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BACKGROUND: Abundant evidence suggests that chronic inflammation is linked to prostate cancer and that infection is a possible cause of prostate cancer. METHODS: To identify microbiota or pathogens associated with prostate cancer, we investigated the transcriptomes of 20 human prostate cancer tissues. We performed de novo assembly of nonhuman sequences from RNA-seq data. RESULTS: We identified four bacteria as candidate microbiota in the prostate, including Moraxella osloensis, Uncultured chroococcidiopsis, Cutibacterium acnes, and Micrococcus luteus. Among these, C. acnes was detected in 19 of 20 prostate cancer tissue samples by immunohistochemistry. We then analyzed the gene expression profiles of prostate epithelial cells infected in vitro with C. acnes and found significant changes in homologous recombination (HR) and the Fanconi anemia pathway. Notably, electron microscopy demonstrated that C. acnes invaded prostate epithelial cells and localized in perinuclear vesicles, whereas analysis of γH2AX foci and HR assays demonstrated impaired HR repair. In particular, BRCA2 was significantly downregulated in C. acnes-infected cells. CONCLUSIONS: These findings suggest that C. acnes infection in the prostate could lead to HR deficiency (BRCAness) which promotes DNA double-strand breaks, thereby increasing the risk of cancer development.
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Células Epiteliales , Próstata , Neoplasias de la Próstata , Masculino , Humanos , Neoplasias de la Próstata/microbiología , Neoplasias de la Próstata/patología , Células Epiteliales/microbiología , Células Epiteliales/patología , Células Epiteliales/metabolismo , Próstata/microbiología , Próstata/patología , Próstata/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Propionibacteriaceae/patogenicidadRESUMEN
The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.
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Lipasa/metabolismo , Fosfolípidos/metabolismo , Membranas Sinápticas/metabolismo , Animales , Transporte de Proteínas , RatasRESUMEN
Mesenchymal stem (stromal) cells (MSCs) are being investigated for treating degenerative and inflammatory disorders because of their reparative and immunomodulatory properties. Intricate mechanisms relate cell death processes with immune responses, which have implications for degenerative and inflammatory conditions. We review the therapeutic value of MSCs in terms of preventing regulated cell death (RCD). When cells identify an insult, specific intracellular pathways are elicited for execution of RCD processes, such as apoptosis, necroptosis, and pyroptosis. To some extent, exacerbated RCD can provoke an intense inflammatory response and vice versa. Emerging studies are focusing on the molecular mechanisms deployed by MSCs to ameliorate the survival, bioenergetics, and functions of unfit immune or nonimmune cells. Given these aspects, and in light of MSC actions in modulating cell death processes, we suggest the use of novel functional in vitro assays to ensure the potency of MSCs for preventing RCD. Such analyses should be associated with existing functional assays measuring the anti-inflammatory capabilities of MSCs in vitro. MSCs selected on the basis of two in vitro functional criteria (i.e., prevention of inflammation and RCD) could possess optimal therapeutic efficacy in vivo. In addition, we underline the implications of these perspectives in clinical studies of MSC therapy, with particular focus on acute respiratory distress syndrome. Stem Cells Translational Medicine 2017;6:713-719.
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Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Animales , Biomarcadores/metabolismo , Muerte Celular , HumanosRESUMEN
The cyst wall of the resting cyst of the ciliated protozoan Colpoda cucullus (Nag-1 strain) is composed of several layers of endocyst, a single layer of ectocyst associated with a mucous layer and lepidosomes composed of a fibrous or crystal-like structure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the ectocyst associated with lepidosomes and mucous materials contained proteins corresponding to 27, 31, 45 kDa and smear bands ranging from 50 to 60 kDa. Liquid chromatography-tandem mass spectrometry of these proteins revealed that the 45-kDa protein (p45) was elongation factor Tu (EF-Tu). Immunofluorescence microscopy with an anti-EF-Tu polyclonal antibody showed that Colpoda EF-Tu (p45) was localized in the lepidosomes. The lepidosomes were stained vividly with Congo red, which is bound to the stacked ß-sheets of amyloid protofibrils. In the presence of puromycin, no cyst wall components including lepidosomes were formed, indicating that cyst wall formation requires synthesis of proteins including EF-Tu. Electron microscopy of encysting cells implied that vesicles which were presumably budded from endoplasmic reticula possibly fuse with a lepidosome-precursor vacuole containing electron-dense fine particles or fibrous structures, and followed by the subsequent fusion with other electron-lucent granules.
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The biological effects of indium-tin-oxide (ITO) are of considerable importance because workers exposed to indium compounds have been diagnosed with interstitial lung disease or pulmonary alveolar proteinosis; however, the pathophysiology of these diseases is undefined. Here, mice intraperitoneally inoculated with ITO-nanoparticles (ITO-NPs) resulted in peritonitis dependent in NLRP3 inflammasome, with neutrophils recruitment and interleukin-1ß (IL-1ß) production. Withal peritoneal macrophages exposed ex vivo to ITO-NPs caused IL-1ß secretion and cytolysis. Further, alveolar macrophages exposed to ITO-NPs in vitro showed ITO-NP endocytosis and production of tumor necrosis factor-α (TNF-α) and IL-1ß, ensued cell death by cytolysis. This cell death was RIPK1-independent but caspase1-dependent, and thus identified as pyroptosis. Endocytosis of ITO-NPs by activated THP-1 cells induced pyroptosis with IL-1ß/TNF-α production and cytolysis, but not in activated THP-1 cells with knockdown of NLRP3, ASC, or caspase1. However, exposing activated THP-1 cells with NLRP3 or ASC knockdown to ITO-NPs resulted in cell death but without cytolysis, with deficiency in IL-1ß/TNF-α, and revealing features of apoptosis. While, mesenchymal stem cells (MSCs) co-cultured with macrophages impaired both inflammation and cell death induced by ITO-NPs. Together, our findings provide crucial insights to the pathophysiology of respiratory diseases caused by ITO particles, and identify MSCs as a potent therapeutic.
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Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasa 1/metabolismo , Macrófagos/metabolismo , Células Madre Mesenquimatosas/fisiología , Piroptosis , Receptores de Superficie Celular/metabolismo , Compuestos de Estaño/metabolismo , Animales , Células Cultivadas , Técnicas de Cocultivo , Endocitosis , Humanos , Interleucina-1beta/metabolismo , Ratones , Nanopartículas/administración & dosificación , Nanopartículas/metabolismo , Neutrófilos/inmunología , Peritonitis/inducido químicamente , Compuestos de Estaño/administración & dosificaciónRESUMEN
MicroRNAs (miRNAs) are involved in the progression and suppression of various diseases through translational inhibition of target mRNAs. Therefore, the alteration of miRNA biogenesis induces several diseases. The nuclear factor 90 (NF90)-NF45 complex is known as a negative regulator in miRNA biogenesis. Here, we showed that NF90-NF45 double-transgenic (dbTg) mice develop skeletal muscle atrophy and centronuclear muscle fibers in adulthood. Subsequently, we found that the levels of myogenic miRNAs, including miRNA 133a (miR-133a), which promote muscle maturation, were significantly decreased in the skeletal muscle of NF90-NF45 dbTg mice compared with those in wild-type mice. However, levels of primary transcripts of the miRNAs (pri-miRNAs) were clearly elevated in NF90-NF45 dbTg mice. This result indicated that the NF90-NF45 complex suppressed miRNA production through inhibition of pri-miRNA processing. This finding was supported by the fact that processing of pri-miRNA 133a-1 (pri-miR-133a-1) was inhibited via binding of NF90-NF45 to the pri-miRNA. Finally, the level of dynamin 2, a causative gene of centronuclear myopathy and concomitantly a target of miR-133a, was elevated in the skeletal muscle of NF90-NF45 dbTg mice. Taken together, we conclude that the NF90-NF45 complex induces centronuclear myopathy through increased dynamin 2 expression by an NF90-NF45-induced reduction of miR-133a expression in vivo.
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Dinamina II/genética , MicroARNs/genética , Músculo Esquelético/patología , Atrofia Muscular/genética , Proteína del Factor Nuclear 45/genética , Proteínas del Factor Nuclear 90/genética , Animales , Línea Celular , Dinamina II/metabolismo , Femenino , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Proteína del Factor Nuclear 45/metabolismo , Proteínas del Factor Nuclear 90/metabolismo , Regulación hacia ArribaRESUMEN
Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle.
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Centrosoma/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Huso Acromático/metabolismo , Antígenos Nucleares/metabolismo , Proteínas de Ciclo Celular , Humanos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismoRESUMEN
A Gram-stain-negative, non-motile, mesophilic, aerobic, rod-shaped bacterium, strain 2-9(T), was isolated from surface seawater at Muroto city, Kochi prefecture, Japan. The strain was transparent on 1/5 strength marine broth plate but became easily visible when the plate was supplemented with pyruvate. Phylogenetic analyses based on the 16S rRNA gene sequence showed that the strain fell within the class Gammaproteobacteria and was most closely related to the genus Arenicella (92.7-93.0 % 16S rRNA gene sequence similarities to type strains of species of this genus) of an unclassified order within this class. The DNA G+C content of strain 2-9(T) was 41.7 mol%. The major fatty acids were C18 : 1ω7c (37.6 %), C16 : 1ω7c and/or iso-C15 : 0 2-OH (summed feature 3; 19.1 %), C18 : 0 (10.8 %), C16 : 0 (10.2 %) and an unidentified fatty acid with an equivalent chain-length value of 11.799 (9.5 %). The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine and three unidentified lipids. Ubiquinone-8 (Q-8) was detected as the sole isoprenoid quinone. From these taxonomic data, it is proposed that strain 2-9(T) represents a novel species of a new genus, Perspicuibacter marinus gen. nov., sp. nov. The type strain of the type species is 2-9(T) (â= NBRC 110144(T)â= KCTC 42196(T)). A new family, Arenicellaceae fam. nov. (type genus Arenicella), and order, Arenicellales ord. nov., of the class Gammaproteobacteria are proposed to accommodate the novel taxon.
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Gammaproteobacteria/clasificación , Filogenia , Agua de Mar/microbiología , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Gammaproteobacteria/genética , Gammaproteobacteria/aislamiento & purificación , Japón , Datos de Secuencia Molecular , Fosfatidiletanolaminas/química , Fosfatidilgliceroles/química , Pigmentación , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Ubiquinona/químicaRESUMEN
In neurons, the plasma membrane is functionally separated into several distinct segments. Neurons form these domains by delivering selected components to and by confining them within each segment of the membrane. Although some mechanisms of the delivery are elucidated, that of the confinement is unclear. We show here that 1-oleoyl-2-palmitoyl-phosphatidylcholine (OPPC), a unique molecular species of phospholipids, is concentrated at the protrusion tips of several neuronal culture cells and the presynaptic area of neuronal synapses of the mouse brain. In PC12 cells, NGF-stimulated neuronal differentiation induces a phospholipase A1 activity at the protrusion tips, which co-localizes with the OPPC domain. Inhibition of the phospholipase A1 activity leads to suppression of phospholipid remodeling in the tip membrane and results in disappearance of the OPPC at the tips. In these cells, confinement of dopamine transporter and Gαo proteins to the tip was also disrupted. These findings link the lateral distribution of the molecular species of phospholipids to the formation of functional segments in the plasma membrane of neurons and to the mechanism of protein confinement at the synapse.
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Membrana Celular/metabolismo , Neuronas/metabolismo , Fosfatidilcolinas/metabolismo , Sinapsis/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Ratones , Factor de Crecimiento Nervioso/farmacología , Neuronas/citología , Células PC12 , Fosfolipasas A1/metabolismo , RatasRESUMEN
While NF90 has been known to participate in transcription, translation and microRNA biogenesis, physiological functions of this protein still remain unclear. To uncover this, we generated transgenic (Tg) mice using NF90 cDNA under the control of ß-actin promoter. The NF90 Tg mice exhibited a reduction in body weight compared with wild-type mice, and a robust expression of NF90 was detected in skeletal muscle, heart and eye of the Tg mice. To evaluate the NF90 overexpression-induced physiological changes in the tissues, we performed a number of analyses including CT-analysis and hemodynamic test, revealing that the NF90 Tg mice developed skeletal muscular atrophy and heart failure. To explore causes of the abnormalities in the NF90 Tg mice, we performed histological and biochemical analyses for the skeletal and cardiac muscles of the Tg mice. Surprisingly, these analyses demonstrated that mitochondria in those muscular tissues of the Tg mice were degenerated by autophagy. To gain further insight into the cause for the mitochondrial degeneration, we identified NF90-associated factors by peptide mass fingerprinting. Of note, approximately half of the NF90-associated complexes were ribosome-related proteins. Interestingly, protein synthesis rate was significantly suppressed by high-expression of NF90. These observations suggest that NF90 would negatively regulate the function of ribosome via its interaction with the factors involved in the ribosome function. Furthermore, we found that the translations or protein stabilities of PGC-1 and NRF-1, which are critical transcription factors for expression of mitochondrial genes, were significantly depressed in the skeletal muscles of the NF90 Tg mice. Taken together, these findings suggest that the mitochondrial degeneration engaged in the skeletal muscle atrophy and the heart failure in the NF90 Tg mice may be caused by NF90-induced posttranscriptional repression of transcription factors such as PGC-1 and NRF-1 for regulating nuclear-encoded genes relevant to mitochondrial function.
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Autofagia/fisiología , Mitofagia/fisiología , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Miocardio/metabolismo , Proteínas del Factor Nuclear 90/metabolismo , Actinas/genética , Animales , Presión Sanguínea , Western Blotting , Peso Corporal/genética , ADN Complementario/genética , Genotipo , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Proteínas del Factor Nuclear 90/genética , Factor Nuclear 1 de Respiración/metabolismo , Regiones Promotoras Genéticas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ribosómicas/metabolismo , Tomografía Computarizada por Rayos X , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: To investigate the histological and clinical relation between eyes with true exfoliation (TEX) and a double-ring sign (DRS). STUDY DESIGN: Retrospective clinical case study. PARTICIPANTS: Twenty-four eyes of 22 patients who underwent cataract surgery at Kochi Medical School Hospital and its affiliated hospitals during the period from April 1994 to September 2007. METHODS: Twenty-four anterior lens capsules excised in cataract surgery underwent a histological examination and their clinical backgrounds were investigated. The TEX group consisted of 13 eyes with capsular delamination on the anterior lens capsule confirmed by slit-lamp microscopy before the surgery, and the DRS group consisted of 11 eyes that showed a double-ring sign during capsulorrhexis. RESULTS: Twenty-two eyes underwent phacoemulsification and aspiration after uneventful capsulorrhexis. Two eyes that showed phacodonesis underwent extracapsular cataract extraction. In all specimens the capsular delamination was confirmed. In some specimens from both groups, vesicle spaces were observed in the capsule and the underlying epithelium. In the TEX group, 8 eyes had capsular complications, such as pseudoexfoliation and phacodonesis, and in the DRS group, 2 eyes had phacodonesis (p = 0.016, student's t test). CONCLUSIONS: In spite of different clinical processes leading to capsular delamination, there were not any specific histological differences distinguishing the TEX group from the DRS group. The incidence of capsular complications was significantly higher in the TEX group than in the DRS group.
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Síndrome de Exfoliación/diagnóstico , Cápsula del Cristalino/patología , Enfermedades del Cristalino/diagnóstico , Anciano , Anciano de 80 o más Años , Capsulorrexis , Femenino , Humanos , Masculino , Facoemulsificación , Estudios RetrospectivosRESUMEN
We report the successful purification of a cloned lysin encoded by the novel Staphylococcus aureus bacteriophage phi MR11. The lysin, designated MV-L, rapidly and completely lysed cells of a number of S. aureus strains tested, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus and a subset of vancomycin-intermediate S. aureus (VISA) in growing conditions. MV-L-mediated killing is specific to S. aureus and not to other species, except for S. simulans. MV-L exerted its staphylocidal effect synergistically with glycopeptide antibiotics against VISA. MV-L efficiently eliminated MRSA that had been artificially inoculated into the nares of mice. The intraperitoneal administration of MV-L also protected mice against MRSA septic death, without any harmful effects. Although MV-L evoked detectable levels of a humoral response in mice, the antibodies did not abolish the bacteriolytic activity. These results indicate that MV-L might be useful as a powerful therapeutic agent against multidrug-resistant S. aureus infections.
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Antibacterianos/administración & dosificación , Toxinas Bacterianas/administración & dosificación , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Proteínas Hemolisinas/administración & dosificación , Proteínas Recombinantes/biosíntesis , Fagos de Staphylococcus/metabolismo , Staphylococcus aureus/efectos de los fármacos , Administración Intranasal , Administración Tópica , Animales , Antibacterianos/aislamiento & purificación , Toxinas Bacterianas/aislamiento & purificación , Modelos Animales de Enfermedad , Endopeptidasas/administración & dosificación , Endopeptidasas/biosíntesis , Femenino , Proteínas Hemolisinas/aislamiento & purificación , Resistencia a la Meticilina/efectos de los fármacos , Ratones , Proteínas Recombinantes/aislamiento & purificación , Staphylococcus aureus/virología , Resistencia a la Vancomicina/efectos de los fármacosRESUMEN
Yellowtail ascites virus (YTAV) is the causative agent of ascites and deformity in fish and causes serious losses to the fish-farming industry of yellowtail fry and fingerling Seriola quinqueradiata in Japan. In 2006, cultured yellowtail died from ascites in Kochi, Japan. We isolated and characterized a virus from the diseased fish. Based on the pathogenicity, culture characteristics, morphological features, RT-PCR results targeting VP2/NS region, phylogeny based on the VP1 amino acid sequence, and immunochemical reactivity of structural proteins, the virus isolate was identified as YTAV (designated as YTAV-06). YTAV-06 was a more virulent isolate than YTAV Y-6, isolated originally from yellowtail with ascites. To our knowledge, this is the first report describing that YTAV isolates may vary in their virulence.
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Ascitis/veterinaria , Birnaviridae/patogenicidad , Virulencia/genética , Animales , Ascitis/virología , Birnaviridae/clasificación , Birnaviridae/genética , Birnaviridae/aislamiento & purificación , Línea Celular , Enfermedades de los Peces/virología , Peces , FilogeniaRESUMEN
Characteristic intracellular organelles of the foam cells in xanthoma are composed of membrane-bound lipid vacuoles, membrane-free lipid vacuoles, cholesterol crystals, multivesicular or multilocular lipid bodies, myelin-like bodies, and ceroid granules. We aimed to clarify the formation of myelin-like bodies and ceroid granules in the foam cells of xanthoma. We ultrastructurally examined mouse peritoneal macrophages incubated with human low-density lipoprotein (LDL) modified by incubation with xanthoma tissue, with xanthoma tissue-extracted LDL density substances, and with homogenized xanthoma tissue-derived crude material. A large number of membrane-bound and membrane-free lipid vacuoles were observed in macrophages incubated with xanthoma tissue-modified LDL. The macrophages incubated with the xanthoma tissue-extracted LDL density substances contained a large number of myelin-like bodies and ceroid granules. The macrophages incubated with the homogenized xanthoma tissue-derived crude material accumulated many vacuoles containing vesicular structures and a small number of myelin bodies and ceroid granules. Membrane-bound lipid vacuoles are derived from lysosomes that accumulate mostly extravasated modified LDL in xanthoma tissue. On the other hand, myelin-like bodies and ceroid granules are mostly derived from LDL density substances derived from xanthoma tissue homogenate.
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Ceroide/metabolismo , Células Espumosas/ultraestructura , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneales/ultraestructura , Vaina de Mielina/metabolismo , Xantomatosis/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Células Espumosas/metabolismo , Humanos , Lisosomas/metabolismo , Lisosomas/ultraestructura , Macrófagos Peritoneales/metabolismo , Ratones , Microscopía Electrónica de Transmisión , Vaina de Mielina/ultraestructura , Conejos , Vacuolas/metabolismo , Vacuolas/ultraestructura , Xantomatosis/patologíaRESUMEN
In IL-5 transgenic mice (C3H/HeN-TgN(IL-5)-Imeg), in which 50% of peripheral blood leukocytes are eosinophils, the development of infection by Leishmania amazonensis was clearly suppressed. To determine mechanistically how this protozoan parasite is killed, we performed in vitro killing experiments. Either IL-4 or IFN-gamma effectively stimulated eosinophils to kill Leishmania amazonensis promastigotes, and most of the killing was inhibited by catalase but not by the NO inhibitor L-N5-(1-iminoethyl)-ornithine, suggesting that hydrogen peroxide is responsible for the killing of L. amazonensis by eosinophils. There was no significant degranulation of eosinophils in the culture, because eosinophil peroxidase was not detected in culture supernatants when L. amazonensis promastigotes were killed by activated eosinophils. Such resistance was also observed in BALB/c mice, which are highly susceptible to L. amazonensis. Expression plasmids for IL-4, IL-5, and IFN-gamma were transferred into muscle by electroporation in vivo starting 1 week before infection. Expression plasmid for IL-5 was most effective in slowing the development of infection among three expression plasmids. Expression plasmid for IL-4 was slightly effective and that for IFN-gamma had no effect on the progress of disease. These results suggest that IL-5 gene transfer into muscle by electroporation is useful as a supplementary protection method against L. amazonensis infection.
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Eosinófilos/inmunología , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-4/inmunología , Interleucina-5/fisiología , Leishmania/patogenicidad , Leishmaniasis/prevención & control , Ornitina/análogos & derivados , Animales , Catalasa/farmacología , Cricetinae , Electroporación , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Eosinófilos/parasitología , Interleucina-4/genética , Interleucina-5/genética , Leishmania/efectos de los fármacos , Leishmania/crecimiento & desarrollo , Leishmaniasis/etiología , Leishmaniasis/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Transgénicos , Ornitina/farmacología , Superóxidos/metabolismoRESUMEN
Marine birnavirus (MABV), a member of the genus Aquabirnavirus, family Birnaviridae, is an unenveloped icosahedral virus with two genomes of double-stranded RNA. The mechanisms of MABV adsorption and penetration are still undetermined. This work examined MABV infection in susceptible and resistant fish cell lines. MABV adsorbed not only onto the cell surfaces of susceptible (CHSE-214 and RSBK-2) cells but also onto resistant (FHM and EPC) cells. Furthermore, the virus entered the cytoplasm through the endocytotic pathway in CHSE-214, RSBK-2 and FHM cells but did not penetrate EPC cells. Thus, restriction of the MABV replication cycle is different between resistant FHM and EPC cells. The virus was found to bind to an around 250 kDa protein on CHSE-214, RSBK-2, FHM and EPC cells. Thus, this 250 kDa protein may be a major MABV receptor that exists in the plasma membranes of all four cell lines examined. This result suggests further that another receptor for virus penetration may exist in CHSE-214, RSBK-2 and FHM cells but not in EPC cells.
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Aquabirnavirus/patogenicidad , Infecciones por Birnaviridae/virología , Peces/virología , Animales , Aquabirnavirus/aislamiento & purificación , Aquabirnavirus/metabolismo , Línea Celular/ultraestructura , Cyprinidae/virología , Susceptibilidad a Enfermedades , Endocitosis , Receptores Virales/metabolismo , Salmón/virología , Dorada/virología , Agua de MarRESUMEN
The cochlear stria vascularis produces the endolymph and generates the endocochlear DC potential, two indispensable ingredients of an auditory transduction process. The marginal cell, one of the several cell types constituting the stria vascularis, is called 'the dark cell' on the basis of its appearance by transmission electron microscopy (TEM). To clarify whether this commonly observed 'dark appearance' is a normal characteristic of marginal cells, as conjectured in the literature, or an experimental artifact, we developed an in vivo fixation method for minimizing ischemic tissue damages. While under sustained systemic circulation with oxygenated blood, the stria vascularis of gerbils was chemically fixed by perilymphatic perfusion with a fixative, and the stria vascularis was observed by TEM. In contrast to a number of previous reports, the cytoplasm of marginal cells was not dark, and quantitative analysis showed that the difference between the cytoplasmic electron density of marginal cells and that of intermediate cells (another type of strial cells) was not statistically significant. For comparison, the gerbils were allowed to undergo 3 min of ischemia following decapitation. Under these conditions, marginal cells showed typical 'dark appearance', as reported previously, and their cytoplasmic electron density was 1.7 times higher than that of the intermediate cells. In addition, the volume of mitochondria in marginal cells undergoing 3 min of ischemia was higher than that fixed in vivo. We therefore conclude that the widely recognized 'dark cell' appearance of marginal cells following conventional fixation procedures reflects cell injury due to ischemia, which is inherent in the standard fixation procedures, but can be avoided by our fixation protocol here introduced.