RESUMO
Transglutaminase 2 (TG2 or TGM2) is a multi-functional enzyme which catalyzes transamidation reactions or acts as a G-protein in intracellular signalling. Tgm2-/- Mice lacking TG2 activity are glucose intolerant and show impairment of insulin secretion, suggesting an important physiological role for TG2 in the pancreatic beta cell. We have previously described a TGM2 heterozygous missense mutation ((c.998A>G, p.N333S) in a 14 year-old patient with insulin-treated diabetes and in his diabetic father. The aim of this study was to further investigate the role of TG2 in early-onset type 2 diabetes. We analysed the TGM2 gene in 205 patients with clinically defined Maturity Onset Diabetes of the Young (MODY) or early-onset type 2 diabetes. We found two novel heterozygous mutations (c.989T>G, p.M330R; c.992T>A, p.I331N), which were not detected in 300 normoglycemic controls. All mutations were in residues which are located close to the catalytic site and impaired transamidating activity in vitro. Gene expression of TGM family genes and localization of TG2 in normal human pancreas indicated that TG2 is the only transglutaminase significantly expressed in human pancreatic islet cells. We conclude that reduced TG2 activity can contribute to disorders of glucose metabolism possibly via an impairment of insulin secretion.
Assuntos
Diabetes Mellitus Tipo 2/genética , Proteínas de Ligação ao GTP/genética , Mutação de Sentido Incorreto , Transglutaminases/genética , Adolescente , Adulto , Idade de Início , Animais , Células COS , Chlorocebus aethiops , Heterozigoto , Humanos , Imuno-Histoquímica , Proteína 2 Glutamina gama-GlutamiltransferaseRESUMO
We measured the tissue concentration of human urokinase receptor (uPAR) in 22 breast carcinomas and 9 benign breast lesions using in vitro quantitative autoradiography. Tissue sections were incubated with increasing concentrations of 125I-pro-urokinase in the presence or absence of unlabeled competitor. Breast carcinomas were found to contain 5 times more uPAR than benign breast lesions with respect to their protein content [523 +/- 72 versus 108 +/- 20 (SE) fmol/mg (P < 0.001)]. Simultaneous quantitation of urokinase (uPA) by immunoenzymatic assay on tissue extracts from the same specimens showed that breast carcinomas also contain 19 times more uPA than benign tumors (611 +/- 134 versus 32 +/- 8 fmol/mg) (P < 0.01). The reliability of quantitative autoradiography measurements was confirmed by uPAR cross-linking assay on membrane fraction from either U937 histiocytic lymphoma cells or breast carcinomas and immunoperoxidase staining with an anti-uPAR antibody on tumor sections. Also, immunoperoxidase staining with an anti-uPA monoclonal antibody showed that uPA is indeed localized on the plasma membrane of epithelial tumor cells in confined areas of breast carcinomas whereas cells from benign breast lesions were devoid of uPA under the same experimental conditions. In conclusion, our findings support the hypothesis that uPAR plays a central role in the acquisition of an invasive phenotye and favor its potential use as a prognostic factor in patients with breast carcinoma.
Assuntos
Doenças Mamárias/enzimologia , Doenças Mamárias/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/ultraestrutura , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adulto , Idoso , Autorradiografia , Neoplasias da Mama/irrigação sanguínea , Divisão Celular , Membrana Celular/ultraestrutura , Feminino , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Neovascularização Patológica , Prognóstico , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
Current evidence regarding the regulation of urokinase-dependent extracellular proteolysis indicates that plasminogen activation is a surface-associated process. We have compared the histological localization of urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR) in breast cancer sections using a panel of monoclonal antibodies. First, the ability of six different anti-uPA monoclonal antibodies to recognize pro-uPA, uPA, and in vitro-formed complexes of uPA with either soluble uPAR or with plasminogen activator inhibitor type 1 was compared. Then the reactivity of the anti-uPAR antibodies was tested, and the occurrence of an uPA receptor of about M(r) 55,000 in samples from breast carcinoma was assessed by immunoprecipitating the uPA receptor from an in vitro 125I-labeled tumor extract. Immunocytochemical data from adjacent sections of 10 tumor specimens showed that antibodies recognizing free and bound uPA mostly stain the cytoplasm and the membrane of epithelial tumor cells in confined areas of the tumor and some fibroblast-like stromal cells. Acid pretreatment of tumor sections, which removes receptor-bound uPA, causes a strong reduction of the immunocytochemical reactivity of epithelial tumor cells, whereas staining of fibroblast-like cells is not considerably affected. Consistent with these results, epithelial tumor cells were mostly unreactive to anti-uPAR antibodies unless pretreated with acidic buffer, whereas fibroblast-like stromal cells showed a faint but acid-resistant staining with all anti-uPARs. In conclusion, these results show that occupied uPA receptors are definitely present on the membrane of epithelial tumor cells and suggest the occurrence of uPA-uPAR-dependent proteolytic activity on the surface of breast cancer cells.
Assuntos
Anticorpos Monoclonais , Neoplasias da Mama/química , Mama/química , Carcinoma Ductal de Mama/química , Carcinoma Lobular/química , Receptores de Superfície Celular/análise , Ativador de Plasminogênio Tipo Uroquinase/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Receptores de Ativador de Plasminogênio Tipo UroquinaseRESUMO
Maturity-onset diabetes of the young (MODY), an autosomal dominant, early-onset form of type-2 diabetes, is caused by mutations in five different genes all leading to defect(s) in the pancreatic beta cell. However, some patients with this form of diabetes do not bear a mutation in any of the known (MODY1-MODY5) loci, a notion prompting the search for new MODY genes. Clinical and genetic data point toward a defect in beta cell function in the majority of patients with MODY, and partners of the glucose-sensing device are reasonable functional candidates. The high-capacity glucose transporter GLUT2 has the ideal kinetic features for performing this task. However, complete GLUT2 deficiency in humans leads to hepato-renal glycogenosis (Fanconi-Bickel syndrome), and heterozygous GLUT2 mutations apparently behave in a recessive manner. Furthermore, in the human beta cell GLUT1 mRNA is predominant when compared to GLUT2 and glucose influx appears to be largely mediated by this low-Km transporter. Thus, we looked for the presence of sequence variants by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) within the GLUT1 gene in 90 Italian pedigrees negative at the search for mutations in glucokinase (MODY2) and hepatocyte nuclear factor-1alpha (MODY3), the two genes responsible for about 60% of MODY cases in Italian children. We found three already described silent mutations and a new single base deletion in position -173 of the 5' regulatory region. The -173de1A variant, which was detected in the heterozygous or homozygous state in 30.8% of MODY patients examined and is located in a Nuclear Factor Y binding sequence, is not associated with hyperglycemia in affected relatives of MODY probands. In conclusion, it appears from these results that the glucose transporter gene GLUT1 is unlikely to play a major role in the etiology of MODY diabetes.
Assuntos
Diabetes Mellitus Tipo 2/genética , Proteínas de Transporte de Monossacarídeos/genética , Polimorfismo Conformacional de Fita Simples , Adolescente , Sequência de Bases , Fator de Ligação a CCAAT/metabolismo , Criança , Pré-Escolar , Sequência Consenso/genética , Éxons/genética , Transportador de Glucose Tipo 1 , Humanos , Itália , Mutação/genética , Reação em Cadeia da Polimerase , Elementos de Resposta/genética , Fatores de Transcrição/metabolismoRESUMO
Since many of the factors involved in tumor growth and progression act through a receptor-mediated mechanism, we applied in vitro receptor imaging techniques to study the intratumoral distribution and concentration of receptor-molecules having experimental biological relevance in such processes. Here we summarize the results of a study concerning the role of urokinase receptor (uPAR) in the acquisition of an invasive phenotype by tumor cells. Independently of the system studied, we demonstrated that in vitro receptor imaging techniques can be used to define the biological characteristics of human solid tumors and can contribute to clarify the complex network of ligand/receptor interactions leading to tumor spread.
Assuntos
Doenças Mamárias/diagnóstico , Neoplasias da Mama/diagnóstico , Receptores de Superfície Celular/metabolismo , Autorradiografia , Doenças Mamárias/enzimologia , Doenças Mamárias/metabolismo , Doenças Mamárias/patologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Humanos , Técnicas Imunoenzimáticas , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Until early 2000, permanent and transient neonatal diabetes mellitus (NDM), defined as diabetes with onset within 6 weeks from birth that requires insulin therapy for at least 2 weeks, were considered exceedingly rare conditions, with a global incidence of 1:500,000-1:400,000 live births. The new definition of NDM recently adopted, that includes patients with diabetes onset within 6 months of age, has prompted studies that have set the incidence of the permanent form alone between 1:210,000 and 1:260,000 live births. Aim of the present work was to ascertain the incidence of NDM (i.e. permanent + transient form) in Italy for years 2005-2010. Patients referred to the Italian reference laboratory for NDM between years 2005 and 2010 and screened for mutations in common NDM genes (KCNJ11, ABCC8, and INS) and for uniparental isodisomy of chromosome 6 (UDP6) were reviewed. A questionnaire aimed at identifying NDM cases investigated in other laboratories was sent to 54 Italian reference centers for pediatric diabetes. Twenty-seven patients with NDM born between 2005 and 2010 were referred to the reference laboratory. In this group, a mutation of either KCNJ11, ABCC8 or INS was found in 18 patients, and a case with UDP6 was identified. Questionnaires revealed 4 additional cases with transient neonatal diabetes due to UDP6. Incidence of NDM was calculated at 1:90,000 (CI: 1:63,000-1:132,000) live births. Thus, with the definition currently in use, about 6 new cases with NDM are expected to be born in Italy each year.
Assuntos
Diabetes Mellitus Tipo 1/epidemiologia , Doenças do Recém-Nascido/epidemiologia , Diabetes Mellitus Tipo 1/genética , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Doenças do Recém-Nascido/genética , Itália/epidemiologia , Nascido Vivo , Masculino , MutaçãoRESUMO
Rabbit immune sera against human alcohol dehydrogenase (ADH) isozymes were found to cross-react with mouse ADH. The immune serum raised against horse ADH and specific for the human isozymes coded at the ADH1, ADH2 and ADH3 loci (class I of ADH isozymes) reacted with the ADH-A2 isozyme from mouse liver and ADH-C2 isozyme from mouse stomach. The two mouse isozymes exhibit partial immunological identity. The immune serum raised against the human chi-ADH isozyme coded at the ADH5 locus (class III of ADH isozymes) reacted with the ADH-B2 isozyme from mouse stomach and liver. The results indicate that the A2 and C2 forms of mouse ADH may be related to the human ADH isozymes coded at the ADH1, ADH2 and ADH3 loci whereas the B2 form may be homologous of the human ADH isozyme coded at the ADH5 locus.
Assuntos
Álcool Desidrogenase/imunologia , Isoenzimas/imunologia , Camundongos Endogâmicos BALB C/imunologia , Álcool Desidrogenase/genética , Álcool Desidrogenase/isolamento & purificação , Animais , Reações Cruzadas , Cavalos/imunologia , Cavalos/metabolismo , Humanos , Soros Imunes , Isoenzimas/isolamento & purificação , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos BALB C/genética , Camundongos Endogâmicos BALB C/metabolismo , Coelhos , Especificidade da Espécie , Estômago/enzimologiaRESUMO
Rabbit immune sera raised against denatured forms of horse liver alcohol dehydrogenase and of human ADH5 isozyme were found to react with the denatured subunits of all the human ADH isozymes regardless of their class. The immune serum against the human ADH isozyme cross-reacted also with horse ADH subunits and, at appropriate dilutions, both the immune sera reacted with denatured yeast ADH, suggesting that common structures have been preserved in these molecules over a long evolutionary period. The immune sera partially reacted also with the respective antigens in their native conformation, indicating that some 'sequential' epitopes are expressed on the surface of the folded proteins.
Assuntos
Álcool Desidrogenase/imunologia , Soros Imunes/imunologia , Isoenzimas/imunologia , Animais , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Epitopos/imunologia , Humanos , Desnaturação Proteica , CoelhosRESUMO
Immune sera raised in rabbits against denatured alcohol dehydrogenase (ADH) isozymes of class I from horse liver and class III from human placenta were found to cross-react with the subunits of other dehydrogenases from various sources. Similarities in the primary structure of some of these enzymes with horse ADH can account for the observed cross-reactivity. The cross-reactivity of the same antisera with other enzymes suggests that some structures within the ADH molecule have been preserved over a long evolutionary period. The immune sera were also found to react with some of the tested enzymes in their native conformations, suggesting that they contained antibodies directed against 'sequential' epitopes expressed on the surface of the proteins when folded in the native state.
Assuntos
Álcool Desidrogenase/imunologia , Epitopos/imunologia , Animais , Reações Cruzadas , Feminino , Cavalos , Humanos , Fígado/enzimologia , Oxirredutases/imunologia , Placenta/enzimologia , Gravidez , Desnaturação Proteica/imunologiaRESUMO
Point mutations in the ryanodine receptor (RYR1) gene are associated with malignant hyperthermia, an autosomal dominant disorder triggered in susceptible people (MHS) by volatile anaesthetics and depolarising skeletal muscle relaxants. To date, 17 missense point mutations have been identified in the human RYR1 gene by screening of the cDNA obtained from muscle biopsies. Here we report single strand conformation polymorphism (SSCP) screening for nine of the most frequent RYR1 mutations using genomic DNA isolated from MHS patients. In addition, the Argl63Cys mutation was analysed by restriction enzyme digestion. We analysed 57 unrelated patients and detected seven of the known RYR1 point mutations. Furthermore, we found a new mutation, Arg2454His, segregating with the MHS phenotype in a large pedigree and a novel amino acid substitution at position 2436 in another patient, indicating a 15.8% frequency of these mutations in Italian patients. A new polymorphic site in intron 16 that causes the substitution of a G at position -7 with a C residue was identified.
Assuntos
Hipertermia Maligna/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Cafeína/metabolismo , Primers do DNA , Feminino , Halotano/metabolismo , Humanos , Itália , Masculino , Músculos/metabolismo , Miopatias da Nemalina/genética , Linhagem , Mutação Puntual , Polimorfismo Genético , Polimorfismo Conformacional de Fita SimplesRESUMO
In this study we show that SV-IV, a major immunomodulatory, anti-inflammatory, and sperm immunoprotective protein secreted from the rat seminal vesicle epithelium, acts in vitro as a substrate of protein kinase C (PKC) competing efficiently with H1 histone, a very well known PKC substrate. Electrospray mass spectrometry (ES-MS) analysis demonstrated that approximately 10% of the native SV-IV molecules were phosphorylated by PKC and that such a modification involved only a single serine residue (Ser58) out of the 22 occurring in the protein. Interestingly, this modification produced a substantial enhancement (approximately 50%) of the native SV-IV's ability to stimulate the activity of both horseradish peroxidase (POD) and selenium-dependent glutathione peroxidase (GPX), an enzyme that is known to protect the mammalian spermatozoa from oxidative stress and loss of motility in the female genital tract following ejaculation. In contrast, the phosphorylation of SV-IV on Ser58 did not produce any effect on the anti-inflammatory properties of SV-IV, as measured by its ability to inhibit the phospholipase A2.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glutationa Peroxidase/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Proteína Quinase C/metabolismo , Proteínas/química , Proteínas/metabolismo , Glândulas Seminais/metabolismo , Serina , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Cinética , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosforilação , Proteínas/isolamento & purificação , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Proteínas de Plasma Seminal , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
We recently reported that in vivo phosphorylation of urokinase-type plasminogen activator on Ser138/303 prevents its catalytic-independent ability to promote myelomonocytic cell adherence and motility. We now show that Ca2+ activated, phospholipid-dependent protein kinase C from rat brain phosphorylates in vitro a peptide corresponding to prourokinase residues 133-143 (DGKKPSSPPEE) and the full-length molecule on Ser138/139. The in vivo involvement of the protein kinase C isoenzyme family is supported by the finding that inhibition of kinase C activity prevents prourokinase phosphorylation on Ser138/303 in A431 human carcinoma cells. Conversely, a short treatment of A431 cells with phorbol myristate acetate increases the extent of phosphorylated prourokinase and, concomitantly, affects its function; under these conditions, the capability of prourokinase to up-regulate U937 monocyte-like cell adherence is severely impaired, although receptor binding is unaltered. By the aid of a "phosphorylation-like" variant (Ser138 to Glu) we show that modification of Ser138 is sufficient to confer to prourokinase the antagonistic properties observed following in vivo stimulation of protein kinase C activity. These observations provide the first evidence that protein kinase C directs the formation of a receptor competitive antagonist by regulating the in vivo phosphorylation state of prourokinase.
Assuntos
Precursores Enzimáticos/metabolismo , Proteína Quinase C/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/enzimologia , Adesão Celular , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Humanos , Isoenzimas/metabolismo , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Fosforilação , Ratos , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Células U937RESUMO
AIMS/HYPOTHESIS: The aim of this study was to assess the prevalence of glucokinase gene mutations in Italian children with MODY and to investigate genotype/phenotype correlations of the mutants. METHODS: Screening for sequence variants in the glucokinase gene was performed by denaturing gradient gel electrophoresis and direct sequencing in 132 children with maturity onset diabetes of the young (MODY) and in 9 children with chronic fasting hyperglycaemia but without laboratory evidence for Type I (insulin-dependent) diabetes mellitus and with normoglycaemic parents ("non-classical" MODY). RESULTS: Altogether 54 mutations were identified in the MODY group (54/132 or 41%) and 3 among the "non-classical" MODY individuals (3/9 or 33%). Paternity testing indicated that the latter mutations have arisen de novo. Mean fasting plasma glucose concentrations of the children with the mutant glucokinase was in the expected impaired fasting glucose range. In contrast, results of the oral glucose tolerance test showed a wide range from normal glucose tolerance (Group 1: 2-h OGTT = 6.7 +/- 1.1 mmol/l; 11 patients) to diabetes (Group 2: 2-h OGTT = 11.5 +/- 0.5 mmol/l; 9 patients), with the remaining in the impaired glucose tolerance range. Disruptive mutations (i.e. nonsense, frameshifts, splice-site) were equally represented in Groups 1 and 2 and were not clearly associated with an impaired first-phase insulin response. Surprisingly, 5 out of 11 children (or 45%) in Group 1 were found to be overweight but no children in Group 2 were overweight. Sensitivity index (SI), calculated by a recently described method, was found to be significantly lower in Group 2 than in Group 1 (SI Group 2 = 0.0013 +/- 0.0009 ml Kg(-1) min(-1)/muU/ml; SI Group 1 = 0.0068 +/- 0.0048, p < 0.0035). CONCLUSION/INTERPRETATION: Mutations in glucokinase are the first cause of MODY among Italian children selected through a low threshold limit of fasting plasma glucose (i. e. > 5.5 mmol). The lack of correlation between the molecular severity of glucokinase mutations, insulin secretion at intravenous glucose tolerance test and differences in glucose tolerance suggests that factors outside the beta cell are also involved in determining post-load glucose concentrations in these subjects. Our results seem to indicate that the differences observed in the 2-h responses at the OGTT among children with MODY 2 could be related to individual differences in insulin sensitivity.