RESUMEN
This work analysed intracellular calcium stores of boar spermatozoa subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced acrosome exocytosis (IVAE). Intracellular calcium was analysed through two calcium markers with different physico-chemical properties, Fluo-3 and Rhod-5N. Indicative parameters of IVC and IVAE were also evaluated. Fluo-3 was located at both the midpiece and the whole head. Rhod-5N was present at the sperm head. This distribution did not change in any of the assayed conditions. Induction of IVC was concomitant with an increase in both head and midpiece Ca(2+) signals. Additionally, while IVC induction was concurrent with a significant (p < 0.05) increase in sperm membrane permeability, no significant changes were observed in O2 consumption and ATP levels. Incubation of boar spermatozoa in the absence of calcium showed a loss of both Ca(2+) labellings concomitantly with the sperm's inability to achieve IVC. The absence of extracellular calcium also induced a severe decrease in the percentage of spermatozoa exhibiting high mitochondrial membrane potential (hMMP). The IVAE was accompanied by a fast increase in both Ca(2+) signalling in control spermatozoa. These peaks were either not detected or much lessened in the absence of calcium. Remarkably, Fluo-3 marking at the midpiece increased after progesterone addition to sperm cells incubated in a medium without Ca(2+) . The simultaneous addition of progesterone with the calcium chelant EGTA inhibited IVAE, and this was accompanied by a significant (p < 0.05) decrease in the intensity of progesterone Ca(2+) -induced peak, O2 consumption and ATP levels. Our results suggest that boar spermatozoa present different calcium deposits with a dynamic equilibrium among them and with the extracellular environment. Additionally, the modulation role of the intracellular calcium in spermatozoa function seems to rely on its precise localization in boar spermatozoa.
Asunto(s)
Reacción Acrosómica , Calcio/metabolismo , Capacitación Espermática , Espermatozoides/metabolismo , Porcinos/metabolismo , Adenosina Trifosfato/metabolismo , Compuestos de Anilina , Animales , Ácido Egtácico , Exocitosis , Masculino , Fluidez de la Membrana , Potencial de la Membrana Mitocondrial , Modelos Biológicos , Oxígeno/metabolismo , Rodaminas , Motilidad Espermática , XantenosRESUMEN
The aim of this work was to determine the existence of a functional Wnt/ß-catenin signaling pathway in boar spermatozoa, which would be linked with the already well-known GSK-3 signaling pathway. This was first confirmed by detecting the presence of the specific Frizzled 3 receptor in these cells. Furthermore, this signaling pathway was activated in boar spermatozoa subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced 'in vitro' acrosome exocytosis (IVAE) by incubating cells with separate concentrations of Wnt1 ligand, the Wnt/ß-catenin signaling pathway-specific effector. Incubation with the Wnt1 ligand increased the rhythm of the time-dependent reduction in sperm viability during the achievement of both IVC and IVAE. This finding was concomitant with an increase in the percentage of spermatozoa with altered membrane fluidity and permeability determined through both merocyanine-540 and YO-PRO-1 stains. While the Wnt1 ligand did not affect total sperm motility during the achievement of the IVC, it induced a fast and transient increase in the overall motility patterns in spermatozoa subjected to IVAE. This IVAE-linked action was related to a decrease in the percentage of cells with high mitochondrial membrane potential and an increase in the percentage of cells with high intracellular Fluo-3-marked Ca(2+) content. In conclusion, our results suggest that the Wnt1 ligand-modulated Wnt/ß-catenin signaling pathway plays a relevant role in the modulation of both IVC and subsequent, progesterone-induced IVAE. Furthermore, our data indicate that the transduction pathways by which the Wnt1 ligand acts on IVC and IVAE are different, and that the Wnt/ß-catenin pathway is independent from GSK-3 activity in the achievement of IVC.
Asunto(s)
Exocitosis , Receptores Frizzled/metabolismo , Capacitación Espermática , Proteína Wnt1/metabolismo , Animales , Técnicas In Vitro , Masculino , PorcinosRESUMEN
The aim of this study is to determine changes in the expression and location of protein serine phosphorylation (pSer) during 'in vitro' capacitation (IVC) and 'in vitro' acrosome exocytosis (IVAE) in boar spermatozoa. This was performed in both mono- and bi-dimensional analyses of protein expression through Western blot, as well as through immunocytochemistry. Furthermore, IVC was induced through incubation in an IVC medium, and afterwards, progesterone-induced IVAE was performed. The mono-dimensional Western blot analysis showed the presence of a predominant pSer band of approximately 70-75 kDa, which was accompanied by fainter bands, especially three with molecular weights of approximately 50, 35 and 32 kDa. Neither IVC nor IVAE significantly modified this pattern. Bi-dimensional analyses showed a more complex pattern, with at least five protein clusters. The attainment of IVC caused the disappearance of the proteins with the highest molecular weight concomitantly with the appearance of pSer proteins of 75-kDa/pI 9.5 and 80-kDa/pI 10. The induction of IVAE caused the appearance of new pSer proteins of a 75-kDa/pI 6.5-7.5 and 75-kDa/pI 10. Immunocytochemistry showed that the main pSer expression in boar expression before the attainment of IVC was located at the midpiece. The IVC induced the appearance of acrosomal pSer, which was greatly increased during IVAE. Our results indicate that the changes in serine protein phosphorylation associated with IVC and IVAE comprise not only the appearance of specific phosphorylated proteins, such as the pSer-75 kDa, but also changes in pI and displacements in the sperm location of phosphorylated proteins, like the specific acrosomal pSer signal induced during IVC.
Asunto(s)
Reacción Acrosómica/efectos de los fármacos , Acrosoma/química , Fosfoserina/análisis , Progesterona/farmacología , Capacitación Espermática/fisiología , Porcinos , Acrosoma/fisiología , Animales , Western Blotting/veterinaria , Inmunohistoquímica , Técnicas In Vitro , Masculino , FosforilaciónRESUMEN
The main scope of this manuscript is to analyse the dynamics of mitochondrial activity in boar sperm subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced 'in vitro' acrosome reaction (IVAR). This was determined after analysis of the rhythm of O(2) consumption and concomitant changes in the mitochondria activity-specific JC-1 staining. Results showed that IVC, and especially IVAR, was concomitant with a peak in O(2) consumption (from 1.61 ± 0.08 nmol O(2)/min/10(7) viable sperm at 0 h of incubation to 2.62 ± 0.12 nmol O(2) /min/10(7) viable sperm after 5 min of IVAR induction). These results were accompanied by parallel changes in the mean intensity of JC-1 staining. Based on JC-1, mitochondrial activation followed a nucleated pattern, with specific, activation starting points at the midpiece from which mitochondrial activation was spread. Moreover, four separate sperm subpopulations were detected following the JC-1 orange-red/green ratio, and the observed changes in the mean JC-1 staining during IVC and IVAR were related to concomitant changes in both the orange-red/green JC-1 ratio and the percentage of sperm included in each subpopulation. All of these results indicate that IVC and the first minutes of IVAR are accompanied by a progressive increase in mitochondrial activity, which reached a peak coincidental with the achievement of IVAR. Moreover, results suggest the presence of separate sperm subpopulations, which show a different mitochondrial sensitivity to IVC and IVAR. Finally, mitochondrial activation, at least under JC-1 staining, seems to originate in concrete nucleation points at the midpiece, thus suggesting thus a well-coordinated pattern in boar-sperm mitochondrial activity modulation.
Asunto(s)
Reacción Acrosómica/fisiología , Mitocondrias/fisiología , Capacitación Espermática/fisiología , Espermatozoides/fisiología , Porcinos/fisiología , Animales , Masculino , Consumo de Oxígeno , Progesterona , Espermatozoides/citología , Espermatozoides/efectos de los fármacosRESUMEN
Interleukin-3 (IL-3) regulates the proliferation, survival and differentiation of haematopoietic cells via interaction with specific cell-surface receptors. IL-3 is expressed in several non-hematopoietic cell types. Studies have demonstrated the presence of IL-3 in the central nervous system, however, its physiological role in these cells is poorly understood. Previously we have been demonstrated that IL-3 prevents neuronal death induced by fibrillary ß amyloid in these cells, by PI 3-kinase and Jak/STAT pathway activation. In this study, we demonstrated that IL-3 significantly reduced Aß-promoted neurite degeneration and toxicity. Thus, this cytokine provides cellular protection against Aß neurotoxicity in primary cortical neuronal cells, by modulating microtubular dynamics and prevention of tau cleavage and hyperphosphorylation. We also demonstrates that IL-3 is expressed in the "in vivo" mouse model of AD, Tg2576, which also expresses human AßPP with the Swedish mutation. In summary, these results suggest that IL-3 could play a neuroprotective role in AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Citoprotección/fisiología , Interleucina-3/fisiología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/prevención & control , Proteínas tau/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/toxicidad , Animales , Células Cultivadas , Citoprotección/efectos de los fármacos , Humanos , Interleucina-3/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Degeneración Nerviosa/patología , Proteínas tau/fisiologíaRESUMEN
Incubation of diluted boar sperm from fresh ejaculates in a previously established "in vitro" capacitation medium induced a significant, time-dependent increase in several mean parameters of sperm motility, such as curvilinear velocity (VCL), linear velocity (VSL), mean velocity (VAP), linearity coefficient (LIN), straightness coefficient (STR) and wobble coefficient (WOB). Furthermore, motile boar-sperm semen samples were structured in four definite subpopulations. Subpopulation 1 showed the lowest values of VCL, VSL and VAP and also low values of linearity. Subpopulation 2 showed the second lowest values of VCL and VAP and higher values of LIN and STR. Subpopulation 3 was characterized by high values of velocity and low values of linearity. Finally, Subpopulation 4 was characterized by high values of velocity and linearity. "In vitro" capacitation and further acrosome reaction induced changes in the motility characteristics of each subpopulation as well as in their percentage distribution, Subpopulations 3 and 4 being those that showed the most significant changes. However, despite these changes, the observed, overall four-subpopulation structure was firmly maintained during the entire "in vitro" capacitation and acrosome-reaction process. Our results suggest that capacitation-induced motility changes are related to specific changes in the percentage of each motile-sperm subpopulation in the ejaculate without losing the overall, specific four-subpopulation structure. In this way, the maintenance of a four-subpopulation structure seems to be important in the control of the whole ejaculate physiology.
Asunto(s)
Reacción Acrosómica/fisiología , Capacitación Espermática/fisiología , Motilidad Espermática , Espermatozoides/clasificación , Espermatozoides/ultraestructura , Porcinos/fisiología , Animales , Tampones (Química) , Técnicas In Vitro , Masculino , Progesterona/farmacología , SolucionesRESUMEN
Up-regulation of the glomerular expression and the activity of vascular endothelial growth factor-A (VEGF) have been identified as an early pathogenic event for the progression of diabetic nephropathy. Currently, however the mediators are not yet clearly recognized. In this study we identified all four adenosine receptor (AR) subtypes, i.e. A(1), A(2A), A(2B) and A(3) in isolated rat kidney glomeruli. We localized the expression of A(2B)AR in podocytes, the primary VEGF producing cells. The ex vivo treatment of kidney glomeruli with adenosine or a general AR agonist NECA, increases VEGF protein content. In addition, NECA treatment elicits VEGF release. These effects were blocked by the A(2B)AR selective antagonist MRS1754 supplementation. Furthermore, we showed that A(2B)AR activation was necessary to promote a higher expression of VEGF in kidney glomeruli upon exposure to high d-glucose concentration, a pathogenic condition like those observed in diabetic nephropathy.
Asunto(s)
Glomérulos Renales/metabolismo , Receptor de Adenosina A2B/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Células Cultivadas , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine capable of stimulating proliferation, maturation and function of haematopoietic cells. Receptors for this cytokine are composed of two subunits, alpha and beta, and are expressed in myeloid progenitors and mature mononuclear phagocytes, monocytes, eosinophils and neutrophils, as well as in other non-haematopoietic cells. We have previously demonstrated that bull spermatozoa express functional GM-CSF receptors that signal for increased glucose and vitamin-C uptake and enhance several parameters of sperm motility in the presence of glucose or fructose substrates. In this study, we have analyzed the expression of GM-CSF receptors in ovine spermatozoa and studied the effect of GM-CSF on sperm viability and motility after the freezing-thawing process. Immunolocalization and immunoblotting analyses demonstrated that ovine spermatozoa (Xisqueta race) expressed GM-CSF receptors. In addition, GM-CSF partially counteracted the impairing action of freezing/thawing on the percentage of total motility, as well as on the specific motility patterns of each of the separate, motile sperm subpopulations of ram ejaculates subjected to this protocol. These results suggest that GM-CSF can play a role in the resistance of ram spermatozoa to environmental thermal stress.
Asunto(s)
Criopreservación/veterinaria , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/análisis , Ovinos , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Animales , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Calor , Immunoblotting , Técnicas de Inmunoadsorción , Masculino , Preservación de Semen/veterinaria , Espermatozoides/química , Espermatozoides/fisiologíaRESUMEN
In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metabolic substrate (l-CCM) was accompanied by a progressive increase of intracellular glycogen during the first 2 h of incubation, which was followed by a subsequent decrease of glycogen levels after up to 4 h of incubation. Lactate from the medium is the source for the observed glycogen synthesis, as the presence of [(14)C]glycogen after the addition to l-CCM with [(14)C]lactate was demonstrated. The existence of functional gluconeogenesis in dog sperm was also sustained by the presence of key enzymes of this metabolic pathway, such as fructose 1,6-bisphophatase and aldolase B. On the other hand, glycogen metabolism from gluconeogenic sources was important in the maintenance of a correct in vitro fertilization after incubation in the l-CCM. This was demonstrated after the addition of phenylacetic acid (PAA) to l-CCM. In the presence of PAA, in vitro capacitation of dog spermatozoa suffered alterations, which translated into changes in capacitation functional markers, like the increase in the percentage of altered acrosomes, a distinct motion pattern, decrease or even disappearance of capacitation-induced tyrosine phosphorylation, and increased heterogeneity of the chlorotetracycline pattern in capacitated cells. Thus, this is the first report indicating the existence of a functional glyconeogenesis in mammalian spermatozoa. Moreover, gluconeogenesis-linked glycogen metabolism seems to be of importance in the maintenance of a correct in vitro capacitation in dog sperm in the absence of hexoses in the medium.
Asunto(s)
Medios de Cultivo/química , Perros/fisiología , Gluconeogénesis/fisiología , Glucógeno/metabolismo , Capacitación Espermática/fisiología , Espermatozoides/fisiología , Animales , Técnicas de Cultivo de Célula , Perros/metabolismo , Glucosa , Ácido Láctico/administración & dosificación , Masculino , Fenilacetatos/farmacología , Capacitación Espermática/efectos de los fármacos , Espermatozoides/enzimología , Espermatozoides/metabolismoRESUMEN
A set of different protein kinases have been involved in tau phosphorylations, including glycogen synthase kinase 3beta (GSK3 beta), MARK kinase, MAP kinase, the cyclin-dependent kinase 5 (Cdk5) system and others. The latter system include the catalytic component Cdk5 and the regulatory proteins p35, p25 and p39. Cdk5 and its neuron-specific activator p35 are essential molecules for neuronal migration and for the laminar configuration of the cerebral cortex. Recent evidence that the Cdk5/p35 complex concentrates at the leading edge of axonal growth cones, together with the involvement of this system in the phosphorylation of neuronal microtubule-asociated proteins (MAPs), provide further support to the role of this protein kinase in regulating axonal extension in developing brain neurons. Although the aminoacid sequence of p35 has little similarity with those of normal cyclins, studies have shown that its activation domain may adopt a conformation of the cyclin-folded structure. The computed structure for Cdk5 is compatible with experimental data obtained from studies on the Cdk5/p35 complex, and has allowed predictions on the protein interacting domains. This enzyme exhibits a wide cell distribution, even though a regulated Cdk5 activity has been shown only in neuronal cells. Cdk5 has been characterized as a proline-directed Ser/Thr protein kinase, that contributes to phosphorylation of human tau on Ser202, Thr205, Ser235 and Ser404. Cdk5 is active in postmitiotic neurons, and it has been implicated in cytoskeleton assembly and its organization during axonal growth. In addition to tau and other MAPs, Cdk5 phosphorylates the high molecular weight neurofilament proteins at their C-terminal domain. Moreover, nestin, a protein that regulates cytoskeleton organization of neuronal and muscular cells during development of early embryos, and several other regulatory proteins appear to be substrates of Cdk5 and are phosphorylated by this kinase. Studies also suggest, that in addition to Cdk5 involvement in neuronal differentiation, its activity is induced during myogenesis, however, the mechanisms of how this activity is regulated during muscular differentiation has not yet been elucidated. Recent studies have shown that the beta-amyloid peptide (A beta) induces a deregulation of Cdk5 in cultured brain cells, and raises the question on the possible roles of this tau-phosphorylating protein kinase in the sequence of molecular events leading to neuronal death triggered by A beta. In this context, there are evidence that Cdk5 is involved in tau hyperphosphorylation promoted by A beta in its fibrillary form. Cdk5 inhibitors protect hippocampal neurons against both tau anomalous phosphorylations and neuronal death. The links between the studies on the Cdk5/p35 system in normal neurogenesis and its claimed participation in neurodegeneration, provide the framework to understand the regulatory relevance of this kinase system, and changes in its regulation that may be implicated in disturbances such as those occurring in Alzheimer disease.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Quinasas Ciclina-Dependientes/metabolismo , Neuronas/citología , Quinasa 5 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/química , Humanos , Conformación ProteicaRESUMEN
We studied the expression and function of the granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in male germ cells. RT-PCR showed expression of mRNAs encoding the alpha- and beta-subunits of the GM-CSF receptor in human testis, and the presence of the alpha- and beta-proteins was confirmed by immunoblotting with anti-alpha and anti-beta-antibodies. Immunolocalization studies showed the level of expression of GM-CSF alpha- and beta-subunits in the germ line in the testis and in ejaculated spermatozoa. Receptor binding studies using radiolabeled GM-CSF revealed that bull spermatozoa have about 105 high-affinity sites with a K(d) of 222 pM and approximately 1100 low-affinity sites with a K(d) of 10 nM. GM-CSF signaled, in a time- and dose-dependent manner, for an increased uptake of glucose and vitamin C.
Asunto(s)
Ácido Ascórbico/metabolismo , Glucosa/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Transducción de Señal , Testículo/metabolismo , Animales , Sitios de Unión , Bovinos , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Agar , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Cinética , Masculino , Unión Proteica , Transporte de Proteínas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semen/metabolismo , Espermatozoides/metabolismo , Factores de TiempoRESUMEN
We analyzed the expression of hexose transporters in human testis and in human, rat, and bull spermatozoa and studied the uptake of hexoses and vitamin C in bull spermatozoa. Immunocytochemical and reverse transcription-polymerase chain reaction analyses demonstrated that adult human testis expressed the hexose transporters GLUT1, GLUT2, GLUT3, GLUT4, and GLUT5. Immunoblotting experiments demonstrated the presence of proteins of about 50-70 kD reactive with anti-GLUT1, GLUT2, GLUT3, and GLUT5 in membranes prepared from human spermatozoa, but no proteins reactive with GLUT4 antibodies were detected. Immunolocalization experiments confirmed the presence of GLUT1, GLUT2, GLUT3, GLUT5, and low levels of GLUT4 in human, rat, and bull spermatozoa. Each transporter isoform showed a typical subcellular localization in the head and the sperm tail. In the tail, GLUT3 and GLUT5 were present at the level of the middle piece in the three species examined, GLUT1 was present in the principal piece, and the localization of GLUT2 differed according of the species examined. Bull spermatozoa transported deoxyglucose, fructose, and the oxidized form of vitamin C, dehydroascorbic acid. Transport of deoxyglucose and dehydroascorbic acid was inhibited by cytochalasin B, indicating the direct participation of facilitative hexose transporters in the transport of both substrates by bull spermatozoa. Transport of fructose was not affected by cytochalasin B, which is consistent for an important role for GLUT5 in the transport of fructose in these cells. The data show that human, rat, and bull spermatozoa express several hexose transporter isoforms that allow for the efficient uptake of glucose, fructose, and dehydroascorbic acid by these cells.
Asunto(s)
Ácido Ascórbico/metabolismo , Hexosas/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Espermatozoides/metabolismo , Animales , Secuencia de Bases , Bovinos , Cartilla de ADN , Humanos , MasculinoRESUMEN
We studied the expression and function of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor in the human prostate carcinoma cell line LNCaP and looked for its presence in normal and neoplastic human prostatic tissue. The GM-CSF receptor is composed of two subunits, alpha and beta. While the isolated alpha subunit binds GM-CSF at low-affinity, the isolated beta subunit does not bind GM-CSF by itself; but complexes with the alpha subunit to form a high-affinity receptor. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed expression of mRNAs encoding the alpha and beta subunits of the GM-CSF receptor in LNCaP cells, and the presence of the alpha and beta proteins was confirmed by immunolocalization with anti-alpha and anti-beta antibodies. Receptor binding studies using radiolabeled GM-CSF showed that LNCaP cells have about 150 high-affinity sites with a kd of 40 pmol/L and approximately 750 low-affinity sites with a kd of 2 nmol/L. GM-CSF signaled, in a time- and dose-dependent manner, for protein tyrosine phosphorylation and induced the proliferation of the LNCaP cells. Immunolocalization studies showed low level expression of GM-CSF alpha and beta subunits in normal prostate tissue, with substantial expression in benign prostatic hyperplasia and prominent expression in neoplastic prostate tissue. Maximal expression of both subunits was observed in prostatic carcinomas metastatic to lymph node and bone. Tumor cells that stained positively with anti-alpha subunit antibodies were also reactive with anti-beta subunit antibodies, indicating that they express high-affinity GM-CSF receptors. Our data show that the LNCaP cells express functional GM-CSF receptors and that prostatic carcinomas have prominent GM-CSF receptor expression. These findings imply that both hyperplastic and neoplastic prostatic tissues may be responsive to GM-CSF.
Asunto(s)
Expresión Génica , Neoplasias de la Próstata/química , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Epitelio/química , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Células HL-60/metabolismo , Humanos , Técnicas para Inmunoenzimas , Masculino , Fosforilación , Fosfotirosina/metabolismo , Reacción en Cadena de la Polimerasa , Hiperplasia Prostática/metabolismo , Neoplasias de la Próstata/metabolismo , ARN Mensajero/análisis , ADN Polimerasa Dirigida por ARN , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/análisis , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Although erythrocytes readily metabolize fructose, it has not been known how this sugar gains entry to the red blood cell. We present evidence indicating that human erythrocytes express the fructose transporter GLUT5, which is the major means for transporting fructose into the cell. Immunoblotting and immunolocalization experiments identified the presence of GLUT1 and GLUT5 as the main facilitative hexose transporters expressed in human erythrocytes, with GLUT2 present in lower amounts. Functional studies allowed the identification of two transporters with different kinetic properties involved in the transport of fructose in human erythrocytes. The predominant transporter (GLUT5) showed an apparent Km for fructose of approximately 10 mmol/L. Transport of low concentrations of fructose was not affected by 2-deoxy-D-glucose, a glucose analog that is transported by GLUT1 and GLUT2. Similarly, cytochalasin B, a potent inhibitor of the functional activity of GLUT1 and GLUT2, did not affect the transport of fructose in human erythrocytes. The functional properties of the fructose transporter present in human erythrocytes are consistent with a central role for GLUT5 as the physiological transporter of fructose in these cells.
Asunto(s)
Eritrocitos/metabolismo , Fructosa/metabolismo , Proteínas de Transporte de Monosacáridos/biosíntesis , Transporte Biológico , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 2 , Transportador de Glucosa de Tipo 5 , Humanos , Inmunohistoquímica , Proteínas de Transporte de Monosacáridos/metabolismoRESUMEN
Vitamin C (ascorbic acid) is required for normal host defense and functions importantly in cellular redox systems. To define the interrelationship between human immunodeficiency virus (HIV) infection and vitamin C flux at the cellular level, we analyzed vitamin C uptake and its effects on virus production and cellular proliferation in HIV-infected and uninfected human lymphoid, myeloid, and mononuclear phagocyte cell lines. Chronic or acute infection of these cell lines by HIV-1 led to increased expression of glucose transporter 1, associated with increased transport and accumulation of vitamin C. Infected cells also showed increased transport of glucose analogs. Exposure to vitamin C had a complex effect on cell proliferation and viral production. Low concentrations of vitamin C increased or decreased cell proliferation depending on the cell line and either had no effect or caused increased viral production. Exposure to high concentrations of vitamin C preferentially decreased the proliferation and survival of the HIV-infected cells and caused decreased viral production. These findings indicate that HIV infection in lymphocytic, monocytic, and myeloid cell lines leads to increased expression of glucose transporter 1 and consequent increased cellular vitamin C uptake. High concentrations of vitamin C were preferentially toxic to HIV-infected host defense cell lines in vitro.
Asunto(s)
Ácido Ascórbico/metabolismo , Infecciones por VIH/sangre , VIH-1 , Linfocitos/virología , Fagocitos/virología , División Celular , Línea Celular , Ácido Deshidroascórbico/metabolismo , Transportador de Glucosa de Tipo 1 , Células HL-60 , Hexosas/metabolismo , Humanos , Linfocitos/metabolismo , Proteínas de Transporte de Monosacáridos/sangre , Fagocitos/metabolismo , Replicación ViralRESUMEN
The localization of fructose 1,6-bisphosphatase (D-Fru-1,6-)2-1-phosphohydrolase, EC 3.1.3.11) in rat kidney and liver was determined immunohistochemically using a polyclonal antibody raised against the enzyme purified from pig kidney. The immunohistochemical analysis revealed that the bisphosphatase was preferentially localized in hepatocytes of the periportal region of the liver and was absent from the perivenous region. Fructose-1,6-bisphosphatase was also preferentially localized in the cortex of the kidney proximal tubules and was absent in the glomeruli, loops of Henle, collecting and distal tubules, and in the renal medulla. As indicated by immunocytochemistry using light microscopy and confirmed with the use of reflection confocal microscopy, the enzyme was preferentially localized in a perinuclear position in the liver and the renal cells. Subcellular fractionation studies followed by enzyme activity assays revealed that a majority of the cellular fructose-1,6-bisphosphatase activity was associated to subcellular particulate structures. Overall, the data support the concept of metabolic zonation in liver as well as in kidney, and establish the concept that the Fructose-1,6-bisphosphatase is a particulate enzyme that can not be considered a soluble enzyme in the classical sense.
Asunto(s)
Fructosa-Bifosfatasa/metabolismo , Riñón/enzimología , Hígado/enzimología , Animales , Western Blotting , Núcleo Celular/metabolismo , Fructosa-Bifosfatasa/inmunología , Inmunohistoquímica , Riñón/citología , Corteza Renal/metabolismo , Glomérulos Renales/metabolismo , Médula Renal/metabolismo , Túbulos Renales Distales/metabolismo , Túbulos Renales Proximales/metabolismo , Hígado/citología , Asa de la Nefrona/metabolismo , Microscopía Confocal , Ratas , Ratas Sprague-DawleyRESUMEN
The primary metabolic characteristic of malignant cells is an increased uptake of glucose and its anaerobic metabolism. We studied the expression and function of the glucose transporters in human breast cancer cell lines and analyzed their expression in normal and neoplastic primary human breast tissue. Hexose uptake assays and immunoblotting experiments revealed that the breast carcinoma cell lines MCF-7 and MDA-468 express the glucose transporters GLUT1 and GLUT2, isoforms expressed in both normal and neoplastic breast tissue. We also found that the breast cancer cell lines transport fructose and express the fructose transporter GLUT5. Immunolocalization studies revealed that GLUT5 is highly expressed in vivo in human breast cancer but is absent in normal human breast tissue. These findings indicate that human breast cancer cells have a specialized capacity to transport fructose, a metabolic substrate believed to be used by few human tissues. Identification of a high-affinity fructose transporter on human breast cancer cells opens opportunities to develop novel strategies for early diagnosis and treatment of breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Mama/metabolismo , Desoxiglucosa/metabolismo , Epitelio/metabolismo , Fructosa/metabolismo , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 2 , Transportador de Glucosa de Tipo 5 , Humanos , Técnicas Inmunológicas , Proteínas de Transporte de Monosacáridos/inmunología , Células Tumorales CultivadasRESUMEN
We performed a detailed kinetic analysis of the uptake of dehydroascorbic acid by HL-60 cells under experimental conditions that enabled the differentiation of dehydroascorbic acid transport from the intracellular reduction/accumulation of ascorbic acid. Immunoblotting and immunolocalization experiments identified GLUT1 as the main glucose transporter expressed in the HL-60 cells. Kinetic analysis allowed the identification of a single functional activity involved in the transport of dehydroascorbic acid in the HL-60 cells. Transport was inhibited in a competitive manner by both 3-O-methyl-D-glucose and 2-deoxy-D-glucose. In turn, dehydroascorbic acid competitively inhibited the transport of both sugars. A second functional component identified in experiments measuring the accumulation of ascorbic acid appears to be associated with the intracellular reduction of dehydroascorbic acid to ascorbic acid and is not directly involved in the transport of dehydroascorbic acid via GLUT1. Transport of dehydroascorbic acid by HL-60 cells was independent of the presence of external Na+, whereas the intracellular accumulation of ascorbic acid was found to be a Na(+)-sensitive process. Thus, the transport of dehydroascorbic acid via glucose transporters is a Na(+)-independent process which is kinetically and biologically separable from the reduction of dehydroascorbic acid to ascorbic acid and its subsequent intracellular accumulation.
Asunto(s)
Ácido Ascórbico/metabolismo , Ácido Deshidroascórbico/metabolismo , 3-O-Metilglucosa , Unión Competitiva , Transporte Biológico Activo , Línea Celular , Desoxiglucosa/metabolismo , Transportador de Glucosa de Tipo 1 , Humanos , Líquido Intracelular/metabolismo , Cinética , Metilglucósidos/metabolismo , Modelos Biológicos , Proteínas de Transporte de Monosacáridos/metabolismo , Oxidación-Reducción , Sodio/metabolismoRESUMEN
Immunoblot analysis of sperm protein from several species revealed the presence of polypeptides recognised by anti-Sm sera obtained from patients with systemic lupus erythematosus. Immunoreactive polypeptides in human, bull, mouse and rat sperm were identified as protein B', B and D as compared with the Sm polypeptides of HeLa cells. In the sperm of rooster, the teleost fish Cyprinus carpio and the mussel Choromytilus chorus, the immunoreactive polypeptide profile was more complex. To ascertain the sperm origin of the Sm antigens, immunolocalisation with anti-Sm serum was carried out. The results demonstrated that in all the species studied staining was confined to the sperm nucleus, confirming that some polypeptides of the small nuclear ribonucleoprotein complex are present in the gamete.
Asunto(s)
Autoantígenos/análisis , Núcleo Celular/inmunología , Ribonucleoproteínas Nucleares Pequeñas/análisis , Espermatozoides/inmunología , Animales , Anticuerpos/inmunología , Western Blotting , Bovinos , Núcleo Celular/química , Pollos , Peces , Células HeLa , Humanos , Inmunohistoquímica , Lupus Eritematoso Sistémico/inmunología , Masculino , Ratones , Microscopía Electrónica , Ratas , Espermatozoides/química , Espermatozoides/ultraestructura , Proteínas Nucleares snRNPRESUMEN
Analysis of epididymal rat sperm RNA strongly suggested the presence of U snRNAs, especially U1 and U2 snRNA. By Northern blot analysis with radiolabeled oligodeoxynucleotide probes, the presence of U1 and U2 snRNA in rat sperm was confirmed. To precisely localize these RNAs, in situ hybridization with antisense and sense oligo probes labeled with digoxigenin was carried out. The results indicate that U1 as well as U2 snRNA are confined to the sperm nucleus.