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A cyclin-dependent kinase (CDK) inhibitor, p57Kip2, is an important molecule involved in bone development; p57Kip2-deficient (p57-/-) mice display neonatal lethality resulting from abnormal bone formation and cleft palate. The modulator 1α,25-dihydroxyvitamin D3 (l,25-(OH)2VD3) has shown the potential to suppress the proliferation and induce the differentiation of normal and tumor cells. The current study assessed the role of p57Kip2 in the 1,25-(OH)2VD3-regulated differentiation of osteoblasts because p57Kip2 is associated with the vitamin D receptor (VDR). Additionally, 1,25-(OH)2VD3 treatment increased p57KIP2 expression and induced the colocalization of p57KIP2 with VDR in the osteoblast nucleus. Primary p57-/- osteoblasts exhibited higher proliferation rates with Cdk activation than p57+/+ cells. A lower level of nodule mineralization was observed in p57-/- osteoblasts than in p57+/+ cells. In p57+/+ osteoblasts, 1,25-(OH)2VD3 upregulated the p57Kip2 and opn mRNA expression levels, while the opn expression levels were significantly decreased in p57-/- cells. The osteoclastogenesis assay performed using bone marrow cocultured with 1,25-(OH)2VD3-treated osteoblasts revealed a decreased efficiency of 1,25-(OH)2VD3-stimulated osteoclastogenesis in p57-/- cells. Based on these results, p57Kip2 might function as a mediator of 1,25-(OH)2VD3 signaling, thereby enabling sufficient VDR activation for osteoblast maturation.
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Receptores de Calcitriol , Vitamina D , Animales , Ratones , Diferenciación Celular , Núcleo Celular/metabolismo , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Osteoblastos/metabolismo , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Vitamina D/metabolismoRESUMEN
Matrix components of growth plate cartilage and mandibular condylar cartilage were immunohistochemically analyzed in cartilage calcification insufficient (CCI) rats, a model for dwarf rats. Reduction in total tibial length, elongation of growth plate, and appearance of noncartilaginous regions in the growth plate were observed in CCI rats. Immunoreactivity for type I collagen and hyaluronic acid (HA) staining were observed in the noncartilaginous region. However, weak immunoreactivity was observed for aggrecan, collagen types II and X, and decorin in this region. Transmission electron microscopy indicated that the noncartilaginous region showed a loose network of thin collagen fibrils, indicating that HA is predominantly involved in capturing space of the noncartilaginous region in the growth plate. Meanwhile, the mandibular condylar cartilage in CCI rats also showed elongation of the cartilaginous region and had a noncartilaginous region, predominantly comprising thick collagen fibrils. The structural difference between the two types of cartilages in CCI rats may be due to the presence of the fibrous cell zone and the fibrocartilaginous nature of the normal condylar cartilage. Additionally, the reduction in mandibular length was relatively less than the reduction in tibial length. The outline of the condylar process showed only slight abnormality. These results suggest that the condylar cartilage compensated its growth by supplying the characteristic noncartilaginous region effectively and may adapt to severe structural changes observed in CCI rats.
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Calcificación Fisiológica , Cartílago/metabolismo , Cartílago/fisiología , Matriz Extracelular/química , Matriz Extracelular/ultraestructura , Placa de Crecimiento/fisiología , Inmunohistoquímica/métodos , Cóndilo Mandibular/metabolismo , Animales , Ratas EndogámicasRESUMEN
OBJECTIVE: The helioxanthin derivative 4-(4-methoxyphenyl)thieno[2,3-b:5,4-c']dipyridine-2-carboxamide (TH) is a low-molecular-weight compound that was identified through screening for osteogenic compounds that enhance the activity of mouse preosteoblastic MC3T3-E1 cells. In the present study, we found that TH suppressed osteoclast differentiation. METHODS: Using the hematopoietic stem cells of ddY mice, TH was added to the culture in the experimental group, and the number of osteoclasts was measured with rhodamine phalloidin staining and TRAP staining. In osteo assay, bone resorption area was compared by the von Kossa staining. RESULTS: Specifically, TH inhibited the cyclic guanosine monophosphate (cGMP)-degrading activity of phosphodiesterase (PDE), promoted nitric oxide (NO) production, and dose-dependently suppressed osteoclast differentiation in an osteoclast formation culture of mouse bone marrow cells. The NO-competitive guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) attenuated the suppressive activity of TH on osteoclast differentiation. Conclusion: Given the previously reported suppressive action of cGMP on osteoclastogenesis, our data suggest that TH negatively impacts osteoclast differentiation at least to some extent by stimulating NO production and inhibiting PDE activity, both of which lead to the upregulation of intracellular cGMP. This study supports the potential use of TH as a novel antiosteoporotic reagent that not only stimulates bone formation but also inhibits bone resorption.
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Objective We evaluated the safety and efficacy of vonoprazan-based amoxicillin and clarithromycin 7-day triple therapy (VAC) in comparison to proton pump inhibitor (PPI)-based (PAC) as a first-line treatment and vonoprazan-based amoxicillin and metronidazole 7-day triple therapy (VAM) in comparison to PPI-based (PAM) as a second-line treatment for the eradication of Helicobacter pylori in Japan. Methods We performed a non-randomized, multi-center, parallel-group study to compare first-line VAC to PAC and second-line VAM to PAM. A pre-planned subgroup analysis on CAM resistance was also performed. Safety was evaluated with an adverse effects questionnaire (AEQ), which was completed by patients during therapy. Results The first-line eradication rates (ER) in the intention-to-treat (ITT) and per protocol (PP) analyses were 84.9% (95% CI: 81.9-87.6%, n=623) and 86.4% (83.5-89.1%, n=612), respectively, for VAC and 78.8% (75.3-82.0%, n=608) and 79.4% (76.0-82.6%, n=603), respectively, for PAC. The ER of VAC was higher than that of PAC in the ITT (p=0.0061) and PP analyses (p=0.0013). The ERs for VAC in patients with CAM-resistant and CAM-susceptible bacteria were 73.2% (59.7-84.2%, n=56) and 88.9% (83.4-93.1%, n=180), respectively. PAC was associated with higher AEQ scores for diarrhea, nausea, headache, and general malaise. In the second-line ITT and PP analyses VAM achieved ERs of 80.5% (74.6-85.6%, n=216) and 82.4% (76.6-87.3%, n=211), respectively, while PAM achieved ERs of 81.5% (74.2-87.4%, n=146) and 82.1% (74.8-87.9%, n=145), respectively. No significant differences were observed in the ITT (p=0.89) or PP (p=1.0) analyses. Conclusion The ER of first-line VAC was higher than that of PAC, but still <90%. No difference was observed between second-line VAM and PAM. Vonoprazan-based triple therapy was safe and well tolerated.
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Amoxicilina/uso terapéutico , Antibacterianos/uso terapéutico , Claritromicina/uso terapéutico , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori/efectos de los fármacos , Inhibidores de la Bomba de Protones/uso terapéutico , Pirroles/uso terapéutico , Sulfonamidas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Quimioterapia Combinada , Femenino , Humanos , Japón , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Adulto JovenRESUMEN
Currently, anti-TNFα antibodies are used to treat Crohn's disease. We report on a 45-year-old Japanese female with Crohn's disease developing SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis) syndrome following exposure to the anti-TNFα antibody adalimumab. Initially, adalimumab induced remission, but the patient showed SAPHO syndrome 11 weeks following the start of adalimumab therapy for the first time. Cutaneous and articular involvement were exacerbating the condition, so adalimumab was discontinued and the patient was put on low-dose methotrexate to control her symptoms. To our knowledge, this is the first report of SAPHO syndrome occurring during anti-TNF therapy, which is thought to be a paradoxical response to adalimumab.
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Nicorandil is a hybrid angina therapeutic agent that has nitric oxide (NO) action and the ability to open ATP-sensitive K+ channels (KATP channels). A transient increase in NO and intracellular Ca2+ has been demonstrated to be highly involved in the differentiation and activation of osteoclasts. The objective of this study was to verify that the pharmacological effect of nicorandil suppresses the differentiation process of osteoclasts in vitro. Although little authentic NO production was detected in the culture medium in osteoclast formation assays, NO production increased only in the presence of nicorandil. The number of osteoclasts decreased markedly at late time-points after nicorandil addition compared with the number at early time-points. Both the number of TRAP-positive multinucleated cells and the number of cells that obtained F-actin rings decreased in the presence of nicorandil in a concentration-dependent manner. The osteo assay showed that the bone resorption area was also reduced with nicorandil in a concentration-dependent manner. An inhibition recovery experiment was conducted by adding a soluble guanylyl cyclase (sGC) inhibitor (ODQ) and a KATP channel-opening inhibitor (glibenclamide) during the osteoclast formation process. In the inhibition recovery experiment, the inhibitory effect of nicorandil on osteoclastogenesis was blocked by the addition of ODQ and glibenclamide. These results suggest that both the NO and KATP channel-opening activity of nicorandil inhibit osteoclast differentiation. Further study of nicorandil may lead to the development of drugs for osteoporosis treatment.
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Diferenciación Celular/efectos de los fármacos , Nicorandil/farmacología , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Humanos , Canales KATP/antagonistas & inhibidores , Ratones , Óxido Nítrico/biosíntesis , Osteoclastos/metabolismo , Oxadiazoles/farmacología , Quinoxalinas/farmacología , Guanilil Ciclasa Soluble/antagonistas & inhibidoresRESUMEN
Three putative deuterolysin (EC 3.4.24.29) genes (deuA, deuB, and deuC) were found in the Aspergillus oryzae genome database ( http://www.bio.nite.go.jp/dogan/project/view/AO ). One of these genes, deuA, was corresponding to NpII gene, previously reported. DeuA and DeuB were overexpressed by recombinant A. oryzae and were purified. The degradation profiles against protein substrates of both enzymes were similar, but DeuB showed wider substrate specificity against peptidyl MCA-substrates compared with DeuA. Enzymatic profiles of DeuB except for thermostability also resembled those of DeuA. DeuB was inactivated by heat treatment above 80° C, different from thermostable DeuA. Transcription analysis in wild type A. oryzae showed only deuB was expressed in liquid culture, and the addition of the proteinous substrate upregulated the transcription. Furthermore, the NaNO3 addition seems to eliminate the effect of proteinous substrate for the transcription of deuB.
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Aspergillus oryzae/genética , Proteínas Fúngicas/genética , Metaloendopeptidasas/genética , Aspergillus oryzae/enzimología , Estabilidad de Enzimas/genética , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Regulación Fúngica de la Expresión Génica , Nitratos/química , Especificidad por Sustrato , TemperaturaRESUMEN
Three extracellular dipeptidyl peptidase genes, dppB, dppE, and dppF, were unveiled by sequence analysis of the Aspergillus oryzae genome. We investigated their differential enzymatic profiles, in order to gain an understanding of the diversity of these genes. The three dipeptidyl peptidases were expressed using Aspergillus nidulans as the host. Each recombinant enzyme was purified and subsequently characterized. The enzymes displayed similar optimum pH values, but optimum temperatures, pH stabilities, and substrate specificities varied. DppB was identified as a Xaa-Prolyl dipeptidyl peptidase, while DppE scissile substrates were similar to the substrates for Aspergillus fumigatus DPPV (AfDPPV). DppF was found to be a novel enzyme that could digest both substrates for A. fumigatus DPPIV and AfDPPV. Semi-quantitative PCR revealed that the transcription of dppB in A. oryzae was induced by protein substrates and repressed by the addition of an inorganic nitrogen source, despite the presence of protein substrates. The transcription of dppE depended on its growth time, while the transcription of dppF was not affected by the type of the nitrogen source in the medium, and it started during the early stage of the fungal growth. Based on these results, we conclude that these enzymes may represent the nutrition acquisition enzymes. Additionally, DppF may be one of the sensor peptidases responsible for the detection of the protein substrates in A. oryzae environment. DppB may be involved in nitrogen assimilation control, since the transcription of dppB was repressed by NaNO3, despite the presence of protein substrates.
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Aspergillus oryzae/enzimología , Aspergillus oryzae/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/química , Aspergillus fumigatus/enzimología , Aspergillus fumigatus/genética , Aspergillus nidulans/enzimología , Aspergillus nidulans/genética , ADN de Hongos/aislamiento & purificación , Concentración de Iones de Hidrógeno , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Osteoclasts are important target cells for osteoporosis treatment. Recently, a real-time cell analysis (RTCA) system was developed to observe cell morphology and adhesion; however, the use of RTCA to study osteoclastogenesis has not been reported. Here, we investigated whether osteoclast formation could be monitored in real-time using RTCA. The cell index determined via electrical impedance using RTCA, and the number of osteoclasts exhibited a significant positive correlation. RTCA was useful for determining the effect of (-)-epigallocatechin-3-gallate on the inhibition of bone resorption. We established a new method of measuring osteoclast formation in real-time using RTCA.
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Sistemas de Computación , Técnicas Citológicas/métodos , Impedancia Eléctrica , Osteoclastos/citología , Resorción Ósea/patología , Resorción Ósea/prevención & control , Catequina/análogos & derivados , Catequina/farmacología , Diferenciación Celular , Células Cultivadas , Humanos , Factor Estimulante de Colonias de Macrófagos , Ligando RANKRESUMEN
Rats with dwarfism accompanied by skeletal abnormalities, such as shortness of the limbs, tail, and body (dwarf rats), emerged in a Jcl-derived Sprague-Dawley rat colony maintained at the Institute for Animal Experimentation, St. Marianna University Graduate School of Medicine. Since the dwarfism was assumed to be due to a genetic mutation based on its frequency, we bred the dwarf rats and investigated their characteristics in order to identify the causative factors of their phenotypes and whether they could be used as a human disease model. One male and female that produced dwarf progeny were selected, and reproduction was initiated by mating the pair. The incidence of dwarfism was 25.8% among the resultant litter, and dwarfism occurred in both genders, suggesting that it was inherited in an autosomal recessive manner. At 12 weeks of age, the body weights of the male and female dwarf rats were 40% and 57% of those of the normal rats, respectively. In soft X-ray radiographic and histological examinations, shortening and hypoplasia of the long bones, such as the tibia and femur, were observed, which were suggestive of endochondral ossification abnormalities. An immunohistochemical examination detected an aggrecan synthesis disorder, which might have led to delayed calcification and increased growth plate thickening in the dwarf rats. We hypothesized that the principal characteristics of the dwarf rats were systemically induced by insufficient cartilage calcification in their long bones; thus, we named them cartilage calcification insufficient (CCI) rats.
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Calcificación Fisiológica , Cartílago/fisiopatología , Enanismo/genética , Enanismo/fisiopatología , Ratas Sprague-Dawley , Agrecanos/biosíntesis , Animales , Huesos/patología , Huesos/fisiopatología , Cartílago/patología , Modelos Animales de Enfermedad , Enanismo/metabolismo , Enanismo/patología , Femenino , Genes Recesivos , Placa de Crecimiento/patología , Masculino , FenotipoRESUMEN
ß-Aminopeptidases exhibit both hydrolytic and aminolytic (peptide bond formation) activities and have only been reported in bacteria. We identified a gene encoding the ß-aminopeptidase homolog from a genome database of the filamentous fungus Aspergillus oryzae. The gene was overexpressed in A. oryzae, and the resulting recombinant enzyme was purified. Apart from bacterial homologs [ß-Ala-para-nitroanilide (pNA)], the enzyme preferred D-Leu-pNA and D-Phe-pNA as substrates. Therefore, we designated this gene as d-stereoselective aminopeptidase A (damA). The purified recombinant DamA was estimated to be a hexamer and was composed of two subunits with molecular masses of 29.5 and 11.5 kDa, respectively. Optimal hydrolytic activity of DamA toward D-Leu-pNA was observed at 50 °C and pH 8.0. The enzyme was stable up to 60 °C and from pH 4.0-11.0. DamA also exhibited aminolytic activity, producing D-Leu-D-Leu-NH2 from D-Leu-NH2 as a substrate. In the presence of 3.0 M NaCl, the amount of pNA liberated from D-Leu-pNA by DamA was 3.1-fold higher than that in the absence of NaCl. Thus, DamA is a halophilic enzyme. The enzyme was utilized to synthesize several hetero-dipeptides containing a D-amino acid at the N-terminus as well as physiologically active peptides.
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Aspergillus oryzae/enzimología , Glutamil Aminopeptidasa/metabolismo , Péptidos/metabolismo , Cloruro de Sodio/farmacología , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Péptidos/química , Estereoisomerismo , Especificidad por SustratoRESUMEN
Aspergillus oryzae has an ortholog of Saccharomyces cerevisiae KEX1, termed kexA. A truncated form of KexA protein showed serine-type carboxypeptidase activity and somewhat broader substrate specificity than Kex1 protease. Furthermore, our results indicated that KexA is required for normal growth of A. oryzae and that it might be involved in hyphal branching.
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Aspergillus oryzae/enzimología , Carboxipeptidasas/metabolismo , Hifa/enzimología , Factores de Virulencia/metabolismo , Aspergillus oryzae/crecimiento & desarrollo , Carboxipeptidasas/química , Hifa/crecimiento & desarrollo , Saccharomyces cerevisiae/enzimología , Especificidad por Sustrato , Factores de Virulencia/químicaRESUMEN
The apsA and apsB genes encoding family M1 aminopeptidases were identified in the industrial fungus Aspergillus oryzae. The apsB was transcriptionally up-regulated up to 2.5-fold in response to the deprivation of nitrogen or carbon sources in growth media, while up-regulation of apsA was less significant. The encoded proteins were bacterially expressed and purified to characterize their enzymatic properties. ApsA and ApsB were optimally active at pH 7.0 and 35 °C and stable at pH ranges of 6-10 and 4-10, respectively, up to 40 °C. The enzymes were inhibited by bestatin and EDTA, as has been reported for family M1 aminopeptidases that characteristically contain a zinc-binding catalytic motif. Both enzymes preferentially liberated N-terminal lysine, which is an essential amino acid and an important additive to animal feed. Enzymes that efficiently release N-terminal lysine from peptides could be useful for food and forage industries. Examination of the reactivity toward peptide substrate of varying length revealed that ApsB exhibited broader substrate specificity than ApsA although the reactivity of ApsB decreased as the length of peptide substrate decreased.
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Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Aspergillus oryzae/enzimología , Aspergillus oryzae/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Aminopeptidasas/química , Proteínas Fúngicas/química , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Matrix metalloproteinases (MMPs) play an important role in degeneration of the matrix associated with bone and cartilage. Regulation of osteoclast activity is essential in the treatment of bone disease, including osteoporosis and rheumatoid arthritis. Polyphenols in green tea, particularly epigallocatechin-3-gallate (EGCG), inhibit MMPs expression and activity. However, the effects of the black tea polyphenol, theaflavin-3,3'-digallate (TFDG), on osteoclast and MMP activity are unknown. Therefore, we examined whether TFDG and EGCG affect MMP activity and osteoclast formation and differentiation in vitro. TFDG or EGCG (10 and 100 µM) was added to cultures of rat osteoclast precursors cells and mature osteoclasts. Numbers of multinucleated osteoclasts and actin rings decreased in polyphenol-treated cultures relative to control cultures. MMP-2 and MMP-9 activities were lower in TFDG- and EGCG-treated rat osteoclast precursor cells than in control cultures. MMP-9 mRNA levels declined significantly in TFDG-treated osteoclasts in comparison to control osteoclasts. TFDG and EGCG inhibited the formation and differentiation of osteoclasts via inhibition of MMPs. TFDG may suppress actin ring formation more effectively than EGCG. Thus, TFDG and EGCG may be suitable agents or lead compounds for the treatment of bone resorption diseases.
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The gdaA gene encoding S12 family glycine-D-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the D-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high D-stereospecificity and efficiently released N-terminal glycine and D-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and D-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and D-alanine aminopeptidase activity detected at a pH range of 6 to 9.
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Alanina/metabolismo , Aminopeptidasas/metabolismo , Aspergillus oryzae/enzimología , Glicina/metabolismo , Oryza/metabolismo , Aminopeptidasas/genética , Aminopeptidasas/aislamiento & purificación , Aspergillus oryzae/genética , Medios de Cultivo/química , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/metabolismo , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Ochrobactrum anthropi/enzimología , Ochrobactrum anthropi/genética , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
Matrix metalloproteinases (MMPs) play an important role in degeneration of the matrix associated with bone and cartilage. Regulation of osteoclast activity is essential in the treatment of bone disease, including osteoporosis and rheumatoid arthritis. Polyphenols in green tea, particularly epigallocatechin-3-gallate (EGCG), inhibit MMPs expression and activity. However, the effects of the black tea polyphenol, theaflavin-3,3'-digallate (TFDG), on osteoclast and MMP activity are unknown. Therefore, we examined whether TFDG and EGCG affect MMP activity and osteoclast formation and differentiation in vitro. TFDG or EGCG (10 and 100 µM) was added to cultures of rat osteoclast precursors cells and mature osteoclasts. Numbers of multinucleated osteoclasts and actin rings decreased in polyphenol-treated cultures relative to control cultures. MMP-2 and MMP-9 activities were lower in TFDG- and EGCG-treated rat osteoclast precursor cells than in control cultures. MMP-9 mRNA levels declined significantly in TFDG-treated osteoclasts in comparison to control osteoclasts. TFDG and EGCG inhibited the formation and differentiation of osteoclasts via inhibition of MMPs. TFDG may suppress actin ring formation more effectively than EGCG. Thus, TFDG and EGCG may be suitable agents or lead compounds for the treatment of bone resorption diseases.
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Biflavonoides/farmacología , Camellia sinensis , Catequina/análogos & derivados , Ácido Gálico/análogos & derivados , Osteoclastos/efectos de los fármacos , Animales , Catequina/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Ácido Gálico/farmacología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Osteoclastos/citología , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
Gene AO090103000153 is unique to Aspergillus oryzae RIB40 and A. flavus NRRL3357, and is speculated to encode a serine-type carboxypeptidase. In this study, we purified and characterized a heterologously expressed gene product of AO090103000153. 5'-Rapid amplification of cDNA ends indicated that the translation start site of the gene is located 1,586 bp downstream of the translation start site predicted by the genome sequencing project. The gene, starting from the revised translation start codon, termed ocpC, was transcribed constantly in A. oryzae RIB40. Purified recombinant OcpC exhibited the enzymatic properties of a serine-type carboxypeptidase. This protease was stable at temperatures below 45°C and a low pH, and had broad substrate specificity for N-acylpeptides, but it exhibited significantly lower specific activity and a lower k(cat) value for substrates than previously reported serine-type carboxypeptidases from A. oryzae.
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Aspergillus oryzae/enzimología , Carboxipeptidasas/metabolismo , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Aspergillus nidulans/genética , Aspergillus oryzae/genética , Carboxipeptidasas/química , Carboxipeptidasas/genética , Carboxipeptidasas/aislamiento & purificación , Vectores Genéticos/genética , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Especificidad de la Especie , Transcripción GenéticaRESUMEN
Cysteinyl dipeptidase from Aspergillus oryzae (CdpA) was produced in Escherichia coli and purified. The enzyme showed activity specific toward cysteine-containing dipeptides, but its substrate specificity was distinct from those of other cysteinyl dipeptidases of the M20 family. It was optimally active at pH 7-8 and stable at pH 6-9 and at up to 40 °C.
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Aspergillus oryzae/enzimología , Cisteína/metabolismo , Dipeptidasas/genética , Dipeptidasas/metabolismo , Escherichia coli/genética , Clonación Molecular , Dipeptidasas/biosíntesis , Dipeptidasas/aislamiento & purificación , Expresión Génica , Humanos , Oligopéptidos/metabolismo , Especificidad por SustratoRESUMEN
Leucine aminopeptidase (LAP), an enzyme used in the food industry, is an exopeptidase that removes an amino acid residue, primarily leucine (Leu), from the N-terminus of peptides and protein substrates. In this study, we focused on the leucine aminopeptidase A (lapA) gene from Aspergillus oryzae RIB40. To purify and characterize the LapA, lapA was overexpressed in A. oryzae RIB40 using the amyB promoter. LAP activity in the culture supernatant of one transformant harboring the lapA expression plasmid was 33 times that of the host strain. LapA was purified from the culture supernatant of this lapA-overexpressing strain by column chromatography. The purified recombinant LapA had a molecular mass of 33 kDa, and its N-terminal amino acid was the tyrosine at position 80 of the deduced amino acid sequence. Optimal enzyme activity was observed at 60°C and pH 8.5, and the enzyme was stable at temperatures up to 60°C and in the pH range 7.5-11. In transcriptional analysis, lapA was induced under alkaline conditions and expressed at a relatively low level under normal conditions. LapA showed maximum hydrolyzing activity for the substrate leucine para-nitroanilide (Leu-pNA), followed by substrates Phe-pNA (39% activity compared with Leu-pNA), Met-pNA, Lys-pNA, and Arg-pNA. In addition, LapA preferentially hydrolyzed peptides longer than tripeptides.
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Aspergillus oryzae/enzimología , Expresión Génica , Leucil Aminopeptidasa/metabolismo , Aspergillus oryzae/genética , Medios de Cultivo/química , Estabilidad de Enzimas , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Concentración de Iones de Hidrógeno , Leucil Aminopeptidasa/química , Leucil Aminopeptidasa/genética , Leucil Aminopeptidasa/aislamiento & purificación , Peso Molecular , Regiones Promotoras Genéticas , Especificidad por Sustrato , TemperaturaRESUMEN
Apopotosis of osteoclasts is regulated by the Bcl-2 family protein Bim. Bim is degraded in the course of osteoclast apoptosis, which is regulated by Caspase-3. Osteoclasts generated from caspase-3 -/- mice exhibited a shorter life span and a higher bone-resorbing activity than those generated from normal littermates. These results suggest the important role of Caspase-3-Bim axis in regulating both apoptosis and activation of osteoclasts.