8 results on '"Serrano-Sánchez, María Isabel"'
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2. Proliferation, apoptosis, and number of Sertoli cells in the Syrian hamster during recrudescence after exposure to short photoperiod†‡
- Author
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Martínez-Hernández, Jesús, primary, Seco-Rovira, Vicente, primary, Beltrán-Frutos, Ester, primary, Ferrer, Concepción, primary, Serrano-Sánchez, María Isabel, primary, and Pastor, Luis Miguel, primary
- Published
- 2019
- Full Text
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3. Regulación de la expresión de acetilcolinesterasa de células Caco-2 de adenocarcinoma colorrectal humano por el sistema colinérgico
- Author
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Serrano Sánchez, María Isabel, Campoy Menéndez, Francisco Javier, Vidal Moreno, Cecilio Jesús, and Universidad de Murcia. Departamento de Bioquímica y Biología Molecular A
- Subjects
Genética molecular ,Citología ,Ciencias - Abstract
Las colinesterasas, acetilcolinesterasa (AChE) y butirilcolinesterasa (BuChE), hidrolizan la acetilcolina (ACh) y ejercen acciones no catalíticas, estando implicadas en los procesos de proliferación, diferenciación, morfogénesis, hematopoyesis, apoptosis y tumorogénesis. En estudios previos habíamos observado un gran aumento de la actividad AChE en las células Caco-2 al ser tratadas con un inhibidor reversible y específico de AChE, el BW284c51, lo que podría reflejar la existencia de un mecanismo regulador de los niveles de AChE activa. Así, el objetivo general de esta Tesis Doctoral fue obtener información sobre el posible mecanismo regulador de la actividad AChE mediado por los receptores de acetilcolina en las células de colon. La metodología incluye el empleo de cultivos de células Caco-2 y SW480 con agonistas y antagonistas colinérgicos, los ensayos colorimétricos para la medida de actividad AChE y los ensayos de reducción del MTT para la medida de la viabilidad celular. La cuantificación relativa de los mensajeros de los componentes del sistema colinérgico se llevó a cabo mediante RT PCR a tiempo real. Se realizaron análisis de sedimentación para determinar el contenido de formas moleculares de AChE y ensayos de Western blot para la detección de proteínas. Además se realizaron tinciones de Hoechst para la detección de células apoptóticas y tinciones histocitoquímicas de AChE. Los resultados de estos estudios revelaron la existencia de un mecanismo regulador de la AChE de las células de colon por el que aumenta la actividad AChE, en respuesta a una exposición prolongada de los receptores de acetilcolina a agonistas colinérgicos como el carbacol. Mediante el uso de cicloheximida se vio que el mecanismo de acción del carbacol requería la síntesis de nueva proteína AChE, además en los ensayos de Western blot se observó un aumento en el contenido de proteína AChE en células Caco-2 tratadas con BW. La variante 3’ del mensajero de AChE más abundante fue la de tipo H; además se observaron variantes 5’ con los exones E1c y E1e. Los cambios que producen carbacol y BW en la expresión del gen ACHE no explican el aumento observado en la actividad AChE. El patrón de formas moleculares de AChE en las células Caco-2 tampoco se vio modificado por el tratamiento con carbacol o con BW, siendo las formas moleculares predominantes en estas células las de tipos G1A y G2A. Por su parte, la tinción con Hoechst mostró que el carbacol no induce apoptosis en las células Caco-2. Además, el teñido histocitoquímico indicó que el aumento de actividad AChE por carbacol se produce de forma similar en todas las células tratadas, descartando que la sobrerregulación de AChE se deba a la inducción de apoptosis. Los tratamientos con quelantes de calcio demostraron un papel del calcio intracelular en el mecanismo de acción del carbacol que da lugar a su efecto sobrerregulador de AChE. Por último, los estudios con agonistas (acetilcolina, carbacol, nicotina, colina, oxotremorina M) mostraron que la activación tanto de los mAChR como de los nAChR produce un aumento de la actividad AChE, lo que sugiere que acetilcolina y carbacol ejercen su acción sobrerreguladora de AChE actuando sobre ambos tipos de receptores, tanto en las células Caco-2 como en las SW480. Los resultados de los ensayos de RT-PCR sugieren que en ambas líneas podrían formarse receptores nicotínicos de tipos homomérico α7 y muscular, junto a posibles heterómeros α3β4α5, α3β2β4α5 y α4β2α5, además de mAChR M3; aunque serán necesarios más estudios para caracterizar los distintos tipos de receptores implicados en este mecanismo sobrerregulador de AChE y las distintas etapas del proceso., Cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), hydrolyze acetylcholine (ACh) and exert non-catalytic actions, being involved in processes of proliferation, differentiation, morphogenesis, hematopoiesis, apoptosis and tumorogenesis. In previous studies we had observed a large increase in AChE activity in Caco-2 cells when treated with a reversible and specific AChE inhibitor, BW284c51, which could reflect the existence of a mechanism regulating active AChE levels. Thus, the general objective of this Doctoral Thesis was to obtain information on the possible regulatory mechanism of AChE activity mediated by acetylcholine receptors in intestine cells. The methodology includes culturing Caco-2 and SW480 cells with cholinergic agonists and antagonists, colorimetric assays for measuring AChE activity and MTT reduction assays for measuring cell viability. The relative quantification of the mRNA of the cholinergic system components was carried out by real-time RT-PCR. Sedimentation analyzes were performed to determine the content of AChE molecular forms and Western blotting assays for protein detection. In addition, Hoechst dye was used for detection of apoptotic cells, and histocytochemical AChE staining assays were also performed. These studies revealed the existence of a regulatory mechanism of AChE activity levels in intestine cells, by which AChE activity increases in response to prolonged exposure of acetylcholine receptors to cholinergic agonists such as carbachol. By using cycloheximide, it was found that the mechanism of action of carbachol requires the synthesis of new AChE protein. In Western blotting assays, an increase in AChE protein content was observed in Caco-2 cells treated with BW. The most abundant 3' variant of AChE mRNA was type H; 5' variants with exons E1c and E1e were also observed. The changes produced by carbachol and BW in ACHE gene expression do not explain the increase in AChE activity. The pattern of AChE molecular forms in Caco-2 cells was not modified by carbachol or BW treatments, the G1A and G2A molecules being the predominant molecular forms in these cells. On the other hand, Hoechst staining showed that carbachol does not induce apoptosis in Caco-2 cells. In addition, histocytochemical staining indicated that the increase in AChE activity after carbachol treatment is shown similarly by all treated cells, discarding that the upregulation of AChE is due to the induction of apoptosis. Calcium chelator treatments demonstrated a role of intracellular calcium in the mechanism of action of carbachol for AChE upregulation. Finally, studies with agonists (acetylcholine, carbachol, nicotine, choline, oxotremorine M) showed that activation of either muscarinic or nicotinic ACh receptors results in an increase in AChE activity. This suggests that acetylcholine and carbachol exert their upregulatory action on AChE by acting on both types of receptors in Caco 2 and SW480 cells. The results of the RT-PCR assays indicate that homomeric α7 as well as muscle type nicotinic receptors may form in both lines, together with possible α3β4α5, α3β2β4α5 and α4β2α5 heteromers, along with M3 mAChR. More studies will be needed to characterize the different types of receptors involved in this AChE upregulatory mechanism and the different steps of the process.
- Published
- 2017
4. Lectin-binding pattern of glycoconjugates during spontaneous testicular recrudescence in Syrian hamster (Mesocricetus auratus) after exposure to short photoperiod
- Author
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Martínez-Hernández, Jesús, primary, Seco-Rovira, Vicente, additional, Beltrán-Frutos, Ester, additional, Ferrer, Concepción, additional, Serrano-Sánchez, María Isabel, additional, and Pastor, Luis Miguel, additional
- Published
- 2018
- Full Text
- View/download PDF
5. Proliferation, apoptosis, and number of Sertoli cells in the Syrian hamster during recrudescence after exposure to short photoperiod†‡.
- Author
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Martínez-Hernández, Jesús, Seco-Rovira, Vicente, Beltrán-Frutos, Ester, Ferrer, Concepción, Serrano-Sánchez, María Isabel, and Pastor, Luis Miguel
- Abstract
The Sertoli cell (Sc) has been described as a quiescent cell once the animal has reached sexual maturity. Syrian hamster is an animal that displays testicular regression due to short photoperiod, during which process germ cells and Sc are removed through apoptosis. The aim of this work was to investigate histochemically whether the spontaneous testicular recrudescence processes after exposure to a short photoperiod lead to an increase in Sc proliferative activity in order to restore the normal population. Three spontaneous recrudescence groups were established: initial (IR), advanced (AR), and total (TR) recrudescence, which were compared with animal undergoing the regression process (mild: MRg, strong: SRg, and total: TRg) and animals in long photoperiod (Controls). Histological sections were submitted to histochemical techniques for detecting apoptotic and proliferative Sc with bright-field and fluorescence microscopy. For each group, the proliferative Sc index (PScI) and apoptotic Sc index (AScI), and the total number of Sc were obtained. The results revealed the existence of Vimentin+/TUNEL+ as well as Vimentin+/PCNA+ cells. The PScI was significantly higher in TRg and IR than in the other groups. The AScI was only significantly higher in MRg and SRg with respect to the other groups. The total number of Sc increased among TRg, IR, and AR, reaching values similar to those of the Controls. In conclusion, the increase in Sc proliferation from final regression and recrudescence, accompanied by a similar rate of apoptosis to the Control group, is the cause of the restoration of the Sc population during spontaneous recrudescence.
- Published
- 2020
- Full Text
- View/download PDF
6. Lectin‐binding pattern of glycoconjugates during spontaneous testicular recrudescence in Syrian hamster (Mesocricetus auratus) after exposure to short photoperiod.
- Author
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Beltrán‐Frutos, Ester, Ferrer, Concepción, Serrano‐Sánchez, María Isabel, Pastor, Luis Miguel, Martínez‐Hernández, Jesús, and Seco‐Rovira, Vicente
- Abstract
Lectin histochemistry was used to characterise glycoconjugates and cellular apoptosis in the seminiferous epithelium and interstitium of hamster testis during spontaneous recrudescence. An increase in the LTA lectin affinity was observed in spermatids in the Golgi phase. An increase in labelling of PNA and Con‐A lectin in acrosome of spermatids (acrosome phase) as well as increased labelling with Con‐A in spermatids (cap phase) was observed. Spermatocytes showed decreased affinity with PNA and AAA lectins and an increase in positivity for LTA and GNA lectins. Spermatogonia showed a slight decrease in positivity to WGA and an increase in labelling with Con‐A and a decreased affinity for the AAA lectin. At the end of recrudescence, all these germinal cells showed a similar pattern to the control. The Sertoli cells showed a gradual decrease in labelling with the GNA lectin and the Leydig cells an increase in labelling with Con‐A and GNA. Particularly unusual was the observation of apoptotic spermatocytes and spermatids positive for PNA, GNA, AAA and Con‐A, together with spermatocytes positive to LTA. In conclusion, the normal lectin pattern is recovered during testis recrudescence and germ cell apoptotic activity is low, as is observed by specific lectins for germ cells in apoptosis. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
7. HSP47 expression in the hamster Sertoli cell: An immunohistochemical study.
- Author
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Seco-Rovira V, Serrano-Sánchez MI, Beltrán-Frutos E, Martínez-Hernández J, Ferrer C, and Pastor LM
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- Male, Animals, Cricetinae, Mesocricetus, Seminiferous Tubules metabolism, Seminiferous Tubules pathology, Testis metabolism, Testis pathology, Aging metabolism, Aging pathology, Photoperiod, Sertoli Cells metabolism, Sertoli Cells pathology, HSP47 Heat-Shock Proteins metabolism, Spermatogenesis physiology, Immunohistochemistry
- Abstract
HSP47, a chaperone whose main function is the maturation of collagen molecules, is considered a marker of fibrotic diseases. Increased collagen synthesis in the testis has been associated with various pathologies leading to seminiferous tubule regression. Our aim was to study whether HSP47 is expressed in hamster Sertoli cells both in the adult and in two physiological situations of seminiferous tubule atrophy: irreversible testicular ageing and testicular regression due to short photoperiod (reversible). Eighteen animals were divided as follows: a group of 6 young animals aged 6 months, a group of 6 animals aged 24 months, which were exposed to a long photoperiod, and a final group of 6 young animals subjected to a short photoperiod. Testicular samples were fixed in methacarn and an immunohistochemical technique was used to detect HSP47. A semiquantitative study of of this protein expresion was performed between tubular sections of aged animals with complete spermatogenesis and arrested spermatogenesis and tubular sections with arrest spermatogenesis of photoinhibited testes. Sertoli cells were positive for HSP47, the intensity being greater in tubular sections with arrested spermatogenesis in both aged and photoinhibited animals. Semiquantitative analysis corroborated this observation in the sense that the expression of this protein differed according to the functional state of the seminiferous tubules. Thus, the radio of immunoreactivity was significantly higher in tubular sections with arrested spermatogenesis in aged animals compared with regressed animals, and in the latter compared with those whose tubular sections showed complete spermatogenesis. In conclusion, HSP47 expression in Sertoli cells was found for the first time in mammals. Moreover, increased expression seemed to be related to the degree of atrophy of the seminiferous epithelium and to the reversible or non-reversible physiological state of the seminiferous epithelium., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)
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- 2024
- Full Text
- View/download PDF
8. Lectin-binding pattern of glycoconjugates during spontaneous testicular recrudescence in Syrian hamster (Mesocricetus auratus) after exposure to short photoperiod.
- Author
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Martínez-Hernández J, Seco-Rovira V, Beltrán-Frutos E, Ferrer C, Serrano-Sánchez MI, and Pastor LM
- Subjects
- Acrosome metabolism, Animals, Cricetinae, Male, Mesocricetus, Recurrence, Seminiferous Epithelium metabolism, Spermatids metabolism, Apoptosis physiology, Glycoconjugates metabolism, Lectins metabolism, Photoperiod, Testis metabolism
- Abstract
Lectin histochemistry was used to characterise glycoconjugates and cellular apoptosis in the seminiferous epithelium and interstitium of hamster testis during spontaneous recrudescence. An increase in the LTA lectin affinity was observed in spermatids in the Golgi phase. An increase in labelling of PNA and Con-A lectin in acrosome of spermatids (acrosome phase) as well as increased labelling with Con-A in spermatids (cap phase) was observed. Spermatocytes showed decreased affinity with PNA and AAA lectins and an increase in positivity for LTA and GNA lectins. Spermatogonia showed a slight decrease in positivity to WGA and an increase in labelling with Con-A and a decreased affinity for the AAA lectin. At the end of recrudescence, all these germinal cells showed a similar pattern to the control. The Sertoli cells showed a gradual decrease in labelling with the GNA lectin and the Leydig cells an increase in labelling with Con-A and GNA. Particularly unusual was the observation of apoptotic spermatocytes and spermatids positive for PNA, GNA, AAA and Con-A, together with spermatocytes positive to LTA. In conclusion, the normal lectin pattern is recovered during testis recrudescence and germ cell apoptotic activity is low, as is observed by specific lectins for germ cells in apoptosis., (© 2018 Blackwell Verlag GmbH.)
- Published
- 2019
- Full Text
- View/download PDF
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