RESUMEN
L-type amino acid transporter 1 (LAT1) is specifically expressed in many malignancies, contributes to the transport of essential amino acids, such as leucine, and regulates the mammalian target of rapamycin (mTOR) signaling pathway. We investigated the expression profile and functional role of LAT1 in prostate cancer using JPH203, a specific inhibitor of LAT1. LAT1 was highly expressed in castration-resistant prostate cancer (CRPC) cells, including C4-2 and PC-3 cells, but its expression level was low in castration-sensitive LNCaP cells. JPH203 significantly inhibited [14C] leucine uptake in CRPC cells but had no effect in LNCaP cells. JPH203 inhibited the proliferation, migration, and invasion of CRPC cells but not of LNCaP cells. In C4-2 cells, Cluster of differentiation (CD) 24 was identified by RNA sequencing as a novel downstream target of JPH203. CD24 was downregulated in a JPH203 concentration-dependent manner and suppressed activation of the Wnt/ß-catenin signaling pathway. Furthermore, an in vivo study showed that JPH203 inhibited the proliferation of C4-2 cells in a castration environment. The results of this study indicate that JPH203 may exert its antitumor effect in CRPC cells via mTOR and CD24.
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Antígeno CD24 , Movimiento Celular , Proliferación Celular , Transportador de Aminoácidos Neutros Grandes 1 , Neoplasias de la Próstata Resistentes a la Castración , Masculino , Humanos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Línea Celular Tumoral , Animales , Proliferación Celular/efectos de los fármacos , Antígeno CD24/metabolismo , Ratones , Movimiento Celular/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Benzoxazoles/farmacología , Leucina/farmacología , Leucina/análogos & derivados , Ratones Desnudos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Tirosina/análogos & derivadosRESUMEN
BACKGROUND: Degenerative cervical myelopathy (DCM) is the most common cause of chronic, progressive spinal cord impairment worldwide. Patients experience substantial pain, functional neurological decline and disability. Health-related quality of life (HRQoL) appears to be particularly poor, even when compared to other chronic diseases. However, the determinants of HRQoL are poorly understood. The objective was to perform a systematic review of the determinants of quality of life of people with DCM. METHODS: A systematic search was conducted in MEDLINE and Embase following PRISMA 2020 guidelines (PROSPERO CRD42018115675). Full-text papers in English, exclusively studying DCM, published before 26 March 2020 were eligible for inclusion and were assessed using the Newcastle-Ottawa Scale and the Cochrane Risk of Bias 2 (RoB 2) tool. Study sample characteristics, patient demographics, cohort type, HRQoL instrument utilised, HRQoL score, and relationships of HRQoL with other variables were qualitatively synthesised. RESULTS: A total of 1176 papers were identified; 77 papers and 13,572 patients were included in the final analysis. A total of 96% of papers studied surgical cohorts and 86% utilised the 36-Item Short Form Survey (SF-36) as a measure of HRQoL. HRQoL determinants were grouped into nine themes. The most common determinant to be assessed was surgical technique (38/77, 49%) and patient satisfaction and experience of pain (10/77, 13%). HRQoL appeared to improve after surgery. Pain was a negative predictor of HRQoL. CONCLUSION: Current data on the determinants of HRQoL in DCM are limited, contradictory and heterogeneous. Limitations of this systematic review include lack of distinction between DCM subtypes and heterogenous findings amongst the papers in which HRQoL is measured postoperatively or post-diagnosis. This highlights the need for greater standardisation in DCM research to allow further synthesis. Studies of greater precision are necessary to account for HRQoL being complex, multi-factorial and both time and context dependent.
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Calidad de Vida , Enfermedades de la Médula Espinal , Humanos , Vértebras Cervicales/cirugía , Enfermedades de la Médula Espinal/cirugía , Enfermedades de la Médula Espinal/diagnóstico , Cuello , Satisfacción del PacienteRESUMEN
Ischemic tolerance is a phenomenon in which resistance to subsequent invasive ischemia is acquired by a preceding noninvasive ischemic application, and is observed in many organs, including the brain, the organ most vulnerable to ischemic insult. To date, much research has been conducted on cerebral ischemic tolerance as a cell-autonomous action of neurons. In this article, we review the essential roles of microglia and astrocytes in the acquisition of ischemic tolerance through neuron-non-autonomous mechanisms, where the two types of glial cells function in a concerted manner to induce ischemic tolerance.
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Isquemia Encefálica , Precondicionamiento Isquémico , Astrocitos/fisiología , Humanos , Isquemia , Microglía/fisiologíaRESUMEN
Currently, the emergence of drug resistance is an important issue in the treatment of hepatitis B virus (HBV). Recently, our collaborating group developed a novel long-acting anti-HBV drug, E-CFCP. However, until this study, the effects of E-CFCP in the kidney have remained unclarified. Using cell viability and uptake assays, we examined the effects of E-CFCP on the function of renal organic anion transporters (OATs). No cytotoxicity was shown related to the E-CFCP in the renal OATs in either assay. Thus, this study suggested that E-CFCP may be a novel, excellent candidate drug for the treatment of drug-resistant HBV.
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Hepatitis B , Transportadores de Anión Orgánico , Humanos , Hepatitis B/tratamiento farmacológico , Virus de la Hepatitis B , Riñón , Proteínas de Transporte de Membrana , Antivirales/farmacología , Antivirales/uso terapéutico , Farmacorresistencia ViralRESUMEN
Amino acid transporters are responsible for the uptake of amino acids, critical for cell proliferation. L-type amino acid transporters play a major role in the uptake of essential amino acids. L-type amino acid transporter 1 (LAT1) exerts its functional properties by forming a dimer with 4F2hc. Utilizing this cancer-specificity, research on diagnostic imaging and therapeutic agents for malignant tumors targeting LAT1 progresses in various fields. In hormone-sensitive prostate cancer, the up-regulation of L-type amino acid transporter 3 (LAT3) through the androgen receptor (AR) has been identified. On the other hand, in castration-resistant prostate cancer, the negative regulation of LAT1 through AR has been determined. Furthermore, 4F2hc: a binding partner of LAT1, was identified as the specific downstream target of Androgen Receptor Splice Variant 7: AR-V7. LAT1 has been suggested to contribute to acquiring castration resistance in prostate cancer, making LAT1 a completely different therapeutic target from anti-androgens and taxanes. Increased expression of LAT1 has also been found in renal and bladder cancers, suggesting a contribution to acquiring malignancy and progression. In Japan, clinical trials of LAT1 inhibitors for solid tumors are in progress, and clinical applications are now underway. This article will summarize the relationship between LAT1 and urological malignancies.
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Transportador de Aminoácidos Neutros Grandes 1 , Neoplasias de la Próstata , Neoplasias Urológicas , Humanos , Masculino , Sistemas de Transporte de Aminoácidos , Transportador de Aminoácidos Neutros Grandes 1/genética , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Receptores Androgénicos/genética , Neoplasias Urológicas/tratamiento farmacológico , Neoplasias Urológicas/genéticaRESUMEN
BACKGROUND: AO Spine RECODE-DCM (Research objectives and common data elements for degenerative cervical myelopathy) has highlighted that the subjective disability reported by people living with DCM is much broader than routinely considered today by most professionals. This includes a description of tremor. The objective of this review was to study the incidence and possible aetiology of tremor in degenerative cervical myelopathy (DCM). METHODS: A systematic review registered in PROSPERO (CRD42020176905) was conducted in Embase and MEDLINE for papers studying tremor and DCM published on or before the 20th of July 2020. All manuscripts describing an association between tremor and DCM in humans were included. Articles relating to non-human animals, and those not available in English were excluded. An analysis was conducted in accordance with PRISMA and SWiM guidelines for systematic reviews. RESULTS: Out of a total of 4402 screened abstracts, we identified 7 case reports and series describing tremor in 9 DCM patients. Papers were divided into three groups for the discussion. The first group includes DCM correctly identified on presentation, with tremor as a described symptom. The second group includes cases where DCM was misdiagnosed, often as Parkinson's disease. The third group includes a single case with a previous history of DCM, presenting with an otherwise unexplained tremor. This grouping allows for the clustering of cases supporting various arguments for the association between tremor and DCM. CONCLUSION: DCM can be associated with tremor. The current evidence is restricted to case series. Further study is warranted to establish tremor prevalence, and its significance to assessment and management.
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Vértebras Cervicales , Enfermedades de la Médula Espinal , Humanos , Incidencia , Cuello , Enfermedades de la Médula Espinal/diagnóstico , Temblor/etiologíaRESUMEN
A sub-lethal ischemic episode (preconditioning [PC]) protects neurons against a subsequent lethal ischemic injury. This phenomenon is known as ischemic tolerance. PC itself does not cause brain damage, but affects glial responses, especially astrocytes, and transforms them into an ischemia-resistant phenotype. P2X7 receptors (P2X7Rs) in astrocytes play essential roles in PC. Although P2X7Rs trigger inflammatory and toxic responses, PC-induced P2X7Rs in astrocytes function as a switch to protect the brain against ischemia. In this review, we focus on P2X7Rs and summarize recent developments on how astrocytes control P2X7Rs and what molecular mechanisms they use to induce ischemic tolerance.
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Astrocitos , Isquemia Encefálica , Isquemia Encefálica/genética , Humanos , Isquemia , Neuronas , Receptores Purinérgicos P2X7/genéticaRESUMEN
We previously showed that noninvasive mild ischemia (preconditioning; PC) induced ischemic tolerance by upregulation of P2X7 receptors in astrocytes via a hypoxia inducible factor-1α (HIF-1α)-dependent mechanism. The P2X7 receptor is known as a low-sensitivity P2 receptor that requires a high extracellular ATP (eATP) concentration for activation. PC increased the eATP level but was not sufficient to activate P2X7 receptors. Here, we show that astrocytes possess an elaborate mechanism for activation of P2X7 receptors, thus contributing to ischemic tolerance. Nicotinamide adenine dinucleotide (NAD+ ) was shown to increase the sensitivity of P2X7 receptors to eATP via ecto-ADP-ribosyltransferase 2 (ARTC2)-catalyzed ADP-ribosylation in peripheral immune cells. Although ARTC2-positive signals were mostly absent in the naïve brain, they were selectively increased in astrocytes by PC. The spatiotemporal pattern of PC-evoked ARTC2 was well associated with that of P2X7 receptors. In the in vitro experiments, NAD+ increased the sensitivity of P2X7 receptors to ATP, and at higher concentrations, NAD+ itself activated P2X7 receptors without eATP in cultured astrocytes. In the in vivo experiments using middle cerebral artery occlusion model mice, the PC-evoked increase in HIF-1α in astrocytes was abolished by the ARTC2 inhibitor S + 16a. S + 16a also abolished PC-evoked ischemic tolerance. Taken together, the results suggested that P2X7 receptors can be sensitized to ATP by NAD+ /ARTC2-catalyzed ADP-ribosylation, which allows astrocytes to drive P2X7 receptor-mediated ischemic tolerance even though PC only slightly increases the amount of eATP.
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Astrocitos , Receptores Purinérgicos P2X7 , ADP Ribosa Transferasas/metabolismo , Adenosina Trifosfato , Animales , Astrocitos/metabolismo , Células Cultivadas , Infarto de la Arteria Cerebral Media , Ratones , NAD/metabolismoRESUMEN
Metabotropic glutamate receptor 5 (mGluR5) in astrocytes is a key molecule for controlling synapse remodeling. Although mGluR5 is abundant in neonatal astrocytes, its level is gradually down-regulated during development and is almost absent in the adult. However, in several pathological conditions, mGluR5 re-emerges in adult astrocytes and contributes to disease pathogenesis by forming uncontrolled synapses. Thus, controlling mGluR5 expression in astrocyte is critical for several diseases, but the mechanism that regulates mGluR5 expression remains unknown. Here, we show that adenosine triphosphate (ATP)/adenosine-mediated signals down-regulate mGluR5 in astrocytes. First, in situ Ca2+ imaging of astrocytes in acute cerebral slices from post-natal day (P)7-P28 mice showed that Ca2+ responses evoked by (S)-3,5-dihydroxyphenylglycine (DHPG), a mGluR5 agonist, decreased during development, whereas those evoked by ATP or its metabolite, adenosine, increased. Second, ATP and adenosine suppressed expression of the mGluR5 gene, Grm5, in cultured astrocytes. Third, the decrease in the DHPG-evoked Ca2+ responses was associated with down-regulation of Grm5. Interestingly, among several adenosine (P1) receptor and ATP (P2) receptor genes, only the adenosine A2B receptor gene, Adora2b, was up-regulated in the course of development. Indeed, we observed that down-regulation of Grm5 was suppressed in Adora2b knockout astrocytes at P14 and in situ Ca2+ imaging from Adora2b knockout mice indicated that the A2B receptor inhibits mGluR5 expression in astrocytes. Furthermore, deletion of A2B receptor increased the number of excitatory synapse in developmental stage. Taken together, the A2B receptor is critical for down-regulation of mGluR5 in astrocytes, which would contribute to terminate excess synaptogenesis during development.
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Astrocitos , Receptor de Adenosina A2B , Receptor del Glutamato Metabotropico 5 , Adenosina/metabolismo , Adenosina/farmacología , Animales , Astrocitos/metabolismo , Proteínas Portadoras/metabolismo , Ratones , Receptor de Adenosina A2B/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismoRESUMEN
Islatravir (ISL; 4'-ethynyl-2-fluoro-2'-deoxyadenosine or EFdA) is a novel reverse transcriptase translocation inhibitor and has a unique structure and high antiviral activity against wild-type and multidrug resistant HIV strains. In this study, we investigated whether islatravir (ISL) can cause kidney damage compared to tenofovir disoproxil fumarate (TDF) and tenofovir (TFV). We also investigated interactions of these drugs with organic anion transporters (OATs). There is a large gap in ISL concentration between the pharmacological dose to proximal tubular cells and the clinical dose. ISL is unlikely to be taken up via OAT1 or OAT3; therefore, OAT1 and OAT3 may not be involved in the injury to tubular cells. Present data strongly suggests that ISL is not toxic to proximal tubules because blood levels of ISL are not high enough to cause kidney damage in the clinical setting.
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Desoxiadenosinas/efectos adversos , Desoxiadenosinas/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Transportadores de Anión Orgánico/metabolismo , Inhibidores de la Transcriptasa Inversa/efectos adversos , Inhibidores de la Transcriptasa Inversa/metabolismo , Lesión Renal Aguda/etiología , Células Cultivadas , Desoxiadenosinas/sangre , Relación Dosis-Respuesta a Droga , HumanosRESUMEN
Hyaluronan is synthesized, secreted, and anchored by hyaluronan synthases (HAS) at the plasma membrane and comprises the backbone of perineuronal nets around neuronal soma and dendrites. However, the molecular targets of hyaluronan to regulate synaptic transmission in the central nervous system have not been fully identified. Here, we report that hyaluronan is a negative regulator of excitatory signals. At excitatory synapses, glutamate is removed by glutamate transporters to turn off the signal and prevent excitotoxicity. Hyaluronan synthesized by HAS supports the activity of glial glutamate transporter 1 (GLT1). GLT1 also retracted from cellular processes of cultured astrocytes after hyaluronidase treatment and hyaluronan synthesis inhibition. A serial knockout study showed that all three HAS subtypes recruit GLT1 to cellular processes. Furthermore, hyaluronidase treatment activated neurons in a dissociated rat hippocampal culture and caused neuronal damage due to excitotoxicity. Our findings reveal that hyaluronan helps to turn off excitatory signals by supporting glutamate clearance. Cover Image for this issue: doi: 10.1111/jnc.14516.
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Sistema de Transporte de Aminoácidos X-AG/metabolismo , Encéfalo/metabolismo , Ácido Hialurónico/biosíntesis , Transmisión Sináptica/fisiología , Animales , Astrocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
AIMS: To investigate circadian gene expressions in the mouse bladder urothelium to establish an experimental model and study the functions of the circadian rhythm. METHODS: The gene expression rhythms of the clock genes, mechano-sensors such as Piezo1 and TRPV4, ATP release mediated molecules (ARMM) such as Cx26 and VNUT were investigated in mouse primary cultured urothelial cells (UCs) of wild-type (WT) and Clock mutant (ClockΔ19/Δ19 ) mice using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and western blotting analysis. The long-term oscillation of the clock genes in UC was investigated by measuring bioluminescence from UC isolated from Period2luciferase knock-in mice (Per2::luc) and Per2::luc with ClockΔ19/Δ19 using a luminometer. The mRNA expression rhythms after treatment with Clock short interfering RNA (siRNA) were also measured to compare differences between Clock point mutations and Clock deficiency. RESULTS: The UCs from WT mice showed the time-dependent gene expressions for clock genes, mechano-sensors, and ARMM. The abundances of the products of these genes also correlated with the mRNA expression rhythms in UCs. The bioluminescence of Per2::Luc in UCs showed a circadian rhythm. By contrast, all the gene expressions rhythms observed in WT mice were abrogated in the ClockΔ19/Δ19 mice. Transfection with Clock siRNA in UCs had the same effect as the Clock mutation. CONCLUSIONS: We demonstrated that the time-dependent gene expressions, including clock genes, mechano-sensors, and ARMM, were reproducible in UCs. These findings demonstrated that UCs have the potential to progress research into the circadian functions of the lower urinary tract regulated by clock genes.
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Proteínas CLOCK/metabolismo , Conexina 26/metabolismo , Canales Iónicos/metabolismo , Proteínas de Transporte de Nucleótidos/metabolismo , Canales Catiónicos TRPV/metabolismo , Urotelio/metabolismo , Animales , Proteínas CLOCK/genética , Células Cultivadas , Ritmo Circadiano/genética , Conexina 26/genética , Expresión Génica , Canales Iónicos/genética , Ratones , Proteínas de Transporte de Nucleótidos/genética , Canales Catiónicos TRPV/genética , Urotelio/citologíaRESUMEN
We recently demonstrated that ischemic tolerance was dependent on astrocytes, for which HIF-1α had an essential role. The mild ischemia (preconditioning; PC) increased HIF-1α in a biphasic pattern, that is, a quick and transient increase in neurons, followed by a slow and sustained increase in astrocytes. However, mechanisms underlying such temporal difference in HIF-1α increase remain totally unknown. Here, we show that unlike a hypoxia-dependent mechanism in neurons, astrocytes increase HIF-1α via a novel hypoxia-independent but P2X7-dependent mechanism. Using a middle cerebral artery occlusion (MCAO) model of mice, we found that the PC (a 15-min MCAO period)-evoked increase in HIF-1α in neurons was quick and transient (from 1 to 3 days after PC), but that in astrocytes was slow-onset and long-lasting (from 3 days to at least 2 weeks after PC). The neuronal HIF-1α increase was dependent on inhibition of PHD2, an oxygen-dependent HIF-1α degrading enzyme, whereas astrocytic one was independent of PHD2. Astrocytes even do not possess this enzyme. Instead, they produced a sustained increase in P2X7 receptors, activation of which resulted in HIF-1α increase. The hypoxia-independent but P2X7-receptor-dependent mechanism could allow astrocytes to cause long-lasting HIF-1α expression, thereby leading to induction of ischemic tolerance efficiently. GLIA 2017;65:523-530.
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Astrocitos/metabolismo , Encéfalo/patología , Regulación de la Expresión Génica/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Infarto de la Arteria Cerebral Media/patología , Precondicionamiento Isquémico/métodos , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Astrocitos/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Lateralidad Funcional/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Receptores Purinérgicos P2X7/deficiencia , Receptores Purinérgicos P2X7/genéticaRESUMEN
AIMS: The pathophysiologies of nocturia (NOC) and nocturnal polyuria (NP) are multifactorial and their etiologies remain unclear in a large number of patients. Clock genes exist in most cells and organs, and the products of Clock regulate circadian rhythms as representative clock genes. Clock genes regulate lower urinary tract function, and a newly suggested concept is that abnormalities in clock genes cause lower urinary tract symptoms. In the present study, we investigated the voiding behavior of Clock mutant (ClockΔ19/Δ19 ) mice in order to determine the effects of clock genes on NOC/NP. METHODS: Male C57BL/6 mice aged 8-12 weeks (WT) and male C57BL/6 ClockΔ19/Δ19 mice aged 8 weeks were used. They were bred under 12 hr light/dark conditions for 2 weeks and voiding behavior was investigated by measuring water intake volume, urine volume, urine volume/void, and voiding frequency in metabolic cages in the dark and light periods. RESULTS: No significant differences were observed in behavior patterns between ClockΔ19/Δ19 and WT mice. ClockΔ19/Δ19 mice showed greater voiding frequencies and urine volumes during the sleep phase than WT mice. The diurnal change in urine volume/void between the dark and light periods in WT mice was absent in ClockΔ19/Δ19 mice. Additionally, functional bladder capacity was significantly lower in ClockΔ19/Δ19 mice than in WT mice. CONCLUSIONS: We demonstrated that ClockΔ19/Δ19 mice showed the phenotype of NOC/NP. The ClockΔ19/Δ19 mouse may be used as an animal model of NOC and NP. Neurourol. Urodynam. 36:1034-1038, 2017. © 2016 Wiley Periodicals, Inc.
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Proteínas CLOCK/genética , Ritmo Circadiano/genética , Nocturia/genética , Poliuria/genética , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Preconditioning (PC) using a preceding sublethal ischemic insult is an attractive strategy for protecting neurons by inducing ischemic tolerance in the brain. Although the underlying molecular mechanisms have been extensively studied, almost all studies have focused on neurons. Here, using a middle cerebral artery occlusion model in mice, we show that astrocytes play an essential role in the induction of brain ischemic tolerance. PC caused activation of glial cells without producing any noticeable brain damage. The spatiotemporal pattern of astrocytic, but not microglial, activation correlated well with that of ischemic tolerance. Interestingly, such activation in astrocytes lasted at least 8 weeks. Importantly, inhibiting astrocytes with fluorocitrate abolished the induction of ischemic tolerance. To investigate the underlying mechanisms, we focused on the P2X7 receptor as a key molecule in astrocyte-mediated ischemic tolerance. P2X7 receptors were dramatically upregulated in activated astrocytes. PC-induced ischemic tolerance was abolished in P2X7 receptor knock-out mice. Moreover, our results suggest that hypoxia-inducible factor-1α, a well known mediator of ischemic tolerance, is involved in P2X7 receptor-mediated ischemic tolerance. Unlike previous reports focusing on neuron-based mechanisms, our results show that astrocytes play indispensable roles in inducing ischemic tolerance, and that upregulation of P2X7 receptors in astrocytes is essential.
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Astrocitos/patología , Isquemia Encefálica/patología , Animales , Astrocitos/metabolismo , Eritropoyetina/biosíntesis , Eritropoyetina/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Infarto de la Arteria Cerebral Media/patología , Ataque Isquémico Transitorio/patología , Precondicionamiento Isquémico , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/fisiología , Receptores Purinérgicos P2X7/biosíntesis , Receptores Purinérgicos P2X7/genéticaRESUMEN
Previously, we demonstrated that when mesenchymal stem cells (MSCs) from mouse ES cells were transplanted into skeletal muscle, more than 60% of them differentiated into muscles in the crush-injured tibialis anterior muscle in vivo, although MSCs neither differentiated nor settled in the intact muscle. Microenvironments, including the extracellular matrix between the injured and intact muscle, were quite different. In the injured muscle, hyaluronan (HA), heavy chains of inter-α-inhibitor (IαI), CD44, and TNF-α-stimulated gene 6 product (TSG-6) increased 24-48 h after injury, although basement membrane components of differentiated muscle such as perlecan, laminin, and type IV collagen increased gradually 4 days after the crush. We then investigated the microenvironments crucial for cell transplantation, using the lysate of C2C12 myotubules for mimicking injured circumstances in vivo. MSCs settled in the intact muscle when they were transplanted together with the C2C12 lysate or TSG6. MSCs produced and released TSG6 when they were cultured with C2C12 lysates in vitro. MSCs pretreated with the lysate also settled in the intact muscle. Furthermore, MSCs whose TSG6 was knocked down by shRNA, even if transplanted or pretreated with the lysate, could not settle in the muscle. Immunofluorescent staining showed that HA and IαI always co-localized or were distributed closely, suggesting formation of covalent complexes, i.e. the SHAP-HA complex in the presence of TSG6. Thus, TSG6, HA, and IαI were crucial factors for the settlement and probably the subsequent differentiation of MSCs.
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Moléculas de Adhesión Celular/biosíntesis , Diferenciación Celular , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Nicho de Células Madre , Animales , Moléculas de Adhesión Celular/genética , Línea Celular , Técnicas de Cocultivo , Células Madre Mesenquimatosas/citología , Ratones , Fibras Musculares Esqueléticas/citologíaRESUMEN
The present study was conducted to examine the feasibility of in vitro embryo production and transfer technologies for producing Middle White piglets. After collection from three retired Middle White sows, a total of 222 oocytes were matured, fertilized and cultured in vitro, and a total of 50 embryos from the 4-cell to blastocyst stage were produced by the 4th or 5th day. These embryos were transferred individually into three recipients along with 5 in vivo-derived Duroc blastocysts. All of the recipients became pregnant, and they farrowed a total of 9 Middle White and 9 Duroc piglets. These results suggest that in vitro embryo production using ovaries from retired sows is useful for reproduction of pigs of pure breeds including the Middle White for breeding activities and conservation/utilization of genetic resources.
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Transferencia de Embrión/veterinaria , Embrión de Mamíferos/citología , Fertilización In Vitro/veterinaria , Porcinos , Animales , Animales Recién Nacidos , Cruzamiento/métodos , Técnicas de Cultivo de Embriones/veterinaria , Estudios de Factibilidad , Femenino , Masculino , Embarazo , Porcinos/embriología , Porcinos/fisiologíaRESUMEN
A sublethal ischemic episode [termed preconditioning (PC)] protects neurons in the brain against a subsequent severe ischemic injury. This phenomenon is known as brain ischemic tolerance and has received much attention from researchers because of its robust neuroprotective effects. We have previously reported that PC activates astrocytes and subsequently upregulates P2X7 receptors, thereby leading to ischemic tolerance. However, the downstream signals of P2X7 receptors that are responsible for PC-induced ischemic tolerance remain unknown. Here, we show that PC-induced P2X7 receptor-mediated lactate release from astrocytes has an indispensable role in this event. Using a transient focal cerebral ischemia model caused by middle cerebral artery occlusion, extracellular lactate levels during severe ischemia were significantly increased in mice who experienced PC; this increase was dependent on P2X7 receptors. In addition, the intracerebroventricular injection of lactate protected against cerebral ischemic injury. In in vitro experiments, although stimulation of astrocytes with the P2X7 receptor agonist BzATP had no effect on the protein levels of monocarboxylate transporter (MCT) 1 and MCT4 (which are responsible for lactate release from astrocytes), BzATP induced the plasma membrane translocation of these MCTs via their chaperone CD147. Importantly, CD147 was increased in activated astrocytes after PC, and CD147-blocking antibody abolished the PC-induced facilitation of astrocytic lactate release and ischemic tolerance. Taken together, our findings suggest that astrocytes induce ischemic tolerance via P2X7 receptor-mediated lactate release.
Asunto(s)
Astrocitos , Precondicionamiento Isquémico , Ácido Láctico , Ratones Endogámicos C57BL , Transportadores de Ácidos Monocarboxílicos , Receptores Purinérgicos P2X7 , Animales , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Precondicionamiento Isquémico/métodos , Ácido Láctico/metabolismo , Ácido Láctico/farmacología , Receptores Purinérgicos P2X7/metabolismo , Masculino , Transportadores de Ácidos Monocarboxílicos/metabolismo , Basigina/metabolismo , Isquemia Encefálica/metabolismo , Simportadores/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Modelos Animales de Enfermedad , Proteínas Musculares/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Ratones , Células Cultivadas , Encéfalo/metabolismo , Ratones NoqueadosRESUMEN
This study was conducted to clarify the feasibility of newly developed vitrification techniques for porcine embryos using the micro volume air cooling (MVAC) method without direct contact with liquid nitrogen (LN2). Expanded blastocysts were vitrified in a solution containing 6 M ethylene glycol, 0.6 M trehalose and 2% (wt/vol) polyethylene glycol in 10% HEPES-buffered PZM-5. The blastocysts were collected from gilts and vitrified using the new device (MVAC) or a Cryotop (CT). Blastocysts were stored in LN2 for at least 1 month. After warming, cryoprotective agents were removed using a single step. Survival of the embryos was assessed by in vitro culture (Experiment 1) and by embryo transfer to recipients (Experiment 2). In Experiment 1, the embryos vitrified by the MVAC or CT and fresh embryos without vitrification (Control) were used. The survival rates of embryos in the MVAC, CT and Control groups were 88.9% (32/36), 91.7% (33/36) and 100% (34/34), respectively, after 48 h culture, and the hatching rates of embryos after 48 h incubation were 69.4% (25/36), 63.9% (23/36) and 94.1% (32/34), respectively. In Experiment 2, 64 vitrified embryos were transferred to 5 recipient gilts, and 8 healthy piglets were produced from 3 recipients in the MVAC group. Similarly, 66 vitrified embryos were transferred to 5 recipient gilts, and 9 healthy piglets were produced from 2 recipients in the CT group. These results indicated that porcine expanded blastocysts can be cryopreserved using the MVAC method without potential pathogen contamination from LN2.
Asunto(s)
Blastocisto , Criopreservación/veterinaria , Ectogénesis , Implantación del Embrión , Transferencia de Embrión/veterinaria , Sus scrofa/fisiología , Vitrificación , Animales , Animales Endogámicos , Blastocisto/efectos de los fármacos , Cruzamientos Genéticos , Criopreservación/instrumentación , Criopreservación/métodos , Crioprotectores/farmacología , Ectogénesis/efectos de los fármacos , Técnicas de Cultivo de Embriones/veterinaria , Implantación del Embrión/efectos de los fármacos , Glicol de Etileno/farmacología , Estudios de Factibilidad , Femenino , Inseminación Artificial/veterinaria , Japón , Nacimiento Vivo/veterinaria , Masculino , Polietilenglicoles/farmacología , Embarazo , Trehalosa/farmacologíaRESUMEN
Xenin is a 25-amino acid peptide identified in human gastric mucosa, which is widely expressed in peripheral and central tissues. It is known that the central or peripheral administration of xenin decreases food intake in rodents. Nesfatin-1/NUCB2 (nesfatin-1) has been identified as an anorexic neuropeptide, it is often found co-localized with many peptides in the central nervous system. After the intracerebroventricular administration of xenin on nesfain-1-like immunoreactivity (LI) neurons, we examined its effects on food intake and water intake in rats. As a result, Fos-LI neurons were observed in the organum vasculosum of the laminae terminalis (OVLT), the median preoptic nucleus (MnPO), the subfornical organ (SFO), the supraoptic nucleus (SON), the paraventricular nucleus (PVN), the arcuate nucleus (Arc), the lateral hypothalamic area (LHA), the central amygdaloid nucleus (CAN), the dorsal raphe nucleus (DR), the locus coeruleus (LC), the area postrema (AP) and the nucleus of the solitary tract (NTS). After the administration, the number of Fos-LI neurons was significantly increased in the LC and the OVLT, the MnPO, the SFO, the SON, the PVN, the Arc, the LHA, the CAN, the DR, the AP and the NTS, compared with the control group. After the administration of xenin, we conducted double immunohistochemistry for Fos and nesfatin-1, and found that the number of nesfatin-1-LI neurons expressing Fos were significantly increased in the SON, the PVN, the Arc, the LHA, the CAN, the DR, the AP and the NTS, compared with the control group. The pretreatment of nesfatin-1 antisense significantly attenuated this xenin-induced feeding suppression, while that of nesfatin-1 missense showed no improvement. These results indicate that central administered xenin may have anorexia effects associated with activated central nesfatin-1 neurons.