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Biomedical subjects

V Zappia

Publications and source records attributed to V Zappia.

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

Behaviour of human osteoblasts cultured on bioactive glass coatings.

Two new formulations of bioactive glasses were used as coatings on titanium alloy (TiAl6V4) implants for prosthetic applications in the orthopaedic field. The biocompatibility of these bioglasses, as well as their osteoconductive properties, were assessed by employing primary cultures of human osteoblasts. A nonbioactive glass, the titanium alloy and polystyrene surface were used as controls. The results obtained demonstrated that the two bioglasses elicited a rapid and strong proliferative response by osteoblasts, which spread, formed a close layer and then expressed the specific osteoblastic marker i.e. osteocalcin. In comparison, cells grew on the nonbioactive glass to a much minor extent, similar to that of polystyrene control, showing individual cellular elements not forming a compact sheet, but expressed levels of osteocalcin clearly higher than both the polystyrene control and the two bioglasses. Finally, a very low proliferative rate of osteoblasts and the synthesis of hardly detectable osteocalcin amounts were observed with the titanium alloy. In conclusion, our studies indicate that the new bioactive glasses are effective in stimulating osteoblast growth and differentiation.

Alloys↗
PubMed1997

Down-regulation of protein kinase CKII activity by sodium butyrate.

Butyrate, a dietary fiber derivative, is a well-known differentiating agent in cultured cell lines. In addition, its antineoplastic activity toward colon-rectum cancers has been documented both in vivo and in vitro. Despite the large amount of information on the potential clinical efficacy of butyrate, its mechanism of action at the molecular level has only been partially investigated. Here, we show that serine/threonine protein kinase CKII is a target of butyrate activity. In the human adenocarcinoma cell line, HT29, treated with 2 mM sodium butyrate, CKII activity decreases 50% at 24 and 48 hours after drug addition. The enzyme down-regulation is not due to changes in protein amount since the levels of the different CKII subunits remain constant during butyrate treatment. The data reported provide the first evidence that CKII down-regulation is involved in the signal transduction pathway started by butyrate.

Adenocarcinoma↗
PubMed1997

Influence of osmotic stress on protein methylation in resealed erythrocytes.

Resealed erythrocytes are a useful means of targeting exogenous proteins or extending their activity. We tested human resealed erythrocytes as a model system for studying protein methyl esterification, a reaction involved in the processing of spontaneously deamidated/isomerized polypeptides. Our results show that resealed erythrocytes are still active in the metabolic processes that lead to the formation of methyl-esterified proteins. The methylation pattern of endogenous membrane proteins appeared to be similar to that of normal erythrocytes, with bands 2.1, 3, 4.1 and 4.2 as the major methyl acceptors. We detected methyl esterification of ovalbumin, as an exogenous substrate trapped within resealed erythrocytes. Methyl incorporation was almost completely inhibited by simultaneously loading red cells with adenosine and homocysteine thiolactone, in vivo precursors of the transmethylation inhibitor S-adenosylhomocysteine. We investigated the effects of repeated resealing procedures on methyl acceptability of endogenous membrane proteins. We found that methyl-incorporation levels increased, despite an apparent conserved protein composition of the membrane. This result suggests that osmotic stress to the membrane may be responsible for increased protein methylation due to the appearance of new sites or an increased accessibility of existing sites.

Binding Sites↗
PubMed1997

Non-thermal effects of microwaves on proteins: thermophilic enzymes as model system.

Two thermophilic and thermostable enzymes, isolated from Sulfolobus solfataricus, S-adenosylhomocysteine hydrolase and 5'-methylthioadenosine phosphorylase, were exposed to 10.4 GHz microwave radiation in order to discriminate between thermal and non-thermal microwave effects. The exposure causes a non-thermal, irreversible and time-dependent inactivation of both enzymes; the inactivation rate is related to the energy absorbed and is independent of the enzyme concentration. The influence of salts on enzyme inactivation has also been investigated. Conformational changes of S-adenosylhomocysteine hydrolase, detected by fluorescence and circular dichroism techniques, suggest that microwaves induce protein structural rearrangements not related to temperature.

Adenosylhomocysteinase↗
PubMed1997

The protective effect of the olive oil polyphenol (3,4-dihydroxyphenyl)-ethanol counteracts reactive oxygen metabolite-induced cytotoxicity in Caco-2 cells.

We investigated the injurious effects of reactive oxygen metabolites on the intestinal epithelium and the possible protective role played by two olive oil phenolic compounds, (3,4-dihydroxyphenyl)ethanol and (p-hydroxyphenyl)ethanol, using the Caco-2 human cell line. We induced oxidative stress in the apical compartment, either by the addition of 10 mmol/L H2O2 or by the action of 10 U/L xanthine oxidase in the presence of xanthine (250 micromol/L); after the incubation, we evaluated the cellular and molecular alterations. Both treatments produced significant decreases in Caco-2 viability as assessed by the neutral red assay. Furthermore, we observed a significant increase in malondialdehyde intracellular concentration and paracellular inulin transport, indicating the occurrence of lipid peroxidation and monolayer permeability changes, respectively. The H2O2-induced alterations were completely prevented by preincubating Caco-2 cells with (3,4-dihydroxyphenyl)ethanol (250 micromol/L); when the oxidative stress was induced by xanthine oxidase, complete protection was obtained at a concentration of polyphenol as small as 100 micromol/L. In contrast, (p-hydroxyphenyl)ethanol was ineffective up to a concentration of 500 micromol/L. Our data demonstrate that (3,4-dihydroxyphenyl)ethanol can act as a biological antioxidant in a cell culture experimental model and that the ortho-dihydroxy moiety of the molecule is essential for antioxidant activity. This study suggests that dietary intake of olive oil polyphenols may lower the risk of reactive oxygen metabolite-mediated diseases such as some gastrointestinal diseases and atherosclerosis.

Antioxidants↗
PubMed1997

Metabolic consequences of folate-induced reduction of hyperhomocysteinemia in uremia.

Plasma homocysteine, a well-recognized risk factor for cardiovascular disease, is elevated in uremic patients on hemodialysis. The authors have recently demonstrated that one consequence is the reduction in red cell membrane protein methylation levels, caused by a rise of intracellular adenosylhomocysteine, a potent inhibitor of methyltransferases. Protein methylation is involved in a repair mechanism of damaged membrane proteins, and an impairment in methylation leads to the accumulation of altered proteins. Therapy with folates, cofactors in the transformation of homocysteine to methionine, is effective in lowering plasma homocysteine. This article details a study on the metabolic effects of oral methyltetrahydrofolate, the active form of folic acid, on 14 uremic hemodialysis patients. Two months of therapy led to a significant reduction of plasma homocysteine levels, with a proportional response to pre-folate levels. In five of 13 patients with homocysteine levels above 20 microM, plasma homocysteine level was reduced to less than 15 microM. After treatment, levels of adenosylmethionine, the methyl donor in transmethylations, had significantly increased; levels of adenosylhomocysteine had increased to a smaller extent. Therefore, the ratio between the two compounds, an excellent indicator of the presence and the degree of methylation inhibition, was significantly ameliorated. Methionine plasma levels increased after treatment in all patients and were correlated with posttreatment adenosylmethionine levels. It was concluded that treatment with methyltetrahydrofolate brings the plasma homocysteine concentration back to an "acceptable" level, and the metabolic consequences are in the direction of an increase in the normal flow of transmethylations, as monitored by an increase in the [adenosylmethionine]/[adenosylhomocysteine] ratio.

Erythrocyte Membrane↗
PubMed1996

Cloning and sequencing of the gene coding for S-adenosylhomocysteine hydrolase in the thermophilic archaeon Sulfolobus solfataricus.

The gene from the thermophilic archaeon Sulfolobus solfataricus (Ss), encoding the S-adenosylhomocysteine hydrolase (AdoHcyHD), has been cloned. Two degenerate oligodeoxyribonucleotide (oligo) probes, synthesized on the basis of amino acid (aa) sequence of cyanogen bromide-peptide fragments of the purified protein, were used to screen a genomic library of Ss cloned into the pGEM7Zf(+) vector. The AdoHcyHD gene (adohcyhd) comprises 1254 nucleotides (nt) and encodes a polypeptide of 417 aa with a deduced molecular mass of 46 kDa, in good agreement with the value directly measured for the purified enzyme. The identity of more than 32% of the deduced aa sequence was confirmed by Edman degradation of peptides. Putative regulatory elements which are in good agreement with the archaeal promoter consensus sequences were identified in the flanking regions. Comparison of the aa sequences of AdoHcyHD from different sources shows a remarkable degree of conservation. Surprisingly, several aa residues, thought important in substrate binding and catalysis, show non-conserved replacements in Ss AdoHcyHD.

Adenosylhomocysteinase↗
PubMed1996

Extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus. Gene cloning and amino acid sequence determination.

A gene encoding an extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase was cloned from the archaeon Sulfolobus solfataricus. Two degenerate oligodeoxyribonucleotide probes synthesized on the basis of the N-terminal amino acid sequence of the protein were used to screen a genomic library of S. solfataricus cloned into the pGEM7Zf(+) vector. The DNA fragment of 2118 bp containing the 5'-methylthioadenosine phosphorylase gene was sequenced. The open reading frame comprises 711 nucleotides, which includes the stop codon, and encodes a protein of 236 residues whose molecular mass is in good agreement with the value determined by gel filtration for the purified enzyme. The N- and C-terminal sequences of the protein and the sequences of the peptides prepared by cyanogen bromide cleavage exactly match with the corresponding sequences deduced from the gene, thus confirming the identity of the 5'-methylthioadenosine phosphorylase gene. Typical archaebacterial regulatory sites were identified in the flanking regions and a potential Shine-Dalgarno-like sequence was recognized around the ATG initiation codon. The deduced amino acid sequence showed 32% identity and 30% identity with Escherichia coli purine-nucleoside phosphorylase and with E, coli uridine phosphorylase, respectively. Evolutionary and structural implications of this similarity are discussed.

Amino Acid Sequence↗
PubMed1996

Biochemical characterization of p16INK4- and p18-containing complexes in human cell lines.

The regulation of the D-type cyclin-dependent kinase (CDK4 and CDK6) activity appears to be the key step in the progression of eukaryotic cells through the G1 cell cycle phase. One of the mechanisms involved in this process is the binding of some small proteic inhibitors, with a molecular mass ranging between 14 and 20 kDa, to these CDKs. We have evaluated the amount of two such inhibitors, namely p16(INK4) and p18, in normal and transformed cells, as well as the biochemical features of the macromolecular complexes containing these proteins. The results obtained indicated that (i) p18 gene expression, unlike p16(INK4) gene, is not regulated by pRb status, (ii) no evident relationship exists between the expression of p16(INK4) and p18 genes, (iii) significant amounts of the two proteins are not bound to CDKs but occur as free molecules, (iv) each inhibitor forms a complex with the CDK protein with a 1:1 stoichiometry, and (v) a competition exists between cyclin D and the inhibitor protein toward the CDK protein resulting in the absence of detectable cellular free kinase. Moreover, employing the human native partially purified p16(INK4)or the pure recombinant protein, we have been able to demonstrate in vitro the dissociation of CDK4-cyclin D1 complex and the formation of CDK4-p16(INK4) bimolecular complex. Our findings suggest that during the cell division cycle the members of the p16(INK4) protein family and cyclin Ds compete for binding to CDK4/CDK6 and that their quantitative ratio is essential for G1 --> S transition.

Carrier Proteins↗
PubMed1996

Purification and characterization of recombinant human 5'-methylthioadenosine phosphorylase: definite identification of coding cDNA.

5'-Methylthioadenosine phosphorylase gene maps on the 9p21 chromosome, strictly linked to the important tumor suppressor gene p16INK4A. Chromosomal deletions encompassing both the phosphorylase and p16INK4A genes cause the complete absence of the enzymatic activity in a large number of tumors, thus resulting in well-defined metabolic differences between malignant and normal cells. Recently, the cloning of the phosphorylase gene has been reported on the basis of indirect evidence. In order to demonstrate definitely the identification of 5'-methylthioadenosine phosphorylase gene, we have cloned the putative enzyme coding sequence in a prokaryotic expression vector and expressed the protein in bacteria. The recombinant phosphorylase has been purified to homogeneity and its physicochemical, immunological and kinetic features have been characterized. The results obtained allowed the conclusive demonstration of 5'-methylthioadenosine phosphorylase gene cloning and the use of recombinant protein for further characterization.

Chromatography, Gel↗
PubMed1996

Myotonic dystrophy: antisense oligonucleotide inhibition of DMPK gene expression in vitro.

Antisense phosphorothioate oligonucleotides, targeted against the first codon starting region of DMPK mRNA, were successfully used in K562 and HepG2 cells to decrease DMPK expression. The most effective antisense oligo, MIO1, when added to K562 cells, shows a 75% reduction of the DMPK gene expression 6 hours after addition. The same molecule, when encapsulated in liposomes, delays myotonin mRNA decrease at 24 hours after cell treatment. This considerable success with such inhibition in vitro could be utilised to generate a cell model to study myotonic dystrophy (DM) chemio-physiological alterations.

Base Sequence↗
PubMed1996

Cytoskeletal behaviour in spectrin and in band 3 deficient spherocytic red cells: evidence for differentiated splenic conditioning role.

Based on quantitative analysis of red cell membrane proteins, hereditary spherocytosis (HS) can be divided into two main groups including isolated or ankyrin combined spectrin deficiency and band 3 reduction. Protein methyl esterification catalysed by protein carboxyl methyl-transferase (PCMT type II; EC 2.1.1.77) is a post-biosynthetic modification which is involved in the metabolism of damaged membrane proteins. We utilized the evaluation of erythrocyte membrane protein methyl esterification as a marker of cytoskeletal disarray in seven HS subjects with spectrin reduction and in seven patients with HS due to band 3 deficiency. Our results support the notion that band 3 deficient erythrocytes are not affected by an extensive cytoskeletal derangement. On the contrary, we found a remarkable increase of membrane methylation in the unsplenectomized, spectrin-deficient. HS patients, suggesting a striking membrane skeleton disarray. This phenomenon was not observed in the spectrin-deficient red cells of splenectomized patients. Therefore in spectrin deficient erythrocytes the induction of cytoskeletal damage, specifically recognized by PCMT type II, could be one of the splenic steps producing conditioned spherocytes.

Adolescent↗
PubMed1996

Biocompatibility studies on glass ionomer cements by primary cultures of human osteoblasts.

Glass ionomer cements (GICs) are materials largely employed in the dental field that have been considered recently as cements in orthopaedic surgery for their proven osteogenic features. The aim of this study was to compare the response of cultured human osteoblastic cells to a number of commercial glass ionomer cements in order to provide indications useful for the further development of formulations that have potential for use as cements or implants in repair and replacement of bone tissue. The GICs tested were: Ketac-Fil Aplicap, lonocem lonocap 1,0, GC Fuji II, GC Fuji II LC and Vitremer 3M. Several features such as plating efficiency, adhesion and morphology of the cells were studied, as well as the only specific biochemical parameter of osteoblastic phenotype, namely osteocalcin production. In addition, the colonisation of materials by osteoblastic cells was verified by means of scanning electron microscopy. Altogether, the results obtained indicate that four of the five glass ionomer cements tested are biocompatible, showing vital cells adhering to the materials, proliferating and expressing the biochemical markers of osteoblastic phenotype, whereas Vitremer 3M, although currently employed in the dental field, exhibits a great cytotoxicity toward the cells. The adverse reaction of this GIC can be attributed to the leaching of at least two components of the polyacidic phase evidenced by protonic magnetic resonance analysis (PMR), namely 2-hydroxyethylmethacrylate (HEMA), and an unidentified acidic species. The addition of pure HEMA at the same concentrations found by means of PMR to cultures of osteoblastic cells resulted in a complete cell death. Our results also show that in vitro methods employing primary cultures of human cells specific to the implant sites of prostheses are appropriate and suitable tools for evaluating biocompatibility of materials. Furthermore, this kind of approach can provide indications useful in the design of novel materials as well as in improving the characteristics of the formulations already available.

Biocompatible Materials↗
PubMed1995

Increased membrane-protein methylation in hereditary spherocytosis. A marker of cytoskeletal disarray.

Protein carboxyl methyltransferase of type II selectively recognizes L-isoaspartyl and D-aspartyl residues spontaneously occurring in proteins and peptide substrates. Membrane protein methylation levels increase with erythrocyte aging in circulation, in parallel with the spontaneous formation of abnormal aspartyl sites, due to protein intrinsic instability. We found that enzymic methyl esterification of erythrocyte membrane proteins in hereditary spherocytosis, a model of cytoskeletal disarray, is significantly increased compared to normal red blood cells. This cannot be explained by an increase in mean age of spherocytes, which are on the contrary significantly younger than control cells. No differences in cytosolic methyltransferase specific activity, as well as in the intracellular concentrations of the methyl donor adenosylmethionine and/or of the methylation inhibitor adenosylhomocysteine were observed. We identified bands 2.1, 4.1 and 4.2 as the main targets for increased methylation, whose levels were correlated with the degree of spectrin deficiency associated with this anemia. Our findings indicate that membrane-protein methyl esterification represents a marker of membrane structural alteration in vivo in spherocytosis. We hypothesize that either an increased accessibility of methylation sites normally not available to the methyltransferase, or accelerated formation of methyl-accepting sites in membrane proteins are present in spherocytosis.

Cytoskeleton↗
PubMed1995

5'-Deoxy-5'-methylthioadenosine phosphorylase and p16INK4 deficiency in multiple tumor cell lines.

5'-Deoxy-5'methylthioadenosine phosphorylase (MTA-Pase) gene is localized at the 9p21 region linked to the recently identified putative tumor suppressor gene, p16INK4, which appears implicated in the control of cell division cycle. The phosphorylase is a housekeeping enzyme involved in the purine and amino acid metabolism whose activity is evidentiable in all the normal tissues. Chromosomal deletions encompassing both MTAPase and p16INK4 genes cause the total absence of the enzymatic activity only in malignant cells, thus resulting in defined metabolic differences between malignant and normal cells. MTAPase deficiency was investigated by direct radiochemical assay method and by immunochemical techniques in 35 different human malignant cell lines established from several tumor types. The enzyme-deficient cells derived from breast, lung, ovary and liver cancer, malignant melanomas, malignant gliomas and liposarcomas. Two of the MTAPase-deficient cell preparations (from a liver carcinoma and from a melanoma) are primary cultures thus directly representing the original cancer genotypes. Several of the MTAPase-negative cells were studied for p16INK4 gene deletions and for p16INK4 protein deficiency. In all the examined samples a full correlation exists between the lack of MTAPase and that of p16INK4. A similar result was obtained analysing extracts of Vero cell line, which is a fibroblast MTAPase-negative cell line established from the kidney of a normal adult monkey. Conversely, Cos cells, which also are fibroblasts derived from monkey kidney, show both MTAPase and p16INK4 protein. These results: (i) demonstrate that the phosphorylase deficiency is distributed among almost all the most important human cancers; (ii) confirm and extend the tumor types were p16INK4 gene inactivation is observable and (iii) suggest that deletions at 9p21 (in humans) or at syntenic chromosomes (in other species) might represent a general mechanism of p16INK4 gene loss of function and possibly, in turn, of cancer development and/or progression.

Animals↗
PubMed1995

Mechanism of erythrocyte accumulation of methylation inhibitor S-adenosylhomocysteine in uremia.

We have recently demonstrated that methyl esterification of erythrocyte membrane proteins, a reaction involved in recognition and repair of specifically damaged proteins, is impaired in uremia. This is accompanied by a significant increase in intracellular S-adenosylhomocysteine (AdoHcy), a potent inhibitor of methyltransferases. AdoHcy accumulation is normally prevented by its enzymatic hydrolysis to homocysteine (Hcy) and adenosine, a reversible reaction catalyzed by AdoHcy hydrolase. To assess the contribution that Hcy offers in the elevation of AdoHcy, we measured plasma and red blood cell Hcy, AdoHcy, adenosine, and S-adenosylmethionine (AdoMet) intracellular concentrations, as well as RBC AdoHcy hydrolase specific activity, in standard hemodialysis patients and normal subjects. Plasma and red blood cell Hcy levels are significantly higher in the dialysis group, and are positively correlated to AdoHcy levels. Adenosine and AdoMet levels, and AdoHcy hydrolase specific activity are not significantly different between the two groups. The enzymatic formation of labeled AdoHcy from Hcy and tracer adenosine appears to be significantly increased, in vitro, in erythrocytes from both control and uremic patients, when 50 microM Hcy (concentration comparable to plasma levels actually found in vivo in uremic patients) is added to the incubation medium. When erythrocytes from uremic patients are incubated in vitro in absence of Hcy, a significant reduction of intracellular AdoHcy is observed with time compared to identical samples incubated in presence of 50 microM Hcy, with a T1/2 of approximately 270 minutes. The results allow us to conclude that plasma and red cell Hcy levels actually found in uremia can be effectively responsible for the intracellular accumulation of the toxic compound AdoHcy.

Adenosine↗