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1.
Protein Sci ; 25(3): 650-61, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26660500

RESUMEN

Cyclic ADP-ribose (cADPR) mobilizes intracellular Ca(2+) stores and activates Ca(2+) influx to regulate a wide range of physiological processes. It is one of the products produced from the catalysis of NAD(+) by the multifunctional CD38/ADP-ribosyl cyclase superfamily. After elimination of the nicotinamide ring by the enzyme, the reaction intermediate of NAD(+) can either be hydrolyzed to form linear ADPR or cyclized to form cADPR. We have previously shown that human CD38 exhibits a higher preference towards the hydrolysis of NAD(+) to form linear ADPR while Aplysia ADP-ribosyl cyclase prefers cyclizing NAD(+) to form cADPR. In this study, we characterized the enzymatic properties of porcine CD38 and revealed that it has a prominent secondary NAD(+) cyclase activity producing cADPR. We also determined the X-ray crystallographic structures of porcine CD38 and were able to observe conformational flexibility at the base of the active site of the enzyme which allow the NAD(+) reaction intermediate to adopt conformations resulting in both hydrolysis and cyclization forming linear ADPR and cADPR respectively.


Asunto(s)
ADP-Ribosil Ciclasa 1/metabolismo , NAD/metabolismo , ADP-Ribosil Ciclasa/química , ADP-Ribosil Ciclasa/metabolismo , ADP-Ribosil Ciclasa 1/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , ADP-Ribosa Cíclica/metabolismo , Humanos , Modelos Moleculares , Dominios Proteicos , Porcinos
2.
Hum Antibodies ; 24(3-4): 45-51, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-28128764

RESUMEN

Objectives were to: 1) induce a lytic IgG antibody (ab) response (via the so called `third vaccination method') against CD38 antigen (ag) residing on the extra-cellular domain of multiple myeloma (MM) cells in recipient rabbits, by combining the CD38 ag with donor-derived anti-CD38 ag lytic IgG ab into an immune complex (IC); and 2) determine whether abs produced would cause complement-mediated lysis (in vitro) of human MM cells containing CD38 ag. The vaccine was created in a two-step process. First, ab (rabbit anti-CD38 ag IgG ab) was raised in donor rabbits by injections of low molecular weight soluble CD38 ag in Freund's complete adjuvant (FCA) and aqueous solution. Second, transfer of pathogenic lytic IgG ab response into recipient rabbits was achieved by injections of ICs composed of CD38 ag and homologous anti-CD38 ag IgG ab. Consequently, recipient rabbits produced the same ab with the same specificity against the target ag as was present in the inoculum, namely agglutinating, precipitating and lytic (as demonstrated in vitro). In an in vitro study, in the presence of complement, donor and recipient rabbits' immune sera caused lysis of CD38 ag associated human MM cells. The most effective lytic ab response causing sera were those from donor rabbits injected with CD38 ag in FCA and those from rabbits injected with ICs, especially when they were administered in adjuvants. These results provided proof of concept that the third vaccination method has good potential as a stand-alone and efficacious method of controlling cancer.


Asunto(s)
ADP-Ribosil Ciclasa 1/inmunología , Complejo Antígeno-Anticuerpo/administración & dosificación , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Mieloma Múltiple/terapia , Vacunación/métodos , ADP-Ribosil Ciclasa 1/administración & dosificación , ADP-Ribosil Ciclasa 1/genética , Pruebas de Aglutinación , Animales , Anticuerpos Antineoplásicos/biosíntesis , Complejo Antígeno-Anticuerpo/genética , Complejo Antígeno-Anticuerpo/inmunología , Antígenos de Neoplasias/administración & dosificación , Antígenos de Neoplasias/genética , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/genética , Línea Celular Tumoral , Proteínas del Sistema Complemento/farmacología , Citotoxicidad Inmunológica , Femenino , Adyuvante de Freund/administración & dosificación , Expresión Génica , Humanos , Sueros Inmunes/farmacología , Inmunoglobulina G/biosíntesis , Mieloma Múltiple/genética , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Conejos
3.
Hum Antibodies ; 24(3-4): 53-57, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-28128765

RESUMEN

Employing passive immunization - using a heterologous anti-CD38 IgG antibody containing serum - in SCID mice injected subcutaneously with human multiple myeloma cells, we have shown that treatments with the antiserum - especially in the presence of complement - significantly decreased cancer growth. However, administered antibody and complement was not sufficient in amount to prevent cancer cell multiplication and cancer growth expansion to a satisfactory degree. Larger volumes of the same components more than likely would have further reduced cancer growth and prolonged the life of mice. In control mice, cancer growth progressed faster proving that lytic immune response against multiple myeloma cells is necessary for cancer cell kill.


Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Vacunas contra el Cáncer/administración & dosificación , Proteínas del Sistema Complemento/administración & dosificación , Sueros Inmunes/administración & dosificación , Mieloma Múltiple/prevención & control , ADP-Ribosil Ciclasa 1/genética , ADP-Ribosil Ciclasa 1/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Proliferación Celular , Femenino , Expresión Génica , Humanos , Sueros Inmunes/química , Inmunización Pasiva/métodos , Inyecciones Subcutáneas , Ratones , Ratones SCID , Mieloma Múltiple/genética , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Conejos , Trasplante Heterólogo , Carga Tumoral
4.
Methods Mol Biol ; 1016: 39-56, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23681571

RESUMEN

Cyclic ADP-ribose (cADPR) is a novel second messenger that releases calcium from intracellular stores. Although first shown to release calcium in the sea urchin egg, cADPR has been shown since to be active in a variety of cells and tissues, from plant to human. cADPR stimulates calcium release via ryanodine receptors although the mechanism is still not completely understood. cADPR is produced enzymatically from NAD by ADP-ribosyl cyclases; several of these proteins have been identified including one isolated from Aplysia californica, two types found in mammals (CD38 and CD157), and three forms in sea urchin. A cyclase activity has been measured in extracts from Arabidopsis thaliana although the protein is still unidentified. Nicotinic acid adenine dinucleotide phosphate (NAADP) is another novel messenger that releases calcium from internal stores and is produced by these same enzymes by an exchange reaction. NAADP targets lysosomal stores whereas cADPR releases calcium from the endoplasmic reticulum. Due to their importance in cell signaling, cADPR and NAADP have been the focus of numerous investigations over the last 25 years. This chapter describes several assay methods for the measurements of cADPR and NAADP concentration and cyclase activity in extracts from cells.


Asunto(s)
ADP-Ribosil Ciclasa/metabolismo , ADP-Ribosa Cíclica/metabolismo , NADP/análogos & derivados , Extractos de Tejidos/metabolismo , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Fluorescencia , Nucleótidos de Guanina , Humanos , NAD/análogos & derivados , NAD+ Nucleosidasa/metabolismo , NADP/metabolismo , Ciclo del Sustrato
5.
Scand J Immunol ; 71(3): 125-33, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20415778

RESUMEN

Public health can be protected most effectively through vaccination programmes. However, while presently available vaccination techniques protects the individual by provoking immune responses against exogenous antigens (ags), such as those associated with certain bacteria and viruses, they cannot protect against or treat mishaps caused by endogenous ag. Recently, Barabas and colleagues have developed a new vaccination method, called modified vaccination technique (MVT), which allows the presentation of disease causing agents in such a way as to initiate and maintain desired immune response outcomes even in the context of mishaps associated with endogenous ag. For example, in an experimental autoimmune kidney disease, the MVT downregulated/terminated pathogenic immune responses that were causing morphological and functional changes of the kidney. The MVT promises, with appropriate case-specific modifications, both preventative and curative applications for ailments, such as endogenous ag initiated mishaps (i.e. autoimmune diseases and cancer) and diseases caused by chronic infection, that are presently only treatable with drugs. To achieve specific immune responses, purified components of the vaccine (ag and antibodies) must be produced and assembled into immune complexes having the potential of inducing predetermined corrective immune response outcomes.


Asunto(s)
Autoantígenos/inmunología , Enfermedades Autoinmunes/prevención & control , Enfermedades Renales/prevención & control , Vacunación/métodos , Animales , Presentación de Antígeno/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Antígenos de Neoplasias/inmunología , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/terapia , Vacunas contra el Cáncer/uso terapéutico , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Enfermedades Renales/inmunología , Enfermedades Renales/terapia , Ratas
6.
Curr Biol ; 18(20): 1612-8, 2008 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-18951023

RESUMEN

Cyclic ADP-ribose is an important Ca(2+)-mobilizing cytosolic messenger synthesized from beta-NAD(+) by ADP-ribosyl cyclases (ARCs). However, the focus upon ectocellular mammalian ARCs (CD38 and CD157) has led to confusion as to how extracellular enzymes generate intracellular messengers in response to stimuli. We have cloned and characterized three ARCs in the sea urchin egg and found that endogenous ARCbeta and ARCgamma are intracellular and located within the lumen of acidic, exocytotic vesicles, where they are optimally active. Intraorganelle ARCs are shielded from cytosolic substrate and targets by the organelle membrane, but this barrier is circumvented by nucleotide transport. We show that a beta-NAD(+) transporter provides ARC substrate that is converted luminally to cADPR, which, in turn, is shuttled out to the cytosol via a separate cADPR transporter. Moreover, nucleotide transport is integral to ARC activity physiologically because three transport inhibitors all inhibited the fertilization-induced Ca(2+) wave that is dependent upon cADPR. This represents a novel signaling mechanism whereby an extracellular stimulus increases the concentration of a second messenger by promoting messenger transport from intraorganelle synthesis sites to the cytosol.


Asunto(s)
ADP-Ribosil Ciclasa/metabolismo , Señalización del Calcio , Exosomas/metabolismo , ADP-Ribosil Ciclasa/genética , Adenosina Difosfato Ribosa/metabolismo , Animales , Transporte Biológico , Clonación Molecular , ADP-Ribosa Cíclica/metabolismo , Citosol/metabolismo , Exocitosis , Fertilización , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Proteínas de Transporte de Nucleótidos , Óvulo/metabolismo , Strongylocentrotus purpuratus/enzimología , Strongylocentrotus purpuratus/genética
7.
Exp Cell Res ; 303(1): 14-21, 2005 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-15572023

RESUMEN

The cell surface antigen, CD38, is a 45-kDa transmembrane protein which is predominantly expressed on hematopoietic cells during differentiation. As a bifunctional ectoenzyme, it catalyzes the synthesis of cyclic ADP-ribose (cADPR) from NAD(+) and hydrolysis of either NAD(+) or cADPR to ADP-ribose. All-trans-retinoic acid (RA) is a potent and specific inducer of CD38 in myeloid cells. In this report, we demonstrate that the nuclei of RA-treated human HL-60 myeloblastic cells reveal enzymatic activities inherent to CD38. Thus, GDP-ribosyl cyclase and NAD(+) glycohydrolase activities in the nuclear fraction increased very significantly in response to incubation with RA. With Western blotting, we detected in the nuclear protein fraction from RA-treated cells a approximately 43-kDa protein band which was reactive with the CD38-specific monoclonal antibody OKT10. The expression of CD38 in HL-60 nuclei was also shown with FACScan analysis. RA treatment gave rise to an increase in in vitro ADP ribosylation of the approximately 43-kDa nuclear protein. Moreover, nuclei isolated from RA-treated HL-60 cells revealed calcium release in response to cADPR, whereas a similar response was not observed in control nuclei. These results suggest that CD38 is expressed in HL-60 cell nuclei during RA-induced differentiation.


Asunto(s)
ADP-Ribosil Ciclasa/inmunología , Antígenos CD/inmunología , Calcio/metabolismo , Diferenciación Celular/fisiología , Núcleo Celular/metabolismo , Tretinoina/farmacología , ADP-Ribosil Ciclasa 1 , Diferenciación Celular/efectos de los fármacos , ADP-Ribosa Cíclica/metabolismo , Células HL-60 , Humanos , Glicoproteínas de Membrana
8.
Comb Chem High Throughput Screen ; 6(4): 367-79, 2003 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12769681

RESUMEN

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are two novel Ca(2+) messengers derived respectively from NAD and NADP. Since their discovery in sea urchin eggs, both have now been shown to serve messenger functions in a wide range of cells from plant to human. In this article, a series of fluorimetric assays for cADPR, NAADP and their metabolic enzymes is compiled. The enzyme assay makes use of an analog of NAD, nicotinamide guanine dinucleotide, which is non-fluorescent but is cyclized by the enzymes to a fluorescent analog of cADPR, cyclic GDP-ribose. Other NAD utilizing enzymes are not capable of catalyzing the cyclization and thus produce no interference. The fluorimetric assays for cADPR and NAADP make use of coupled-enzyme amplification and can readily detect nanomolar concentrations of either messenger. All the assays described can be performed in multi-well format, allowing ready automation and use in high throughput screening. An added advantage of these assays is that all the required reagents are commercially available, facilitating general adoption of the techniques by all those who are interested in the physiology and enzymology of the novel Ca(2+) signaling pathways mediated by cADPR and NAADP.


Asunto(s)
ADP-Ribosil Ciclasa/análisis , ADP-Ribosa Cíclica/análisis , Glicósido Hidrolasas/análisis , N-Glicosil Hidrolasas , NADP/análogos & derivados , NADP/análisis , ADP-Ribosil Ciclasa/metabolismo , Antígenos CD/análisis , Antígenos CD/metabolismo , Señalización del Calcio/fisiología , Ciclización , Fluorometría/métodos , Glicósido Hidrolasas/metabolismo , Células HL-60 , Humanos , NAD/análogos & derivados , NAD/análisis
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