantation (OLT). MATERIAL AND METHODS: Twenty-one patients, 13 males and 8 females aged 24 to 63, awaiting OLT were examined using the three modalities. B-mode US was performed as a preliminary study to identify the districts of interest and the profile of the diaphragm wall, searching for any related alterations. This was followed by M-mode US, with an approach along the left and right posterior axillary lines during spontaneous and forced maximal expiration, to calculate and document the curve representing diaphragm mobility. All patients were also studied pre- and postoperatively by standard chest X-ray double projections. The parameters evaluated by US were diaphragmatic inspiratory slant and diaphragm range while the standard chest X-ray was used to assess hypoventilation, diaphragm range and pleural effusion. RESULTS: For each parameter considered we obtained the following results: presence or absence of pleural effusion (sensitivity: 100% with US vs 64% with chest X-ray) and diaphragmatic hypomobility with related hypoventilatory phenomena (sensitivity: 85% with US, with 15% false negatives). In 15 cases the chest X-ray revealed a clear elevation of the diaphragm, a finding supported by US in 11 cases. In 7 cases US showed a reduction in the diaphragm range curve without, however, any radiological evidence of any ventilatory dysfunction of the lung base and/or elevation of the corresponding hemidiaphragm. CONCLUSION: Our results suggest that radiology, B-mode US and color Doppler US, which are widely used for monitoring OLT patients, can be usefully integrated by M-mode US to evaluate diaphragmatic mobility both pre- and post-operatively. This method is fast, easy to use and widely available.

Adult↗
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

Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis.

The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)+ individuals (CD4+ T- lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34+ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single- or multiple-color cell staining and logical gating strategies. These can be accomplished using single- or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources.

Antigens, CD34↗
PubMed2000

TNF-related apoptosis-inducing ligand (TRAIL) as a negative regulator of normal human erythropoiesis.

The impact of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal hematopoietic development was investigated using adult peripheral blood CD34(+) hematopoietic progenitor cells, induced to differentiate along the erythroid, megakaryocytic, granulocytic, and monocytic lineages by the addition of specific cytokine cocktails. TRAIL selectively reduced the number of erythroblasts, showing intermediate levels of glycophorin A (glycophorin A(interm)) surface expression, which appeared in liquid cultures supplemented with stem cell factor + interleukin 3 + erythropoietin at days 7-10. However, neither immature (day 4) glycophorin A(dim) erythroid cells nor mature (day 14) glycophorin A(bright) erythroblasts were sensitive to TRAIL-mediated apoptosis. Moreover, pre-exposure to TRAIL significantly decreased the number and size of erythroid colonies in semisolid assays. These adverse effects of TRAIL were selective for erythropoiesis, as TRAIL did not significantly influence the survival of cells differentiating along the megakaryocytic, granulocytic, or monocytic lineages. Furthermore, TRAIL was detected by Western blot analysis in lysates obtained from normal bone marrow mononuclear cells. These findings indicate that TRAIL acts in a lineage- and stage of differentiation-specific manner, as a negative regulator of normal erythropoiesis. (Blood. 2000;95:3716-3724)

Adult↗
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

Coupling of electron transfer with proton transfer at heme a and Cu(A) (redox Bohr effects) in cytochrome c oxidase. Studies with the carbon monoxide inhibited enzyme.

A study is presented on the coupling of electron transfer with proton transfer at heme a and Cu(A) (redox Bohr effects) in carbon monoxide inhibited cytochrome c oxidase isolated from bovine heart mitochondria. Detailed analysis of the coupling number for H(+) release per heme a, Cu(A) oxidized (H(+)/heme a, Cu(A) ratio) was based on direct measurement of the balance between the oxidizing equivalents added as ferricyanide to the CO-inhibited fully reduced COX, the equivalents of heme a, Cu(A), and added cytochrome c oxidized and the H(+) released upon oxidation and all taken up back by the oxidase upon rereduction of the metal centers. One of two reductants was used, either succinate plus a trace of mitochondrial membranes (providing a source of succinate-c reductase) or hexaammineruthenium(II) as the chloride salt. The experimental H(+)/heme a, Cu(A) ratios varied between 0.65 and 0.90 in the pH range 6.0-8.5. The pH dependence of the H(+)/heme a, Cu(A) ratios could be best-fitted by a function involving two redox-linked acid-base groups with pK(o)-pK(r) of 5.4-6.9 and 7.3-9.0, respectively. Redox titrations in the same samples of the CO-inhibited oxidase showed that Cu(A) and heme a exhibited superimposed E'(m) values, which decreased, for both metals, by around 20 mV/pH unit increase in the range 6.0-8.5. A model in which oxido-reduction of heme a and Cu(A) are both linked to the pK shifts of the two acid-base groups, characterized by the analysis of the pH dependence of the H(+)/heme a, Cu(A) ratios, provided a satisfactory fit for the pH dependence of the E'(m) of heme a and Cu(A). The results presented are consistent with a primary involvement of the redox Bohr effects shared by heme a and Cu(A) in the proton-pumping activity of cytochrome c oxidase.

Animals↗
PubMed2000

Lineage-related sensitivity to apoptosis in human tumor cells undergoing hyperthermia.

In this study the role of hyperthermia as an apoptotic trigger was analyzed in four human tumor cell lines: HL60, U937, DOHH2, and K562. These cell lines were chosen because of their well known and different expression of bcl-2 and bcr-abl genes, the expression of which is known to be an antiapoptotic condition. HL60 and U937 cells were strongly susceptible to heat exposure, while DOHH2 cells were weakly sensitive and K562 cells were resistant, thus suggesting a possible gene involvement in this type of programmed cell death. The mechanisms underlying this apoptosis were investigated by flow cytometry, agarose gel electrophoresis, and light and electron microscopy. A subdiploid peak and DNA laddering, both of which are parameters specifically correlated to programmed cell death, were present in HL60 and U937 and, even if less evident, in DOHH2 cells undergoing hyperthermic treatment, and were absent in K562 cells. In addition, DNA single-strand cleavage was revealed by in situ nick translation, observed by confocal microscopy. Morphological analysis confirmed these results and revealed the typical chromatin changes, followed by the appearance of micronuclei and apoptotic bodies.

Apoptosis↗
PubMed2000

Chronic low dose ethanol intake: biochemical characterization of liver mitochondria in rats.

Liver mitochondria were isolated from male rats exposed for 2 months to low doses of ethanol (3% v/v in drinking water), a condition not associated with tolerance or dependence. The results show no significant changes in the content of reduced or oxidized glutathione in the liver mitochondria of ethanol treated rats with respect to controls. However, a slight but significant increase in lipid peroxidation, accompanied by an increased content of oxidized proteins, was found in ethanol exposed animals. Mitochondrial content of cytochrome complexes was not significantly affected by ethanol intake. The specific enzymatic activity of cytochrome oxidase showed, however, a significant decrease in ethanol-treated rats. The slight mitochondrial alterations found in the liver of rats exposed chronically to low doses of ethanol might represent the beginning of a more extensive damage previously observed in rats exposed to high doses of this substance.

Animals↗
PubMed2000

Programmed cell death in 2',3'-dideoxycytidine-resistant human monoblastoid U937 cells.

2',3'-Dideoxycytidine is a powerful in vitro inhibitor of human immunodeficiency virus and is currently used in the treatment of acquired immunodeficiency syndrome. A long-term exposure of U937 monoblastoid cells to dideoxycytidine induces the selection of drug-resistant cells (U937-R). In previous studies, we investigated some important biochemical properties and functional activities, such as basal respiration, protein kinase C activity, superoxide anion release, and the level of reduced glutathione, which were found to be higher in the drug-resistant cell line, compared to the parental one. In the present study, we evaluated the response of the two cell lines to the induction of apoptosis by treatment with staurosporine and okadaic acid, which interfere with the protein kinase and phosphatase pathways, respectively. Moreover, knowing that GSH plays a crucial role in the regulation of nitric oxide-dependent apoptosis, U937-R and parental lines have been treated with SIN-1, which is known to generate significant amounts of O2 and nitric oxide. Resistant and parental cells have been analysed by light and electron microscopy and agarose gel electrophoresis of isolated DNA has been performed. The obtained results demonstrate a different susceptibility of U937-R cell line to apoptosis induced with the three triggers. U937-R cells show more advanced apoptotic features if compared with parental cells, after staurosporine treatment. Differently, the okadaic acid does not induce a different behaviour in the two models. On the contrary, the agent SIN-1 determines an increased number of apoptotic cells in the U937 line. The results suggest that a higher level of protein kinase C and glutathione could prevent programmed cell death in U937-R.

Anti-HIV Agents↗
PubMed2000

The structural and functional connection between the catalytic and proton translocating sectors of the mitochondrial F1F0-ATP synthase.

The structural and functional connection between the peripheral catalytic F1 sector and the proton-translocating membrane sector F0 of the mitochondrial ATP synthase is reviewed. The observations examined show that the N-terminus of subunit gamma, the carboxy-terminal and central region of F0I-PVP(b), OSCP, and part of subunit d constitute a continuous structure, the lateral stalk, which connects the peripheries of F1 to F0 and surrounds the central element of the stalk, constituted by subunits gamma and delta. The ATPase inhibitor protein (IF1) binds at one side of the F1F0 connection. The carboxy-terminal segment of IF1 apparently binds to OSCP. The 42L-58K segment of IF1, which is per se the most active domain of the protein, binds at the surface of one of the three alpha/beta pairs of F1, thus preventing the cyclic interconversion of the catalytic sites required for ATP hydrolysis.

Animals↗
PubMed2000

Matrix metalloproteinases expression in HL-60 promyelocytic leukemia cells during apoptosis.

Human promyelocytic leukemia HL-60 cells have been used as a model to study both the expression of matrix-metalloproteinases and the mechanisms of programmed cell death. In the present study we examined the expression of these proteases in HL-60 cells stimulated by different apoptotic triggers. As shown by zymography, HL-60 cells released three major isofroms of the matrix-degrading proteases; when the leukemic cells were grown in serum-free conditions, as well as after hyperthermia and methotrexate treatment, we found a significant loss of the constitutive production of the 92 kDa matrixmetalloprotease, with an unequivocable molecular and ultrastructural evidence of programmed cell death. These results suggest that in HL-60 cells the expression/release of matrix metalloproteases can be down-regulated in the presence of the apoptotic-induced alterations, and that the decreased matrix-degrading capacity of this leukemic cell line during apoptosis may reduce its invasive potential.

Apoptosis↗
PubMed2000

Reduction of intra- and interlaboratory variation in CD34+ stem cell enumeration using stable test material, standard protocols and targeted training. DK34 Task Force of the European Working Group of Clinical Cell Analysis (EWGCCA).

The European Working Group on Clinical Cell Analysis (EWGCCA) has, in preparation for a multicentre peripheral blood stem cell clinical trial, developed a single-platform flow cytometric protocol for the enumeration of CD34+ stem cells. Using this protocol, stabilized blood and targeted training, the EWGCCA have attempted to standardize CD34+ stem cell enumeration across 24 clinical sites. Results were directly compared with participants in the UK National External Quality Assessment Scheme (NEQAS) for CD34+ Stem Cell Quantification that analysed the same specimens using non-standardized methods. Two bead-counting systems, Flow-Count and TruCount, were also evaluated by the EWGCCA participants during trials 2 and 3. Using Flow-Count, the intralaboratory coefficient of variation (CV) was </= 5% in 39% of the laboratories (trial 1), increasing to 65% by trial 3. Interlaboratory variation was reduced from 23.3% (trial 1) to 10.8% in trial 3. In trial 2, 70% of laboratories achieved an intralaboratory CV </= 5% using TruCount, increasing to 74% for trial 3; the interlaboratory CV was reduced from 23.4% to 9.5%. Comparative analysis of the EWGCCA and the UK NEQAS cohorts revealed that EWGCCA laboratories, using the standardized approach, had lower interlaboratory variation. Thus, the use of a common standardized protocol and targeted training significantly reduced intra- and interlaboratory CD34+ cell count variation.

Antigens, CD34↗
PubMed2000

F1 and F0 connections in the bovine mitochondrial ATP synthase: the role of the of alpha subunit N-terminus, oligomycin-sensitivity conferring protein (OCSP) and subunit d.

We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F1F0 complex and isolated F1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross-linking of oligomycin-sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated. The results obtained show that the alpha subunit N-terminus is essential for the correct, functional connection of F1 to F0. The alpha-subunit N-terminus contacts OSCP which, in turn, contacts the F0I-PVP(b) and the F0-d subunits. The N-terminus of subunit alpha, OSCP, a segment of subunit d and the C-terminal and central region of F0I-PVP(b) subunits are peripherally located with respect to subunits gamma and delta which are completely shielded in the F1F0 complex against trypsin digestion. This qualifies the N-terminus of subunit alpha, OSCP, subunit d and F0I-PVP(b) as components of the lateral element of the stalk. These subunits, rather than being confined at one side of the complex which would leave most of the central part of the gamma subunit uncovered, surround the gamma and the delta subunits located in the central stalk.

Adenosine Triphosphatases↗
PubMed2000

Prostate-specific antigen synthesis and secretion by human placenta: a physiological kallikrein source during pregnancy.

Prostate-specific antigen (PSA), a kallikrein-like serine protease until recently thought to be prostate specific, has been demonstrated in various nonprostatic tissues and body fluids. PSA has been also found in human endometrium and amniotic fluids, even if the significance of this novel expression is unclear. In this study, we have demonstrated by multiple techniques that human placental tissue, obtained at delivery from normal full-term pregnancies, synthesizes and secretes PSA. RT-PCR showed the presence of PSA messenger ribonucleic acid; biochemical, chromatographic, and immunological studies revealed the expression of both free and complexed PSA forms; immunoelectron microscopy indicated the syncytiotrophoblast as the site of PSA synthesis and secretion. Moreover, in vitro experiments demonstrated that PSA production and secretion are up-regulated by 17beta-estradiol, a pregnancy-related steroid hormone. These results suggest that human placenta is a source of the PSA present in amniotic fluid and maternal serum during pregnancy.

Adult↗