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Embryonic stem cells (ESCs) are metabolically distinct from their differentiated counterparts. ESC mitochondria are less complex and fewer in number than their differentiated progeny. However, few studies have examined the proteins responsible for differences in mitochondrial structure and function between ESCs and somatic cells. Therefore, in this study, we aimed to investigate the differences between mitochondrial proteins in these two cell types. We demonstrate that HSP60 is more abundant in mouse ESC mitochondria than in mouse embryonic fibroblasts. Depletion of HSP60 inhibited mouse ESC proliferation and self-renewal, characterized by decreased OCT4 expression. HSP60 depletion also enhanced apoptosis during mouse ESC differentiation into embryoid bodies. Our results suggest that HSP60 expression has an essential role in ESC self-renewal and survival of differentiated cells from ESCs.
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BACKGROUND: Humanized mouse models are still under development, and various protocols exist to improve human cell engraftment and function. METHODS: Fourteen NOD/SCID/IL-2Rγnull (NSG) mice (4‒5 wk old) were conditioned with busulfan and injected with human umbilical cord blood (hUCB)-derived CD34+ hematopoietic stem cells (HSC) via retro-orbital sinuses. The bone marrow (BM), spleen, and peripheral blood (PB) were analyzed 8 and 12 weeks after HSC transplantation. RESULTS: Most of the NSG mice tolerated the regimen well. The percentage of hCD45+ and CD19+ cells rose significantly in a time-dependent manner. The median percentage of hCD45+cells in the BM was 55.5% at week 8, and 67.2% at week 12. The median percentage of hCD45+ cells in the spleen at weeks 8 and 12 was 42% and 51%, respectively. The median percentage of hCD19+ cells in BM at weeks 8 and 12 was 21.5% and 39%, respectively (P=0.04). Similarly, the median percentage of hCD19+ cells in the spleen at weeks 8 and 12 was 10% and 24%, respectively (P=0.04). The percentage of hCD19+ B cells in PB was 23% at week 12. At week 8, hCD3+ T cells were barely detectable, while hCD7+ was detected in the BM and spleen. The percentage of hCD3+ T cells was 2‒3% at week 12 in the BM, spleen, and PB of humanized NSG mice. CONCLUSION: We adopted a simplified protocol for establishing humanized NSG mice. We observed a higher engraftment rate of human CD45+ cells than earlier studies without any significant toxicity. And human CD45+ cell engraftment at week 8 was comparable to that of week 12.
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Animais , Humanos , Camundongos , Linfócitos B , Medula Óssea , Bussulfano , Sangue Fetal , Células-Tronco Hematopoéticas , Baço , Linfócitos T , Cordão UmbilicalRESUMO
PURPOSE: Stress is associated with depression, which induces many psychiatric disorders. Serotonin, also known as 5-hydroxy-tryptamine (5-HT), acts as a biochemical messenger and regulator in the brain. It also mediates several important physiological functions. Depression is closely associated with an overactive bladder. In the present study, we investigated the effect of treadmill exercise on stress-induced depression while focusing on the expression of 5-HT 1A (5-H(1A)) receptors in the dorsal raphe. METHODS: Stress was induced by applying a 0.2-mA electric foot shock to rats. Each set of electric foot shocks comprised a 6-second shock duration that was repeated 10 times with a 30-second interval. Three sets of electric foot shocks were applied each day for 7 days. For the confirmation of depressive state, a forced swimming test was performed. To visualize the expression of 5-HT and tryptophan hydroxylase (TPH), immunohistochemistry for 5-HT and TPH in the dorsal raphe was performed. Expression of 5-H(1A) receptors was determined by western blot analysis. RESULTS: A depressive state was induced by stress, and treadmill exercise alleviated the depression symptoms in the stress-induced rats. Expressions of 5-HT, TPH, and HT 1A in the dorsal raphe were reduced by the induction of stress. Treadmill exercise increased 5-HT, TPH, and HT 1A expressions in the stress-induced rats. CONCLUSIONS: Treadmill exercise enhanced 5-HT synthesis through the up-regulation of 5-HT(1A) receptors, and improved the stress-induced depression. In the present study, treadmill exercise improved depression symptoms by enhancing 5-HT(1A) receptor expression. The present results suggest that treadmill exercise might be helpful for the alleviation of overactive bladder and improve sexual function.
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Animais , Ratos , Western Blotting , Encéfalo , Depressão , Teste de Esforço , Exercício Físico , Pé , Imuno-Histoquímica , Esforço Físico , Receptor 5-HT1A de Serotonina , Serotonina , Choque , Estresse Psicológico , Triptofano Hidroxilase , Regulação para Cima , Bexiga Urinária HiperativaRESUMO
PURPOSE: To evaluate the usefulness of quantitative analysis of the facial nerve using contrast-enhanced three-dimensional (CE 3D) fluid-attenuated inversion recovery-volume isotopic turbo spin echo acquisition (FLAIR-VISTA) for the diagnosis of Bell's palsy in pediatric patients. MATERIALS AND METHODS: Twelve patients (24 nerves) with unilateral acute facial nerve palsy underwent MRI from March 2014 through March 2015. The unaffected sides were included as a control group. First, for quantitative analysis, the signal intensity (SI) and relative SI (RSI) for canalicular, labyrinthine, geniculate ganglion, tympanic, and mastoid segments of the facial nerve on CE 3D FLAIR images were measured using regions of interest (ROI). Second, CE 3D FLAIR and CE T1-SE images were analyzed to compare their diagnostic performance by visual assessment (VA). The sensitivity, specificity, and accuracy of RSI measurement and VA were compared. RESULTS: The absolute SI of canalicular and mastoid segments and the sum of the five mean SI (total SI) were higher in the palsy group than in the control group, but with no significant differences. The RSI of the canalicular segment and the total SI were significantly correlated with the symptomatic side (P = 0.028 and 0.015). In 11/12 (91.6%) patients, the RSI of total SI resulted in accurate detection of the affected side. The sensitivity, specificity, and accuracy for detecting Bell's palsy were higher with RSI measurement than with VA of CE 3D FLAIR images, while those with VA of CE T1-SE images were higher than those with VA of CE 3D FLAIR images. CONCLUSION: Quantitative analysis of the facial nerve using CE 3D FLAIR imaging can be useful for increasing the diagnostic performance in children with Bell's palsy when difficult to diagnose using VA alone. With regard to VA, the diagnostic performance of CE T1-SE imaging is superior to that of CE 3D FLAIR imaging in children. Further studies including larger populations are necessary.
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Criança , Humanos , Paralisia de Bell , Diagnóstico , Nervo Facial , Gânglio Geniculado , Imageamento por Ressonância Magnética , Processo Mastoide , Paralisia , Sensibilidade e EspecificidadeRESUMO
Streptococcus pneumoniae is a major human pathogen that is involved in community-acquired pneumonia. Tumor necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine that activates immune responses against infection, invasion, injury, or inflammation. To study the role of TNF-alpha during S. pneumoniae infection, a murine pneumococcal pneumonia model was used. We intranasally infected C57BL/6J wild-type (WT) and TNF-alpha knockout (KO) mice with S. pneumoniae D39 serotype 2. In TNF-alpha KO mice, continuous and distinct loss of body weight, and low survival rates were observed. Bacterial counts in the lungs and blood of TNF-alpha KO mice were significantly higher than those in WT mice. Histopathological lesions in the spleen of TNF-alpha KO mice were more severe than those in WT mice. In TNF-alpha KO mice, severe depletion of white pulp was observed and the number of apoptotic cells was significantly increased. Interferon-gamma (IFN-gamma), IL-12p70 and IL-10 levels in serum were significantly increased in TNF-alpha KO mice. TNF-alpha is clearly involved in the regulation of S. pneumoniae infections. Early death and low survival rates of TNF-alpha KO mice were likely caused by a combination of impaired bacterial clearance and damage to the spleen. Our findings suggest that TNF-alpha plays a critical role in protecting the host from systemic S. pneumoniae infection.
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Animais , Humanos , Camundongos , Carga Bacteriana , Peso Corporal , Inflamação , Interferon gama , Interleucina-10 , Pulmão , Pneumonia , Pneumonia Pneumocócica , Baço , Streptococcus pneumoniae , Taxa de Sobrevida , Fator de Necrose Tumoral alfaRESUMO
Streptococcus pneumoniae is a major pathogen that causes various diseases, including pneumonia and sepsis, as millions of people suffer from S. pneumoniae infection worldwide. To better understand the immune and inflammatory responses to S. pneumoniae, we produced murine models. To investigate the differences between intranasal and intratracheal infection, BALB/c mice were infected with S. pneumoniae D39 intranasally or intratracheally. Mice showed no significant differences in survival rates, body weight changes, and bacterial loads. To investigate resistance and susceptibility among mouse strains, BALB/c, C57BL/6J, tumor necrosis factor-alpha (TNF-alpha) knockout, and interleukin-10 (IL-10) knockout mice were infected with S. pneumoniae D39 via intranasal or intravenous routes. In this study, BALB/c and C57BL/6J mice were resistant, IL-10 knockout mice were intermediate, and TNF-alpha knokout mice were susceptible to S. pneumoniae infection. These data show that intranasal and intratracheal infection induced similar results after S. pneumoniae infection, and the genetic background of mice must be considered when studying S. pneumoniae infection in vivo.
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Animais , Camundongos , Carga Bacteriana , Alterações do Peso Corporal , Interleucina-10 , Camundongos Knockout , Infecções Pneumocócicas , Pneumonia , Sepse , Streptococcus , Streptococcus pneumoniae , Taxa de Sobrevida , Fator de Necrose Tumoral alfaRESUMO
Nitric oxide (NO) is a reactive free radical and a messenger molecule in many physiological functions. However, excessive NO is believed to be a mediator of neurotoxicity. The medicinal plant Coriolus versicolor is known to possess anti-tumor and immune-potentiating activities. In this study, we investigated whether Coriolus versicolor possesses a protective effect against NO donor sodium nitroprusside (SNP)-induced apoptosis in the human neuroblastoma cell line SK-N-MC. We utilized 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, DNA fragmentation assay, reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and caspase-3 enzyme activity assay in SK-N-MC cells. MTT assay showed that SNP treatment significantly reduces the viability of cells, and the viabilities of cells pre-treated with the aqueous extract of Coriolus versicolor cultivated in citrus extract (CVEcitrus) was increased. However, aqueous extract of Coriolus versicolor cultivated in synthetic medium (CVEsynthetic) showed no protective effect and aqueous citrus extract (CE) had a little protective effect. The cell treated with SNP exhibited several apoptotic features, while those pre-treated for 1 h with CVEcitrus prior to SNP expose showed reduced apoptotic features. The cells pre-treated for 1 h with CVEcitrus prior to SNP expose inhibited p53 and Bax expressions and caspase-3 enzyme activity up-regulated by SNP. We showed that CVEcitrus exerts a protective effect against SNP-induced apoptosis in SK-N-MC cells. Our study suggests that CVEcitrus has therapeutic value in the treatment of a variety of NO-induced brain diseases.
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Humanos , Apoptose , Western Blotting , Encefalopatias , Caspase 3 , Linhagem Celular , Citrus , Fragmentação do DNA , DNA Nucleotidilexotransferase , Citometria de Fluxo , Indóis , Neuroblastoma , Óxido Nítrico , Nitroprussiato , Plantas Medicinais , Sais de Tetrazólio , Tiazóis , Doadores de TecidosRESUMO
The various murine models have contributed to the study of human atopic dermatitis (AD). However limitations of the models involve low reproducibility and long time to develop AD. In an attempt to overcome these limitations and establish an atopic dermatitis murine model, we repeated the application of 2, 4-dinitrochlorobenzene (DNCB) patch in NC/Nga and BALB/c mice, which has advantages in reproduction and cost. For the sensitization, a 1 cm2 gauze-attached patch, where 1% or 0.2% DNCB was periodically attached on the back of NC/Nga and BALB/c mice. To estimate how homologous our model was with human atopic dermatitis, clinical, histological and immunological alterations were evaluated. Both strains showed severe atopic dermatitis, increase in subiliac lymph node weight, mast cells, epidermal hyperplasia and serum IgE levels. Though both exhibited a high IL-4/IFN-gamma and IL-4/TNF-beta ratio in the expression of mRNA, the shifting of DNCB-treated BALB/c mice was increased to more than double that of NC/Nga mice. These results suggest that our DNCB patched model using BALB/c mice were more suitable than NC/Nga mice in demonstrating the immune response. We anticipate that our novel model may be successfully used for pathogenesis of atopic dermatitis and assessment of therapeutic approaches.
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Animais , Humanos , Camundongos , Dermatite Atópica , Dinitroclorobenzeno , Hiperplasia , Imunoglobulina E , Linfonodos , Mastócitos , Reprodução , RNA MensageiroRESUMO
Peripheral nerve injuries are a commonly encountered clinical problem and often result in a chronic pain and severe functional deficits. c-Fos expression is sometimes used as a marker of increased neuronal activity. We have developed herbal bath "HAC" for pain control using the following herbs: Harpagophytum procumbens, Atractylodes japonica, and Corydalis tuber. In the present study, we investigated the effects of herbal bath "HAC" on the recovery rate of the locomotor function and the expression of c-Fos in the ventrolateral periaqueductal gray (vlPAG) region of brain following sciatic crushed nerve injury in rats. Walking track analysis for the evaluation of functional recovery and immunohistochemistry for the c-Fos expression were used for this study. In the present results, characteristic gait change with dropping of the sciatic function index (SFI) was observed and c-Fos expression in the vlPAG was suppressed following sciatic crushed nerve injury in rats. Immersion into herbal bath "HAC" enhanced SFI value and restored c-Fos expression in the vlPAG to the control value. These results suggest that herbal bath "HAC" might activate neurons in the vlPAG, and it facilitates functional recovery from peripheral nerve injury. Here we showed that herbal bath "HAC" could be used as a new therapeutic intervention for pain control and functional recovery from peripheral nerve injury.
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Animais , Ratos , Atractylodes , Banhos , Encéfalo , Dor Crônica , Corydalis , Marcha , Harpagophytum , Imersão , Imuno-Histoquímica , Neurônios , Substância Cinzenta Periaquedutal , Traumatismos dos Nervos Periféricos , Atletismo , CaminhadaRESUMO
Fucoidan, a sulfated polyanionic polymer of L-fucose, is obtained from brown marine macroalgae. In the present study, neuroprotective effect of fucoidan against N-methyl- D-aspartate (NMDA)-induced excitotoxicity in the hippocampus was investigated. The patch clamp study revealed that fucoidan significantly inhibited NMDA receptor-activated ion current in the acutely dissociated hippocampal CA1 neurons. In an organotypic hippocampal slice culture, fucoidan inhibited NMDA-induced neuronal cell death in a dose-dependent manner. The present study showed that fucoidan possesses a neuroprotective effect against NMDA-induced excitotoxicity, and that the suppressive effect of fucoidan on the NMDA-induced ion current can be suggested as being the underlying neuroprotective mechanism of fucoidan.
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Animais , Ratos , Morte Celular , Ácido D-Aspártico , Hipocampo , N-Metilaspartato , Neurônios , Fármacos Neuroprotetores , Polímeros , Polissacarídeos , Alga MarinhaRESUMO
It has been focused on the importance of the host inflammatory response in periodontal pathogenesis and progression, treatment has been introduced to control the host response and the method, which diminishes production and activity of MMP by doxycycline, has been used in periodontal field. MMP is a proteolytic enzyme which plays a major role in tissue destruction and MMP-1 is secreted in the periodontally healthy tissue, while MMP-8, 9, 13, etc in the inflammatory state. Among these, MMP-13 has been discovered lately and reported to degrade primarily type II collagen. Periodontal ligament (PDL) cell plays a role in destruction of periodontal tissue. This study was to evaluate the effect of doxycycline and mefenamic acid, non-steroidal antiinflammatory drug on MMP-13 mRNA expression in the rat PDL cell. Doxycycline concentration of 1~100 microgram/ml was added rat PDL cell and cell activity was measured by MTT assay at day 1 and 3. MMP-13 gene expression was evaluated by RT-PCR after PDL cells were pre-treated for 1 hour with doxycycline (50 microgram/ml) alone or with mefenamic acid (10(-6)M), then added IL-1beta(1.0 ng/ml) and incubated for 16 -18 hours. The results are as follows: 1. Cell activity decreased significantly at 24 and 72 hours in 100 microgram/ml (p<0.05). 2. Level of MMP-13 mRNA was in 202% increase by IL-1beta and in pre-treating doxycycline group, expression of IL-1beta induced MMP-13 mRNA was inhibited by 31% than IL-1beta treated only. 3. Mefenamic acid did not inhibit on the expression of IL-1beta induced MMP-13 mRNA, while mefenamic acid in combination with doxycycline inhibited the expression by 41% compared to only IL-1beta stimulation. These results suggest that doxycycline synergistically inhibit the expression of IL-1beta induced MMP-13 mRNA in combination with mefenamic acid.