RESUMO
Iron overload has been recognized as a risk factor for liver disease; however, little is known about its pathological role in the modification of liver injury. The purpose of this study is to investigate the influence of iron overload on liver injury induced by two hepatotoxicants with different pathogenesis in rats. Rats were fed a control (Cont), 0.8% high-iron (0.8% Fe), or 1% high-iron diet (1% Fe) for 4 weeks and were then administered with saline, thioacetamide (TAA), or carbon tetrachloride (CCl4). Hepatic and systemic iron overload were seen in the 0.8% and 1% Fe groups. Twenty-four hours after administration, hepatocellular necrosis induced by TAA and hepatocellular necrosis, degeneration, and vacuolation induced by CCl4, as well as serum transaminase values, were exacerbated in the 0.8% and 1% Fe groups compared to the Cont group. On the other hand, microvesicular vacuolation induced by CCl4 was decreased in 0.8% and 1% Fe groups. Hepatocellular DNA damage was increased by iron overload in both models, whereas a synergistic effect of oxidative stress by excess iron and hepatotoxicant was only present in the CCl4 model. The data showed that dietary iron overload exacerbates TAA- and CCl4-induced acute liver injury with different mechanisms.
Assuntos
Tetracloreto de Carbono , Doença Hepática Induzida por Substâncias e Drogas , Sobrecarga de Ferro , Fígado , Tioacetamida , Animais , Tioacetamida/toxicidade , Ratos , Tetracloreto de Carbono/toxicidade , Masculino , Doença Hepática Induzida por Substâncias e Drogas/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Estresse Oxidativo/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Ratos Sprague-Dawley , Ferro/toxicidadeRESUMO
Interleukin (IL-19) belongs to the IL-10 family of cytokines and plays diverse roles in inflammation, cell development, viral responses, and lipid metabolism. Acute lung injury (ALI) is a severe respiratory condition associated with various diseases, including severe pneumonia, sepsis, and trauma, lacking established treatments. However, the role of IL-19 in acute inflammation of the lungs is unknown. We reported the impact of IL-19 functional deficiency in mice crossed with an ALI model using HCl. Lungs damages, neutrophil infiltration, and pulmonary edema induced by HCl were significantly worse in IL-19 knockout (KO) mice than in wild-type (WT) mice. mRNA expression levels of C-X-C motif chemokine ligand 1 (CXCL1) and IL-6 in the lungs were significantly higher in IL-19 KO mice than in WT mice. Little apoptosis was detected in lung injury in WT mice, whereas apoptosis was observed in exacerbated area of lung injury in IL-19 KO mice. These results are the first to show that IL-19 is involved in acute inflammation of the lungs, suggesting a novel molecular mechanism in acute respiratory failures. If it can be shown that neutrophils have IL-19 receptors and that IL-19 acts directly on them, it would be a novel drug target.
Assuntos
Lesão Pulmonar Aguda , Ácido Clorídrico , Interleucinas , Camundongos Knockout , Animais , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/genética , Interleucinas/genética , Interleucinas/metabolismo , Camundongos Endogâmicos C57BL , Interleucina-6/metabolismo , Interleucina-6/genética , Modelos Animais de Doenças , Infiltração de Neutrófilos , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Masculino , Pulmão/patologia , Pulmão/metabolismo , Apoptose/genética , Apoptose/efeitos dos fármacos , Camundongos , Neutrófilos , Edema Pulmonar/etiologia , Expressão GênicaRESUMO
Lipopolysaccharide (LPS) has dose-dependent biphasic functions (cell protective versus cell toxic). To clarify the different effects of LPS on liver homeostasis or liver diseases, comparisons were made between low and high doses of LPS, in terms of the mutual relation of hepatic macrophages, autophagy, and damage-associated molecular patterns (DAMPs) in male F344/DuCrlCrlj rats. Rats injected with low dose (0.1 mg/kg) or high dose (2.0 mg/kg) of LPS were examined at 6, 10, and 24 hours following single injections. Histologically, focal hepatocellular necrosis was occasionally present in high-dose animals, whereas there were no significant changes in low-dose animals. In low-dose animals, Kupffer cells reacting to CD163 and CD204 were hypertrophic and regarded as M2 macrophages, which promote resolution of inflammation and tissue repair, whereas in high-dose animals, infiltration of M1 macrophages expressing CD68 and major histocompatibility complex class II, which enhance cell injury, was seen. Hepatocytes with high-mobility-group box-1 (HMGB1) (one of DAMPs)-positive cytoplasmic granules appeared more frequently in high-dose animals than in low-dose animals, indicating the translocation of nuclear HMGB1 into the cytoplasm. However, although light-chain 3 beta-positive autophagosomes in hepatocytes increased in both doses, abnormally vacuolated autophagosomes were only seen in injured hepatocytes in the high-dose group, indicating possible extracellular release of HMGB1, which might result in cell injury and inflammation. These findings suggested that low-dose LPS induced a favorable mutual relationship among hepatic macrophages, autophagy, and DAMPs leading to cytoprotection of hepatocytes, whereas failures of the relationship in high-dose LPS caused hepatocyte injury.
Assuntos
Proteína HMGB1 , Hepatopatias , Masculino , Ratos , Animais , Lipopolissacarídeos/toxicidade , Proteína HMGB1/farmacologia , Ratos Endogâmicos F344 , Fígado/patologia , Macrófagos/patologia , Hepatopatias/patologia , Hepatopatias/veterinária , Inflamação/patologia , Inflamação/veterinária , AutofagiaRESUMO
Canine induced pluripotent stem cells (ciPSCs) provide a platform for regenerative veterinary medicine, disease modeling, and drug discovery. However, in the conventional method, ciPSCs are maintained using chemically-undefined media containing unknown animal components under on-murine embryonic fibroblast feeder conditions, which were reported to modify cell surface of iPSCs and increases the risk of immune rejection when the cells are transplanted into patients. Moreover, in the conventional method, ciPSCs are mechanically passaged, which requires much time and effort. Therefore, the large-scale expansion of ciPSCs is difficult, which should be resolved for using ciPSCs in clinical application and research. Here, it was shown that StemFit® AK02N and iMatrix-511 could maintain the pluripotency of ciPSCs using conventional culture method. Furthermore, it was demonstrated that the feeder-free and chemically-defined ciPSC culture systems using StemFit® AK02N and iMatrix-511 could stably maintain and allow the easy expansion of ciPSCs generated using N2B27 and StemFit® AK02N, without causing karyotype abnormalities. ciPSCs expressed several pluripotency markers and formed teratomas, including cells derived from three germ layers.
Assuntos
Técnicas de Cultura de Células , Meios de Cultura/farmacologia , Cães/anatomia & histologia , Células-Tronco Pluripotentes Induzidas/citologia , Cultura Primária de Células/métodos , Animais , Biomarcadores , Adesão Celular , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Células Cultivadas , Células Clonais , Técnicas de Cocultura , Meios de Cultura/análise , Camadas Germinativas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/transplante , Cariotipagem , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Teratoma/etiologia , Teratoma/patologiaRESUMO
Using auto-erasable Sendai virus vector, we generated ciPSC line. After several passages, virus was not present in ciPSCs by RT-PCR. ciPSCs from canine PBMCs had pluripotent state, differentiated all three germ layers in vitro, and had normal 78 XX karyotype. These results proved that PBMCs were one of the good cell sources to generate ciPSC lines from companion and patient dogs.
Assuntos
Cães , Células-Tronco Pluripotentes Induzidas/fisiologia , Leucócitos Mononucleares/fisiologia , Cultura Primária de Células , Vírus Sendai/fisiologia , Animais , Diferenciação Celular/genética , Linhagem Celular Transformada , Transformação Celular Viral/genética , Reprogramação Celular/genética , Feminino , Vetores Genéticos/genética , Células-Tronco Pluripotentes Induzidas/citologia , Cariótipo , Leucócitos Mononucleares/citologia , Cultura Primária de Células/métodos , Cultura Primária de Células/veterinária , Vírus Sendai/genéticaRESUMO
A monoclonal antibody (A3) was generated by using rat malignant fibrous histiocytoma (MFH) cells as the antigen. Generally, MFH is considered to be a sarcoma derived from undifferentiated mesenchymal cells. Molecular biological analyses using the lysate of rat MFH cells revealed that A3 is a conformation specific antibody recognizing both N-glycan and peptide. A3-labeled cells in bone marrow were regarded as somatic stem cells, because the cells partly coexpressed CD90 and CD105 (both immature mesenchymal markers). In the hair follicle cycle, particularly the anagen, the immature epithelial cells (suprabasal cells) near the bulge and some immature mesenchymal cells in the disassembling dermal papilla and regenerating connective tissue sheath/hair papilla reacted to A3. In the cutaneous wound-healing process, A3-labeled epithelial cells participated in re-epithelialization in the wound bed, and apparently, the labeled cells were derived from the hair bulge; in addition, A3-labeled immature mesenchymal cells in the connective tissue sheath of hair follicles at the wound edge showed the expansion of the A3 immunolabeling. A3-labeled immature epithelial and mesenchymal cells contributed to morphogenesis in the hair cycle and tissue repair after a cutaneous wound. A3 could become a unique antibody to identify somatic stem cells capable of differentiating both epithelial and mesenchymal cells in rat tissues.
Assuntos
Folículo Piloso/citologia , Células-Tronco Mesenquimais/fisiologia , Reepitelização , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Folículo Piloso/fisiologia , Masculino , Células-Tronco Mesenquimais/metabolismo , Peptídeos/imunologia , Polissacarídeos/imunologia , Ratos , Ratos Endogâmicos F344RESUMO
The Noda epileptic rat (NER) exhibits generalized tonic-clonic seizures (GTCS). A genetic linkage analysis identified two GTCS-associated loci, Ner1 on Chr 1 and Ner3 on Chr 5. The wild-type Ner1 and Ner3 alleles suppressed GTCS when combined in double-locus congenic lines, but not when present in single-locus congenic lines. Global expression analysis revealed that cholecystokinin B receptor (Cckbr) and suppressor of tumorigenicity 5 (St5), which map within Ner1, and PHD finger protein 24 (Phf24), which maps within Ner3, were significantly downregulated in NER. De novo BAC sequencing detected an insertion of an endogenous retrovirus sequence in intron 2 of the Phf24 gene in the NER genome, and PHF24 protein was almost absent in the NER brain. Phf24 encodes a Gαi-interacting protein involved in GABAB receptor signaling pathway. Based on these findings, we conclude that Cckbr, St5, and Phf24 are strong candidate genes for GTCS in NER.
Assuntos
Epilepsia Tônico-Clônica/genética , Receptor de Colecistocinina B/genética , Proteínas Supressoras de Tumor/genética , Animais , Cromossomos de Mamíferos/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Eletroencefalografia/métodos , Eletroencefalografia/veterinária , Epilepsia/genética , Ligação Genética/genética , Loci Gênicos/genética , Dedos de Zinco PHD/genética , Ratos , Ratos Wistar/genética , Receptor de Colecistocinina B/fisiologia , Convulsões/genéticaRESUMO
INTRODUCTION: Liver steatosis is the most frequent liver disease and may further develop into non-alcoholic steatohepatitis (NASH), liver cirrhosis, and finally hepatocellular carcinoma. Adipophilin (Adp) is localized on lipid droplet membrane in cytoplasm, and its increased expression is related to development of steatosis and NASH. The relationship between M1-/M2-macrophage polarization and Adp-rich hepatocyte-consisting pseudolobules (PLs) was investigated in thioacetamide (TAA)-induced rat cirrhosis. MATERIALS AND METHOD: F344 rats were injected twice weekly with TAA (100mg/kg bodyweight) and sacrificed at post-first injection (PFI) weeks 5, 10, 15, 20, 25 and 32. Macrophage immunophenotypes and Adp-containing hepatocytes were analyzed by single immunolabeling. Adp and M1-/M2-related factors were analyzed by real -time RT-PCR. RESULTS: PLs consisting exclusively of Adp-containing hepatocytes (Adp-positive) and PLs consisting of few Adp-containing hepatocytes (Adp-negative) were clearly distinguishable at PFI week 20 onwards. The numbers of M1-macrophages (reacting to CD68 and Iba1) and M2- macrophages (reacting to CD163, CD204 and Gal-3) were considerably greater in Adp-positive PLs. Expressions for both M1 (TNF-α, MCP-1, and Iba1)- and M2 (IL-4, TGF-ß1, Gal-3, and Hsp25)-related factors were markedly higher in Adp-positive PLs at PFI week 25. Interestingly, MHC class II-positive macrophages/dendritic cells were increased in Adp-positive clusters/foci at the early stages at PFI weeks 5 and 10, and the level was gradually decreased thereafter. CONCLUSIONS: M1-/M2-macrophages may simultaneously participate in the pathogenesis of steatosis in TAA-induced cirrhosis through M1- and M2-related factors. MHC class II cells may be responsible for steatosis at early stages, suggesting different functions from the above M1-/M2-macropahges.
Assuntos
Polaridade Celular , Hepatócitos/metabolismo , Hepatócitos/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Macrófagos/patologia , Perilipina-2/metabolismo , Animais , Antígenos CD/metabolismo , Apoptose , Proliferação de Células , Galectina 3/metabolismo , Regulação da Expressão Gênica , Imuno-Histoquímica , Imunofenotipagem , Cinética , Fígado/patologia , Cirrose Hepática/genética , Masculino , Perilipina-2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Endogâmicos F344 , TioacetamidaRESUMO
Lacaziosis, formerly called as lobomycosis, is a zoonotic mycosis, caused by Lacazia loboi, found in humans and dolphins, and is endemic in the countries on the Atlantic Ocean, Indian Ocean and Pacific Ocean of Japanese coast. Susceptible Cetacean species include the bottlenose dolphin (Tursiops truncatus), the Indian Ocean bottlenose dolphin (T. aduncus), and the estuarine dolphin (Sotalia guianensis); however, no cases have been recorded in other Cetacean species. We diagnosed a case of Lacaziosis in a Pacific white-sided dolphin (Lagenorhynchus obliquidens) nursing in an aquarium in Japan. The dolphin was a female estimated to be more than 14 years old at the end of June 2015 and was captured in a coast of Japan Sea in 2001. Multiple, lobose, and solid granulomatous lesions with or without ulcers appeared on her jaw, back, flipper and fluke skin, in July 2014. The granulomatous skin lesions from the present case were similar to those of our previous cases. Multiple budding and chains of round yeast cells were detected in the biopsied samples. The partial sequence of 43-kDa glycoprotein coding gene confirmed by a nested PCR and sequencing, which revealed a different genotype from both Amazonian and Japanese lacaziosis in bottlenose dolphins, and was 99 % identical to those derived from Paracoccidioides brasiliensis; a sister fungal species to L. loboi. This is the first case of lacaziosis in Pacific white-sided dolphin.
Assuntos
Antígenos de Fungos/genética , Golfinhos , Proteínas Fúngicas/genética , Glicoproteínas/genética , Lacazia/isolamento & purificação , Lobomicose/veterinária , Saccharomycetales/isolamento & purificação , Animais , Animais de Zoológico , Biópsia , Feminino , Histocitoquímica , Japão , Arcada Osseodentária/patologia , Lacazia/classificação , Lacazia/genética , Lobomicose/microbiologia , Lobomicose/patologia , Pulmão/diagnóstico por imagem , Pulmão/patologia , Microscopia , Reação em Cadeia da Polimerase , Radiografia Torácica , Saccharomycetales/classificação , Saccharomycetales/genética , Análise de Sequência de DNA , Homologia de Sequência , Pele/patologiaRESUMO
Glial fibrillary acidic protein (GFAP), a type III intermediate filament protein, is expressed in hepatic stellate cells (HSCs), the principal fibrogenic cell type in the liver. Further, GFAP could be a marker for hepatic progenitor cells (HPCs). In this study, the participation of GFAP-expressing cells in HPC expansion/ductular reaction was investigated in a rat model of liver cirrhosis. Six-week-old male F344 rats were injected intraperitoneally with thioacetamide (100mg/kg BW, twice a week) and examined at post-first injection weeks 5, 10, 15, 20 and 25. Fibrosis-related proliferation of ductular cells was observed as demonstrated by CK19 immunostaining. Some of these cells were stained with GFAP. No co-staining was observed between CK19 and α-smooth muscle actin (α-SMA; myofibroblast marker). There were proliferating ductular cells stained with α-fetoprotein or ß-catenin; the ductular reaction was related to increased expression of hepatocarcinogenesis-related factors (Wnt2, Wnt4 and glypican-3). These results for the first time show the participation of GFAP-positive HPCs in ductular reaction in a chemically induced rodent model. Though the ductular cells were chaperoned by myofibroblasts, they show no direct evidence for epithelial to mesenchymal transition. These findings shed new light in understanding the roles of GFAP-expressing HPCs in liver cirrhosis and provide further evidence of interaction between newly-formed bile ductules and HSCs, suggesting that both cells could be in the common lineage of HPCs.
Assuntos
Fibrose/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Células Estreladas do Fígado/patologia , Actinas/genética , Actinas/metabolismo , Animais , Proliferação de Células , Transição Epitelial-Mesenquimal , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Fibrose/etiologia , Fibrose/metabolismo , Proteína Glial Fibrilar Ácida/genética , Glipicanas/genética , Glipicanas/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/fisiologia , Masculino , Miofibroblastos/metabolismo , Ratos , Ratos Endogâmicos F344 , Tioacetamida/toxicidade , Proteína Wnt2/genética , Proteína Wnt2/metabolismo , Proteína Wnt4/genética , Proteína Wnt4/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The vacuole formation (VF) rat is an autosomal recessive myelin mutant characterized by generalized tremor, hypomyelination, and periaxonal vacuole formation of the central nervous system (CNS). Here, we report the most likely causative gene for neurological disease in the VF rat and pursue its roles in the development and maintenance of the CNS myelin. We identified a nonsense mutation in the dopey family member 1 (Dopey1) located on rat chromosome 8. Expression level of Dopey1 mRNA was decreased and DOPEY1 protein was undetectable both in the white and gray matter of the spinal cords in the VF rats. Double immunohistochemistry demonstrated that DOPEY1 was mainly expressed in neurons and oligodendrocytes in the wild-type rats, whereas no positive cells were detected in the VF rats. We also demonstrated a marked reduction in myelin components both at mRNA and protein levels during myelinogenesis in the VF rats. In addition, proteolipid protein and myelin-associated glycoprotein accumulated in oligodendrocyte cell body, suggesting that Dopey1 is likely to be involved in the traffic of myelin components. Our results highlighted the importance of Dopey1 for the development and maintenance of the CNS myelin.
Assuntos
Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/fisiopatologia , Bainha de Mielina/genética , Bainha de Mielina/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Animais , Doenças do Sistema Nervoso Central/patologia , Códon sem Sentido , Complexo de Golgi/patologia , Complexo de Golgi/fisiologia , Substância Cinzenta/patologia , Substância Cinzenta/fisiopatologia , Proteína Proteolipídica de Mielina/metabolismo , Bainha de Mielina/patologia , Glicoproteína Associada a Mielina/metabolismo , Neurônios/patologia , Neurônios/fisiologia , Oligodendroglia/patologia , Oligodendroglia/fisiologia , RNA Mensageiro/metabolismo , Ratos , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Tremor/genética , Tremor/patologia , Tremor/fisiopatologia , Vacúolos/genética , Vacúolos/patologia , Vacúolos/fisiologia , Substância Branca/patologia , Substância Branca/fisiopatologiaRESUMO
INTRODUCTION: Resident and exudate macrophages play an important role in the development of liver cirrhosis. Ionized calcium binding adaptor molecule 1(+) (Iba1(+)) and galectin-3(+) (Gal-3(+)) macrophages regulate liver fibrosis probably through pro-inflammatory and pro-fibrotic factors. Macrophages show polarized functions in liver fibrosis; however, M1-/M2-polarization of Iba1(+) and Gal-3(+) macrophages remains obscured. This study investigated the M1-/M2-polarized properties of Iba1(+) and Gal-3(+) macrophages in chemical-induced liver cirrhosis. MATERIALS AND METHODS: Cirrhosis was induced in F344 rats by repeated injections of thioacetamide (100mg/kg BW, twice a week for 25 weeks). Liver samples were collected from post-first-injection (PFI) week 5 to 25. Macrophage immunophenotypes and myofibroblasts in the fibrous bridges (FBs) and pseudolobules (PLs) were analyzed by immunohistochemistry. Expressions of M1- and M2-related factors were analyzed with RT-PCR, separately in FBs and PLs. RESULTS: Activation of myofibroblasts was most pronounced in livers at week 15. CD68(+) (M1), CD204(+) (M2), Iba1(+) and Gal-3(+) macrophages in the FBs increased gradually and peaked at week 15, consistent with the upregulation of both M1-(MCP-1, IFN-γ, IL-1ß, IL-6, and TNF-α) and M2-(TGF-ß1, IL-4, and IL-10) related factors. Iba1(+) and Gal-3(+) macrophages showed both M1- and M2-immunophenotypes. CD163(+) macrophages showed a persistent increase, consistent with TGF-ß1 upregulation. MHC class II(+) macrophages increased in the developing fibrotic lesions, and then reduced in the advanced stage cirrhosis. CONCLUSION: Both M1- and M2-macrophage polarizations occur during development of liver cirrhosis. Iba1(+) and Gal-3(+) macrophages participate in liver cirrhosis through production of both M1- and M2-related factors.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Cirrose Hepática/patologia , Macrófagos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Tioacetamida/toxicidade , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte/genética , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Proteínas da Matriz Extracelular/genética , Perfilação da Expressão Gênica , Imuno-Histoquímica , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Macrófagos/patologia , Masculino , Proteínas dos Microfilamentos/genética , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344 , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Canavan disease (CD) is a fatal hereditary neurological disorder caused by a mutation in the aspartoacylase (ASPA) gene and characterized by neurological signs and vacuolation in the central nervous system (CNS). The mutation inhibits the hydrolysis of N-acetyl-aspartate (NAA) resulting in accumulation of NAA in the CNS. A new Aspa-knockout rat was generated by transcription activator-like effector nuclease (TALEN) technology. Herein we describe the pathological and morphometrical findings in the brain and spinal cords of Aspa-knockout rats. Although Aspa-knockout rats did not show any neurological signs, vacuolation with swollen axons, hypomyelination, and activated swollen astrocytes were observed mainly in the brainstem reticular formation, ascending and descending motor neuron pathway, and in the olfactory tract. Morphometrical analysis revealed no obvious change in the number of neurons. These changes in the CNS are similar to human CD, suggesting that this animal model would be useful for further study of treatment and understanding the pathophysiology of human CD.
Assuntos
Amidoidrolases , Doença de Canavan , Modelos Animais de Doenças , Bainha de Mielina , Animais , Doença de Canavan/genética , Amidoidrolases/genética , Amidoidrolases/metabolismo , Bainha de Mielina/patologia , Bainha de Mielina/metabolismo , Medula Espinal/patologia , Encéfalo/patologia , Ratos , Técnicas de Inativação de Genes , Masculino , Astrócitos/patologia , Astrócitos/metabolismoRESUMO
Neuroaxonal dystrophy (NAD) is a neurodegenerative disease characterized by spheroid (swollen axon) formation in the nervous system. In the present study, we focused on a newly established autosomal recessive mutant strain of F344-kk/kk rats with hind limb gait abnormalities and ataxia from a young age. Histopathologically, a number of axonal spheroids were observed throughout the central nervous system, including the spinal cord (mainly in the dorsal cord), brain stem, and cerebellum in F344-kk/kk rats. Transmission electron microscopic observation of the spinal cord revealed accumulation of electron-dense bodies, degenerated abnormal mitochondria, as well as membranous or tubular structures in the axonal spheroids. Based on these neuropathological findings, F344-kk/kk rats were diagnosed with NAD. By a positional cloning approach, we identified a missense mutation (V95E) in the Hspa8 (heat shock protein family A (Hsp70) member 8) gene located on chromosome 8 of the F344-kk/kk rat genome. Furthermore, we developed the Hspa8 knock-in (KI) rats with the V95E mutation using the CRISPR-Cas system. Homozygous Hspa8-KI rats exhibited ataxia and axonal spheroids similar to those of F344-kk/kk rats. The V95E mutant HSC70 protein exhibited the significant but modest decrease in the maximum hydrolysis rate of ATPase when stimulated by co-chaperons DnaJB4 and BAG1 in vitro, which suggests the functional deficit in the V95E HSC70. Together, our findings provide the first evidence that the genetic alteration of the Hspa8 gene caused NAD in mammals.
RESUMO
Although it is in its early stages, canine induced pluripotent stem cells (ciPSCs) hold great potential for innovative translational research in regenerative medicine, developmental biology, drug screening, and disease modeling. However, almost all ciPSCs were generated from fibroblasts, and available canine cell sources for reprogramming are still limited. Furthermore, no report is available to generate ciPSCs under feeder-free conditions because of their low reprogramming efficiency. Here, we reanalyzed canine pluripotency-associated genes and designed canine LIN28A, NANOG, OCT3/4, SOX2, KLF4, and C-MYC encoding Sendai virus vector, called 159cf. and 162cf. We demonstrated that not only canine fibroblasts but also canine urine-derived cells, which can be isolated using a noninvasive and straightforward method, were successfully reprogrammed with or without feeder cells. ciPSCs existed in undifferentiated states, differentiating into the three germ layers in vitro and in vivo. We successfully generated ciPSCs under feeder-free conditions, which can promote studies in veterinary and consequently human regenerative medicines.
Assuntos
Células-Tronco Pluripotentes Induzidas , Animais , Cães , Humanos , Reprogramação Celular/genética , Vírus Sendai/genética , Fator 4 Semelhante a Kruppel , Células Alimentadoras , Fibroblastos , Diferenciação Celular/genéticaRESUMO
The Hiyama cross-coupling reaction of (E)-trimethyl(3,3,3-trifluoroprop-1-enyl)silane (1) with 2-iodoaniline (2) proceeded without any protection of the amino group. The coordination of copper(II) fluoride to 2,2'-bipyridyl provided the fluoride source required to trigger this reaction, affording (E)-2-(3,3,3-trifluoroprop-1-enyl)aniline (3). In the presence of a stoichiometric amount of [Cu(OTf)]2·C6H6, the treatment of 3 with an aryl aldehyde at 200 °C provided the 2-aryl-3-trifluoromethylquinoline (4) via the oxidative cyclization of an in situ-generated imine substructure.
Assuntos
Compostos de Anilina/química , Quinolinas/síntese química , Silanos/química , 2,2'-Dipiridil/química , Aldeídos/química , Catálise , Cobre/química , Ciclização , Fluoretos/química , Estrutura Molecular , EstereoisomerismoRESUMO
A reaction between (E)-trimethyl(3,3,3-trifluoroprop-1-en-1-yl)silane (1) and arylaldehydes 2 was triggered by fluoride anions to afford aryl 3,3,3-trifluoropropyl ketones 3 in moderate to good yield. A mechanistic study of this reaction indicated that it occurred via an allyl alkoxide (4). A subsequent 1,3-proton shift of the benzylic proton of 4 forms 3. This reaction involves oxidative 3,3,3-trifluoropropylation of an arylaldehyde to afford 4,4,4-trifluoro-1-arylbutan-1-one.
RESUMO
Rodent coat color genes have been studied as a bioresource to understand developmental and cellular processes. The Downunder rat is a fancy variety with a marking on its belly that runs from the neck to the breech and appears to mirror the dorsal hooded marking. Here, we established a congenic strain carrying the Downunder (Du) gene in an F344 genetic background. In addition to the ventral marking, Du/+ rats exhibit anophthalmia or microphthalmia with incomplete penetrance. Du/Du embryos die in the early stages of organogenesis. Genetic linkage analysis mapped the Du gene to rat chromosome 3 and haplotype mapping with congenic rats localized the Du locus to a 3.9-Mb region. The Du locus includes two functional genes, glycosyltransferase-like domain-containing 1 (Gtdc1) and zinc finger E-box binding homeobox 2 (Zeb2). Although we found no functional variation within any of Zeb2's exons or intron-exon boundaries, Zeb2 mRNA levels were significantly lower in Du/+ rats compared with wild-type rats. It is known that melanocyte-specific Zeb2 deletion results in the congenital loss of hair pigmentation in mice. Taken together, our results indicate that the Du mutation exerts pleiotropic effects on hair pigmentation, eye morphology, and development. Moreover, the Zeb2 gene is a strong candidate for the Du mutation.
Assuntos
Cromossomos Humanos Par 3 , Pigmentação , Humanos , Ratos , Camundongos , Animais , Fenótipo , Ratos Endogâmicos F344 , Mutação , Pigmentação/genética , Glicosiltransferases/genéticaRESUMO
Herein, we describe the management of nasopharyngitis caused by Schizophyllum commune infection in a captive cheetah. Computed tomography revealed a nodule in the nasal cavity and pharynx, and an endoscopic biopsy was performed. As a result, the nodule was surgically resected because of a suspected carcinoma. However, the surgical specimen was histologically re-evaluated and a fungal granuloma was diagnosed. Sequence analysis of DNA from formalin-fixed, paraffin-embedded samples revealed S. commune infection. The cheetah was administered fluconazole orally for 73 days. However, the drug was ineffective and itraconazole was administered for 14 days. Symptoms such as nasal discharge and sneezing have completely resolved for 4 years.
Assuntos
Acinonyx , Micoses , Nasofaringite , Schizophyllum , Animais , Schizophyllum/genética , Nasofaringite/veterinária , Micoses/diagnóstico , Micoses/tratamento farmacológico , Micoses/cirurgia , Micoses/veterinária , Itraconazol/uso terapêuticoRESUMO
Na+/Ca2+ exchangers (NCX) are an exchange transporter of Na+ and Ca2+ ions on the plasma membrane. There are three types of NCX: NCX1, NCX2, and NCX3. We have been working for many years to understand the role of NCX1 and NCX2 in gastrointestinal motility. In this study, we focused on the pancreas, an organ closely related to the gastrointestinal tract, and used a mouse model of acute pancreatitis to investigate a possible role for NCX1 in the pathogenesis of pancreatitis. We characterized a model of acute pancreatitis induced by excessive doses of L-arginine. We administered the NCX1 inhibitor SEA0400 (1 mg/kg) 1 hr prior to L-arginine-induced pancreatitis and evaluated pathological changes. Mice treated with NCX1 inhibitors show exacerbation of the disease with decreased survival and increased amylase activity in response to L-arginine-induced experimental acute pancreatitis, and this exacerbation correlates with increased autophagy mediated by LC3B and p62. These results suggest that NCX1 has a role in regulating pancreatic inflammation and acinar cell homeostasis.