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BACKGROUND: Seven peptides with low molecular weights in blood have been identified as possible biomarkers of hypertensive disorders of pregnancy (HDP). A history of HDP is known to be associated with a high risk of cardiovascular disease in the later life of women with HDP. However, it remains to be determined whether HDP-related peptides are useful biomarkers of cardiovascular disease in the general population. The purpose of this study was to determine the relationships between these peptides and cardiometabolic risk in adult men. METHODS: We investigated the relationships between HDP-related peptides and two recent indices of cardiometabolic risk, hematometabolic index (HMI) and lipid accumulation product (LAP), in male workers aged 35 to 69 years. Concentrations of the HDP-related seven peptides with mass/charge ratios (m/z) of 2081 (P-2081), 2091 (P-2091), 2127 (P-2127), 2209 (P-2209), 2378 (P-2378), 2858 (P-2858), and 3156 (P-3156) were measured simultaneously by using a mass spectrometer. Standardized partial regression coefficients (ß) were obtained in multivariable linear regression analysis, and mean levels of the log-transformed HMI and LAP were compared in tertile groups of each peptide in the analysis of covariance with adjustment for age, habits of smoking and alcohol drinking, history of diabetes, and medication therapy for dyslipidemia. RESULTS: There was a significant positive correlation between the HMI and the serum level of P-2378 (ß = 0.310), a fragment of complement component 4, while a significant inverse correlation (ß = -0.389) was obtained between the LAP and the serum level of P-3156, a fragment of inter-α-trypsin inhibitor heavy chain H4. Other peptides (P-2081, P-2091, P-2127, P-2209, and P-2858) did not show significant correlations with the HMI or LAP. The log-transformed HMI tended to be higher with an increase in the tertile for P-2378. The mean level of log-transformed LAP in the first tertile group of P-3156 was significantly higher than those in the second and third tertile groups of P-3156. CONCLUSION: The HDP-related peptides with m/z of 2378 and m/z of 3156 were shown to be associated with the HMI and LAP, respectively, which are recent indices reflecting cardiometabolic risk. Therefore, the peptides with m/z of 2378 and m/z of 3156 were thought to be potential biomarkers for discrimination of cardiovascular risk in adult men. Further studies on the relationships between the peptides and cardiovascular risk factors in non-pregnant women are needed to confirm the findings of this study.
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BACKGROUND AND AIMS: Seven circulating peptides, consisting of 18-28 amino acids, were identified as possible biomarkers of hypertensive disorders of pregnancy (HDP) in our previous study. However, it is unknown whether these peptides are relevant to cardiovascular disease. The purpose of this study was to clarify the relationships between serum levels of these peptides and leg arterial blood flow in patients with lower extremity arterial disease (LEAD). METHODS: The subjects were 165 outpatients with LEAD. Patients with advanced LEAD (stages 5 and 6 of the Rutherford classification) were not included. Leg arterial blood flow was evaluated by ankle brachial pressure index (ABI) and % decrease in ABI after leg exercise induced by a leg loader or treadmill. Concentrations of the seven peptides with m/z 2081 (P-2081), 2091 (P-2091), 2127 (P-2127), 2209 (P-2209), 2378 (P-2378), 2858 (P-2858) and 3156 (P-3156) were measured simultaneously with a mass spectrometer. RESULTS: P-2081, P-2127 and P-2209 levels showed significant positive correlations with leg arterial blood flow, while P-2091, P-2378 and P-2858 levels showed significant inverse correlations with leg arterial blood flow. There was no significant correlation between P-3156 levels and leg arterial blood flow. The above positive and inverse associations between peptide levels and leg arterial blood flow were also found in logistic regression analysis using tertile groups divided by the concentrations of each peptide. CONCLUSIONS: Serum levels of six HDP-related peptides (P-2081, P-2091, P-2127, P-2209, P-2378 and P-2858) were associated with lower extremity arterial blood flow in patients with LEAD, and thus these peptides are possible biomarkers for severity of LEAD.
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Hipertensión Inducida en el Embarazo , Embarazo , Femenino , Humanos , Extremidad Inferior , Índice Tobillo Braquial , Arterias , BiomarcadoresRESUMEN
We previously identified seven peptides in serum that are associated with hypertensive disorders of pregnancy (HDP). However, the significance of these peptides in the general population is unknown. The aim of this study was to clarify the relationships of HDP-associated peptides with hypertension and other cardiovascular risks in adult men. We investigated the relationships of peptide levels with cardiovascular risk factors, including adiposity, blood pressure, blood lipids and glycemic status, in men (mean age: 46.4 years) who were receiving annual health checkups at their workplace. The concentrations of the abovementioned seven peptides in serum were measured simultaneously using a mass spectrometer. Among the seven peptides, only a peptide with m/z 2091 (P-2091) derived from fibrinogen-α showed a significant correlation with diastolic blood pressure (Spearman's rank correlation coefficient [r], -0.446). Another peptide with m/z 2378 (P-2378) originating from complement component 4 showed a significant positive correlation with body mass index (r, 0.273) and a significant inverse correlation with HDL cholesterol (r, -0.336). In addition, a peptide with m/z 3156 (P-3156) derived from an inter-α-trypsin inhibitor showed significant inverse correlations with body mass index (r, -0.258) and triglycerides (r, -0.334). There was no significant correlation of the levels of any of the seven peptides with hemoglobin A1c. Among the seven peptides related to HDP, P-2091, P-2378 and P-3156 were inversely associated with diastolic blood pressure, HDL cholesterol and triglycerides, respectively. Therefore, these peptides are possible biomarkers for discriminating cardiovascular risk in a general population.
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Enfermedades Cardiovasculares , Hipertensión Inducida en el Embarazo , Adulto , Presión Sanguínea , Índice de Masa Corporal , Enfermedades Cardiovasculares/etiología , Femenino , Factores de Riesgo de Enfermedad Cardiaca , Humanos , Masculino , Persona de Mediana Edad , Péptidos , Embarazo , Factores de RiesgoRESUMEN
Liposomal fasudil as a treatment for cerebral ischemia/reperfusion (I/R) injury has been demonstrated to be effective in animal models due to the high accumulation of liposomes in damaged brain tissue. However, it is still unclear what effect drug release rate has on the treatment of I/R injury, where pathology progresses dramatically in a short time. In the present study, we assessed four formulations of liposomal fasudil. The results of an in vitro drug release assay showed that the release properties of fasudil were changed by varying the lipid composition and internal phase of the liposomes. Based on these results, differences in the transition of fasudil plasma concentration were monitored after the administration of each type of liposomal fasudil in normal rats. A pharmacokinetic study showed that higher levels of drug retention in liposomal fasudil resulted in higher fasudil plasma concentration. Finally, treatment of I/R injury model rats with liposomal fasudil revealed that a mid-level release rate of fasudil from liposomes resulted in the greatest therapeutic effect among the formulations. In conclusion, these results demonstrate that an optimized drug release rate from liposomes enhances the therapeutic effect of fasudil for the treatment of cerebral I/R injury.
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1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Liposomas/química , Daño por Reperfusión/tratamiento farmacológico , 1,2-Dipalmitoilfosfatidilcolina/química , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/sangre , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacocinética , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Sulfato de Amonio/química , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Ácido Cítrico/química , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Composición de Medicamentos , Sistemas de Liberación de Medicamentos/métodos , Liberación de Fármacos , Liposomas/farmacocinética , Masculino , Fosfatidilcolinas/química , Compuestos de Amonio Cuaternario/química , Ratas Wistar , Daño por Reperfusión/patología , Resultado del TratamientoRESUMEN
Hypertensive disorders of pregnancy (HDP) is the most common and widely known as serious complication of pregnancy. As this syndrome is a major leading cause of maternal, fetal, and neonatal morbidity/mortality worldwide, many studies have sought to identify candidate molecules as potential disease biomarkers (DBMs) for use in clinical examinations. Accumulating evidence over the past 2 decades that the many proteolytic peptides appear in human humoral fluids, including peripheral blood, in association with an individual's health condition. This review provides the potential utility of peptidomic analysis for monitoring for pathophysiological status in HDP, and presents an overview of current status of peptide quantification technology. Especially, the technical limitations of the methods used for DBM discovery in the blood are discussed.
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Hipertensión/sangre , Péptidos/sangre , Complicaciones Cardiovasculares del Embarazo/sangre , Biomarcadores/sangre , Femenino , Humanos , Hipertensión/complicaciones , Embarazo , Complicaciones Cardiovasculares del Embarazo/fisiopatologíaRESUMEN
NF-κB plays a key role in the transcriptional regulation of genes involved in immunity, inflammation, cell proliferation, and oncogenesis. The NF-κB activation process includes nuclear translocation, followed by association with basal transcription machinery. These steps are tightly regulated by posttranslational modification of the proteins involved in this pathway. We recently reported that NF-κB transactivation activity is enhanced by knockdown of diacylglycerol kinase ζ (DGKζ), which belongs to an enzyme family that phosphorylates lipidic second messenger diacylglycerol to phosphatidic acid. To investigate details of the regulatory mechanism exerted by DGKζ, we identified DEAD-box RNA helicase DDX5 as a novel DGKζ-interacting protein and examined functional role of DDX5 in NF-κB transactivation activity. Here we show that DDX5 knockdown exerts no significant effect on nuclear translocation, but specifically attenuates Ser311 phosphorylation of p65 subunit. Luciferase reporter assay reveals that the NF-κB transcriptional activity is repressed in DDX5-knockdown cells. Furthermore, we found that DDX5 knockdown selectively downregulates the expression level of Bcl-2 of the NF-κB-inducible anti-apoptotic factors upon TNF-α stimulation. Considering the evidence collectively, we can infer that DGKζ-interacting multi-protein complex modulates the NF-κB transactivation activity in a negative and positive manner under conditions in which the expression level of a component of the complex is altered.
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Apoptosis , ARN Helicasas DEAD-box/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Serina/metabolismo , Factor de Transcripción ReIA/metabolismo , Apoptosis/efectos de los fármacos , Cicloheximida/farmacología , Diacilglicerol Quinasa/metabolismo , Células HEK293 , Células HeLa , Humanos , Modelos Biológicos , Fosforilación/efectos de los fármacos , Unión Proteica , Fracciones Subcelulares/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Purpose We previously attempted to develop quantitative enzyme-linked immunosorbent assay (ELISA) systems for the PDA039/044/071 peptides, potential serum disease biomarkers (DBMs) of pregnancy-induced hypertension (PIH), primarily identified by a peptidomic approach (BLOTCHIP®-mass spectrometry (MS)). However, our methodology did not extend to PDA071 (cysteinyl α2-HS-glycoprotein341-367), due to difficulty to produce a specific antibody against the peptide. The aim of the present study was to establish an alternative PDA071 quantitation system using liquid chromatography-multiple reaction monitoring (LC-MRM)/MS, to explore the potential utility of PDA071 as a DBM for PIH. Methods We tested heat/acid denaturation methods in efforts to purify serum PDA071 and developed an LC-MRM/MS method allowing for specific quantitation thereof. We measured serum PDA071 concentrations, and these results were validated including by three-dimensional (3D) plotting against PDA039 (kininogen-1439-456)/044 (kininogen-1438-456) concentrations, followed by discriminant analysis. Results PDA071 was successfully extracted from serum using a heat denaturation method. Optimum conditions for quantitation via LC-MRM/MS were developed; the assayed serum PDA071 correlated well with the BLOTCHIP® assay values. Although the PDA071 alone did not significantly differ between patients and controls, 3D plotting of PDA039/044/071 peptide concentrations and construction of a Jackknife classification matrix were satisfactory in terms of PIH diagnostic precision. Conclusions Combination analysis using both PDA071 and PDA039/044 concentrations allowed PIH diagnostic accuracy to be attained, and our method will be valuable in future pathophysiological studies of hypertensive disorders of pregnancy.
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Hipertensión Inducida en el Embarazo/sangre , Péptidos/sangre , alfa-2-Glicoproteína-HS/metabolismo , Biomarcadores/sangre , Cromatografía Liquida/métodos , Femenino , Humanos , Espectrometría de Masas/métodos , EmbarazoRESUMEN
We previously established an anti-mouse sperm auto-monoclonal antibody, Ts4, which shows immunoreactivity against several kinds of glycoproteins in the acrosomal region of epididymal spermatozoa, testicular germ cells, and early embryo, via binding to an epitope containing a common N-linked oligosaccharide (OS) chain on the molecules. In mice, we have already demonstrated that the OS chain in the epitope for Ts4 is a fucosylated agalacto-complex-type biantennary glycan carrying bisecting N-acetylglucosamine. In the testis, one of the specific OS chain-conjugated molecules is TEX101, a germ cell-marker glycoprotein, which is expressed in spermatocytes, spermatids, and testicular spermatozoa, but not in epididymal spermatozoa. In this study, we identified a Ts4-reactive glycoprotein in mouse cauda epididymal sperm. An immunoprecipitation method together with liquid chromatography-tandem mass spectrometry showed that alpha-N-acetylglucosaminidase (Naglu; a degradation enzyme of heparan sulfate) is one of the glycoproteins recognized by Ts4 in the epididymal spermatozoa. Western blot and immunohistochemical analyses revealed that mouse Naglu exists in two forms (82 and 77kDa) and is expressed in the acrosomal region and the flagellum of cauda epididymal sperm. Of the two Naglu-forms expressed in sperm, Ts4 immunoreacted against only the 82-kDa form located on the acrosomal region. The Ts4 mAb and anti-Naglu pAb negatively affected mouse fertilization in vitro. In addition, Ts4 inhibited sperm acrosome reaction induced by heparan sulfate. The Ts4-recognized fucosylated agalactobiantennary complex-type glycan with bisecting N-acetylglucosamine and Naglu on cauda epididymal spermatozoa may play a role in the process of fertilization.
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Acrosoma/metabolismo , Epidídimo/patología , Epítopos/metabolismo , Infertilidad/inmunología , Espermatozoides/metabolismo , Acrosoma/inmunología , Reacción Acrosómica/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Autoanticuerpos/metabolismo , Autoinmunidad , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Espermatozoides/inmunología , Espermatozoides/patologíaRESUMEN
BACKGROUND: We previously reported peptide candidates of disease biomarkers for pregnancy-induced hypertension syndrome using a novel peptidomic analytical method, BLOTCHIP®-MS. The aim of this study was to establish a sandwich enzyme-linked immunosorbent assay system for quantitation of such peptides and to validate their usefulness as disease biomarkers of pregnancy-induced hypertension syndrome including gestational hypertension/pre-eclampsia. METHODS: We focused on three peptide fragments, kininogen-1439-456 (PDA039), kininogen-1438-456 (PDA044) and cysteinyl α2-HS-glycoprotein341-367 (PDA071). Using polyclonal antibodies specific for each peptide, suitable conditions for the sandwich enzyme-linked immunosorbent assay system were investigated. The quantitative enzyme-linked immunosorbent assay values were confirmed by quantitative matrix assisted laser desorption/ionization time-of-flight MS analyses. Using the established enzyme-linked immunosorbent assay systems, serum samples from gestational hypertension/pre-eclampsia patients and paired serum samples from healthy pregnant females were analysed. RESULTS: The optimum sandwich enzyme-linked immunosorbent assay conditions for PDA039/044 quantitation were developed. Quantitation of PDA071 by enzyme-linked immunosorbent assay failed, presumably due to issues with polyclonal antibody specificity for the native peptide. Bland-Altman plots showed a satisfactory correlation between the serum PDA039/044 concentration by enzyme-linked immunosorbent assay and that by quantitative MS analysis. Although the PDA044 concentration showed no significant change during pregnancy, including gestational hypertension/pre-eclampsia patients, the serum PDA039 concentration was significantly increased (P < 0.0001) in the patients. CONCLUSIONS: The simple quantitation technology for PDA039 by enzyme-linked immunosorbent assay was established for the first time. PDA039 confirmed its clinical utility as a disease biomarker for gestational hypertension/pre-eclampsia by the enzyme-linked immunosorbent assay system using clinical samples. The information provided from the present study would be a new valuable addition in the field of gestational hypertension/pre-eclampsia research.
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Hipertensión Inducida en el Embarazo/sangre , Péptidos/sangre , Proteómica/métodos , Adulto , Biomarcadores/sangre , Femenino , Humanos , Hipertensión Inducida en el Embarazo/diagnóstico , Preeclampsia/sangre , Preeclampsia/diagnóstico , Embarazo , Adulto JovenRESUMEN
Various types of polycyclic aromatic compounds (PACs) in diesel exhaust particles are thought to contribute to carcinogenesis in mammals. Although the carcinogenicity, mutagenicity and tumour-initiating activity of these compounds have been evaluated, their tumour-promoting activity is unclear. In the present study, to determine the tumour-inducing activity of PACs, including previously known mutagenic compounds in atmospheric environments, a transformation assay for promoting activity mediated by the release of contact inhibition was conducted for six polycyclic aromatic hydrocarbons (PAHs), seven oxygenated PAHs (oxy-PAHs) and seven nitrated PAHs (nitro-PAHs) using mouse embryonic fibroblast cells transfected with the v-Ha-ras gene (Bhas 42 cells). Of these, two PAHs [benzo[k]fluoranthene (B[k]FA) and benzo[b]fluoranthene (B[b]FA)], one oxy-PAH [6H-benzo[cd]pyren-6-one (BPO)] and two nitro-PAHs (3-nitro-7H-benz[de]anthracen-7-one and 6-nitrochrysene) were found to exhibit particularly powerful tumour-promoting activity (≥10 foci following exposure to <100nM). In addition, clear mRNA expression of CYP1A1, which is associated with aryl hydrocarbon receptor (AhR)-mediated activation, was observed following the exposure of cells to two PAHs (B[k]FA and B[b]FA) and three oxy-PAHs (1,2-naphthoquinone, 11H-benzo[b]fluoren-11-one and BPO). Further, an HO-1 antioxidant response activation was observed following exposure to B[k]FA, B[b]FA and BPO, suggesting that the induction of tumour-promoting activity in these compounds is correlated with the dysfunction of signal transduction via AhR-mediated responses and/or oxidative stress responses.
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Carcinógenos/química , Carcinógenos/toxicidad , Transformación Celular Neoplásica/inducido químicamente , Hidrocarburos Policíclicos Aromáticos/química , Hidrocarburos Policíclicos Aromáticos/toxicidad , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Ratones , Mutágenos/toxicidad , ARN Mensajero/genética , Emisiones de Vehículos/toxicidadRESUMEN
Lactosylceramide (LacCer), which is essential for many cellular processes, is highly expressed on the plasma membranes of human neutrophils and mediates innate immune functions. Less is known, however, about the properties and biological functions of LacCer in mouse neutrophils. This study therefore analyzed the properties of mouse neutrophil LacCer. LacCer was observed on the surface of these cells, with flow cytometry indicating that mouse neutrophil LacCer could be detected by the anti-LacCer mAb T5A7, but not by the anti-LacCer antibodies Huly-m13 and MEM-74. The molecular species of LacCer were nearly identical in mouse and human neutrophils, including C24:0 and C24:1 fatty acid chain-containing species, although the LacCer content in plasma membranes was â¼ 20-fold lower in mouse than in human neutrophils. Surface plasmon resonance analysis revealed that T5A7 bound to a lipid monolayer composed of LacCer, DOPC, cholesterol and sphingomyelin (molar ratio 0.1 : 10 : 10 : 1), whereas Huly-m13 did not. T5A7 induced neutrophil migration, which was abolished by inhibitors of Src-family kinases, PI-3 kinases, and trimeric G (o/i) proteins. T5A7 also inhibited phagocytosis of non-opsonized zymosans by neutrophils. Taken together, these findings suggest that in mouse neutrophils, (i) LacCer is expressed as LacCer-enriched microdomains in cell surface plasma membranes, (ii) these microdomains are recognized by T5A7 but not by other known anti-LacCer antibodies and (iii) LacCer is involved in cell migration and phagocytosis.
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Antígenos CD/inmunología , Antígenos CD/metabolismo , Lactosilceramidos/inmunología , Lactosilceramidos/metabolismo , Neutrófilos/química , Animales , Anticuerpos Monoclonales/inmunología , Reacciones Antígeno-Anticuerpo , Antígenos CD/biosíntesis , Calcio/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Voluntarios Sanos , Humanos , Lactosilceramidos/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/citología , Neutrófilos/inmunología , Neutrófilos/metabolismoRESUMEN
OBJECTIVE: We have shown that connective tissue growth factor (CTGF) plays an important role in the pathogenesis of rheumatoid arthritis (RA). Insulin-like growth factor binding proteins (IGFBPs) are modules of CTGF. IGFBPs bind IGF-I and IGF-II. IGF-I plays a role in the regulation of immunity, bone metabolism and inflammation. Therefore, we investigated how the IGF system is associated with RA disease progression. METHODS: Serum samples were collected from RA patients. IGF-I and IGFBP-3 production were evaluated by enzyme-linked immunosorbent assay, real-time RT-PCR and indirect immunofluorescence microscopy. Osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase staining, a bone resorption assay and osteoclast-specific enzyme production. Angiogenesis was examined by a tube formation assay using human umbilical vein endothelial cells. RESULTS: The serum concentrations of IGFBP-3 in RA patients were greater than those in normal controls. IGF-I and IGFBP-3 were produced primarily by macrophages in the RA synovium. Furthermore, tumor necrosis factor-α could induce aberrant IGF-I and IGFBP-3 production in synovial fibroblasts. IGF-I and IGFBP-3 promoted the induction of osteoclast generation and morphological changes, in combination with M-colony stimulating factor and the receptor activator of NF-κB ligand. In addition, IGF-I and IGFBP-3 induced angiogenesis, as determined by the tube formation assay. These effects were neutralized by anti-IGF-IR monoclonal antibody (mAb). CONCLUSIONS: These results indicate that aberrant IGF-I and IGFBP-3 production plays a role in abnormal osteoclastic activation and angiogenesis in RA. This work supports future clinical exploration of anti-IGF-IR mAb in drug repositioning as a new treatment for RA.
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Artritis Reumatoide/metabolismo , Somatomedinas/antagonistas & inhibidores , Adulto , Anciano , Anticuerpos Monoclonales/farmacología , Artritis Reumatoide/sangre , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Proteína C-Reactiva/metabolismo , Línea Celular , Progresión de la Enfermedad , Femenino , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Metaloproteinasa 3 de la Matriz/metabolismo , Persona de Mediana Edad , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Osteoclastos/metabolismo , Ligando RANK/metabolismo , Receptor IGF Tipo 1 , Receptores de Somatomedina/antagonistas & inhibidores , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismoRESUMEN
To date, numerous studies have searched for candidate molecules or clinical examination methods as potential biomarkers for monitoring intractable diseases, such as carcinomas. Evidence accumulated over the past decade shows that many proteolytic peptides appear in human humoral fluids, including peripheral blood, in association with an individual's health condition. Although an analysis of the whole peptide (the 'peptidome') using mass spectrometry is thought to be one of the most powerful and promising experimental approaches, it has failed to identify biomarkers in the clinical blood samples, presumably due to the methodological limitations. In general, commonly used techniques for proteomic analysis of blood require the removal of large amounts of serum/plasma proteins prior to mass spectrometry analysis, and this step seems to have resulted in the overlooking of important biomarkers during the analytical process. Here, we provide a brief overview of a new quantitative peptidomic analysis by a one-step direct transfer technology without depletion of major blood proteins. Using this technology, we herein report experimental data on serum peptidomic analysis for patients with pregnancy-induced hypertension as a clinical model. In addition, we refer to the potential utility of this approach for the monitoring of pathophysiological status in female reproductive system disorders in general.
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Enfermedades de los Genitales Femeninos/metabolismo , Proteómica/tendencias , Femenino , Enfermedades de los Genitales Femeninos/fisiopatología , Humanos , Hipertensión Inducida en el Embarazo/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transferencia de TecnologíaRESUMEN
Rheumatoid arthritis (RA) is a chronic inflammatory disorder of the synovial membrane that results in the destruction of bone and cartilage in affected joints. Tocilizumab is a biological agent and an anti-interleukin-6 (IL-6) receptor monoclonal antibody that blocks IL-6-mediated inflammatory processes in RA patients. In order to identify novel disease-related proteins and candidate biomarkers, we analyzed the changes in the serum proteome profiles of patients with RA who were treated with tocilizumab. Serum samples were collected from the RA patients before and after tocilizumab treatment. Following immunodepletion of major proteins, the proteins were digested and labeled with isobaric tag, iTRAQ reagent. The proteins were identified and quantified using liquid chromatography-tandem mass spectrometry. Among a total of 311 proteins identified, seven were decreased and 16 were increased by tocilizumab treatment. Although some of the proteins are known to be related to RA, several are currently unknown with respect to their relationship to RA and may be involved in the development of this disease. This study is the first to perform a comparative serum proteomic analysis of RA patients treated with tocilizumab. Our results may contribute to the identification of novel disease-related proteins and enhance the understanding of the pathogenesis of RA.
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Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Artritis Reumatoide/sangre , Artritis Reumatoide/tratamiento farmacológico , Proteoma/metabolismo , Receptores de Interleucina-6/antagonistas & inhibidores , Suero/metabolismo , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Persona de Mediana Edad , Receptores de Interleucina-6/metabolismoRESUMEN
OBJECTIVE: We have shown that connective tissue growth factor (CTGF) plays an important role in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to evaluate the effects of blockade of the CTGF pathway on the development of collagen-induced arthritis (CIA) in mice. METHODS: Arthritis was induced in DBA/1J mice by immunization with a combination of type II collagen (CII) and Freund's complete adjuvant. We evaluated the development of arthritis in mice with CIA left untreated versus treated with neutralizing anti-CTGF monoclonal antibody (mAb). RESULTS: Inhibition of CTGF in mice treated with neutralizing anti-CTGF mAb significantly ameliorated arthritis compared to the untreated mice with CIA. Serum levels of matrix metalloproteinase 3 were reduced by anti-CTGF mAb treatment. Moreover, blockade of CTGF decreased interleukin-17 expression on purified CD4+ T lymphocytes. Although the expression of the retinoic acid receptor-related orphan receptor γt gene was not suppressed by anti-CTGF mAb treatment, that of interferon regulatory factor 4 (IRF-4) and IκBζ (Nfkbiz), which are other important molecules for the differentiation of Th17 cells, was suppressed. In addition, blockade of CTGF inhibited pathologic proliferation of T lymphocytes in response to CII restimulation in vitro. Moreover, aberrant osteoclastogenesis in mice with CIA was restored by anti-CTGF mAb treatment. CONCLUSION: Our findings indicate that blockade of CTGF prevents the progression of arthritis in mice with CIA. Anti-CTGF mAb treatment suppresses pathologic T cell function and restores aberrant osteoclastogenesis in mice with CIA. CTGF may become a new target for the treatment of RA.
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Anticuerpos Monoclonales/farmacología , Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Factor de Crecimiento del Tejido Conjuntivo/antagonistas & inhibidores , Activación de Linfocitos/efectos de los fármacos , Animales , Anticuerpos Monoclonales/inmunología , Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Immunoblotting , Inmunohistoquímica , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos DBA , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
AIM: Rheumatoid arthritis (RA) is a chronic inflammatory disorder of the synovium resulting in the destruction of affected joint cartilage and bone structures. Etanercept is a biological agent that blocks the tumor necrosis factor-α (TNF-α)-mediated inflammatory processes in RA patients, and has a regenerative effect on cartilage. In order to identify novel disease-related proteins and candidate biomarkers, we performed proteomic profiling of the serum in patients with RA who were treated with etanercept. METHOD: Serum samples were obtained from eight RA patients before and after etanercept treatment. The low molecular weight proteins in the serum were concentrated and analyzed by liquid chromatography-tandem mass spectrometry. The results before and after etanercept treatment were compared by the spectrum count method. RESULTS: Among a total of 477 proteins identified, 12 were found to be decreased and five were increased by etanercept treatment. Some of the changed proteins were known to be related to RA, and most of the other changed proteins may play possible roles in the TNF-α signaling pathway or the state of cartilage and extracellular matrix. CONCLUSION: The present proteomic study identified several proteins that could be involved in the pathogenesis of RA. These findings could thus lead to the identification of novel candidate disease-related protein biomarkers for RA, or indicate new targets for therapy.
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Artritis Reumatoide/sangre , Artritis Reumatoide/tratamiento farmacológico , Inmunoglobulina G/uso terapéutico , Proteoma/metabolismo , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Adulto , Factor D del Complemento/metabolismo , Encefalinas/sangre , Etanercept , Proteínas de la Matriz Extracelular/sangre , Femenino , Humanos , Oxidorreductasas Intramoleculares/sangre , Quininógenos/sangre , Lipocalinas/sangre , Persona de Mediana Edad , Precursores de Proteínas/sangre , Proteoglicanos/sangreRESUMEN
AIM: To elucidate the role of neuropilin-1 (Nrp-1) and semaphorin 3A (Sema3A) in sinusoidal remodeling during liver regeneration in rats. METHODS: Male Wistar/ST rats at 7 wk of age, weighing about 200 g, were used for all animal experiments. In vivo, at 24, 48, 72, 96, 144 and 192 h after two-thirds partial hepatectomy (PHx), the remnant livers were removed. Liver tissues were immunohistochemically stained for Nrp-1, Sema3A and SE-1, a liver sinusoidal endothelial cell (SEC) marker. Total RNA of the liver tissue was extracted and reversely transcribed into cDNA. The mRNA expression of Sema3A was analyzed by quantitative real-time polymerase chain reaction and normalized to that of ribosomal protein S18. In vitro, SECs were isolated from rat liver and cultured in endothelial growth medium containing 20 ng/mL vascular endothelial cell growth factor. Migration of SECs in primary culture was assessed by cell transwell assay with or without recombinant Sema3A. Apoptotic cells were determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method. RESULTS: In vitro, immunohistochemistry study revealed that Sema3A and Nrp-1 were constitutively expressed in hepatocytes and SECs, respectively, in normal rat liver tissues. Nrp-1 expression in SECs was quantified by the percentage of immunostained area with anti-Nrp-1 antibody in relation to the area stained with SE-1. Between 24 h and 96 h following resection of liver, Nrp-1 expression in SECs was transiently increased. Compared with the baseline (5.2% ± 0.1%), Nrp-1 expression in SECs significantly increased at 24 h (17.3% ± 0.7%, P < 0.05), 48 h (39.1% ± 0.6%, P < 0.01), 72 h (46.9% ± 4.5%, P < 0.01) and 96 h (29.9% ± 3.8%, P < 0.01) after PHx, then returned to the basal level at termination of liver regeneration. Interestingly, the expression of Sema3A was inversely associated with that of Nrp-1 in liver after PHx. Sema3A mRNA expression was significantly reduced by about 75% over the period 24-144 h after PHx (P < 0.05), and returned to basal levels at 192 h after PHx. In vitro, SECs isolated from rats after PHx (PHx-SECs) were observed to migrate to the lower chamber of the cell transwell system after incubation for 24 h, but not cells from normal rats (CONT-SECs), indicating that mobility of PHx-SECs increases as compared with that of CONT-SECs. Moreover, recombinant Sema3A significantly attenuated migration in PHx-SECs in primary culture (vehicle-treated 100% ± 7.9% vs Sema3A-treated 42.6% ± 5.4%, P < 0.01), but not in CONT-SECs. Compared with CONT-SECs, the apoptotic rate of PHx-SECs decreased by 78.3% (P < 0.05). There was no difference in apoptosis between CONT-SECs that were treated with vehicle and Sema3A. However, in PHx-SECs, apoptosis was induced by the presence of 5 nmol Sema3A for 24 h (vehicle-treated 21.7% ± 7.6% vs Sema3A-treated 104.3% ± 8.9%, P < 0.05). In addition, immunohistochemistry confirmed the increased expression of Nrp-1 in PHx-SECs, while it was noted to a lesser extent in CONT-SECs. CONCLUSION: The interplay of Nrp-1 and Sema3A shown in our results may lead to a better understanding of interaction between sinusoidal remodeling and SECs during liver regeneration.
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Hepatectomía , Regeneración Hepática/fisiología , Neuropilina-1/fisiología , Semaforina-3A/fisiología , Animales , Apoptosis , Movimiento Celular , Células Cultivadas , Masculino , Neuropilina-1/análisis , Ratas , Ratas Wistar , Semaforina-3A/análisisRESUMEN
Recent reports have described the involvement of the diacylglycerol kinase (DGK) family in various pathological conditions. In an animal model of transient ischemia, DGKζ containing a nuclear localization signal (NLS) is shown to translocate quickly from the nucleus to the cytoplasm in hippocampal neurons and to disappear gradually after reperfusion. Those neurons die a delayed neuronal death because of glutamate excitotoxicity. This study investigated the molecular mechanism and functional relation linking DGKζ and neuronal death. In primary cultured neurons, transient exposure to excitotoxic concentration of glutamate led to cytoplasmic accumulation of DGKζ followed by its down-regulation. Results showed that DGKζ down-regulation was caused by proteolytic degradation through the ubiquitin-proteasome system (UPS) rather than transcriptional inhibition. DGKζ polyubiquitination was inhibited in the presence of nuclear export inhibitor leptomycin B. Furthermore, NLS-deleted mutant DGKζΔNLS, which mainly localizes to the cytoplasm, was ubiquitinated more heavily than wild-type DGKζ. From a functional perspective, in vitro gene silencing of DGKζ via specific siRNA enhanced DNA fragmentation in cultured neurons after glutamate exposure. At the organismal level, hippocampal neurons of DGKζ-deficient mice showed vulnerability to kainate-induced seizures. In addition, DGKζ-deficient hippocampus exhibited a significant increase in Ser807/811 phosphorylated retinoblastoma protein levels together with up-regulation of the expression of type D and E cyclins, indicative of cell cycle reentry. Collectively, these results suggest that 1) glutamate excitotoxicity induces nucleocytoplasmic translocation of DGKζ followed by its degradation through the cytoplasmic UPS in hippocampal neurons and that 2) DGKζ-deficient neurons do not succumb directly to apoptosis, although they are more vulnerable to excitotoxicity because of aberrant cell cycle reentry.
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Apoptosis , Ciclo Celular , Citoplasma/metabolismo , Diacilglicerol Quinasa/metabolismo , Neuronas/citología , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Diacilglicerol Quinasa/deficiencia , Ácido Glutámico/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
Diacylglycerol kinase (DGK) plays an important role in phosphoinositide signaling cascade by regulating the intracellular level of diacylglycerol and phosphatidic acid. The DGK family is involved in various pathophysiological responses that are mediated through unique binding partners in different tissues and cells. In this study, we identified a small GTPase effector protein, IQGAP1, as a novel DGKζ-associated complex protein. A bacterial endotoxin, lipopolysaccharide (LPS), facilitated the complex formation in macrophages. Both proteins co-localized at the edge and phagocytic cup of the cell. Furthermore, RNA interference-mediated knockdown of DGKζ or IQGAP1 impaired LPS-induced Rac1 activation. Primary macrophages derived from DGKζ(-/-) mice attenuated LPS-induced phagocytosis of bacteria. These results suggest that DGKζ is involved in IQGAP1/Rac1-mediated phagocytosis upon LPS stimulation in macrophages.