RESUMEN
Caveolins form complexes of various sizes that deform membranes into polyhedral shapes. However, the recent structure of the 8S complex was disk-like with a flat membrane-binding surface. How can a flat complex deform membranes into nonplanar structures? Molecular dynamics simulations revealed that the 8S complex rapidly takes the form of a suction cup. Simulations on implicit membrane vesicles determined that binding is stronger when E140 gets protonated. In that case, the complex binds much more strongly to 5- and 10-nm-radius vesicles. A concave membrane-binding surface readily explains the membrane-deforming ability of caveolins by direct scaffolding. We propose that the 8S complex sits at the vertices of the caveolar polyhedra, rather than at the center of the polyhedral faces.
Asunto(s)
Caveolinas , Proteínas de la Membrana , Caveolinas/análisis , Caveolinas/metabolismo , Proteínas de la Membrana/química , Caveolina 1/metabolismo , Membranas/metabolismo , Membrana Celular/metabolismoRESUMEN
Caveolae are plasma membrane invaginations with a distinct lipid composition. Membrane lipids cooperate with the structural components of caveolae to generate a metastable surface domain. Recent studies have provided insights into the structure of essential caveolar components and how lipids are crucial for the formation, dynamics, and disassembly of caveolae. They also suggest new models for how caveolins, major structural components of caveolae, insert into membranes and interact with lipids.
Asunto(s)
Caveolas , Lípidos de la Membrana , Caveolas/química , Caveolas/metabolismo , Lípidos de la Membrana/análisis , Lípidos de la Membrana/metabolismo , Caveolinas/análisis , Caveolinas/metabolismo , EndocitosisRESUMEN
Background and Objectives: The saphenous vein is one of the most common used grafts (SVG) for surgical revascularization. The mechanism of the SVGs occlusion is still unknown. Surgical preparation techniques have an important role in the early and late graft occlusion. Our study analyzed the influence of the three different surgical techniques on the histological and immunohistochemical characteristics of the vein grafts. Methods: Between June 2019 and December 2020, 83 patients who underwent surgical revascularization were prospectively randomly assigned to one of the three groups, according to saphenous vein graft harvesting (conventional (CVH), no-touch (NT) and endoscopic (EVH)) technique. The vein graft samples were sent on the histological (hematoxylin-eosin staining) and immunohistochemical (CD31, Factor VIII, Caveolin and eNOS) examinations. Results: The CVH, NT, and EVH groups included 27 patients (mean age 67.66 ± 5.6), 31 patients (mean age 66.5 ± 7.4) and 25 patients (mean age 66 ± 5.5), respectively. Hematoxylin-eosin staining revealed a lower grade of microstructural vein damage in the NT group (2, IQR 1-2) in comparison with CVH and EVH (3, IQR 2-4), (4, IQR 2-4) respectively (p < 0.001). Immunohistochemical examination revealed a high grade of staining in the NT group compared to the CVH and EVH group (CD 31 antibody p = 0.02, FVIII, p < 0.001, Caveolin, p = 0.001, and eNOS, p = 0.003). Conclusion: The best preservation of the structural vein integrity was in the NT group, while the lowest rate of leg wound complication was in the EVH group. These facts increase the interest in developing and implementing the endoscopic no-touch technique.
Asunto(s)
Puente de Arteria Coronaria , Vena Safena , Anciano , Humanos , Persona de Mediana Edad , Caveolinas/análisis , Puente de Arteria Coronaria/métodos , Endoscopía , Vena Safena/química , Vena Safena/patología , Vena Safena/trasplante , Grado de Desobstrucción VascularRESUMEN
Abstract Caveolin, the protein of the caveolar membrane, interacts and binds with endothelial nitric oxide synthase (eNOS), forming a caveolin-eNOS complex leading to suppression of the eNOS activity. Caveolin, therefore, maintains eNOS in the inactivated state leading to reduced nitric oxide (NO) production. Ischemic preconditioning disrupts the caveolin-eNOS complex leading to activation of the eNOS and thus results in cardioprotection. During ischemic preconditioning, NO produces cardioprotection by the opening of the KATP channel, and the caveolin forms a suitable signalling platform facilitating the interaction of NO with the KATP channel. Estrogen deficiency has been reported to upregulate caveolin-1 expression. The article aims to review the various mechanisms that placed the women at the risk of coronary artery diseases after postmenopausal estrogen deficiency and their role in the cardioprotective effect of ischemic preconditioning.
Asunto(s)
Rol , Mujeres , Enfermedad de la Arteria Coronaria/complicaciones , Posmenopausia/metabolismo , Caveolinas/análisis , Precondicionamiento Isquémico/efectos adversos , Óxido NítricoRESUMEN
BACKGROUND: TGF-ß plays an important role in growth and development but is also involved in scarring and fibrosis. Differences for this growth factor are known between scarless fetal wound healing and adult wound healing. Nonetheless, most of the data in this area are from animal studies or in vitro studies and, thus, information about the human situation is incomplete and scarce. OBJECTIVE: The aim of this study was to compare the canonical TGF-ß signaling in unwounded human fetal and adult skin. METHODS: Q-PCR, immunohistochemistry, Western Blot and Luminex assays were used to determine gene expression, protein levels and protein localization of components of this pathway in healthy skin. RESULTS: All components of the canonical TGF-ß pathway were present in unwounded fetal skin. Compared to adult skin, fetal skin had differential concentrations of the TGF-ß isoforms, had high levels of phosphorylated receptor-Smads, especially in the epidermis, and had low expression of several fibrosis-associated target genes. Further, the results indicated that the processes of receptor endocytosis might also differ between fetal and adult skin. CONCLUSION: This descriptive study showed that there are differences in gene expression, protein concentrations and protein localization for most components of the canonical TGF-ß pathway between fetal and adult skin. The findings of this study can be a starting point for further research into the role of TGF-ß signaling in scarless healing.
Asunto(s)
Expresión Génica , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Piel/metabolismo , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adolescente , Adulto , Caveolinas/análisis , Caveolinas/genética , Clatrina/análisis , Clatrina/genética , Colágeno Tipo III/genética , Factor de Crecimiento del Tejido Conjuntivo/genética , Decorina/genética , Endocitosis/fisiología , Feto , Edad Gestacional , Humanos , Proteínas de Unión a TGF-beta Latente/genética , Proteínas de Unión a TGF-beta Latente/metabolismo , Persona de Mediana Edad , Fosforilación , Inhibidor 1 de Activador Plasminogénico/genética , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/análisis , Receptores de Factores de Crecimiento Transformadores beta/genética , Piel/química , Proteínas Smad/análisis , Proteínas Smad/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Factor de Crecimiento Transformador beta/análisis , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta1/análisis , Factor de Crecimiento Transformador beta2/análisis , Factor de Crecimiento Transformador beta3/análisis , Adulto JovenRESUMEN
Orientia tsutsugamushi is an intracellular parasite that causes scrub typhus. After entering the cytoplasm by induced phagocytosis, O. tsutsugamushi escapes from the primary phagosome into the host cytosol, where it replicates slowly. Subsequently, it is released from the host cells by a process resembling viral budding with a remaining bacterial aggregate near the nucleus. Lipid rafts have been implicated in the life cycle of a wide variety of pathogenic microorganisms. We have observed that proteins of O. tsutsugamushi were co-fractionated with the lipid rafts over a sucrose density gradient, suggesting the possible involvement of lipid rafts during the intracellular life cycle of O. tsutsugamushi. The entry of O. tsutsugamushi into the host cells was shown to be independent on lipid rafts as judged by the inability of lipid raft-disrupting agents to inhibit bacterial entry and no co-localization of bacterial proteins with caveolin. To our interest, a 47-kDa protein (HtrA) was observed to be co-localized with caveolin at the cell membrane at 72 h after infection, when bacterial particles move to the cell membrane and initiate the exit into the extracellular environment. Our results suggest that O. tsutsugamushi involves lipid rafts of the host cells in the budding-like process to exit from host cells.
Asunto(s)
Exocitosis , Microdominios de Membrana/metabolismo , Orientia tsutsugamushi/fisiología , Proteínas Bacterianas/análisis , Caveolinas/análisis , Membrana Celular/química , Microscopía Confocal , Microscopía Fluorescente , UltracentrifugaciónRESUMEN
Caveolin-rich lipid rafts (CLRs) are thickened sections of the cell membrane that are composed of the integral membrane proteins caveolins together with saturated long chain fatty acids, cholesterol and lipids. Membrane proteins - lipid raft proteins in particular - may play important roles in cell signaling and cell-cell interaction. Due to their unique structure, CLRs seem to be the preferred docking site for specific proteins involved in focal adhesion and cancer metastasis. Our objective was thus to identify and quantify CLR proteins from primary and metastatic colorectal cancer (CRC) clones. We found differential expression of nine CLR proteins from primary and metastatic CRC clones. Among the identified proteins, an immune system inhibiting protein was significantly overexpressed in the metastatic clone, while cell adhesion and transport molecules were among the overexpressed proteins in the primary clone. All the identified CRL proteins are involved in tumorigenesis, specifically metastasis, and may thus serve as therapeutic targets. A novel concept for identification and quantification of CLR proteins with label-free mass spectrometry method was specifically examined in this study. Validation of the method against immunoblotting and FACS analysis indicates that it can be applied for the identification of novel biomarkers for cancer and metastasis.
Asunto(s)
Caveolinas/análisis , Neoplasias Colorrectales/metabolismo , Microdominios de Membrana/química , Metástasis de la Neoplasia/fisiopatología , Proteínas Portadoras/inmunología , Caveolina 1/análisis , Adhesión Celular , Transformación Celular Neoplásica/metabolismo , Células Clonales , Neoplasias Colorrectales/patología , Humanos , Proteínas Mitocondriales/inmunología , Metástasis de la Neoplasia/patología , Proteómica , Espectrometría de Masas en Tándem , Células Tumorales CultivadasRESUMEN
Voltage-gated calcium channels (Ca(v)s) comprise a pore-forming α1 with auxiliary α2δ and ß subunits which modulate Ca(v) function and surface expression. Ca(v)α1 and α2δ are present in signalling complexes termed lipid rafts but it is unclear whether α2δ is obligatory for targeting Ca(v)s to rafts or to what extent this influences cell surface organisation of Ca(v)s. Here, we have used imaging, biochemistry and electrophysiology to determine localisation and raft-partitioning of WT and functionally active HA-epitope tagged α2δ-1 and Ca(v)2.2 subunits expressed in COS-7 cells. We show that α2δ-1 not only partitions into lipid rafts itself but also mediates raft-partitioning of Ca(v)2.2/ß(1b) complexes. Ca(v)α2δ-1, Ca(v)2.2/ß(1b) and Ca(v)2.2/ß(1b)/α2δ-1 complexes are all organised into cell surface clusters although only in the presence of α2δ-1 do they co-localise with raft markers, caveolin and flotillin. Such clusters persist in the presence of 3-methyl-ß-cyclodextrin even though the raft markers disperse. However, clustering is profoundly sensitive to disruption of the actin-based cytoskeleton by cytochalasin-D. We conclude that α2δ-1, and likely other α2δ subunits, is necessary and sufficient for targeting Ca(v)s to lipid rafts. However, formation of clusters supporting "hotspots" of Ca(v) activity requires aggregation of macromolecular complexes containing raft components, stabilised by interactions with the cytoskeleton.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Microdominios de Membrana/metabolismo , Actinas/metabolismo , Animales , Células COS , Canales de Calcio Tipo N/análisis , Caveolinas/análisis , Caveolinas/metabolismo , Chlorocebus aethiops , Citocalasina D/química , Citocalasina D/metabolismo , Citoesqueleto , Fenómenos Electrofisiológicos , Microdominios de Membrana/química , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Técnicas de Placa-Clamp , Subunidades de Proteína/análisis , Subunidades de Proteína/fisiología , Ratas , beta-Ciclodextrinas/farmacologíaRESUMEN
Las caveolas participan en múltiples procesos celulares tales como el transportevesicular, homeostasis del colesterol, regulación de la señalización intracelular,por integrinas y proliferación celular. Sin embargo, su función en el hígado no estábien establecida. La expresión de caveolina 1 (Cav), la proteína más abundante enlas caveolas, está bien descrita en el hígado y en varias líneas de hepatocitos y enhígado cirrótico humano y en carcinoma hepatocelular. Sin embargo, el papel deCav-1 en la fisiopatología hepática es controvertido, ya que se ha propuesto un papel crítico en el proceso de regeneración tras hepatectomía parcial (HP). Contrariamentea esta observación, nuestros datos sugieren que Cav-1 aumenta en elhígado regenerante, con una re-distribución de la proteína desde las caveolas haciadominios no caveolares. Además, la Cav-1 localizada en estas fracciones está fosforiladaen la tirosina 14. A pesar de ello, el gen de la Cav-1 es dispensable parala regeneración hepática tras HP, tal como se deduce de animales que carecen deeste gen. En conjunto, estos datos muestran un papel dinámico de la Cav-1 en laproliferación hepática tras HP y en líneas hepáticas en cultivo, pero con mínimasimplicaciones en el proceso regenerativo
Although caveolae participate in many cellular processes such as vesicular transport,cholesterol homeostasis, regulation of signal transduction, integrin signalingand cell growth, their role in liver remains elusive. Expression of caveolin 1 (Cav),the most abundant protein of caveolae, has been reported in liver and in differenthepatocyte cell lines, in human cirrhotic liver and in hepatocellular carcinomas.However, the role of Cav-1 in liver pathophysiology remains controversial and acritical role in regeneration after partial hepatectomy (PH) has been reported.Opposite to this observation, our data support the view that Cav-1 increases inliver after PH with a redistribution of the protein from the caveolae enricheddomain to the noncaveolar fraction. Moreover, the Cav-1 located in the noncaveolarfraction is phosphorylated in tyrosine 14 (Tyr14). Even though, the Cav-1 geneis dispensable for liver regeneration after PH as deduced from data obtained withcommercially available animals lacking this gene. Taken together these resultssupport a dynamic role for Cav-1 in liver proliferation both in vivo after PH, andin vitro in cultured hepatic cell lines, but with minimal implications in the liverregeneration process
Asunto(s)
Caveolinas/química , Caveolinas/farmacología , Regeneración Hepática , Hígado/química , Hepatectomía/métodos , Hepatectomía/rehabilitación , Caveolinas/análisis , Caveolinas/síntesis química , Caveolinas/farmacocinética , Regeneración Hepática/inmunología , Regeneración Hepática/fisiología , Caveolas/química , Caveolas , Hígado , Factor de Crecimiento de Hepatocito/síntesis química , Factor de Crecimiento de Hepatocito/farmacologíaRESUMEN
The caveolar cycle is thought to be regulated by synchronised function of kinases and phosphatases. Using ocadaic acid--a serine/threonine protein phosphatase inhibitor--and an inhibitor of tyrosine phosphatase (sodium orthovanadate) we have followed the internalisation of caveolae. Since albumin binding to its receptor (gp60) can induce pinching off of caveolae from the plasma membrane, we also used this physiological ligand to induce the internalisation. Our confocal microscopic results show that both ocadaic acid and vanadate treatments have significantly decreased caveolin (caveolin-1 and -2) labelling on the cell surface, while the cytoplasmic labelling became much stronger. Quite often large, strongly labelled "granules" appear at the perinuclear region. Very strong caveolin labelling was detected along the actin-cytoskeleton suggesting that caveolae might move along these filaments. Our electron microscopic results also show an intensive caveolae pinching off from the plasma membrane. After ocadaic acid and vanadate treatments the number of surface connected vesicles (caveolae) decreases. At the same time, large multivesicular bodies (termed caveosomes) appear in the perinuclear area of the cytoplasm. By immunoprecipitation and Western blot analysis we detect an increased tyrosine phosphorylation of a approximately 29kDa protein in ocadaic acid and vanadate treated samples. This protein was identified as caveolin-2. No significant change in the tyrosine phosphorylation of caveolin-1 was found. From these data we can conclude that caveolae internalisation is regulated by phosphorylation of caveolin-2.
Asunto(s)
Caveolas/metabolismo , Monoéster Fosfórico Hidrolasas/fisiología , Fosfotransferasas/fisiología , Western Blotting , Caveolinas/análisis , Línea Celular Tumoral , Membrana Celular/química , Citoplasma/química , Inhibidores Enzimáticos/farmacología , Humanos , Inmunohistoquímica , Inmunoprecipitación , Microscopía Confocal , Microscopía Electrónica de Transmisión , Modelos Biológicos , Ácido Ocadaico/farmacología , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Vanadatos/farmacologíaRESUMEN
We show here that the GTPase RhoG is involved in caveolar trafficking. Wild-type RhoG moves sequentially to the plasma membrane, intracellular vesicles, and the Golgi apparatus along markers of this endocytic pathway. Such translocation is associated with changes in RhoG GDP/GTP levels and is highly dependent on lipid raft integrity and on the function of the GTPase dynamin2. In addition, the constitutively active RhoG(Q61L) mutant is preferentially located in endocytic vesicles that can be decorated with markers of the caveola-derived endocytic pathway. RhoG(Q61L), but not the analogous Rac1 mutant protein, affects caveola internalization and the subsequent delivery of endocytic vesicles to the Golgi apparatus. The expression of RhoG/Rac1 chimeric proteins and RhoG(Q61L) effector mutants in cells induces alterations in the internalization of caveolae and severe changes in vesicle structure, respectively. However, the knockdown of endogenous rhoG transcripts using small interfering RNAs does not affect significantly the trafficking of caveola-derived vesicles, suggesting that RhoG function is dispensable for this endocytic process or, alternatively, that its function is compensated by other molecules. Taken together, these observations assign a novel function to RhoG and suggest that caveolar trafficking, as previously shown for other endocytic routes, is modulated by GTPases of the Ras superfamily.
Asunto(s)
Caveolas/fisiología , Endocitosis/fisiología , Fluoresceína-5-Isotiocianato/análogos & derivados , Proteínas de Unión al GTP rho/fisiología , Animales , Células CHO , Caveolinas/análisis , Caveolinas/fisiología , Toxina del Cólera/análisis , Clatrina/análisis , Clatrina/fisiología , Cricetinae , Cricetulus , Dinamina II/genética , Dinamina II/farmacología , Activación Enzimática , Fluoresceína-5-Isotiocianato/metabolismo , Colorantes Fluorescentes/análisis , Microscopía Fluorescente , Mutación Puntual , Transporte de Proteínas , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes de Fusión/fisiología , Fracciones Subcelulares/química , Transfección , Proteínas de Unión al GTP rho/genéticaRESUMEN
Blood stages of Plasmodium vivax induce the development of caveolae and caveola-vesicle complexes (CVC) in the membrane of their host erythrocyte. Caveolae are found in almost all types of cells and are involved in endogenous processes as calcium and cholesterol homeostasis, cell signalling, transporting, ligand internalization and transcytosis of serum components. Major structural components of caveolae are the proteins caveolins and flotillins. The functional role of caveolae in the P. vivax-infected erythrocyte is not properly understood. As these organelles have been shown to contain malaria antigens, it has been suggested that they are involved in the transport and release of specific parasite antigens from the infected erythrocyte and in the uptake of plasma proteins. Using specific antibodies to classical caveolae proteins and an immunolocalization approach, we found caveolin-2, caveolin-3, and flotillin-2 in the vesicle profiles and some CVC of P. vivax-infected erythrocytes. Caveolin-1-3 were not found in uninfected erythrocytes. This is the first report of identification and localization of caveolins in the CVC present in erythrocytes infected with P. vivax, thereby providing evidence of the role of this particular organelle in the protein-trafficking pathway that connect parasite-encoded proteins with the erythrocyte cytoplasm and the cell surface throughout the asexual blood cycle of vivax malaria parasite.
Asunto(s)
Caveolas/metabolismo , Caveolinas/análisis , Eritrocitos/parasitología , Proteínas de la Membrana/análisis , Plasmodium vivax/química , Plasmodium vivax/patogenicidad , Animales , Caveolas/ultraestructura , Membrana Eritrocítica/parasitología , Membrana Eritrocítica/ultraestructura , Eritrocitos/ultraestructura , Humanos , Estadios del Ciclo de Vida , Malaria Vivax/parasitología , Plasmodium vivax/crecimiento & desarrolloRESUMEN
BACKGROUND: Caveolin-1 is the principal protein that composes caveolae, which are vesicular invaginations present on the plasma membrane of different cell types. Caveolae are involved in a variety of cellular functions including regulation of proliferation rate and resistance to chemotherapeutic drugs. Chemotherapy frequently induces alopecia which is reversible most probably due to the low proliferative rate of hair follicle stem cells and due to the expression of proteins which confer resistance. OBJECTIVES: Using a specific animal model and immunohistochemistry, we analysed the expression of both caveolin-1 and the cell proliferation marker beta-catenin, at different stages of the hair follicle cycle, both before and after doxorubicin (DXR) -induced alopecia. METHODS: Seven-week-old C57BL/6 mice were depilated in order to synchronize hair follicle cycle in the anagen phase. Chemotherapy with DXR 15 mg kg(-1) was used to induce alopecia. Control and treated mice were then sacrificed at precise time points and caveolin-1 expression in hairs at different stages of the cycle were analysed by immunohistochemistry. By double immunofluorescence, colocalization of caveolin-1 and cytokeratin-15 was confirmed in the bulge region. The state of proliferation of cells composing hair follicle was assessed by beta-catenin immunohistochemistry. RESULTS: Caveolin-1 was expressed by the cells of the bulge area, the multipotent compartment of the hair follicle, during all phases of growth (anagen), regression (catagen) and resting (telogen). During the anagen phases, nuclear beta-catenin labelling was not observed in bulge cells, but rather in the deeper portion of the follicle. Damaged hair follicles from DXR-treated mice presented bulge cells which still expressed caveolin-1, suggesting that this protein might play a role in their drug resistance. As expected, no beta-catenin nuclear staining was detectable in DXR-treated hair follicles, indicating the complete lack of proliferative processes. The differential localization of caveolin-1 and beta-catenin suggests that the mutually exclusive expression of these proteins is useful for correct hair regrowth, whether during the physiological cycle or after chemotherapy-induced alopecia. CONCLUSIONS: Expression of caveolin-1 within the multipotent cell compartment of the hair follicle can explain the resistance of bulge cells to many chemotherapeutics, suggested by the reversibility of chemotherapy-induced alopecia.
Asunto(s)
Alopecia/metabolismo , Caveolinas/análisis , Folículo Piloso/patología , Células Madre Multipotentes/metabolismo , Alopecia/inducido químicamente , Animales , Antineoplásicos/efectos adversos , Biomarcadores/análisis , Caveolina 1 , Caveolinas/metabolismo , Proliferación Celular , Proteínas del Citoesqueleto/análisis , Doxorrubicina/efectos adversos , Resistencia a Medicamentos , Remoción del Cabello , Inmunohistoquímica/métodos , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Células Madre Multipotentes/efectos de los fármacos , Transactivadores/análisis , beta CateninaRESUMEN
Proteins implicated in the "SNARE hypothesis" for membrane fusion have been characterized in the acrosome of several mammalian species, and a functional role for these proteins during the acrosome reaction has been proposed. We have investigated the presence of SNAREs in equine sperm, using semen samples obtained from stallions with varying fertility. Immunocytochemical analysis revealed that members of different SNARE families can be detected on the acrosome of equine sperm, notably in the acrosomal cap and equatorial segment. These proteins include the t-SNARE syntaxin, the v-SNARE synaptobrevin/VAMP, the calcium sensor synaptotagmin, and the ATPase NSF. Also present is caveolin-1, a component of lipid rafts. Stallions with fertility problems presented the worst quality of sperm and acrosomal membrane, and had less sperm cells stained positively for SNAREs and caveolin-1, than sperm from fertile donors (p < 0.001). Ubiquitin surface staining was also performed and it seemed to inversely correlate with stallion fertility, supporting data obtained with the negative staining technique. A male-related problem was confirmed when mares that had failed to impregnate with samples from an infertile stallion were successfully inseminated with sperm from a fertile donor. Furthermore NSF, synaptotagmin and caveolin-1 staining seemed to be useful in predicting stallion fertility, i.e. significantly more sperm cells stained positively for these proteins in samples from fertile males. Although these results need to be expanded on a larger scale, they suggest that acrosomal and surface staining of equine sperm with novel probes may constitute useful tools in predicting stallion fertility.
Asunto(s)
Caveolinas/fisiología , Fertilidad , Caballos , Espermatozoides/química , Espermatozoides/fisiología , Proteínas de Transporte Vesicular/fisiología , Acrosoma/química , Acrosoma/ultraestructura , Reacción Acrosómica , Animales , Proteínas de Unión al Calcio/análisis , Caveolina 1 , Caveolinas/análisis , Femenino , Inmunohistoquímica , Inseminación Artificial/veterinaria , Masculino , Glicoproteínas de Membrana/análisis , Proteínas de la Membrana/análisis , Proteínas Sensibles a N-Etilmaleimida , Proteínas del Tejido Nervioso/análisis , Proteínas Qa-SNARE , Proteínas R-SNARE , Proteínas SNARE , Sinaptotagminas , Ubiquitina/análisis , Ubiquitina/metabolismo , Proteínas de Transporte Vesicular/análisisRESUMEN
We describe here--presumably for the first time--a Cajal-like type of tubal interstitial cells (t-ICC), resembling the archetypal enteric ICC. t-ICC were demonstrated in situ and in vitro on fresh preparations (tissue cryosections and primary cell cultures) using methylene-blue, crystal-violet, Janus-Green B or MitoTracker-Green FM Probe vital stainings. Also, t-ICC were identified in fixed specimens by light microscopy (methylene-blue, Giemsa, trichrome stainings, Gomori silver-impregnation) or transmission electron microscopy (TEM). The positive diagnosis of t-ICC was strengthened by immunohistochemistry (IHC; CD117/c-kit+ and other 14 antigens) and immunofluorescence (IF; CD117/c-kit+ and other 7 antigens). The spatial density of t-ICC (ampullar-segment cryosections) was 100-150 cells/mm2. Non-conventional light microscopy (NCLM) of Epon semithin-sections revealed a network-like distribution of t-ICC in lamina propria and smooth muscle meshwork. t-ICC appeared located beneath of epithelium, in a 10-15 microm thick 'belt', where 18+/-2% of cells were t-ICC. In the whole lamina propria, t-ICC were about 9%, and in muscularis approximately 7%. In toto, t-ICC represent ~8% of subepithelial cells, as counted by NCLM. In vitro, t-ICC were 9.9+/-0.9% of total cell population. TEM showed that the diagnostic 'gold standard' (Huizinga et al., 1997) is fulfilled by 'our' t-ICC. However, we suggest a 'platinum standard', adding a new defining criterion- characteristic cytoplasmic processes (number: 1-5; length: tens of microm; thickness: < or =0.5 microm; aspect: moniliform; branching: dichotomous; organization: network, labyrinthic-system). Quantitatively, the ultrastructural architecture of t-ICC is: nucleus, 23.6+/-3.2% of cell volume, with heterochromatin 49.1+/-3.8%; mitochondria, 4.8+/-1.7%; rough and smooth endoplasmic-reticulum (1.1+/-0.6%, 1.0+/-0.2%, respectively); caveolae, 3.4+/-0.5%. We found more caveolae on the surface of cell processes versus cell body, as confirmed by IF for caveolins. Occasionally, the so-called 'Ca2+-release units' (subplasmalemmal close associations of caveolae+endoplasmic reticulum+mitochondria) were detected in the dilations of cell processes. Electrophysiological single unit recordings of t-ICC in primary cultures indicated sustained spontaneous electrical activity (amplitude of membrane potentials: 57.26+/-6.56 mV). Besides the CD117/c-kit marker, t-ICC expressed variously CD34, caveolins 1&2, alpha-SMA, S-100, vimentin, nestin, desmin, NK-1. t-ICC were negative for: CD68, CD1a, CD62P, NSE, GFAP, chromogranin-A, PGP9.5, but IHC showed the possible existence of (neuro)endocrine cells in tubal interstitium. We call them 'JF cells'. In conclusion, the identification of t-ICC might open the door for understanding some tubal functions, e.g. pace-making/peristaltism, secretion (auto-, juxta- and/or paracrine), regulation of neurotransmission (nitrergic/purinergic) and intercellular signaling, via the very long processes. Furthermore, t-ICC might even be uncommitted bipotential progenitor cells.
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Células del Tejido Conectivo/citología , Trompas Uterinas/citología , Actinas/análisis , Antígenos CD34/análisis , Membrana Basal/citología , Membrana Basal/ultraestructura , Vasos Sanguíneos/ultraestructura , Antígenos CD57/análisis , Caveolas/ultraestructura , Caveolinas/análisis , Recuento de Células , Núcleo Celular/ultraestructura , Extensiones de la Superficie Celular/ultraestructura , Células Cultivadas , Cromogranina A , Cromograninas/análisis , Células del Tejido Conectivo/química , Células del Tejido Conectivo/ultraestructura , Citoplasma/ultraestructura , Electrofisiología , Trompas Uterinas/química , Trompas Uterinas/ultraestructura , Femenino , Histocitoquímica , Humanos , Uniones Intercelulares/ultraestructura , Proteínas de Filamentos Intermediarios/análisis , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Mitocondrias/ultraestructura , Membrana Mucosa/citología , Músculo Liso/citología , Músculo Liso/ultraestructura , Fibras Nerviosas/ultraestructura , Proteínas Proto-Oncogénicas c-kit/análisis , Proteínas S100/análisis , Coloración y Etiquetado , Ubiquitina Tiolesterasa/análisisRESUMEN
Stimulation of endothelin receptors (ETRs) leads to activation of the extracellular signal-regulated protein kinase (ERK) cascade. It is unclear whether compartmentalization to lipid rafts is necessary for proper endothelin signaling, as methodologies employed to isolate and study caveolae involve detergent extraction, which may induce aggregation of membrane-associated proteins. The present study was to determine if components of the endothelin-1 (ET-1) pathway leading to ERK activation localize to caveolae and constitute preformed signalosomes. Microsomes were prepared from intact ventricular myocardium, in the absence of detergents, and fractionated by differential and sucrose-density gradient centrifugation to determine if caveolins and components of the ETRs post-receptor signaling cascade were in vesicles having similar physical properties. Confocal fluorescence microscopy, followed by digital deconvolution, was employed to determine if the signaling proteins colocalized with caveolin within intact, freshly isolated adult myocytes. With the exception of ET(A)Rs, proteins from the ET-1 pathway copurified in part or entirely (Galpha(11)), with caveolin-1 and caveolin-3. In contrast, with the exception of Galpha(q/11), Galpha(i3) and Gbeta G-protein subunits, most of the proteins studied showed little colocalization with caveolin-3. Thus, although components of the ET-1 signaling pathway may exist in vesicles having similar characteristics to vesicles containing caveolin, these proteins did not associate with caveolae in intact myocytes. The lack of detectable colocalization of caveolin-3 with proteins within the endothelin post-receptor signaling system in intact myocytes argues against the presence of a preformed, caveolae-associated signalosome.
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Caveolas/química , Caveolinas/análisis , Miocardio/química , Miocitos Cardíacos/química , Receptores de Endotelina/análisis , Transducción de Señal , Animales , Caveolas/metabolismo , Caveolina 1 , Caveolina 3 , Caveolinas/metabolismo , Perros , Proteínas de Unión al GTP/metabolismo , Ventrículos Cardíacos/citología , Isoenzimas/metabolismo , Microsomas/química , Microsomas/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Fosfolipasa C beta , Proteína Quinasa C/metabolismo , Proteína Quinasa C-epsilon , Ratas , Receptores de Endotelina/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
Decreased endothelial NO synthase (eNOS)-derived NO bioavailability and impaired vasomotor control are crucial factors in cardiovascular disease pathogenesis. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a vascular disorder associated with ENDOGLIN (ENG) haploinsufficiency and characterized by venous dilatations, focal loss of capillaries, and arteriovenous malformations (AVMs). We report that resistance arteries from Eng+/- mice display an eNOS-dependent enhancement in endothelium-dependent dilatation and impairment in the myogenic response, despite reduced eNOS levels. We have found that eNOS is significantly reduced in endoglin-deficient endothelial cells because of decreased eNOS protein half-life. We demonstrate that endoglin can reside in caveolae and associate with eNOS, suggesting a stabilizing function of endoglin for eNOS. After Ca2+-induced activation, endoglin-deficient endothelial cells have reduced eNOS/Hsp90 association, produce less NO, and generate more eNOS-derived superoxide (O2-), indicating that endoglin also facilitates eNOS/Hsp90 interactions and is an important regulator in the coupling of eNOS activity. Treatment with an O2- scavenger reverses the vasomotor abnormalities in Eng(+/-) arteries, suggesting that uncoupled eNOS and resulting impaired myogenic response represent early events in HHT1 pathogenesis and that the use of antioxidants may provide a novel therapeutic modality.
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Péptidos y Proteínas de Señalización Intracelular/fisiología , Óxido Nítrico Sintasa/fisiología , Resistencia Vascular/fisiología , Acetilcolina/farmacología , Animales , Antígenos CD , Presión Sanguínea/efectos de los fármacos , Caveolina 1 , Caveolinas/análisis , Células Cultivadas/efectos de los fármacos , Regulación hacia Abajo , Endoglina , Endotelio Vascular/fisiología , Activación Enzimática/fisiología , Proteínas HSP90 de Choque Térmico/análisis , Proteínas HSP90 de Choque Térmico/fisiología , Heterocigoto , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Hígado/enzimología , Microdominios de Membrana/química , Microdominios de Membrana/enzimología , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/enzimología , Arterias Mesentéricas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/fisiología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Nitroprusiato/farmacología , Fenilefrina/farmacología , Receptores de Superficie Celular , Transducción de Señal/fisiología , Superóxido Dismutasa/farmacología , Telangiectasia Hemorrágica Hereditaria/enzimología , Telangiectasia Hemorrágica Hereditaria/genética , Venas Umbilicales/citología , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/fisiología , Resistencia Vascular/genética , Vasodilatación/efectos de los fármacosRESUMEN
BACKGROUND: Over-expression of the oncogene c-Myc has been implicated in the development and progression of human prostate carcinoma. However, previous assessments of c-Myc expression have not revealed its potential for predicting prostate carcinoma progression. Caveolin-1 is associated with prostate carcinoma progression and is a downstream target gene of c-Myc. The observation that caveolin-1 can suppress c-Myc-induced apoptosis suggested the potential for cooperation between c-Myc and caveolin-1 in malignant progression. In this study, the authors evaluated the prognostic potential of combined c-Myc and caveolin-1 expression in human prostate carcinoma progression. METHODS: Immunostaining with c-Myc and caveolin-1-specific antibodies was performed on paraffin sections from 104 radical prostatectomy specimens from men with lymph node negative prostate carcinoma. Combined c-Myc and caveolin-1 immunostaining scores were related with the clinical and pathologic features and the probability of prostate-specific antigen recurrence after surgery. RESULTS: The combination of c-Myc and caveolin-1 immunopositivity correlated positively with Gleason score (rho = 0.219; P = 0.0253) and positive surgical margin (rho = 0.333; P = 0.0006). The combination of positive c-Myc and caveolin-1 in patients with clinically confined prostate carcinoma was a significant prognostic marker for the time to disease progression after surgery in both univariate analysis (P = 0.0039; hazard ratio, 3.035) and multivariate analysis (P = 0.0114; hazard ratio, 2.916). CONCLUSIONS: The coexpression of c-Myc and caveolin-1 showed potential as a useful prognostic marker for human prostate carcinoma. The current results suggest interactions between c-Myc and caveolin-1 in the progression of human prostate carcinoma.
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Adenocarcinoma/patología , Biomarcadores de Tumor/metabolismo , Caveolinas/análisis , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Adenocarcinoma/cirugía , Distribución por Edad , Anciano , Biopsia con Aguja , Caveolina 1 , Estudios de Cohortes , Intervalos de Confianza , Progresión de la Enfermedad , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Probabilidad , Pronóstico , Modelos de Riesgos Proporcionales , Prostatectomía/métodos , Neoplasias de la Próstata/mortalidad , Neoplasias de la Próstata/cirugía , Medición de Riesgo , Sensibilidad y Especificidad , Análisis de Supervivencia , Técnicas de Cultivo de Tejidos , Resultado del TratamientoRESUMEN
The vascular endothelium is richly endowed with caveolae, which are specialized membrane microdomains that facilitate the integration of specific cellular signal transduction processes. We found that the large conductance Ca(2+)-activated K+ (BK) channels are associated with caveolin-1 in bovine aortic endothelial cells (BAECs). OptiPrep gradient cell fractionation demonstrated that BK channels were concentrated in the caveolae-rich fraction in BAECs. Immunofluorescence imaging showed co-localization of caveolin-1 and BK channels in the BAEC membrane. Immunoprecipitation and glutathione S-transferase pull-down assay results indicated that caveolin-1 and BK channels are physically associated. However, whole cell patch clamp recordings could not detect BK (iberiotoxin-sensitive) currents in cultured BAECs under baseline conditions, even though the presence of BK mRNA and protein expression was confirmed by reverse transcription-PCR and Western blots. Cholesterol depletion redistributed the BK channels to non-caveolar fractions of BAECs, resulting in BK channel activation (7.3 +/- 1.6 pA/picofarad (pF), n = 5). BK currents were also activated by isoproterenol (ISO, 1 microM, 6.9 +/- 2.4 pA/pF, n = 6). Inclusion of a caveolin-1 scaffolding domain peptide (10 microM) in the pipette solution completely abrogated the effects of ISO on BK channel activation, whereas inclusion of the scrambled control peptide (10 microM) did not inhibit the ISO effects. We have also found that caveolin-1 knockdown by small interference RNA activated BK currents (5.3 +/- 1.4 pA/pF, n = 6). We conclude that: 1) BK channels are targeted to caveolae microdomains in vascular endothelial cells; 2) caveolin-1 interacts with BK channels and exerts a negative regulatory effect on channel functions; and 3) BK channels are inactive under control conditions but can be activated by cholesterol depletion, knockdown of caveolin-1 expression, or ISO stimulation. These novel findings may have important implications for the role of BK channels in the regulation of endothelial function.
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Caveolas/fisiología , Células Endoteliales/fisiología , Canales de Potasio Calcio-Activados/fisiología , Secuencia de Aminoácidos , Animales , Bovinos , Caveolina 1 , Caveolinas/análisis , Caveolinas/fisiología , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio , Datos de Secuencia Molecular , Canales de Potasio Calcio-Activados/análisis , Canales de Potasio Calcio-Activados/química , Canales de Sodio/fisiologíaRESUMEN
Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)-deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1-independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.