RESUMO
Escherichia coli 6-carboxytetrahydropterin synthase (eCTPS), a homologue of 6-pyruvoyltetrahydropterin synthase (PTPS), possesses a much stronger catalytic activity to cleave the side chain of sepiapterin in vitro compared with genuine PTPS activity and catalyzes the conversion of dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterin in vivo. Crystal structures of wild-type apo eCTPS and of a Cys27Ala mutant eCTPS complexed with sepiapterin have been determined to 2.3 and 2.5 Å resolution, respectively. The structures are highly conserved at the active site and the Zn(2+) binding site. However, comparison of the eCTPS structures with those of mammalian PTPS homologues revealed that two specific residues, Trp51 and Phe55, that are not found in mammalian PTPS keep the substrate bound by stacking it with their side chains. Replacement of these two residues by site-directed mutagenesis to the residues Met and Leu, which are only found in mammalian PTPS, converted eCTPS to the mammalian PTPS activity. These studies confirm that these two aromatic residues in eCTPS play an essential role in stabilizing the substrate and in the specific enzyme activity that differs from the original PTPS activity. These aromatic residues Trp51 and Phe55 are a key signature of bacterial PTPS enzymes that distinguish them from mammalian PTPS homologues.
Assuntos
Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Mamíferos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenilalanina/química , Fósforo-Oxigênio Liases/genética , Filogenia , Conformação Proteica , Pterinas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Due to some severe side effects or lack of efficacy of currently used synthetic drugs, such as bisphosphonates (BPs), the search for new therapeutic agents that can more effectively prevent and treat osteoporosis (OP) has been an increasingly important topic of research. In this study, the low-molecular weight hyaluronan (LMW-HA, 50 kDa) produced by enzymatic degradation of high-molecular weight hyaluronan (HMW-HA, 1922 kDa) from Streptococcus zooepidemicus was evaluated in vitro for its anti-osteoclastogenic potentials using RAW 264.7 murine macrophage cells. LMW-HA (25-200 µg/ml) dose-dependently inhibited the receptor activator of NF-κB ligand (RANKL)-induced tartrate-resistance acid phosphatase (TRAP) activity and the formation of multinucleated osteoclasts. Western blot analysis showed that LMW-HA reduced the RANKL-induced expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), gelsolin and c-Src-proline-rich tyrosine kinase 2 suggesting that it could inhibit actin ring formation of osteoclast cells. In addition, LMW-HA inhibited the bone resorption activity of osteoclastic cells by dose-dependently attenuating the RANKL-induced expression of carbonic anhydrase II and integrin ß3. RT-PCR analysis showed that LMW-HA dose-dependently decreased the expression of osteoclast-specific genes, such as matrix metalloproteinase 9 (MMP-9) and cathepsin K, suggesting that it has potential to inhibit the differentiation of osteoclastic cells. Taken collectively, these results suggested that LMW-HA (50 kDa) has significant anti-osteoporotic activity in vitro and may be used as a potent functional ingredient in health beneficial foods or as a therapeutic agent to prevent or treat OP.
Assuntos
Ácido Hialurônico/farmacologia , Osteoporose/tratamento farmacológico , Fosfatase Ácida/metabolismo , Animais , Catepsina K/biossíntese , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Ácido Hialurônico/uso terapêutico , Isoenzimas/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/biossíntese , Camundongos , Peso Molecular , Osteoclastos/citologia , Osteoclastos/metabolismo , Ligante RANK/antagonistas & inibidores , Fator 6 Associado a Receptor de TNF/biossíntese , Fosfatase Ácida Resistente a TartaratoRESUMO
Tetrahydrobiopterin (BH4) is an essential cofactor in NO synthesis by endothelial nitric oxide synthase (eNOS) enzymes. It has been previously suggested that reduced intrahepatic BH4 results in a decrease in intrahepatic NO and contributes to increased hepatic vascular resistance and portal pressure in animal models of cirrhosis. The main aim of the present study was to evaluate the relationship between BH4 and portal hypertension (PHT). One hundred ninety-three consecutive patients with chronic liver disease were included in the study. Liver biopsy, measurement of BH4 and hepatic venous pressure gradient (HVPG) were performed. Hepatic fibrosis was classified using the Laennec fibrosis scoring system. BH4 levels were determined in homogenized liver tissues of patients using a high performance liquid chromatography (HPLC) system. Statistical analysis was performed to evaluate the relationship between BH4 and HVPG, grade of hepatic fibrosis, clinical stage of cirrhosis, Child-Pugh class. A positive relationship between HVPG and hepatic fibrosis grade, clinical stage of cirrhosis and Child-Pugh class was observed. However, the BH4 level showed no significant correlation with HVPG or clinical features of cirrhosis. BH4 concentration in liver tissue has little relation to the severity of portal hypertension in patients with chronic liver disease.
Assuntos
Biopterinas/análogos & derivados , Cromatografia Líquida de Alta Pressão , Hipertensão Portal/diagnóstico , Hepatopatias/diagnóstico , Adulto , Idoso , Biopterinas/análise , Doença Crônica , Técnicas de Imagem por Elasticidade , Feminino , Veias Hepáticas/fisiologia , Humanos , Hipertensão Portal/complicações , Hipertensão Portal/metabolismo , Fígado/patologia , Cirrose Hepática/diagnóstico por imagem , Hepatopatias/complicações , Hepatopatias/metabolismo , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Pressão na Veia Porta , Análise de Regressão , Índice de Gravidade de DoençaRESUMO
We analyzed the effects of inactivating the pteridine glycosyltransferase gene (pgtA) on the photomovement of the cyanobacterium Synechocystis sp. PCC 6803 under different light conditions. The pgtA mutant displayed abnormal photomovement under UV-A/blue light. In particular, the pgtA mutant showed a negative phototactic response under UV-A (315-400 nm), whereas the wild-type did not show any photomovement. Inhibition of pterin biosynthesis by N-acetylserotonin (NAS), an inhibitor of sepiapterin reductase, also inhibited a positive phototactic response of the wild-type under white and blue light. In addition, negative phototaxis of the pgtA mutant was observed under UV-A/blue light in the presence of NAS. These results indicated that the product of the PgtA enzyme, cyanopterin, is involved in the inhibition of the negative phototaxis of the wild-type by sensing the UV-A. However, 2,4-diamino-6-hydroxypyrimidine-mediated inhibition of GTP cyclohydrolase I, the rate-limiting enzyme for pterin biosynthesis, significantly increased the positive phototaxis toward UV-A in the wild-type and the pgtA mutant. Furthermore, we measured the action spectrum of phototaxis in vivo for the wild-type and pgtA mutant. Maximal activity of the wild-type was at 300, 380 and 440 nm, indicating absorption by pterins and flavin. In particular, the UV-A/ blue peak at 380 and 440 nm obtained from the action spectrum of phototaxis was found to be closely correlated with the in vitro absorption spectrum previously reported for the cyanobacterial cryptochrome DASH. By investigating the photomovement of the wild-type and pgtA mutant to UV and blue light, we suggest that pterin can function as the chromophore of putative UV/blue photoreceptor(s) in cyanobacterial phototaxis.
Assuntos
Proteínas de Bactérias/metabolismo , Dissacarídeos/biossíntese , Glicosiltransferases/metabolismo , Transdução de Sinal Luminoso , Synechocystis/efeitos da radiação , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Glicosiltransferases/genética , Mutação , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Pterinas , Synechocystis/genética , Synechocystis/fisiologia , Raios UltravioletaRESUMO
Here, we report cloning of cyanobacterial genes encoding pteridine glycosyltransferases that catalyze glucosyl or xylosyl transfer from UDP-sugars to tetrahydrobiopterin. The genes were cloned by PCR amplification from genomic DNA which was isolated from culture and environmental samples and overexpressed in Escherichia coli for an in vitro activity assay.
Assuntos
Biopterinas/análogos & derivados , Cianobactérias/enzimologia , Microbiologia Ambiental , Glucose/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Xilose/metabolismo , Sequência de Aminoácidos , Biopterinas/metabolismo , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Análise por Conglomerados , Cianobactérias/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Expressão Gênica , Glicosiltransferases/isolamento & purificação , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The quantitative determination of tetrahydrobiopterin (BH4) and its oxidized forms (dihydrobiopterin and biopterin) is important in searching for possible markers of neuropsychiatric and cardiovascular disorders as well as in diagnosing BH4 deficiencies. Currently, two high-performance liquid chromatography (HPLC) methods are available, although both have some limitations. We developed an enzymatic method to distinguish BH4 from the oxidized forms by employing BH4:UDP-glucose alpha-glucosyltransferase (BGluT), which catalyzes glucosyl transfer from UDP-glucose to BH4. The recombinant BGluT isolated from Escherichia coli converted essentially all of the BH4 in a mixture containing oxidized biopterins to the glucoside while leaving the oxidized forms intact. Therefore, acidic iodine oxidation of the reaction mixture followed by single fluorescence HPLC permitted the determination of biopterin and biopterin-glucoside, which represent oxidized biopterins and BH4, respectively. The validity of the method was evaluated using authentic biopterins and animal samples such as human urine, rat plasma, and rat liver. The BGluT-catalyzed reaction not only would reduce the burden of chromatographic separation but also would promise non-HPLC analysis of BH4.
Assuntos
Biopterinas/análogos & derivados , Glicosiltransferases/metabolismo , Uridina Difosfato Glucose/metabolismo , Animais , Biopterinas/sangue , Biopterinas/metabolismo , Biopterinas/urina , Glicosiltransferases/genética , Humanos , Oxirredução , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Dictyostelium discoideum phenylalanine hydroxylase (DicPAH; residues 1-415) was expressed in Escherichia coli and purified for structural analysis. Apo DicPAH and DicPAH complexed with dihydrobiopterin (BH(2)) and Fe(III) were crystallized using 0.06 M PIPES pH 7.0, 26%(w/v) PEG 2000 by the hanging-drop vapour-diffusion method. Crystals of apo DicPAH and the DicPAH-BH(2)-Fe(III) complex diffracted to 2.6 and 2.07 A resolution, respectively, and belonged to space group P2(1), with unit-cell parameters a = 70.02, b = 85.43, c = 74.86 A, beta = 110.12 degrees and a = 70.97, b = 85.33, c = 74.89 A, beta = 110.23 degrees , respectively. There were two molecules in the asymmetric unit. The structure of DicPAH has been solved by molecular replacement.
Assuntos
Biopterinas/análogos & derivados , Dictyostelium/enzimologia , Compostos Férricos/química , Fenilalanina Hidroxilase/química , Biopterinas/química , Biopterinas/metabolismo , Cristalização , Cristalografia por Raios X , Compostos Férricos/metabolismo , Fenilalanina Hidroxilase/isolamento & purificação , Fenilalanina Hidroxilase/metabolismo , Ligação ProteicaRESUMO
Diabetic cardiomyopathy (DCM) is a major cause of mortality/morbidity in diabetes mellitus patients. Although tetrahydrobiopterin (BH4) shows therapeutic potential as an endogenous cardiovascular target, its effect on myocardial cells and mitochondria in DCM and the underlying mechanisms remain unknown. Here, we determined the involvement of BH4 deficiency in DCM and the therapeutic potential of BH4 supplementation in a rodent DCM model. We observed a decreased BH4:total biopterin ratio in heart and mitochondria accompanied by cardiac remodeling, lower cardiac contractility, and mitochondrial dysfunction. Prolonged BH4 supplementation improved cardiac function, corrected morphological abnormalities in cardiac muscle, and increased mitochondrial activity. Proteomics analysis revealed oxidative phosphorylation (OXPHOS) as the BH4-targeted biological pathway in diabetic hearts as well as BH4-mediated rescue of down-regulated peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) signaling as a key modulator of OXPHOS and mitochondrial biogenesis. Mechanistically, BH4 bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and activated downstream AMP-activated protein kinase/cAMP response element binding protein/PGC-1α signaling to rescue mitochondrial and cardiac dysfunction in DCM. These results suggest BH4 as a novel endogenous activator of CaMKK2.
Assuntos
Biopterinas/análogos & derivados , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Cardiomiopatias Diabéticas/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/genética , Animais , Biopterinas/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Diabetes Mellitus/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/fisiopatologia , Coração/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Contração Miocárdica , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Biogênese de Organelas , Fosforilação Oxidativa , Ratos , Ratos Long-Evans , Transdução de Sinais/fisiologiaRESUMO
To identify the new targets for hypertension, we analyzed the protein expression profiles of aortic smooth muscle in spontaneously hypertensive rats (SHR) of various ages during the development of hypertension, as well as in age-matched normotensive Wistar-Kyoto (WKY) rats, using a proteomic analysis. The expressions of seven proteins were altered in SHR compared with WKY rats. Of these proteins, NADH dehydrogenase 1alpha, GSTomega1, peroxi-redoxin I and transgelin were upregulated in SHR compared with WKY rats. On the other hand, the expression of HSP27 and Ran protein decreased in SHR. The diminution of dihydrobiopterin reductase, an enzyme located in the regeneration pathways of tetrahydrobiopterin (BH4), was also prominent in SHR. The results from a PCR analysis revealed that the expression of BH4 biosynthesis enzymes - GTP cyclohydrolase-1 and sepiapterin reductase - decreased and increased, respectively, in SHR compared with WKY rats. The level of BH4 was less in aortic strips from SHR than from WKY rats. Moreover, treatment with BH4 inhibited aortic smooth muscle contraction induced by serotonin. These results suggest that the deficiency in BH4 regeneration produced by diminished dihydrobiopterin reductase expression is involved in vascular disorders in hypertensive rats.
Assuntos
Aorta/enzimologia , Di-Hidropteridina Redutase/metabolismo , Hipertensão/enzimologia , Músculo Liso/enzimologia , Sequência de Aminoácidos , Animais , Biomarcadores/metabolismo , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Pressão Sanguínea , Di-Hidropteridina Redutase/química , Di-Hidropteridina Redutase/genética , Regulação Enzimológica da Expressão Gênica , Hipertensão/fisiopatologia , Masculino , Dados de Sequência Molecular , Proteômica , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Tetrahydrobiopterin (BH4) shows therapeutic potential as an endogenous target in cardiovascular diseases. Although it is involved in cardiovascular metabolism and mitochondrial biology, its mechanisms of action are unclear. We investigated how BH4 regulates cardiovascular metabolism using an unbiased multiple proteomics approach with a sepiapterin reductase knock-out (Spr-/-) mouse as a model of BH4 deficiency. Spr-/- mice exhibited a shortened life span, cardiac contractile dysfunction, and morphological changes. Multiple proteomics and systems-based data-integrative analyses showed that BH4 deficiency altered cardiac mitochondrial oxidative phosphorylation. Along with decreased transcription of major mitochondrial biogenesis regulatory genes, including Ppargc1a, Ppara, Esrra, and Tfam, Spr-/- mice exhibited lower mitochondrial mass and severe oxidative phosphorylation defects. Exogenous BH4 supplementation, but not nitric oxide supplementation or inhibition, rescued these cardiac and mitochondrial defects. BH4 supplementation also recovered mRNA and protein levels of PGC1α and its target proteins involved in mitochondrial biogenesis (mtTFA and ERRα), antioxidation (Prx3 and SOD2), and fatty acid utilization (CD36 and CPTI-M) in Spr-/- hearts. These results indicate that BH4-activated transcription of PGC1α regulates cardiac energy metabolism independently of nitric oxide and suggests that BH4 has therapeutic potential for cardiovascular diseases involving mitochondrial dysfunction.
Assuntos
Biopterinas/análogos & derivados , Fármacos Cardiovasculares/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Animais , Biopterinas/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/metabolismo , Biogênese de Organelas , Transdução de Sinais/efeitos dos fármacosRESUMO
6-Pyruvoyltetrahydropterin synthase from E. coli (ePTPS) has been crystallized using the hanging-drop vapour-diffusion method. Hexagonal- and rectangular-shaped crystals were obtained. Diffraction data were collected from the hexagonal and rectangular crystals to 3.0 and 2.3 A resolution, respectively. The hexagonal plate-shaped crystals belonged to space group P321, with unit-cell parameters a = b = 112.59, c = 68.82 A , and contained two molecules in the asymmetric unit. The rectangular crystals belonged to space group I222, with unit-cell parameters a = 112.76, b = 117.66, c = 153.57 A , and contained six molecules in the asymmetric unit. The structure of ePTPS in both crystal forms has been determined by molecular replacement.
Assuntos
Escherichia coli/enzimologia , Fósforo-Oxigênio Liases/química , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Conformação ProteicaRESUMO
Dihydropteridine reductase from Dictyostelium discoideum (dicDHPR) can produce D-threo-BH(4) [6R-(1'R,2'R)-5,6,7,8-tetrahydrobiopterin], a stereoisomer of L-erythro-BH(4), in the last step of tetrahydrobiopterin (BH(4)) recycling. In this reaction, DHPR uses NADH as a cofactor to reduce quinonoid dihydrobiopterin back to BH(4). To date, the enzyme has been purified to homogeneity from many sources. In this report, the dicDHPR-NAD complex has been crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as a precipitant. Rectangular-shaped crystals were obtained. Crystals grew to maximum dimensions of 0.4 x 0.6 x 0.1 mm. The crystal belonged to space group P2(1), with unit-cell parameters a = 49.81, b = 129.90, c = 78.76 A, beta = 100.00 degrees , and contained four molecules in the asymmetric unit, forming two closely interacting dicDHPR-NAD dimers. Diffraction data were collected to 2.16 A resolution using synchrotron radiation. The crystal structure has been determined using the molecular-replacement method.
Assuntos
Dictyostelium/enzimologia , Di-Hidropteridina Redutase/química , Proteínas de Protozoários/química , Animais , Cristalização/métodos , Cristalografia por Raios X , Di-Hidropteridina Redutase/isolamento & purificação , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas de Protozoários/isolamento & purificaçãoRESUMO
Sepiapterin reductase from Chlorobium tepidum (cSR) catalyzes the synthesis of a distinct tetrahydrobiopterin (BH4), L-threo-BH4, different from the mammalian enzyme product. The 3-D crystal structure of cSR has revealed that the product configuration is determined solely by the substrate binding mode within the well-conserved catalytic triads. In cSR, the sepiapterin is stacked between two aromatic side chains of Phe-99 and Trp-196 and rotated approximately 180 degrees C around the active site from the position in mouse sepiapterin reductase. To confirm their roles in substrate binding, we mutated Phe-99 and/or Trp-196 to alanine (F99A, W196A) by site-directed mutagenesis and comparatively examined substrate binding of the purified proteins by kinetics analysis and differential scanning calorimetry. These mutants had higher Km values than the wild type. Remarkably, the W196A mutation resulted in a higher Km increase compared with the F99A mutation. Consistent with the results, the melting temperature (Tm) in the presence of sepiapterin was lower in the mutant proteins and the worst was W196A. These findings indicate that the two residues are indispensable for substrate binding in cSR, and Trp-196 is more important than Phe-99 for different stereoisomer production.
Assuntos
Oxirredutases do Álcool/química , Biopterinas/análogos & derivados , Chlorobium/enzimologia , Modelos Químicos , Modelos Moleculares , Fenilalanina/química , Triptofano/química , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Biopterinas/síntese química , Simulação por Computador , Ativação Enzimática , Estabilidade Enzimática , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
Although ginseng marc is a by-product obtained during manufacturing of various commercial ginseng products and has been routinely discarded as a waste, it still contains considerable amounts of potential bioactive compounds, including saponins and polysaccharides. Previously, we reported that ginseng oligosaccharides derived from ginseng marc polysaccharides by enzymatic hydrolysis exert immunostimulatory activities in macrophages and these activated macrophages are in turn able to inhibit the growth of skin melanoma cells by inducing apoptosis. In the present study, a more detailed investigation of the immunostimulatory activity and underlying action mechanisms of an enzymatic hydrolysate (GEH) containing these oligosaccharides derived from ginseng marc polysaccharides was performed. The levels of proinflammatory cytokines and anti-inflammatory cytokines were measured in GEH-stimulated RAW264.7 macrophages using RT-PCR analysis and ELISA. The expression levels of Toll-like receptor 2 (TLR2) and TLR4, Dectin-1, and MerTK were measured by RT-PCR analysis or western blot analysis, and the phagocytic activities of GEH-challenged bone marrow-derived macrophages toward apoptotic Jurkat cells were assayed using fluorescence microscopy. GEH induced the production of both proinflammatory cytokines TNF-α and IL-6, and anti-inflammatory cytokine IL-10 in RAW 264.7 cells. The expression of the TLR2 and MerTK mRNAs was increased upon GEH treatment. Phagocytosis of apoptotic Jurkat cells was enhanced in GEH-treated macrophages. Based on the results, this enzymatic hydrolysate (GEH) containing oligosaccharides exerts immunostimulatory effects by maintaining the balance between M1 and M2 cytokines, facilitating macrophage activation and contributing to the efficient phagocytosis of apoptotic cells. Therefore, the GEH could be developed as value-added, health-beneficial food materials with immunostimulatory effects.
Assuntos
Fatores Imunológicos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Oligossacarídeos/metabolismo , Panax/química , Proteínas Tirosina Quinases/metabolismo , Receptor 2 Toll-Like/metabolismo , Animais , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Fatores Imunológicos/isolamento & purificação , Camundongos , Oligossacarídeos/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Successful bone tissue engineering using scaffolds is primarily dependent on the properties of the scaffold, including biocompatibility, highly interconnected porosity, and mechanical integrity. METHODS: In this study, we propose new composite scaffolds consisting of mesoporous magnesium silicate (m_MS), polycaprolactone (PCL), and wheat protein (WP) manufactured by a rapid prototyping technique to provide a micro/macro porous structure. Experimental groups were set based on the component ratio: (1) WP0% (m_MS:PCL:WP =30:70:0 weight per weight; w/w); (2) WP15% (m_MS:PCL:WP =30:55:15 w/w); (3) WP30% (m_MS:PCL:WP =30:40:30 w/w). RESULTS: Evaluation of the properties of fabricated scaffolds indicated that increasing the amount of WP improved the surface hydrophilicity and biodegradability of m_MS/PCL/WP composites, while reducing the mechanical strength. Moreover, experiments were performed to confirm the biocompatibility and osteogenic differentiation of human mesenchymal stem cells (MSCs) according to the component ratio of the scaffold. The results confirmed that the content of WP affects proliferation and osteogenic differentiation of MSCs. Based on the last day of the experiment, ie, the 14th day, the proliferation based on the amount of DNA was the best in the WP30% group, but all of the markers measured by PCR were the most expressed in the WP15% group. CONCLUSION: These results suggest that the m_MS/PCL/WP composite is a promising candidate for use as a scaffold in cell-based bone regeneration.
Assuntos
Materiais Biocompatíveis/farmacologia , Silicatos de Magnésio/farmacologia , Osteogênese , Proteínas de Plantas/farmacologia , Poliésteres/farmacologia , Alicerces Teciduais/química , Triticum/química , Absorção Fisico-Química , Fosfatase Alcalina/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Força Compressiva , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Silicatos de Magnésio/química , Camundongos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , PorosidadeRESUMO
In Dictyostelium discoideum Ax2 l-erythro-tetrahydrobiopterin (BH4) is produced in much smaller amount than its stereoisomer d-threo-tetrahydrobiopterin (DH4), both of which are catalyzed by sepiapterin reductase (SR) at the terminal steps. In order to investigate their putative function and biosynthetic regulation, we performed quantitative analysis of not only the intracellular pteridines by HPLC but also the biosynthetic enzymes (GTP cyclohydrolase I, 6-pyruvoyltetrahydropterin synthase, SR, and aldose reductase-like enzyme) by Northern blot analysis and activity assay. We found that both SR transcript and activity increased in parallel with a remarkable decline in aldose reductase-like enzyme activity when BH4 increased transiently in the early development. Through in vitro assay of BH4/DH4 synthesis and in vivo rescue experiment of SR knockout mutant, we demonstrated that Dictyostelium SR favors DH4 synthesis while human SR does BH4 synthesis. The results suggest that Dictyostelium SR prefers 1'-oxo-2'-d-hydroxypropyl-tetrahydropterin to 6-pyruvoyltetrahydropterin as a substrate, thereby maintaining dominant production of DH4 over BH4 in sufficient supply of AR-like enzyme, while allowing increase of BH4 when SR prevails quantitatively over aldose reductase-like enzyme. On the other hand, a transient increase of BH4 may imply that BH4 has an independent function from DH4 in Dictyostelium.
Assuntos
Oxirredutases do Álcool/metabolismo , Dictyostelium/classificação , Dictyostelium/metabolismo , Pteridinas/metabolismo , Animais , Catálise , Cromatografia Líquida de Alta Pressão , Dictyostelium/enzimologia , Dictyostelium/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Pteridinas/química , Estereoisomerismo , Especificidade por Substrato , Fatores de TempoRESUMO
A putative cellular function of tetrahydropteridines (l-erythro-tetrahydrobiopterin and d-threo-tetrahydrobiopterin) was investigated in Dictyostelium discoideum Ax2 using a mutant disrupted in the gene encoding sepiapterin reductase (SR). The SR mutant, which produces about 3% of tetrahydropteridines if compared to wild-type, was elucidated to have several functional defects related to mitochondria and oxidative stress: retarded growth, poor spore viability, impaired mitochondrial function, and increased susceptibility to oxidative stress induced by hydroxylamine or cumene-hydroperoxide. However, the physiological defects were almost completely rescued by extrachromosomal expression of Dictyostelium SR. The results strongly suggested that tetrahydropteridines in Dictyostelium are associated with mitochondrial function, probably via direct protection against oxidative stress.
Assuntos
Dictyostelium/efeitos dos fármacos , Dictyostelium/metabolismo , Mitocôndrias/metabolismo , Pteridinas/farmacologia , Animais , Dictyostelium/crescimento & desenvolvimento , Estresse Oxidativo , Esporos de Protozoários/efeitos dos fármacos , Esporos de Protozoários/crescimento & desenvolvimentoRESUMO
The pteridine glycosyltransferase (PGT) found in Chlorobium tepidum (CtPGT) catalyzes the conversion of L-threo-tetrahydrobiopterin to 1-O-(L-threo-biopterin-2'-yl)-ß-N-acetylglucosamine using UDP-N-acetylglucosamine. The gene for CtPGT was cloned, and selenomethionine-derivatized protein was overexpressed and purified using various chromatographic techniques. The protein was crystallized by the hanging-drop vapour-diffusion method using 0.24â M triammonium citrate pH 7.0, 14%(w/v) PEG 3350 as a reservoir solution. Multiple-wavelength anomalous diffraction data were collected to 2.15â Å resolution from a single CtPGT crystal. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 189.61, b = 79.98, c = 105.92â Å, ß = 120.5°.
Assuntos
Chlorobium/enzimologia , Glicosiltransferases/química , Pteridinas/metabolismo , Cristalização , Cristalografia por Raios X , Glicosiltransferases/metabolismo , Conformação ProteicaRESUMO
We investigated the use of Polycaprolactone (PCL)/ ß-tricalcium phosphate (ß-TCP) composites with applied mechanical stimulation as scaffold for bone tissue engineering. PCL-based three-dimensional (3D) structures were fabricated in a solvent-free process using a 3D-printing technique. The mass fraction of ß-TCP was varied in the range 0-30%, and the structure and compressive modulus of the specimens was characterized. The shape and interconnectivity of the pores was found to be satisfactory, and the compressive modulus of the specimens was comparable with that of human trabecular bone. Human mesenchymal stem cells were seeded on the composites, and various biological evaluations were performed over 9 days. With a mass fraction of ß-TCP of 30%, differentiation began earlier; however, the cell proliferation rate was lower. Through the use of mechanical stimulation, however, the proliferation rate recovered, and was comparable with that of the other groups. This stimulation effect was also observed in ECM generation and other biological assays. With mechanical stimulation, expression of osteogenic markers was lower on samples with a ß-TCP content of 10 wt% than without ß-TCP; however, with mechanical stimulation, the sample with a ß-TCP content of 30 wt% exhibited significantly greater expression of those markers than the other samples. We found that mechanical stimulation and the addition of ß-TCP interacted closely, and that a mass fraction of ß-TCP of 30% was particularly useful as a bone tissue scaffold when accompanied by mechanical stimulation.
RESUMO
6-Pyruvoyltetrahydropterin synthase (PTPS) catalyzes the second step of tetrahydrobiopterin (BH4) synthesis. We previously identified PTPS orthologs (bPTPS-Is) in bacteria which do not produce BH4. In this study we disrupted the gene encoding bPTPS-I in Synechococcus sp. PCC 7942, which produces BH4-glucoside. The mutant was normal in BH4-glucoside production, demonstrating that bPTPS-I does not participate in BH4 synthesis in vivo and bringing us a new PTPS ortholog (bPTPS-II) of a bimodular polypeptide. The recombinant Synechococcus bPTPS-II was assayed in vitro to show PTPS activity higher than human enzyme. Further computational analysis revealed the presence of mono and bimodular bPTPS-II orthologs mostly in green sulfur bacteria and cyanobacteria, respectively, which are well known for BH4-glycoside production. In summary we found new bacterial PTPS orthologs, having either a single or dual domain structure and being responsible for BH4 synthesis in vivo, thereby disclosing all the bacterial PTPS homologs.