RESUMEN

Pathogen-induced cancers account for 15% of human tumors and are a growing concern for endangered wildlife. Fibropapillomatosis is an expanding virally and environmentally co-induced sea turtle tumor epizootic. Chelonid herpesvirus 5 (ChHV5) is implicated as a causative virus, but its transmission method and specific role in oncogenesis and progression is unclear. We applied environmental (e)DNA-based viral monitoring to assess viral shedding as a direct means of transmission, and the relationship between tumor burden, surgical resection and ChHV5 shedding. To elucidate the abundance and transcriptional status of ChHV5 across early, established, regrowth and internal tumors we conducted genomics and transcriptomics. We determined that ChHV5 is shed into the water column, representing a likely transmission route, and revealed novel temporal shedding dynamics and tumor burden correlations. ChHV5 was more abundant in the water column than in marine leeches. We also revealed that ChHV5 is latent in fibropapillomatosis, including early stage, regrowth and internal tumors; higher viral transcription is not indicative of poor patient outcome, and high ChHV5 loads predominantly arise from latent virus. These results expand our knowledge of the cellular and shedding dynamics of ChHV5 and can provide insights into temporal transmission dynamics and viral oncogenesis not readily investigable in tumors of terrestrial species.

Asunto(s)

ADN Ambiental/análisis , Herpesviridae/genética , Tortugas/virología , Verrugas/transmisión , Animales , Carcinogénesis/genética , ADN/genética , Monitoreo del Ambiente/métodos , Genómica/métodos , Herpesviridae/patogenicidad , Sanguijuelas/genética , Sanguijuelas/patogenicidad , Papiloma/etiología , Papiloma/virología , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/virología , Tortugas/genética , Esparcimiento de Virus/genética , Verrugas/veterinaria , Verrugas/virología

RESUMEN

Sea turtle populations are under threat from an epizootic tumor disease (animal epidemic) known as fibropapillomatosis. Fibropapillomatosis continues to spread geographically, with prevalence of the disease also growing at many longer-affected sites globally. However, we do not yet understand the precise environmental, mutational and viral events driving fibropapillomatosis tumor formation and progression.Here we perform transcriptomic and immunohistochemical profiling of five fibropapillomatosis tumor types: external new, established and postsurgical regrowth tumors, and internal lung and kidney tumors. We reveal that internal tumors are molecularly distinct from the more common external tumors. However, they have a small number of conserved potentially therapeutically targetable molecular vulnerabilities in common, such as the MAPK, Wnt, TGFß and TNF oncogenic signaling pathways. These conserved oncogenic drivers recapitulate remarkably well the core pan-cancer drivers responsible for human cancers. Fibropapillomatosis has been considered benign, but metastatic-related transcriptional signatures are strongly activated in kidney and established external tumors. Tumors in turtles with poor outcomes (died/euthanized) have genes associated with apoptosis and immune function suppressed, with these genes providing putative predictive biomarkers.Together, these results offer an improved understanding of fibropapillomatosis tumorigenesis and provide insights into the origins, inter-tumor relationships, and therapeutic treatment for this wildlife epizootic.

Asunto(s)

Biomarcadores de Tumor , Proliferación Celular , Recurrencia Local de Neoplasia/veterinaria , Papiloma/veterinaria , Neoplasias Cutáneas/veterinaria , Infecciones Tumorales por Virus/veterinaria , Tortugas , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Inmunohistoquímica , Papiloma/genética , Papiloma/metabolismo , Papiloma/cirugía , Transducción de Señal , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/cirugía , Transcriptoma , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/cirugía

RESUMEN

Thioredoxin 2 (TXN2) is a small redox protein found in nearly all organisms. As a mitochondrial member of the thioredoxin antioxidant defense system, TXN2 interacts with peroxiredoxin 3 (PRDX3) to remove hydrogen peroxide. Accordingly, TXN2 is thought to play an important role in maintaining the appropriate mitochondrial redox environment and protecting the mitochondrial components against oxidative stress. In the current study, we investigated the effects of Txn2 haplodeficiency on cochlear antioxidant defenses, auditory function, and cochlear cell loss across the lifespan in wild-type (WT) and Txn2 heterozygous knockout (Txn2+/-) mice backcrossed onto CBA/CaJ mice, a well-established model of age-related hearing loss. Txn2+/- mice displayed a 58% decrease in TXN2 protein levels in the mitochondria of the inner ears compared to WT mice. However, Txn2 haplodeficiency did not affect the thioredoxin or glutathione antioxidant defense in both the mitochondria and cytosol of the inner ears of young mice. There were no differences in the levels of mitochondrial biogenesis markers, mitochondrial DNA content, or oxidative DNA and protein damage markers in the inner ears between young WT and Txn2+/- mice. In a mouse inner ear cell line, knockdown of Txn2 did not affect cell viability under hydrogen peroxide treatment. Consistent with the tissue and cell line results, there were no differences in hair cell loss or spiral ganglion neuron density between WT and Txn2+/- mice at 3-5 or 23-25 months of age. Furthermore, Txn2 haplodeficiency did not affect auditory brainstem response threshold, wave I latency, or wave I amplitude at 3-5, 15-16, or 23-25 months of age. Therefore, Txn2 haplodeficiency does not affect cochlear antioxidant defenses, accelerate degeneration of cochlear cells, or affect auditory function in mice across the lifespan.

Asunto(s)

Antioxidantes , Pérdida Auditiva , Animales , Umbral Auditivo , Cóclea , Potenciales Evocados Auditivos del Tronco Encefálico , Pérdida Auditiva/genética , Longevidad , Ratones , Ratones Endogámicos CBA

RESUMEN

Cisplatin is one of the most widely used chemotherapeutic drugs for the treatment of cancer. Unfortunately, one of its major side effects is permanent hearing loss. Here, we show that glutathione transferase α4 (GSTA4), a member of the Phase II detoxifying enzyme superfamily, mediates reduction of cisplatin ototoxicity by removing 4-hydroxynonenal (4-HNE) in the inner ears of female mice. Under cisplatin treatment, loss of Gsta4 results in more profound hearing loss in female mice compared to male mice. Cisplatin stimulates GSTA4 activity in the inner ear of female wild-type, but not male wild-type mice. In female Gsta4-/- mice, cisplatin treatment results in increased levels of 4-HNE in cochlear neurons compared to male Gsta4-/- mice. In CBA/CaJ mice, ovariectomy decreases mRNA expression of Gsta4, and the levels of GSTA4 protein in the inner ears. Thus, our findings suggest that GSTA4-dependent detoxification may play a role in estrogen-mediated neuroprotection.

Asunto(s)

Cisplatino/efectos adversos , Glutatión Transferasa/metabolismo , Ototoxicidad/enzimología , Animales , Umbral Auditivo/efectos de los fármacos , Capilares/patología , Cóclea/enzimología , Cóclea/patología , Cóclea/fisiopatología , Cruzamientos Genéticos , Daño del ADN/genética , Potenciales Evocados Auditivos del Tronco Encefálico/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión Transferasa/deficiencia , Pérdida Auditiva/complicaciones , Pérdida Auditiva/enzimología , Pérdida Auditiva/fisiopatología , Masculino , Ratones Endogámicos CBA , Ototoxicidad/complicaciones , Ototoxicidad/patología , Ototoxicidad/fisiopatología , Estrés Oxidativo/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ganglio Espiral de la Cóclea/efectos de los fármacos , Ganglio Espiral de la Cóclea/patología

RESUMEN

Adult human neural progenitor and stem cells have been implicated as a potential source of brain cancer causing cells, but specific events that might cause cells to progress towards a transformed phenotype remain unclear. The L1CAM (L1) cell adhesion/recognition molecule is expressed abnormally by human glioma cancer cells and is released as a large extracellular ectodomain fragment, which stimulates cell motility and proliferation. This study investigates the effects of ectopic overexpression of the L1 long ectodomain (L1LE; ˜180 kDa) on the motility, proliferation, and differentiation of human neural progenitor cells (HNPs). L1LE was ectopically expressed in HNPs using a lentiviral vector. Surprisingly, overexpression of L1LE resulted in reduced HNP motility in vitro, in stark contrast to the effects on glioma and other cancer cell types. L1LE overexpression resulted in a variable degree of maintenance of HNP proliferation in media without added growth factors but did not increase proliferation. In monolayer culture, HNPs expressed a variety of differentiation markers. L1LE overexpression resulted in loss of glutamine synthetase (GS) and ß3-tubulin expression in normal HNP media, and reduced vimentin and increased GS expression in the absence of added growth factors. When co-cultured with chick embryonic brain cell aggregates, HNPs show increased differentiation potential. Some HNPs expressed p-neurofilaments and oligodendrocytic O4, indicating differentiation beyond that in monolayer culture. Most HNP-L1LE cells lost their vimentin and GFAP (glial fibrillary acidic protein) staining, and many cells were positive for astrocytic GS. However, these cells rarely were positive for neuronal markers ß3-tubulin or p-neurofilaments, and few HNP oligodendrocyte progenitors were found. These results suggest that unlike for glioma cells, L1LE does not increase HNP cell motility, but rather decreases motility and influences the differentiation of normal brain progenitor cells. Therefore, the effect of L1LE on increasing motility and proliferation appears to be limited to already transformed cells.

Asunto(s)

Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Células-Madre Neurales/metabolismo , Línea Celular , Preescolar , Expresión Génica Ectópica , Humanos , Masculino , Molécula L1 de Adhesión de Célula Nerviosa/genética , Células-Madre Neurales/citología

RESUMEN

Mitochondrial DNA (mtDNA) mutations are thought to have a causal role in a variety of age-related neurodegenerative diseases, including age-related hearing loss (AHL). In the current study, we investigated the roles of mtDNA deletions and point mutations in AHL in mitochondrial mutator mice (Polgmut/mut) that were backcrossed onto CBA/CaJ mice, a well-established model of late-onset AHL. mtDNA deletions accumulated significantly with age in the inner ears of Polgmut/mut mice, while there were no differences in mtDNA deletion frequencies in the inner ears between 5 and 17 months old Polg+/+ mice or 5 months old Polg+/+ and Polgmut/mut mice. mtDNA deletions also accumulated significantly in the inner ears of CBA/CaJ mice during normal aging. In contrast, 5 months old Polgmut/mut mice displayed a 238-fold increase in mtDNA point mutation frequencies in the inner ears compared to age-matched Polg+/+ mice, but there were no differences in mtDNA point mutation frequencies in the inner ears between 5 and 17 months old Polgmut/mut mice. Seventeen-month-old Polgmut/mut mice also displayed early-onset severe hearing loss associated with a significant reduction in neural output of the cochlea, while age-matched Polg+/+ mice displayed little or no hearing impairment. Consistent with the physiological and mtDNA deletion test result, 17-month-old Polgmut/mut mice displayed a profound loss of spiral ganglion neurons in the cochlea. Thus, our data suggest that a higher burden of mtDNA point mutations from a young age and age-related accumulation of mtDNA deletions likely contribute to early-onset AHL in mitochondrial mutator mice.

Asunto(s)

ADN Polimerasa gamma/genética , ADN Mitocondrial/química , Presbiacusia/genética , Animales , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Mutación Puntual , Presbiacusia/patología , Eliminación de Secuencia , Ganglio Espiral de la Cóclea/patología

RESUMEN

Isocitrate dehydrogenase (IDH) 2 participates in the TCA cycle and catalyzes the conversion of isocitrate to α-ketoglutarate and NADP+ to NADPH. In the mitochondria, IDH2 also plays a key role in protecting mitochondrial components from oxidative stress by supplying NADPH to both glutathione reductase (GSR) and thioredoxin reductase 2 (TXNRD2). Here, we report that loss of Idh2 accelerates age-related hearing loss, the most common form of hearing impairment, in male mice. This was accompanied by increased oxidative DNA damage, increased apoptotic cell death, and profound loss of spiral ganglion neurons and hair cells in the cochlea of 24-month-old Idh2-/- mice. In young male mice, loss of Idh2 resulted in decreased NADPH redox state and decreased activity of TXNRD2 in the mitochondria of the inner ear. In HEI-OC1 mouse inner ear cell lines, knockdown of Idh2 resulted in a decline in cell viability and mitochondrial oxygen consumption. This was accompanied by decreased NADPH redox state and decreased activity of TXNRD2 in the mitochondria of the HEI-OC1 cells. Therefore, IDH2 functions as the principal source of NADPH for the mitochondrial thioredoxin antioxidant defense and plays an essential role in protecting hair cells and neurons against oxidative stress in the cochlea of male mice.

Asunto(s)

Envejecimiento/genética , Pérdida Auditiva/genética , Isocitrato Deshidrogenasa/genética , Tiorredoxina Reductasa 2/genética , Envejecimiento/patología , Animales , Apoptosis/genética , Supervivencia Celular/genética , Cóclea/metabolismo , Cóclea/patología , Modelos Animales de Enfermedad , Glutatión Reductasa/genética , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patología , Pérdida Auditiva/metabolismo , Pérdida Auditiva/patología , Humanos , Masculino , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , NADP/metabolismo , Neuronas/metabolismo , Neuronas/patología , Estrés Oxidativo/genética , Consumo de Oxígeno/genética , Ganglio Espiral de la Cóclea/metabolismo , Ganglio Espiral de la Cóclea/patología

RESUMEN

Glutathione reductase (GSR), a key member of the glutathione antioxidant defense system, converts oxidized glutathione (GSSG) to reduced glutathione (GSH) and maintains the intracellular glutathione redox state to protect the cells from oxidative damage. Previous reports have shown that Gsr deficiency results in defects in host defense against bacterial infection, while diquat induces renal injury in Gsr hypomorphic mice. In flies, overexpression of GSR extended lifespan under hyperoxia. In the current study, we investigated the roles of GSR in cochlear antioxidant defense using Gsr homozygous knockout mice that were backcrossed onto the CBA/CaJ mouse strain, a normal-hearing strain that does not carry a specific Cdh23 mutation that causes progressive hair cell degeneration and early onset of hearing loss. Gsr-/- mice displayed a significant decrease in GSR activity and GSH/GSSG ratios in the cytosol of the inner ears. However, Gsr deficiency did not affect ABR (auditory brainstem response) hearing thresholds, wave I amplitudes or wave I latencies in young mice. No histological abnormalities were observed in the cochlea of Gsr-/- mice. Furthermore, there were no differences in the activities of cytosolic glutathione-related enzymes, including glutathione peroxidase and glutamate-cysteine ligase, or the levels of oxidative damage markers in the inner ears between WT and Gsr-/- mice. In contrast, Gsr deficiency resulted in increased activities of cytosolic thioredoxin and thioredoxin reductase in the inner ears. Therefore, under normal physiological conditions, GSR is not essential for the maintenance of antioxidant defenses in mouse cochlea. Given that the thioredoxin system is known to reduce GSSG to GSH in multiple species, our findings suggest that the thioredoxin system can support GSSG reduction in the mouse peripheral auditory system.

Asunto(s)

Cóclea/metabolismo , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Glutatión Reductasa/deficiencia , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Tiorredoxinas/metabolismo , Animales , Antioxidantes/metabolismo , Femenino , Expresión Génica , Glutamato-Cisteína Ligasa/genética , Glutamato-Cisteína Ligasa/metabolismo , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/genética , Masculino , Ratones , Ratones Endogámicos CBA , Ratones Noqueados , Estrés Oxidativo , Reductasa de Tiorredoxina-Disulfuro/genética , Tiorredoxinas/genética

RESUMEN

Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP+ to NADPH and is thought to be the principal source of NADPH for the cytosolic glutathione and thioredoxin antioxidant defense systems. We investigated the roles of G6PD in the cytosolic antioxidant defense in the cochlea of G6pd hypomorphic mice that were backcrossed onto normal-hearing CBA/CaJ mice. Young G6pd-deficient mice displayed a significant decrease in cytosolic G6PD protein levels and activities in the inner ears. However, G6pd deficiency did not affect the cytosolic NADPH redox state, or glutathione or thioredoxin antioxidant defense in the inner ears. No histological abnormalities or oxidative damage was observed in the cochlea of G6pd hemizygous males or homozygous females. Furthermore, G6pd deficiency did not affect auditory brainstem response hearing thresholds, wave I amplitudes or wave I latencies in young males or females. In contrast, G6pd deficiency resulted in increased activities and protein levels of cytosolic isocitrate dehydrogenase 1, an enzyme that catalyzes the conversion of isocitrate to α-ketoglutarate and NADP+ to NADPH, in the inner ear. In a mouse inner ear cell line, knockdown of Idh1, but not G6pd, decreased cell growth rates, cytosolic NADPH levels, and thioredoxin reductase activities. Therefore, under normal physiological conditions, G6pd deficiency does not affect the cytosolic glutathione or thioredoxin antioxidant defense in mouse cochlea. Under G6pd deficiency conditions, isocitrate dehydrogenase 1 likely functions as the principal source of NADPH for cytosolic antioxidant defense in the cochlea.SIGNIFICANCE STATEMENT Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP+ to NADPH and is thought to be the principal source of NADPH for the cytosolic glutathione and thioredoxin antioxidant defense systems. In the current study, we show that, under normal physiological conditions, G6pd deficiency does not affect the cytosolic glutathione or thioredoxin antioxidant defense in the mouse cochlea. However, under G6pd deficiency conditions, isocitrate dehydrogenase 1 likely functions as the principal source of NADPH for cytosolic antioxidant defense in the cochlea.

Asunto(s)

Antioxidantes/metabolismo , Percepción Auditiva/fisiología , Cóclea/fisiopatología , Deficiencia de Glucosafosfato Deshidrogenasa/fisiopatología , Glutatión/metabolismo , Tiorredoxinas/metabolismo , Animales , Citosol/metabolismo , Femenino , Masculino , Ratones , Ratones Transgénicos

RESUMEN

In the mosquito midgut, luminal pH regulation and cellular ion transport processes are important for the digestion of food and maintenance of cellular homeostasis. pH regulation in the mosquito gut is affected by the vectorial movement of the principal ions including bicarbonate/carbonate and protons. As in all metazoans, mosquitoes employ the product of aerobic metabolism carbon dioxide in its bicarbonate/carbonate form as one of the major buffers of cellular and extracellular pH. The conversion of metabolic carbon dioxide to bicarbonate/carbonate is accomplished by a family of enzymes encoded by the carbonic anhydrase gene family. This study characterizes Aedes aegypti carbonic anhydrases using bioinformatic, molecular, and immunohistochemical methods. Our analyses show that there are fourteen Aedes aegypti carbonic anhydrase genes, two of which are expressed as splice variants. The carbonic anhydrases were classified as either integral membrane, peripheral membrane, mitochondrial, secreted, or soluble cytoplasmic proteins. Using polymerase chain reaction and Western blotting, one of the carbonic anhydrases, Aedes aegypti carbonic anhydrase 9, was analyzed and found in each life stage, male/female pupae, male/female adults, and in the female posterior midgut. Next, carbonic anhydrase 9 was analyzed in larvae and adults using confocal microscopy and was detected in the midgut regions. According to our analyses, carbonic anhydrase 9 is a soluble cytoplasmic enzyme found in the alimentary canal of larvae and adults and is expressed throughout the life cycle of the mosquito. Based on previous physiological analyses of adults and larvae, it appears AeCA9 is one of the major carbonic anhydrases involved in producing bicarbonate/carbonate which is involved in pH regulation and ion transport processes in the alimentary canal. Detailed understanding of the molecular bases of ion homeostasis in mosquitoes will provide targets for novel mosquito control strategies into the new millennium.

Asunto(s)

Aedes/metabolismo , Anhidrasa Carbónica IX/metabolismo , Tracto Gastrointestinal/metabolismo , Animales , Transporte Biológico , Anhidrasas Carbónicas/metabolismo , Concentración de Iones de Hidrógeno , Larva

RESUMEN

The expression of Basigin gene products and monocarboxylate transporter-1 (MCT1) has been investigated within the mammalian neural retina and suggests a role for these proteins in cellular metabolism within that tissue. The purpose of the present study was to investigate the expression of these same proteins in the pineal gland of the mouse brain. Mouse pineal gland and neural retina RNA and protein were subjected to quantitative reverse transcription-polymerase chain reaction and immunoblotting analyses. In addition, paraffin-embedded sections of each tissue were analyzed for expression of Basigin gene products and MCT1 via immunohistochemistry. The results indicate that MCT1 and Basigin variant-2, but not Basigin variant-1, are expressed within the mouse pineal gland. The expression of Basigin variant-2 and MCT1 was localized to the capsule surrounding the gland. The position and relative amounts of the gene products suggest that they play a much less prominent role within the pineal gland than in the neural retina.

Asunto(s)

Basigina/genética , Regulación de la Expresión Génica , Glándula Pineal/metabolismo , Animales , Basigina/metabolismo , Ratones Endogámicos C57BL , Transportadores de Ácidos Monocarboxílicos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Simportadores/metabolismo

RESUMEN

Regular physical exercise reduces the risk for obesity, cardiovascular diseases, and disability and is associated with longer lifespan expectancy (Taylor et al., 2004; Pahor et al., 2014; Anton et al., 2015; Arem et al., 2015). In contrast, decreased physical function is associated with hearing loss among older adults (Li et al., 2013; Chen et al., 2015). Here, we investigated the effects of long-term voluntary wheel running (WR) on age-related hearing loss (AHL) in CBA/CaJ mice, a well established model of AHL (Zheng et al., 1999). WR activity peaked at 6 months of age (12,280 m/d) and gradually decreased over time. At 24 months of age, the average WR distance was 3987 m/d. Twenty-four-month-old runners had less cochlear hair cell and spiral ganglion neuron loss and better auditory brainstem response thresholds at the low and middle frequencies compared with age-matched, non-WR controls. Gene ontology (GO) enrichment analysis of inner ear tissues from 6-month-old controls and runners revealed that WR resulted in a marked enrichment for GO gene sets associated with immune response, inflammatory response, vascular function, and apoptosis. In agreement with these results, there was reduced stria vascularis (SV) atrophy and reduced loss of capillaries in the SV of old runners versus old controls. Given that SV holds numerous capillaries that are essential for transporting oxygen and nutrients into the cochlea, our findings suggest that long-term exercise delays the progression of AHL by reducing age-related loss of strial capillaries associated with inflammation. SIGNIFICANCE STATEMENT: Nearly two-thirds of adults aged 70 years or older develop significant age-related hearing loss (AHL), a condition that can lead to social isolation and major communication difficulties. AHL is also associated with decreased physical function among older adults. In the current study, we show that regular exercise slowed AHL and cochlear degeneration significantly in a well established murine model. Our data suggest that regular exercise delays the progression of AHL by reducing age-related loss of strial capillaries associated with inflammation.

Asunto(s)

Envejecimiento , Cóclea/fisiología , Terapia por Ejercicio/métodos , Condicionamiento Físico Animal/métodos , Presbiacusia/prevención & control , Presbiacusia/fisiopatología , Animales , Cóclea/patología , Pérdida Auditiva , Masculino , Ratones , Ratones Endogámicos DBA , Esfuerzo Físico , Presbiacusia/patología , Resultado del Tratamiento

RESUMEN

Hearing gradually declines with age in both animals and humans, and this condition is known as age-related hearing loss (AHL). Here, we investigated the effects of deficiency of Sirt1, a member of the mammalian sirtuin family, on age-related cochlear pathology and associated hearing loss in C57BL/6 mice, a mouse model of early-onset AHL. Sirt1 deficiency reduced age-related oxidative damage of cochlear hair cells and spiral ganglion neurons and delayed the early onset of AHL. In cultured mouse inner ear cell lines, Sirt1 knockdown increased cell viability under oxidative stress conditions, induced nuclear translocation of Foxo3a, and increased acetylation status of Foxo3a. This resulted in increased activity of the antioxidant enzyme catalase. In young wild-type mice, both Sirt1 and Foxo3a proteins resided in the cytoplasm of the supporting cells within the organ of Corti of the cochlea. Therefore, our findings suggest that SIRT1 promotes early-onset AHL through suppressing FOXO3a-mediated oxidative stress resistance in the cochlea of C57BL/6 mice.

Asunto(s)

Envejecimiento/patología , Envejecimiento/fisiología , Células Ciliadas Auditivas/patología , Pérdida Auditiva/etiología , Pérdida Auditiva/prevención & control , Sirtuina 1/deficiencia , Sirtuina 1/fisiología , Acetilación , Animales , Catalasa/metabolismo , Línea Celular , Supervivencia Celular , Modelos Animales de Enfermedad , Oído Interno/citología , Oído Interno/metabolismo , Femenino , Proteína Forkhead Box O3/metabolismo , Técnicas de Silenciamiento del Gen , Pérdida Auditiva/patología , Masculino , Ratones Endogámicos C57BL , Estrés Oxidativo

RESUMEN

BACKGROUND: Aquaporin (AQP) water channels are important for water homeostasis in all organisms. Malaria transmission is dependent on Anopheles mosquitoes. Water balance is a major factor influencing mosquito survival, which may indirectly affect pathogen transmission. METHODOLOGY/PRINCIPAL FINDINGS: We obtained full-length mRNA sequences for Anopheles gambiae aquaporin 1 (AgAQP1) and identified two splice variants for the gene. In vitro expression analysis showed that both variants transported water and were inhibited by Hg(2+). One splice variant (AgAQP1A) was exclusively expressed in adult female ovaries indicating a function in mosquito reproduction. The other splice variant (AgAQP1B) was expressed in the midgut, malpighian tubules and the head in adult mosquitoes. Immunolabeling showed that in malpighian tubules, AgAQP1 is expressed in principal cells in the proximal portion and in stellate cells in the distal portion. Moreover, AgAQP1 is expressed in Johnston's organ (the "ear"), which is important for courtship behavior. CONCLUSIONS AND SIGNIFICANCE: These results suggest that AgAQP1 may play roles associated with mating (courtship) and reproduction in addition to water homeostasis in this important African malaria vector.

Asunto(s)

Anopheles/metabolismo , Acuaporinas/metabolismo , Proteínas de Insectos/metabolismo , Empalme Alternativo , Animales , Acuaporinas/genética , Proteínas de Insectos/genética , Insectos Vectores , Malaria/transmisión

RESUMEN

Saltwater tolerance is a trait that carries both ecological and epidemiological significance for Anopheles mosquitoes that transmit human malaria, as it plays a key role in determining their habitat use and ecological distribution, and thus their local contribution to malaria transmission. Here, we lay the groundwork for genetic dissection of this trait by quantifying saltwater tolerance in three closely related cryptic species and malaria vectors from the Afrotropical Anopheles gambiae complex that are known to differ starkly in their tolerance to salinity: the obligate freshwater species A. gambiae and A. coluzzii, and the saltwater-tolerant species A. merus. We performed detailed comparisons of survivorship under varying salinities, using multiple strains of A. gambiae, A. coluzzii and A. merus, as well as F1 progeny from reciprocal crosses of A. merus and A. coluzzii. Additionally, using immunohistochemistry, we compared the location of three ion regulatory proteins (Na(+)/K(+)-ATPase, carbonic anhydrase and Na(+)/H(+)-antiporter) in the recta of A. coluzzii and A. merus reared in freshwater or saline water. As expected, we found that A. merus survives exposure to high salinities better than A. gambiae and A. coluzzii. Further, we found that exposure to a salinity level of 15.85 g NaCl l(-1) is a discriminating dose that kills all A. gambiae, A. coluzzii and A. coluzzii-A. merus F1 larvae, but does not negatively impact the survival of A. merus. Importantly, phenotypic expression of saltwater tolerance by A. merus is highly dependent upon the developmental time of exposure, and based on immunohistochemistry, salt tolerance appears to involve a major shift in Na(+)/K+-ATPase localization in the rectum, as observed previously for the distantly related saline-tolerant species A. albimanus.

Asunto(s)

Anopheles/efectos de los fármacos , Anopheles/crecimiento & desarrollo , Salinidad , Cloruro de Sodio/administración & dosificación , Cloruro de Sodio/farmacología , Adaptación Fisiológica/efectos de los fármacos , Animales , Bioensayo , Femenino , Agua Dulce , Humanos , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Masculino , Pupa/efectos de los fármacos , Pupa/crecimiento & desarrollo , Especificidad de la Especie , Análisis de Supervivencia

RESUMEN

BACKGROUND: Mitochondrial short and long-range movements are necessary to generate the energy needed for synaptic signaling and plasticity. Therefore, an effective mechanism to transport and anchor mitochondria to pre- and post-synaptic terminals is as important as functional mitochondria in neuronal firing. Mitochondrial movement range is regulated by phosphorylation of cytoskeletal and motor proteins in addition to changes in mitochondrial membrane potential. Movement direction is regulated by serotonin and dopamine levels. However, data on mitochondrial movement defects and their involvement in defective signaling and neuroplasticity in relationship with mood disorders is scarce. We have previously reported the effects of lithium, valproate and a new antipsychotic, paliperidone on protein expression levels at the synaptic level. HYPOTHESIS: Mitochondrial function defects have recently been implicated in schizophrenia and bipolar disorder. We postulate that mood stabilizer treatment has a profound effect on mitochondrial function, synaptic plasticity, mitochondrial migration and direction of movement. METHODS: Synaptoneurosomal preparations from rat pre-frontal cortex were obtained after 28 daily intraperitoneal injections of lithium, valproate and paliperidone. Phosphorylated proteins were identified using 2D-DIGE and nano LC-ESI tandem mass spectrometry. RESULTS: Lithium, valproate and paliperidone had a substantial and common effect on the phosphorylation state of specific actin, tubulin and myosin isoforms as well as other proteins associated with neurofilaments. Furthermore, different subunits from complex III and V of the electron transfer chain were heavily phosphorylated by treatment with these drugs indicating selective phosphorylation. CONCLUSIONS: Mood stabilizers have an effect on mitochondrial function, mitochondrial movement and the direction of this movement. The implications of these findings will contribute to novel insights regarding clinical treatment and the mode of action of these drugs.

Asunto(s)

Afecto/efectos de los fármacos , Antipsicóticos/farmacología , Fosfoproteínas/metabolismo , Corteza Prefrontal/citología , Proteoma/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Actinas/metabolismo , Animales , Línea Celular Tumoral , Isoxazoles/farmacología , Litio/farmacología , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Palmitato de Paliperidona , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Factores de Tiempo , Tubulina (Proteína)/metabolismo , Ácido Valproico/farmacología

RESUMEN

In an effort to study the mode of action of Cry11Ba, we identified toxin binding proteins in Anopheles gambiae larval midgut and investigated their receptor roles. Previously, an aminopeptidase (AgAPN2) and an alkaline phosphatase (AgALP1) were identified as receptors for Cry11Ba toxin in A. gambiae. However, an A. gambiae cadherin (AgCad1) that bound Cry11Ba with low affinity (K(d) = 766 nM) did not support a receptor role of AgCad1 for Cry11Ba. Here, we studied a second A. gambiae cadherin (AgCad2) that shares 14% identity to AgCad1. Immunohistochemical study showed that the protein is localized on A. gambiae larval midgut apical membranes. Its cDNA was cloned and the protein was analyzed as a transmembrane protein containing 14 cadherin repeats. An Escherichia coli expressed CR14MPED fragment of AgCad2 bound Cry11Ba with high affinity (K(d) = 11.8 nM), blocked Cry11Ba binding to A. gambiae brush border vesicles and reduced Cry11Ba toxicity in bioassays. Its binding to Cry11Ba could be completely competed off by AgCad1, but only partially competed by AgALP1. The results are evidence that AgCad2 may function as a receptor for Cry11Ba in A. gambiae larvae.

Asunto(s)

Anopheles/crecimiento & desarrollo , Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Cadherinas/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insectos/metabolismo , Larva/metabolismo , Secuencia de Aminoácidos , Animales , Anopheles/química , Anopheles/genética , Anopheles/metabolismo , Bacillus thuringiensis/química , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cadherinas/química , Cadherinas/genética , Endotoxinas/química , Endotoxinas/genética , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Proteínas de Insectos/química , Proteínas de Insectos/genética , Mucosa Intestinal/metabolismo , Intestinos/química , Cinética , Larva/química , Larva/genética , Larva/crecimiento & desarrollo , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas

RESUMEN

The gut and Malpighian tubules of insects are the primary sites of active solute and water transport for controlling hemolymph and urine composition, pH, and osmolarity. These processes depend on ATPase (pumps), channels and solute carriers (Slc proteins). Maturation of genomic databases enables us to identify the putative molecular players for these processes. Anion transporters of the Slc4 family, AE1 and NDAE1, have been reported as HCO(3)(-) transporters, but are only part of the story. Here we report Dipteran (Drosophila melanogaster (d) and Anopheles gambiae (Ag)) anion exchangers, belonging to the Slc26 family, which are multi-functional anion exchangers. One Drosophila and two Ag homologues of mammalian Slc26a5 (Prestin) and Slc26a6 (aka, PAT1, CFEX) were identified and designated dPrestin, AgPrestinA and AgPrestinB. dPrestin and AgPrestinB show electrogenic anion exchange (Cl(-)/nHCO(3)(-), Cl(-)/SO(4)(2-) and Cl(-)/oxalate(2-)) in an oocyte expression system. Since these transporters are the only Dipteran Slc26 proteins whose transport is similar to mammalian Slc26a6, we submit that Dipteran Prestin are functional and even molecular orthologues of mammalian Slc26a6. OSR1 kinase increases dPrestin ion transport, implying another set of physiological processes controlled by WNK/SPAK signaling in epithelia. All of these mRNAs are highly expressed in the gut and Malpighian tubules. Dipteran Prestin proteins appear suited for central roles in bicarbonate, sulfate and oxalate metabolism including generating the high pH conditions measured in the Dipteran midgut lumen. Finally, we present and discuss Drosophila genetic models that integrate these processes.

Asunto(s)

Anopheles/metabolismo , Antiportadores de Cloruro-Bicarbonato/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Animales , Anopheles/genética , Antiportadores de Cloruro-Bicarbonato/genética , Drosophila/genética , Proteínas de Drosophila/genética , Transporte Iónico , Filogenia , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Xenopus

RESUMEN

Brush border membrane vesicles from whole Aedes aegypti larvae (AaBBMVw) are confirmed to be valid preparations for membrane transport studies. The Abdul-Rauf and Ellar method was used to isolate AaBBMVw that were frozen, stored for several months, transported to a distant site, thawed and used to study Na(+)-coupled, (3)H-labeled, phenylalanine (Phe) uptake. The affinity for all components of the uptake was very high with half maximal values in the sub-micromolar range. By contrast a K(0.5)(Phe) of 0.2mM and a K(0.5)(Na) of 26 mM were calculated from Phe-induced electrical currents in Xenopus oocytes that were heterologously expressing the Anopheles gambiae symporter (co-transporter), AgNAT8, in a buffer with 98 mM Na(+). What accounts for the >1000-fold discrepancy in affinity for substrates between the BBMV and oocyte experiments? Is it because Ae. aegypti were used to isolate BBMVw whereas An. gambiae were used to transfect oocytes? More likely, it is because BBMVw were exposed to [Na(+)] in the micromolar range with the transporter(s) being surrounded by native lipids. By contrast, the oocyte measurements were made at [Na(+)] 100,000 times higher with AgNAT8 surrounded by foreign frog lipids. The results show that AaBBMVw are osmotically sealed; the time-course has a Na(+)-induced overshoot, the pH optimum is ∼7 and the K(0.5) values for Phe and Na(+) are very low. The transport is virtually unchanged when Na(+) is replaced by K(+) or Li(+) but decreased by Rb(+). This approach to resolving discrepancies between electrical data on solute transporters such as AgNAT8 that are over-expressed in oocytes and flux data on corresponding transporters that are highly expressed in native membrane vesicles, may serve as a model for similar studies that add membrane biochemistry to molecular biology in efforts to identify targets for the development of new methods to control disease-vector mosquitoes.

Asunto(s)

Aedes/metabolismo , Microvellosidades/metabolismo , Fenilalanina/metabolismo , Vesículas Transportadoras/metabolismo , Aedes/ultraestructura , Animales , Concentración de Iones de Hidrógeno , Cinética , Larva/metabolismo , Larva/ultraestructura , Ósmosis , Sodio/metabolismo , Cloruro de Sodio/metabolismo , Tritio , Xenopus

RESUMEN

Mosquito larvae exhibit luminal pH extremes along the axial length of their alimentary canal that range from very alkaline (pH>10) in the anterior midgut to slightly acid in the hindgut. The principal buffer in the system is thought to be bicarbonate and/or carbonate, because the lumen is known to contain high levels of bicarbonate/carbonate and is surrounded by various epithelial cell types which express a variety of carbonic anhydrases. However, the precise mechanisms responsible for the transport of bicarbonate/carbonate into and out of the lumen are unclear. In the present study, we test the hypothesis that SLC4-like anion transporters play a role in bicarbonate/carbonate accumulation in the larval mosquito alimentary canal. Molecular, physiological and immnuohistochemical characterizations of Slc4-like transporters in the gut of larval mosquitoes (Aedes aegypti and Anopheles gambiae) demonstrate the presence of both a Na(+)-independent chloride/bicarbonate anion exchanger (AE) as well as a Na(+)-dependent anion exchanger (NDAE). Notably, immunolocalization experiments in Malpighian tubules show that the two proteins can be located in the same tissue, but to different cell types. Immunolabeling experiments in the gastric caecae show that the two proteins can be found in the same cells, but on opposite sides (basal vs. apical). In summary, our results indicate that the alimentary canal of larval mosquitoes exhibits robust expression of two SLC4-like transporters in locations that are consistent with a role in the regulation of luminal pH. The precise physiological contributions of each transporter remain to be determined.

Asunto(s)

Aedes/metabolismo , Proteínas de Transporte de Anión/metabolismo , Anopheles/metabolismo , Antiportadores de Cloruro-Bicarbonato/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Transporte de Anión/genética , Anopheles/genética , Antiportadores de Cloruro-Bicarbonato/genética , Femenino , Tracto Gastrointestinal/metabolismo , Larva/metabolismo , Datos de Secuencia Molecular , Xenopus