RESUMEN
BACKGROUND: CircMMP11 is a characterized circRNA with oncogenic function in breast cancer. In this study, we explored the involvement of circMMP11 in non-small cell lung cancer (NSCLC). METHODS: Paired cancer and non-cancer tissues were collected from 66 NSCLC patients, and the expression of circMMP11 and miR-143 in these tissues were detected using RT-qPCRs. Overexpression levels of circMMP11 and miR-143 were performed by transfection, and their crosstalk was analyzed by RT-qPCRs. The effect of circMMP11 overexpression on miR-143 methylation was analyzed by methylation-specific PCR. CCK-8 assay was performed to analyze the roles of miR-143 and circMMP11 in regulating NSCLC cell proliferation. RESULTS: We found that circMMP11 was overexpressed in NSCLC and predicted patients' poor survival. Moreover, a close correlation between circMMP11 and miR143 was observed. In NSCLC cells, circMMP11 overexpression reduced miR-143 expression and increased miR-143 methylation. CCK-8 assay analysis showed that miR-143 reversed the enhancing effects of circMMP11 overexpression on cell proliferation. CONCLUSIONS: CircMMP11 is overexpressed in NSCLC and predicts poor survival. In addition, circMMP11 may downregulate miR-143 through methylation to suppress cell proliferation.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , MicroARNs , ARN Complementario/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Proliferación Celular , Humanos , Neoplasias Pulmonares/genética , Metilación , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
Eight-segmented, negative-sense, single-stranded genomic RNAs of influenza A virus are terminated with 5' and 3' untranslated regions (UTRs). All segments have highly conserved extremities of 13 and 12 nucleotides at the 5' and 3' UTRs, respectively, constructing the viral RNA (vRNA) promoter. Adjacent to the duplex stem of 3 base pairs (bps) between the two conserved strands, additional 1-4 bps are existing in a segment-specific manner. We investigated the roles of the matrix (M) segment-specific base pair between the 14th nucleotide uridine (U14') of the 5' UTR and the 13th nucleotide adenosine (A13) of the 3' UTR by preparing possible vRNA promoters, named vXY, as well as cRNA promoters, named cYX. We analysed their RNA-dependent RNA replication efficiency using the minigenome replicon system and an enzyme assay system in vitro with synthetic RNA promoters. Notably, in contrast to vAC(s) that is a synthetic vRNA promoter with A14' and C13, base-pair disruption at the complementary RNA (cRNA) promoter in cAC(s), which has A13' and C14, not only reduced viral RNA replication in cells but also impaired de novo initiation of unprimed vRNA synthesis. Reverse genetics experiments confirmatively exhibited that this breakage in the cRNA promoter affected the rescue of infectious virus. The present study suggests that the first segment-specific base pair plays an essential role in generating infectious viruses by regulating the promoter activity of cRNA rather than vRNA. It could provide insights into the role of the segment-specific nucleotides in viral genome replication for sustainable infection.
Asunto(s)
Virus de la Influenza A/genética , ARN Complementario/genética , ARN Viral/genética , Regiones no Traducidas 3'/genética , Animales , Perros , Regulación Viral de la Expresión Génica , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Conformación de Ácido Nucleico , Nucleótidos/química , Nucleótidos/genética , Regiones Promotoras Genéticas/genética , Transcripción GenéticaRESUMEN
Influenza viruses encode a viral RNA-dependent RNA polymerase (FluPol), which is responsible for transcribing and replicating the negative-sense viral RNA (vRNA) genome. FluPol transcribes vRNA using a host-capped mRNA primer and replicates it by synthesizing a positive-sense cRNA intermediate, which is copied back into vRNA. To carry out these functions, FluPol interacts with vRNA and cRNA using conserved promoter elements at the 5' and 3' termini. Recent structural studies have identified a new surface binding site for the 3' vRNA and cRNA promoters on FluPol, referred to as the mode B site. However, the role of this binding site in FluPol function is unknown. In this study, we used a combination of cell-based and biochemical assays to show that the mode B site is important for both viral genome transcription and replication in influenza A virus. Furthermore, we show that the mode B site is not needed for initiating transcription in vitro but is required to synthesize a full-length product. This is consistent with a model in which the 3' terminus of the vRNA template binds in the mode B site during elongation. Our data provide the first functional insights into the role of the mode B site on FluPol, which advances our understanding of FluPol function and influenza virus replication.IMPORTANCE Influenza viruses are responsible for up to 650,000 deaths per year through seasonal epidemics, and pandemics have caused tens of millions of deaths in the past. Most current therapeutics suffer from widespread resistance, creating a need for new drug targets against influenza virus. The virus encodes an RNA-dependent RNA polymerase, which replicates and transcribes the vRNA genome. The polymerase interacts with vRNA and the complementary replicative intermediate cRNA using several specific binding sites; however, the functions associated with these binding sites remain unknown. Here, we functionally characterize a binding site for the 3' vRNA and cRNA promoters. Our data offer insight into the mechanism of viral genome transcription by the influenza virus polymerase and may be applicable to other related viruses.
Asunto(s)
Virus de la Influenza A/genética , Regiones Promotoras Genéticas/genética , ARN Polimerasa Dependiente del ARN/genética , Regiones no Traducidas 3'/genética , Sitios de Unión/genética , Genoma Viral/genética , Células HEK293 , Humanos , Virus de la Influenza A/metabolismo , Virus de la Influenza A/patogenicidad , Mutación/genética , Orthomyxoviridae/genética , Proteínas con Motivos de Reconocimiento de ARN , ARN Complementario/genética , ARN Complementario/metabolismo , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Transcripción Genética/genética , Replicación Viral/genéticaRESUMEN
The involvement of miRNAs in the pathogenesis of various diseases, including cancer, poses the need for developing miRNA inhibitors. Previously, using unmodified DNA, we designed LidNA, which inhibited miRNA function more potently than 2'-O-methylated RNA and locked nucleic acid. LidNA consists of a complementary sequence to miRNA flanked by two structured DNAs. Alterations in the connected sequences between the complementary region and structured region modestly affect miRNA inhibition activity. Surprisingly, variations in the mismatched insertion sequence in the center of the complementary sequence significantly affect activity. The central insertion sequence xxxA is required for the potent miRNA inhibitory effects of LidNA. This suggests that both the structure and insertion sequence of LidNA and other miRNA inhibitors should be considered for maximal miRNA inhibitory activity.
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ADN/genética , MicroARNs/genética , ARN Complementario/genética , Proteínas Argonautas/metabolismo , Secuencia de Bases , Sitios de Unión/genética , ADN/química , MicroARNs/química , ARN Complementario/químicaRESUMEN
SmartFlare technology allows detection of mRNA in single living cells. We studied the possibility of using SmartFlare nanoprobes for detection of small subsets of CD4+ lymphocytes. It was found that SmartFlare allows detection of transcriptional master regulators of major CD4+T helper subsets in living human lymphocytes. Nanoprobes labeled with Cy5 fluorophore were better detected by flow cytometry than nanoprobes labeled with Cy3. Appropriate time of lymphocyte incubation with SmartFlare probes was 24 h.
Asunto(s)
Citometría de Flujo/métodos , Colorantes Fluorescentes/química , ARN Complementario/genética , ARN Mensajero/genética , Subgrupos de Linfocitos T/citología , Linfocitos T Colaboradores-Inductores/citología , Carbocianinas/química , Oro/química , Humanos , Nanopartículas del Metal/químicaRESUMEN
BACKGROUND: Vast amounts of next generation sequencing RNA data has been deposited in archives, accompanying very diverse original studies. The data is readily available also for other purposes such as genome annotation or transcriptome assembly. However, selecting a subset of available experiments, sequencing runs and reads for this purpose is a nontrivial task and complicated by the inhomogeneity of the data. RESULTS: This article presents the software VARUS that selects, downloads and aligns reads from NCBI's Sequence Read Archive, given only the species' binomial name and genome. VARUS automatically chooses runs from among all archived runs to randomly select subsets of reads. The objective of its online algorithm is to cover a large number of transcripts adequately when network bandwidth and computing resources are limited. For most tested species VARUS achieved both a higher sensitivity and specificity with a lower number of downloaded reads than when runs were manually selected. At the example of twelve eukaryotic genomes, we show that RNA-Seq that was sampled with VARUS is well-suited for fully-automatic genome annotation with BRAKER. CONCLUSIONS: With VARUS, genome annotation can be automatized to the extent that not even the selection and quality control of RNA-Seq has to be done manually. This introduces the possibility to have fully automatized genome annotation loops over potentially many species without incurring a loss of accuracy over a manually supervised annotation process.
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Bases de Datos Genéticas , ARN Complementario/genética , Análisis de Secuencia de ARN/métodos , Programas Informáticos , Algoritmos , Animales , Drosophila melanogaster/genética , Eucariontes/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Intrones/genética , Anotación de Secuencia Molecular , Transcriptoma/genéticaRESUMEN
BACKGROUND: Blood flukes of the genus Schistosoma cause schistosomiasis-a neglected tropical disease (NTD) that affects more than 200 million people worldwide. Studies of schistosome genomes have improved our understanding of the molecular biology of flatworms, but most of them have focused largely on protein-coding genes. Small non-coding RNAs (sncRNAs) have been explored in selected schistosome species and are suggested to play essential roles in the post-transcriptional regulation of genes, and in modulating flatworm-host interactions. However, genome-wide small RNA data are currently lacking for key schistosomes including Schistosoma haematobium-the causative agent of urogenital schistosomiasis of humans. METHODOLOGY: MicroRNAs (miRNAs) and other sncRNAs of male and female adults of S. haematobium and small RNA transcription levels were explored by deep sequencing, genome mapping and detailed bioinformatic analyses. PRINCIPAL FINDINGS: In total, 89 transcribed miRNAs were identified in S. haematobium-a similar complement to those reported for the congeners S. mansoni and S. japonicum. Of these miRNAs, 34 were novel, with no homologs in other schistosomes. Most miRNAs (n = 64) exhibited sex-biased transcription, suggestive of roles in sexual differentiation, pairing of adult worms and reproductive processes. Of the sncRNAs that were not miRNAs, some related to the spliceosome (n = 21), biogenesis of other RNAs (n = 3) or ribozyme functions (n = 16), whereas most others (n = 3798) were novel ('orphans') with unknown functions. CONCLUSIONS: This study provides the first genome-wide sncRNA resource for S. haematobium, extending earlier studies of schistosomes. The present work should facilitate the future curation and experimental validation of sncRNA functions in schistosomes to enhance our understanding of post-transcriptional gene regulation and of the roles that sncRNAs play in schistosome reproduction, development and parasite-host cross-talk.
Asunto(s)
ARN Complementario/genética , ARN Pequeño no Traducido/genética , Schistosoma haematobium/genética , Esquistosomiasis Urinaria/parasitología , Animales , Biología Computacional , Cricetinae , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , ARN Complementario/metabolismo , ARN Pequeño no Traducido/metabolismo , Schistosoma haematobium/metabolismo , Análisis de Secuencia de ARNRESUMEN
l-Cysteine is an endogenous sulfur-containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson's and Alzheimer's disease. l-Cysteine can modulate the activity of ionic channels, including voltage-gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l-cysteine on responses mediated by homomeric GABAA ρ1 receptors, which are known for mediating tonic γ-aminobutyric acid (GABA) responses in retinal neurons. GABAA ρ1 receptors were expressed in Xenopus laevis oocytes and GABA-evoked chloride currents recorded by two-electrode voltage-clamp in the presence or absence of l-cysteine. l-Cysteine antagonized GABAA ρ1 receptor-mediated responses; inhibition was dose-dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration-response curves for GABA were shifted to the right in the presence of l-cysteine without a substantial change in the maximal response. l-Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N-ethyl maleimide. Our results suggest that redox modulation is not involved during l-cysteine actions and that l-cysteine might be acting as a competitive antagonist of the GABAA ρ1 receptors.
Asunto(s)
Cisteína/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Receptores de GABA-A/efectos de los fármacos , Animales , Unión Competitiva , Cloruros/metabolismo , Cistina/farmacología , Relación Dosis-Respuesta a Droga , Etilmaleimida/farmacología , Homocisteína/farmacología , Humanos , Transporte Iónico/efectos de los fármacos , Oocitos , Técnicas de Placa-Clamp , ARN Complementario/genética , Receptores de GABA-A/fisiología , Proteínas Recombinantes/metabolismo , Xenopus laevis , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
The spindle assembly checkpoint (SAC) prevents chromosome missegregation by coupling anaphase onset with correct chromosome attachment and tension to microtubules. It does this by generating a diffusible signal from free kinetochores into the cytoplasm, inhibiting the anaphase-promoting complex (APC). The volume in which this signal remains effective is unknown. This raises the possibility that cell volume may be the reason the SAC is weak, and chromosome segregation error-prone, in mammalian oocytes. Here, by a process of serial bisection, we analyzed the influence of oocyte volume on the ability of the SAC to inhibit bivalent segregation in meiosis I. We were able to generate oocytes with cytoplasmic volumes reduced by 86% and observed changes in APC activity consistent with increased SAC control. However, bivalent biorientation remained uncoupled from APC activity, leading to error-prone chromosome segregation. We conclude that volume is one factor contributing to SAC weakness in oocytes. However, additional factors likely uncouple chromosome biorientation with APC activity.
Asunto(s)
Tamaño de la Célula , Segregación Cromosómica , Microtúbulos/metabolismo , Oocitos/metabolismo , Huso Acromático/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/genética , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Animales , Femenino , Regulación de la Expresión Génica , Gonadotropinas Equinas/farmacología , Cinetocoros/efectos de los fármacos , Cinetocoros/metabolismo , Cinetocoros/ultraestructura , Meiosis/genética , Ratones , Ratones Endogámicos C57BL , Microinyecciones , Microtúbulos/efectos de los fármacos , Microtúbulos/ultraestructura , Milrinona/farmacología , Nocodazol/farmacología , Oocitos/efectos de los fármacos , Oocitos/ultraestructura , ARN Complementario/genética , ARN Complementario/metabolismo , Huso Acromático/efectos de los fármacos , Huso Acromático/ultraestructuraRESUMEN
BACKGROUND: Subsyndromal symptomatic depression (SSD) is a subtype of subthreshold depressive and can lead to significant psychosocial functional impairment. Although the pathogenesis of major depressive disorder (MDD) and SSD still remains poorly understood, a set of studies have found that many same genetic factors play important roles in the etiology of these two disorders. Nowadays, the differential gene expression between MDD and SSD is still unknown. In our previous study, we compared the expression profile and made the classification with the leukocytes by using whole-genome cRNA microarrays among drug-free first-episode subjects with SSD, MDD and matched healthy controls (8 subjects in each group), and finally determined 48 gene expression signatures. Based on these findings, we further clarify whether these genes mRNA was different expressed in peripheral blood in patients with SSD, MDD and healthy controls (60 subjects respectively). METHOD: With the help of the quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR), we gained gene relative expression levels among the three groups. RESULTS: We found that there are three of the forty eight co-regulated genes had differential expression in peripheral blood among the three groups, which are CD84, STRN, CTNS gene (F = 3.528, p = 0.034; F = 3.382, p = 0.039; F = 3.801, p = 0.026, respectively) while there were no significant differences for other genes. CONCLUSION: CD84, STRN, CTNS gene may have significant value for performing diagnostic functions and classifying SSD, MDD and healthy controls.
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Depresión/genética , Trastorno Depresivo Mayor/genética , Expresión Génica/genética , Adulto , Estudios de Casos y Controles , Femenino , Humanos , Leucocitos/metabolismo , Masculino , ARN Complementario/genética , ARN Mensajero/genética , Índice de Severidad de la EnfermedadRESUMEN
Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus that evokes intracellular calcium (Ca2+) oscillations that are essential for the initiation of egg activation during mammalian fertilisation. A recent genetic study reported a male infertility case that was directly associated with a point mutation in the PLCζ C2 domain, where an isoleucine residue had been substituted with a phenylalanine (I489F). Here, we have analysed the effect of this mutation on the in vivo Ca2+ oscillation-inducing activity and the in vitro biochemical properties of human PLCζ. Microinjection of cRNA or recombinant protein corresponding to PLCζI489F mutant at physiological concentrations completely failed to cause Ca2+ oscillations and trigger development. However, this infertile phenotype could be effectively rescued by microinjection of relatively high (non-physiological) amounts of recombinant mutant PLCζI489F protein, leading to Ca2+ oscillations and egg activation. Our in vitro biochemical analysis suggested that the PLCζI489F mutant displayed similar enzymatic properties, but dramatically reduced binding to PI(3)P and PI(5)P-containing liposomes compared with wild-type PLCζ. Our findings highlight the importance of PLCζ at fertilisation and the vital role of the C2 domain in PLCζ function, possibly due to its novel binding characteristics.
Asunto(s)
Dominios C2 , Calcio/metabolismo , Infertilidad Masculina/genética , Fosfoinositido Fosfolipasa C/química , Mutación Puntual , Sustitución de Aminoácidos , Animales , Señalización del Calcio , Bovinos , Femenino , Fertilización , Expresión Génica , Humanos , Isoleucina/química , Isoleucina/metabolismo , Liposomas/química , Liposomas/metabolismo , Masculino , Ratones , Microinyecciones , Oocitos/citología , Oocitos/metabolismo , Fenilalanina/química , Fenilalanina/metabolismo , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoinositido Fosfolipasa C/genética , Fosfoinositido Fosfolipasa C/metabolismo , Unión Proteica , ARN Complementario/administración & dosificación , ARN Complementario/genética , ARN Complementario/metabolismo , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espermatozoides/metabolismo , Espermatozoides/patologíaRESUMEN
This protocol describes the isolation of recombinant human and mammalian membrane proteins expressed in Xenopus laevis frog oocytes for structural studies. The cDNA-derived cRNA of the desired genes is injected into several hundreds of oocytes, which are incubated for several days to allow protein expression. Recombinant proteins are then purified via affinity chromatography. The novelty of this method comes from the design of a plasmid that produces multi-tagged proteins and, most importantly, the development of a protocol for efficiently discarding lipids, phospholipids, and lipoproteins from the oocyte egg yolk, which represent the major contaminants in protein purifications. Thus, the high protein purity and good yield obtained from this method allows protein structure determination by transmission electron microscopy of single detergent-solubilized protein particles and of 2D crystals of membrane protein embedded in lipid bilayers. Additionally, a radiotracer assay for functional analysis of the expressed target proteins in oocytes is described. Overall, this method is a valuable option for structural studies of mammalian and particularly human proteins, for which other expression systems often fail.
Asunto(s)
Mamíferos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Xenopus laevis/genética , Animales , Cromatografía de Afinidad , Clonación Molecular , ADN Complementario/genética , Femenino , Vectores Genéticos/administración & dosificación , Humanos , Mamíferos/genética , Proteínas de la Membrana/genética , Oocitos/metabolismo , Plásmidos/genética , Conformación Proteica , ARN Complementario/genética , Proteínas Recombinantes/metabolismo , Xenopus laevis/metabolismoRESUMEN
Hemorrhagic fever with renal syndrome (HFRS) is a severe, viral zoonotic disease which occurs worldwide, particularly in Asia and Europe. In China, the Hantaan virus (HTNV) and the Seoul virus (SEOV) are known to be the most prevalent causative agents of HFRS. Since no protective vaccines or effective treatments are available for human use, accurate and reliable diagnostic methods are essential for disease surveillance. In the present study, the viral loads in cell culture supernatant, infected mice blood and clinical serum samples were quantified using the SYBRGreen I-based reverse transcription-quantitiative polymerase chain reaction (RT-qPCR) assay, which targeted the S gene sequence of the HTNV and SEOV genomes. The cRNA of these two viruses were synthesized as a positive control and 10-fold serially diluted from 1x105 to 1x100 copies/µl. Standard curves were generated by plotting the mean cycle threshold (Ct) values versus copy numbers. The standard curve of HTNV had a correlation coefficient (R2) of 0.994, efficiency of amplification (E) of 101.9%, and the slope of -3.278, whereas that of SEOV had an R2 of 0.993, E of 104.8%, and the slope of -3.212. The minimum detection limit of the RT-qPCR assay for HTNV and SEOV was 101 copies/µl. Two qPCR assays were successfully established for the detection of HTNV and SEOV, respectively. Taken together, these findings demonstrate that using the SYBRGreen I-based RT-qPCR assay, the HTNV and SEOV may be genotyped precisely without cross-reactivity. Furthermore, viral RNA may be detected and quantified in cells, mice and infected individuals, which may be useful in epidemiological studies as well as for early monitoring and further preventative treatment against SEOV and HTNV-induced diseases.
Asunto(s)
Virus Hantaan/genética , Fiebre Hemorrágica con Síndrome Renal/diagnóstico , Compuestos Orgánicos/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Virus Seoul/genética , Animales , Benzotiazoles , Diaminas , Dosificación de Gen , Genoma Viral/genética , Genotipo , Virus Hantaan/fisiología , Fiebre Hemorrágica con Síndrome Renal/sangre , Fiebre Hemorrágica con Síndrome Renal/virología , Interacciones Huésped-Patógeno , Humanos , Ratones , Quinolinas , ARN Complementario/sangre , ARN Complementario/química , ARN Complementario/genética , ARN Viral/sangre , ARN Viral/química , ARN Viral/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Virus Seoul/fisiología , Especificidad de la EspecieRESUMEN
Egg activation refers to events required for transition of a gamete into an embryo, including establishment of the polyspermy block, completion of meiosis, entry into mitosis, selective recruitment and degradation of maternal mRNA, and pronuclear development. Here we show that zinc fluxes accompany human egg activation. We monitored calcium and zinc dynamics in individual human eggs using selective fluorophores following activation with calcium-ionomycin, ionomycin, or hPLCζ cRNA microinjection. These egg activation methods, as expected, induced rises in intracellular calcium levels and also triggered the coordinated release of zinc into the extracellular space in a prominent "zinc spark." The ability of the gamete to mount a zinc spark response was meiotic-stage dependent. Moreover, chelation of intracellular zinc alone was sufficient to induce cell cycle resumption and transition of a meiotic cell into a mitotic one. Together, these results demonstrate critical functions for zinc dynamics and establish the zinc spark as an extracellular marker of early human development.
Asunto(s)
Óvulo/metabolismo , Zinc/metabolismo , Ionóforos de Calcio/farmacología , Quelantes/química , Diaminas/química , Etilenos/química , Femenino , Humanos , Ionomicina/farmacología , Meiosis , Microinyecciones , Microscopía Fluorescente , Óvulo/efectos de los fármacos , Fosfoinositido Fosfolipasa C/genética , Compuestos Policíclicos/química , ARN Complementario/genética , ARN Complementario/metabolismo , Zinc/químicaRESUMEN
In this study, we demonstrate the upregulation in the expression of caspases 1 and 11 by SJL/J mouse brain astrocytes infected with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV). The upregulation of both proteases hints at protection of astrocytic cells from apoptotic death. We therefore looked for the reason of the demonstrated absence of programmed cell death in BeAn-infected SJL/J astrocytes. Complementary RNA (cRNA) from mock- and TMEV-infected cells was hybridized to the whole murine genome U74v2 DNA microarray from Affymetrix. Those experiments demonstrated the upregulation of gene expression for caspases 1 and 11 in infected cells. We further confirmed and validated their messenger RNA (mRNA) increase by reverse transcriptase quantitative real-time PCR (qPCR). The presence of both enzymatically active caspases 1 and 11 was demonstrated in cell lysates using a colorimetric and fluorymetric assay, respectively. We also show that overexpressed caspase 11 activated caspase 1 after preincubation of cytosol in vitro following a time-dependent process. This induction was neutralized by an anti-caspase 11 polyclonal antibody. These results demonstrate the activation of the caspase 1 precursor by caspase 11 and suggest a new mechanism of protection of BeAn-infected astrocytes from apoptosis. The direct experimental evidence that the protection effect demonstrated in this article was mediated by caspase 1, is provided by the fact that its specific inhibitor Z-WEHD-FMK induced de novo apoptotic death.
Asunto(s)
Astrocitos/virología , Infecciones por Cardiovirus/virología , Caspasa 1/genética , Caspasas/genética , Interacciones Huésped-Patógeno , Theilovirus/genética , Clorometilcetonas de Aminoácidos/farmacología , Animales , Animales Recién Nacidos , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Infecciones por Cardiovirus/patología , Caspasa 1/metabolismo , Inhibidores de Caspasas/farmacología , Caspasas/metabolismo , Caspasas Iniciadoras , Regulación de la Expresión Génica , Ratones , Cultivo Primario de Células , ARN Complementario/genética , ARN Complementario/metabolismo , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Theilovirus/efectos de los fármacos , Theilovirus/metabolismoRESUMEN
USP18 (Ubiquitin-like specific protease 18) is an enzyme cleaving ubiquitin from target proteins. USP18 plays a pivotal role in antiviral and antibacterial immune responses. On the other hand, ubiquitination participates in the regulation of several ion channels and transporters. USP18 sensitivity of transporters has, however, never been reported. The present study thus explored, whether USP18 modifies the activity of the peptide transporters PEPT1 and PEPT2, and whether the peptide transporters are sensitive to the ubiquitin ligase Nedd4-2. To this end, cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding USP18. Electrogenic peptide (glycine-glycine) transport was determined by dual electrode voltage clamp. As a result, in Xenopus laevis oocytes injected with cRNA encoding PEPT1 or PEPT2, but not in oocytes injected with water or with USP18 alone, application of the dipeptide gly-gly (2 mM) was followed by the appearance of an inward current (Igly-gly). Coexpression of USP18 significantly increased Igly-gly in both PEPT1 and PEPT2 expressing oocytes. Kinetic analysis revealed that coexpression of USP18 increased maximal Igly-gly. Conversely, overexpression of the ubiquitin ligase Nedd4-2 decreased Igly-gly. Coexpression of USP30 similarly increased Igly-gly in PEPT1 expressing oocytes. In conclusion, USP18 sensitive cellular functions include activity of the peptide transporters PEPT1 and PEPT2.
Asunto(s)
Dipéptidos/metabolismo , Endopeptidasas/metabolismo , Simportadores/metabolismo , Animales , Transporte Biológico , Dipéptidos/farmacología , Endopeptidasas/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Femenino , Humanos , Inyecciones , Canal de Potasio KCNQ1/genética , Canal de Potasio KCNQ1/metabolismo , Mediciones Luminiscentes/métodos , Potenciales de la Membrana/efectos de los fármacos , Ubiquitina-Proteína Ligasas Nedd4 , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oocitos/fisiología , Técnicas de Placa-Clamp , Transportador de Péptidos 1 , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , ARN Complementario/administración & dosificación , ARN Complementario/genética , Conejos , Simportadores/genética , Ubiquitina Tiolesterasa , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Xenopus , Xenopus laevisRESUMEN
BACKGROUND/AIMS: Kinases involved in the regulation of epithelial transport include SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1). SPAK and OSR1 are both regulated by WNK (with-no-K(Lys)) kinases. The present study explored whether SPAK and/or OSR1 influence the excitatory amino acid transporter EAAT3, which accomplishes glutamate and aspartate transport in kidney, intestine and brain. METHODS: cRNA encoding EAAT3 was injected into Xenopus laevis oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, wild-type OSR1, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1 and catalytically inactive (D164A)OSR1. Glutamate-induced current was taken as measure of electrogenic glutamate transport and was quantified utilizing dual electrode voltage clamp. Furthermore, Ussing chamber was employed to determine glutamate transport in the intestine from gene-targeted mice carrying WNK insensitive SPAK (spak(tg/tg)) and from corresponding wild-type mice (spak(+/+)). RESULTS: EAAT3 activity was significantly decreased by wild-type SPAK and (T233E)SPAK, but not by (T233A)SPAK and (D212A)SPAK. SPAK decreased maximal transport rate without affecting significantly affinity of the carrier. Similarly, EAAT3 activity was significantly downregulated by wild-type OSR1 and (T185E)OSR1, but not by (T185A)OSR1 and (D164A)OSR1. Again OSR1 decreased maximal transport rate without affecting significantly affinity of the carrier. Intestinal electrogenic glutamate transport was significantly lower in spak(+/+) than in spak(tg/tg) mice. CONCLUSION: Both, SPAK and OSR1 are negative regulators of EAAT3 activity.
Asunto(s)
Transportador 3 de Aminoácidos Excitadores/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Ácido Aspártico/metabolismo , Transportador 3 de Aminoácidos Excitadores/genética , Ácido Glutámico/metabolismo , Humanos , Ratones , Oocitos/metabolismo , Técnicas de Placa-Clamp , Proteínas Serina-Treonina Quinasas/genética , ARN Complementario/biosíntesis , ARN Complementario/genética , Agua/metabolismo , Xenopus laevisRESUMEN
The endometrium is a dynamic tissue, demonstrating cyclical growth/remodelling in preparation for implantation. In mice, seminal constituents trigger mechanisms to prepare the endometrium, a process dubbed 'seminal priming' that modifies immune system components and mediates endometrial remodelling in preparation for pregnancy. An array of cytokines has been reported to mediate this interaction, although much of the literature relates to in vitro studies on isolated endometrial epithelial cells. This study measured changes in immune-related gene expression in endometrial epithelial and stromal cells in vivo following natural mating. CD1 mice were naturally mated and sacrificed over the first 4 days post-coitum (n=3 each day). Endometrial epithelial and stromal compartments were isolated by laser capture microdissection. Labelled cRNA was generated and hybridised to genome-wide expression microarrays. Pathway analysis identified several immune-related pathways active within epithelial and stromal compartments, in particular relating to cytokine networks, matrix metalloproteinases and prostaglandin synthesis. Cluster analysis demonstrated that the expression of factors involved in immunomodulation/endometrial remodelling differed between the epithelial and stromal compartments in a temporal fashion. This study is the first to examine the disparate responses of the endometrial epithelial and stromal compartments to seminal plasma in vivo in mice, and demonstrates the complexity of the interactions between these two compartments needed to create a permissive environment for implantation.
Asunto(s)
Endometrio/inmunología , Epitelio/inmunología , Inmunidad/fisiología , Células del Estroma/inmunología , Animales , Citocinas/biosíntesis , Citocinas/genética , Implantación del Embrión/fisiología , Endometrio/citología , Femenino , Expresión Génica/inmunología , Estudio de Asociación del Genoma Completo , Inmunidad/genética , Masculino , Metaloproteinasas de la Matriz/biosíntesis , Metaloproteinasas de la Matriz/genética , Ratones , Análisis por Micromatrices , Microdisección , Embarazo , Prostaglandinas/biosíntesis , ARN Complementario/biosíntesis , ARN Complementario/genética , Semen/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Útero/citología , Útero/metabolismoRESUMEN
Capsaicin is an activator of the heat-sensitive TRPV1 (transient receptor potential vanilloid 1) ion channels and has been used as a local analgesic. We found that activation of TRPV1 channels with capsaicin either in dorsal root ganglion neurons or in a heterologous expression system inhibited the mechanosensitive Piezo1 and Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its precursor phosphatidylinositol 4-phosphate [PI(4)P] from the plasma membrane through Ca(2+)-induced phospholipase Cδ (PLCδ) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLCß indicated that inhibition of Piezo channels required depletion of both PI(4)P and PI(4,5)P2. The mechanically activated current amplitudes decreased substantially in the excised inside-out configuration, where the membrane patch containing Piezo1 channels is removed from the cell. PI(4,5)P2 and PI(4)P applied to these excised patches inhibited this decrease. Thus, we concluded that Piezo channel activity requires the presence of phosphoinositides, and the combined depletion of PI(4,5)P2 and PI(4)P reduces channel activity. In addition to revealing a role for distinct membrane lipids in mechanosensitive ion channel regulation, these data suggest that inhibition of Piezo2 channels may contribute to the analgesic effect of capsaicin.
Asunto(s)
Capsaicina/farmacología , Ganglios Espinales/citología , Canales Iónicos/metabolismo , Fosfatidilinositoles/metabolismo , Canales Catiónicos TRPV/metabolismo , Análisis de Varianza , Animales , Membrana Celular/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Ganglios Espinales/metabolismo , Células HEK293 , Humanos , Ratones , Microscopía Fluorescente , Neuronas/metabolismo , Oocitos/metabolismo , Técnicas de Placa-Clamp , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipasa C delta/metabolismo , ARN Complementario/genética , Xenopus laevisRESUMEN
UNLABELLED: Natural oestrogens, which are degraded but not completely removed in wastewater treatment plants, are suspected of causing the endocrine disruption of aquatic organisms in the receiving water body. While several bacterial isolates were reported to be oestrogen-degrading bacteria, our previous study implied that only the unidentified rod-shaped Betaproteobacteria in chains were responsible for estrone (E1) degradation by activated sludge especially at the sub-milligram per litre level. The Betaproteobacteria were suspected to be related to genera Sphaerotilus and Leptothrix according to morphological observations. Probe Spha823 was newly developed to target 16S rRNA gene clones obtained from activated sludge and closely related to the above genera. [(3) H]E1-incubated sludge samples showed that most of the (3) H-labelled cells hybridized with probe Spha823 by microautoradiography (MAR) fluorescent in situ hybridization. Spha823-defined cells were present in all three activated sludge samples tested, where they accounted for up to 3% of the total microbial biomass. Spha823-defined cells comprised 59·5-80·1% of the total MAR-positive cells, which suggested that the Sphaerotilus-Leptothrix-related bacteria were the most abundant micro-organisms involved in E1 degradation (at 200 µg l(-1) ) in the activated sludge samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Estrone (E1) is one of the natural estrogens, which can be degraded but is not always completely removed in wastewater treatment plants. E1 is suspected of causing the endocrine disruption of aquatic organisms in the receiving water body. We identified dominant E1-incorporating bacteria, which should include E1-degrading bacteria, in activated sludge treating domestic wastewater. Sphaerotilus-Leptothrix-related bacteria, which had never been reported in the previous attempts based on culture-dependent approach, occupied 60-80% of the E1-incorporating bacteria. This study demonstrates the identification of functionally active bacteria to degrade micro-pollutants at sub-milligram per litre level.