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The aim of this study was to determine the proportion of near-miss dispensing errors in hospital pharmacies in Japan. A prospective multi-center observational study was conducted between December 2018 and March 2019. The primary objective was to determine the proportion of near-miss dispensing errors in hospital pharmacy departments. The secondary objective was to determine the predictive factors for near-miss dispensing errors using multiple logistic regression analysis. The study was approved by the ethical committee at The Institute of Medical Sciences, University of Tokyo, Japan. A multi-center prospective observational study was conducted in 20 hospitals comprising 8862 beds. Across the 20 hospitals, we assessed data from 553 pharmacists and 53039 prescriptions. A near-miss dispensing error proportion of 0.87% (n = 461) was observed in the study. We found predictive factors for dispensing errors in day-time shifts: a higher number of drugs in a prescription, higher number of quantified drugs, such as liquid or powder formula, in a prescription, and higher number of topical agents in a prescription; but we did not observe for career experience level for clinical pharmacists. For night-time and weekend shifts, we observed a negative correlation of near-miss dispensing errors with clinical pharmacist experience level. We found an overall incidence of near-miss dispensing errors of 0.87%. Predictive factors for errors in night-time and weekend shifts was inexperienced pharmacists. We recommended that pharmacy managers should consider education or improved work flow to avoid near-miss dispensing errors by younger pharmacists, especially those working night or weekend shifts.
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Potencial Evento Adverso , Farmacias , Hospitales , Humanos , Japón , Errores de Medicación/prevención & control , Farmacéuticos , Polvos , Estudios ProspectivosRESUMEN
BACKGROUND: Antimicrobial peptides (AMPs) play important roles in the innate immune system of all life forms and recently have been characterized as multifunctional peptides that have a variety of biological roles such as anticancer agents. However, detailed mechanism of antimicrobial peptides on cancer cells is still largely unknown. METHODS: miRNA array and real-time qPCR were performed to reveal the behavior of miRNA in colon cancer HCT116 cells during the growth suppression induced by the AMPs. Establishment of miR-663a over-expressing HCT116 cells was carried out for the evaluation of growth both in vitro and in vivo. To identify the molecular mechanisms, we used western blotting analysis. RESULTS: miR-663a is upregulated by administration of the human cathelicidin AMP, LL-37, and its analogue peptide, FF/CAP18, in the colon cancer cell line HCT116. Over-expression of miR-663a caused anti-proliferative effects both in vitro and in vivo. We also provide evidence supporting the view that these effects are attributed to suppression of the expression of the chemokine receptor CXCR4, resulting in the abrogation of phosphorylation of Akt and cell cycle arrest in G2/M via p21 activation. CONCLUSIONS: This study contributes to the understanding of the AMPs' mediated anti-cancer mechanisms in colon cancer cells and highlights the possibility of using AMPs and miRNAs towards developing future strategies for cancer therapy.
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Péptidos Catiónicos Antimicrobianos/administración & dosificación , Antineoplásicos/administración & dosificación , Neoplasias del Colon/tratamiento farmacológico , MicroARNs/genética , Receptores CXCR4/genética , Animales , Péptidos Catiónicos Antimicrobianos/farmacología , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Humanos , Ratones , Fosforilación , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba , Ensayos Antitumor por Modelo de Xenoinjerto , CatelicidinasRESUMEN
Nonhuman primates are useful animal models for the study of human diseases. However, the number of established cell lines from nonhuman primates is quite limited compared with the number established from other experimental animals. The establishment of nonhuman primate cell lines would allow drug testing on those cell lines before moving experiments into primates. In this study, we established nonhuman primate primary cell lines by introducing the genes for CDK4R24C, cyclin D1, and hTERT. These cell lines proliferated more rapidly than primary cells and bypassed cellular senescence. Karyotype analysis showed that the chromosome patterns were intact in the immortalized cell lines. Furthermore, we showed that the expression of introduced genes could be precisely controlled through the Tet-Off system with the addition of doxycycline. The present study shows that introduction of the CDK4R24C, cyclin D1, and hTERT genes are effective methods of establishing nonhuman primate cell lines.
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Proteínas de Ciclo Celular/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica/genética , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Transformada , Proliferación Celular/genética , Células Cultivadas , Senescencia Celular/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Doxiciclina/farmacología , Femenino , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/genética , Humanos , Immunoblotting , Cariotipo , Lentivirus/genética , Macaca , Masculino , Mutación Missense , Telomerasa/genética , Telomerasa/metabolismo , TransfecciónRESUMEN
Leptospiral lipopolysaccharide (L-LPS) has shown potency in activating toll-like receptor 2 (TLR2) in pig fibroblasts (PEFs_NCC1), and causes the expression of proinflammatory cytokines. However, the stimulation by L-LPS was weak eliciting the function of TLR2 sufficiently in pig innate immunity responses during Leptospira infection. In this study, the immune response of pig embryonic fibroblast cell line (PEFs_SV40) was investigated and was found to be the high immune response, thus TLR2 is the predominate receptor of L-LPS in pig cells. Further, we found a strategy using the antibody against L-LPS, to prevent L-LPS interaction with TLR2 in pig cells which could impact on immune activation.
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Induced pluripotent stem (iPS) cells have proven to be an effective technology in regenerative medicine; however, the low efficiency of reprogramming is a major obstacle to the successful generation of iPS cell lines. One of the most important characteristics of a high-quality iPS cell line is the inactivation of transgenes driven by a retrovirus-derived long terminal repeat promoter. In this study, we established a novel marker system containing three kinds of proteins: secreted-type luciferase (MetLuc), copepod Pontellina plumata green fluorescent protein (copGFP), and an antibiotic-resistant gene product (Neo(r)). The introduction of MetLuc-copGFP-Neo(r) in mouse embryonic fibroblasts (MEFs) allowed us to monitor the reporter expression changes as an indicator of the state of silencing during reprogramming. Transformation of iPS cells induced a remarkable reduction in reporter activity, indicating that the retroviral silencing was detected successfully. Our system enables us to monitor the silencing status of transgenes and to efficiently select iPS cell lines that can be used for further applications.
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Silenciador del Gen , Genes Reporteros , Ingeniería Genética/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Lentivirus/genética , Animales , Diferenciación Celular , Reprogramación Celular/genética , Embrión de Mamíferos , Fibroblastos/citología , Fibroblastos/metabolismo , Vectores Genéticos , Proteínas Fluorescentes Verdes/antagonistas & inhibidores , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/citología , Luciferasas/antagonistas & inhibidores , Luciferasas/genética , Luciferasas/metabolismo , Ratones , TransgenesRESUMEN
Antimicrobial peptides of the cathelicidin family play a central role in the host defense system. Our group has reported previously that cathelicidin-related or cathelicidin-modified antimicrobial peptides, such as FF/CAP-18, have antiproliferative effects on the squamous cell carcinoma cell line SAS-H1 and colon cancer-derived cell line HCT116. Ceragenin CSA-13, which mimics the hydrophobic and cationic morphology of cathelicidin-related peptides, was developed to reduce synthetic costs and resolve stability issues in the presence of proteases. In this study, we evaluated the antiproliferative effect of CSA-13 on HCT116 cells. We evaluated the effects of CSA-13 in HCT116 cells by measuring cell growth, detecting apoptosis, analyzing the cell cycle, and examining mitochondrial membrane depolarization. Treatment with CSA-13 suppressed HCT116 cell proliferation in a dose-dependent manner, increasing the incidence of apoptosis detected by the binding of Annexin V. Furthermore, cell cycle analysis showed that the cell cycle of CSA-13-treated wild-type and p53 null mutant HCT116 cells was arrested at the G1/S phase, indicating that CSA-13 affects the cell cycle by a p53-independent pathway. Our study showed that CSA-13 exerts an antiproliferative effect in cancer cells similar to that of FF/CAP-18, suggesting that membrane-permeabilizing capability is the common underlying mechanism for anticancer and antimicrobial effects of CSA-13 and anitimicrobial peptides.
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Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/patología , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Mutación , Esteroides/farmacología , Proteína p53 Supresora de Tumor/genética , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Activación Enzimática , Células HCT116 , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Factores de TiempoRESUMEN
The maintenance of stem cells often requires the support of feeder cells. Primary mouse embryonic fibroblasts (MEFs) have traditionally been used as feeder cells, and although these MEF-derived feeder cells have exhibited a reasonable performance, they require repeated cell isolation, since MEFs cannot expand indefinitely. To overcome this limitation, immortalized cells, such as STO cells, have been used. However, one major disadvantage is that previously reported immortalized cells can only support stem cell cultures for a relatively short period, typically 4 to 7â¯days. In this study, we found that our newly established rat-derived fibroblasts immortalized by the expression of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase, can function as feeder cells for relatively long cell culture periods of approximately 14â¯days. The rat-derived immortalized cells developed in this study should be a useful source of feeder cells to support stem cell research.
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Ciclina D/biosíntesis , Quinasa 4 Dependiente de la Ciclina/biosíntesis , Células Nutrientes/metabolismo , Fibroblastos/metabolismo , Mutación , Células Madre/metabolismo , Telomerasa/biosíntesis , Animales , Línea Celular Transformada , Técnicas de Cocultivo , Ciclina D/genética , Quinasa 4 Dependiente de la Ciclina/genética , Células Nutrientes/citología , Fibroblastos/citología , Ratones , Ratas , Células Madre/citología , Telomerasa/genéticaRESUMEN
In March 2011, an accident at the Fukushima Daiichi Nuclear Power Plant led to major problems, including the release of radionuclides such as Cesium (Cs)-137 into the environment. Ever since this accident, Cs-137 in foods has become a serious problem. In this study, we determined the concentration of Cs-137 in the feces, urine, and ruminal contents of cattle and demonstrated the possibility of its elimination from the body by intestinal bacteria. The results revealed a high Cs-137 concentration in the feces; in fact, this concentration was higher than that in skeletal muscles and other samples from several animals. Furthermore, intestinal bacteria were able to trap Cs-137, showing an uptake ratio within the range of 38-81% in vitro. This uptake appeared to be mediated through the sodium-potassium (Na+-K+) ion pump in the bacterial cell membrane. This inference was drawn based on the fact that the uptake ratio of Cs-137 was decreased in media with high potassium concentration. In addition, it was demonstrated that intestinal bacteria hindered the trapping of Cs-137 by the animal. Cattle feces showed high concentration of Cs-137 and intestinal bacteria trapped Cs-137. This study is the first report showing that intestinal bacteria contribute to the elimination of Cs-137 from the body.
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Antimicrobial peptides (AMPs) are multifunctional factors with an important role in the innate immune system. Our previous studies revealed that the human cathelicidin LL37 and its analog, FF/CAP18, limit the proliferation of colon cancer cell lines. In the present study, the exosomes released by HCT116 cells treated with FF/CAP18 were analyzed. After the treatment, exosomes were isolated from the culture supernatant by ultrafiltration and using the miRCURY™ Exosome Isolation Kit. Membrane vesicles 40100nm expressing CD63 and CD81 were identified before and after FF/CAP18 treatment. Exosome concentration in the culture supernatant was increased after treatment with FF/CAP18. Exosomes formed in HCT116 cells treated with FF/CAP18 induced growth suppression of the cells in a dosedependent manner. By contrast, the exosomes formed in nontreated HCT116 cells did not affect cell viability. Microarray analysis of miRNA expression indicated that FF/CAP18 treatment induced increases in the expression of three miRNAs (miR5845p, miR1202 and miR31625p) in both HCT116 cells and exosomes. These results suggest that FF/CAP18 treatment increases exosome formation, and that exosomeencapsulated miRNAs suppress HCT116 cell proliferation. Exosomal miRNAs are considered to be involved in the dissemination of cell signals to control local cellular microenvironments. The present findings suggest that FF/CAP18 regulates cancer growth by modulating celltocell communication. AMPs localize in the cytoplasm of cancer cells and enhance the expression of growthsuppressing miRNAs. These miRNAs are also transported to other cancer cells via exosomes. Therefore, transportation of these miRNAs has the potential to suppress cancer growth. AMPs exert their effects directly by targeting cancer cells and indirectly via exosomes.
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Péptidos Catiónicos Antimicrobianos/farmacología , Antineoplásicos/farmacología , Exosomas/efectos de los fármacos , Exosomas/genética , MicroARNs/genética , Anexina A7/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Exosomas/ultraestructura , Perfilación de la Expresión Génica , Células HCT116 , Humanos , Transporte de Proteínas , CatelicidinasRESUMEN
BACKGROUND: Antimicrobial peptides (AMPs) are considered promising candidates for the development of novel anti-infective agents. In arthropods such as ticks, AMPs form the first line of defense against pathogens in the innate immune response. Persulcatusin (IP) was found in the Ixodes persulcatus midgut, and its amino acid sequence was reported. However, the complete structure of IP has not been identified. We evaluated the relation between structural features and antimicrobial activity of IP, and its potential as a new anti-methicillin-resistant Staphylococcus aureus (MRSA) agent. METHODS: The structure of IP was predicted using homology modeling and molecular dynamics. IP and other tick AMPs were synthesized using a solid-phase method and purified by high-performance liquid chromatography. Methicillin-susceptible S. aureus (MSSA) and MRSA were used for the minimum inhibitory concentration (MIC) test and short-time killing assay of IP and other tick peptides. The influence of IP on mammalian fibroblasts and colon epithelial cells and each cell DNA and its hemolytic activity towards human erythrocytes were also examined. RESULTS: In the predicted IP structure, the structure with an S-S bond was more stable than that without an S-S bond. The MIC after 24 h of incubation with IP was 0.156-1.25 µg/mL for MSSA and 0.625-2.5 µg/mL for MRSA. Compared with the mammalian antimicrobial peptide and other tick peptides, IP was highly effective against MRSA. Moreover, IP showed a dose-dependent bactericidal effect on both MSSA and MRSA after 1 h of incubation. IP had no observable effect on mammalian cell growth or morphology, on each cell DNA and on human erythrocytes. CONCLUSIONS: We predicted the three-dimensional structure of IP and found that the structural integrity was maintained by three S-S bonds, which were energetically important for the stability and for forming α helix and ß sheet. IP has cationic and amphipathic properties, which might be related to its antimicrobial activity. Furthermore, the antimicrobial activity of IP against MRSA was stronger than that of other antimicrobial peptides without apparent damage to mammalian and human cells, demonstrating its possible application as a new anti-MRSA medicine.
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Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas de Insectos/aislamiento & purificación , Proteínas de Insectos/farmacología , Ixodes/química , Staphylococcus aureus/efectos de los fármacos , Animales , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/química , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Eritrocitos/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Hemólisis , Humanos , Proteínas de Insectos/síntesis química , Proteínas de Insectos/química , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Conformación ProteicaRESUMEN
The prairie vole (Microtus ochrogaster) shows social behaviors such as monogamy and parenting of infants with pair bonding. These social behaviors are specific to the prairie vole and have not been observed in other types of voles, such as mountain voles. Although the prairie vole has several unique characteristics, an in vitro cell culture system has not been established for this species. Furthermore, establishment of cultured cells derived from the prairie vole may be beneficial based on the three Rs (i.e., Replacement, Reduction, and Refinement) concept. Therefore, in this study, we attempted to establish an immortalized cell line derived from the prairie vole. Our previous research has shown that transduction with mutant forms of cyclin-dependent kinase 4 (CDK4), cyclin D, and telomerase reverse transcriptase (TERT) could efficiently immortalize cells from multiple species, including humans, cattle, pigs, and monkeys. Here, we introduced these three genes into prairie vole-derived muscle fibroblasts. The expression of mutant CDK4 and cyclin D proteins was confirmed by western blotting, and telomerase activity was detected in immortalized vole muscle-derived fibroblasts (VMF-K4DT cells or VMFs) by stretch PCR. Population doubling analysis showed that the introduction of mutant CDK4, cyclin D, and TERT extended the lifespan of VMFs. To the best of our knowledge, this is the first report describing the establishment of an immortalized cell line derived from the prairie vole through the expression of mutant CDK4, cyclin D, and human TERT.
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Arvicolinae , Ciclina D/genética , Quinasa 4 Dependiente de la Ciclina/genética , Técnicas Citológicas , Vectores Genéticos , Lentivirus , Telomerasa/genética , Transducción Genética/métodos , Animales , Western Blotting , Línea Celular , Fibroblastos , Expresión Génica , Humanos , Músculos/citología , Reacción en Cadena de la PolimerasaRESUMEN
Antimicrobial peptides (AMPs) play a critical role in innate host defense against microbial pathogens in many organisms. The human cathelicidin, LL-37, has a net positive charge and is amphiphilic, and can eliminate pathogenic microbes directly via electrostatic attraction toward negatively charged bacterial membranes. A number of studies have shown that LL-37 participates in various host immune systems, such as inflammatory responses and tissue repair, in addition to its antibacterial properties. Moreover, recent evidence suggests that it is also involved in the regulation of cancer. Indeed, previous studies have suggested that human LL-37 is involved in carcinogenesis via multiple reporters, such as FPR2 (FPRL1), epidermal growth factor receptor, and ERBb2, although LL-37 and its fragments and analogs also show anticancer effects in various cancer cell lines. This discrepancy can be attributed to peptide-based factors, host membrane-based factors, and signal regulation. Here, we describe the association between AMPs and cancer with a focus on anticancer peptide functions and selectivity in an effort to understand potential therapeutic implications.
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Pigs throughout the world are afflicted with leptospirosis, causing serious economic losses and potential hazards to human health. Although it has been known that leptospiral lipopolysaccharide (L-LPS) is involved in an immunological reaction between an antigen and a host cell, little is known about how the immune system of pigs can respond to L-LPS. Here, we stimulated pig fibroblasts by L-LPS and then quantitatively measured gene and protein expression levels of two toll-like receptors (TLRs), TLR2 and TLR4, by real-time PCR and Western blotting. As a result, expression of TLR2 was found to be significantly up-regulated within 24 h after L-LPS stimulation whereas induction of TLR4 expression was relatively weak. We also revealed that of myeloid differentiation primary response gene 88 (MyD88), interleukin 6 (IL-6) and IL-8 gene expressions were markedly up-regulated by L-LPS stimulation. These results may suggest that the pig cell can activate TLR2 rather than TLR4 by L-LPS stimulation, thereby inducing expression of cytokines.
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Fibroblastos/metabolismo , Mediadores de Inflamación/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Lipopolisacáridos/efectos adversos , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Animales , Células Cultivadas , Expresión Génica/efectos de los fármacos , Leptospira interrogans/química , Lipopolisacáridos/aislamiento & purificación , Datos de Secuencia Molecular , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Estimulación Química , Porcinos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Metabolic reprogramming is one of the hallmarks of cancer and can be targeted by therapeutic agents. We previously reported that cathelicidin-related or modified antimicrobial peptides, such as FF/CAP18, have antiproliferative effects on the squamous cell carcinoma cell line SAS-H1, and the colon carcinoma cell line HCT116. Although antimicrobial peptides have potential use in the development of new therapeutic strategies, their effects on the metabolism of cancer cells are poorly understood. Here, we investigated changes in the levels of metabolites in HCT116 cells caused by FF/CAP18, via capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Analysis of the 177 intracellular metabolites and 113 metabolites in conditioned medium that were detected by CE-TOFMS, revealed dramatic changes in the metabolic profile of HCT116 cells after treatment with FF/CAP18. The metabolic profile showed that the levels of most metabolites in the major metabolic pathways supported the rapid proliferation of cancer cells. Purine metabolism, glycolysis, and the TCA cycle, were altered in FF/CAP18-treated cells in a dose-dependent manner. Our present study provides mechanistic insights into the anticancer effects of antimicrobial peptides that show great potential as new therapies for colon cancer.
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Péptidos Catiónicos Antimicrobianos/farmacología , Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Metaboloma , Apoptosis , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Células HCT116 , Humanos , Espectrometría de Masas/métodos , Metabolómica/métodos , CatelicidinasRESUMEN
Cattle are the economically important animals in human society. They are essential for the production of livestock products such as milk and meats. The production efficiency of livestock products is negatively impacted by infection with zoonotic pathogens. To prevent and control infectious diseases, it is important to understand the interaction between cattle tissue and pathogenic bacteria. In this study, we established an in vitro infection model of an immortalized bovine colon-derived epithelial cell line by transducing the cells with lentiviral vectors containing genes encoding cell cycle regulators cyclin D1, mutant cyclin dependent kinase 4 (CDK4), and human telomerase reverse transcriptase (TERT). The established cell line showed continuous cell proliferation, expression of epithelial markers, and an intact karyotype, indicating that the cells maintained their original nature as colon-derived epithelium. Furthermore, we exposed the established cell line to two strains of Salmonella enterica and EHEC. Interestingly, S. Typhimurium showed higher affinity for the established cell line and invaded the cytoplasm than S. Enteritidis. Quantitative RT-PCR revealed that gene expression of Toll-like receptor 1 (TLR1), TLR 2 and TLR 3, whereas TLR 4, 5 and 6 were not detectable in established cells. Our established immortalized colon-derived epithelial cell should be a useful tool for studies evaluating the molecular mechanisms underlying bacterial infection.
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Colon/citología , Ciclina D1/genética , Quinasa 4 Dependiente de la Ciclina/genética , Escherichia coli Enterohemorrágica/fisiología , Mucosa Intestinal/citología , Salmonella enterica/fisiología , Telomerasa/genética , Animales , Adhesión Bacteriana , Biomarcadores/metabolismo , Bovinos , División Celular , Línea Celular , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Mucosa Intestinal/metabolismo , Cariotipo , Masculino , Mutación , Fenotipo , Receptores Toll-Like/genéticaRESUMEN
Embryonic stem cells and induced pluripotent stem (iPS) cells are usually maintained on feeder cells derived from mouse embryonic fibroblasts (MEFs). In recent years, the cell culture of iPS cells under serum- and feeder-free conditions is gaining attention in overcoming the biosafety issues for clinical applications. In this study, we report on the use of multiple small-molecular inhibitors (i.e., CHIR99021, PD0325901, and Thiazovivin) to efficiently cultivate mouse iPS cells without feeder cells in a chemically-defined and serum-free condition. In this condition, we showed that mouse iPS cells are expressing the Nanog, Oct3/4, and SSEA-1 pluripotent markers, indicating that the culture condition is optimized to maintain the pluripotent status of iPS cells. Without these small-molecular inhibitors, mouse iPS cells required the adaptation period to start the stable cell proliferation. The application of these inhibitors enabled us the shortcut culture method for the cellular adaptation. This study will be useful to efficiently establish mouse iPS cell lines without MEF-derived feeder cells.
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Cattle and pigs comprise the most economically important livestock. Despite their importance, cultured cells from these species, which are useful for physiological analyses, are quite limited in cell banks. One of the reasons for the limited number of cell lines is the difficulty in their establishment. To overcome limitations in cell-line establishment, we attempted to immortalize bovine and porcine fibroblasts by transduction of multiple cell cycle regulators (mutant cyclin dependent kinase 4, cyclin D and telomerase reverse transcriptase). The transduced cells continued to display a stable proliferation rate and did not show cellular senescence. Furthermore, cell cycle assays showed that induction of these exogenous genes enhanced turnover of the cell cycle, especially at the G1-S phase. Furthermore, our established cell lines maintained normal diploid karyotypes at 98-100%. Our study demonstrated that bypassing p16/Rb-mediated cell arrest and activation of telomerase activity enabled efficient establishment of immortalized bovine- and porcine-derived fibroblasts. The high efficiency of establishing cell lines suggests that the networks of cell cycle regulators, especially p16/Rb-associated cell cycle arrest, have been conserved during evolution of humans, cattle, and pigs.
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Línea Celular , Ciclina D/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/citología , Telomerasa/metabolismo , Animales , Bovinos , Ciclo Celular , Proliferación Celular , Ciclina D/genética , Quinasa 4 Dependiente de la Ciclina/genética , Diploidia , Fibroblastos/metabolismo , Cariotipo , Porcinos , Telomerasa/genética , Transducción GenéticaRESUMEN
A bovine myeloid antimicrobial peptide antimicrobial peptide (BMAP-28) is a member of the cathelicidin family and acts as a component of innate immunity. There are few reports of susceptibility difference of methicillin-resistant Staphylococcus aureus (MRSA) and susceptible strains (MSSA) against BMAP-28. This study aims to clarify how a few amino acid substitutions of BMAP-28 are related to its antimicrobial activity using four analog peptides of BMAP-28. We also compared cellular fatty acid components of MSSA and MRSA using gas chromatography. We found that a few amino acid substitutions of BMAP-28 do not change antimicrobial activity. It was also revealed that the percentage of cis-11-eicosenoic acid in total detected fatty acids of MRSA was significantly higher than that of MSSA. In addition, the percentage of palmitic acid in total detected fatty acids of MRSA tended to be lower than that of MSSA. Our results will provide new information to deal with the question of differences in bacterial susceptibility against BMAP-28.
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Antibacterianos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Proteínas/farmacología , Staphylococcus aureus/efectos de los fármacos , Sustitución de Aminoácidos , Antibacterianos/química , Cromatografía de Gases , Farmacorresistencia Bacteriana , Ácidos Grasos/metabolismo , Ácidos Grasos Monoinsaturados/metabolismo , Staphylococcus aureus Resistente a Meticilina/metabolismo , Ácido Palmítico/metabolismo , Proteínas/química , Staphylococcus aureus/metabolismo , Relación Estructura-ActividadRESUMEN
The cathelicidin family is one of the several families of antimicrobial peptides (AMPs). A bovine myeloid antimicrobial peptide (BMAP-28) belongs to this family. Recently, the emergence of drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) has become a big problem. AMPs are expected to be leading compounds of new antibiotics against drug-resistant bacteria. In this study, we focused on the activity of BMAP-28 against bacterial cell surfaces. First, we observed morphological change of MRSA caused by BMAP-28 using a scanning probe microscope. We also studied activities of BMAP-28 against adherence of S. aureus to fibronectin, collagen type I, collagen type IV. We confirmed whether BMAP-28 can bind to lipoteichoic acid (LTA) of S. aureus. BMAP-28 was indicated as damaging the cell surface of MRSA. In a particular range of concentrations, BMAP-28 promoted adherence of S. aureus against fibronectin and collagens. It was revealed that BMAP-28 and LTA of S. aureus bound with each other. Our study showed the potential of BMAP-28 which can damage MRSA and interact with LTA of S. aureus but promote its adherence in some concentrations. This study provides new points of which to take notice when we use AMPs as medicines.
Asunto(s)
Adhesión Bacteriana/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Proteínas/farmacología , Staphylococcus aureus Resistente a Meticilina/citología , Microscopía Electrónica de RastreoRESUMEN
BACKGROUND AND AIMS: Large-scale epidemiological studies have shown that drinking more than two cups of coffee per day reduces the risks of hepatitis and liver cancer. However, the heterogeneity of the human genome requires studies of experimental animal models with defined genetic backgrounds to evaluate the coffee effects on liver diseases. We evaluated the efficacy of coffee consumption with one of experimental animal models for human disease. METHOD: We used the Long Evans Cinnamon (LEC) rat, which onsets severe hepatitis and high incidence of liver cancer, due to the accumulation of copper and iron in livers caused by the genetic mutation in Atp7B gene, and leading to the continuous oxidative stress. We determined the expression of inflammation related genes, and amounts of copper and iron in livers, and incidence of the pre-neoplastic foci in the liver tissue of LEC rats. RESULTS: Coffee administration for 25 weeks delayed the occurrence of hepatitis by two weeks, significantly improved survival, reduced the expression of inflammatory cytokines, and reduced the incidence of small pre-neoplastic liver foci in LEC rats. There was no significant difference in the accumulation of copper and iron in livers, indicating that coffee administration does not affect to the metabolism of these metals. These findings indicate that drinking coffee potentially prevents hepatitis and liver carcinogenesis through its anti-inflammatory effects. CONCLUSION: This study showed the efficacy of coffee in the prevention of hepatitis and liver carcinogenesis in the LEC model.