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1.
Virus Genes ; 55(4): 550-556, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31161411

RESUMO

Japanese encephalitis virus SA14-14-2 (JEV SA14-14-2) is a widely used vaccine in China and other southeastern countries to prevent Japanese encephalitis in children. In this study, a stable infectious cDNA clone of JEV SA14-14-2 with a low copy number pACYC177 vector dependent on the T7 promoter and T7 terminator was developed. Two introns were inserted into the capsid gene and envelope gene of JEV cDNA for gene stability. Hepatitis delta virus ribozyme (HDVr) was engineered into the 3' UTR cDNA of JEV for authentic 3' UTR transcription. The rescued virus showed biological properties indistinguishable from those of the parent strain (JEV SA14-14-2). The establishment of a JEV SA14-14-2 reverse genetics system lays the foundation for the further development of other flavivirus vaccines and viral pathogenesis studies.

2.
Genes (Basel) ; 10(3)2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30866582

RESUMO

Coat color is one of the most important characteristics for distinguishing Chinese indigenous pig breeds. In Wuzhishan pigs, the animals have black on the back and white on the abdomen. However, the molecular genetic basis of this phenotype is unclear. In this study, we used high-throughput RNA sequencing to compare expression profiles of coding and non-coding RNAs from white and black skin samples obtained from individual Wuzhishan pigs. The expression profiling revealed that 194 lncRNAs (long non-coding RNAs), 189 mRNAs (messenger RNAs), and 162 miRNAs (microRNAs) had significantly different levels of expression (|log2 fold change| > 1, p-value < 0.05) in white and black skin. Compared to RNA levels in black skin, white skin had higher levels of expression of 185 lncRNAs, 181 mRNAs, and 23 miRNAs and lower levels of expression of 9 lncRNAs, 8 mRNAs, and 139 miRNAs. Functional analysis suggested that the differentially expressed transcripts are involved in biological processes such as melanin biosynthesis, pigmentation and tyrosine metabolism. Several key genes involved in melanogenesis, including MLANA, PMEL, TYR, TYRP1, DTC, TRPM1 and CAMK2A, had significantly different levels of expression in the two skin tissues. Potential lncRNA⁻miRNA⁻gene interactions were also examined. A total of 15 lncRNAs, 11 miRNAs and 7 genes formed 23 lncRNA⁻miRNA⁻gene pairs, suggesting that complex regulatory networks of coding and non-coding genes underlie the coat color trait in Wuzhishan pigs. Our study provides a foundation for understanding how lncRNA, miRNA and genes interact to regulate coat color in black-back/white-belly pigs. We also constructed lncRNA⁻miRNA⁻gene interaction networks to elucidate the complex molecular mechanisms underlying skin physiology and melanogenesis. The results extend our knowledge about the diversity of coat color among different domestic animals and provide a foundation for studying novel mechanisms that control coat color in Chinese indigenous pigs.

3.
Artigo em Inglês | MEDLINE | ID: mdl-30017777

RESUMO

Recently there is increasing concern over the association between sleep deprivation (S-Dep) and depression. Mounting evidence suggests that S-Dep might be a risk factor for depression. However, underlying molecular mechanism remains elusive and currently there is no effective therapy to negate the effects of S-Dep. In this study, we aimed to examine whether subchronic treatment of citalopram (CTM), an antidepressant, can attenuate the negative effects of S-Dep in mice. Three-month-old C57BL/6J mice were divided into control, S-Dep, CTM control and CTM + S-Dep groups. CTM and CTM + S-Dep group treated with citalopram for 5 consecutive days at a dose of 10 mg/kg per day before experimental procedure. S-Dep and CTM + S-Dep group mice were sleep deprived for 24 h using an automated treadmill method. Our results revealed that S-Dep animals displayed an increased depressive-like behavior in forced swim, tail suspension and sucrose preference test and anxiety-like behavior in the open field and elevated plus maze, as well as disrupted spatial memory in Morris water maze. Western blotting analysis revealed that S-Dep caused reductions in the levels of the plasticity- and memory-related signaling molecules i.e. pCaMKII and pCREB in the hippocampus. Moreover, S-Dep animals showed synaptic plasticity deficits in the Schaffer collateral pathway. Interestingly, subchronic CTM treatment prevented S-Dep-induced decrease in pCaMKII and pCREB levels in the hippocampus. Furthermore, CTM treatment prevented S-Dep-induced deficits in synaptic plasticity, spatial memory, depressive-like behavior in sucrose preference test and anxiety-like behavior in open field test but not in force swim, tail suspension and elevated plus maze test. This data suggests differential effects of CTM on S-Dep-associated behavioral alterations and cognitive impairments.


Assuntos
Antidepressivos de Segunda Geração/uso terapêutico , Citalopram/uso terapêutico , Depressão/tratamento farmacológico , Depressão/etiologia , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/etiologia , Privação do Sono/complicações , Animais , Antidepressivos de Segunda Geração/farmacologia , Proteína de Ligação a CREB/metabolismo , Citalopram/farmacologia , Modelos Animais de Doenças , Potenciais Evocados/efeitos dos fármacos , Comportamento Exploratório/efeitos dos fármacos , Feminino , Preferências Alimentares/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Resposta de Imobilidade Tônica/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Sacarose/administração & dosagem , Natação/psicologia
4.
Connect Tissue Res ; : 1-10, 2018 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-29447019

RESUMO

Lysophosphatidic acid is a serum-derived growth factor that is involved in wound healing. Although in its infancy, a growing body of evidence has demonstrated that lysophosphatidic acid exerts a potentially significant role in regulating bone cell biology. However, previous studies mainly focused on the osteoinductive potential of lysophosphatidic acid, its effects on bone tissue vascularization, another essential element during bone regeneration, remains ill-defined so far. Here in this study, we examined the effects of lysophosphatidic acid on osteogenic differentiation as well as the angiogenesis-inducing capacity of pre-osteoblasts, a cell population that coordinates osteogenic and angiogenic processes in bone regenerating niche. Our results showed that treatment of MC3T3-E1 pre-osteoblastic cells with lysophosphatidic acid enhanced alkaline phosphatase activity and matrix mineralization, demonstrating in vitro osteoblastic differentiation. Of particular importance was the finding that vascular endothelial growth factor secretion also increased after lysophosphatidic acid treatment. Lysophosphatidic acid conditioned media of MC3T3-E1 cells was capable of promoting angiogenic behavior of endothelial cells, as evidenced by stimulating proliferation, migration, and tube formation. Besides, inhibition of LPA1/3 receptor abolished lysophosphatidic acid-induced elevation of the osteogenic and angiogenic capability of pre-osteoblasts. Our research demonstrated the important role of lysophosphatidic acid in coupling osteogenesis and angiogenesis during bone remodeling through orchestrating pre-osteoblast behavior, and implications therein for novel and effective treatment strategies for bone regeneration success.

5.
J Sci Food Agric ; 96(12): 4207-14, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26777397

RESUMO

BACKGROUND: The objective of the present study was to investigate the chronic effect of transgenic maize lines by the insertion of the cry1Ac gene from Bacillus thuringiensis (Bt) on the growth performance, immune response and health using a Wuzhishan miniature pig model through a 196-day feeding study. RESULTS: Based on the gender and weight, 72 Wuzhishan miniature pigs were randomly assigned one of the diets containing 65% non-transgenic isogenic corn or Bt corn at three stages of growth (day 0-69, 70-134 and 135-196). The potential toxicological effects of transgenic corn on pigs were explored. No difference between the diet treatments for growth performance and haematology parameters at any stages of growth. Although subtle differences in serum content of alanine aminotransferase, relative kidney weight and some immune response were observed between the Bt group and isogenic group, they were not considered as diet treatment-related. CONCLUSION: Long-term feeding Bt corn carrying cry1Ac genes to Wuzhishan miniature pigs did not indicate adverse effects on the growth, immune response and health indicators at any stages of growth. © 2016 Society of Chemical Industry.


Assuntos
Ração Animal/efeitos adversos , Proteínas de Bactérias/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Plantas Geneticamente Modificadas/efeitos adversos , Plantas Geneticamente Modificadas/genética , Porco Miniatura/crescimento & desenvolvimento , Zea mays/efeitos adversos , Zea mays/genética , Animais , Bacillus thuringiensis/genética , Linhagem Celular , Feminino , Rim/anatomia & histologia , Rim/patologia , Masculino , Modelos Animais , Plantas Geneticamente Modificadas/química , Suínos , Porco Miniatura/sangue , Porco Miniatura/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Testes de Toxicidade , Ganho de Peso , Zea mays/química
6.
Bone ; 74: 182-90, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25549868

RESUMO

Mechanical stimulation plays an important role in bone cell metabolic activity. However, bone cells lose their mechanosensitivity upon continuous mechanical stimulation (desensitization) and they can recover the sensitivity with insertion of appropriate rest period into the mechanical loading profiles. The concrete molecular mechanism behind the regulation of cell mechanosensitivity still remains unclear. As one kind of mechanosensitive cell to react to the mechanical stimulation, osteoblasts respond to fluid shear stress (FSS) with actin cytoskeleton reorganization, and the remodeling of actin cytoskeleton is closely associated with the alteration of cell mechanosensitivity. In order to find out whether inhibiting the actin cytoskeleton reorganization by silencing LIM-kinase 2 (LIMK2) gene would increase the mechanosensitivity of primary osteoblasts, we attenuated the formation of actin stress fiber under FSS in a more specific way: inhibiting the LIMK2 expression by RNA interference. We found that inhibition of LIMK2 expression by RNA interference attenuated the formation of FSS-induced actin stress fiber, and simultaneously maintained the integrity of actin cytoskeleton in primary osteoblasts. We confirmed that the decreased actin cytoskeleton reorganization in response to LIMK2 inhibition during FSS increased the mechanosensitivity of the osteoblasts, based on the increased c-Fos and COX-2 expression as well as the enhanced proliferative activity in response to FSS. These data suggest that osteoblasts can increase their mechanosensitivity under continuous mechanical stimulation by reducing the actin stress fiber formation through inhibiting the LIMK2 expression. This study provides us with a new and more specific method to regulate the osteoblast mechanosensitivity, and also a new therapeutic target to cure bone related diseases, which is of importance in maintaining bone mass and promoting osteogenesis.


Assuntos
Inativação Gênica , Quinases Lim/genética , Mecanotransdução Celular , Reologia , Estresse Mecânico , Citoesqueleto de Actina , Animais , Animais Recém-Nascidos , Ciclo Celular , Proliferação de Células , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Citometria de Fluxo , Quinases Lim/metabolismo , Camundongos Endogâmicos BALB C , Osteoblastos/citologia , Osteoblastos/enzimologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Regulação para Cima
7.
Virol J ; 9: 288, 2012 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-23176172

RESUMO

BACKGROUND: The VP3 protein of goose parvovirus (GPV) or Muscovy duck parvovirus (MDPV), a major structural protein, can induce neutralizing antibodies in geese and ducks, but monoclonal antibodies (MAbs) against VP3 protein has never been characterized. RESULTS: Three hybridoma cell lines secreting anti-GPV VP3 MAbs were obtained and designated 4A8, 4E2, and 2D5. Immunoglobulin subclass tests differentiated them as IgG2b (4A8 and 4E2) and IgG2a (2D5). Dot blotting assays showed that three MAbs reacted with His-VP3 protein in a conformation-independent manner. A competitive binding assay indicated that the MAbs delineated two epitopes, A and B of VP3. Immunofluorescence assay showed that MAbs 4A8, 4E2, and 2D5 could specifically bind to goose embryo fibroblast cells (GEF) or duck fibroblast cells (DEF) infected with GPV and MDPV. Dot blotting also showed that the MAbs recognized both nature GPV and MDPV antigen. Western blotting confirmed that the MAbs recognized VP3 proteins derived from purified GPV and MDPV particles. The MAbs 4A8 and 2D5 had universal reactivity to heterologous GPV and MDPV tested in an antigen-capture enzyme-linked immunosorbent assay. CONCLUSIONS: Preparation and characterization of these the MAbs suggests that they may be useful for the development of a MAb-capture ELISA for rapid detection of both GPV and MDPV. Virus isolation and PCR are reliable for detecting GPV and MDPV infection, but these procedures are laborious, time-consuming, and requiring instruments. These diagnosis problems highlight the ongoing demand for rapid, reproducible, and automatic methods for the sensitive detection of both GPV and MDPV infection.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Dependovirus/imunologia , Patos/virologia , Gansos/virologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Western Blotting , Imunofluorescência , Camundongos , Camundongos Endogâmicos BALB C
8.
Nan Fang Yi Ke Da Xue Xue Bao ; 31(2): 304-7, 2011 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-21354917

RESUMO

OBJECTIVE: To isolate the wild-type virulent phage of Helicobacter pylori (Hp) and simulate the treatments in vitro to investigate the methods for oral Hp-assisted penetration of the phage through the gastric barrier and offspring phage release for infection and treatment of gastrointestinal Hp. METHODS: The Hp strain was cultured with the candle cylinder method and the virulent phage was isolated by single plate or double plate experiment. A simulated gastric juice was applied and the bactericidal effect of the phage was tested with double flats experiment. RESULTS: After a 1.5-h treatment in simulated gastric juice, the orally derived Hp-borne phage was still capable of forming plaques while the control phage was not. CONCLUSION: The oral Hp can help the phage resist the gastric juice and then infect the gastrointestinal Hp.


Assuntos
Bacteriófagos/isolamento & purificação , Bacteriófagos/fisiologia , Trato Gastrointestinal/microbiologia , Infecções por Helicobacter/terapia , Helicobacter pylori/virologia , Humanos , Virulência
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