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1.
PLoS One ; 14(9): e0222234, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31509576

RESUMO

Prostatic acid phosphatase (PAP), which is secreted by prostate, increases in some diseases such as prostate cancer. PAP is also present in the central nervous system. In this study we reveal that α-synuclein (Snca) gene is co-deleted/mutated in PAP null mouse. It is indicated that mice deficient in transmembrane PAP display neurological alterations. By using immunohistochemistry, cerebellar cortical neurons and zone and stripes pattern were studied in Pap-/- ;Snca-/- mouse cerebellum. We show that the Pap-/- ;Snca-/- cerebellar cortex development appears to be normal. Compartmentation genes expression such as zebrin II, HSP25, and P75NTR show the zone and stripe phenotype characteristic of the normal cerebellum. These data indicate that although aggregation of PAP and SNCA causes severe neurodegenerative diseases, PAP -/- with absence of the Snca does not appear to interrupt the cerebellar architecture development and zone and stripe pattern formation. These findings question the physiological and pathological role of SNCA and PAP during cerebellar development or suggest existence of the possible compensatory mechanisms in the absence of these genes.


Assuntos
Fosfatase Ácida/metabolismo , Córtex Cerebelar/metabolismo , alfa-Sinucleína/metabolismo , Fosfatase Ácida/genética , Fosfatase Ácida/fisiologia , Animais , Cerebelo/metabolismo , Expressão Gênica/genética , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/genética , Atividade Motora/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Transcriptoma/genética , alfa-Sinucleína/genética , alfa-Sinucleína/fisiologia
2.
Protein Sci ; 27(7): 1151-1165, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29493036

RESUMO

Misfolding and amyloid aggregation of intrinsically disordered proteins (IDPs) are implicated in a variety of diseases. Studies have shown that membrane plays important roles on the formation of intermediate structures of IDPs that can initiate (and/or speed-up) amyloid aggregation to form fibers. The process of amyloid aggregation also disrupts membrane to cause cell death in amyloid diseases like Alzheimer's disease and type-2 diabetes. On the other hand, recent studies reported the membrane fusion properties of amyloid fibers. Remarkably, amyloid-fibril formation by short peptide fragments of highly abundant prostatic acidic-phosphatase (PAP) in human semen and are capable of boosting the rate of HIV infection up to 400,000-fold during sexual contact. Unlike the least toxic fully matured fibers of most amyloid proteins, the semen-derived enhancer of virus infection (SEVI) amyloid-fibrils of PAP peptide fragments are highly potent in rendering the maximum rate of HIV infection. This unusual property of amyloid fibers has witnessed increasing number of studies on the biophysical aspects of fiber formation and fiber-membrane interactions. NMR studies have reported a highly disordered partial helical structure in a membrane environment for the intrinsically disordered PAP peptide that promotes the fusion of the viral membrane with that of host cells. The purpose of this review article is to unify and integrate biophysical and immunological research reported in the previous studies on SEVI. Specifically, amyloid aggregation, dramatic HIV infection enhancing properties, membrane fusion properties, high resolution NMR structure, and approaches to eliminate the enhancement of HIV infection of SEVI peptides are discussed.


Assuntos
Amiloide/química , Infecções por HIV/epidemiologia , Sêmen/metabolismo , Doenças Virais Sexualmente Transmissíveis/metabolismo , Fosfatase Ácida/química , Fosfatase Ácida/fisiologia , Amiloide/fisiologia , Infecções por HIV/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/fisiologia , Masculino , Modelos Moleculares , Estrutura Secundária de Proteína
3.
Plant Cell Environ ; 39(10): 2247-59, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27411391

RESUMO

Under phosphate (Pi ) starvation, plants increase the secretion of purple acid phosphatases (PAPs) into the rhizosphere to scavenge organic phosphorus (P) for plant use. To date, only a few members of the PAP family have been characterized in crops. In this study, we identified a novel secreted PAP in rice, OsPAP10c, and investigated its role in the utilization of external organic P. OsPAP10c belongs to a monocotyledon-specific subclass of Ia group PAPs and is specifically expressed in the epidermis/exodermis cell layers of roots. Both the transcript and protein levels of OsPAP10c are strongly induced by Pi starvation. OsPAP10c overexpression increased acid phosphatase (APase) activity by more than 10-fold in the culture media and almost fivefold in both roots and leaves under Pi -sufficient and Pi -deficient conditions. This increase in APase activity further improved the plant utilization efficiency of external organic P. Moreover, several APase isoforms corresponding to OsPAP10c were identified using in-gel activity assays. Under field conditions with three different Pi supply levels, OsPAP10c-overexpressing plants had significantly higher tiller numbers and shorter plant heights. This study indicates that OsPAP10c encodes a novel secreted APase that plays an important role in the utilization of external organic P in rice.


Assuntos
Fosfatase Ácida/fisiologia , Oryza/metabolismo , Fósforo/metabolismo , Proteínas de Plantas/fisiologia , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Produtos Agrícolas/enzimologia , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Regulação da Expressão Gênica de Plantas , Oryza/enzimologia , Oryza/genética , Fósforo/farmacologia , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , RNA Mensageiro/metabolismo
4.
J Exp Bot ; 67(14): 4141-54, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27194738

RESUMO

Stylo (Stylosanthes spp.) is a pasture legume predominant in tropical and subtropical areas, where low phosphorus (P) availability is a major constraint for plant growth. Therefore, stylo might exhibit superior utilization of the P pool on acid soils, particularly organic P. However, little is known about mechanisms of inorganic phosphate (Pi) acquisition employed by stylo. In this study, the utilization of extracellular deoxy-ribonucleotide triphosphate (dNTP) and the underlying physiological and molecular mechanisms were examined for two stylo genotypes with contrasting P efficiency. Results showed that the P-efficient genotype, TPRC2001-1, was superior to the P-inefficient genotype, Fine-stem, when using dNTP as the sole P source. This was reflected by a higher dry weight and total P content for TPRC2001-1 than for Fine-stem, which was correlated with higher root-associated acid phosphatase (APase) activities in TPRC2001-1 under low P conditions. Subsequently, three PAP members were cloned from TPRC2001-1: SgPAP7, SgPAP10, and SgPAP26 Expression levels of these three SgPAPs were up-regulated by Pi starvation in stylo roots. Furthermore, there was a higher abundance of transcripts of SgPAP7 and SgPAP10 in TPRC2001-1 than in Fine-stem. Subcellular localization analysis demonstrated that these three SgPAPs were localized on the plasma membrane. Overexpression of these three SgPAPs could result in significantly increased root-associated APase activities, and thus extracellular dNTP utilization in bean hairy roots. Taken together, the results herein suggest that SgPAP7, SgPAP10, and SgPAP26 may differentially contribute to root-associated APase activities, and thus control extracellular dNTP utilization in stylo.


Assuntos
Fosfatase Ácida/metabolismo , Desoxirribonucleotídeos/metabolismo , Fabaceae/enzimologia , Glicoproteínas/metabolismo , Fosfatase Ácida/genética , Fosfatase Ácida/fisiologia , Fabaceae/genética , Fabaceae/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Genótipo , Glicoproteínas/genética , Glicoproteínas/fisiologia , Filogenia , Raízes de Plantas/enzimologia , Raízes de Plantas/metabolismo
5.
PLoS One ; 10(6): e0130144, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26091289

RESUMO

In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas' disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia.


Assuntos
Fosfatase Ácida/fisiologia , Catepsina D/fisiologia , Atresia Folicular , Hemípteros/enzimologia , Proteínas de Insetos/fisiologia , Insetos Vetores/enzimologia , Fosfatase Ácida/química , Animais , Catepsina D/química , Doença de Chagas/parasitologia , Corpo Adiposo/enzimologia , Feminino , Expressão Gênica , Hemípteros/parasitologia , Hemolinfa/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Insetos/química , Insetos Vetores/parasitologia , Células MCF-7 , Masculino , Especificidade de Órgãos , Ovário/enzimologia , Proteólise , Trypanosoma cruzi/fisiologia , Vitelinas/química , Vitelinas/metabolismo
6.
Rheumatology (Oxford) ; 54(10): 1913-22, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25998451

RESUMO

OBJECTIVE: Diseases associated with calcium-containing crystal deposition can lead to local bone erosion. We aimed to determine whether calcium-containing crystal-hydroxyapatite, ß-tricalcium phosphate and CPPD enhanced osteoclastogenesis and to define underlying mechanisms of action. METHODS: Osteoclastogenesis was studied by culturing murine RAW 264.7 osteoclast precursor cells with RANK ligand (RANKL)/ M-CSF and/or calcium-containing crystals, and observing the tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and TRAP activity. Resorption pit formation was used to evaluate osteoclast activity. Real-time RT-PCR analysis revealed osteoclast marker genes, including TRAP, cathepsin K and calcitonin receptor (CTR). Western blotting was used to analyse the phosphorylation levels of signal transduction molecules. RESULTS: Three kinds of calcium-containing crystal significantly enhanced RANKL/M-CSF-induced osteoclastogenesis in RAW 264.7 cells, as evidenced by the increased number of TRAP-positive multinucleated cells, TRAP activity and resorption pit formation in a dose-dependent manner. Hydroxyapatite, ß-tricalcium phosphate and CPPD treatments significantly enhanced RANKL/M-CSF-induced mRNA expression of TRAP, cathepsin K and CTR. Moreover, the three kinds of calcium-containing crystal enhanced the phosphorylation of extracellular-signal-regulated kinase and p38 in RANKL/M-CSF-treated cells. CONCLUSION: We concluded that calcium-containing crystals can promote osteoclastogenesis and bone resorption through the extracellular-signal-regulated kinase and p38 pathways. Together with synovial activation, this mechanism may be important in the pathogenesis of destructive arthropathies triggered by calcium-containing crystals.


Assuntos
Cálcio/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/fisiologia , Osteoclastos/citologia , Osteogênese/efeitos dos fármacos , Ligante RANK/fisiologia , Fosfatase Ácida/fisiologia , Animais , Reabsorção Óssea/fisiopatologia , Cálcio/química , Fosfatos de Cálcio/farmacologia , Catepsina K/fisiologia , Linhagem Celular , Células Cultivadas , Cristalização , Durapatita/farmacologia , Técnicas In Vitro , Isoenzimas/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Modelos Animais , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Osteogênese/fisiologia , Receptores da Calcitonina/fisiologia , Fosfatase Ácida Resistente a Tartarato
7.
J Vet Med Sci ; 77(7): 875-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25755022

RESUMO

Blood samples were obtained sequentially from 10 dairy cows around the time of parturition to assess plasma fluctuations in estradiol-17ß (E2) levels in association with those of several bone resorption markers. Plasma E2 concentration increased sharply a few days prepartum and decreased quickly after parturition. In terms of bone resorption markers, the plasma level of tartrate-resistant acid phosphatase isoform 5b (TRAP5b) rose significantly, commencing 1 week prepartum, and was maintained at this level to a few days postpartum. The plasma concentration of carboxyterminal collagen cross-links of type-I collagen (CTx) increased significantly after parturition. These observations suggest that osteoclast-mediated bone resorption was activated after parturition when plasma E2 concentrations decreased.


Assuntos
Fosfatase Ácida/sangue , Bovinos/sangue , Colágeno Tipo I/sangue , Estradiol/sangue , Isoenzimas/sangue , Parto/sangue , Peptídeos/sangue , Fosfatase Ácida/fisiologia , Animais , Biomarcadores/sangue , Reabsorção Óssea/sangue , Reabsorção Óssea/fisiopatologia , Reabsorção Óssea/veterinária , Cálcio/sangue , Bovinos/fisiologia , Colágeno Tipo I/fisiologia , Estradiol/fisiologia , Feminino , Hidroxiprolina/sangue , Isoenzimas/fisiologia , Hormônio Paratireóideo/sangue , Parto/fisiologia , Peptídeos/fisiologia , Fosfatase Ácida Resistente a Tartarato
8.
Endocrinology ; 155(11): 4521-30, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25093463

RESUMO

Uteroferrin (UF) is a progesterone-induced acid phosphatase produced by uterine glandular epithelia in mammals during pregnancy and targeted to sites of hematopoiesis throughout pregnancy. The expression pattern of UF is coordinated with early fetal hematopoietic development in the yolk sac and then liver, spleen, and bone to prevent anemia in fetuses. Our previous studies suggested that UF exerts stimulatory impacts on hematopoietic progenitor cells. However, the precise role and thereby the mechanism of action of UF on hematopoiesis have not been investigated previously. Here, we report that UF is a potent regulator that can greatly enhance fetal erythropoiesis. Using primary fetal liver hematopoietic cells, we observed a synergistic stimulatory effect of UF with erythropoietin and other growth factors on both burst-forming unit-erythroid and colony-forming unit-erythroid formation. Further, we demonstrated that UF enhanced erythropoiesis at terminal stages using an in vitro culture system. Surveying genes that are crucial for erythrocyte formation at various stages revealed that UF, along with erythropoietin, up-regulated transcription factors required for terminal erythrocyte differentiation and genes required for synthesis of hemoglobin. Collectively, our results demonstrate that UF is a cytokine secreted by uterine glands in response to progesterone that promotes fetal erythropoiesis at various stages of pregnancy, including burst-forming unit-erythroid and colony-forming unit-erythroid progenitor cells and terminal stages of differentiation of hematopoietic cells in the erythroid lineage.


Assuntos
Fosfatase Ácida/fisiologia , Eritropoese/genética , Feto , Isoenzimas/fisiologia , Fosfatase Ácida/farmacologia , Sequência de Aminoácidos , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Embrião de Mamíferos , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/fisiologia , Eritropoese/efeitos dos fármacos , Feminino , Feto/efeitos dos fármacos , Feto/fisiologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Isoenzimas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Gravidez , Progesterona/farmacologia , Homologia de Sequência , Fosfatase Ácida Resistente a Tartarato
9.
PLoS Genet ; 10(1): e1004061, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24391523

RESUMO

Phosphorus (P) is essential for all living cells and organisms, and low-P stress is a major factor constraining plant growth and yield worldwide. In plants, P efficiency is a complex quantitative trait involving multiple genes, and the mechanisms underlying P efficiency are largely unknown. Combining linkage analysis, genome-wide and candidate-gene association analyses, and plant transformation, we identified a soybean gene related to P efficiency, determined its favorable haplotypes and developed valuable functional markers. First, six major genomic regions associated with P efficiency were detected by performing genome-wide associations (GWAs) in various environments. A highly significant region located on chromosome 8, qPE8, was identified by both GWAs and linkage mapping and explained 41% of the phenotypic variation. Then, a regional mapping study was performed with 40 surrounding markers in 192 diverse soybean accessions. A strongly associated haplotype (P = 10(-7)) consisting of the markers Sat_233 and BARC-039899-07603 was identified, and qPE8 was located in a region of approximately 250 kb, which contained a candidate gene GmACP1 that encoded an acid phosphatase. GmACP1 overexpression in soybean hairy roots increased P efficiency by 11-20% relative to the control. A candidate-gene association analysis indicated that six natural GmACP1 polymorphisms explained 33% of the phenotypic variation. The favorable alleles and haplotypes of GmACP1 associated with increased transcript expression correlated with higher enzyme activity. The discovery of the optimal haplotype of GmACP1 will now enable the accurate selection of soybeans with higher P efficiencies and improve our understanding of the molecular mechanisms underlying P efficiency in plants.


Assuntos
Fosfatase Ácida/genética , Fósforo/metabolismo , Soja/genética , Estresse Fisiológico/genética , Fosfatase Ácida/fisiologia , Mapeamento Cromossômico , Cromossomos de Plantas , Regulação da Expressão Gênica de Plantas , Estudo de Associação Genômica Ampla , Haplótipos , Dados de Sequência Molecular , Fenótipo , Locos de Características Quantitativas/genética , Soja/crescimento & desenvolvimento
10.
Wei Sheng Wu Xue Bao ; 54(12): 1446-52, 2014 Dec 04.
Artigo em Chinês | MEDLINE | ID: mdl-25876330

RESUMO

OBJECTIVE: Expression level of laccase POXA1c would be increased by screening effective signal peptide in Pichia pastoris. METHODS: According to 2D-gel and profile of P. pastoris genome sequence, seven signal peptides from high secreted endogenous proteins of P. pastoris X-33 were chosen to evaluate their secreted ability by using POXA1c as reporter protein. RESULTS: Compared with POXA1c's native signal peptide, the signal peptide of repressible acid phosphatases PHO5 and lectin-like protein FLO10 showed 2. 5-fold and 2-fold increase of laccase activity. Furthermore, PHO5-αpro and FLO10-αpro were constructed by fusing signal peptide of PHO5 and FLO10 with pro-peptide of α-MF respectively. The laccase activity under the leading of FLO10-αpro and PHO5-αpro showed 3-fold and 3. 5- fold laccase activity higher than native signal peptide, and showed 20% and 40% increase compared with saccharomyces cerevisiae α-MF signal respectively. CONCLUSION: Signal peptides from high secreted endogenous proteins of P. pastoris X-33 could be effectively used to lead laccase expression in P. pastoris. The activity of POXA1c under the leading of the PHO5-αpro signal peptide was 57.98 U/mL after high density fermentation.


Assuntos
Fosfatase Ácida/fisiologia , Lacase/genética , Pichia/genética , Sinais Direcionadores de Proteínas/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Fermentação , Lacase/metabolismo , Dados de Sequência Molecular
11.
J Biomech ; 47(2): 518-25, 2014 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-24275439

RESUMO

A multi-well fluid loading (MFL) system was developed to deliver oscillatory subphysiologic to supraphysiologic fluid shear stresses to cell monolayers in vitro using standard multi-well culture plates. Computational fluid dynamics modeling with fluid-structure interactions was used to quantify the squeeze film fluid flow between an axially displaced piston and the well plate surface. Adjusting the cone angle of the piston base modulated the fluid pressure, velocity, and shear stress magnitudes. Modeling results showed that there was near uniform fluid shear stress across the well with a linear drop in pressure across the radius of the well. Using the MFL system, RAW 264.7 osteoclastic cells were exposed to oscillatory fluid shear stresses of 0, 0.5, 1.5, 4, 6, and 17 Pa. Cells were loaded 1 h per day at 1 Hz for two days. Compared to sub-physiologic and physiologic levels, supraphysiologic oscillatory fluid shear induced upregulation of osteoclastic activity as measured by tartrate-resistant acid phosphatase activity and formation of mineral resorption pits. Cell number remained constant across all treatment groups.


Assuntos
Hidrodinâmica , Osteoclastos/fisiologia , Estresse Mecânico , Fosfatase Ácida/metabolismo , Fosfatase Ácida/fisiologia , Animais , Reabsorção Óssea/enzimologia , Reabsorção Óssea/fisiopatologia , Células Cultivadas , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Camundongos , Osteoclastos/citologia , Osteoclastos/enzimologia , Fosfatase Ácida Resistente a Tartarato , Regulação para Cima/fisiologia
12.
Oncogene ; 33(11): 1395-406, 2014 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-23604121

RESUMO

Tartrate-resistant acid phosphatase 5 (ACP5), which is essential for bone resorption and osteoclast differentiation, promotes cell motility through the modulation of focal adhesion kinase phosphorylation. However, whether ACP5 contributes to the metastasis and progression of hepatocellular carcinoma (HCC) remains unknown. In this paper, a complementary DNA microarray, serial deletion, site-directed mutagenesis and a chromatin immunoprecipitation assays confirmed that ACP5 is a direct transcriptional target of Forkhead box M1 (FoxM1). ACP5 expression was markedly higher in HCC tissues compared with adjacent noncancerous tissues. ACP5 overexpression was correlated with microvascular invasion, poor differentiation and higher tumor-node-metastasis stage. HCC patients with positive ACP5 expression had poorer prognoses than those with negative ACP5 expression. A multivariate analysis revealed that ACP5 expression was an independent and significant risk factor for disease recurrence and reduced-patient survival following curative resection. Transwell assays and an orthotopic metastatic model showed that the upregulation of ACP5 promoted HCC invasion and lung metastasis, whereas ACP5 knockdown inhibited these processes. The knockdown of ACP5 significantly attenuated FoxM1-enhanced invasion and lung metastasis. Immunohistochemistry revealed that ACP5 expression was positively correlated with FoxM1 expression in human HCC tissues, and their coexpression was associated with poor prognoses. In summary, ACP5 is a direct transcriptional and functional target of FoxM1. This novel FoxM1/ACP5 signaling pathway promotes HCC metastasis and may be a candidate biomarker for prognosis and a target for new therapies.


Assuntos
Fosfatase Ácida/fisiologia , Carcinoma Hepatocelular/patologia , Fatores de Transcrição Forkhead/metabolismo , Isoenzimas/fisiologia , Neoplasias Hepáticas/patologia , Metástase Neoplásica , Fosfatase Ácida/metabolismo , Adulto , Feminino , Proteína Forkhead Box M1 , Humanos , Isoenzimas/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Fosfatase Ácida Resistente a Tartarato
13.
Int J Mol Med ; 28(1): 73-9, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21491081

RESUMO

The effects of mechanical stress release on osteoclastogenesis may be as important as those of mechanical stress application. However, the direct effects of mechanical stress on the behavior of osteoclasts has not been thoroughly investigated and there is limited information on the results of the release from mechanical stress. In this study, the effects of mechanical stress application and its release on osteoclast differentiation were examined. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts derived from RAW264.7 cells were measured and the expression of osteoclast differentiation genes, which was altered in response to the release from mechanical stress according to the Flexercell tension system was evaluated by real-time PCR. Osteoclast differentiation and fusion were suppressed by mechanical stress application and were rapidly induced after mechanical stress release. The mRNA expression of the osteoclast specific genes, TRAP, matrix metalloproteinase-9 (MMP-9), cathepsin-K (cath-k), calcitonin receptor (CTR), ATPase H+ transporting vacuolar proton pump member I (ATP6i), chloride channel-7 (ClC7) and dendritic cell-specific transmembrane protein (DC-STAMP) was decreased with mechanical stress application, and increased up to 48 h after the release from it. These alterations in gene mRNA expression were associated with the number of osteoclasts and large osteoclasts. Inducible nitric oxide synthetase (iNOS) mRNA was increased with mechanical stress and decreased after its release. Nitric oxide (NO) production was increased with mechanical stress. Nuclear factor of activated T cells cytoplasmic (NFATc) family mRNAs were not altered with mechanical stress, but were up-regulated up to 48 h after the release from it. These findings indicate that the suppression of osteoclast differentiation and fusion induced by mechanical stress is the result of NO increase via iNOS, and that the promotion of osteoclast differentiation and fusion after the release from mechanical stress is related to the NFATc family genes, whose expression remained constant during mechanical stress but was up-regulated after the release from mechanical stress.


Assuntos
Diferenciação Celular , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico/metabolismo , Osteoclastos/fisiologia , Estresse Mecânico , Fosfatase Ácida/genética , Fosfatase Ácida/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Catepsina K/genética , Catepsina K/fisiologia , Linhagem Celular , Canais de Cloreto/genética , Canais de Cloreto/fisiologia , Expressão Gênica/genética , Expressão Gênica/fisiologia , Isoenzimas/genética , Isoenzimas/fisiologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/fisiologia , Osteoclastos/citologia , Pressão , Receptores da Calcitonina/genética , Receptores da Calcitonina/fisiologia , Fosfatase Ácida Resistente a Tartarato , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/fisiologia
15.
J Immunol ; 184(9): 5141-50, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20348422

RESUMO

Francisella tularensis contains four putative acid phosphatases that are conserved in Francisella novicida. An F. novicida quadruple mutant (AcpA, AcpB, AcpC, and Hap [DeltaABCH]) is unable to escape the phagosome or survive in macrophages and is attenuated in the mouse model. We explored whether reduced survival of the DeltaABCH mutant within phagocytes is related to the oxidative response by human neutrophils and macrophages. F. novicida and F. tularensis subspecies failed to stimulate reactive oxygen species production in the phagocytes, whereas the F. novicida DeltaABCH strain stimulated a significant level of reactive oxygen species. The DeltaABCH mutant, but not the wild-type strain, strongly colocalized with p47(phox) and replicated in phagocytes only in the presence of an NADPH oxidase inhibitor or within macrophages isolated from p47(phox) knockout mice. Finally, purified AcpA strongly dephosphorylated p47(phox) and p40(phox), but not p67(phox), in vitro. Thus, Francisella acid phosphatases play a major role in intramacrophage survival and virulence by regulating the generation of the oxidative burst in human phagocytes.


Assuntos
Fosfatase Ácida/fisiologia , Francisella tularensis/enzimologia , Francisella tularensis/imunologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Fagócitos/enzimologia , Fagócitos/microbiologia , Fosfatase Ácida/genética , Animais , Células Cultivadas , Francisella tularensis/crescimento & desenvolvimento , Humanos , Líquido Intracelular/enzimologia , Líquido Intracelular/imunologia , Líquido Intracelular/microbiologia , Isoenzimas/genética , Isoenzimas/fisiologia , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/biossíntese , Neutrófilos/enzimologia , Neutrófilos/imunologia , Neutrófilos/microbiologia , Fagócitos/imunologia , Fosforilação/imunologia , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória/imunologia
16.
Calcif Tissue Int ; 85(6): 465-83, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19915788

RESUMO

Type 5 tartrate-resistant acid phosphatase (TRAP) has been a clinically relevant biomarker for about 50 years. It has always been a reliable and specific cytochemical marker for hairy cell leukemia and for differentiated cells of monocytic lineage. Only recently has the test for serum TRAP activity been accepted as sensitive and specific enough for clinical use as a marker of osteoclasts and bone resorption. This has come about through steady advances in knowledge about TRAP enzymology, structure, function, and molecular regulation and a consequent appreciation that TRAP isoforms 5a and 5b have very different clinical significance. As a measure of osteoclast number and bone resorption, TRAP 5b has diagnostic and prognostic applications in osteoporosis, cancers with bone metastasis, chronic renal failure, and perhaps other metabolic and pathologic bone diseases. Serum TRAP 5a, on the other hand, has no relationship to bone metabolism but seems instead to be a measure of activated macrophages and chronic inflammation. Exploration of the real clinical usefulness of serum TRAP 5a for diagnosis and disease management in a wide variety of chronic inflammatory diseases is only now beginning. This perspective traces the important basic scientific developments that have led up to the refinement of serum TRAP isoform immunoassays and their validation as biomarkers of disease. Many unanswered questions remain, providing a wealth of opportunity for continued research of this multifaceted enzyme.


Assuntos
Fosfatase Ácida/fisiologia , Isoenzimas/fisiologia , Fosfatase Ácida/sangue , Fosfatase Ácida/metabolismo , Animais , Biomarcadores/sangue , Reabsorção Óssea/metabolismo , Humanos , Isoenzimas/sangue , Isoenzimas/metabolismo , Macrófagos/metabolismo , Modelos Biológicos , Osteoclastos/citologia , Osteoclastos/metabolismo , Isoformas de Proteínas/metabolismo , Fosfatase Ácida Resistente a Tartarato
17.
J Invertebr Pathol ; 102(3): 238-44, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19679133

RESUMO

The use of Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as a biological control method against free living larvae of gastrointestinal nematodes of livestock animals. This fungus captures and infects the nematode by cuticle penetration, immobilization and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. This report characterizes the acid phosphatase activity during the interaction of D. flagrans with the free-living nematode Panagrellus sp. The optimum pH for the hydrolysis of the acid phosphatase substrate p-nitrophenyl phosphate was 2.2, 2.8 and 5.4 from D. flagrans alone and 2.2 and 5.4 for Panagrellus sp alone, fungus-nematode interaction in liquid medium and fungus-nematode interaction in solid medium. Different acid phosphatase activity bands were detected by SDS-PAGE. Maximum acid phosphatase activity of the fungus or nematode alone and of the fungus-nematode interaction occurred within 70min of incubation in the presence of the substrate 4-methylumbelliferyl phosphate. The activity of this enzyme was significantly higher for the fungus-nematode interaction when compared to the organisms alone, indicating a synergistic response. Furthermore, structures appeared in the hyphae after 30min, nematodes were observed adhered after 40min and many were captured by the typical fungus traps after 70min of interaction. The participation of acid phosphatase activity and its importance during the interaction of the fungus with the nematode were discussed.


Assuntos
Fosfatase Ácida/metabolismo , Ascomicetos/enzimologia , Rabditídios/microbiologia , Fosfatase Ácida/fisiologia , Animais , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Cinética , Rabditídios/enzimologia , Fatores de Tempo
18.
Clin Oral Implants Res ; 20(11): 1254-64, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19531103

RESUMO

OBJECTIVES: Little information is available on the molecular events that occur during graft incorporation over time. The calvarial bone (Cb) grafts have been reported to produce greater responses compared with other donor regions in maxillofacial reconstructions, but the scientific evidences for this are still lacking. The objectives of this study are (1) to study the morphological pattern of Cb onlay bone grafts and compare them with the biological events through immunohistochemical responses and (2) to establish the effects of perforations in maintaining the volume and bone density of the receptor bed. MATERIAL AND METHODS: Sixty New Zealand White rabbits were submitted to Cb onlay bone grafts on the mandible. In 30 rabbits, the receptor bed was perforated (perforated group), while for the remaining animals the bed was kept intact (non-perforated group). Six animals from each group were sacrificed at 5, 7, 10, 20 and 60 days after surgery. Histological sections from the grafted area were prepared for immunohistochemical and histological analyses. Immuno-labeling was found for proteins Osteoprotegerin (OPG), receptor activator of nuclear factor-kappabeta ligand (RANKL), alkaline phosphatase (ALP), osteopontin (OPN), vascular endothelial growth factor (VEGF), tartrate-resistant acid phosphatase (TRAP), Type I collagen (COL I) and osteocalcin (OC). The tomography examination [computerized tomography (CT) scan] was conducted just after surgery and at the sacrifice. RESULTS: The histological findings revealed that the perforations contributed to higher bone deposition during the initial stages at the graft-receptor bed interface, accelerating the graft incorporation process. The results of the CT scan showed lower resorption for the perforated group (P

Assuntos
Transplante Ósseo/fisiologia , Sobrevivência de Enxerto/fisiologia , Osseointegração/fisiologia , Osteogênese/fisiologia , Crânio/cirurgia , Fosfatase Ácida/fisiologia , Fosfatase Alcalina/fisiologia , Animais , Reabsorção Óssea/metabolismo , Transplante Ósseo/métodos , Colágeno Tipo I/fisiologia , Imuno-Histoquímica , Isoenzimas/fisiologia , Masculino , Osteocalcina/fisiologia , Osteopontina/fisiologia , Osteoprotegerina/fisiologia , Ligante RANK/fisiologia , Coelhos , Estatísticas não Paramétricas , Fosfatase Ácida Resistente a Tartarato , Tomografia Computadorizada por Raios X , Fator A de Crescimento do Endotélio Vascular/fisiologia
19.
Trends Biochem Sci ; 34(6): 273-8, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19467874

RESUMO

Although classical protein tyrosine phosphatase (PTP) superfamily members are cysteine-dependent, emerging evidence shows that many acid phosphatases (AcPs) function as histidine-dependent PTPs in vivo. These AcPs dephosphorylate phospho-tyrosine substrates intracellularly and could have roles in development and disease. In contrast to cysteine-dependent PTPs, they utilize histidine, rather than cysteine, for substrate dephosphorylation. Structural analyses reveal that active site histidine, but not cysteine, faces towards the substrate and functions as the phosphate acceptor. Nonetheless, during dephosphorylation, both histidine-dependent and cysteine-dependent PTPs use their active site arginine and aspartate for substrate binding and proton donation, respectively. Thus, we propose that they should be referred to as a distinct group of 'histidine-dependent PTPs' within the PTP superfamily.


Assuntos
Fosfatase Ácida/fisiologia , Histidina/química , Proteínas Tirosina Fosfatases/fisiologia , Fosfatase Ácida/metabolismo , Animais , Domínio Catalítico , Cisteína/química , Humanos , Modelos Biológicos , Neurônios/metabolismo , Fosfatos/química , Filogenia , Plantas/metabolismo , Conformação Proteica , Proteínas Tirosina Fosfatases/metabolismo , Especificidade da Espécie
20.
Proc Natl Acad Sci U S A ; 105(43): 16590-5, 2008 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-18940929

RESUMO

Most newly synthesized proteins destined for the lysosome reach this location via a specific intracellular pathway. In the Golgi, a phosphotransferase specifically labels lysosomal proteins with mannose 6-phosphate (Man-6-P). This modification is recognized by receptors that target the lysosomal proteins to the lysosome where, in most cell types, the Man-6-P recognition marker is rapidly removed. Despite extensive characterization of this pathway, the enzyme responsible for the removal of the targeting modification has remained elusive. In this study, we have identified this activity. Preliminary investigations using a cell-based bioassay were used to follow a dephosphorylation activity that was associated with the lysosomal fraction. This activity was high in the liver, where endogenous lysosomal proteins are efficiently dephosphorylated, but present at a much lower level in the brain, where the modification persists. This observation, combined with an analysis of the expression of lysosomal proteins in different tissues, led us to identify acid phosphatase 5 (ACP5) as a candidate for the enzyme that removes Man-6-P. Expression of ACP5 in N1E-115 neuroblastoma cells, which do not efficiently dephosphorylate lysosomal proteins, significantly decreased the steady state levels of Man6-P glycoproteins. Analysis of ACP5-deficient mice revealed that levels of Man-6-P glycoproteins were highly elevated in tissues that normally express ACP5, and this resulted from a failure to dephosphorylate lysosomal proteins. These results indicate a central role for ACP5 in removal of the Man-6-P recognition marker and open up new avenues to investigate the importance of this process in cell biology and medicine.


Assuntos
Fosfatase Ácida/metabolismo , Isoenzimas/metabolismo , Manosefosfatos/metabolismo , Proteínas/metabolismo , Fosfatase Ácida/fisiologia , Animais , Linhagem Celular Tumoral , Glicoproteínas/análise , Humanos , Isoenzimas/fisiologia , Camundongos , Camundongos Knockout , Neuroblastoma/patologia , Fosforilação , Processamento de Proteína Pós-Traducional , Fosfatase Ácida Resistente a Tartarato
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