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S Formisano

Publications and source records attributed to S Formisano.

At least 37 records · Page 2Linked to original sources
PubMed1998

Effects of wortmannin on human neutrophil respiratory burst and phagocytosis.

Modulation of neutrophil response to naturally occurring stimuli is important to avoid host tissue injure. Both soluble and particulate stimuli may induce superoxide anion generation in human polymorphonuclear leukocytes. Recently wortannin has been shown to inhibit the N-formyl-methionyl-leucyl-phenylalanine (fMLP) induced activation of respiratory burst via phosphatidylinositol 3-kinase. However no data are available about the effect of the inhibitor on the respiratory burst induced by a particulate stimulus. In this paper we studied the effect of wortmannin on E. coli induced respiratory burst and phagocytosis by flow cytometry, which allows the quantitation of both H2O2 production and ingested bacteria in whole blood samples without the need of purification and concomitant manipulation of the cells. The effects of worthmannin on fMLP-induced chemotaxis was also examined by the under agarose method. Neither the E. coli nor the fMLP-induced responses were blocked by wortmannin, suggesting that PI 3-kinase activity is not required to activate these neutrophil functions. Since it is known that the respiratory burst elicited by fMLP is blocked by wortmannin, our results suggest that the generation of oxygen radicals is controlled via different signal transduction pathways, depending on the agonist used.

Androstadienes↗
PubMed1998

Fanconi's anemia cells are relatively resistant to H2O2-induced damage.

BACKGROUND AND OBJECTIVE: Fanconi's anemia (FA) is a rare autosomal recessive syndrome characterized by skeletal abnormalities, late onset bone marrow failure and susceptibility to neoplasias. Reduced defense against oxidative stress is thought to be one of the cell damaging mechanisms. We investigated in vitro the effects of oxidative stress on red blood cells (RBC) and on hematopoietic progenitor growth of normal donors and of FA patients. DESIGN AND METHODS: The effects of hydrogen peroxide (H2O2) on RBC and hematopoietic progenitors were studied in vitro by erythrophagocytosis assay and by hematopoietic progenitor colony assay, respectively. RESULTS: In an erythrophagocytosis assay using normal monocytes, RBC from nine FA patients showed increased binding index (defined as the percentage of monocytes with adherent or phagocytosed RBC) compared to that obtained with RBC from nine normal controls. Upon exposure to H2O2, the binding index of normal RBC increased, while that of FA RBC remained unchanged. In a set of different experiments, H2O2 treatment of peripheral blood mononuclear cells (PBMNC) caused a significant decrease of the number of colonies from circulating progenitor cells in all normal subjects; the inhibition was dose-dependent and direct as proven by using normal purified CD34+ cells. In nine FA patients colony assays from intact cells showed a decreased number of circulating progenitors as compared to normal subjects; however, H2O2 treatment of FA PBMNC did not cause any further decrease of the plating efficiency. INTERPRETATION AND CONCLUSIONS: Untreated FA cells behave as normal cells after exposure to the toxic effects of H2O2. However, since H2O2 exposure is inoffensive to circulating FA RBC and hematopoietic progenitors, it seems that a selection for cells resistant to further oxidative stress has taken place in the residual hematopoiesis of FA patients. We may surmise that the survival of cells that have suffered from oxidative damage may have increased the risk of their leukemic transformation.

Adolescent↗
PubMed1997

Perturbation of cellular calcium delays the secretion and alters the glycosylation of thyroglobulin in FRTL-5 cells.

Treatment of FRTL-5 cells with a Ca2+ ionophore, A23187, or a specific inhibitor of the endoplasmic reticulum Ca2+ ATPases, thapsigargin, delayed thyroglobulin secretion. The secreted thyroglobulin showed an increased electrophoretic mobility and a reduced sensitivity to neuraminidase. Only thyroglobulin that was still in the endoplasmic reticulum was sensitive to the Ca(2+)-perturbant drugs as shown by experiments in which the drugs were added at different times during a chase. Analysis of the carbohydrate chains by BioGel P4 showed that thyroglobulin secreted in the presence of the Ca(2+)-perturbants displayed an increased ratio high mannose/complex chains.

Animals↗
PubMed1997

Biphasic control of NF-kappa B activation induced by the triggering of HLA-DR antigens expressed on B cells.

The regulation of NF-kappa B activation following the triggering of HLA-DR antigens by mAb L243 has been studied at various times in Raji cells. Electrophoretic mobility shift assays demonstrated a strong increase of NF-kappa B DNA binding after triggering of HLA-DR antigens. Using TNF-alpha-activity neutralizing antibodies, the authors demonstrated that the upregulation of NF-kappa B was found to depend, at later time point, on an autocrine effect of TNF-alpha secreted following triggering of HLA-DR antigens. In contrast, it was found to be TNF-alpha independent in the early time point. Moreover, the upregulation of NF-kappa B binding activity is regulated by the triggering of selected epitopes of HLA-DR antigens. In fact, mAb L243 but not the staphylococcal superantigens, staphylococcal exotoxin toxic shock syndrome toxin-I or staphylococcal enterotoxin B, regulate the NF-kappa B binding activity.

Antibodies, Monoclonal↗
PubMed1997

Decreased low-density lipoprotein oxidation after repeated selective apheresis in homozygous familial hypercholesterolemia.

Familial hypercholesterolemia was the first genetic disorder recognized to cause myocardial infarction. Patients with homozygous familial hypercholesterolemia have rapidly progressive coronary atherosclerosis with angina pectoris, myocardial infarction, or sudden death at a young age. Selective apheresis on dextran sulfate cellulose columns reduces mortality and may induce regression of coronary lesions. These patients have both increased levels and prolonged circulation residence time of low-density lipoprotein (LDL), which is not removed by cellular receptor. LDL oxidation may play a pivotal role in atherogenesis. LDL undergoes oxidation before being taken up by macrophages and then transformed into arterial wall foam cells. The aim of this study was to investigate LDL oxidation in eight homozygous patients with familial hypercholesterolemia during repeated LDL apheresis. LDL lipid peroxidation, estimated by conjugated-diene absorbance at 234 nm, lipid peroxides, and malondialdehyde showed an increased resistance against oxidation after repeated LDL apheresis. This phenomenon was also observed in the oxidative indexes of protein moiety of LDL (apolipoprotein-B100 fragmentation, trinitrobenzenesulfonic acid reactivity, and electrophoresis agarose mobility). Similarly, cholesteryl esterification was decreased after LDL apheresis. Thus selective LDL apheresis not only decreases the pool of LDL, but it also induces changes that render LDL less susceptible to oxidation. This phenomenon might contribute to reduce coronary atherosclerosis and thus mortality of these particular patients.

Apolipoprotein B-100↗
PubMed1997

Hydrogen-deuterium exchange studies of the rat thyroid transcription factor 1 homeodomain.

The 1H NMR solution structure of the rat thyroid transcription factor 1 homeodomain (TTF-1 HD) showed that the molecule folds like classical homeodomains. The C-terminal extension of helix III (fragment 51-59) appeared to adopt a helical geometry, albeit not as rigid as the preceding portion, but the hydrogen-deuterium exchange of backbone amides and the NOE data provided evidence of a discontinuity between the two moieties of helix III at the highly conserved fragment Asn51-His52-Arg53. Analysis of quantitative measurements of isotope exchange rates allows one to recognize the general occurrence, in that region of HD motifs, of opposite effects to helix III stability. Asparagine, histidine and arginine residues occur most frequently at the beginning and end of protein helices. In TTF-1 HD a local fluctuation is observed in the fragment 51-53 which either kinks or tightens the alpha-helix. A search through the protein structure database reveals that the three most common variants of HD fragments 51-53 are often involved in helices and, frequently, in helix initiation or termination. For homeodomains in general, the nature of the fragment 51-53 may be related to the conformational dynamics of their DNA-recognition helix (helix III). Besides the specific results on fragment 51-53, the complete isotope exchange analysis of TTF-1 HD data shows that the partially solvent-exposed recognition helix is stabilized by hydrophobic interactions, like most of the structured regions of the molecule. Hydrophobic stabilization of the contacting regions meets the requirements of a DNA-interaction mechanism which, as shown with other DNA-protein complexes, should entail negative heat capacity variations due to changes in solvent exposure of the nonpolar protein surface.

Amides↗
PubMed1996

Analysis of the solution structure of the homeodomain of rat thyroid transcription factor 1 by 1H-NMR spectroscopy and restrained molecular mechanics.

The solution structure of the rat thyroid transcription factor 1 (TTF-1) homeodomain has been elucidated by 1H-NMR and restrained modeling. The TTF-1 homeodomain folds in the same manner as classical homeodomains, with three helices, a loose loop between the first two helices, and a tight turn between helix II and helix III. The typical assembly of the hydrophobic core is maintained and N-capping motifs are identified in helix I and helix III. The N-terminal stretch of helix II exhibits some mobility, similar to the preceding loop region, which may be related to its anomalous capping. The N-terminal decapeptide and the C-terminal octapeptide of the molecule (68 residues long) are disordered. All the previous characteristics are shared by all known isolated homeodomain structures. An important difference among these structures occurs at the C-terminal extension of helix III, which is either disordered or helically folded. In the TTF-1 homeodomain, the C-terminal extension of helix III (residues 51-59) appears structured, albeit not as rigidly as the preceding portion. Analysis of the NOEs and hydrogendeuterium exchange of backbone amides provides evidence for discontinuity between the two moieties of helix III, which is introduced by a tightening or a kink of residues 51-53.

Animals↗
PubMed1996

A molecular code dictates sequence-specific DNA recognition by homeodomains.

Most homeodomains bind to DNA sequences containing the motif 5'-TAAT-3'. The homeodomain of thyroid transcription factor 1 (TTF-1HD) binds to sequences containing a 5'-CAAG-3' core motif, delineating a new mechanism for differential DNA recognition by homeodomains. We investigated the molecular basis of the DNA binding specificity of TTF-1HD by both structural and functional approaches. As already suggested by the three-dimensional structure of TTF-1HD, the DNA binding specificities of the TTF-1, Antennapedia and Engrailed homeodomains, either wild-type or mutants, indicated that the amino acid residue in position 54 is involved in the recognition of the nucleotide at the 3' end of the core motif 5'-NAAN-3'. The nucleotide at the 5' position of this core sequence is recognized by the amino acids located in position 6, 7 and 8 of the TTF-1 and Antennapedia homeodomains. These data, together with previous suggestions on the role of amino acids in position 50, indicate that the DNA binding specificity of homeodomains can be determined by a combinatorial molecular code. We also show that some specific combinations of the key amino acid residues involved in DNA recognition do not follow a simple, additive rule.

Animals↗
PubMed1996

In the TTF-1 homeodomain the contribution of several amino acids to DNA recognition depends on the bound sequence.

The thyroid transcription factor-1 homeodomain (TTF-1HD) shows a peculiar DNA binding specificity, preferentially recognizing sequences containing the 5'-CAAG-3' core motif. Most other homeodomains instead recognize sites containing the 5'-TAAT-3' core motif. Here, we show that TTF-1HD efficiently recognizes another sequence, called D1, devoid of the 5'-CAAG-3' core motif. Different experimental approaches indicate that TTF-1HD contacts the D1 sequence in a manner which is different to that used to interact with sequences containing the 5'-CAAG-3' core motif. The binding activities that mutants of TTF-1HD display with the D1 sequence or with the sequence containing the 5'-CAAG-3' core motif indicate that the role of several DNA-contacting amino acids is different. In particular, during recognition of the D1 sequence, backbone-interacting amino acids not relevant in binding to sequences containing the 5'-CAAG-3' core motif play an important role. In the TTF-1HD, therefore, the contribution of several amino acids to DNA recognition depends on the bound sequence. These data indicate that although a common bonding network exists in all of the HD/DNA complexes, peculiarities important for DNA recognition may occur in single cases.

Amino Acids↗
PubMed1996

IL-10 inhibits nuclear factor-kappa B/Rel nuclear activity in CD3-stimulated human peripheral T lymphocytes.

IL-10 markedly reduces nuclear factor (NF)-kappa B/Rel nuclear activity induced in PBMC by stimulation with the anti-CD3 mAb OKT3. The inhibition is exerted specifically on the NF-kappa B/Rel activation induced by mAb OKT3, and not that produced by PMA. As judged by supershifting the DNA-protein complexes with Abs recognizing specific components of the NF-kappa B/Rel protein family, the p50/p65 (Rel A) heterodimeric form of NF-kappa B is primarily affected. The maximal effect is observed at the IL-10 concentration of 20 U/ml. IL-10 inhibitory activity is exerted on T lymphocytes and is mediated by monocytes. Indeed, monocytes pretreated with IL-10 are able so inhibit NF-kappa B nuclear activity in purified T lymphocytes stimulated with OKT3. Soluble factors do not appear to be involved in the mechanism of inhibition. On the other hand, the up-regulation of CD80 Ag, found on monocytes obtained from PBMC incubated with OKT3, is not detected after addition of IL-10, and the anti-CD28 mAb CLB-CD28/1 restores the NF-kappa B/Rel nuclear activity in IL-10-inhibited lymphocytes. Therefore, the NF-kappa B/Rel inhibition might be ascribed to a lack of cooperation between accessory cells and T lymphocytes, resulting from down-regulation of a costimulatory molecule, such as CD80, produced by IL-10 on activated monocytes. Our results demonstrate that IL-10 can inhibit the induction of NF-kappa B/Rel nuclear activity in CD3-stimulated T lymphocytes. Since inappropriate activation of kappa B-driven genes has a physiopathologic role in a number of diseases, such as HIV infection, our findings support the possibility of using this cytokine to suppress an undesirable activation of these transcription factors.

Base Sequence↗
PubMed1995

Expression, purification and functional characterisation of a Kunitz-type module from chicken type VI collagen.

The primary amino acid sequence of the carboxyl-terminal portion of the alpha 3 chain of chicken type VI collagen (K-VI) presents a 58-residue motif with a high degree of homology with members of the Kunitz serine-proteinase inhibitors family. This module was cloned, expressed in E. coli, purified and compared to the bovine pancreatic trypsin inhibitor (BPTI) in an inhibition profile assay of two serine proteases, trypsin and plasmin. We found that recombinant K-VI is not endowed with inhibitory activity but it slightly activates both plasmin and trypsin, differently from other members of the family. Moreover, the ability to inhibit the serine protease activity is also lacking in the intact type VI collagen molecule.

Animals↗
PubMed1995

Serum withdrawal induces apoptotic cell death in Ki-ras transformed but not in normal differentiated thyroid cells.

Thyroid cells transformed by the Kirsten-ras oncogene become tumorigenic in syngeneic animals. Their growth is no longer dependent on TSH but becomes dependent on serum. Combining morphological and biochemical evidence, we show that serum withdrawal induces apoptotic cell death in Kirsten and Harvey-ras transformed thyroid cell. On the other hand, neither serum nor TSH withdrawal induce apoptosis in differentiated FRTL-5 cells. The induction of apoptosis by serum withdrawal is rapid and not triggered at a specific phase of the cell cycle. We suggest that induction of apoptosis following growth factor deprivation is an additional important characteristic, besides TSH-independence for growth and dedifferentiation, of the thyroid transformed phenotype.

Animals↗
PubMed1994

Analysis of the conformation and stability of rat TTF-1 homeodomain by circular dichroism.

The conformational stability of TTF-1HD has been determined by CD-monitored thermal denaturation and isothermal urea unfolding studies. The Gibbs free energy of stabilization found are 1.44 and 1.26 kcal.mol-1, respectively. TTF-1HD exhibits a Tm of 42 degrees C and a delta Cp of 80 cal.mol-1.K-1 indicating that TTF-1HD, when free in solution, is a mobile flexible segment folded into loose helices. Such a flexibility would be relevant for the DNA-binding function of this homeodomain. In fact, a small reduction of the alpha-helical content of TTF-1HD significantly modifies its DNA-binding activity.

Animals↗
PubMed1994

Sequence-specific DNA recognition by the thyroid transcription factor-1 homeodomain.

The molecular basis for the DNA binding specificity of the thyroid transcription factor 1 homeodomain (TTF-1HD) has been investigated. Methylation and ethylation interference experiments show that the TTF-1HD alone recapitulates the DNA binding properties of the entire protein. Studies carried out with mutant derivatives of TTF-1HD indicate a precise correspondence of some of its amino acid residues with specific bases in its binding site, allowing a crude orientation of the TTF-1HD within the protein-DNA complex. TTF-1HD shows an overall geometry of interaction with DNA similar to that previously observed for Antennapedia class HDs, even though the binding specificities of these two types of HDs are distinct. We demonstrate that the crucial difference between the binding sites of Antennapedia class and TTF-1 HDs is in the motifs 5'-TAAT-3', recognized by Antennapedia, and 5'-CAAG-3', preferentially bound by TTF-1. Furthermore, the binding of wild type and mutants TTF-1 HD to oligonucleotides containing either 5'-TAAT-3' or 5'-CAAG-3' indicate that only in the presence of the latter motif the Gln50 in TTF-1 HD is utilized for DNA recognition. Since the Gln at position 50 is an essential determinant for DNA binding specificity for several other HDs that bind to 5'-TAAT-3' containing sequences, we suggest that utilization by different HDs of key residues may depend on the sequence context and probably follows a precise hierarchy of contacts.

Antennapedia Homeodomain Protein↗
PubMed1994

Presence of dityrosine bridges in thyroglobulin and their relationship with iodination.

The presence of dimeric thyroglobulin (19S), after reduction and alkylation, suggests that within thyroglobulin there may be intermolecular cross-links, other than disulphide bridges. However, the nature of these intermolecular cross-links is still unknown. In this study, we show the presence of 3-3' dityrosine bridges in the molecule of bovine thyroglobulin by NMR and fluorescence studies. Also, we evaluated the role of iodination in dityrosine formation in vivo and in vitro.

Animals↗
PubMed1994

Regulation of NF-kappa B nuclear activity in peripheral blood mononuclear cells: role of CD28 antigen.

The NF-kappa B/rel family of transcription factors regulates the expression of a number of genes, including interleukin 2 (IL-2), IL-2 receptor alpha chain (Tac), and others, controlling T lymphocyte activation. The CD28 antigen is involved in regulation of T cell activation. To investigate whether CD28 antigen regulates NF-kappa B factors, we analyzed the effect of an anti-CD28 monoclonal antibody (mAb), CLB-CD28/1, on the nuclear activity of NF-kappa B complexes in resting and CD3-activated peripheral blood mononuclear cells (PBMC) of 11 donors. Cells were incubated with or without the anti-CD3 mAb OKT3 and/or the mAb CLB- CD28/1. Then nuclear extracts were obtained and analyzed for their binding to a 32P-labeled oligonucleotide, corresponding to the NF-kappa B 5'-CAACGGCAGGGGAATCTCCCTCTCCTT-3' consensus sequence in electrophoretic mobility shift assays. PBMC incubated with control medium did not appear to contain significant levels of NF-kappa B nuclear activity. The anti-CD28 mAb did not induce any detectable NF-kappa B nuclear activity in PBMC when used alone, except for two cases. However, cells incubated with the anti-CD3 mAb displayed NF-kappa B nuclear activity in 7 of the 11 cases. The addition of anti-CD28 to the anti-CD3 mAb-stimulated cells enhanced the levels of NF-kappa B activity in eight PBMC, while it did not modify PBMC in one sample and partially inhibited the induction of NF-kappa B in the remaining two samples. The stimulatory effect of anti-CD28 mAb on NF-kappa B nuclear activity was detected also on CD3-stimulated purified T lymphocytes. By analysis with antisera recognizing the p50 and p65 components of the NF-kappa B/rel family, NF-kappa B complexes of CD3+CD28-stimulated PBMC were found to contain both p50 and p65 proteins. An enhanced production of IL-2 was detected in cultures of CD3+CD28-stimulated PBMC. Our results indicate that CD28 triggering can modulate the activity on NF-kappa B nuclear complexes in T lymphocytes stimulated via CD3. Such an effect appeared not to require the presence of accessory cells (AC) or AC-derived cytokines.

Antibodies, Monoclonal↗
PubMed1993

Structural study of rat thyroid transcription factor 1 homeodomain (TTF-1 HD) by nuclear magnetic resonance.

The 500 MHz 1H NMR spectrum of a 68-residue peptide, encompassing the rat thyroid transcription factor 1 homeodomain (TTF-1 HD), was fully assigned using standard 2D NMR methodology. The secondary structure elements and their spatial organization were determined and led to a structure very similar to that previously described for other homeodomains and expected also for TTF-1 HD from homology modeling predictions. The three-dimensional arrangement of the three helix fragments of TTF-1 HD preserves the helix-turn-helix motif commonly occurring in many classes of DNA-binding proteins.

Amino Acid Sequence↗