RESUMEN
Mitochondrial dysfunction, oxidative stress, inflammation and glucose dysmetabolism are pathological signs of Alzheimer's disease (AD). Dietary aluminum (Al) overload is often used to induce AD in rodents and trigger the onset of oxidative-stress hallmarks resembling those of the human disease. The Nuclear factor erythroid 2-related factor 2 (Nrf2), owing to its key role in redox homeostasis, mitochondrial function and inflammation, is a promising drug target for neurological disorders, but only a few data are available on its modulatory effects on glucose transporter expression levels. While it has been found that the protective effect of Conjugated linoleic acid (CLA) occurs through the activation of an Nrf2-mediated adaptive response, its beneficial effect on the considered pathological signs in the Al-induced model has not been established yet. Thirty-five male BalbC mice were divided into 5 groups: two Al-intoxicated groups were treated for 5 weeks with low or high Al doses (8 or 100 mg/kg/day in drinking water, respectively; L or H). Two groups of animals, orally supplemented with CLA (600 mg/kg bw/day) for 7 weeks (2 preliminary weeks plus the 5-week treatment with Al; CLA + L, CLA + H) were used to investigate its protective effect, while untreated mice were used as control (Cntr). We provide evidence that mitochondrial dysfunction, Nrf2 alteration, inflammation and Acetylcholinesterase (AChE) hyperactivation can occur even from L exposure. Interestingly, animal pre-treatment with an allometric CLA dose led to significant downregulation of the toxic effects elicited by L or H, likely through the activation of an adaptive response. In conclusion, CLA ability to increase the level of glucose transporters - along with its antioxidant and anti-inflammatory effect - expands the therapeutic targets of these molecules and comes out as an intriguing suitable candidate for the treatment of multifactorial disease.
Asunto(s)
Enfermedad de Alzheimer , Encéfalo , Ácidos Linoleicos Conjugados , Acetilcolinesterasa/metabolismo , Aluminio/toxicidad , Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Animales , Antiinflamatorios/farmacología , Antioxidantes/farmacología , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Ácidos Linoleicos Conjugados/farmacología , Masculino , Ratones , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés OxidativoRESUMEN
Huntington's disease (HD) has traditionally been described as a disorder purely of the brain; however, evidence indicates that peripheral abnormalities are also commonly seen. Among others, severe unintended body weight loss represents a prevalent and often debilitating feature of HD pathology, with no therapies available. It correlates with disease progression and significantly affects the quality of life of HD patients. Curcumin, a naturally occurring polyphenol with multiple therapeutic properties, has been validated to exert important beneficial effects under health conditions as well as in different pathological settings, including neurodegenerative and gastrointestinal (GI) disorders. Here, we investigated the potential therapeutic action that curcumin-supplemented diet may exert on central and peripheral dysfunctions in R6/2 mice, a well-characterized HD animal model which recapitulates some features of human pathology. Maintenance of normal motor function, protection from neuropathology and from GI dysfunction and preservation of GI emptying and conserved intestinal contractility, proved the beneficial role of life-long dietary curcumin in HD and corroborated the potential of the compound to be exploited to alleviate very debilitating symptoms associated with the disease.
Asunto(s)
Conducta Animal/efectos de los fármacos , Curcumina/administración & dosificación , Enfermedad de Huntington/dietoterapia , Pérdida de Peso/efectos de los fármacos , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Curcumina/farmacología , Suplementos Dietéticos , Modelos Animales de Enfermedad , Femenino , Enfermedad de Huntington/fisiopatología , Masculino , Ratones , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , FenotipoRESUMEN
Oogenesis was examined in nine species of Antarctic fish to verify the existence of morphological peculiarities. The analyses were carried out on specimens belonging to three different families of Notothenioids (Nototheniidae, Channichthyidae and Bathydraconidae), all captured in the Ross Sea, in front of the Italian Station of Terra Nova Bay. Following dissection, the ovaries were processed and examined at the light and electron microscopes to determine the oocyte gross and fine morphology. The attention, in particular, was focused on the presence of cytoplasmic round bodies and on the organization of the cortical alveoli and the vitelline envelope. Results reveal significant specie-specific differences that could be partly correlated to the phylogenetic radiation but not to the peculiar environmental conditions being essentially comparable to those observed among temperate species.
Asunto(s)
Frío , Oocitos/ultraestructura , Oogénesis , Perciformes/fisiología , Animales , Membrana Basal/ultraestructura , Nucléolo Celular/ultraestructura , Núcleo Celular/ultraestructura , Femenino , Microscopía Electrónica de Transmisión , Perciformes/anatomía & histología , Especificidad de la Especie , Membrana Vitelina/ultraestructuraRESUMEN
Glucose-6-phosphate dehydrogenase-deleted embryonic stem (ES) cells (G6pd Delta) proliferate in vitro without special requirements, but when challenged with oxidants fail to sustain glutathione disulphide reconversion to reduced glutathione (GSH), entering a condition of oxidative stress. Here, we investigate the signalling events downstream of GSH oxidation in G6pd Delta and wild-type (wt) ES cells. We found that G6pd Delta ES cells are very sensitive to oxidants, activating an apoptotic pathway at oxidant concentrations otherwise sublethal for wt ES cells. We show that the apoptotic pathway activated by low oxidant concentrations is accompanied by mitochondria dysfunction, and it is therefore blocked by the overexpression of Bcl-X(L). Bcl-X(L) does not inhibit the decrease in cellular GSH and reactive oxygen species formation following oxidant treatment. We also found that oxidant treatment in ES cells is followed by the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. Interestingly, ERK activation has opposite outcomes in G6pd Delta ES cells compared to wt, which has a proapoptotic function in the first and a prosurvival function in the latter. We show that this phenomenon can be regulated by the cellular GSH level.
Asunto(s)
Apoptosis/fisiología , Glucosafosfato Deshidrogenasa/metabolismo , Glutatión/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Células Madre/citología , Animales , Apoptosis/efectos de los fármacos , Caspasas , Diamida/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa 4 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Mitocondrias/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células Madre/efectos de los fármacos , Reactivos de Sulfhidrilo/farmacología , Proteína bcl-XRESUMEN
The authors studied the otoliths of the Nototheniid Trematomus bernacchii with scanning electron microscopy and X-ray diffraction analysis. Results obtained reveal that three otoliths are present: a large sagitta, a lapillus and a fragile asteriscus. Their sensorial faces appear finely decorated as shown by scanning electron microscopy (SEM). The sagitta and the lapillus are aragonitic while the asteriscus is vateritic, as demonstrated by X-ray diffraction.
Asunto(s)
Membrana Otolítica/diagnóstico por imagen , Membrana Otolítica/ultraestructura , Perciformes/anatomía & histología , Animales , Carbonato de Calcio/análisis , Cristalografía por Rayos X , Microanálisis por Sonda Electrónica , Femenino , Masculino , Microscopía Electrónica de Rastreo , Radiografía , Difracción de Rayos XRESUMEN
The onset of resistance to drug-induced apoptosis of tumour cells is a major problem in cancer therapy. We studied a drug-selected clone of promyelocytic HL-60 cells, called HCW-2, which display a complex resistance to a wide variety of apoptosis-inducing agents and we found that these cells show a dramatic increase in the expression of heat shock proteins (Hsps) 70 and 27, while the parental cell line does not. It is known that stress proteins such as Hsps can confer resistance to a variety of damaging agents other than heat shock, such as TNF-alpha, monocyte-induced cytotoxicity, and also play a role in resistance to chemotherapy. This elevated expression of Hsps is paralleled by an increased activity of mitochondrial metabolism and pentose phosphate pathway, this latter leading to high levels of glucose-6-phosphate dehydrogenase and, consequently, of glutathione. Thus, the apoptotic-deficient phenotype is likely because of the presence of high levels of stress response proteins and GSH, which may confer resistance to apoptotic agents, including chemotherapy drugs. Moreover, the fact that in HCW-2 cells Hsp70 are mainly localised in mitochondria may account for the increased performances of mitochondrial metabolism. These observations could have some implications for the therapy of cancer, and for the design of combined strategies that act on antioxidant defences of the neoplastic cell.
Asunto(s)
Apoptosis , Mitocondrias/metabolismo , Oxidación-Reducción , Células Clonales , ADN Mitocondrial/análisis , Resistencia a Múltiples Medicamentos/genética , Glucosafosfato Deshidrogenasa/metabolismo , Glutatión/biosíntesis , Células HL-60 , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Mitocondrias/ultraestructura , Vía de Pentosa Fosfato , Fenotipo , Proteínas Proto-Oncogénicas c-bcl-2/análisisRESUMEN
To investigate the regulation of Chionodraco hamatus metallothionein (MT) encoding genes about 1000-bp regions of both MT-I and MT-II gene promoters were cloned and sequenced. Both promoters were rich in A-T content, and lacked the canonical TATA box; several putative cis-regulatory sequences were also present. In the MT-I promoter, four MREs were identified within the first 300 bp from the ATG codon. In the MT-II promoter, seven MREs were organized into two clusters, one containing three MREs located close to the ATG codon, and the other consisting of four MREs lying 500-900 bp upstream of the transcription starting point. The alignment of the MT-I and MT-II promoter regions showed 57% identity, which increased to 87% in the 300-bp region upstream of the ATG. Only the three proximal putative MREs identified were conserved both in position and sequence. Functional analysis of MT-I and MT-II promoters was performed by introducing deletion mutants of the 5'-flanking regions into vector pGL-3, directly upstream of the firefly luciferase reporter gene. Each construct was tested in the HepG2 cell lines in the absence or presence of zinc or cadmium ions. Maximum inducibility of the MT-II gene promoter was achieved with a construct containing both the proximal and the distal MRE clusters. The lack of the most distally located MRE dramatically affected MT-II promoter sensitivity to metals; removal of the distal cluster of MREs also reduced metal inducibility. The MT-I promoter was more compact, since maximal activity and metal inducibility depended on the presence of the proximal cluster of four MREs. This study suggests that the different organization of the MT-I and MT-II gene promoter regions might account for the observed differences in the basal and metal-induced expression of MT-I and MT-II isoforms in the C. hamatus liver.
Asunto(s)
Peces/genética , Metalotioneína/genética , Metales/farmacología , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Secuencia de Bases , Cadmio/farmacología , Clonación Molecular , ADN/química , ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Luciferasas/efectos de los fármacos , Luciferasas/genética , Luciferasas/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Eliminación de Secuencia , Homología de Secuencia de Ácido Nucleico , Células Tumorales Cultivadas , Zinc/farmacologíaRESUMEN
The present paper reports the full nucleotide sequence of a cloned cDNA prepared from RNA of lizard ovaries. The open reading frame consists of 2019 nucleotides, which encodes a protein of 673 amino acids belonging to the G protein-coupled receptor superfamily with a large extracellular N-terminal domain involved in hormone recognition. The transmembrane domain ends with a short intracytoplasmic COOH-terminal domain involved in effector activation. Phylogenetic analysis showed that the lizard receptor belongs to the family of follicle-stimulating hormone (FSH) receptors. The hydrophobicity profile is similar to that observed for mammalian and avian FSH receptors. Northern blot analysis of total RNA revealed that the FSH receptor is expressed at high levels in the ovary. In situ hybridization experiments demonstrate that FSH receptor mRNA is specifically localized within the small cells of the follicular epithelium surrounding the oocyte.
Asunto(s)
Lagartos/genética , Receptores de HFE/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Femenino , Expresión Génica , Hibridación in Situ , Datos de Secuencia Molecular , Ovario/metabolismo , Filogenia , ARN/genética , ARN/metabolismo , Análisis de Secuencia de ADN , Distribución TisularRESUMEN
Recombinant purine nucleoside phosphorylase (PNPase) from Escherichia coli was prepared in high yield in order to facilitate its use in coupled assays to measure the kinetics of phosphate-liberating enzymes. The E. coli enzyme was overexpressed in E. coli by inserting the genomic fragment containing the deoD gene downstream of the isopropyl beta-d-thiogalactoside-inducible promotor of pSE380 expression vector. The recombinant protein was purified to approximately 90% homogeneity and with a yield of approximately 9000 units of activity/L of culture, using an efficient one-column procedure. A continuous spectrophotometric assay coupling P(i) release to the phosphorolysis of the nucleoside analogue 7-methylinosine (m(7)Ino) was recently described. Here, we report the steady-state kinetic parameters of the recombinant E. coli PNPase catalyzed reaction with m(7)Ino and P(i) as substrates and compare these parameters with those of a bacterial PNPase commercially available for use in coupled assays. Under the assay conditions described, the recombinant E. coli protein is active at higher pH values and is stable up to a temperature of approximately 55 degrees C and following multiple freeze-thaw cycles. It is activated by high ionic strength but loses some activity following dialysis or concentration under pressure. Finally, a new procedure for the synthesis of m(7)Ino from inosine is described which is safe and cost effective, making the use of this methylated nucleoside in PNPase-coupled P(i) assays more attractive.
Asunto(s)
Escherichia coli/enzimología , Escherichia coli/genética , Inosina/análogos & derivados , Purina-Nucleósido Fosforilasa/genética , Purina-Nucleósido Fosforilasa/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Actinomycetales/enzimología , Dicroismo Circular , Clonación Molecular , Estabilidad de Enzimas , Vectores Genéticos/síntesis química , Concentración de Iones de Hidrógeno , Inosina/síntesis química , Inosina/metabolismo , Cinética , Peso Molecular , Concentración Osmolar , Fosfatos/metabolismo , Estructura Secundaria de Proteína , Purina-Nucleósido Fosforilasa/biosíntesis , Purina-Nucleósido Fosforilasa/química , Proteínas Recombinantes/química , Especificidad por SustratoRESUMEN
The present work was carried out to clarify the nature and origin of the yolk DNA present in vitellogenic oocytes of the lizard Podarcis sicula. Morphological and biochemical evidences indicate that it has an intrafollicular origin, from the apoptotic bodies resulting from follicle cells regression at the end of previtellogenesis. This conclusion is reinforced by the observation that the oocyte membrane, in in vitro experiments, is unpermeable to exogenous DNA. Biochemical evidences reveal that the yolk DNA has a low (200bp) molecular weight and this suggests that it is produced by the endonucleases typically involved in apoptotic DNA laddering. Indeed, immunocytochemical analyses demonstrate that follicle cells contain significant amounts of DNAse I. In immunoblots, carried out during different periods of the ovarian cycle, the enzyme shows a MW of about 33, 66 or 100 kDa thus indicating that its activity in the follicle of Podarcis is modulated by dimerization and/or binding to regulatory factors. Mol. Reprod. Dev. 59: 422-430, 2001.
Asunto(s)
ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Yema de Huevo/metabolismo , Lagartos/fisiología , Oocitos/metabolismo , Folículo Ovárico/fisiología , Animales , Apoptosis , Yema de Huevo/ultraestructura , Electroforesis en Gel de Poliacrilamida , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Femenino , Oocitos/enzimología , Oocitos/ultraestructura , Folículo Ovárico/citología , Folículo Ovárico/enzimología , Folículo Ovárico/ultraestructuraRESUMEN
OBJECTIVE: To elucidate the molecular basis of G6PD deficiency in the Han and Li nationalities in Hainan, China. METHODS: Polymerase chain reaction and restriction enzyme digestion were used to screen the mutations 1388G-->A, 1360C-->T, 1024C-->T, 592C-->T, 517T-->C, 493A-->G, 487G-->A, 392G-->T and 95A-->G. Single strand conformation polymorphism analysis was used to screen the other mutations followed by DNA sequencing to characterize the mutations of the samples with abnormal SSCP bands. RESULTS: Of the fifty-nine Han cases with G6PD deficiency, fourteen with 1388G-->A (23.7%), three with 871G-->A(5.1%), one with 835A-->T(1.7%), one with 517T-->C (1.7%), three with 392G-->T(5.1%), and four with 95A-->G(6.8%) were found. Of the thirty-two Li cases with G6PD deficiency, six with 1388G-->A(18.8%), three with 871G-->A(9.4%), and two with 95A-->G(6.3%) were found. A new mutation 835A-->G which causes the substitution of Ala for Thr at 279 in a Han case was identified and named as G6PD Haikou. The enzyme activity of the variant is about 10% of the normal and lower than the activity of the variant 835A-->T with about 40% of the normal. Analysis of the 3D model of human G6PD has revealed that the hydroxyl group of Thr at 279 is a group in maintaining the interaction of the G6PD subunits. CONCLUSION: The most common mutations of G6PD deficiency in Han and Li nationalities in Hainan are similar. Compared with the mutation spectrum of G6PD gene in the populations in other regions of China, the results indicate that some G6PD gene mutations are widespread in the populations of different regions in the southern part of China. The hydroxyl group of the Thr at 279 of human G6PD may be a necessary group for maintaining the interaction of the G6PD subunits and the enzyme activity.
Asunto(s)
Deficiencia de Glucosafosfato Deshidrogenasa/genética , Mutación , Niño , Preescolar , China/etnología , Femenino , Humanos , Lactante , MasculinoRESUMEN
To determine whether oogonial proliferation and oocyte recruitment are under control of hypophyseal and/or ovarian factors, we carried out a series of investigations using Podarcis sicula, a lizard inhabiting the temperate lowlands of Europe in which oocyte recruitment occurs throughout the year, as animal model. Germinal beds containing oogonia and oocytes in prefollicular stages were cocultured with different ovarian compartments in presence/absence of FSH, and the effects of different treatments were evaluated by counting the number of prelepto-leptotene oocytes. Results revealed that oocyte recruitment from the pool of oogonia is under the control of a factor released by follicle cells while FSH has an indirect effect on modulating oogonial proliferation. SDS-PAGE analyses carried out on media conditioned by follicles suggest that the factor involved in the control of oocyte recruitment may be a small protein (about 21 kDa) and that its release is dependent on the period of the ovarian cycle but apparently not on the circulating levels of FSH.
Asunto(s)
Factores Biológicos/metabolismo , Oocitos/citología , Oogénesis/fisiología , Folículo Ovárico/metabolismo , Animales , Factores Biológicos/aislamiento & purificación , Factores Biológicos/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Demecolcina/farmacología , Difusión , Electroforesis en Gel de Poliacrilamida , Femenino , Hormona Folículo Estimulante/farmacología , Hormona Folículo Estimulante/fisiología , Células de la Granulosa/metabolismo , Lagartos , Meiosis , Peso Molecular , Oocitos/efectos de los fármacos , Oogénesis/efectos de los fármacos , Folículo Ovárico/citología , Células Tecales/metabolismoRESUMEN
The study of environmental factors affecting vertebrate reproduction has long interested both developmental and evolutionary biologists. Although photoperiod has been considered to be an important environmental parameter for vertebrates such as birds, temperature is probably a primary external factor responsible for reproductive cyclicity in reptiles. In spite of the progress made in the understanding of reptilian reproductive strategies and adaptations, much remains to be learned about the interplay between endocrine physiological factors, such as hormones, and environmental parameters. In this report, we have examined the effects of in vivo administered FSH on oocyte recruitment during the most significant periods of the reproductive cycle of the lizard, Podarcis sicula. The results show that when FSH is administered in proximity to the reproductive period, it stimulates oocyte growth and ovulation; when the hormone is administered at the beginning of the winter stasis it affects ovarian activity without inducing ovulation. Ovarian adenylate cyclase activity is moderately sensitive to in vitro FSH stimulation during the pre- and post-reproductive periods. The sensitivity to hormone stimulation increases significantly during the reproductive period and winter stasis. We have also tested the hypothesis that environmental temperature affects the responsiveness of ovarian adenylate cyclase to FSH stimulation. For such a purpose, we exposed animals to 28 degrees C or 4 degrees C in different periods of the ovarian cycle. The results show that, whenever the temperature applied mimics the thermal regime of the coming season, adenylate cyclase sensitivity to FSH shifts towards levels that anticipate the natural responsiveness.
Asunto(s)
Adenilil Ciclasas/metabolismo , Lagartos/fisiología , Ovario/enzimología , Estaciones del Año , Temperatura , Análisis de Varianza , Animales , Femenino , Hormona Folículo Estimulante/farmacología , Lagartos/metabolismo , Oocitos/efectos de los fármacos , Oocitos/crecimiento & desarrollo , Ovario/efectos de los fármacos , Reproducción/fisiologíaRESUMEN
This article describes a new organelle found in the cytoplasm of the growth stage fish oocytes. In particular, we describe its organization at the morphological level and investigate its composition by different cytochemical and immunocytochemical approaches with both light and electron microscope. The conclusion is that the body is a peculiar protein scaffold functioning as a temporary trap for the storage of rRNA in the mid to late growth stage oocytes. Its presence would be related to the reorganization of the mass of amplified rDNA in micronucleoli and to the consequent temporary stop in the rRNA synthesis.
Asunto(s)
Oncorhynchus mykiss , Oocitos/metabolismo , ARN Ribosómico/metabolismo , Vitelogénesis/fisiología , Animales , Femenino , Oocitos/crecimiento & desarrollo , Oocitos/ultraestructuraRESUMEN
Full-length zebrafish cDNAs encoding two aspartic proteinases were cloned and sequenced. One of the two cDNAs was a 1708 bp product with an open reading frame of 398 amino acid residues corresponding to a cathepsin D. The other was a 1383 bp product encoding a polypeptide chain of 416 amino acids homologous to nothepsin, an aspartic proteinase first identified by us in the liver of Antarctic Notothenioidei. Gene expression assessed by RT-PCR and northern blot hybridization of RNA from different tissues showed that the expression was tissue- and sex-specific. Whereas the cathepsin D gene was expressed in all the tissues examined independently of the sex, the nothepsin gene was expressed exclusively in female livers.
Asunto(s)
Ácido Aspártico Endopeptidasas/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Catepsina D/genética , ADN Complementario/química , ADN Complementario/genética , Femenino , Regulación Enzimológica de la Expresión Génica , Masculino , Datos de Secuencia Molecular , Filogenia , ARN/genética , ARN/metabolismo , Análisis de Secuencia de ADN , Factores Sexuales , Distribución TisularRESUMEN
Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway that is responsible for the generation of NADPH, which is required in many detoxifying reactions. We have recently demonstrated that G6PD expression is induced by a variety of chemical agents acting at different steps in the biochemical pathway controlling the intracellular redox status. Although we obtained evidence that the oxidative stress-mediated enhancement of G6PD expression is a general phenomenon, the functional significance of such G6PD induction after oxidant insult is still poorly understood. In this report, we used a GSH-depleting drug that determines a marked decrease in the intracellular pool of reduced glutathione and a gradual but notable increase in G6PD expression. Both effects are seen soon after drug addition. Once G6PD activity has reached the maximum, the GSH pool is restored. We suggest and also provide the first direct evidence that G6PD induction serves to maintain and regenerate the intracellular GSH pool. We used HeLa cell clones stably transfected with the human G6PD gene that display higher G6PD activity than the parent HeLa cells. Although the activities of glutathione peroxidase, glutathione reductase, and catalase were comparable in all strains, the concentrations of GSH were significantly higher in G6PD-overexpressing clones. A direct consequence of GSH increase in these cells is a decreased reactive oxygen species production, which makes these cells less sensitive to the oxidative burst produced by external stimuli. Indeed, all clones that constitutively overexpress G6PD exhibited strong protection against oxidants-mediated cell killing. We also observe that NF-kappaB activation, in response to tumor necrosis factor-alpha treatment, is strongly reduced in human HeLa cells overexpressing G6PD.
Asunto(s)
Glucosafosfato Deshidrogenasa/biosíntesis , Glutatión/metabolismo , Antioxidantes/farmacología , Butionina Sulfoximina/farmacología , Catalasa/metabolismo , Diamida/farmacología , Citometría de Flujo , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Células HeLa , Humanos , Pirrolidinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Tiocarbamatos/farmacología , Células Tumorales CultivadasRESUMEN
During vitellogenesis, the oocytes of oviparous species accumulate in the cytoplasm a large amount of proteic nutrients synthetized in the liver. Once incorporated into the oocytes, these nutrients, especially represented by vitellogenin (VTG) and very low-density lipoprotein (VLDL), are cleaved into a characteristic set of polypeptides forming yolk platelets. We have studied the molecular mechanisms involved in yolk formation in a reptilian species Podarcis sicula, a lizard characterized by a seasonal reproductive cycle. Our results demonstrate the existence in the lizard ovary of an aspartic proteinase having a maximal activity at acidic pH and a molecular mass of 40 kDa. The full-length aspartic proteinase cDNA produced from total RNA by RT-PCR is 1,442 base pairs long and encodes a protein of 403 amino acids. A comparison of the proteic sequence with aspartic proteinases from various sources demonstrates that the lizard enzyme is a cathepsin D. Lizard ovarian cathepsin D activity is maximal in June, in coincidence with vitellogenesis and ovulation, and is especially abundant in vitellogenic follicles and in eggs. Ovarian cathepsin D activity can be enhanced during the resting period by treatment with FSH in vivo. Northern blot analysis shows that cathepsin D mRNA is exceedingly abundant during the reproductive period, and accumulates preferentially in previtellogenic oocytes.
Asunto(s)
Catepsina D/genética , Lagartos , Ovario/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catepsina D/aislamiento & purificación , Catepsina D/metabolismo , Clonación Molecular , ADN Complementario , Femenino , Expresión Génica , Gónadas , Humanos , Datos de Secuencia Molecular , ARN Mensajero , Reproducción , Homología de Secuencia de AminoácidoRESUMEN
In the present study we analyse the nature and the functional significance of the spherical and fibrillo-granular structures appearing in the oocyte nucleus of the lizard Podarcis sicula, following the disappearance of the typical nucleolus. By LM and TEM approaches, we demonstrate that the fibrillo-granuli, containing DNA, RNA and nucleolar proteins, are micronucleoli transcriptionally active and that their DNA is probably derived from nucleolar fragmentation. By contrast, we could not explain the origin and role of the so-called spherical bodies, appearing earlier in oocyte growth; these, in fact, do not contain nucleic acids or nucleolar proteins and do not incorporate uridine. Different possible explanations of their significance are discussed.
Asunto(s)
Nucléolo Celular/ultraestructura , Lagartos/metabolismo , Oocitos/crecimiento & desarrollo , Animales , ADN/análisis , Femenino , Microscopía Electrónica , Oocitos/ultraestructura , ARN/análisis , Tinción con Nitrato de Plata/veterinaria , Transcripción Genética , Uridina/metabolismo , Vitelogénesis/genéticaRESUMEN
Gonadotropins (FSH and LH) affect several mammalian gonadal functions. In particular, FSH stimulates oogonial proliferation and oocyte growth, while LH regulates ovulation and progesterone secretion. In lacertilian reptiles, gonadal function is also regulated by pituitary gonadotropins, but which hormone controls ovarian activities and the mechanisms of action are unknown. The present study aimed to clarify mechanisms of action of pituitary gonadotropins on the ovary of Podarcis sicula (Lacertilia). The data demonstrate that mammalian gonadotropins FSH and LH produce a threefold stimulation of adenylate cyclase activity in follicular membranes, while hCG and TSH are less effective, causing a twofold increase in adenylate cyclase activity. Neurotransmitters such as dopamine, serotonin, and catecholamines have no effect on enzyme activity. The action of mammalian FSH and LH on the ovary mimics the effect of homologous hormones: in lizard ovaries incubated in vitro in the presence of isolated homologous pituitary glands, the intracellular cAMP level increased by 50% with respect to control ovaries. Mammalian gonadotropins appear homologous to lizard gonadotropin(s): Southern blot analyses show that the lizard genome contains nucleotide sequences homologous to those encoding for mammalian beta FSH and beta LH. Both homologous and heterologous desensitization of adenylate cyclase activity occurs in the lizard ovary. In fact, responsiveness of adenylate cyclase to gonadotropin stimulation is abolished in animals 2 hr after in vivo treatment with FSH. Sensitivity to gonadotropin stimulation is restored 2 weeks after the beginning of the in vivo treatment. Desensitization was also observed in ovaries incubated in vitro with mammalian FSH or with isolated pituitary glands.