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1.
Orthod Craniofac Res ; 23(4): 501-508, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-32562339

RESUMEN

OBJECTIVE: To determine if Pyk2 deficiency increases midpalatal suture bone mass and preserves sutural integrity after maxillary expansion. SETTING AND SAMPLE: Thirty-six male Pyk2 knockout (KO) and control (WT) mice at 6 weeks of age. MATERIALS AND METHODS: Mice received nickel-titanium spring expanders delivering 0 g (no intervention control), 10 or 20 g force for 14 days. High-resolution micro-CT was used to determine bone volume/tissue volume (BV/TV), sutural width and intermolar width. Effects on osteoclasts, chondrocytes and suture morphology were determined by histomorphometry. RESULTS: Pyk2-KO controls (0 g) had 7% higher BV/TV compared with WT controls. Expanded Pyk2-KO maxillae also exhibited 12% (10 g) and 18% (20 g) higher BV/TV than WT mice. Although bone loss following expansion occurred in both genotypes, BV/TV was decreased to a greater extent in WT maxillae (-10% at 10g; -22% at 20 g) compared with Pyk2-KO maxillae (-11% only at 20 g). Expanded WT maxillae also showed a greater increase in sutural width, intermolar width and fibrous connective tissue width compared with expanded Pyk2-KO maxillae. Moreover, osteoclast number was increased 77% (10 g) and 132% (20 g) in expanded WT maxillae, but remained unchanged in expanded Pyk2-KO, compared to their respective controls. Cartilage area and chondrocyte number were increased to the same extent in expanded WT and Pyk2-KO sutures. CONCLUSIONS: These findings suggest that midpalatal suture expansion increases osteoclast formation in WT but not Pyk2-KO mice, leading to higher BV/TV in expanded Pyk2-KO maxillae. These studies suggest Pyk2-targeted strategies may be beneficial to increase bone density and preserve sutural integrity during maxillary expansion.


Asunto(s)
Suturas Craneales , Quinasa 2 de Adhesión Focal , Animales , Densidad Ósea , Suturas Craneales/diagnóstico por imagen , Masculino , Ratones , Técnica de Expansión Palatina , Suturas
2.
Mol Cell Endocrinol ; 474: 35-47, 2018 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-29428397

RESUMEN

Bone remodeling is controlled by the actions of bone-degrading osteoclasts and bone-forming osteoblasts (OBs). Aging and loss of estrogen after menopause affects bone mass and quality. Estrogen therapy, including selective estrogen receptor modulators (SERMs), can prevent bone loss and increase bone mineral density in post-menopausal women. Although investigations of the effects of estrogen on osteoclast activity are well advanced, the mechanism of action of estrogen on OBs is still unclear. The proline-rich tyrosine kinase 2 (Pyk2) is important for bone formation and female mice lacking Pyk2 (Pyk2-KO) exhibit elevated bone mass, increased bone formation rate and reduced osteoclast activity. Therefore, in the current study, we examined the role of estrogen signaling on the mechanism of action of Pyk2 in OBs. As expected, Pyk2-KO OBs showed significantly higher proliferation, matrix formation, and mineralization than WT OBs. In addition we found that Pyk2-KO OBs cultured in the presence of either 17ß-estradiol (E2) or raloxifene, a SERM used for the treatment of post-menopausal osteoporosis, showed a further robust increase in alkaline phosphatase (ALP) activity and mineralization. We examined the possible mechanism of action and found that Pyk2 deletion promotes the proteasome-mediated degradation of estrogen receptor α (ERα), but not estrogen receptor ß (ERß). As a consequence, E2 signaling via ERß was enhanced in Pyk2-KO OBs. In addition, we found that Pyk2 deletion and E2 stimulation had an additive effect on ERK phosphorylation, which is known to stimulate cell differentiation and survival. Our findings suggest that in the absence of Pyk2, estrogen exerts an osteogenic effect on OBs through altered ERα and ERß signaling. Thus, targeting Pyk2, in combination with estrogen or raloxifene, may be a novel strategy for the prevention and/or treatment of bone loss diseases.


Asunto(s)
Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Estrógenos/farmacología , Quinasa 2 de Adhesión Focal/deficiencia , Osteoblastos/citología , Clorhidrato de Raloxifeno/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Biomarcadores/metabolismo , Recuento de Células , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/agonistas , Receptor beta de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Eliminación de Gen , Leupeptinas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Osteoblastos/metabolismo , Proteolisis/efectos de los fármacos
3.
J Cell Biochem ; 118(8): 2231-2240, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28067429

RESUMEN

The Lnk adapter protein negatively regulates the signaling of thrombopoietin (TPO), the main megakaryocyte (MK) growth factor. Lnk-deficient (-/-) mice have increased TPO signaling and increased MK number. Interestingly, several mouse models exist in which increased MK number leads to a high bone mass phenotype. Here we report the bone phenotype of these mice. MicroCT and static histomorphometric analyses at 20 weeks showed the distal femur of Lnk-/- mice to have significantly higher bone volume fraction and trabecular number compared to wild-type (WT) mice. Notably, despite a significant increase in the number of osteoclasts (OC), and decreased bone formation rate in Lnk-/- mice compared to WT mice, Lnk-/- mice demonstrated a 2.5-fold greater BV/TV suggesting impaired OC function in vivo. Additionally, Lnk-/- mouse femurs exhibited non-significant increases in mid-shaft cross-sectional area, yet increased periosteal BFR compared to WT femurs was observed. Lnk-/- femurs also had non-significant increases in polar moment of inertia and decreased cortical bone area and thickness, resulting in reduced bone stiffness, modulus, and strength compared to WT femurs. Of note, Lnk is expressed by OC lineage cells and when Lnk-/- OC progenitors are cultured in the presence of TPO, significantly more OC are observed than in WT cultures. Lnk is also expressed in osteoblast (OB) cells and in vitro reduced alkaline phosphatase activity was observed in Lnk-/- cultures. These data suggest that both direct effects on OB and OC as well as indirect effects of MK in regulating OB contributes to the observed high bone mass. J. Cell. Biochem. 118: 2231-2240, 2017. © 2017 Wiley Periodicals, Inc.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismo , Trombopoyetina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Western Blotting , Células de la Médula Ósea/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , Femenino , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Megacariocitos/metabolismo , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis/genética , Osteogénesis/fisiología , Células RAW 264.7 , Trombopoyetina/genética , Microtomografía por Rayos X
4.
J Cell Biochem ; 117(6): 1396-406, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26552846

RESUMEN

Osteoblast differentiation and migration are necessary for bone formation during bone remodeling. Mice lacking the proline-rich tyrosine kinase Pyk2 (Pyk2-KO) have increased bone mass, in part due to increased osteoblast proliferation. Megakaryocytes (MKs), the platelet-producing cells, also promote osteoblast proliferation in vitro and bone-formation in vivo via a pathway that involves Pyk2. In the current study, we examined the mechanism of action of Pyk2, and the role of MKs, on osteoblast differentiation and migration. We found that Pyk2-KO osteoblasts express elevated alkaline phosphatase (ALP), type I collagen and osteocalcin mRNA levels as well as increased ALP activity, and mineralization, confirming that Pyk2 negatively regulates osteoblast function. Since Pyk2 Y402 phosphorylation is important for its catalytic activity and for its protein-scaffolding functions, we expressed the phosphorylation-mutant (Pyk2(Y402F) ) and kinase-mutant (Pyk2(K457A) ) in Pyk2-KO osteoblasts. Both Pyk2(Y402F) and Pyk2(K457A) reduced ALP activity, whereas only kinase-inactive Pyk2(K457A) inhibited Pyk2-KO osteoblast migration. Consistent with a role for Pyk2 on ALP activity, co-culture of MKs with osteoblasts led to a decrease in the level of phosphorylated Pyk2 (pY402) as well as a decrease in ALP activity. Although, Pyk2-KO osteoblasts exhibited increased migration compared to wild-type osteoblasts, Pyk2 expression was not required necessary for the ability of MKs to stimulate osteoblast migration. Together, these data suggest that osteoblast differentiation and migration are inversely regulated by MKs via distinct Pyk2-dependent and independent signaling pathways. Novel drugs that distinguish between the kinase-dependent or protein-scaffolding functions of Pyk2 may provide therapeutic specificity for the control of bone-related diseases.


Asunto(s)
Quinasa 2 de Adhesión Focal/genética , Quinasa 2 de Adhesión Focal/metabolismo , Megacariocitos/citología , Osteoblastos/citología , Animales , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Técnicas de Cocultivo , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Megacariocitos/metabolismo , Ratones , Osteoblastos/metabolismo , Fosforilación , Transducción de Señal
5.
J Cell Biochem ; 117(4): 959-69, 2016 04.
Artículo en Inglés | MEDLINE | ID: mdl-26375403

RESUMEN

C-Mpl is the receptor for thrombopoietin (TPO), the main megakaryocyte (MK) growth factor, and c-Mpl is believed to be expressed on cells of the hematopoietic lineage. As MKs have been shown to enhance bone formation, it may be expected that mice in which c-Mpl was globally knocked out (c-Mpl(-/-) mice) would have decreased bone mass because they have fewer MKs. Instead, c-Mpl(-/-) mice have a higher bone mass than WT controls. Using c-Mpl(-/-) mice we investigated the basis for this discrepancy and discovered that c-Mpl is expressed on both osteoblasts (OBs) and osteoclasts (OCs), an unexpected finding that prompted us to examine further how c-Mpl regulates bone. Static and dynamic bone histomorphometry parameters suggest that c-Mpl deficiency results in a net gain in bone volume with increases in OBs and OCs. In vitro, a higher percentage of c-Mpl(-/-) OBs were in active phases of the cell cycle, leading to an increased number of OBs. No difference in OB differentiation was observed in vitro as examined by real-time PCR and functional assays. In co-culture systems, which allow for the interaction between OBs and OC progenitors, c-Mpl(-/-) OBs enhanced osteoclastogenesis. Two of the major signaling pathways by which OBs regulate osteoclastogenesis, MCSF/OPG/RANKL and EphrinB2-EphB2/B4, were unaffected in c-Mpl(-/-) OBs. These data provide new findings for the role of MKs and c-Mpl expression in bone and may provide insight into the homeostatic regulation of bone mass as well as bone loss diseases such as osteoporosis.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogénesis/genética , Receptores de Trombopoyetina/genética , Trombopoyetina/genética , Animales , Animales Recién Nacidos , Densidad Ósea , Recuento de Células , Diferenciación Celular , División Celular , Efrina-B2/genética , Efrina-B2/metabolismo , Homeostasis/genética , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Megacariocitos/citología , Megacariocitos/metabolismo , Ratones , Ratones Noqueados , Osteoblastos/citología , Osteoclastos/citología , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligando RANK/genética , Ligando RANK/metabolismo , Receptor EphB2/genética , Receptor EphB2/metabolismo , Receptor EphB4/genética , Receptor EphB4/metabolismo , Receptores de Trombopoyetina/deficiencia , Transducción de Señal , Cráneo/citología , Cráneo/metabolismo , Trombopoyetina/metabolismo
6.
Int J Biochem Cell Biol ; 46: 9-18, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24387844

RESUMEN

Bone formation is controlled by osteoblasts, but the signaling proteins that control osteoblast differentiation and function are still unclear. We examined if the dynamin GTPase, which is associated with actin remodeling and migration in other cells, plays a role in osteoblast differentiation and migration. Dynamin mRNA was expressed in primary osteoblasts throughout differentiation (0-21 days). However, alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was decreased in osteoblasts over-expressing dynamin. Conversely, ALP activity was increased following shRNA-mediated knockdown of dynamin and in osteoblasts treated with the dynamin inhibitor, dynasore. Dynasore also reduced c-fos and osterix expression, markers of early osteoblasts, suggesting a role for dynamin in pre-osteoblast to osteoblast differentiation. Since dynamin GTPase activity is regulated by tyrosine phosphorylation, we examined the mechanism of dynamin dephosphorylation in osteoblasts. Dynamin formed a protein complex with the tyrosine phosphatase PTP-PEST and inhibition of phosphatase activity increased the level of phosphorylated dynamin. Further, PTP-PEST blocked the Src-mediated increase in the phosphorylation and GTPase activity of wild-type dynamin but not the phosphorylation mutant dynY231F/Y597F. Although ALP activity was increased in osteoblasts expressing GTPase-defective dynK44A, and to a lesser extent dynY231F/Y597F, osteoblast migration was significantly inhibited by dynK44A and dynY231F/Y597F. These studies demonstrate a novel role for dynamin GTPase activity and phosphorylation in osteoblast differentiation and migration, which may be important for bone formation.


Asunto(s)
Movimiento Celular/fisiología , Dinaminas/metabolismo , Osteoblastos/citología , Osteoblastos/enzimología , Animales , Diferenciación Celular/fisiología , Dinaminas/biosíntesis , Dinaminas/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Ratones , Fosforilación
7.
Bone ; 60: 235-45, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24380811

RESUMEN

Bone homeostasis is maintained by the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation in the activity of the bone cells can lead to osteoporosis, a disease characterized by low bone mass and an increase in bone fragility and risk of fracture. Kalirin is a novel GTP-exchange factor protein that has been shown to play a role in cytoskeletal remodeling and dendritic spine formation in neurons. We examined Kalirin expression in skeletal tissue and found that it was expressed in osteoclasts and osteoblasts. Furthermore, micro-CT analyses of the distal femur of global Kalirin knockout (Kal-KO) mice revealed significantly reduced trabecular and cortical bone parameters in Kal-KO mice, compared to WT mice, with significantly reduced bone mass in 8, 14 and 36week-old female Kal-KO mice. Male mice also exhibited a decrease in bone parameters but not to the level seen in female mice. Histomorphometric analyses also revealed decreased bone formation rate in 14week-old female Kal-KO mice, as well as decreased osteoblast number/bone surface and increased osteoclast surface/bone surface. Consistent with our in vivo findings, the bone resorbing activity and differentiation of Kal-KO osteoclasts was increased in vitro. Although alkaline phosphatase activity by Kal-KO osteoblasts was increased in vitro, Kal-KO osteoblasts showed decreased mineralizing activity, as well as decreased secretion of OPG, which was inversely correlated with ERK activity. Taken together, our findings suggest that deletion of Kalirin directly affects osteoclast and osteoblast activity, leading to decreased OPG secretion by osteoblasts which is likely to alter the RANKL/OPG ratio and promote osteoclastogenesis. Therefore, Kalirin may play a role in paracrine and/or endocrine signaling events that control skeletal bone remodeling and the maintenance of bone mass.


Asunto(s)
Huesos/metabolismo , Huesos/patología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Animales , Biomarcadores/sangre , Remodelación Ósea , Huesos/diagnóstico por imagen , Recuento de Células , Diferenciación Celular , Femenino , Fémur/diagnóstico por imagen , Fémur/metabolismo , Fémur/patología , Eliminación de Gen , Factores de Intercambio de Guanina Nucleótido/deficiencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos , Osteoblastos/patología , Osteoclastos/patología , Microtomografía por Rayos X
8.
Protein Expr Purif ; 91(1): 20-9, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23827208

RESUMEN

This work describes the design and expression of a stereoselective Fab that possesses binding properties comparable to those displayed by the parent monoclonal antibody. Utilizing mRNA from hybridoma clones that secrete a stereoselective anti-l-amino acid antibody, a corresponding biotechnologically produced Fab was generated. For that, appropriate primers were designed based on extensive literature and databank searches. Using these primers in PCR resulted in successful amplification of the VH, VL, CL and CH1 gene fragments. Overlap PCR was utilized to combine the VH and CH1 sequences and the VL and CL sequences, respectively, to obtain the genes encoding the HC and LC fragments. These sequences were separately cloned into the pEXP5-CT/TOPO expression vector and used for transfection of BL21(DE3) cells. Separate expression of the two chains, followed by assembly in a refolding buffer, yielded an Fab that was demonstrated to bind to l-amino acids but not to recognize the corresponding d-enantiomers.


Asunto(s)
Aminoácidos/inmunología , Antígenos/biosíntesis , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Aminoácidos/química , Aminoácidos/aislamiento & purificación , Aminoácidos/metabolismo , Animales , Antígenos/química , Antígenos/inmunología , Antígenos/metabolismo , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Ratones , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Estereoisomerismo
9.
J Signal Transduct ; 2012: 296450, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22888421

RESUMEN

Cell adhesion to the extracellular matrix (ECM) is essential for cell migration, proliferation, and embryonic development. Cells can contact the ECM through a wide range of matrix contact structures such as focal adhesions, podosomes, and invadopodia. Although they are different in structural design and basic function, they share common remodeling proteins such as integrins, talin, paxillin, and the tyrosine kinases FAK, Pyk2, and Src. In this paper, we compare and contrast the basic organization and role of focal adhesions, podosomes, and invadopodia in different cells. In addition, we discuss the role of the tyrosine kinases, FAK, Pyk2, and Src, which are critical for the function of the different adhesion structures. Finally, we discuss the essential role of these tyrosine kinases from the perspective of human diseases.

10.
J Biol Chem ; 287(21): 17257-17268, 2012 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-22447931

RESUMEN

The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton.


Asunto(s)
Actinas/metabolismo , Caspasas/metabolismo , Quinasa 2 de Adhesión Focal/biosíntesis , Regulación Enzimológica de la Expresión Génica/fisiología , Megacariocitos/metabolismo , Osteoblastos/metabolismo , Empalme Alternativo , Animales , Diferenciación Celular/fisiología , Proliferación Celular , Células Cultivadas , Adhesiones Focales/metabolismo , Isoenzimas/biosíntesis , Megacariocitos/citología , Ratones , Osteoblastos/citología
11.
Int J Biochem Cell Biol ; 44(5): 790-800, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22342188

RESUMEN

Bone loss is caused by the dysregulated activity of osteoclasts which degrade the extracellular bone matrix. The tyrosine kinase Pyk2 is highly expressed in osteoclasts, and mice lacking Pyk2 exhibit an increase in bone mass, in part due to impairment of osteoclast function. Pyk2 is activated by phosphorylation at Y402 following integrin activation, but the mechanisms leading to Pyk2 dephosphorylation are poorly understood. In the current study, we examined the mechanism of action of the dynamin GTPase on Pyk2 dephosphorylation. Our studies reveal a novel mechanism for the interaction of Pyk2 with dynamin, which involves the binding of Pyk2's FERM domain with dynamin's plextrin homology domain. In addition, we demonstrate that the dephosphorylation of Pyk2 requires dynamin's GTPase activity and is mediated by the tyrosine phosphatase PTP-PEST. The dephosphorylation of Pyk2 by dynamin and PTP-PEST may be critical for terminating outside-in integrin signaling, and for stabilizing cytoskeletal reorganization during osteoclast bone resorption.


Asunto(s)
Resorción Ósea/enzimología , Citoesqueleto/enzimología , Dinaminas/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Osteoclastos/enzimología , Proteína Tirosina Fosfatasa no Receptora Tipo 12/metabolismo , Animales , Sitios de Unión , Resorción Ósea/patología , Adhesión Celular , Línea Celular , Citoesqueleto/patología , Humanos , Integrinas/metabolismo , Ratones , Osteoclastos/patología , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Transducción de Señal , Transfección
12.
Chirality ; 17 Suppl: S9-18, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-15612044

RESUMEN

This article describes the production of stereoselective antibodies using both classical immunological and modern molecular biological techniques. Stereoselective antibodies against alpha-hydroxy acids were raised in rabbits and mice and compared with previously produced anti-alpha-amino acid antibodies. It was found that both types of antibodies combine stereoselectivity with class-specificity. Sequence analyses revealed that antibodies with opposing stereoselectivities can be formed during the affinity maturation process from a common progenitor or independently using nonhomologous binding sites. For the first time, phage display was employed to obtain stereoselective antibody fragments. The versatility of stereoselective antibodies as chiral selectors was demonstrated by applying them in several immunosensors and in chiral chromatography. A simple, membrane-based optical sensor allowed detection of enantiomeric impurities at the 1/2,000 level (99.9% ee). Silica-based antibody chiral stationary phases could be used for enantiomer separation of aliphatic amino acids in standard-sized columns, while miniaturized columns allowed interfacing with an MS-detector.


Asunto(s)
Formación de Anticuerpos , Especificidad de Anticuerpos , Hidroxiácidos/química , Hidroxiácidos/inmunología , Secuencia de Aminoácidos , Aminoácidos/química , Aminoácidos/inmunología , Animales , Cromatografía de Afinidad/métodos , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Ratones , Datos de Secuencia Molecular , Biblioteca de Péptidos , Conejos , Homología de Secuencia de Aminoácido , Estereoisomerismo
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