RESUMEN
The aim of this study is to present a new method for determining the root-derived extracellular acid phosphomonoesterase (EAPM) activity fraction within the total EAPM activity of soil. EAPM activity was determined for roots, organic and mineral soil. Samples were collected using paired PVC cylinders, inserted to a depth of 15 cm, within seven selected forest stands. Root-derived EAPM formed between 4 and18% of the total EAPM activity of soil from forests of differing maturity. A new approach, presented in this work, enables separation of root-derived EAPM activity from total soil EAPM. Separation of root-derived EAPM from soil provides a better understanding of its role in P-cycling in terrestrial ecosystems. The method presented in this work is a first step towards the separation of root- and microbe-derived EAPM in soils, which are thought to possess different kinetic properties and different sensitivity to environmental change.
Asunto(s)
Ecosistema , Fosfodiesterasa I/análisis , Raíces de Plantas/enzimología , Suelo/química , Árboles/enzimología , República ChecaAsunto(s)
Enfermedad de Alzheimer/líquido cefalorraquídeo , Proteínas del Líquido Cefalorraquídeo/análisis , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/líquido cefalorraquídeo , Enfermedad de Parkinson/líquido cefalorraquídeo , Proteómica/métodos , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/fisiopatología , Apolipoproteínas E/análisis , Apolipoproteínas E/líquido cefalorraquídeo , Biomarcadores/análisis , Biomarcadores/líquido cefalorraquídeo , Encéfalo/metabolismo , Encéfalo/fisiopatología , Complemento C4a/análisis , Complemento C4a/líquido cefalorraquídeo , Diagnóstico Diferencial , Proteínas del Ojo/análisis , Proteínas del Ojo/líquido cefalorraquídeo , Humanos , Complejos Multienzimáticos/análisis , Complejos Multienzimáticos/líquido cefalorraquídeo , Factores de Crecimiento Nervioso/análisis , Factores de Crecimiento Nervioso/líquido cefalorraquídeo , Enfermedad de Parkinson/diagnóstico , Enfermedad de Parkinson/fisiopatología , Fosfodiesterasa I/análisis , Fosfodiesterasa I/líquido cefalorraquídeo , Hidrolasas Diéster Fosfóricas , Valor Predictivo de las Pruebas , Pirofosfatasas/análisis , Pirofosfatasas/líquido cefalorraquídeo , Serpinas/análisis , Serpinas/líquido cefalorraquídeo , Superóxido Dismutasa/análisis , Superóxido Dismutasa/líquido cefalorraquídeo , Superóxido Dismutasa-1 , Regulación hacia Arriba/fisiologíaRESUMEN
Autotaxin (ATX) is a tumour cell motility-stimulating factor originally isolated from melanoma cell supernatants. ATX is identical to lysophospholipase D, which produces a bioactive lipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine. ATX is overexpressed in various malignancies, including Hodgkin lymphoma, and ATX may stimulate tumour progression via LPA production. The present study measured the serum ATX antigen levels in patients with haematological malignancies using a recently developed automated enzyme immunoassay. The serum ATX antigen levels in patients with B-cell neoplasms, especially follicular lymphoma (FL), were higher than those in healthy subjects. Serum ATX antigen levels in FL patients were associated with tumour burden and changed in parallel with the patients' clinical courses. The serum ATX antigen levels were little affected by inflammation, unlike the soluble interleukin-2 receptor and beta2-microglobulin levels. As expected, the plasma LPA levels in FL patients were correlated with the serum ATX antigen levels. Given that leukaemic tumour cells from FL patients expressed ATX, the shedding of ATX from lymphoma cells probably leads to the elevation of serum ATX antigen levels. Our results suggest that the serum ATX antigen level may be a promising and novel marker for FL.
Asunto(s)
Biomarcadores de Tumor/sangre , Linfoma Folicular/sangre , Complejos Multienzimáticos/sangre , Fosfodiesterasa I/sangre , Pirofosfatasas/sangre , Adulto , Anciano , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Citometría de Flujo/métodos , Humanos , Linfocitos/química , Lisofosfolipasa/sangre , Masculino , Persona de Mediana Edad , Complejos Multienzimáticos/análisis , Fosfodiesterasa I/análisis , Hidrolasas Diéster Fosfóricas , Pirofosfatasas/análisis , Estadísticas no ParamétricasRESUMEN
BACKGROUND: It is now established that the bioactive lipid lysophosphatidic acid (LPA) contributes to cancer initiation, progression and metastasis, including those of prostate cancer. LPA is produced in the serum and plasma mainly by conversion from lysophospholipids through the action of lysophospholipase D (lysoPLD), which is identical to the soluble form of autotaxin (ATX) originally isolated as a tumour cell motility-stimulating factor. In this study, we evaluated the usefulness of lysoPLD/ATX activity as a diagnostic marker. METHODS: The serum lysoPLD activity, assessed by measuring choline liberation from the substrate lysophosphatidylcholine, was measured in patients with prostate cancer and compared with the concentrations of prostate-specific antigen (PSA) and conventional nutritional assessment markers. RESULTS: The serum lysoPLD activity in prostate cancer patients was not statistically different from that in the controls. Consistent with this, there was no correlation between the serum lysoPLD activity and the serum PSA concentrations. However, the lysoPLD/ATX activity did decrease after operation in the prostate cancer patients and seemed to reflect the postoperative damage or the nutritional status. CONCLUSIONS: We postulate that while the serum lysoPLD/ATX may not be a marker of prostate cancer, it promises to instead be a new marker of nutritional status.
Asunto(s)
Biomarcadores de Tumor/sangre , Complejos Multienzimáticos/sangre , Evaluación Nutricional , Trastornos Nutricionales/diagnóstico , Fosfodiesterasa I/sangre , Hidrolasas Diéster Fosfóricas/sangre , Neoplasias de la Próstata/sangre , Pirofosfatasas/sangre , Biomarcadores de Tumor/análisis , Hemoglobinas/análisis , Humanos , Masculino , Complejos Multienzimáticos/análisis , Trastornos Nutricionales/sangre , Trastornos Nutricionales/etiología , Fosfodiesterasa I/análisis , Hidrolasas Diéster Fosfóricas/análisis , Prealbúmina/análisis , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/complicaciones , Pirofosfatasas/análisis , Albúmina Sérica/análisis , Factores de TiempoRESUMEN
Lysophosphatidic acid (LPA) is involved in a broad spectrum of biological activities, including wound healing and cancer metastasis. Autotaxin (ATX), originally isolated from a melanoma supernatant as a tumor cell motility-stimulating factor, has been shown to be molecularly identical to lysophospholipase D (lysoPLD), which is the main enzyme in the production of LPA. Although ATX/lysoPLD is known to be widely expressed in normal human tissues, the exact distribution of ATX-producing cells has not been fully investigated. In this study, we evaluated ATX/lysoPLD expression by immunohistochemical staining using a rat anti-ATX mAb in the human gastrointestinal tract and found that submucosal mast cells (MC) highly expressed this enzyme. This was confirmed by immunofluorescent double staining using mAbs to tryptase and chymase. Then, we isolated MC from human gastric tissue by an immunomagnetic method using CD117-microbeads and showed that a subpopulation of CD203c-positive MC showed positive staining for intracellular ATX/lysoPLD on flowcytometry. This was confirmed by Western blotting of the isolated cells. Moreover, a significant level of ATX/lysoPLD release could be detected in the culture supernatants of human MC by Western blot analysis. Our data suggest that submucosal MC play significant roles in various aspects of pathophysiology in the gastrointestinal tract by locally providing bioactive LPA through the production of ATX/lysoPLD.
Asunto(s)
Tracto Gastrointestinal/metabolismo , Lisofosfolípidos/biosíntesis , Mastocitos/metabolismo , Complejos Multienzimáticos/metabolismo , Fosfodiesterasa I/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/metabolismo , Quimasas/análisis , Tracto Gastrointestinal/citología , Humanos , Mastocitos/clasificación , Complejos Multienzimáticos/análisis , Complejos Multienzimáticos/fisiología , Fosfodiesterasa I/análisis , Fosfodiesterasa I/fisiología , Hidrolasas Diéster Fosfóricas/análisis , Hidrolasas Diéster Fosfóricas/fisiología , Pirofosfatasas/análisis , Pirofosfatasas/fisiología , Triptasas/análisis , Cicatrización de HeridasRESUMEN
Levels of lysophosphatidic acid (LPA), an important phospholipid mediator, in serum and ascitic fluid from ovarian cancer patients were shown to be higher than those from healthy women and from patients with other type of cancer, respectively. Although LPA in human serum seems mainly to be generated by lysophospholipase D (lysoPLD), the source and pathway for LPA in the ascitic fluid remain still obscure. In this study, we examined whether lysoPLD activity producing bioactive LPA in human peritoneal fluid was significantly elevated under pathological statuses. Lysophospholipase D activity in human peritoneal fluids was measured by quantifying choline released from exogenous lysophosphatidylcholine on their incubation at 37 degrees C. We also compared the activity of lysoPLD in sera from patients with different gynecologic diseases. We found relatively high lysoPLD activity in peritoneal fluids from patients with ovarian cancer, dermoid cyst or mucinous cystadenoma, whereas there were no significant differences in the serum lysoPLD activity among clinical groups and healthy subjects. The lysoPLD in the peritoneal fluid was found to have similar substrate specificity and metal ion requirement to those of serum lysoPLD, that has been identified as autotaxin, a tumor cell-motility stimulating protein. Our results suggest that increased lysoPLD activity in peritoneal fluid from patients with certain gynecologic tumors might be relevant to its potential of tumor progression.
Asunto(s)
Líquido Ascítico/enzimología , Biomarcadores de Tumor/análisis , Cistoadenoma Mucinoso/enzimología , Quiste Dermoide/enzimología , Neoplasias Ováricas/enzimología , Hidrolasas Diéster Fosfóricas/análisis , Adulto , Biomarcadores de Tumor/sangre , Colina/análisis , Cistoadenoma Mucinoso/sangre , Quiste Dermoide/sangre , Femenino , Humanos , Lisofosfolípidos/análisis , Lisofosfolípidos/sangre , Complejos Multienzimáticos/análisis , Complejos Multienzimáticos/sangre , Neoplasias Ováricas/sangre , Fosfodiesterasa I/análisis , Fosfodiesterasa I/sangre , Hidrolasas Diéster Fosfóricas/sangre , Pirofosfatasas/análisis , Pirofosfatasas/sangreRESUMEN
[reaction: see text] Lysophospholipase D (lysoPLD), also known as autotaxin (ATX), is an important source of the potent mitogen lysophosphatidic acid (LPA). Two fluorogenic substrate analogues for lysoPLD were synthesized in nine steps from (S)-PMB-glycerol. The substrates (FS-2 and FS-3) show significant increases in fluorescence when treated with recombinant ATX and have potential applications in screening for this emerging drug target.
Asunto(s)
Colorantes Fluorescentes/síntesis química , Complejos Multienzimáticos/análisis , Fosfodiesterasa I/análisis , Hidrolasas Diéster Fosfóricas/análisis , Pirofosfatasas/análisis , Colorantes Fluorescentes/metabolismo , Lisofosfolípidos , Estructura Molecular , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Fosfodiesterasa I/genética , Fosfodiesterasa I/metabolismo , Fosfolípidos/química , Fosfolípidos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/genética , Pirofosfatasas/metabolismoRESUMEN
Intestinal alk-SMase (alkaline sphingomyelinase) is an ectoenzyme related to the NPP (nucleotide phosphodiesterase) family. It has five potential N-glycosylation sites and predicated transmembrane domains at both the N- and C-termini. The amino acid residues forming the two metal-binding sites in NPP are conserved, and those of the active core are modified. We examined the functional changes of the enzyme induced by deglycosylation and mutagenesis. Treating alk-SMase cDNA-transfected COS-7 cells with tunicamycin rendered the expressed enzyme completely inactive. Mutations of the five potential N-glycosylation sites individually and in combination showed that these sites were all glycosylated and deficient glycosylation decreased the enzyme activity. Immunogold labelling showed that the wild-type enzyme was mainly located in the plasma membrane, whereas the C-terminal domain-truncated enzyme was released into the medium. Deglycosylation blocked the release of the enzyme that accumulated in endosome-like structures. The enzyme activity was also decreased by mutations of the residues forming the putative metal-binding sites and the active core. Substitution of the active core sequence with that of NPP or mutation of T75 in the core abolished the enzyme activity against sphingomyelin but failed to render the enzyme NPP active. Our results indicate that alk-SMase activity is severely affected by defective N-glycosylation and structural alterations of the putative metal-binding sites and the predicted active core.
Asunto(s)
Cationes Bivalentes/metabolismo , Intestinos/enzimología , Procesamiento Proteico-Postraduccional , Esfingomielina Fosfodiesterasa/química , Animales , Sitios de Unión , Células COS/enzimología , Membrana Celular/enzimología , Cricetinae , Cricetulus , Medios de Cultivo Condicionados , Endosomas/enzimología , Glicosilación/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Proteínas de la Membrana/análisis , Mutagénesis Sitio-Dirigida , Fosfodiesterasa I/análisis , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Esfingomielina Fosfodiesterasa/análisis , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielinas/metabolismo , Transfección , Tunicamicina/farmacologíaRESUMEN
BACKGROUND: Insulin resistance characterizes type 1 diabetes mellitus with nephropathy. The molecular mechanisms of insulin resistance are not completely understood. Recently some advances have been made in identification of transmembrane glycoprotein PC-1 as a potential factor of insulin resistance. METHODS: We measured urinary excretion of PC-1 (alkaline phosphodiesterase I), a potential factor of insulin resistance, and N-acetyl-beta-D-glucosaminidase (NAGA) in 62 type 1 diabetic patients with different damage to the kidney. RESULTS: In newly detected type 1 diabetes patients, before insulin therapy, urine PC-1 excretion was significantly increased (P<0.05) over the control level. However, in patients after 12.4 years of therapy, urinary PC-1 was significantly decreased (P<0.05). Decreased urine PC-1 activity (P<0.05) was found also in type 1 diabetes patients with microalbuminuria and manifest nephropathy, including those with renal failure. Urinary NAGA excretion was found to be significantly increased (P=0.001) in all but the group of type 1 diabetes patients without nephropathy. CONCLUSION: This study of urinary PC-1 in patients with type 1 diabetes shows increased excretion in newly detected patients with poor glycaemic control, but decreased excretion in patients with micro-/macroalbuminuria as well as in those without apparent kidney damage. In patients with primary glomerulonephritis, urinary excretion of PC-1 was significantly decreased and that of NAGA significantly increased compared with the excretion in healthy controls.