RESUMO
The a disintegrin and metalloprotease (ADAM) family proteins comprise a group of membrane-anchored proteins. ADAM32 is expressed specifically in testis and is closely related phylogenetically to ADAM2 and ADAM3, which are known to be critical for fertilization in mice. To assess the biological role of ADAM32, we analyzed Adam32-mutant mice. We found that male mice lacking ADAM32 have normal fertility, testicular integrity, and sperm characteristics. ADAM32 was found to exist at lower levels than ADAM2 and ADAM3 in wild-type testis and sperm, respectively. The present study demonstrates that ADAM32 is dispensable for fertility and appears to be functionally unrelated to ADAM2 and ADAM3 in mice.
Assuntos
Proteínas ADAM/deficiência , Proteínas ADAM/fisiologia , Fertilidade/fisiologia , Expressão Gênica/fisiologia , Testículo/metabolismo , Proteínas ADAM/análise , Proteínas ADAM/genética , Animais , Cruzamento , Epididimo/anatomia & histologia , Feminino , Fertilinas/análise , Masculino , Glicoproteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/química , Espermatozoides/fisiologia , Testículo/anatomia & histologia , Testículo/químicaRESUMO
We have found that A Disintegrin And Metalloproteinase-9 (ADAM9) localises to cell-cell junctions with VE-Cadherin in confluent endothelial monolayers. Co-cultures of cells separately transfected with ADAM9-EGFP or ADAM9-HA showed expression is required in two adjacent cells for localisation to cell-cell junctions suggesting the ADAM9 ectodomain may self-associate. A direct interaction between ADAM9 ectodomains was confirmed using recombinant proteins and an ELISA based method. As the ADAM9 ectodomain can also exist as a soluble form physiologically, we examined if this could inhibit endothelial functions dependent on cell-cell junctions. The soluble ADAM9 ectodomain could not increase endothelial monolayer permeability or inhibit monocyte-endothelial adhesion, but could inhibit monocyte-endothelial transmigration. These novel findings point to ADAM9 playing an important role in endothelial cell biology that is distinct from the other ADAMs.
Assuntos
Proteínas ADAM/metabolismo , Células Endoteliais/citologia , Junções Intercelulares/metabolismo , Proteínas de Membrana/metabolismo , Monócitos/citologia , Migração Transendotelial e Transepitelial , Proteínas ADAM/análise , Animais , Linhagem Celular , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Junções Intercelulares/ultraestrutura , Proteínas de Membrana/análise , Camundongos , Monócitos/metabolismo , Domínios ProteicosRESUMO
Although A Disintegrin And Metalloproteinase 8 (ADAM8) is not crucial for tissue development and homeostasis, it has been implicated in various inflammatory diseases by regulating processes like immune cell recruitment and activation. ADAM8 expression has been associated with human atherosclerosis development and myocardial infarction, however a causal role of ADAM8 in atherosclerosis has not been investigated thus far. In this study, we examined the expression of ADAM8 in early and progressed human atherosclerotic lesions, in which ADAM8 was significantly upregulated in vulnerable lesions. In addition, ADAM8 expression was most prominent in the shoulder region of human atherosclerotic lesions, characterized by the abundance of foam cells. In mice, Adam8 was highly expressed in circulating neutrophils and in macrophages. Moreover, ADAM8 deficient mouse macrophages displayed reduced secretion of inflammatory mediators. Remarkably, however, neither hematopoietic nor whole-body ADAM8 deficiency in mice affected atherosclerotic lesion size. Additionally, except for an increase in granulocyte content in plaques of ADAM8 deficient mice, lesion morphology was unaffected. Taken together, whole body and hematopoietic ADAM8 does not contribute to advanced atherosclerotic plaque development, at least in female mice, although its expression might still be valuable as a diagnostic/prognostic biomarker to distinguish between stable and unstable lesions.
Assuntos
Proteínas ADAM/análise , Proteínas ADAM/deficiência , Aterosclerose/fisiopatologia , Proteínas de Membrana/análise , Proteínas de Membrana/deficiência , Placa Aterosclerótica/patologia , Animais , Antígenos CD , Artérias Carótidas/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Macrófagos/química , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
BACKGROUND: Disintegrin-like and Metalloproteinase with Thrombospondin Motifs (ADAMTS) proteins that are fundamentally located in the extracellular matrix (ECM) have critical roles on different cellular processes by altering the ECM architecture. It has been known that expression of some members of these proteinases increases in aneurismal and dissectional aortic tissue. The purpose of this study is to investigate ADAMTS1, 5, 16 and tissue inhibitors of metalloproteinases-1, -2 (TIMP-1, -2) levels in aortic tissue obtained from patients with thoracic aortic aneurysms and dissections and to achieve new insights about the function of ADAMTS family members. METHODS: We investigated ADAMTS1, 5, and 16 expression in human thoracic aortic aneurysms (TAA) (n = 22), thoracic aortic dissections (TAD) (n = 12), and thoracic aortas from age-matched control organ donors (n = 6) (a total number of 34 cases and 6 controls). The expression levels of ADAMTS proteins were determined by Western blot technique using anti-ADAMTS1, ADAMTS5, ADAMTS16, TIMP-1 and TIMP-2 antibodies. RESULTS: ADAMTS1, 5, and 16 protein expressions were significantly higher in thoracic aortic aneurysm and dissection tissues compared to control aortic tissues. Furthermore, TIMP-1 protein levels decreased in TAA and TAD tissues, TIMP-2 did not change. CONCLUSIONS: Under the light of our findings, increased expression of ADAMTS1, 5, and 16 proteins may promote deceleration in thoracic aortic aneurysm progression. This is the first study that demonstrates ADAMTS5 and ADAMTS16 proteolytic activity in aneurysm and dissection.
Assuntos
Proteínas ADAM/análise , Aneurisma da Aorta Torácica/metabolismo , Dissecção Aórtica/metabolismo , Proteínas ADAMTS , Proteína ADAMTS1 , Proteína ADAMTS5 , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibidor Tecidual de Metaloproteinase-1/análise , Inibidor Tecidual de Metaloproteinase-2/análiseRESUMO
A fluorescent resonance energy transfer substrate with improved sensitivity for ADAM17, -10, and -9 (where ADAM represents a disintegrin and metalloproteinase) has been designed. The new substrate, Dabcyl-Pro-Arg-Ala-Ala-Ala-Homophe-Thr-Ser-Pro-Lys(FAM)-NH2, has specificity constants of 6.3 (±0.3) × 10(4) M(-1) s(-1) and 2.4 (±0.3) × 10(3) M(-1) s(-1) for ADAM17 and ADAM10, respectively. The substrate is more sensitive than widely used peptides based on the precursor tumor necrosis factor-alpha (TNF-alpha) cleavage site, PEPDAB010 or Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(FAM)-NH2 and Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Arg-NH2. ADAM9 also processes the new peptide more than 18-fold better than the TNF-alpha-based substrates. The new substrate has a unique selectivity profile because it is processed less efficiently by ADAM8 and MMP1, -2, -3, -8, -9, -12, and -14. This substrate provides a unique tool in which to assess ADAM17, -10, and -9 activities.
Assuntos
Proteínas ADAM/análise , Proteínas ADAM/metabolismo , Membrana Celular/metabolismo , Corantes Fluorescentes/química , Proteínas ADAM/química , Linhagem Celular Tumoral , Transferência Ressonante de Energia de Fluorescência , Humanos , Hidrólise , Análise de Regressão , SolubilidadeRESUMO
OBJECTIVE: Preeclampsia (PE), can be classified according to the timing of disease onset: early-onset PE occurs before the 34th gestational week and late-onset PE occurs in the 34th gestational week or later. The aim of this study was to determine whether total antioxidant status (TAS), and total oxidant status (TOS), ADAMTS-12 and decorin levels differ among early-onset severe PE (EOS-PE), late-onset severe PE (LOS-PE) and uncomplicated pregnancies. METHODS: In this case-control study, placental samples obtained from 25 pregnant patients with EOS-PE, 26 pregnant patients with LOS-PE and 28 healthy patients with uncomplicated pregnancies (control group). RESULTS: Placenta levels of decorin and TOS were significantly higher and TAS was significantly lower in the EOS-PE and LOS-PE groups than in the control group. These alterations were more prominent in patients with EOS-PE than in patients with LOS-PE. There were no significant differences in the ADAMTS-12 levels of the groups. CONCLUSION: The distinctly higher rate of negative perinatal outcomes in both EOS-PE and LOS-PE patients is well evidenced. However, the main questions that need to be answered are whether the only difference between these two diseases is the time of their onset and whether the only difference between them with respect to fetal morbidity and mortality is prematurity.
Assuntos
Proteínas ADAM/análise , Decorina/análise , Placenta/química , Pré-Eclâmpsia/metabolismo , Proteínas ADAM/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Idade Gestacional , Humanos , Transtornos de Início Tardio , Oxirredução , Placenta/metabolismo , Gravidez , Adulto JovemRESUMO
The metalloprotease ADAM10 mediates the shedding of the ectodomain of various cell membrane proteins, including APP, the precursor of the amyloid peptide Aß, and Notch receptors following ligand binding. ADAM10 associates with the members of an evolutionary conserved subgroup of tetraspanins, referred to as TspanC8, which regulate its exit from the endoplasmic reticulum. Here we show that 4 of these TspanC8 (Tspan5, Tspan14, Tspan15 and Tspan33) which positively regulate ADAM10 surface expression levels differentially impact ADAM10-dependent Notch activation and the cleavage of several ADAM10 substrates, including APP, N-cadherin and CD44. Sucrose gradient fractionation, single molecule tracking and quantitative mass-spectrometry analysis of the repertoire of molecules co-immunoprecipitated with Tspan5, Tspan15 and ADAM10 show that these two tetraspanins differentially regulate ADAM10 membrane compartmentalization. These data represent a unique example where several tetraspanins differentially regulate the function of a common partner protein through a distinct membrane compartmentalization.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Receptor Notch1/metabolismo , Tetraspaninas/metabolismo , Proteínas ADAM/análise , Proteínas ADAM/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/análise , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Humanos , Receptores de Hialuronatos/metabolismo , Imunoprecipitação , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Microscopia Confocal , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor Notch1/genética , Especificidade por Substrato , Espectrometria de Massas em Tandem , Tetraspaninas/antagonistas & inibidores , Tetraspaninas/genéticaRESUMO
Pre-eclampsia (PE) complicates 2%-8% of all pregnancies and is an important cause of perinatal morbidity and mortality worldwide. In order to reduce these complications and to develop possible treatment modalities, it is important to identify women at risk of developing PE. The use of biomarkers in early pregnancy would allow appropriate stratification into high and low risk pregnancies for the purpose of defining surveillance in pregnancy and to administer interventions. We used formal methods for a systematic review and meta-analyses to assess the accuracy of all biomarkers that have been evaluated so far during the first and early second trimester of pregnancy to predict PE. We found low predictive values using individual biomarkers which included a disintegrin and metalloprotease 12 (ADAM-12), inhibin-A, pregnancy associated plasma protein A (PAPP-A), placental growth factor (PlGF) and placental protein 13 (PP-13). The pooled sensitivity of all single biomarkers was 0.40 (95% CI 0.39-0.41) at a false positive rate of 10%. The area under the Summary of Receiver Operating Characteristics Curve (SROC) was 0.786 (SE 0.02). When a combination model was used, the predictive value improved to an area under the SROC of 0.893 (SE 0.03). In conclusion, although there are multiple potential biomarkers for PE their efficacy has been inconsistent and comparisons are difficult because of heterogeneity between different studies. Therefore, there is an urgent need for high quality, large-scale multicentre research in biomarkers for PE so that the best predictive marker(s) can be identified in order to improve the management of women destined to develop PE.
Assuntos
Pré-Eclâmpsia/diagnóstico , Proteínas ADAM/análise , Proteína ADAM12 , Biomarcadores/análise , Feminino , Galectinas/análise , Humanos , Inibinas/análise , Proteínas de Membrana/análise , Fator de Crescimento Placentário , Gravidez , Proteínas da Gravidez/análise , Proteína Plasmática A Associada à Gravidez/análiseRESUMO
A wide variety of biological processes including differentiation, regeneration, and cancer progression are regulated by shedding of membrane-anchored proteins. One of the major sheddases is A Disintegrin And Metalloprotease-17 (ADAM17) whose extracellular region consists of a pro-, a catalytic, a disintegrin-, and a membrane-proximal domain (MPD) as well as a short juxtamembrane segment of 17 amino acid residues that has been named "Conserved ADAM-seventeeN Dynamic Interaction Sequence" (CANDIS). This segment is involved in substrate recognition. Key mediators of inflammation including interleukin-6 receptor (IL-6R) and tumor necrosis factor (TNF-α) are substrates of ADAM17. The shedding activity of ADAM17 is regulated by the conformation of the membrane-proximal domain preceding the CANDIS segment. Here, we show that CANDIS, besides being involved in substrate recognition, is able to interact with lipid bilayers in vitro and that this property could be involved in regulating ADAM17 shedding activity.
Assuntos
Proteínas ADAM/metabolismo , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas ADAM/análise , Proteínas ADAM/genética , Proteína ADAM17 , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Hep G2 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Dados de Sequência Molecular , Mutação , Domínios e Motivos de Interação entre Proteínas , Especificidade por SubstratoRESUMO
Synaptic degeneration and accumulation of the neurotoxic amyloid ß-peptide (Aß) in the brain are hallmarks of Alzheimer disease. Aß is produced by sequential cleavage of the amyloid precursor protein (APP), by the ß-secretase ß-site APP cleaving enzyme 1 (BACE1) and γ-secretase. However, Aß generation is precluded if APP is cleaved by the α-secretase ADAM10 instead of BACE1. We have previously shown that Aß can be produced locally at the synapse. To study the synaptic localization of the APP processing enzymes we used western blotting to demonstrate that, compared to total brain homogenate, ADAM10 and BACE1 were greatly enriched in synaptic vesicles isolated from rat brain using controlled-pore glass chromatography, whereas Presenilin1 was the only enriched component of the γ-secretase complex. Moreover, we detected ADAM10 activity in synaptic vesicles and enrichment of the intermediate APP-C-terminal fragments (APP-CTFs). We confirmed the western blotting findings using in situ proximity ligation assay to demonstrate close proximity of ADAM10 and BACE1 with the synaptic vesicle marker synaptophysin in intact mouse primary hippocampal neurons. In contrast, only sparse co-localization of active γ-secretase and synaptophysin was detected. These results indicate that the first step of APP processing occurs in synaptic vesicles whereas the final step is more likely to take place elsewhere.
Assuntos
Proteínas ADAM/análise , Secretases da Proteína Precursora do Amiloide/análise , Ácido Aspártico Endopeptidases/análise , Proteínas de Membrana/análise , Vesículas Sinápticas/química , Proteína ADAM10 , Animais , Células Cultivadas , Hipocampo/química , Hipocampo/citologia , Camundongos , Camundongos Endogâmicos C57BL , RatosRESUMO
BACKGROUND: This study aimed to evaluate the association of a disintegrin and metalloproteinase-33 protein ('ADAM-33') expression in vocal polyp formation and to determine its correlation with clinical characteristics. METHODS: Medical charts and histological sections of 32 patients diagnosed with vocal polyps who underwent surgery were analysed. Controls were histopathologically normal vocal fold tissues obtained from 36 patients who underwent surgery for laryngeal squamous cell carcinoma. Immunohistochemical staining was performed to detect ADAM-33 expression in epithelial cells, stroma and vessels. RESULTS: All epithelial, stromal and vascular staining scores were significantly greater in polyp tissue than in controls (p < 0.001). Stromal ADAM-33 staining scores were higher in vocal polyp patients with a symptom duration of less than six months (p < 0.05). Vocal overuse or the presence of reflux symptoms, sinonasal symptoms or allergy did not affect ADAM-33 immunostaining scores (p = 0.05). CONCLUSION: In this study, ADAM-33 immunostaining was significantly increased in vocal polyps. Therefore, over-expression of this protein may be associated with vocal polyp pathogenesis.
Assuntos
Proteínas ADAM/análise , Desintegrinas/análise , Doenças da Laringe/metabolismo , Pólipos/química , Prega Vocal , Adolescente , Adulto , Idoso , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
OBJECTIVE: To evaluate the potential of ADAMTS-4 (aggrecanase -1) activity in synovial fluid (SF) as a biomarker of knee injury and joint disease. DESIGN: We have measured ADAMTS-4 activity in the synovial fluid of 170 orthopaedic patients with different degrees of joint pathology, using a commercial ADAMTS-4 fluorescence resonance energy transfer (FRET) substrate assay. Patients were classified at arthroscopy as (i) macroscopically normal, (ii) with an injury of the meniscus, anterior cruciate ligament or chondral/osteochondral defects or (iii) with osteoarthritis, and the influence of independent factors (age, patient group, effusion and synovial inflammation) on ADAMTS-4 activity levels was assessed. RESULTS: In most patients (106/170) ADAMTS-4 activity was undetectable; ADAMTS-4 ranged from 0 to 2.8 ng/mL in synovial fluid from patients with an injury, 0-4.1 ng/mL in osteoarthritic patients and 4.0-12.3 ng/mL in patients with large effusions. Four independent variables each significantly influenced ADAMTS-4 activity in synovial fluid (all P < 0.001): age (concordance = 0.69), presence of osteoarthritis (OA) (concordance = 0.66), level of effusion (concordance = 0.78) and inflammation (concordance = 0.68). Not only did effusion influence the amount of ADAMTS-4 activity most strongly, but it also did this in an ordered manner (P < 0.001). CONCLUSIONS: The main finding of this study is that ADAMTS-4 levels in synovial fluid are most strongly correlated with inflammation and severity of effusion in the knee. Further study is required to determine if it could provide a useful tool to aid clinical diagnoses, indicate treatment, to monitor progression of joint degeneration or OA or alternatively the success of treatment.
Assuntos
Proteínas ADAM/análise , Artropatias/enzimologia , Traumatismos do Joelho/enzimologia , Osteoartrite do Joelho/enzimologia , Pró-Colágeno N-Endopeptidase/análise , Líquido Sinovial/química , Proteína ADAMTS4 , Adulto , Biomarcadores/análise , Transferência Ressonante de Energia de Fluorescência , Humanos , Pessoa de Meia-IdadeRESUMO
AIMS: Although many markers were associated with abdominal aortic aneurysm (AAA), there is no clear consensus on which marker is of the most value. Studies have implicated the role of Notch signaling in the pathogenesis of AAA. We investigate the value of plasma Jagged1, Notch receptors and tumor necrosis factor-α converting enzyme (TACE) in identifying AAA. MAIN METHODS: 42 patients with AAA and 36 controls were enrolled in our study. The concentrations of plasma Jagged1, Notch receptors and TACE were measured by enzyme-linked immunosorbent assay (ELISA). The diagnostic value of plasma Notch1 and TACE was assessed by logistic regression and receiver operator characteristic (ROC) curve. Double immunofluorescence staining was used to investigate the distribution of Notch1 and TACE in AAA tissue specimens. KEY FINDINGS: The concentrations of plasma Notch1 and TACE were significantly higher in AAA than in the controls, respectively (Notch1: P < 0.001; TACE: P = 0.0001). The area under the curve (AUC) from ROC curve of plasma Notch1 and TACE in determining the presence of AAA was 0.878 and 0.804, respectively. Combining detection of plasma Notch1 and TACE could improve the accuracy in detecting AAA (AUC 0.984, P < 0.0001). The predicted probability cutoff of 0.70 gave a sensitivity of 90.5% and a specificity of 100% for combining detection of plasma Notch1 and TACE in predicting AAA. SIGNIFICANCE: This is the first report revealing that plasma Notch1 and TACE are highly expressed in AAA. Combining detection of plasma Notch1 and TACE may be reliable for identifying the presence of AAA.
Assuntos
Proteínas ADAM/análise , Aneurisma da Aorta Abdominal/diagnóstico , Biomarcadores/análise , Receptor Notch1/análise , Proteínas ADAM/sangue , Proteína ADAM17 , Idoso , Idoso de 80 Anos ou mais , Aneurisma da Aorta Abdominal/sangue , Biomarcadores/sangue , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ligação ao Cálcio/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/análise , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Proteína Jagged-1 , Masculino , Proteínas de Membrana/análise , Proteínas de Membrana/sangue , Pessoa de Meia-Idade , Receptor Notch1/sangue , Receptores Notch/análise , Receptores Notch/sangue , Reprodutibilidade dos Testes , Proteínas Serrate-JaggedRESUMO
BACKGROUND: Genome-wide association studies have established ADAMTS7 as a locus for coronary artery disease in humans. However, these studies fail to provide directionality for the association between ADAMTS7 and coronary artery disease. Previous reports have implicated ADAMTS7 in the regulation of vascular smooth muscle cell migration, but a role for and the direction of impact of this gene in atherogenesis have not been shown in relevant model systems. METHODS AND RESULTS: We bred an Adamts7 whole-body knockout mouse onto both the Ldlr and Apoe knockout hyperlipidemic mouse models. Adamts7(-/-)/Ldlr(-/-) and Adamts7(-/-)/Apoe(-/-) mice displayed significant reductions in lesion formation in aortas and aortic roots compared with controls. Adamts7 knockout mice also showed reduced neointimal formation after femoral wire injury. Adamts7 expression was induced in response to injury and hyperlipidemia but was absent at later time points, and primary Adamts7 knockout vascular smooth muscle cells showed reduced migration in the setting of tumor necrosis factor-α stimulation. ADAMTS7 localized to cells positive for smooth muscle cell markers in human coronary artery disease lesions, and subcellular localization studies in cultured vascular smooth muscle cells placed ADAMTS7 at the cytoplasm and cell membrane, where it colocalized with markers of podosomes. CONCLUSIONS: These data represent the first in vivo experimental validation of the association of Adamts7 with atherogenesis, likely through modulation of vascular cell migration and matrix in atherosclerotic lesions. These results demonstrate that Adamts7 is proatherogenic, lending directionality to the original genetic association and supporting the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans, making it an attractive target for novel therapeutic interventions.
Assuntos
Proteínas ADAM/análise , Proteínas ADAM/fisiologia , Aterosclerose/prevenção & controle , Doença das Coronárias/enzimologia , Neointima/enzimologia , Remodelação Vascular/fisiologia , Proteínas ADAM/deficiência , Proteínas ADAM/genética , Proteína ADAMTS7 , Sequência de Aminoácidos , Animais , Aorta/enzimologia , Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/etiologia , Aterosclerose/genética , Aterosclerose/patologia , Divisão Celular , Movimento Celular , Células Cultivadas , Doença das Coronárias/patologia , Dieta Ocidental/efeitos adversos , Células Endoteliais/metabolismo , Feminino , Artéria Femoral/lesões , Artéria Femoral/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Hiperlipidemias/complicações , Hiperlipidemias/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/ultraestrutura , Neointima/patologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Receptores de LDL/deficiência , Receptores de LDL/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
ADAMTS13 is a metalloproteinase that cleaves von Willebrand factor (VWF) into smaller multimers in vivo. This cleavage creates both the typical multimeric size distribution and the characteristic triplet band distribution of VWF. Here we analysed ADAMTS13 content, VWF multimeric size distribution and VWF triplet structure in five commercial VWF/factor VIII (FVIII) concentrates. The relative distribution of ADAMTS13 activity values corresponded well to the ADAMTS13 antigen values for all examined concentrates except Haemate HS®, which had markedly higher ADAMTS13 antigen/activity ratio, with Fanhdi® and Haemate HS® displaying the most intense ADAMTS13 signal. Interestingly, ADAMTS13 levels did not correlate with the high molecular weight multimer content of the concentrates, but did correlate with VWF triplet distribution. Densitometric quantification showed that Wilate®, Immunate® and Willfact® displayed human plasma-like VWF triplet distribution, whereas Fanhdi® and Haemate HS® showed enhanced content of the faster migrating triplet band, which corresponded well to their higher ADAMTS13 content. In summary, Immunate®, Willfact® and Wilate® had lower levels of ADAMTS13 antigen and activity and exhibited a plasma-like VWF triplet structure. Fanhdi® and Haemate HS® had higher ADAMTS13 content and an altered triplet structure. The possible impact of these observations on function and clinical efficacy of VWF/FVIII concentrates is discussed.
Assuntos
Proteínas ADAM/análise , Fator VIII/química , Complexos Multiproteicos/química , Fator de von Willebrand/química , Proteína ADAMTS13 , HumanosRESUMO
BACKGROUND: ADAM28, ADAM33, IL-13, IL-4 and other cytokines (IL-6 and IL-10) seem to play important roles in the persistence and maintenance of acute inflammatory processes that ultimately lead to lung remodeling and pulmonary fibrosis, which may be responsible for the high morbidity and mortality rates associated with non-pandemic acute viral pneumonias in childhood. OBJECTIVES: The aim of this study was to evaluate the roles of ADAM33, ADAM28, IL4, IL6, IL10 and IL13 in the development of inflammation and alveolar fibrosis due to lethal acute respiratory infections of the lower airway in a pediatric population, especially in those with viral etiology. STUDY DESIGN: For this study, 193 cases were selected, and samples from the cases were processed for viral antigen detection by immunohistochemistry and then separated into two groups: virus-positive (n=68) and virus-negative (n=125). Immunohistochemistry was performed to assess the presence of metalloproteinases (ADAM33 and ADAM28) and inflammatory cytokines (IL-4, IL-13, IL-6, IL-10) in the alveolar septa. RESULTS: The virus-positive group showed stronger immunolabeling for ADAM33, ADAM28, IL-4 and IL-13 (p<0.0001 for all variables). The staining intensities for ADAM33 and ADAM28 were directly proportional to the intensities for IL-4 and IL-13 (p<0.0001). CONCLUSIONS: The results of this study suggest that these proteins play important roles in pulmonary inflammatory reactions elicited against etiological viral agents. In addition, these mediators may affect the process of lung remodeling and the development of pulmonary fibrosis.
Assuntos
Proteínas ADAM/análise , Lesão Pulmonar Aguda/patologia , Interleucina-13/análise , Interleucina-4/análise , Pneumonia Viral/patologia , Lesão Pulmonar Aguda/imunologia , Antígenos Virais/análise , Pré-Escolar , Feminino , Fibrose/patologia , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Pulmão/patologia , Masculino , Pneumonia Viral/imunologiaRESUMO
Tendons play a crucial role in musculoskeletal functioning because they physically connect bones and muscles making the movement of articular joints possible. The molecular composition of tendons mostly include collagen I fibrils, which aggregate together to form fibers to form a fascicle. A complex network composed of resident cells (i.e., tenocytes) and extracellular matrix macromolecules (glycosaminoglycans, proteoglycans, glycoproteins and other non collagenous proteins) interact and define the structure of tendons and their properties. Development, renewal and remodeling of tendons composition occur at all ages of living organisms so the homeostasis of proteolytic systems is a critical issue. A major role is played by Metalloproteinases, a family of Zn(2+)-dependent endopeptidases involved in the catabolism of several components of the extracellular matrix, such as collagens, proteoglycans, fibronectin and many others. Among these, two main classes are mostly involved in tendon pathophysiology, namely the Matrix Metalloproteinases (MMPs) and a Disintegrin-like and Metalloproteinase domain with Thrombospondin motifs (ADAMTSs). This study analyses the various aspects of the roles played by Metalloproteinases in the physiological and pathological processes of tendons.
Assuntos
Proteínas ADAM/metabolismo , Metaloproteinases da Matriz/metabolismo , Tendões/enzimologia , Tendões/fisiopatologia , Proteínas ADAM/análise , Animais , Humanos , Metaloproteinases da Matriz/análise , Modelos Moleculares , Conformação Proteica , Tendões/patologia , Tendões/ultraestruturaRESUMO
Mammalian fertilization is completed by direct interaction between sperm and egg. This process is primarily mediated by both adhesion and membrane-fusion proteins found on the gamete surface. ADAM1, 2, and 3 are members of the ADAMs protein family, and have been involved in sperm-egg binding. In this study, we demonstrate the proteolytic processing of ADAM15 during epididymal maturation of guinea pig spermatozoa to produce a mature form a size of 45âkDa. We find that the size of the mature ADAM15, 45âkDa, in cauda epididymal spermatozoa indicates that the pro-domain and metalloprotease domain are absent. In addition, using indirect immunofluorescence, ADAM15 was found throughout the acrosome, at the equatorial region and along the flagellum of guinea pig spermatozoa. After acrosome reaction, ADAM15 is lost from the acrosomal region and retained in the equatorial region and flagellum. In this study, we also report the first evidence of a complex between ADAM15 and acrogranin. By immunoprecipitation, we detected a protein band of 65âkDa which co-immunoprecipated together ADAM15. Analysis of the N-terminal sequence of this 65âkDa protein has revealed its identity as acrogranin. In addition, using cell-surface labeling, ADAM15 was found to be present on the cell surface. Assays of heterologous fertilization showed that the antibody against acrogranin inhibited the sperm-egg adhesion. Interestingly, ADAM15 and acrogranin were also found associated in two breast cancer cell lines. In conclusion, our results demonstrated that ADAM15 and acrogranin are present on and associated with the surface of guinea pig spermatozoa; besides both proteins may play a role during sperm-egg binding.
Assuntos
Proteínas ADAM/fisiologia , Fertilização/fisiologia , Cobaias/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Proteínas ADAM/análise , Animais , Neoplasias da Mama/química , Neoplasias da Mama/patologia , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/análise , Masculino , Fusão de Membrana/fisiologia , Proteínas de Membrana/análise , Capacitação Espermática/fisiologiaRESUMO
BACKGROUND: Asthma is a complex disease influenced by multiple genetic and environmental factors. While Madeira has the highest prevalence of asthma in Portugal (14.6%), the effect of both genetic and environmental factors in this population has never been assessed. We categorized 98 asthma patients according to the Global Initiative for Asthma (GINA) guidelines, established their sensitization profile, and measured their forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) indexes. Selected single nucleotide polymorphisms (SNPs) were analysed as potential markers for asthma susceptibility and severity in the interleukin 4 (IL4), interleukin 13 (IL13), beta-2-adrenergic receptor (ADRB2), a disintegrin and metalloprotease 33 (ADAM33), gasdermin-like (GSDML) and the signal transducer and activator of transcription 6 (STAT6) genes comparatively to a population reference set. RESULTS: Although mites are the major source of allergic sensitization, no significant difference was found amongst asthma severity categories. IL4-590*CT/TT and IL4-RP2*253183/183183 were found to predict the risk (2-fold) and severity (3 to 4-fold) of asthma and were associated with a lower FEV1 index. ADRB2-c.16*AG is a risk factor (3.5-fold), while genotype GSDML-236*TT was protective (4-fold) for moderate-severe asthma. ADAM33-V4*C was associated to asthma and mild asthma by the transmission disequilibrium test (TDT). Finally, ADAM33-V4*CC and STAT6-21*TT were associated with higher sensitization (mean wheal size ≥10 mm) to house dust (1.4-fold) and storage mite (7.8-fold). CONCLUSION: In Madeira, IL4-590C/T, IL4-RP2 253/183, GSDML-236C/T and ADAM33-V4C/G SNPs are important risk factors for asthma susceptibility and severity, with implications for asthma healthcare management.
Assuntos
Asma/genética , Polimorfismo Genético/genética , Proteínas ADAM/análise , Proteínas ADAM/genética , Adolescente , Biomarcadores , Estudos de Casos e Controles , Criança , Desintegrinas/análise , Desintegrinas/genética , Feminino , Volume Expiratório Forçado/genética , Genótipo , Humanos , Interleucina-13/análise , Interleucina-13/genética , Interleucina-4/análise , Interleucina-4/genética , Masculino , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Polimorfismo de Nucleotídeo Único/genética , Portugal , Receptores Adrenérgicos beta 2/análise , Receptores Adrenérgicos beta 2/genética , Fatores de Risco , Fator de Transcrição STAT6/análise , Fator de Transcrição STAT6/genética , Índice de Gravidade de Doença , Estatísticas não Paramétricas , Capacidade Vital/genéticaRESUMO
Thrombotic thrombocytopenic purpura (TTP) is a rare condition, but important to consider in case of thrombocytopenia and haemolysis. It is imperative to proceed with the correct treatment, in order to ensure a satisfactory outcome. TTP is either acquired or idiopathic. This case report shows that a 14-year-old boy has acquired TTP due to an infection with Campylobacter jejuni and Samonella szentes. Plasma exchange plays an essential role in the treatment of TTP.