RESUMEN
Understanding the pharmacodynamic effects of platelet inhibitors is standard for developing more effective antithrombotic therapies. An example is the antithrombotic treatment of acute coronary syndrome (ACS), in particular ST-elevated myocardial infarction (STEMI) patients who are in need for rapid acting strong antithrombotic therapy despite the use of aspirin and oral P2Y12-inhibitors. In this study, we evaluated two injectable platelet inhibitors under clinical development (the P2Y12 antagonist selatogrel and the GPIIb-IIIa antagonist zalunfiban) that may be amenable to pre-hospital treatment of STEMI patients. Platelet reactivity was assessed at inhibitor concentrations that represent clinically relevant levels of platelet inhibition (IC20-50%, 1/2Cmax, and Cmax). Light transmission aggregometry (LTA), was used to evaluate the initial rate of aggregation (primary slope, PS) and maximal aggregation (MA). Both adenosine diphosphate (ADP) and thrombin receptor agonist peptide (TRAP) were used as agonists. Zalunfiban demonstrated similar inhibition of platelet aggregation when blood was collected in PPACK or TSC, whereas selatogrel demonstrated greater inhibition in PPACK. In this study, using PPACK anticoagulant, selatogrel and zalunfiban affected PS in response to ADP equivalently at all drug concentrations tested. In contrast, zalunfiban had significantly greater potency at its Cmax concentration compared to selatogrel using TRAP as agonist. Upon evaluation of MA responses at lower doses, selatogrel had greater inhibition of MA in response to ADP than zalunfiban; however, at concentrations that represent Cmax, the drugs were equivalent. Zalunfiban also had greater inhibition of MA in response to TRAP at the Cmax dose. These data suggest that zalunfiban may provide greater protection in reducing thrombus formation than selatogrel, especially since thrombin is an early, key primary agonist in the pathophysiology of thrombotic events.
RESUMEN
Although stallion spermatozoa produce significant quantities of reactive oxygen species, a lag between 4-hydroxynonenal (4HNE) adduction and the loss of motility in stallion spermatozoa suggests the presence of a robust aldehyde detoxification mechanism. Because there is a paucity of studies characterizing the role of aldehyde dehydrogenase (ALDH) in sperm functionality, the aim of this study was to ascertain the relationship between 4HNE production and motility and ALDH expression by stallion spermatozoa. PCR analysis revealed the presence of the ALDH1A3, ALDH1B1, and ALDH2 isoforms in these cells. Strong correlations (P < 0.001) were found between ALDH expression and various motility parameters of stallion spermatozoa including the percentage of progressive (r = 0.79) and rapidly motile (r = 0.79) spermatozoa, whereas repeated measurements over 24 h revealed highly significant correlations among progressive motility loss, 4HNE accumulation, and ALDH expression (P ≤ 0.001). ALDH inhibition resulted in a spontaneous increase in 4HNE levels in viable cells (21.1 ± 5.8% vs. 42.6 ± 5.2%; P ≤ 0.05) and a corresponding decrease in total motility (41.7 ± 6.2% vs. 6.4 ± 2.6%; P ≤ 0.001) and progressive motility (17.0 ± 4.1% vs. 0.7 ± 0.4%; P ≤ 0.001) of stallion spermatozoa over 24 h. Similarly, inhibition of ALDH in 4HNE-challenged spermatozoa significantly reduced total motility over 4 h (35.4 ± 9.7% vs. 15.3 ± 5.1%, respectively; P ≤ 0.05). This study contributes valuable information about the role of the ALDH enzymes in the maintenance of stallion sperm functionality, with potential diagnostic and in vitro applications for assisted reproductive technologies.
Asunto(s)
Aldehído Deshidrogenasa/metabolismo , Aldehídos/metabolismo , Caballos/metabolismo , Motilidad Espermática , Espermatozoides/enzimología , Animales , Glutatión Transferasa/metabolismo , Isoenzimas , Peroxidación de Lípido , MasculinoRESUMEN
Stallion spermatozoa continue to present scientific and clinical challenges with regard to the biological mechanisms responsible for their survival and function. In particular, deeper understanding of sperm energy metabolism, defence against oxidative damage and cell-cell interactions should improve fertility assessment and the application of advanced reproductive technologies in the equine species. In this study, we used highly sensitive LC-MS/MS technology and sequence database analysis to identify and characterise the proteome of Percoll-isolated ejaculated equine spermatozoa, with the aim of furthering our understanding of this cell's complex biological machinery. We were able to identify 9883 peptides comprising 1030 proteins, which were subsequently attributed to 975 gene products. Gene ontology analysis for molecular and cellular processes revealed new information about the metabolism, antioxidant defences and receptors of stallion spermatozoa. Mitochondrial proteins and those involved in catabolic processes constituted dominant categories. Several enzymes specific to ß-oxidation of fatty acids were identified, and further experiments were carried out to ascertain their functional significance. Inhibition of carnitine palmitoyl transferase 1, a rate-limiting enzyme of ß-oxidation, reduced motility parameters, indicating that ß-oxidation contributes to maintenance of motility in stallion spermatozoa.
Asunto(s)
Cromatografía Liquida , Caballos/metabolismo , Proteoma/análisis , Espermatozoides/metabolismo , Espectrometría de Masas en Tándem , Animales , Masculino , Motilidad Espermática/fisiologíaRESUMEN
Pattern recognition receptors (PRR), Toll-like receptors (TLR), and nucleotide-oligomerization domain-containing proteins (NOD) play critical roles in mediating inflammation and modulating functions in white adipocytes in obesity. However, the role of PRR activation in brown adipocytes, which are recently found to be present in adult humans, has not been studied. Here we report that mRNA of TLR4, TLR2, NOD1, and NOD2 is upregulated, paralleled with upregulated mRNA of inflammatory cytokines and chemokines in the brown adipose tissue (BAT) of the obese mice. During brown adipocyte differentiation, mRNA and protein expression of NOD1 and TLR4, but not TLR2 and NOD2, is also increased. Activation of TLR4, TLR2, or NOD1 in brown adipocytes induces activation of NF-κB and MAPK signaling pathways, leading to inflammatory cytokine/chemokine mRNA expression and/or protein secretion. Moreover, activation of TLR4, TLR2, or NOD1 attenuates both basal and isoproterenol-induced uncoupling protein 1 (UCP-1) expression without affecting mitochondrial biogenesis and lipid accumulation in brown adipocytes. Cellular bioenergetics measurements confirm that attenuation of UCP-1 expression by PRR activation is accompanied by suppression of both basal and isoproterenol-stimulated oxygen consumption rates and isoproterenol-induced uncoupled respiration from proton leak; however, maximal respiration and ATP-coupled respiration are not changed. Further, the attenuation of UCP-1 by PRR activation appears to be mediated through downregulation of the UCP-1 promoter activities. Taken together, our results demonstrate the role of selected PRR activation in inducing inflammation and downregulation of UCP-1 expression and mitochondrial respiration in brown adipocytes. Our results uncover novel targets in BAT for obesity treatment and prevention.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo Pardo/metabolismo , Canales Iónicos/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Obesidad/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Adipocitos/efectos de los fármacos , Adipocitos/patología , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/patología , Animales , Línea Celular , Citocinas/genética , Citocinas/metabolismo , Dieta Alta en Grasa , Transporte de Electrón/efectos de los fármacos , Regulación de la Expresión Génica , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Canales Iónicos/genética , Isoproterenol/farmacología , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/genética , Proteína Adaptadora de Señalización NOD2/metabolismo , Obesidad/metabolismo , Obesidad/patología , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genética , Proteína Desacopladora 1RESUMEN
BACKGROUND: Ticagrelor is a reversible oral P2Y12 platelet inhibitor used to treat patients with acute coronary syndromes, prior myocardial infarction, high-risk coronary artery disease, transient ischemic attack, or ischemic stroke. A healthy volunteer study showed that the intravenous monoclonal antibody bentracimab rapidly reverses ticagrelor, but the effect in patients was unknown. METHODS: In a prespecified interim analysis of a single-arm, prospective study, bentracimab was evaluated in ticagrelor-treated patients who required urgent surgery or had major hemorrhage. The extent of reversal was determined using the VerifyNow P2Y12 assay. Clinical hemostasis was assessed by central adjudication using validated criteria. Treatment-emergent safety events were evaluated. The trial is ongoing and will enroll approximately 200 patients with evaluable data. RESULTS: Of 150 enrolled patients, 142 required urgent surgery and 8 had major hemorrhage. For the end-point analysis, 129 patients had analyzable platelet data; 122 had data on adjudicated hemostasis. Bentracimab provided a rapid reversal of ticagrelor's antiplatelet effects within 5 to 10 minutes. The reversal was sustained for more than 24 hours, as measured with the VerifyNow P2Y12 and vasodilator-stimulated phosphoprotein phosphorylation assays (P<0.001, with both assays and in all subgroups). Adjudicated hemostasis was achieved for more than 90% of patients (P<0.001); approximately 5% of patients had thrombotic events. No allergic or infusion-related reactions were reported. CONCLUSIONS: Bentracimab provided immediate and sustained reversal of the antiplatelet effects of ticagrelor in patients undergoing surgical procedures. (Funded by PhaseBio Pharmaceuticals, Inc.; ClinicalTrials.gov number, NCT04286438.)
Asunto(s)
Adenosina , Antagonistas del Receptor Purinérgico P2Y , Ticagrelor , Humanos , Ticagrelor/uso terapéutico , Ticagrelor/efectos adversos , Masculino , Femenino , Persona de Mediana Edad , Estudios Prospectivos , Anciano , Antagonistas del Receptor Purinérgico P2Y/uso terapéutico , Antagonistas del Receptor Purinérgico P2Y/efectos adversos , Antagonistas del Receptor Purinérgico P2Y/administración & dosificación , Adenosina/análogos & derivados , Adenosina/efectos adversos , Adenosina/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/administración & dosificación , Inhibidores de Agregación Plaquetaria/uso terapéutico , Inhibidores de Agregación Plaquetaria/efectos adversos , Inhibidores de Agregación Plaquetaria/administración & dosificaciónRESUMEN
Evidence is presented for expression of the insulin receptor on the surface of mammalian spermatozoa as well as transcripts for the receptor substrate adaptor proteins (IRS1-4) needed to mediate insulin action. Exposure to this hormone resulted in insulin receptor phosphorylation (pTyr972), activation of AKT (pSer473) and the stimulation of sperm motility. Intriguingly, the male germ line is also shown to be capable of generating insulin, possessing the relevant mRNA transcript and expressing strong immunocytochemical signals for both insulin and C-peptide. Insulin could be released from the spermatozoa by sonication in a concentration-dependent manner but was not secreted in response to glucose, fructose or stimulation with progesterone. However, insulin release could be induced by factors present in human uterine lavages. Furthermore, the endometrium was also shown to possess the machinery for insulin production and action (mRNA, insulin, C-peptide, proprotein convertase and insulin receptor), releasing insulin into the uterine lumen prior to ovulation. These studies emphasize the fundamental importance of extra-pancreatic insulin in regulating the reproductive process, particularly in the support of spermatozoa on their perilous voyage to the site of fertilization.
Asunto(s)
Insulina/biosíntesis , Páncreas/metabolismo , Espermatozoides/citología , Espermatozoides/metabolismo , Animales , Péptido C/metabolismo , Supervivencia Celular , Endometrio/metabolismo , Epitelio/metabolismo , Femenino , Células Germinativas/metabolismo , Humanos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Secreción de Insulina , Masculino , Mamíferos/metabolismo , Ratones , Isoformas de Proteínas/metabolismo , Ratas , Receptor de Insulina/metabolismo , Útero/metabolismoRESUMEN
PURPOSE:: The use of cyanoacrylate for intravenous catheter securement is of interest to clinicians and patients, because of the superior adhesive strength and hemostatic effect of cyanoacrylate compared to current securement devices. The purpose of this study is to use novel in vitro and in vivo testing methods to analyze the hemostatic effect of a catheter securement cyanoacrylate (cyanoacrylate). METHODS:: An unprecedented in vitro method was performed to determine the effects of a cyanoacrylate on a customized modified activated clotting time assay and blood flow inhibition assay by exposing blood or plasma to either one or three drops of cyanoacrylate. For the in vivo testing, full-thickness incisions were made on swine, and the bleeding was scored prior to treatment and at 3, 6, 9, and 12 min after treatment. RESULTS:: The cyanoacrylate rapidly achieved hemostasis in the presence of anticoagulated whole blood, platelet-poor plasma, and non-anticoagulated whole blood, in vitro. The cyanoacrylate achieved hemostasis 12-fold faster than thromboplastin in the modified activated clotting time assay. The cyanoacrylate does not alter normal blood clotting, as measured by prothrombin time. In vivo, the bleeding score of cyanoacrylate prior to treatment and at 3, 6, 9, and 12 min after treatment were 2.3 ± 1.0, 0.3 ± 0.5, 0.2 ± 0.5, 0.2 ± 0.4, and 0.2 ± 0.4, respectively. CONCLUSION:: This study indicates that cyanoacrylate demonstrates a potent mechanical hemostatic effect and cyanoacrylate in the presence of anticoagulated whole blood has an activated clotting time that is 12 times quicker than thromboplastin. The cyanoacrylate was found to be significantly equivalent to two known hemostatic agents, in vivo.
Asunto(s)
Cianoacrilatos/administración & dosificación , Hemorragia/prevención & control , Hemostasis/efectos de los fármacos , Hemostáticos/administración & dosificación , Piel/irrigación sanguínea , Piel/efectos de los fármacos , Adhesivos Tisulares/administración & dosificación , Heridas y Lesiones/tratamiento farmacológico , Administración Cutánea , Alginatos/administración & dosificación , Animales , Pruebas de Coagulación Sanguínea , Modelos Animales de Enfermedad , Femenino , Esponja de Gelatina Absorbible/administración & dosificación , Hemorragia/sangre , Humanos , Sus scrofa , Factores de Tiempo , Heridas y Lesiones/sangreRESUMEN
Previous studies from this laboratory demonstrate that dietary leucine protects against high fat diet-induced mitochondrial impairments and stimulates mitochondrial biogenesis and energy partitioning from adipocytes to muscle cells through SIRT1-mediated mechanisms. Moreover, ß-hydroxy-ß-methyl butyrate (HMB), a metabolite of leucine, has been reported to activate AMPK synergistically with resveratrol in C2C12 myotubes. Therefore, we hypothesize that leucine-induced activation of SIRT1 and AMPK is the central event that links the upregulated mitochondrial biogenesis and fatty acid oxidation in skeletal muscle. Thus, C2C12 myotubes were treated with leucine (0.5 mM), alanine (0.5 mM), valine (0.5 mM), EX527 (SIRT1 inhibitor, 25 µM), and Compound C (AMPK inhibitor, 25 µM) alone or in combination to determine the roles of AMPK and SIRT1 in leucine-modulation of energy metabolism. Leucine significantly increased mitochondrial content, mitochondrial biogenesis-related genes expression, fatty acid oxidation, SIRT1 activity and gene expression, and AMPK phosphorylation in C2C12 myotubes compared to the controls, while EX527 and Compound C markedly attenuated these effects. Furthermore, leucine treatment for 24 hours resulted in time-dependent increases in cellular NAD(+), SIRT1 activity, and p-AMPK level, with SIRT1 activation preceding that of AMPK, indicating that leucine activation of SIRT1, rather than AMPK, is the primary event.
RESUMEN
Defective sperm function is the largest single defined cause of human infertility and one of the major reasons we are witnessing an exponential increase in the uptake of assisted conception therapy in the developed world. A major characteristic of defective human spermatozoa is the presence of large amounts of DNA damage, which is, in turn, associated with reduced fertility, increased rates of miscarriage, and an enhanced risk of disease in the offspring. This DNA damage is largely oxidative and is closely associated with defects in spermiogenesis. To explain the origins of this DNA damage, we postulate that spermiogenesis is disrupted by oxidative stress, leading to the creation of defective gametes with poorly remodeled chromatin that are particularly susceptible to free radical attack. To compound the problem, these defective cells have a tendency to undergo an unusual truncated form of apoptosis associated with high amounts of superoxide generation by the sperm mitochondria. This leads to significant oxidative DNA damage that eventually culminates in the DNA fragmentation we see in infertile patients. In light of the significance of oxidative stress in the etiology of defective sperm function, a variety of antioxidant therapies are now being assessed for their therapeutic potential.
Asunto(s)
Daño del ADN , Células Germinativas/fisiología , Infertilidad Masculina/etiología , Oxidación-Reducción , Capacitación Espermática/fisiología , Espermatozoides/fisiología , Animales , Antioxidantes/metabolismo , Apoptosis , Humanos , Masculino , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Inyecciones de Esperma Intracitoplasmáticas/efectos adversos , Espermatogénesis/fisiología , Espermatozoides/citología , Espermatozoides/metabolismo , Testículo/citología , Testículo/metabolismoRESUMEN
The purpose of this study was to examine the impact of prolactin (PRL) on human sperm function, in light of a recent proteomic analysis indicating that these cells express the PRL receptor (PRLR). Immunocytochemical analyses confirmed the presence of PRLR in human spermatozoa and localized this receptor to the postacrosomal region of the sperm head as well as the neck, midpiece, and principal piece of the sperm tail. Nested PCR analysis indicated that these cells possess four splice variants of the PRLR: the long form and three short isoforms, one of which is reported for the first time. A combination of Western blot analyses and immunocytochemistry demonstrated that PRL inhibited sperm capacitation in a dose-dependent manner, suppressing SRC kinase activation and phosphotyrosine expression, two hallmarks of this process. The suppression of sperm capacitation was accompanied by a powerful prosurvival effect, supporting the prolonged motility of these cells and preventing the formation of spontaneous DNA strand breaks via mechanisms that involved the concomitant suppression of caspase activation. Western blot analyses indicated that the prosurvival effect of PRL on human spermatozoa involved the stimulation of Akt phosphorylation, whereas inhibitors of phosphatidylinositol-3-OH kinase and Akt negated this effect, as did the direct induction of sperm capacitation with cAMP analogues. We conclude that PRL is a prosurvival factor for human spermatozoa that prevents these cells from defaulting to an intrinsic apoptotic pathway associated with cell senescence. These findings have implications for preservation of sperm integrity in vivo and in vitro.
Asunto(s)
Prolactina/metabolismo , Receptores de Prolactina/metabolismo , Capacitación Espermática , Motilidad Espermática , Espermatozoides/fisiología , Caspasas/metabolismo , Muerte Celular , Supervivencia Celular , Roturas del ADN , Regulación hacia Abajo , Activación Enzimática , Genitales Masculinos/metabolismo , Humanos , Masculino , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Familia-src Quinasas/metabolismoRESUMEN
A transcript encoding a rat homologue of DZIP1 (DAZ-interacting protein) was isolated from testis RNA. Like human DZIP1, it contains a C(2)H(2) zinc finger domain. A predicted mouse homologue of DZIP1 was found in the GenBank database. Genome analysis indicated that while DZIP1 and mouse Dzip1 contain 22 and 20 exons, respectively, the rat sequence was intronless, confirmed by PCR on genomic DNA. This rat Dzip1 sequence is homologous to mouse Dzip1 exons 1-6 and DZIP1 exons 5-9. As this rat sequence was shorter than DZIP1 it was designated rat Dzip1S. The rat genome also contained a further predicted homologue of DZIP1 displaying conserved linkage homology with mouse Dzip1 and DZIP1. This sequence, if expressed, is the true rat homologue of DZIP1, designated rat Dzip1. Rat Dzip1S mRNA was present in all tissues examined by qualitative RT-RCR, and in situ hybridization of rat testis confirmed that expression of rat Dzip1S mRNA was confined to the spermatogenic lineage, specifically premeiotic spermatogonia.
Asunto(s)
Proteínas de Unión al ADN/genética , Exones , Expresión Génica , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Proteínas de Unión al ADN/química , Humanos , Hibridación in Situ , Masculino , Ratones , Datos de Secuencia Molecular , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Testículo/metabolismoRESUMEN
Lucigenin-dependent chemiluminescence together with 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt (WST-1) reduction can be detected following addition of NADH to many cell types, including human sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other oxygen detecting metabolite probes, such as MCLA and luminol, do not produce a chemiluminescent signal in this model system. The enzyme responsible for NADH-dependent lucigenin chemiluminescence was purified and identified as cytochrome-b5 reductase. In support of this concept, COS-7 cells overexpressing cytochrome-b5 reductase displayed at least a 3-fold increase in the previously mentioned activity compared with mock-transfected cells. Fractions containing cytochrome-b5 reductase were capable of inducing both lucigenin-dependent chemiluminescence and WST-1 reduction. Oxygen radicals clearly did not mediate the cytochrome b5-mediated activation of these probes in vitro since neither luminol nor MCLA gave a chemiluminescence response in the presence of the enzyme and the cofactor NADH. These results emphasize the importance of the direct NADH-dependent reduction of these putative superoxide-sensitive probes by cytochrome-b5 reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.
Asunto(s)
Acridinas/metabolismo , Citocromo-B(5) Reductasa/metabolismo , NAD/metabolismo , Espermatozoides/enzimología , Animales , Western Blotting , Células COS , Chlorocebus aethiops , Colorantes/farmacología , Citocromo-B(5) Reductasa/genética , Humanos , Imidazoles/farmacología , Mediciones Luminiscentes , Masculino , Oxidación-Reducción , Pirazinas/farmacología , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Fluorescencia , Espermatozoides/metabolismo , Sales de Tetrazolio/farmacología , TransfecciónRESUMEN
Lucigenin-dependent chemiluminescence and WST-1 reduction can be detected following addition of NADPH to many cell types, including rat epididymal sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other probes-such as MCLA and luminol-that are capable of detecting reactive oxygen metabolites do not produce a chemiluminescent signal in this model system. Our aim was to purify and identify the enzyme catalyzing the NADPH-dependent lucigenin and WST-1 reduction from rat epididymal spermatozoa preparations. Here, we show the identity of this enzyme as cytochrome P450-reductase. In support of this, a homogenous preparation of this protein was capable of reducing lucigenin and WST-1 in the presence of NADPH. Moreover, COS-7 cells overexpressing cytochrome P450-reductase displayed a 3-fold increase in the aforementioned activity compared with mock-transfected cells. Immunolocalization studies and biochemical analysis suggest that the majority of the NADPH-lucigenin activity is localized to the epithelial cells present within the epididymis. These results emphasize the importance of the direct NADPH-dependent reduction of superoxide-sensitive probes by cytochrome P450-reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.
Asunto(s)
Acridinas/metabolismo , Epidídimo , NADPH-Ferrihemoproteína Reductasa/metabolismo , NADP/fisiología , Espermatozoides/metabolismo , Sales de Tetrazolio/metabolismo , Acridinas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Mediciones Luminiscentes , Masculino , Datos de Secuencia Molecular , NADP/farmacología , NADPH-Ferrihemoproteína Reductasa/genética , Oxidación-Reducción/efectos de los fármacos , Ratas , Sales de Tetrazolio/aislamiento & purificación , Distribución TisularRESUMEN
A gene encoding a novel RhoGAP of 1146 amino acids was isolated from rat testis RNA. Analysis of this protein identified two conserved domains, a RhoGAP domain and an RA domain. Thus the gene was named RARhoGAP. The RhoGAP domain contained conserved residues critical for RhoGAP activity, suggesting this domain is involved in the down-regulation of Rho GTPases. The presence of the RA domain suggests that RARhoGAP also functions as an effector for Ras- or Ral-like GTPases. RT-PCR analysis showed the transcript was ubiquitous in extragonadal tissues; however, Northern analysis indicated highest expression was in the testis. Homologues of rat RARhoGAP were found in mouse and human and were found expressed in testis by nested RT-PCR. In situ hybridization confirmed the specific expression of RARhoGAP in differentiating male germ cells. We postulate that RARhoGAP may be involved in rearrangements of the cytoskeleton and cell signaling events that occur during spermatogenesis.
Asunto(s)
Proteínas Activadoras de GTPasa/genética , Espermatozoides/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia Conservada , Exones/genética , Proteínas Activadoras de GTPasa/química , Perfilación de la Expresión Génica , Humanos , Hibridación in Situ , Masculino , Ratones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Alineación de Secuencia , Espermatozoides/citología , Testículo/citología , Testículo/metabolismoRESUMEN
Cytochrome b5/cytochrome b5 reductase (cb5/cb5r) is a cytosolic fusion protein between the hemoprotein cytochrome b5 and the flavoprotein cytochrome b5 reductase. We describe the identification and characterization of a novel splice variant of cb5/cb5r in the mouse and rat and show that expression of the variant is conserved in both species but is not expressed in human tissue. Characterization of the exon structure of cb5/cb5r indicated that the variant was due to the deletion of the whole of exon 12, thus the variant was named cb5/cb5rdelta12. Exon 12 codes for the flavin-adenine dinucleotide binding domain of cb5/cb5r. Expression analysis revealed the transcript of cb5/cb5rdelta12 in mouse and rat testis, brain, and skeletal muscle and also in the male germ line. We postulate that cb5/cb5rdelta12 may function in a dominant negative fashion, limiting the amount of damage caused by the production of reactive oxygen species by cb5/cb5r.