RESUMO
OBJECTIVE: The influence of prostatic acid phosphatase (PAP) and human chorionic gonadotropin (HCG), tumor markers have been investigated on human erythrocyte carbonic anhydrase (HCA-I and HCA-II) and bovine erythrocyte (BCA) and bovine lung carbonic anhydrase (CA-IV) in vitro. BACKGROUND: Tumor markers are substances that can often be detected in higher-than-normal amounts in the blood, urine, or body tissues of some patients with certain types of cancer. Tumor markers are produced either by the tumor itself or by the body in response to the presence of cancer or certain benign (noncancerous) conditions. In addition to their role in cancer diagnosis, some tumor marker levels are measured before treatment to help doctors plan appropriate therapy. RESULTS AND CONCLUSION: All of the tumor markers were determined to have inhibition effect, on human CA-I, CA-II, bovine erythrocyte CA (BCA) and bovine lung CA-IV isoenzymes. The effect of each tumor marker on CA was investigated by Wilbur-Andersen method modified by Rickly et al Inhibition effects of two different tumor markers on human CA-I, CA-II, bovine erythrocyte CA (BCA) and bovine lung CA-IV isoenzymes were determined by using the CO2-Hydratase method by plotting activity % vs (tumor markers). I50 values of tumor markers exhibiting inhibition effects were found by means of these graphs (Tab.1, Fig. 2, Ref. 20).
Assuntos
Fosfatase Ácida/farmacologia , Biomarcadores Tumorais/farmacologia , Anidrase Carbônica II/efeitos dos fármacos , Anidrase Carbônica IV/efeitos dos fármacos , Anidrase Carbônica I/efeitos dos fármacos , Inibidores da Anidrase Carbônica/farmacologia , Gonadotropina Coriônica/farmacologia , Animais , Anidrase Carbônica I/antagonistas & inibidores , Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica IV/antagonistas & inibidores , Anidrases Carbônicas/efeitos dos fármacos , Bovinos , Ensaios Enzimáticos , Eritrócitos/enzimologia , Humanos , Técnicas In Vitro , Pulmão/enzimologiaRESUMO
PAP248-286 is a 39-residue fragment (residues 248 to 286) derived from protease cleavage of prostatic acidic phosphatase in semen. The amyloid fibrils formed in vitro by PAP248-286 can dramatically enhance human immunodeficiency virus (HIV) infection. To our knowledge, we present the first report that the HIV-enhancing potency of fibrils formed by PAP248-286 is morphology dependent. We identified pleomorphic fibrils by transmission electron microscopy in two buffer conditions. Our solid-state NMR data showed that these fibrils consist of molecules in distinct conformations. In agreement with NMR, fluorescence measurements confirmed that they are assembled along different pathways, with distinct molecular structures. Furthermore, our cell-based infectivity tests detected distinct HIV-enhancing potencies for fibrils in distinct morphologies. In addition, our transmission electron microscopy and NMR results showed that semen-derived enhancer of viral infection fibrils formed in sodium bicarbonate buffer remain stable over time, but semen-derived enhancer of viral infection fibrils formed in phosphate buffered saline keep evolving after the initial 7 days incubation period. Given time, most of the assemblies in phosphate buffered saline will turn into elongated thin fibrils. They have similar secondary structure but different packing than thin fibrils formed initially after 7 days incubation.
Assuntos
Fosfatase Ácida/farmacologia , Amiloide/farmacologia , HIV-1/patogenicidade , Fosfatase Ácida/química , Amiloide/química , Linhagem Celular Tumoral , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Virulência/efeitos dos fármacosRESUMO
Immunotherapy is one of the attractive treatment strategies for advanced prostate cancer. The US Food and Drug Administration (FDA) previously approved the therapeutic vaccine, sipuleucel-T, which is composed of autologous antigen-presenting cells cultured with a fusion protein [prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GMCSF)]. Although sipuleucel-T has been shown to prolong the median survival of patients for 4.1 months, more robust therapeutic effects may be expected by modifying the vaccination protocol. In the present study, we aimed to develop and validate a novel vaccination strategy using multiple PAP-fused cytokines for prostate cancer treatment. Using a super gene expression (SGE) system that we previously established to amplify the production of a recombinant protein, significant amounts of PAP-fused cytokines [human GMCSF, interleukin-2 (IL2), IL4, IL7 and mouse GMCSF and IL4] were obtained. We examined the activity of the fusion proteins in vitro to validate their cytokine functions. A significant upregulation of dendritic cell differentiation from monocytes was achieved by PAP-GMCSF when used with the other PAP-fused cytokines. The PAP-fused human IL2 significantly increased the proliferation of lymphocytes, as determined by flow cytometry. We also investigated the in vivo therapeutic effects of multiple PAP-fused cytokines in a mouse prostate cancer model bearing prostate-specific antigen (PSA)- and PAP-expressing tumors. The simultaneous intraperitoneal administration of PAP-GMCSF, -IL2, -IL4 and -IL7 significantly prevented tumor induction and inhibited the tumor growth in the PAP-expressing tumors, yet not in the PSA-expressing tumors. The in vivo therapeutic effects with the multiple PAP-fused cytokines were superior to the effects of PAP-GMCSF alone. We thus demonstrated the advantages of the combined use of multiple PAP-fused cytokines including PAP-GMCSF, and propose a promising prostatic antigen-vaccination strategy to enhance the therapeutic effects.
Assuntos
Fosfatase Ácida/uso terapêutico , Adenocarcinoma/tratamento farmacológico , Vacinas Anticâncer/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Imunoterapia Ativa , Interleucinas/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Fosfatase Ácida/farmacologia , Adenocarcinoma/patologia , Animais , Antígenos de Neoplasias/imunologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Expressão Gênica , Genes Sintéticos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucinas/genética , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Monócitos/efeitos dos fármacos , Plasmídeos/genética , Neoplasias da Próstata/patologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
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 TartaratoRESUMO
A previous study showed that BMP-2 (bone morphogenetic protein-2) and wear debris can separately support osteoclast formation induced by the receptor activator of NF-κB ligand (RANKL). However, the effect of BMP-2 on wear debris-induced osteoclast formation is unclear. In this study, we show that neither titanium particles nor BMP-2 can induce osteoclast formation in RAW 264.7 mouse leukemic monocyte macrophage cells but that BMP-2 synergizes with titanium particles to enhance osteoclast formation in the presence of RANKL, and that at a low concentration, BMP-2 has an optimal effect to stimulate the size and number of multinuclear osteoclasts, expression of osteoclast genes, and resorption area. Our data also clarify that the effects caused by the increase in BMP-2 on phosphorylated SMAD levels such as c-Fos expression increased throughout the early stages of osteoclastogenesis. BMP-2 and titanium particles stimulate the expression of p-JNK, p-P38, p-IkB, and P50 compared with the titanium group. These data suggested that BMP-2 may be a crucial factor in titanium particle-mediated osteoclast formation.
Assuntos
Animais , Camundongos , /farmacologia , Diferenciação Celular/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Osteoclastos/metabolismo , Ligante RANK/farmacologia , Titânio/farmacologia , Fosfatase Ácida/farmacologia , Western Blotting , Reabsorção Óssea/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Sinergismo Farmacológico , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Isoenzimas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Smad/metabolismo , Fator de Necrose Tumoral alfa/isolamento & purificaçãoRESUMO
A previous study showed that BMP-2 (bone morphogenetic protein-2) and wear debris can separately support osteoclast formation induced by the receptor activator of NF-κB ligand (RANKL). However, the effect of BMP-2 on wear debris-induced osteoclast formation is unclear. In this study, we show that neither titanium particles nor BMP-2 can induce osteoclast formation in RAW 264.7 mouse leukemic monocyte macrophage cells but that BMP-2 synergizes with titanium particles to enhance osteoclast formation in the presence of RANKL, and that at a low concentration, BMP-2 has an optimal effect to stimulate the size and number of multinuclear osteoclasts, expression of osteoclast genes, and resorption area. Our data also clarify that the effects caused by the increase in BMP-2 on phosphorylated SMAD levels such as c-Fos expression increased throughout the early stages of osteoclastogenesis. BMP-2 and titanium particles stimulate the expression of p-JNK, p-P38, p-IkB, and P50 compared with the titanium group. These data suggested that BMP-2 may be a crucial factor in titanium particle-mediated osteoclast formation.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Diferenciação Celular/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Osteoclastos/metabolismo , Ligante RANK/farmacologia , Titânio/farmacologia , Fosfatase Ácida/farmacologia , Animais , Western Blotting , Reabsorção Óssea/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Sinergismo Farmacológico , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Isoenzimas/farmacologia , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Smad/metabolismo , Fosfatase Ácida Resistente a Tartarato , Fator de Necrose Tumoral alfa/isolamento & purificaçãoRESUMO
It is well documented that macrophages and eosinophils play important roles in normal murine pubertal mammary gland development. Although it is accepted that estrogen (E) and progesterone (P) are key players in mammary gland development, the roles these hormones might play in regulating the actions of leukocytes in that process is an understudied area. We show here that P and E, respectively, induce unique, but overlapping, sets of proinflammatory and angiogenic cytokines and chemokines, in the pubertal female BALB/c mammary gland, as well as induce infiltration of macrophages and eosinophils to the mammary periepithelium. This extends earlier studies showing P induction of proinflammatory products in pubertal and adult mammary epithelial organoids and P-induced in vivo infiltration of leukocytes to the adult mammary periepithelium. Importantly, epidermal growth factor receptor-signaling, which is likely mediated by amphiregulin (Areg), a downstream mediator of E and P, is both necessary and sufficient for both E- and P-induced recruitment of macrophages and eosinophils to the pubertal mammary periepithelium. We further show that receptor activator of nuclear factor κB ligand (RANKL), although not sufficient of itself to cause macrophage and eosinophil recruitment, contributes to an optimal response to P. The potency of Areg is highlighted by the fact that it is sufficient to induce macrophage and eosinophil recruitment at levels equivalent to that induced by either E or P. Our finding of a dominant role for Areg in hormonally induced leukocyte recruitment to the pubertal mammary gland parallels its dominance in regulating ductal outgrowth and its role in P-induced proliferation in the pubertal gland.
Assuntos
Receptores ErbB/metabolismo , Estrogênios/farmacologia , Leucócitos/citologia , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Progesterona/farmacologia , Fosfatase Ácida/farmacologia , Animais , Receptores ErbB/genética , Feminino , Imunofluorescência , Isoenzimas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Fosfatase Ácida Resistente a TartaratoRESUMO
The p38 mitogen-activated protein kinase (p38 MAPK) pathway is involved in the osteoclast differentiation. The aim of the study was to investigate whether SB203580, a p38 MAPK inhibitor, inhibits wear-debris-induced inflammatory osteolysis in mice. Forty-five mice were implanted with calvaria bone from syngeneic littermates; then, titanium (Ti) particles were injected into established air pouches to provoke inflammatory osteolysis. At 14 days after bone/Ti implantation, pouch membranes with intact bone implants underwent histological and molecular analysis. SB203580 had less effect on MMP-9 and TNF-α expression under wear-debris-induced conditions. SB203580, by inhibiting the expression of p38 MAPK and phospho-p38 MAPK, inhibited Ti particle wear-debris-induced inflammatory osteolysis. It also remarkably decreased the number of tartrate-resistant acid phosphatase positive cells in Ti-particle-induced pouch tissues. Results suggest that p38 MAPK may be critical in a murine osteolysis model. SB203580 may notably inhibit wear-debris-induced inflammatory osteolysis by down-regulating expression of MMP-9 and TNF-α via the p38 MAPK pathway.
Assuntos
Inflamação/metabolismo , Osteoclastos/efeitos dos fármacos , Osteólise/imunologia , Titânio/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Fosfatase Ácida/farmacologia , Animais , Osso e Ossos/metabolismo , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Feminino , Imidazóis/farmacologia , Isoenzimas/farmacologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteólise/metabolismo , Osteólise/patologia , Piridinas/farmacologia , RNA Mensageiro/biossíntese , Fosfatase Ácida Resistente a Tartarato , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Abruption-induced thrombin generation and inflammation/infection induced cytokine production have both been associated with fetal membrane (FM) weakening and preterm premature rupture of the fetal membranes (PPROM). Using our in vitro model system we have demonstrated that thrombin, and separately the cytokines, tumor necrosis factor-alpha (TNFα) and interleukin-1-beta (IL-1ß), remodel and weaken full thickness FM. Additionally, we have reported that the anti-oxidant and NFκB inhibitor, alpha-lipoic acid (LA), blocks these thrombin and cytokine induced effects. The purpose of these studies was to determine whether thrombin and cytokines directly weaken the amnion membrane (AM), the major load-bearing component of FM. Isolated AM or full thickness FM fragments from unlabored Cesarean deliveries were incubated with thrombin, TNFα, or IL-1ß, for 48 h. Rupture strength (breaking force) of each fragment was thereafter determined using our published methodology. Biochemical evidence of remodeling and apoptosis; immunoreactive Matrix Metalloproteinase 9 (MMP9), Tissue Inhibitor of Matrix Metalloproteinase 3 (TIMP3) and cleaved poly (ADP-ribose) polymerase (C-PARP) levels in tissue extracts, were determined by western blot and densitometry. Thrombin induced a dose-dependent weakening of isolated AM (P < 0.001) coupled with dose dependent increases in PARP cleavage, and reciprocal increases and decreases, respectively, in MMP9 and TIMP3 protein (all P < 0.01). Thrombin receptor activating peptide-6 (TRAP) also weakened isolated AM. Neither TNFα nor IL-1ß weakened isolated AM. However, both cytokines weakened AM when it was incubated together with the choriodecidua as part of full thickness FM (P < 0.001). Cytokine-conditioned choriodecidua medium also weakened isolated AM (P < 0.001). Under conditions in which cytokines weakened the AM, the changes in MMP9, TIMP3 and PARP cleavage were consistent with those seen after thrombin incubation. LA blocked the FM weakening and remodeling effects. In summary, thrombin weakens AM directly whereas cytokines weaken AM indirectly by causing the release of soluble intermediates from the choriodecidua.
Assuntos
Âmnio/fisiopatologia , Ruptura Prematura de Membranas Fetais/fisiopatologia , Interleucina-1beta/fisiologia , Trombina/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Fosfatase Ácida/farmacologia , Apoptose/fisiologia , Fenômenos Biomecânicos/fisiologia , Western Blotting , Densitometria , Feminino , Humanos , Técnicas In Vitro , Isoenzimas/farmacologia , Metaloproteinase 9 da Matriz/fisiologia , Glicoproteínas de Membrana/fisiologia , Gravidez , Proteínas de Protozoários/fisiologia , Fosfatase Ácida Resistente a Tartarato , Ácido Tióctico/farmacologia , Inibidor Tecidual de Metaloproteinase-3/fisiologiaRESUMO
Our aim was to assess the change in platelet activity along the menstrual cycle. We conducted a prospective observational study. The study group included 16 healthy women with regular menstrual cycles, which were compared to a control group of 14 healthy males. Exclusion criteria were age <18 years or >45 years, use of oral contraceptives or any other forms of hormonal therapy and medical disorders or medications that might affect platelet aggregation. Blood samples were taken from each of the women at four different phases of the menstrual cycle: day 1 +/- 1, day 7 +/- 1, day 14 +/- 1, and day 21 +/- 1. A single blood sample was taken from the males. Platelet aggregation was assessed in whole blood samples using the Multiplate analyzer with three different agonists (ADP, arachidonic acid (AA), and thrombin-receptor activating peptide (TRAP)). Platelet aggregation for each of the women at each of the phases of the menstrual cycle was expressed as the percentage change from the day 1 +/- 1 value. A total of 390 aggregation assays were performed. The mean aggregation activity was significantly higher in females compared with males, irrespective of the agonist used. For the TRAP and the ADP agonists, the relative platelet activity decreased along the menstrual cycle from day 1 towards day 21 and from day 7 towards day 21, respectively, although differences reached statistical significance only for day 21 (-12.4% +/- 3.2%, P < 0.05 for TRAP, and -9.5% +/- 3.9%, P < 0.05 for ADP). When using AA to induce platelet aggregation, the relative platelet activity was highest around the time of ovulation (11.0% +/- 4.7%) and was significantly lower on day 21 (-8.5% +/- 6.7%, P < 0.05). In conclusion, platelet aggregation activity is higher in females compared with males. The association between the phase of the menstrual cycle and platelet activity appears to vary with the type of agonist, but platelet aggregation is consistently lowest in the mid-luteal phase irrespective of the agonist used.
Assuntos
Ciclo Menstrual/sangue , Agregação Plaquetária/fisiologia , Fosfatase Ácida/farmacologia , Difosfato de Adenosina/farmacologia , Adulto , Ácido Araquidônico/farmacologia , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Fase Folicular/sangue , Humanos , Isoenzimas/farmacologia , Fase Luteal/sangue , Masculino , Agregação Plaquetária/efeitos dos fármacos , Estudos Prospectivos , Fosfatase Ácida Resistente a TartaratoRESUMO
Circulating platelets are heterogeneous in size and structure. Whether this translates into differences in platelet function and efficacy of antiplatelet therapy is unclear. Hence, we decided to investigate the activation patterns among different platelet populations differentiated by size, and to compare the inhibitory effects of aspirin in these populations. Circulating platelets from 9 healthy volunteers were separated by size and stratified into the largest and smallest quintiles. Platelets were stimulated with 75 µM arachidonic acid (AA), 10 µM ADP or 25 µM TRAP. Alpha-granule protein secretion and expression (P-selectin, VWF, fibrinogen), surface-protein activation (activated integrin αIIbß3) were assessed. Platelet thromboxane B(2) (TxB(2)) synthesis following AA stimulation was measured in vitro before and after incubation with 265 µM aspirin. Reticulated (juvenile) platelets were assessed using thiazole orange staining. A greater number of large platelets in the largest quintile were reticulated compared with the smallest quintile (6.1 ± 2.8% vs. 1.2 ± 1.5% respectively, p < 0.001). Larger platelets also synthesized more TxB(2) than small platelets both before (1348 ± 276 pg/mL vs. 1023 ± 214 pg/mL, respectively, p = 0.01) and after aspirin (1029 ± 190 pg/mL vs. 851 ± 159 pg/mL, respectively, p = 0.03). After stimulation with each agonist, a greater proportion of large platelets bound fibrinogen, VWF, P-selectin and activated integrin αIIbß3 than small platelets both in the presence and in the absence of in vitro aspirin. In an in vitro setting, large platelets appear to be more active than small platelets and continue to be more active even after in vitro aspirin. Platelets exhibit heterogeneity in size and structure. Whether this translates into platelet function and efficacy of antiplatelet therapy is unclear. We evaluated platelet functional properties and the effects of aspirin on separated platelet subpopulations in an in vitro setting. Platelets were sorted into the largest and smallest size quintiles using flow cytometry forward scatter. Alpha-granule protein release, dense granule content, surface protein activation and thromboxane synthesis were significantly greater in large platelets compared with small platelets, before and after stimulation with arachidonic acid, ADP and TRAP. Even after incubation with aspirin, large platelets continued to be more active than small platelets. In conclusion, large platelets are more active than small platelets and aspirin fails to eliminate these differential activation properties.
Assuntos
Aspirina/farmacologia , Plaquetas/citologia , Tamanho Celular , Ativação Plaquetária/efeitos dos fármacos , Fosfatase Ácida/farmacologia , Difosfato de Adenosina/farmacologia , Ácido Araquidônico/farmacologia , Células Cultivadas , Fibrinogênio/análise , Humanos , Isoenzimas/farmacologia , Selectina-P/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/análise , Fosfatase Ácida Resistente a Tartarato , Tromboxano B2/análise , Fator de von Willebrand/análiseAssuntos
Fosfatase Ácida/farmacologia , Difosfato de Adenosina/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Isoenzimas/farmacologia , Receptores 5-HT3 de Serotonina/sangue , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Ativação Plaquetária/efeitos dos fármacos , Receptores 5-HT3 de Serotonina/classificação , Fosfatase Ácida Resistente a TartaratoRESUMO
The aims of the study were to prepare polyelectrolyte nanocapsules effected by acid phosphatease (ACP) and to study prolonged-releasing performance of the nanocapsules in vitro. Using the layer by layer (LbL) self-assembly technique, polyelectrolyte-beta-glycerophosphoric acid nanocapsules were prepared. The morphologies of the nanocapsules were characterized by transmission electron microscopy (TEM) and biocompatibility was well examined by cell-culture method. The drug adriamycin would be loaded in nanocapsules for concentration gradient, the encapsulation efficiency could be calculated. Nanocapsules were reacted with acid phosphatease standard and HepG2 cells that express the ACP, respectively. The prolonged-releasing of adriamycin was verified and tumor cells apoptosis were measured. TEM images showed that the nanocapsule sizes were between 200-300 nm. The material biocompatibility was good until the concentration of nanacapsule was up to 250 microg/mL. The drug encapsulation efficiency reached 68.12%. The release rate of polyelectrolyte (PAH/PSS-beta-glycerophosphoric acid)s nanocapsules was higher than in the control nanocapsules at 48 h (38% Vs 15%) after its reaction to the ACP standard (P < 0.05). Compared with the control, nanocapsules could significantly inhibit the growth of HepG2 cells that expressed the ACP, and the efficiency of cell apoptosis was 7.59% higher at 24 h (13.73 Vs 6.14, P < 0.05). Polyelectrolytes (PAH/PSS-beta-glycerophosphoric acid) nanocapsules in vitro have response to acid phosphatease by which prolonged-releasing can be affected. This property can be used for treatment of some malignant and benign diseases with elevated acid phosphatease level.
Assuntos
Fosfatase Ácida/farmacologia , Doxorrubicina/administração & dosagem , Eletrólitos/química , Nanocápsulas , Antibióticos Antineoplásicos/administração & dosagem , Preparações de Ação Retardada/síntese química , Células Hep G2 , HumanosRESUMO
The P2X7 nucleotide receptor is an ATP-gated ion channel that plays an important role in bone cell function. Here, we investigated the effects of L: -tyrosine derivatives 1-3 as potent P2X7 antagonists on human primary osteoclasts. We found that the level of expression of P2X7 receptor increased after treatment with the derivatives 1-3, together with the induction of high levels of apoptosis. This effect is associated with activation of caspase-3 and inhibition of expression of IL-6. Interestingly, no pro-apoptotic effect of compounds 1-3 was found on human osteoblasts. Our results suggest that the development of specific P2X7 receptor antagonists may be considered a useful tool to modulate apoptosis of human osteoclasts. Since bone loss due to osteoclast-mediated resorption represents one of the major unsolved problem in osteopenic disorders, the identification of molecules able to induce apoptosis of osteoclasts is of great interest for the development of novel therapeutic strategies.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Apoptose , Inibidores Enzimáticos/farmacologia , Osteoclastos/patologia , Piperazinas/química , Antagonistas do Receptor Purinérgico P2 , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Fosfatase Ácida/farmacologia , Doenças Ósseas Metabólicas/patologia , Osso e Ossos/metabolismo , Caspase 3 , Caspases/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Ativação Enzimática , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Interleucina-6/metabolismo , Isoenzimas/farmacologia , Modelos Químicos , NF-kappa B/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Receptores Purinérgicos P2X7 , Fosfatase Ácida Resistente a Tartarato , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Fatores de TempoRESUMO
UNLABELLED: Stromal/osteoblastic cell expression of RANKL and M-CSF regulates osteoclastogenesis. We show that aging is accompanied by increased RANKL and M-CSF expression, increased stromal/osteoblastic cell-induced osteoclastogenesis, and expansion of the osteoclast precursor pool. These changes correlate with age-related alterations in the relationship between osteoblasts and osteoclasts in cancellous bone. INTRODUCTION: Bone mass is maintained through a balance between osteoblast and osteoclast activity. Osteoblasts regulate the number and activity of osteoclasts through expression of RANKL, osteoprotegerin (OPG), and macrophage-colony stimulation factor (M-CSF). To determine whether age-related changes in stromal/osteoblastic cell expression of RANKL, OPG, and M-CSF are associated with stimulation of osteoclastogenesis and whether the osteoclast precursor pool changes with age, we studied cultures of stromal/osteoblastic cells and osteoclast precursor cells from animals of different ages and examined how aging influences bone cell populations in vivo. MATERIALS AND METHODS: Osteoclast precursors from male C57BL/6 mice of 6 weeks (young), 6 months (adult), and 24 months (old) of age were either co-cultured with stromal/osteoblastic cells from young, adult, or old mice or treated with M-CSF, RANKL, and/or OPG. Osteoclast precursor pool size was determined by fluorescence-activated cell sorting (FACS), and osteoclast formation was assessed by measuring the number of multinucleated TRACP(+) cells and pit formation. The levels of mRNA for RANKL, M-CSF, and OPG were determined by quantitative RT-PCR, and transcription was measured by PCR-based run-on assays. Osteoblast and osteoclast numbers in bone were measured by histomorphometry. RESULTS: Osteoclast formation increased dramatically when stromal/osteoblastic cells from old compared with young donors were used to induce osteoclastogenesis. Regardless of the origin of the stromal/osteoblastic cells, the number of osteoclasts formed from the nonadherent population of cells increased with increasing age. Stromal/osteoblastic cell expression of RANKL and M-CSF increased, whereas OPG decreased with aging. Exogenously administered RANKL and M-CSF increased, dose-dependently, osteoclast formation from all donors, but the response was greater in cells from old donors. Osteoclast formation in vitro positively, and the ratio of osteoblasts to osteoclasts in vivo negatively, correlated with the ratio of RANKL to OPG expression in stromal/osteoblastic cells for all ages. The effects of RANKL-induced osteoclastogenesis in vitro were blocked by OPG, suggesting a causal relationship between RANKL expression and osteoclast-inducing potential. The osteoclast precursor pool and expression of RANK and c-fms increased with age. CONCLUSIONS: Our results show that aging significantly increases stromal/osteoblastic cell-induced osteoclastogenesis, promotes expansion of the osteoclast precursor pool and alters the relationship between osteoblasts and osteoclasts in cancellous bone.
Assuntos
Envelhecimento , Regulação da Expressão Gênica , Osteoblastos/citologia , Células Estromais/citologia , Fosfatase Ácida/farmacologia , Fatores Etários , Animais , Reabsorção Óssea , Osso e Ossos/metabolismo , Proteínas de Transporte/biossíntese , Núcleo Celular/metabolismo , Separação Celular , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Citometria de Fluxo , Glicoproteínas/metabolismo , Separação Imunomagnética , Isoenzimas/farmacologia , Fator Estimulador de Colônias de Macrófagos/biossíntese , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/citologia , Masculino , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteoporose , Osteoprotegerina , Reação em Cadeia da Polimerase , Ligante RANK , RNA Mensageiro/metabolismo , Receptor Ativador de Fator Nuclear kappa-B , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fosfatase Ácida Resistente a Tartarato , Fatores de TempoRESUMO
The G-protein alpha-subunit G(s)alpha is required for the intracellular cAMP responses to hormones and other agonists. G(s)alpha is known to mediate the cAMP response to parathyroid hormone and other hormones and cytokines in bone and cartilage. To analyze the in vivo role of G(s)alpha signaling in osteoblasts, we developed mice with osteoblast/osteocyte-specific G(s)alpha deficiency (BGsKO) by mating G(s)alpha-floxed mice with collagen Ialpha1 promoter-Cre recombinase transgenic mice. Early skeletal development was normal in BGsKO mice, because formation of the initial cartilage template and bone collar was unaffected. The chondrocytic zones of the growth plates also appeared normal in BGsKO mice. BGsKO mice had a defect in the formation of the primary spongiosa with reduced immature osteoid (new bone formation) and overall length, which led to reduced trabecular bone volume. In contrast, cortical bone was thickened with narrowing of the bone marrow cavity. This was probably due to decreased cortical bone resorption, because osteoclasts were markedly reduced on the endosteal surface of cortical bone. In addition, the expression of alkaline phosphatase, an early osteoblastic differentiation marker, was normal, whereas the expression of the late osteoblast differentiation markers osteopontin and osteocalcin was reduced, suggesting that the number of mature osteoblasts in bone is reduced. Expression of the osteoclast-stimulating factor receptor activator of NF-kappaB ligand was also reduced. Overall, our findings have similarities to parathyroid hormone null mice and confirm that the differential effects of parathyroid hormone on trabecular and cortical bone are primarily mediated via G(s)alpha in osteoblasts. Osteoblast-specific G(s)alpha deficiency leads to reduced bone turnover.
Assuntos
Osso e Ossos/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Osteoblastos/metabolismo , Fosfatase Ácida/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/metabolismo , Proteínas de Transporte/metabolismo , Cartilagem/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , AMP Cíclico/metabolismo , Citocinas/metabolismo , Primers do DNA/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Isoenzimas/farmacologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Modelos Genéticos , Osteoblastos/citologia , Osteocalcina/metabolismo , Osteopontina , Fenótipo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/metabolismo , Transdução de Sinais , Crânio/metabolismo , Fosfatase Ácida Resistente a Tartarato , Fatores de TempoRESUMO
Hyaluronic acid (HA) is a component of the extracellular matrix that has been shown to play an important role in bone formation, resorption, and mineralization both in vivo and in vitro. We examined the effects of HA at several molecular weights on osteoclast formation and function induced by RANKL (receptor activator of NF-kappa B ligand) in a mouse monocyte cell line (RAW 264.7). HA at M(r) < 8,000 (low molecular weight HA (LMW-HA)) enhanced tartrate-resistant acid phosphatase-positive multinucleated cell formation and tartrate-resistant acid phosphatase activity induced by RANKL in a dose-dependent manner, whereas HA at M(r) > 900,000 (high molecular weight HA (HMW-HA)) showed no effect on osteoclast differentiation. LMW-HA enhanced pit formation induced by RAW 264.7 cells, whereas HMW-HA did not, and LMW-HA stimulated the expression of RANK (receptor activator of NF-kappa B) protein in RAW 264.7 cells. In addition, we found that LMW-HA enhanced the levels of c-Src protein and phosphorylation of ERKs and p38 MAPK in RAW 264.7 cells stimulated with RANKL, whereas the p38 MAPK inhibitor SB203580 inhibited RANKL-induced osteoclast differentiation. This enhancement of c-Src and RANK proteins induced by LMW-HA was inhibited by CD44 function-blocking monoclonal antibody. These results indicate that LMW-HA plays an important role in osteoclast differentiation and function through the interaction of RANKL and RANK.
Assuntos
Ácido Hialurônico/farmacologia , Osteoclastos/metabolismo , Fosfatase Ácida/farmacologia , Animais , Anticorpos Monoclonais/metabolismo , Western Blotting , Reabsorção Óssea , Proteína Tirosina Quinase CSK , Proteínas de Transporte/metabolismo , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Matriz Extracelular/metabolismo , Receptores de Hialuronatos/biossíntese , Imidazóis/farmacologia , Isoenzimas/farmacologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Peso Molecular , Monócitos/metabolismo , Osteoclastos/efeitos dos fármacos , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Piridinas/farmacologia , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Transdução de Sinais , Fosfatase Ácida Resistente a Tartarato , Fatores de Tempo , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases da Família srcRESUMO
Phosphorus (P) is the second most important macronutrient for plant growth. Plants exhibit numerous physiological and metabolic adaptations in response to seasonal variations in phosphorus content. Activities of acid and alkaline phosphatases, ATPase and ATP content were studied in summer, rainy and winter seasons at two different developmental stages (28 and 58 days after sowing) in Okra. Activities of both acid and alkaline phosphatases increased manifold in winter to cope up with low phosphorus content. ATP content and ATPase activity were high in summer signifying an active metabolic period. Phosphorus deficiency is characterized by low ATP content and ATPase activity (which are in turn partly responsible for a drastic reduction in growth and yield) and enhanced activities of acid and alkaline phosphatases which increase the availability of P in P-deficient seasons.
Assuntos
Abelmoschus/fisiologia , Fosfatase Ácida/farmacologia , Adenosina Trifosfatases/farmacologia , Fosfatase Alcalina/farmacologia , Fósforo/metabolismo , Fosfatase Ácida/análise , Adaptação Fisiológica , Adenosina Trifosfatases/análise , Fosfatase Alcalina/análise , Estações do AnoRESUMO
The sensitivity of protozoa, and particularly ciliated protozoa, to environmental changes suggested a study on the physiological responses arising from exposure to toxic compounds. Tetrahymena pyriformis was used as a test organism in a set of miniaturized assays. The physiological response of this ciliate was assessed in terms of adenosine-5'-triphosphate content and acid phosphatase activity after exposure of the cultures of T. pyriformis to four toxicants: copper, zinc, Triton X-100, and cycloheximide. In the range of concentrations used, stimulation and inhibition of these two parameters were observed. The correlation between the two parameters is analyzed.
Assuntos
Fosfatase Ácida/farmacologia , Trifosfato de Adenosina/análise , Antifúngicos/toxicidade , Cobre/toxicidade , Cicloeximida/toxicidade , Detergentes/toxicidade , Octoxinol/toxicidade , Tetrahymena/fisiologia , Zinco/toxicidade , Trifosfato de Adenosina/metabolismo , Animais , Relação Dose-Resposta a Droga , Tetrahymena/efeitos dos fármacosRESUMO
DNase II is involved in DNA fragmentation induced by a variety of treatments. However, according to past reports DNase II does not directly generate TUNEL (in situ DNA end labeling)-positive cells. The purpose of this study was to investigate the participation of acid phosphatase in the generation of TUNEL-positive cells. DNase II-like proteins, whose molecular weights were 32-kDa, were detected in nuclear extracts of HL60 human myeloid leukemia cells post gamma-irradiation by SDS-PAGE and immunohistochemistry. Acidic nuclease activity was especially active in 32-kDa bands TUNEL assay was positive post gamma-irradiation. From measurements of the activity of acid phosphatase, the activity in nuclear extracts increased remarkably post gamma-irradiation. Gamma-irradiation can directly or indirectly activate DNase II. Once DNase II and acid phosphatase have been translocated from lysosomes into the nuclei, DNase II generates TUNEL reactive ends in combination with acid phosphatase.