RESUMO
Nowadays, asbestos-containing wastes (ACW) still represent an important environmental problem and a severe health hazard due to the well known pulmonary diseases derived from asbestos fibers inhalation. Except for a very few cases, ACW are currently confined in controlled landfills, giving rise to increasingly high amounts of still hazardous wastes. A promising alternative to landfill confinement is represented by ACW inertization, but the high cost of the inertization processes so far proposed by the scientific community have hampered the creation of actually operative plants. In this paper, we explore the possibility to use an innovative process that ensures the obtainment of asbestos-free inert material in an exceptionally short processing time, thus greatly reducing cost-related problems. The efficacy of the inertization process has been verified through accurate mineralogical investigations on both chrysotile and crocidolite de-activated fibers, through X-ray diffraction, scanning and transmission electron microscopy. Overall mineralogical, microstructural and granulometric characteristics of the inert bulk material suggest that it could be successfully re-used as a secondary raw material in ceramic industries. This innovative inertization procedure could therefore provide an effective and economically sustainable solution for ACW management.
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OBJECTIVE: To test the reliability of a new enzyme-linked immunosorbent assay (ELISA) to identify anti-RNA polymerase III (RNAP III) positive sera from Italian patients with Systemic Sclerosis (SSc) and other chronic inflammatory disorders. METHODS: A comparison between the new ELISA for anti-RNAP III and the gold standard technique, immunoprecipitation (IP), was first performed on 106 SSc patients, 16 patients with other connective tissue diseases and 10 healthy subjects. A further ELISA evaluation was performed on 224 SSc patients, 120 subjects with other rheumatic or infectious diseases, and 81 healthy controls. RESULTS: Plotting ELISA and IP data in a Receiver Operator Characteristic curve, the ELISA cut-off value providing the best specificity (99.1%) and sensibility (100%) was 28 U/ml (AUC=0.999; p<0.0001). Using this cut-off in the second analysis, anti-RNAP III positive results were found in 41 (18.3%) SSc patients, all negative for anticentromere or anti-topoisomerase I antibodies, while only 3 subjects tested positive among the 120 sera collected from other patients. All the healthy subjects were negative. CONCLUSION: This new ELISA for anti-RNAP III is highly accurate when a proper cut-off value is employed and represents a valid substitute to IP in a clinical setting.
Assuntos
Anticorpos/sangue , Ensaio de Imunoadsorção Enzimática/métodos , RNA Polimerase III/imunologia , Escleroderma Sistêmico/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Imunoprecipitação/métodos , Itália , Pessoa de Meia-Idade , Curva ROC , Valores de Referência , Reprodutibilidade dos Testes , Escleroderma Sistêmico/sangue , Escleroderma Sistêmico/etnologia , Sensibilidade e EspecificidadeRESUMO
Lung fibrosis is a major cause of mortality and morbidity in systemic sclerosis (SSc). However, its pathogenesis still needs to be elucidated. We examined whether the alteration of certain proteins in bronchoalveolar lavage fluid (BALF) might have a protective or a causative role in the lung fibrogenesis process. For this purpose we compared the BALF protein profile obtained from nine SSc patients with lung fibrosis (SScFib+) with that obtained from six SSc patients without pulmonary fibrosis (SScFib-) by two-dimensional gel electrophoresis (2-DE). Only spots and spot-trains that were consistently expressed in a different way in the two study groups were taken into consideration. In total, 47 spots and spot-trains, corresponding to 30 previously identified proteins in human BALF, showed no significant variation between SScFib+ patients and SScFib- patients, whereas 24 spots showed a reproducible significant variation in the two study groups. These latter spots corresponded to 11 proteins or protein fragments, including serum albumin fragments (13 spots), 5 previously recognized proteins (7 spots), and 4 proteins (3 spots) that had not been previously described in human BALF maps, namely calumenin, cytohesin-2, cystatin SN, and mitochondrial DNA topoisomerase 1 (mtDNA TOP1). Mass analysis did not determine one protein-spot. The two study groups revealed a significant difference in BALF protein composition. Whereas levels of glutathione S-transferase P (GSTP), Cu-Zn superoxide dismutase (SOD) and cystatin SN were downregulated in SScFib+ patients compared with SScFib- patients, we observed a significant upregulation of alpha1-acid glycoprotein, haptoglobin-alpha chain, calgranulin (Cal) B, cytohesin-2, calumenin, and mtDNA TOP1 in SScFib+ patients. Some of these proteins (GSTP, Cu-Zn SOD, and cystatin SN) seem to be involved in mechanisms that protect lungs against injury or inflammation, whereas others (Cal B, cytohesin-2, and calumenin) seem to be involved in mechanisms that drive lung fibrogenesis. Even if the 2-DE analysis of BALF did not provide an exhaustive identification of all BALF proteins, especially those of low molecular mass, it allows the identification of proteins that might have a role in lung fibrogenesis. Further longitudinal studies on larger cohorts of patients will be necessary to assess their usefulness as predictive markers of disease.
Assuntos
Líquido da Lavagem Broncoalveolar/química , Proteínas/análise , Proteoma/análise , Fibrose Pulmonar/metabolismo , Escleroderma Sistêmico/metabolismo , Eletroforese em Gel Bidimensional , Feminino , Humanos , Fibrose Pulmonar/etiologia , Escleroderma Sistêmico/complicaçõesRESUMO
BACKGROUND: In 1994, the American Association on Mental Retardation with the DSM-IV has come to a final definition of pervasive developmental disorders (PDD), in agreement with the ICD-10. Prevalence of PDD in the general population is 0.1-0.15% according to the DSM-IV. PDD are more frequent in people with severe intellectual disability (ID). There is a strict relationship between ID and autism: 40% of people with ID also present a PDD, on the other hand, nearly 70% of people with PDD also have ID. We believe that in Italy PDD are underestimated because there is no agreement about the classification system and diagnostic instruments. METHOD: Our aim is to assess the prevalence of PDD in the Italian population with ID. The Scale of Pervasive Developmental Disorder in Mentally Retarded Persons (PDD-MRS) seems to be a very good instrument for classifying and diagnosing PDD. RESULTS: The application of the PDD-MRS and a clinical review of every individual case on a sample of 166 Italian people with ID raised the prevalence of PDD in this population from 7.8% to 39.2%. CONCLUSIONS: The study confirms the relationship between ID and autism and suggests a new approach in the study of ID in order to elaborate a new integrated model for people with ID.
Assuntos
Transtorno Autístico/epidemiologia , Deficiência Intelectual/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Transtorno Autístico/diagnóstico , Manual Diagnóstico e Estatístico de Transtornos Mentais , Feminino , Humanos , Deficiência Intelectual/diagnóstico , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência , Inquéritos e QuestionáriosRESUMO
OBJECTIVE: To assess the frequency and clinical correlates of the systemic sclerosis-related autoantibodies to RNA polymerases in Italian patients. METHODS: Sera from 115 patients with systemic sclerosis (SSc) and 10 patients with systemic sclerosis-overlap syndromes recruited from a single center in northern Italy were investigated for antibodies to RNA polymerase I, II, and III by means of immunoprecipitation using 35S-labeled HeLa cell antigen extract. Twenty-five normal volunteers and 91 patients with different connective tissue diseases were studied as a control group. RESULTS: Antibodies to RNA-polymerases were found in 14/115 SSc patients (12.1%). None of the normal controls and none of the patients with other connective tissue diseases, including overlap syndromes, were positive. Antibodies reacting with RNA-polymerase I and III (+/- RNA-polymerase II) were found in 9/115 patients (7.8%) and were mutually exclusive with respect to other scleroderma-related autoantibodies. Isolated anti-RNA polymerase II reactivity was found in 5 patients and was associated with anti-topoisomerase I antibodies in 4 cases. Anti-RNA-polymerase I and III antibodies were associated with diffuse cutaneous involvement and male gender. Only two patients from our series had scleroderma renal crisis, and one of them had anti-RNA polymerase antibodies. CONCLUSIONS: Anti-RNA-polymerase antibodies appear to be less frequent in Italian patients than in Caucasian patients from the United Kingdom or USA. This might be associated with the lower frequency of scleroderma renal crisis.
Assuntos
Anticorpos Anti-Idiotípicos/sangue , Autoanticorpos/análise , RNA Polimerases Dirigidas por DNA/metabolismo , Escleroderma Sistêmico/imunologia , Adulto , Idoso , Autoanticorpos/sangue , Estudos de Casos e Controles , RNA Polimerases Dirigidas por DNA/análise , Feminino , Imunofluorescência , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Probabilidade , Valores de Referência , Medição de Risco , Estudos de Amostragem , Escleroderma Sistêmico/sangue , Escleroderma Sistêmico/diagnóstico , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Estatísticas não ParamétricasRESUMO
We attempted to determine whether beta1,3-galactosyltransferase beta3Gal-T5 is involved in the biosynthesis of a specific subset of type 1 chain carbohydrates and expressed in a cancer-associated manner. We transfected Chinese hamster ovary (CHO) cells expressing Fuc-TIII with beta3Gal-T cDNAs and studied the relevant glycoconjugates formed. beta3Gal-T5 directs synthesis of Lewis type 1 antigens in CHO cells more efficiently than beta3Gal-T1, whereas beta3Gal-T2, -T3, and -T4 are almost unable to direct synthesis. In the clone expressing Fuc-TIII and beta3Gal-T5 (CHO-FT-T5), sialyl-Lewis a synthesis is strongly inhibited by swainsonine but not by benzyl-alpha-GalNAc, and sialyl-Lewis x is absent, although it is detected in the clones expressing Fuc-TIII and beta3Gal-T1 (CHO-FT-T1) or Fuc-TIII and beta3Gal-T2 (CHO-FT-T2). Endo-beta-galactosidase treatment of N- glycans prepared from clone CHO-FT-T5 releases (+/-NeuAcalpha2-->3)Galbeta1-->3[Fucalpha1-->4]GlcNAcbeta1-->3Gal but not GlcNAcbeta1-->3Gal or type 2 chain oligosaccharides, which are found in CHO-FT-T1 cells. This result indicates that beta3Gal-T5 expression prevents poly-N-acetyllactosamine and sialyl-Lewis x synthesis on N-glycans. Kinetic studies confirm that beta3Gal-T5 prefers acceptors having the GlcNAcbeta1-->3Gal end, including lactotriosylceramide. Competitive reverse transcriptase mediated-polymerase chain reaction shows that the beta3Gal-T5 transcript is expressed in normal colon mucosa but not or poorly in adenocarcinomas. Moreover, recombinant carcinoembryonic antigen purified from a CHO clone expressing Fuc-TIII and beta3Gal-T5 reacts with anti-sialyl-Lewis a and carries type 1 chains on oligosaccharides released by endo-beta-galactosidase. We conclude that beta3Gal-T5 down-regulation plays a relevant role in determining the cancer-associated glycosylation pattern of N-glycans.
Assuntos
Adenocarcinoma/enzimologia , Antígeno Carcinoembrionário/metabolismo , Neoplasias do Colo/enzimologia , Galactosiltransferases/metabolismo , Glicoproteínas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Idoso , Animais , Células CHO , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Cricetinae , Regulação para Baixo , Feminino , Fucosiltransferases/biossíntese , Galactosiltransferases/genética , Mucosa Gástrica/metabolismo , Glicosilação , Humanos , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Masculino , Pessoa de Meia-Idade , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Antígeno Sialil Lewis X , TransfecçãoRESUMO
A series of observations has suggested that one or more digoxin-like immunoreactive substances (DLIS) in biological fluids is able to cross-react with the antidigoxin antibody. Whether this substance is the endogenous inhibitor of Na+/K+ ATPase has not been well established. The aim of this study was to identify and characterize DLIS from human urine. Treated urine from healthy men was run on an affinity chromatography column at a flow rate of 1 mL/min in which the ligand was an antibody (antiserum) to digoxin. Eluates from affinity chromatography were applied onto analytical reversed-phase HPLC. The active material was eluted with a linear gradient of acetonitrile (from 350 to 650 mL/L) and water. A second step in HPLC was carried out isocratically with 280 mL/L acetonitrile in water. We found a single peak showing cross-reactivity with antidigoxin antibody as measured by RIA. It showed the same retention time as that of a digoxin calibrator. This highly purified substance is able to displace [3H]ouabain from dog kidney-derived Na+/K+ ATPase, to inhibit Na+/K+ ATPase activity as measured by the 86Rb+ uptake in red blood cells and by coupled enzyme assay. Our results are consistent with the hypothesis that DLIS, as isolated by this particular digoxin antibody, is a single substance and an inhibitor of Na+/K+ ATPase.
Assuntos
Cromatografia de Afinidade , Inibidores Enzimáticos/urina , Saponinas/urina , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Anticorpos/imunologia , Ligação Competitiva , Cardenolídeos , Gatos , Cromatografia Líquida de Alta Pressão , Digoxina/imunologia , Cães , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Eritrócitos/metabolismo , Humanos , Rim/enzimologia , Masculino , Ouabaína/metabolismo , Radioimunoensaio , Radioisótopos de Rubídio , Saponinas/imunologia , Saponinas/isolamento & purificação , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
OBJECTIVE: To evaluate adverse reactions frequency following fluorescein i.v. administration to perform retinoic angiography (ARF). DESIGN: Retrospective study. SETTING: Eye Clinic of the University of Florence. MATERIALS AND METHODS: 6524 patients undergoing 10,003 ARF. PARAMETERS EVALUATED: For each patient age, sex, concomitant diseases, previous ARF, allergic history and any adverse reaction were evaluated. RESULTS: Adverse experiences were described in 7.5% of 6524 patients. Most of the observed reactions were mild: nausea (3.8%), vomiting (0.43%), vasovagal phenomena (1.18%), anaphylactoid reactions (1.84%). A major rate of adverse reactions were observed in patients previously submitted to ARF who reported in the anamnesis a previous adverse reaction, and in those with a positive allergic history. Severe reactions occurred in 0.27% of patients, but only in 2 of 6524 patients a rianimatory treatment (0.03) was necessary. CONCLUSION: From the data retrospective analysis it was observed that the manifestation of mild adverse effects was to be expected in many cases. It was therefore possible to point out classes of patients at risk (patients who reported a previous adverse reaction after ARF and patients with positive allergic history). Unfortunately it was not possible to expect and so to prevent the severe effects. Thus it is recommended to perform ARF in an adequately equipped room.
Assuntos
Angiofluoresceinografia/efeitos adversos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Proteoglycans (PGs) from bovine cornea showed a protective effect on liposome peroxidation induced by Fe2+. Both chondroitin sulfate, dermatan sulfate-containing PG (CS,DS-PG) and keratan sulfate-containing PG (KS-PG) inhibited thiobarbituric acid-reactive substance formation when incubated with liposomes and Fe2+, CS,DS-PG being more effective than KS-PG. The native structure of PGs contributed markedly to antioxidant activity. Papain digestion of core protein reduced the protective effect of CS,DS-PG, whereas it abolished completely that of KS-PG. Apparently only hexuronate-containing glycosaminoglycan (GAG) chains may exert a significant antioxidant activity and this was confirmed using standard GAGs. Quasielastic laser light scattering was used to evaluate the structural consequence of peroxidative damage induced by Fenton reagent on liposomes. After exposure to the free-radical-generating system, a bimodal distribution of liposomes was observed, probably depending on the loss of native structure and fragmentation. Both CS,DS-PG and KS-PG prevented liposome breakdown. Again, free KS chains were ineffective against liposome damage, whereas DS and CS maintained the normal distribution of liposome size. These data support the hypothesis that PGs may represent part of the antioxidant mechanisms of organisms and suggest that modifications of PG content and/or composition might affect tissue sensitivity to oxidative stress.
Assuntos
Córnea , Peroxidação de Lipídeos/efeitos dos fármacos , Lipossomos , Fosfatidilcolinas , Proteoglicanas/farmacologia , Animais , Antioxidantes , Bovinos , Elasticidade , Radicais Livres , Peróxido de Hidrogênio , Ferro , Luz , Proteoglicanas/química , Proteoglicanas/isolamento & purificação , Espalhamento de Radiação , Substâncias Reativas com Ácido Tiobarbitúrico/análiseRESUMO
Different proteoglycan (PG) populations were isolated from normal human aorta by extraction of minced tissue with 4M GuHCl and by further digestion of the residue with collagenase. Dissociative extraction induced a complete disappearance of Alcian Blue positive material, which was demonstrable by transmission electron microscopy before the treatment around collagen fibrils and in pericellular areas. However, 4M GuHCl extraction solubilized only an average of 60% of aorta total hexuronate content. Collagenase treatment of the residue resulted in a complete loss of collagen fibril organization, which was coupled with a further hexuronate recovery, accounting for about one third of total tissue content. The bulk of PGs obtained in collagenase digest was retained by Sepharose CL-4B column. Their sulphated glycosaminoglycan (GAG) composition differed from PGs extracted with 4M GuHCl, containing only chondroitin sulphate (CS) and heparan sulphate (HS), without detectable traces of dermatan sulphate (DS). Moreover, they contained hyaluronic acid. The results obtained by agarose polyacrylamide gel electrophoresis (APGE) and Octyl-Sepharose chromatography, followed by further APGE and Sepharose CL-4B gel-filtration, carried out before and after treatment with Chondroitinase ABC and AC and Heparinase I and III, suggested that collagenase digest contained different PG populations, carrying mainly either CS or HS chains. Moreover, HS containing PGs showed higher hydrodynamic size and stronger properties of hydrophobic interactions than CS containing PGs.
Assuntos
Aorta Torácica/química , Proteoglicanas/isolamento & purificação , Adulto , Idoso , Idoso de 80 Anos ou mais , Aorta Torácica/patologia , Cromatografia em Gel , Clostridium/enzimologia , Colagenases , Eletroforese em Gel de Poliacrilamida , Feminino , Guanidina , Guanidinas , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Desnaturação Proteica , Proteoglicanas/análise , Proteoglicanas/fisiologiaRESUMO
The properties of proteoglycans (PGs) produced by normal human skin fibroblasts were investigated with increasing passage. The increase of subculture number was associated with a constant increase in PG molecular size, which was particularly evident in cell layer extracts. In the cell layer, the ratio of DS-PGs/HS-PGs was markedly higher in early passage cultures. Moreover, the cell layer from young cells contained lower amounts of radioactivity incorporated into the most hydrophobic PG populations, suggesting that the PG core protein might also undergo significant modification with increasing subcultures. There was no significant difference in energy charge value between early and late passage cultures, whereas the NAD/NADH ratio was found to decrease markedly in senescent cells.
Assuntos
Senescência Celular , Fibroblastos/metabolismo , Proteoglicanas/metabolismo , Pele/metabolismo , Divisão Celular , Células Cultivadas , Pré-Escolar , Metabolismo Energético , Humanos , Masculino , NAD/metabolismo , Oxirredução , Pele/citologiaRESUMO
Adhesion properties of rat embryo fibroblast cultures and proteoglycans (PGs) produced both in the growth medium and in the cell layer were investigated with increasing passages. Both cell-cell and cell-substrate adhesion increased with increasing subculture number. Cell adhesion properties were improved by cell treatment with chondroitinase ABC. The increase in subculture number was coupled with a constant increase of PG molecular size, which was particularly evident in cell layer extracts. The ratio HS-PGs/DS-PGs increased with increasing passages. PG modifications are likely to represent evidence of changes in extracellular matrix organization and could play a role in the increase of cell adhesion properties.
Assuntos
Adesão Celular , Fibroblastos/fisiologia , Proteoglicanas/química , Animais , Comunicação Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Matriz Extracelular/química , Matriz Extracelular/fisiologia , Fibroblastos/química , Fibroblastos/efeitos dos fármacos , Cinética , Proteoglicanas/análise , RatosRESUMO
Calf cornea slices were incubated with [U-14C]glucose, in varying pO2 or lactate concentrations. Acid glycosaminoglycans were separated by ion-exchange chromatography after papain digestion. The percentage radioactivity incorporated into keratan sulphate increased markedly with decreased oxygen tension, whereas a concomitant relative decrease of the biosynthesis of glycosaminoglycuronans occurred. Similar results were obtained with increased lactate concentration. Our findings support the idea that keratan sulphate is a functional substitute for chondroitin sulphate in conditions of oxygen lack (Scott, J.E. and Haigh, M. (1988) J. Anat. 158, 95-108).
Assuntos
Sulfatos de Condroitina/biossíntese , Córnea/metabolismo , Sulfato de Queratano/biossíntese , Lactatos/farmacologia , Oxigênio/farmacologia , Animais , Radioisótopos de Carbono , Bovinos , Cromatografia por Troca Iônica , Córnea/efeitos dos fármacos , Glucose/metabolismo , Glicosaminoglicanos/biossíntese , Glicosaminoglicanos/isolamento & purificação , Ácido Láctico , Papaína/metabolismoRESUMO
The quality of liquid oxygen in domiciliary medical systems used for oxygen therapy was investigated during the emptying cycle under simulated breathing conditions. The increase in the levels of contaminants during the use of these systems was evaluated by gas chromatography and two sampling methods. The quality of liquid oxygen from storage tanks located in hospitals and in the production or in distribution plants was also investigated.
Assuntos
Oxigenoterapia , Oxigênio/análise , Cromatografia Gasosa/métodos , Contaminação de Medicamentos , Humanos , Metano/análise , Oxigenoterapia/métodos , Oxigenoterapia/normasRESUMO
Pseudoxanthoma elasticum is a genetic disease characterized by progressive mineralization of elastic fibers. Previous studies suggested that other components, apart from elastin, might be involved in the alterations of this connective tissue disorder (Martinez-Hernandez and Huffer, 1974; Pasquali Ronchetti et al., 1981; 1986). Evidence is presented that proteoglycan metabolism is altered in PXE-affected patient. Urinary GAGs suggests an increased degradation of glucosamine-containing GAGs in the patient. Pulse and chase experiments on in vitro skin fibroblasts indicated a decreased rate of synthesis of [35SO4] containing GAGs or an increase of their turnover rate in PXE. Moreover, when PGs produced from skin fibroblasts were identified by ultracentrifugation and gel filtration in associative conditions, PXE fibroblasts produced a significantly higher amount of the high molecular weight fraction of sulfated PGs. This high molecular weight material was present both in the medium and in the matrix and disappeared under dissociative conditions or after treatment with hyaluronidase or with pancreas elastase. By electron microscopy, PXE fibroblasts appeared to produce and secrete an enormous amount of toluidine blue 0 positive material organized as filaments and amorphous masses. These data are in agreement with previous observations of the presence of abnormal masses of microfilaments, in the dermis of PXE patients, which were sensitive to hyaluronidase and partially to trypsin and elastase (Pasquali Ronchetti et al., 1986). The results seem to confirm that at least some of the alterations of connective tissues in PXE are due to abnormal PGs metabolism and to their tendency to form abnormal aggregates in the extracellular space.
Assuntos
Fibroblastos/ultraestrutura , Glicosaminoglicanos/metabolismo , Proteoglicanas/metabolismo , Pseudoxantoma Elástico/patologia , Adulto , Biópsia , Células Cultivadas , Elastina , Feminino , Fibroblastos/análise , Glicosaminoglicanos/análise , Glicosaminoglicanos/isolamento & purificação , Humanos , Hialuronoglucosaminidase/farmacologia , Microscopia Eletrônica , Elastase Pancreática/farmacologia , Proteoglicanas/análise , Proteoglicanas/isolamento & purificação , Pseudoxantoma Elástico/metabolismo , PeleRESUMO
Some biochemical characteristics of collagen extracted from granulation tissue were studied and compared with those of normal skin and scar. By using electrophoretic techniques the type III collagen content was confirmed to be significantly greater in granulation tissue and lower in scar with respect to normal skin. The chromatographic determination of hydroxylysine (Hyl) glycosides in collagen extracted from granulation tissue showed a significant increase in both the degree of Hyl glycosylation and in the di-/monoglycoside ratio, while both parameters turned out to be lower in scar. These data suggest that the degree of Hyl glycosylation and the di-/monoglycoside ratio could represent an index of the degree of collagen fiber maturation.
Assuntos
Cicatriz/metabolismo , Colágeno/metabolismo , Granuloma/metabolismo , Colágeno/classificação , Glicosilação , Humanos , Hidroxilisina/metabolismo , Pele/metabolismoRESUMO
Kinetic parameters and regulatory properties of UDPGDH extracted from cultured human skin fibroblasts were determined and compared with those of UDPGDH from cornea and epiphysial-plate cartilage. Fibroblast enzyme showed an affinity for UDPG 7 times higher than cartilage enzyme and 42 times higher than cornea enzyme. UDP-xylose acted as a co-operative allosteric inhibitor, but under the same experimental conditions fibroblast enzyme was significantly less inhibited. These results were in agreement with the different GAG production of the cells we studied. Fibroblast UDPGDH activity was regulated by the NAD/NADH ratio and it was also affected by modifications of extracellular matrix composition. A significant increase of UDPGDH affinity for UDPG was observed after the treatment of the monolayers with Chase ABC.
Assuntos
Fibroblastos/metabolismo , Uridina Difosfato Ácido Glucurônico/biossíntese , Açúcares de Uridina Difosfato/biossíntese , Células Cultivadas , Condroitina Liases/farmacologia , Fibroblastos/efeitos dos fármacos , Glicosaminoglicanos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , NAD/farmacologia , Pele/citologia , Uridina Difosfato Ácido Glucurônico/fisiologia , Uridina Difosfato Xilose/farmacologia , Ácidos Urônicos/biossínteseRESUMO
The effect of modifications of the extracellular matrix on the biosynthesis of glycosaminoglycans was investigated in human skin fibroblast cultures by studying UDPGDH activity in order to evaluate: a), the histoenzymological and biochemical modifications induced by chondroitinase ABC treatment (new experimental conditions were developed in order to obtain minimum cell damage); b), the reversibility of these modifications; c), the effect of growing the cells in the presence of chondroitinsulfate; d), the specificity of the modifications induced. The results demonstrated that our experimental conditions specifically affected intracellular UDPGDH activity. Chondroitinase ABC treatment induced a reversible increase of UDP-glucuronic acid synthesis. On the contrary, the presence of chondroitinsulfate in the growth medium completely inhibited UDPGDH activity.