RESUMO
An acidic environment in bone is essential for bone metabolism and the production of decarboxylated osteocalcin, which functions as a regulatory hormone of glucose metabolism. Here, we describe the high-resolution X-ray crystal structure of decarboxylated osteocalcin under acidic conditions. Decarboxylated osteocalcin at pH 2.0 retains the α-helix structure of native osteocalcin with three γ-carboxyglutamic acid residues at neutral pH. This implies that decarboxylated osteocalcin is stable under an acidic environment in bone. In addition, site-directed mutagenesis revealed that Glu17 and Glu21 are important for the adiponectin-inducing activity of decarboxylated osteocalcin. These findings suggest that the receptor of decarboxylated osteocalcin responds to the negative charge in helix 1 of osteocalcin.
Assuntos
Adiponectina , Osso e Ossos , Osteocalcina/metabolismo , Osso e Ossos/metabolismo , Ácido 1-CarboxiglutâmicoRESUMO
Diabetes mellitus (DM) is a chronic metabolic disease, mainly characterized by increased blood glucose and insulin dysfunction. In response to the persistent systemic hyperglycemic state, numerous metabolic and physiological complications have already been well characterized. However, its relationship to bone fragility, cognitive deficits and increased risk of dementia still needs to be better understood. The impact of chronic hyperglycemia on bone physiology and architecture was assessed in a model of chronic hyperglycemia induced by a single intraperitoneal administration of streptozotocin (STZ; 55 mg/kg) in Wistar rats. In addition, the bone-to-brain communication was investigated by analyzing the gene expression and methylation status of genes that encode the main osteokines released by the bone [Fgf23 (fibroblast growth factor 23), Bglap (bone gamma-carboxyglutamate protein) and Lcn2 (lipocalin 2) and their receptors in both, the bone and the brain [Fgfr1 (fibroblast growth factor receptor 1), Gpr6A (G-protein coupled receptor family C group 6 member A), Gpr158 (G protein-coupled receptor 158) and Slc22a17 (Solute carrier family 22 member 17)]. It was observed that chronic hyperglycemia negatively impacted on bone biology and compromised the balance of the bone-brain endocrine axis. Ultrastructural disorganization was accompanied by global DNA hypomethylation and changes in gene expression of DNA-modifying enzymes that were accompanied by changes in the methylation status of the osteokine promoter region Bglap and Lcn2 (lipocalin 2) in the femur. Additionally, the chronic hyperglycemic state was accompanied by modulation of gene expression of the osteokines Fgf23 (fibroblast growth factor 23), Bglap (bone gamma-carboxyglutamate protein) and Lcn2 (lipocalin 2) in the different brain regions. However, transcriptional regulation mediated by DNA methylation was observed only for the osteokine receptors, Fgfr1(fibroblast growth factor receptor 1) in the striatum and Gpr158 (G protein-coupled receptor 158) in the hippocampus. This is a pioneer study demonstrating that the chronic hyperglycemic state compromises the crosstalk between bone tissue and the brain, mainly affecting the hippocampus, through transcriptional silencing of the Bglap receptor by hypermethylation of Gpr158 gene.
Assuntos
Fator de Crescimento de Fibroblastos 23 , Hiperglicemia , Receptores Acoplados a Proteínas G , Animais , Ratos , Ácido 1-Carboxiglutâmico/genética , Ácido 1-Carboxiglutâmico/metabolismo , Osso e Ossos/metabolismo , Encéfalo/metabolismo , Repressão Epigenética , Hipocampo/metabolismo , Homeostase , Hiperglicemia/metabolismo , Lipocalina-2/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Ratos Wistar , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Biocompatible hydrogels are promising approaches for bone repair and engineering. A novel therapeutic nanocomposite hydrogel was designed based on triblock copolymer poly e-caprolactone (PCL)-polyethylene glycol-PCL and natural gelatin (PCEC/GEL) and reinforced with halloysite nanotube (HNT). Gentamicin (GM) loaded HNT was immobilized in polymeric hydrogel matrix to fabricate scaffolds using the freeze-drying method. Scaffolds were characterized via Fourier transform infrared (FT-IR), x-ray powder diffraction, and scanning electron microscope (SEM) methods. The swelling ratio, density, porosity, degradation, and mechanical behavior were evaluated to investigate the effects of HNT on the physicochemical properties of the composite. Cell viability and cell attachment were investigated by microculture tetrazolium (MTT) assay and SEM. Cell proliferation was observed without any cytotoxicity effect on human dental pulp-derived mesenchymal stem cells (h-DPSCs). Alizarin red staining and real-time reverse transcription polymerase chain reaction (QRT-PCR) assay were carried out to monitor the osteoconductivity of scaffolds on h-DPSCs which were seeded drop wise onto the top of scaffolds. The quantification of the messenger RNA (mRNA) expression of osteogenic marker genes, bone morphogenetic protein 2, SPARK, bone gamma-carboxyglutamate protein and runt-related transcription factor 2 over a period of 21 d of cell seeding, demonstrated that cell-encapsulating PCEC/GEL/HNT-GM hydrogel scaffolds supported osteoblast differentiation of h-DPSCs into osteogenic cells through the up-regulation of related genes along with moderate effects on cell viability. Moreover, the antibiotics loading reduced bacterial growth while maintaining the osteogenic properties of the scaffold. Therefore, the bactericidal PCEC/GEL/HNT-GM hydrogel nanocomposite, with enhanced durability, maintenance the functionality of seeded cellsin vitrothat can be a remarkable dual-functional candidate for hard tissue reconstruction and customized bone implants fabrication via the direct incorporation of bactericidal drug to prevent infection.
Assuntos
Hidrogéis , Nanocompostos , Ácido 1-Carboxiglutâmico/farmacologia , Antibacterianos/farmacologia , Proteína Morfogenética Óssea 2/farmacologia , Regeneração Óssea , Diferenciação Celular , Proliferação de Células , Argila , Subunidade alfa 1 de Fator de Ligação ao Core , Gelatina , Gentamicinas , Humanos , Hidrogéis/química , Nanocompostos/química , Nanogéis , Polietilenoglicóis , RNA Mensageiro/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
Carboxylative enzymes are involved in many pathways and their regulation plays a crucial role in many of these pathways. In particular, γ-glutamylcarboxylase (GGCX) converts glutamate residues (Glu) into γ-carboxyglutamate (Gla) of the vitamin K-dependent proteins (VKDPs) activating them. VKDPs include at least 17 proteins involved in processes such as blood coagulation, blood vessels calcification, and bone mineralization. VKDPs are activated by the reduced form of vitamin K, naturally occurring as vitamin K1 (phylloquinone) and K2 (menaquinones, MKs). Among these, MK7 is the most efficient in terms of bioavailability and biological effect. Similarly to other trans isomers, it is produced by natural fermentation or chemically in both trans and cis. However, the efficacy of the biological effect of the different isomers and the impact on humans are unknown. Our study assessed carboxylative efficacy of trans and cis MK7 and compared it with other vitamin K isomers, evaluating both the expression of residues of carboxylated Gla-protein by western blot analysis and using a cell-free system to determine the GGCX activity by HPLC. Trans MK7H2 showed a higher ability to carboxylate the 70 KDa GLA-protein, previously inhibited in vitro by warfarin treatment. However, cis MK7 also induced a carboxylation activity albeit of a small extent. The data were confirmed chromatographically, in which a slight carboxylative activity of cis MK7H2 was demonstrated, comparable with both K1H2 and oxidized trans MK7 but less than trans MK7H2 . For the first time, a difference of biological activity between cis and trans configuration of menaquinone-7 has been reported.
Assuntos
Vitamina K 1 , Vitamina K , Ácido 1-Carboxiglutâmico , Humanos , Vitamina K/farmacologia , Vitamina K 1/metabolismo , Vitamina K 1/farmacologia , Vitamina K 2/metabolismo , Vitamina K 2/farmacologia , Varfarina/farmacologiaRESUMO
Periodontitis is a chronic inflammation caused by the deposition of dental plaque on the tooth surface. Human periodontal ligament stem cells (hPDLSCs) have the potential of osteogenic differentiation. Long non-coding RNAs (lncRNAs) are collectively involved in periodontitis. This study was designed to explore the roles of Linc01133 in osteogenic differentiation of hPDLSCs. hPDLSCs obtained from the periodontal ligament (PDL) of patients with periodontitis were used to collect Linc01133, microRNA-30c (miR-30c), and bone gamma-carboxyglutamate protein (BGLAP) expression data, and their expression changes were traced during osteogenic differentiation of hPDLSCs. Quantitative reverse-transcription polymerase chain reaction as well as western blotting were used to analyze the levels of RNAs and proteins. Dual-luciferase reporter and RNA pull-down assays demonstrated the relationship between Linc01133, miR-30c, and BGLAP. Furthermore, alkaline phosphatase (ALP) staining and alizarin red staining were applied to evaluate the degree of osteogenic differentiation. Linc01133 was downregulated in the PDL of patients with periodontitis. Upregulated Linc01133 promoted osteogenic differentiation of hPDLSCs. Linc01133 could inhibit miR-30c expression by sponging miR-30c. miR-30c suppressed osteogenic differentiation. Additionally, miR-30c targeted BGLAP. Knockdown of BGLAP abrogated the effects of decreased miR-30c on osteogenic differentiation of hPDLSCs. Linc01133 acted as a ceRNA to regulate osteogenic differentiation of hPDLSCs via the miR-30c/BGLAP axis. Therefore, Linc01133 may participate in the progress of periodontitis.
Assuntos
MicroRNAs , Periodontite , RNA Longo não Codificante , Ácido 1-Carboxiglutâmico/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Osteogênese/genética , Ligamento Periodontal , Periodontite/genética , Periodontite/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Células-TroncoRESUMO
BACKGROUND: Osteoarthritis (OA) is a common disease of older individuals that impacts detrimentally on the quality and the length of life. It is characterised by the painful loss of articular cartilage and is polygenic and multifactorial. Genome-wide association scans have highlighted over 90 osteoarthritis genetic signals, some of which reside within or close to highly plausible candidate genes. An example is an association to polymorphisms within and adjacent to the matrix Gla protein gene MGP. We set out to undertake a functional study of this gene. METHODS: Nucleic acid was extracted from cartilage, infrapatellar fat pad, synovium, trabecular bone, trapezium and peripheral whole blood from OA patients and also from mesenchymal stem cells (MSCs) subjected to chondrogenesis. Expression of MGP was measured by quantitative PCR (qPCR), RNA-sequencing and allelic expression imbalance (AEI) analysis. Matrix Gla protein was depleted in chondrocytes by knocking down MGP expression using RNA interference (RNAi) and the effect on a range of genes assessed by qPCR. RESULTS: MGP is expressed in joint tissues, blood and chondrocytes cultured from MSCs. There is a higher expression in diseased versus non-diseased cartilage. Polymorphisms that are associated with OA also correlate with the expression of MGP, with the OA risk-conferring allele showing significantly reduced expression in cartilage, fat pad and synovium but increased expression in blood. Depletion of Matrix Gla protein had a significant effect on the majority of genes tested, with an increased expression of catabolic genes that encode enzymes that degrade cartilage. CONCLUSIONS: MGP expression is subject to cis-acting regulators that correlate with the OA association signal. These are active in a range of joint tissues but have effects which are particularly strong in cartilage. An opposite effect is observed in blood, highlighting the context-specific nature of the regulation of this gene's expression. Recapitulation of the genetic deficit in cartilage chondrocytes is pro-catabolic.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Condrócitos/metabolismo , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla/métodos , Osteoartrite/genética , RNA/genética , Ácido 1-Carboxiglutâmico , Alelos , Proteínas de Ligação ao Cálcio/biossíntese , Condrócitos/patologia , Proteínas da Matriz Extracelular/biossíntese , Genótipo , Humanos , Osteoartrite/metabolismo , Osteoartrite/patologia , Proteína de Matriz GlaRESUMO
Secreted recombinant activated clotting factor VII activated (rFVIIa) in cell culture media missing gamma-carboxyglutamic acid (Gla) domain as a result of failure in gamma-carboxylation or cell lysis is called Gla-domainless impurity which has less negative charge compared to native rFVIIa. Based on risk assessment, this type of impurity is considered as critical drug product quality attribute of rFVIIa and its quantitative analysis in product batches is a critical issue in quality control laboratories. Analysis of Gla-domainless impurity is accomplished by Strong Anion Exchange Chromatography (SAX) in recombinant factor VIIa using Tris and Bis-Tris propane salt buffers as equilibrating buffers and high concentration ammonium acetate as an eluent. Appearance of ghost peaks with notable intensity during elution time of Gla-domainless impurity caused distortion of the related peak and interference with robust and accurate quantification of this impurity. Subsequently, the ghost peak was analyzed by LC-ESI-MS to determine the structure which showed the m/z values at 905.27, 623.53 and 341.60 and 563.73. To find the source of these ghost peaks, quality of water, buffer salts and Chelex-100 together with ionic strength of mobile phase A (addition of 25 mM NaCl) were considered as affecting parameters and several experiments designed with DOE software to optimize the best condition of highest quality the method with lowest signal of ghost peak noises. By interpretation of DOE result, it is concluded that high grade water and buffer salt along with high quality Chelex-100 resins are important factors to achieve a method with lowest ghost peaks. However, addition of 25 mM NaCl to mobile phase A with either lower quality buffer salts or lower water grade yields high quality chromatogram peak with acceptable ghost peaks. LC/MS analysis indicates that macrostructures of Bis-Tris propane made up as a result of hydrogen bonds with each other or Tris molecules can be the source of ghost peaks.
Assuntos
Ácido 1-Carboxiglutâmico/análise , Cromatografia por Troca Iônica/normas , Contaminação de Medicamentos , Fator VIIa/normas , Espectrometria de Massas por Ionização por Electrospray/normas , Trometamina/análogos & derivados , Soluções Tampão , Química Farmacêutica , Proteínas Recombinantes/normas , Trometamina/químicaRESUMO
BACKGROUND: Basic and clinical researches have suggested that type 2 diabetes (T2DM) is associated with cognitive impairment, and diabetes mellitus increases the risk of cognitive impairment and dementia. Recently, some reports found that undercarboxylated osteocalcin (ucOC) could affect brain functions, and decreased in patients with T2DM. We aimed to investigate the association of serum ucOC with cognitive impairment in T2DM patients. METHODS: A total of 196 male T2DM patients without medications known to affect bone metabolism or history of bone fracture, aged ≥18years were recruited and divided into impaired cognition group and normal cognition group. We use the scores of Minimum Mental State Examination (MMSE) to evaluate the subjects' cognitive function. Detailed cognitive performance was also evaluated by the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). Serum ucOC was measured by Enzyme-Linked Immunosorbent Assay (ELISA) kit. RESULTS: Compared to male T2DM patients with normal cognition, the mean osteocalcin concentrations were significantly lower in male T2DM patients with impaired cognition (P<0.05). RBANS total and all indexes scores were also lower in patients with impaired cognition (all P<0.05). After adjusted effects of confounding factors, serum ucOC was positively correlated with a variety indexes of RBANS except visuospatial/constructional. CONCLUSIONS: The serum ucOC is positively correlated with RBANS scores in male T2DM patients. It suggests that serum ucOC may be involved in the development and progression of cognitive dysfunction in T2DM patients.
Assuntos
Ácido 1-Carboxiglutâmico/metabolismo , Disfunção Cognitiva/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Osteocalcina/metabolismo , Adulto , Idoso , Biomarcadores/sangue , Glicemia/análise , Glicemia/metabolismo , Estudos de Casos e Controles , Disfunção Cognitiva/epidemiologia , Disfunção Cognitiva/etiologia , Complicações do Diabetes/sangue , Complicações do Diabetes/epidemiologia , Complicações do Diabetes/psicologia , Diabetes Mellitus Tipo 2/psicologia , Hemoglobinas Glicadas/análise , Hemoglobinas Glicadas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Osteocalcina/sangue , Fatores de RiscoRESUMO
Essentials Membrane-binding GLA domains of coagulation factors are essential for proper clot formation. Factor X (FX) is specific to phosphatidylserine (PS) lipids through unknown atomic-level interactions. Molecular dynamics simulations were used to develop the first membrane-bound model of FX-GLA. PS binding modes of FX-GLA were described, and potential PS-specific binding sites identified. SUMMARY: Background Factor X (FX) binds to cell membranes in a highly phospholipid-dependent manner and, in complex with tissue factor and factor VIIa (FVIIa), initiates the clotting cascade. Experimental information concerning the membrane-bound structure of FX with atomic resolution has remained elusive because of the fluid nature of cellular membranes. FX is known to bind preferentially to phosphatidylserine (PS). Objectives To develop the first membrane-bound model of the FX-GLA domain to PS at atomic level, and to identify PS-specific binding sites of the FX-GLA domain. Methods Molecular dynamics (MD) simulations were performed to develop an atomic-level model for the FX-GLA domain bound to PS bilayers. We utilized a membrane representation with enhanced lipid mobility, termed the highly mobile membrane mimetic (HMMM), permitting spontaneous membrane binding and insertion by FX-GLA in multiple 100-ns simulations. In 14 independent simulations, FX-GLA bound spontaneously to the membrane. The resulting membrane-bound models were converted from HMMM to conventional membrane and simulated for an additional 100 ns. Results The final membrane-bound FX-GLA model allowed for detailed characterization of the orientation, insertion depth and lipid interactions of the domain, providing insight into the molecular basis of its PS specificity. All binding simulations converged to the same configuration despite differing initial orientations. Conclusions Analysis of interactions between residues in FX-GLA and lipid-charged groups allowed for potential PS-specific binding sites to be identified. This new structural and dynamic information provides an additional step towards a full understanding of the role of atomic-level lipid-protein interactions in regulating the critical and complex clotting cascade.
Assuntos
Membrana Celular/metabolismo , Fator X/metabolismo , Fosfatidilserinas/metabolismo , Ácido 1-Carboxiglutâmico/metabolismo , Animais , Sítios de Ligação , Bovinos , Fator X/química , Cinética , Simulação de Acoplamento Molecular , Fosfatidilserinas/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-AtividadeRESUMO
Conantokins (con) are short γ-carboxyglutamate (Gla)-containing polypeptides expressed by marine snails that function as antagonists of N-methyl-d-aspartate receptor (NMDAR) ion channels. The Gla residues govern structural conformations and antagonistic activities of the conantokins. In addition to Gla, some conantokins, e.g., conRl-B, also contain a hydroxyproline (HyP or O) residue, which in this case is centrally located in the peptide at position 10. Because conRl-B specifically inhibits ion channels of GluN2B subunit-containing heterotetrameric NMDARs, we evaluated the unusual role of HyP10 in this effect. To accomplish this goal, we examined synthetic variants of conRl-B in which HyP10 was either deleted (conRl-B[ΔO10]) or replaced with alanine (conRl-B[O10A]) or proline (conRl-B[O10P]). The solution structures of these variants were determined by nuclear magnetic resonance spectroscopy. Deletion of HyP10, or replacement of HyP10 with Ala10, attenuated the distortion in the central region of the apo-conRl-B helix and allowed Mg2+-complexed end-to-end α-helix formation. The inhibitory properties of these variants were assessed by measuring NMDA/Gly-stimulated intracellular Ca2+ influx in mice neurons. ConRl-B[O10P] retained its NMDAR ion channel inhibitory activity in wild-type (WT) neurons but lost its GluN2B specificity, whereas conRl-B[ΔO10] showed overall diminished inhibitory function. ConRl-B[O10A] showed attenuated inhibitory function but retained its GluN2B specificity. Thus, HyP10 plays a critical role in maintaining the structural integrity of conRl-B, which can be correlated with its GluN2B subunit-selective inhibition. Weakened inhibition by conRl-B was also observed in neurons lacking either the GluN2C or GluN2D subunit, compared to WT neurons. This suggests that GluN2C and GluN2D are also required for inhibition by conRl-B.
Assuntos
Ácido 1-Carboxiglutâmico/química , Hidroxiprolina/química , Peptídeos/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Ácido 1-Carboxiglutâmico/genética , Ácido 1-Carboxiglutâmico/metabolismo , Alanina/química , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Células Cultivadas , Caramujo Conus/química , Hidroxiprolina/genética , Hidroxiprolina/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos Knockout , Modelos Moleculares , Mutação , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Peptídeos/química , Peptídeos/genética , Prolina/química , Prolina/genética , Prolina/metabolismo , Multimerização Proteica , Estrutura Secundária de Proteína , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , SoluçõesRESUMO
In this issue of Blood, Tie et al report the development of a cleverly engineered, cell-based system for studying mutations in γ-glutamyl carboxylase (GGCX), the enzyme responsible for converting glutamate residues in certain proteins to γ-carboxyglutamate (Gla). They use this cell-based assay system to help explain the clinical manifestations of some otherwise puzzling GGCX gene mutations in humans that cause phenotypes ranging from severe bleeding to Keutel syndrome.
Assuntos
Ácido 1-Carboxiglutâmico , Fenótipo , Ácido Glutâmico , Hemorragia , Humanos , Mutação , Vitamina KRESUMO
Fish collagen has recently been reported to be a novel biomaterial for cell and tissue culture as an alternative to conventional mammalian collagens such as bovine and porcine collagens. Fish collagen could overcome the risk of zoonosis, such as from bovine spongiform encephalopathy. Among fish collagens, tilapia collagen, the denaturing temperature of which is near 37°C, is appropriate for cell and tissue culture. In this study, we investigated chondrogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on tilapia scale collagen fibrils compared with porcine collagen and non-coated dishes. The collagen fibrils were observed using a scanning electronic microscope. Safranin O staining, glycosaminoglycans (GAG) expression, and real-time PCR were examined to evaluate chondrogenesis of hMSCs on each type of collagen fibril. The results showed that hMSCs cultured on tilapia scale collagen showed stronger Safranin O staining and higher GAG expression at day 6. Results of real-time PCR indicated that hMSCs cultured on tilapia collagen showed earlier SOX9 expression on day 4 and higher AGGRECAN and COLLAGEN II expression on day 6 compared with on porcine collagen and non-coated dishes. Furthermore, low mRNA levels of bone gamma-carboxyglutamate, a specific marker of osteogenesis, showed that tilapia collagen fibrils specifically enhanced chondrogenic differentiation of hMSCs in chondrogenic medium, as well as porcine collagen. Accordingly, tilapia scale collagen may provide an appropriate collagen source for hMSC chondrogenesis in vitro.
Assuntos
Diferenciação Celular , Condrogênese , Colágeno/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Tilápia , Ácido 1-Carboxiglutâmico/genética , Agrecanas/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Condrogênese/genética , Colágeno/ultraestrutura , Colágeno Tipo II/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Osteogênese/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOX9/metabolismo , Suínos , Tilápia/anatomia & histologiaRESUMO
Osteogenesis imperfecta (OI) is a group of genetic disorders characterized by bone fragility and deformity. OI type VI is unique owing to the mineralization defects observed in patient biopsies. Furthermore, it has been reported to respond less well to standard therapy with bisphosphonates [1]. Others and we have previously identified SERPINF1 mutations in patients with OI type VI. SERPINF1 encodes pigment epithelium derived factor (PEDF), a secreted collagen-binding glycoprotein that is absent in the sera of patients with OI type VI. Serpinf1 null mice show increased osteoid and decreased bone mass, and thus recapitulate the OI type VI phenotype. We tested whether restoration of circulating PEDF in the blood could correct the phenotype of OI type VI in the context of protein replacement. To do so, we utilized a helper-dependent adenoviral vector (HDAd) to express human SERPINF1 in the mouse liver and assessed whether PEDF secreted from the liver was able to rescue the bone phenotype observed in Serpinf1(-/-) mice. We confirmed that expression of SERPINF1 in the liver restored the serum level of PEDF. We also demonstrated that PEDF secreted from the liver was biologically active by showing the expected metabolic effects of increased adiposity and impaired glucose tolerance in Serpinf1(-/-) mice. Interestingly, overexpression of PEDF in vitro increased mineralization with a concomitant increase in the expression of bone gamma-carboxyglutamate protein, alkaline phosphatase and collagen, type I, alpha I, but the increased serum PEDF level did not improve the bone phenotype of Serpinf1(-/-) mice. These results suggest that PEDF may function in a context-dependent and paracrine fashion in bone homeostasis.
Assuntos
Osso e Ossos/fisiologia , Proteínas do Olho/sangue , Proteínas do Olho/genética , Fígado/metabolismo , Fatores de Crescimento Neural/sangue , Fatores de Crescimento Neural/genética , Osteogênese Imperfeita/fisiopatologia , Osteogênese Imperfeita/terapia , Serpinas/sangue , Serpinas/genética , Ácido 1-Carboxiglutâmico/genética , Adenoviridae/genética , Fosfatase Alcalina/genética , Animais , Densidade Óssea , Colágeno Tipo I/genética , Técnicas de Transferência de Genes , Intolerância à Glucose , Células HEK293 , Homeostase , Humanos , Camundongos , Camundongos Knockout , Mutação , Fatores de Crescimento Neural/deficiência , Fenótipo , Serpinas/deficiênciaRESUMO
New protein nanocages are designed bearing two functional proteins, γ-carboxyglutamic acid of protein C (PC-Gla) and thrombin receptor agonist peptide (TRAP), and have an anti-septic response. These nanoparticles reduce sepsis-induced organ injury and septic mortality in vivo. Noting that there are currently no medications for severe sepsis, these results show that novel nanoparticles can be used to treat sepsis.
Assuntos
Anti-Inflamatórios/administração & dosagem , Nanoestruturas , Fragmentos de Peptídeos/administração & dosagem , Proteína C/administração & dosagem , Receptores de Trombina/agonistas , Sepse/tratamento farmacológico , Ácido 1-Carboxiglutâmico/química , Animais , Anti-Inflamatórios/química , Colagenases/metabolismo , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Ferritinas/química , Sangue Fetal , Células-Tronco Embrionárias Humanas , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/fisiologia , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Nanoestruturas/química , Proteína C/química , Receptor PAR-1/metabolismo , Sepse/metabolismo , Sepse/mortalidadeAssuntos
Osteocalcina/sangue , Ácido 1-Carboxiglutâmico/metabolismo , Animais , Humanos , CamundongosRESUMO
AIM: Des-γ-carboxyprothrombin (DCP) has been used as a tumor marker for hepatocellular carcinoma (HCC). Recently the DCP/NX-DCP ratio, calculated by dividing DCP by NX-DCP, has been reported useful in detecting HCC. The purpose of this study is to clarify the significance of DCP and NX-DCP expression in HCC tissues. METHODS: HCC and non-HCC tissue samples were obtained from 157 patients and were immunohistochemically examined for DCP and NX-DCP expression using anti-DCP antibody and anti-NX-DCP antibody. DCP and NX-DCP expression scores were calculated by multiplying staining intensity grade by percentage of stained area. Serum DCP and NX-DCP levels were determined in 89 patients. We evaluated the relationship between tumor expression, serum level, and pathomorphological findings. RESULTS: Intrahepatic metastasis (im) was significantly more frequent in cases with high DCP expression than in cases with low DCP expression. High NX-DCP expression was associated with significantly lower histological grade, and less frequent im or portal vein invasion (vp) than low NX-DCP expression. Serum DCP was correlated with DCP expression, but serum NX-DCP was not correlated with NX-DCP expression. DCP-positive (≥40 mAU/L), NX-DCP-positive (≥90 mAU/L), and DCP/NX-DCP ratio-positive (≥1.5) cases were associated with significantly larger tumor size and more frequent vp than negative cases. DCP was rarely expressed, but NX-DCP was frequently expressed in non-cancerous liver tissues. Patients with NX-DCP expression-negative tumors showed a lower survival rate than those with NX-DCP expression-positive tumors (p = 0.04), whereas the survival in serum NX-DCP-positive cases was lower than that of serum negative cases (p = 0.02). CONCLUSIONS: DCP and NX-DCP were produced in HCC tissues, but differed in expression level and biological properties. DCP expression, serum DCP or NX-DCP level, and DCP/NX-DCP ratio were closely related to malignant properties of HCC.
Assuntos
Ácido 1-Carboxiglutâmico , Biomarcadores/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Precursores de Proteínas/metabolismo , Protrombina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Biomarcadores/química , Carcinoma Hepatocelular/sangue , Feminino , Humanos , Fígado/metabolismo , Neoplasias Hepáticas/sangue , Masculino , Pessoa de Meia-Idade , Precursores de Proteínas/sangue , Precursores de Proteínas/química , Protrombina/química , Estudos RetrospectivosRESUMO
This study uses high-pressure size exclusion chromatography (HPSEC) to quantify divalent metal ion (X(2+))-induced compaction found in vitamin K-dependent (VKD) proteins. Multiple X(2+) binding sites formed by the presence of up to 12 γ-carboxyglutamic acid (Gla) residues are present in plasma-derived FIX (pd-FIX) and recombinant FIX (r-FIX). Analytical ultracentrifugation (AUC) was used to calibrate the Stokes radius (R) measured by HPSEC. A compaction of pd-FIX caused by the filling of Ca(2+) and Mg(2+) binding sites resulted in a 5 to 6% decrease in radius of hydration as observed by HPSEC. The filling of Ca(2+) sites resulted in greater compaction than for Mg(2+) alone where this effect was additive or greater when both ions were present at physiological levels. Less X(2+)-induced compaction was observed in r-FIX with lower Gla content populations, which enabled the separation of biologically active r-FIX species from inactive ones by HPSEC. HPSEC was sensitive to R changes of approximately 0.01nm that enabled the detection of FIX compaction that was likely cooperative in nature between lower avidity X(2+) sites of the Gla domain and higher avidity X(2+) sites of the epidermal growth factor 1 (EGF1)-like domain.
Assuntos
Ácido 1-Carboxiglutâmico/química , Cromatografia em Gel/métodos , Fator IX/química , Fator IX/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Humanos , Magnésio/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Vitamina K/metabolismoRESUMO
The pH low insertion peptide (pHLIP) offers the potential to deliver drugs selectively to the cytoplasm of cancer cells based on tumor acidosis. The WT pHLIP inserts into membranes with a pH50 of 6.1, while most solid tumors have extracellular pH (pH(e)) of 6.5-7.0. To close this gap, a SAR study was carried out to search for pHLIP variants with improved pH response. Replacing Asp25 with α-aminoadipic acid (Aad) adjusts the pH50 to 6.74, matching average tumor acidity, and replacing Asp14 with γ-carboxyglutamic acid (Gla) increases the sharpness of pH response (transition over 0.5 instead of 1â pH unit). These effects are additive: the Asp14Gla/Asp25Aad double variant shows a pH50 of 6.79, with sharper transition than Asp25Aad. Furthermore, the advantage of the double variant over WT pHLIP in terms of cargo delivery was demonstrated in turn-on fluorescence assays and anti-proliferation studies (using paclitaxel as cargo) in A549 lung cancer cells at pHâ 6.6.
Assuntos
Aminoácidos/química , Proteínas de Membrana/metabolismo , Ácido 1-Carboxiglutâmico/química , Sequência de Aminoácidos , Técnicas Biossensoriais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Portadores de Fármacos/química , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana/química , Dados de Sequência Molecular , Paclitaxel/química , Paclitaxel/toxicidade , Espectrometria de Fluorescência , Relação Estrutura-AtividadeRESUMO
Several drugs have been associated with an increased risk of osteoporosis when used chronically. Coumarins (warfarin, acenocoumarol, phenprocoumon, and fluindione) are oral anticoagulants widely used for the prevention and treatment of arterial and venous thromboembolic diseases. These drugs are vitamin K antagonists that interfere with γ-carboxyglutamate formation, and consequently inhibit the carboxylation of glutamate residues of proteins that are synthesized in the bone. These effects on bone turnover and dietary restrictions in patients on anticoagulation are possible mechanisms inducing osteoporosis in coumarin users. However, conflicting evidence is available concerning the risk of osteoporosis and bone fractures in patients on treatment with these drugs. This risk is likely to be clinically relevant in long-term (more than 1 year) coumarin users. Novel direct oral anticoagulants, recently introduced in clinical practice, exert reduced interference on bone metabolism; however, limited in vitro and animal data are currently available, and their long-term effects will only become apparent in time.