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Bone marrow-derived cells (BMDCs) infiltrate hypoxic tumors at a pre-angiogenic state and differentiate into mature macrophages, thereby inducing pro-tumorigenic immunity. A critical factor regulating this differentiation is activation of SREBP2-a well-known transcription factor participating in tumorigenesis progression-through unknown cellular mechanisms. Here, we show that hypoxia-induced Golgi disassembly and Golgi-ER fusion in monocytic myeloid cells result in nuclear translocation and activation of SREBP2 in a SCAP-independent manner. Notably, hypoxia-induced SREBP2 activation was only observed in an immature lineage of bone marrow-derived cells. Single-cell RNA-seq analysis revealed that SREBP2-mediated cholesterol biosynthesis was upregulated in HSCs and monocytes but not in macrophages in the hypoxic bone marrow niche. Moreover, inhibition of cholesterol biosynthesis impaired tumor growth through suppression of pro-tumorigenic immunity and angiogenesis. Thus, our findings indicate that Golgi-ER fusion regulates SREBP2-mediated metabolic alteration in lineage-specific BMDCs under hypoxia for tumor progression.
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Monocitos , Neoplasias , Humanos , Monocitos/metabolismo , Médula Ósea , Colesterol/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , HipoxiaRESUMEN
BACKGROUND: The heart comprises many types of cells such as cardiomyocytes, endothelial cells (ECs), fibroblasts, smooth muscle cells, pericytes, and blood cells. Every cell type responds to various stressors (eg, hemodynamic overload and ischemia) and changes its properties and interrelationships among cells. To date, heart failure research has focused mainly on cardiomyocytes; however, other types of cells and their cell-to-cell interactions might also be important in the pathogenesis of heart failure. METHODS: Pressure overload was imposed on mice by transverse aortic constriction and the vascular structure of the heart was examined using a tissue transparency technique. Functional and molecular analyses including single-cell RNA sequencing were performed on the hearts of wild-type mice and EC-specific gene knockout mice. Metabolites in heart tissue were measured by capillary electrophoresis-time of flight-mass spectrometry system. The vaccine was prepared by conjugating the synthesized epitope peptides with keyhole limpet hemocyanin and administered to mice with aluminum hydroxide as an adjuvant. Tissue samples from heart failure patients were used for single-nucleus RNA sequencing to examine gene expression in ECs and perform pathway analysis in cardiomyocytes. RESULTS: Pressure overload induced the development of intricately entwined blood vessels in murine hearts, leading to the accumulation of replication stress and DNA damage in cardiac ECs. Inhibition of cell proliferation by a cyclin-dependent kinase inhibitor reduced DNA damage in ECs and ameliorated transverse aortic constriction-induced cardiac dysfunction. Single-cell RNA sequencing analysis revealed upregulation of Igfbp7 (insulin-like growth factor-binding protein 7) expression in the senescent ECs and downregulation of insulin signaling and oxidative phosphorylation in cardiomyocytes of murine and human failing hearts. Overexpression of Igfbp7 in the murine heart using AAV9 (adeno-associated virus serotype 9) exacerbated cardiac dysfunction, while EC-specific deletion of Igfbp7 and the vaccine targeting Igfbp7 ameliorated cardiac dysfunction with increased oxidative phosphorylation in cardiomyocytes under pressure overload. CONCLUSIONS: Igfbp7 produced by senescent ECs causes cardiac dysfunction and vaccine therapy targeting Igfbp7 may be useful to prevent the development of heart failure.
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Insuficiencia Cardíaca , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Ratones Noqueados , Animales , Insuficiencia Cardíaca/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Ratones , Humanos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratones Endogámicos C57BL , Masculino , Modelos Animales de EnfermedadRESUMEN
The phosphanoxy-substituted phosphaalkene bearing the P=C-O-P skeleton can be prepared from diphosphene Mes*P=PMes* (Mes*=2,4,6-tBu3 C6 H2 ), and their use for catalysis is of interest. In this paper, complexation of the phosphanoxy-substituted phosphaalkenes with gold are investigated, and the catalytic activity of the mono- and bis(chlorogold) complexes are subsequently evaluated. Reaction of the P=C-O-P compound with (tht)AuCl (tht=tetrahydrothiophene) showed dominant coordination on the sp3 phosphorus, and complete coordination on the sp2 phosphorus required removal of tetrahydrothiophene. Atoms In Molecules (AIM) analysis based on the X-ray structure of the mono(chlorogold) complex indicated a pseudo coordinating interaction between the gold center and the P=C unit. The bis(chlorogold) complexes displayed conformational isomerism, and catalyzed the cycloisomerization/alkoxycyclization of 1,6-enyne and for hydration of terminal alkyne without activation treatment. Even the mono(chlorogold) complexes catalyzed the alkoxycyclization reactions without a silver co-catalyst, indicating that the alcohols were effective in activating the AuCl unit.
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An artificial cell membrane that is composed of bilayer lipid membranes (BLMs) with transmembrane proteins incorporated within them represents a well-defined system for the analysis of membrane proteins, especially ion channel proteins that are major targets for drug design. Because the BLM system has a high compatibility with recently developed cell-free expression systems, it has attracted attention as a next-generation drug screening system. However, three issues associated with BLM systems, i. e., their instability, the need for non-volatile organic solvents and a low efficiency of ion channel incorporation, have limited their use as a drug screening platform. In this personal account, we discuss our recent approaches to address these issues based on microfabrication. We also discuss the potential for using the BLM system combined with cell-free expression systems as a drug screening system for future personalized medicine.
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Canales Iónicos/química , Membrana Dobles de Lípidos/química , Evaluación Preclínica de MedicamentosRESUMEN
Iron-catalyzed cross-coupling difluoromethylations of the Grignard reagents with difluoroiodomethane provide various aromatic difluoromethyl products in good yields, not employing sterically demanding ligands. Difluoromethylations proceed within 30 min at -20 °C with 2.0 equiv of the Grignard reagents and FeCl3 or Fe(acac)3 (2.5 mol %). Mechanistic investigations clarify difluoromethyl radical intervention; Fe(0) ate is initially generated. Single-electron transfer from Fe(0) ate to difluoroiodomethane takes place. Recombination with aryl groups gives Ar-CF2Hs. The catalyst can be regenerated by the Grignard reagents.
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Posttranslational modifications of histones are well-established epigenetic modifications that play an important role in gene expression and regulation. These modifications are partly mediated by the Trithorax group (TrxG) complex, which regulates the induction or maintenance of gene transcription. We investigated the role of Menin, a component of the TrxG complex, in the acquisition and maintenance of Th2 cell identity using T cell-specific Menin-deficient mice. Our gene expression analysis revealed that Menin was involved in the maintenance of the high expression of the previously identified Th2-specific genes rather than the induction of these genes. This result suggests that Menin plays a role in the maintenance of Th2 cell identity. Menin directly bound to the Gata3 gene locus, and this Menin-Gata3 axis appeared to form a core unit of the Th2-specific gene regulatory network. Consistent with the phenotype of Menin-deficient Th2 cells observed in vitro, Menin deficiency resulted in the attenuation of effector Th2 cell-induced airway inflammation. In addition, in memory Th2 (mTh2) cells, Menin was found to play an important role in the maintenance of the expression of Th2-specific genes, including Gata3, Il4, and Il13 Consequently, Menin-deficient mTh2 cells showed an impaired ability to recruit eosinophils to the lung, resulting in the attenuation of mTh2 cell-induced airway inflammation. This study confirmed the critical role of Menin in Th2 cell-mediated immune responses.
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Epigénesis Genética , Memoria Inmunológica , Proteínas Proto-Oncogénicas/metabolismo , Células Th2/inmunología , Animales , Diferenciación Celular , Eosinófilos/inmunología , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Inmunidad Celular , Inflamación , Interleucina-13/genética , Interleucina-13/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Pulmón/inmunología , Ratones , Unión Proteica , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/inmunología , Células Th2/metabolismo , Transcripción GenéticaRESUMEN
We introduce the principle of a new technique to isolate glycosphingolipids (GSLs) from phospholipids. Neutral and acidic GSLs in organic solvent bind to titanium dioxide under neutral pH and can be eluted with 5 mg/ml of 2,5-dihydroxybenzoic acid in methanol. This special property is applicable for eliminating phospholipids, including sphingomyelin, which cannot be eliminated by a typical mild alkaline treatment. By using this technique, we demonstrated the rapid separation of minor components of GSLs, namely sulfatide and gangliosides from rabbit serum and liver, respectively. The minor GSL components were effectively purified despite both sources containing tremendous amount of phospholipids and simple lipids such as cholesterol, cholesteryl esters and triglycerides.
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Glicoesfingolípidos/aislamiento & purificación , Titanio/química , Animales , Mezclas Complejas/aislamiento & purificación , Ácidos Grasos/análisis , Humanos , Hígado/metabolismoRESUMEN
Artificial bilayer lipid membranes (BLMs) provide well-defined systems for investigating the fundamental properties of membrane proteins, including ion channels, and for screening the effect of drugs that act on them. However, the application of this technique is limited due to the low stability and low reconstitution efficiency of the process. We previously reported on improving the stability of BLM based on the fabrication of microapertures having a tapered edge in SiO2/Si3N4 septa and efficient ion channel incorporation based on vesicle fusion accelerated by a centrifugal force. Although the BLM stability and incorporation probability were dramatically improved when these approaches were used, some BLMs were ruptured when subjected to a centrifugal force. To further improve the BLM stability, we investigated the effect of modifying the surface of the SiO2/Si3N4 septa on the stability of BLM suspended in the septa. The modified surfaces were characterized in terms of hydrophobicity, lipophobicity, and surface roughness. Diffusion coefficients of the lipid monolayers formed on the modified surfaces were also determined. Highly fluidic lipid monolayers were formed on the amphiphobic substrates that had been modified with long-chain perfluorocarbons. Free-standing BLMs formed in amphiphobic septa showed a much higher mechanical stability, including tolerance to water movement and applied centrifugal forces with and without proteoliposomes, than those formed in the septa that had been modified with a short alkyl chain. These results demonstrate that highly stable BLMs are formed when the surface of the septa has amphiphobic properties. Because highly fluidic lipid monolayers that are formed on the septa seamlessly connect with BLMs in a free-standing region, the high fluidity of the lipids contributes to decreasing potential damage to BLMs when mechanical stresses are applied. This approach to improve the BLM stability increases the experimental efficiency of the BLM systems and will contribute to the development of high-throughput platforms for functional assays of ion channel proteins.
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Membrana Dobles de Lípidos/química , Canales Iónicos/química , Fusión de Membrana , Dióxido de Silicio/química , Estrés MecánicoRESUMEN
Gata3 acts as a master regulator for T helper 2 (Th2) cell differentiation by inducing chromatin remodeling of the Th2 cytokine loci, accelerating Th2 cell proliferation, and repressing Th1 cell differentiation. Gata3 also directly transactivates the interleukin-5 (Il5) gene via additional mechanisms that have not been fully elucidated. We herein identified a mechanism whereby the methylation of Gata3 at Arg-261 regulates the transcriptional activation of the Il5 gene in Th2 cells. Although the methylation-mimicking Gata3 mutant retained the ability to induce IL-4 and repress IFNγ production, the IL-5 production was selectively impaired. We also demonstrated that heat shock protein (Hsp) 60 strongly associates with the methylation-mimicking Gata3 mutant and negatively regulates elongation of the Il5 transcript by RNA polymerase II. Thus, arginine methylation appears to play a pivotal role in the organization of Gata3 complexes and the target gene specificity of Gata3.
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Arginina/genética , Metilación de ADN , Factor de Transcripción GATA3/genética , Interleucina-5/genética , Células Th2/metabolismo , Activación Transcripcional , Secuencia de Aminoácidos , Animales , Arginina/metabolismo , Western Blotting , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Chaperonina 60/antagonistas & inhibidores , Chaperonina 60/genética , Chaperonina 60/metabolismo , Ensamble y Desensamble de Cromatina , Inmunoprecipitación de Cromatina , Citocinas/genética , Citocinas/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Factor de Transcripción GATA3/metabolismo , Inmunoprecipitación , Interleucina-5/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/inmunologíaRESUMEN
GATA binding protein 3 (Gata3) is a GATA family transcription factor that controls differentiation of naïve CD4 T cells into T helper 2 (Th2) cells. However, it is unknown how Gata3 simultaneously activates Th2-specific genes while repressing those of other Th lineages. Here we show that chromodomain helicase DNA-binding protein 4 (Chd4) forms a complex with Gata3 in Th2 cells that both activates Th2 cytokine transcription and represses the Th1 cytokine IFN-γ. We define a Gata3/Chd4/p300 transcriptional activation complex at the Th2 cytokine loci and a Gata3/Chd4-nucleosome remodeling histone deacetylase repression complex at the Tbx21 locus in Th2 cells. We also demonstrate a physiological role for Chd4 in Th2-dependent inflammation in an in vivo model of asthmatic inflammation. Thus, Gata3/Chd4 forms functionally distinct complexes, which mediate both positive and negative gene regulation to facilitate Th2 cell differentiation.
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Diferenciación Celular/inmunología , ADN Helicasas/metabolismo , Factor de Transcripción GATA3/inmunología , Complejos Multiproteicos/inmunología , Células Th2/inmunología , Transcripción Genética/inmunología , Animales , Asma/genética , Asma/inmunología , Asma/patología , ADN Helicasas/genética , Modelos Animales de Enfermedad , Factor de Transcripción GATA3/genética , Sitios Genéticos/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Interferón gamma/genética , Interferón gamma/inmunología , Ratones , Ratones Endogámicos BALB C , Complejos Multiproteicos/genética , Nucleosomas/genética , Nucleosomas/inmunología , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/inmunología , Células Th2/patología , Transcripción Genética/genéticaRESUMEN
GATA-binding protein 3 (Gata3) controls the differentiation of naive CD4 T cells into T helper 2 (Th2) cells by induction of chromatin remodeling of the Th2 cytokine gene loci, direct transactivation of Il5 and Il13 genes, and inhibition of Ifng. Gata3 also facilitates Th2 cell proliferation via additional mechanisms that are far less well understood. We herein found that Gata3 associates with RuvB-like protein 2 (Ruvbl2) and represses the expression of a CDK inhibitor, cyclin-dependent kinase inhibitor 2c (Cdkn2c) to facilitate the proliferation of Th2 cells. Gata3 directly bound to the Cdkn2c locus in an Ruvbl2-dependent manner. The defect in the proliferation of Gata3-deficient Th2 cells is rescued by the knockdown of Cdkn2c, indicating that Cdkn2c is a key molecule involved in the Gata3-mediated induction of Th2 cell proliferation. Ruvbl2-knockdown Th2 cells showed decreased antigen-induced expansion and caused less airway inflammation in vivo. We therefore have identified a functional Gata3/Ruvbl2 complex that regulates the proliferation of differentiating Th2 cells through the repression of a CDK inhibitor, Cdkn2c.
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Proliferación Celular , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/metabolismo , ADN Helicasas/inmunología , Factor de Transcripción GATA3/inmunología , Regulación de la Expresión Génica/inmunología , Complejos Multiproteicos/inmunología , Células Th2/citología , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Bromodesoxiuridina , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Técnicas de Silenciamiento del Gen , Immunoblotting , Inmunoprecipitación , Luciferasas , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Pneumonia ranks as the third leading cause of death in Japan. About 97% of patients who die because of pneumonia are elderly, with aspiration generally thought to be involved in the majority of cases of pneumonia in elderly. Once an elderly individual contracts pneumonia, their physical function often declines and their activities of daily living diminish with hospital admission, even in individuals with no underlying disorders. Prolonged confinement to a bed and immobility leads to weakening of the legs and back, making it difficult for elderly patients to attend daily outpatient clinics, often leading to admission to nursing facilities for the aged instead of returning to their own home, even after curative treatment for pneumonia. Most such patients repeatedly develop pneumonia and repeated antibiotic treatment enhances the risk of the emergence of resistant organisms. It is beyond doubt, therefore, that prevention of pneumonia is of vital importance in the elderly.
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Macrólidos/uso terapéutico , Neumonía/prevención & control , Actividades Cotidianas , Anciano , Anciano de 80 o más Años , Trastornos de Deglución , Humanos , PronósticoRESUMEN
BACKGROUND: Early treatment of disseminated intravascular coagulation (DIC) can be associated with improved patient outcomes. The Japanese Ministry of Health and Welfare (JMHW) and the International Society on Thrombosis and Haemostasis (ISTH) criteria are the most specific for diagnosis of septic DIC. The revised Japanese Association for Acute Medicine (JAAM) criteria are able to diagnose sepsis-induced DIC in the early stage. Recombinant human soluble thrombomodulin (rhTM) has recently been used for treating DIC. Previous studies have shown a benefit of using rhTM for D,IC diagnosed by the JMHW or ISTH criteria, but not the JAAM criteria. The purpose of this study was to sequentially evaluate coagulation biomarkers and the DIC score after giving rhTM treatment to patients with sepsis-induced DIC diagnosed according to the JAAM criteria. METHODS: We performed a retrospective cohort study. Critically ill patients were included if diagnosed with sepsis-induced DIC according to the JAAM criteria. They were either treated without rhTM (control group) or with rhTM (treatment group). The primary outcome was the DIC score on day 7. The secondary outcome was 28-day mortality from the start of DIC treatment. Changes in the results of coagulation tests were assessed over time from the start of treatment to day 7. RESULTS: Twelve and 23 patients were assigned to the treatment and control groups, respectively. The DIC score on day 7 was significantly higher in the treatment group (3.3 ± 1.4) than in the control group (4.9 ± 1.8, p < 0.05). Estimated survival showed lower in treatment group than control group. There was significant difference between the control group and the treatment group (p < 0.05). The D-dimer level on day 7 was significantly lower in the treatment group (7.5 ± 4.1 µg/mL) than in the control group (30.9 ± 33.6 µg/mL, p < 0.05). Life-threatening bleeding did not occur. Our results indicated that rhTM improved sepsis-induced DIC and mortality. CONCLUSIONS: Recombinant human soluble thrombomodulin may improve sepsis-induced DIC diagnosed according to the JAAM criteria without an increased bleeding risk.
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An acidic tumor microenvironment plays a critical role in tumor progression. However, understanding of metabolic reprogramming of tumors in response to acidic extracellular pH has remained elusive. Using comprehensive metabolomic analyses, we demonstrated that acidic extracellular pH (pH 6.8) leads to the accumulation of N1-acetylspermidine, a protumor metabolite, through up-regulation of the expression of spermidine/spermine acetyltransferase 1 (SAT1). Inhibition of SAT1 expression suppressed the accumulation of intra- and extracellular N1-acetylspermidine at acidic pH. Conversely, overexpression of SAT1 increased intra- and extracellular N1-acetylspermidine levels, supporting the proposal that SAT1 is responsible for accumulation of N1-acetylspermidine. While inhibition of SAT1 expression only had a minor effect on cancer cell growth in vitro, SAT1 knockdown significantly decreased tumor growth in vivo, supporting a contribution of the SAT1-N1-acetylspermidine axis to protumor immunity. Immune cell profiling revealed that inhibition of SAT1 expression decreased neutrophil recruitment to the tumor, resulting in impaired angiogenesis and tumor growth. We showed that antineutrophil-neutralizing antibodies suppressed growth in control tumors to a similar extent to that seen in SAT1 knockdown tumors in vivo. Further, a SAT1 signature was found to be correlated with poor patient prognosis. Our findings demonstrate that extracellular acidity stimulates recruitment of protumor neutrophils via the SAT1-N1-acetylspermidine axis, which may represent a metabolic target for antitumor immune therapy.
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Peyer's patches (PPs) are specialized gut-associated lymphoid tissues that initiate follicular helper T (Tfh)-mediated immunoglobulin A (IgA) response to luminal antigens derived from commensal symbionts, pathobionts, and dietary sources. IgA-producing B cells migrate from PPs to the small intestinal lamina propria and secrete IgA across the epithelium, modulating the ecological balance of the commensal microbiota and neutralizing pathogenic microorganisms. α-glucosidase inhibitors (α-GIs) are antidiabetic drugs that inhibit carbohydrate digestion in the small intestinal epithelium, leading to alterations in the commensal microbiota composition and metabolic activity. The commensal microbiota and IgA responses exhibit bidirectional interactions that modulate intestinal homeostasis and immunity. However, the effect of α-GIs on the intestinal IgA response remains unclear. We investigated whether α-GIs affect IgA responses by administering voglibose and acarbose to mice via drinking water. We analyzed Tfh cells, germinal center (GC) B cells, and IgA-producing B cells in PPs by flow cytometry. We also assessed pathogen-specific IgA responses. We discovered that voglibose and acarbose induced Tfh cells, GCB cells, and IgA-producing B cells in the PPs of the proximal small intestine in mice. This effect was attributed to the modification of the microbiota rather than a shortage of monosaccharides. Furthermore, voglibose enhanced secretory IgA (S-IgA) production against attenuated Salmonella Typhimurium. Our findings reveal a novel mechanism by which α-GIs augment antigen-specific IgA responses by stimulating Tfh-GCB responses in PPs, and suggest a potential therapeutic application as an adjuvant for augmenting mucosal vaccines.
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Inhibidores de Glicósido Hidrolasas , Inmunoglobulina A , Animales , Ratones , Inhibidores de Glicósido Hidrolasas/farmacología , Inhibidores de Glicósido Hidrolasas/metabolismo , Ganglios Linfáticos Agregados , Acarbosa/metabolismo , Antígenos/metabolismoRESUMEN
Severe fever with thrombocytopenia syndrome (SFTS) is an emerging life-threatening infectious disease caused by the tickborne SFTS virus (SFTSV), first identified in China in 2009 and then in Japan in 2013. Human SFTS cases were reported to be concentrated in western Japan, but the epidemiological conditions of SFTSV infection in a specific region are still obscure. We performed an epidemiological study of SFTSV in Oita Prefecture on the island of Kyushu, located in western Japan. For our research, we collected sera from wild and domestic animals (deer, wild boars, raccoons, cats, and dogs) and ticks from January 2010 to November 2020 in Oita. The anti-SFTSV antibody positivity rate of deer in 2014 was significantly higher than that in 2011 (65% versus 27%, P < 0.001). The anti-SFTSV antibody positivity rates of deer, wild boars, raccoons, wild dogs, domestic dogs, and domestic cats were 55%, 12%, 27%, 1.8%, 0.53%, and 1.4%, respectively. Moreover, RT-PCR could not detect SFTSV in any tick sample. Of the six areas of Oita Prefecture, only the Eastern area showed no incidence or possibility of SFTSV infection among wild and domestic animals, ticks, and human beings. Further investigation is required to assess whether local seroepidemiology in animals will help assess the risk of SFTSV infections in inhabitants.
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Ribosome biogenesis is an energetically expensive program that is dictated by nutrient availability. Here we report that nutrient deprivation severely impairs precursor ribosomal RNA (pre-rRNA) processing and leads to the accumulation of unprocessed rRNAs. Upon nutrient restoration, pre-rRNAs stored under starvation are processed into mature rRNAs that are utilized for ribosome biogenesis. Failure to accumulate pre-rRNAs under nutrient stress leads to perturbed ribosome assembly upon nutrient restoration and subsequent apoptosis via uL5/uL18-mediated activation of p53. Restoration of glutamine alone activates p53 by triggering uL5/uL18 translation. Induction of uL5/uL18 protein synthesis by glutamine is dependent on the translation factor eukaryotic elongation factor 2 (eEF2), which is in turn dependent on Raf/MEK/ERK signaling. Depriving cells of glutamine prevents the activation of p53 by rRNA synthesis inhibitors. Our data reveals a mechanism that tumor cells can exploit to suppress p53-mediated apoptosis during fluctuations in environmental nutrient availability.
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Glutamina , Neoplasias , Glutamina/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico , Precursores del ARN/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Micafungin, a newly developed echinocandin-type antifungal agent, is widely used for the treatment of deep-seated fungal infections including those of Candida species and Aspergillus species. In the present study, the possible alterations in the pharmacokinetics and biliary excretion of micafungin were investigated in endotoxemic rats induced by Klebsiella pneumoniae endotoxin. Endotoxin (2 mg/kg) was injected intraperitoneally 24 h before an intravenous injection of micafungin (1 mg/kg). No significant differences in the plasma concentration-time curves and pharmacokinetic parameters of micafungin were observed between endotoxin-treated and endotoxin-untreated rats. When endotoxin-treated rats received a constant-rate infusion of micafungin, the biliary clearance of micafungin was significantly decreased, whereas the steady-state plasma concentration did not change. By protein immunoblot analysis, a significant decrease in the expression of hepatic multidrug resistance-associated protein 2 (ABCC2/Mrp2), which is an efflux protein for micafungin, was observed in endotoxin-treated rats. These results suggest that endotoxin-induced decrease in the hepatobiliary excretion of micafungin is caused, at least in part, by the reduction of Mrp2-mediated hepatobiliary transport ability. The present study may provide information suggesting that micafungin can be used for patients with endotoxemia without the need for dosage adjustment.
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Sistema Biliar/metabolismo , Equinocandinas/farmacocinética , Endotoxinas/farmacología , Lipopéptidos/farmacocinética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Animales , Sistema Biliar/efectos de los fármacos , Transporte Biológico , Equinocandinas/química , Equinocandinas/metabolismo , Endotoxemia/tratamiento farmacológico , Endotoxemia/microbiología , Endotoxinas/administración & dosificación , Endotoxinas/metabolismo , Klebsiella pneumoniae/patogenicidad , Lipopéptidos/química , Lipopéptidos/metabolismo , Masculino , Micafungina , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Ratas , Ratas Sprague-DawleyRESUMEN
In this study, the involvement of sulfate conjugation and drug efflux transporter multidrug resistance-associated protein 2 (Mrp2) in sex-related differences in the pharmacokinetics of a new quinolone antimicrobial agent, garenoxacin, was investigated in Sprague-Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBRs) lacking Mrp2. The disappearance of garenoxacin from plasma in female SD rats was significantly faster than that in male SD rats after a single intravenous injection of garenoxacin (5 mg/kg). The systemic clearance of garenoxacin in female rats was approximately threefold larger than that of male rats (2.43 ± 0.31 and 0.87 ± 0.06 l/h/kg, respectively), suggesting the existence of sex-related differences in the pharmacokinetics of garenoxacin. When rats received a constant-rate infusion of garenoxacin, the contribution of biliary and renal excretion of garenoxacin was small, and no significant difference in the biliary (CL(BILE)) clearance of garenoxacin was observed between male and female SD rats. The metabolic clearance [CL(M (SULF))] of garenoxacin to garenoxacin sulfate conjugate (which is mainly excreted into the bile) in female SD rats was 8.5-fold larger than that in male SD rats (27.9 ± 2.94 and 3.28 ± 0.07 ml/h/kg, respectively). The CL(BILE) of garenoxacin was decreased in male and female EHBRs by approximately 50% compared with that in male and female SD rats. These results suggest that sulfate conjugation, but not Mrp2, is mainly involved in the sex-related differences in the pharmacokinetics of garenoxacin.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Antibacterianos/farmacocinética , Fluoroquinolonas/farmacocinética , Transportadoras de Casetes de Unión a ATP/genética , Análisis de Varianza , Animales , Antibacterianos/sangre , Antibacterianos/química , Bilis/química , Bilis/metabolismo , Femenino , Fluoroquinolonas/sangre , Fluoroquinolonas/química , Hiperbilirrubinemia/metabolismo , Masculino , Tasa de Depuración Metabólica , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Factores Sexuales , Sulfatos/química , Sulfatos/metabolismoRESUMEN
A fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen fragment-size compounds that weakly and slowly inhibit protein-peptide interactions was established. The interactions were detected by the increased diffusion time of a fluorescently labeled peptide probe after binding to its interacting protein. We analyzed the interactions between the c-Cbl TKB domain and phosphopeptides derived from ZAP-70, APS, and EGFR with the FCS assay and obtained 6 hit fragments that bound to the c-Cbl interaction sites. The binding amounts of the fragments were measured by direct binding measurements using surface plasmon resonance, and 5 fragments were found to bind selectively. The effect of 2 of the 5 fragments on the interaction with c-Cbl and the peptide exhibited strong time dependency. Furthermore, the inhibition by the selected 5 fragments on the protein-peptide interaction was confirmed by their effect on pull-down assays of c-Cbl with the biotin-conjugated interaction peptides. These results indicate the advantage of our FCS-based assay to study the time-dependent binding of compounds to their target protein.