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Objective To investigate the effects of 72 h sleep deprivation(SD)on circadian clock gene expression in the rat liver.Methods Twelve rats were randomly divided into control group and SD group.An SD instrument was used to deprive the rats′sleep for 72 h.Then the abdominal cavity was exposed to obtain liver,and the expression of clock genes was detected by RT-PCR and Western blotting analysis, respectively.Results Compared with the control group, the mRNA levels of clock,npas2 and rev-erbαstrikingly decreased in the livers of the SD group rats.However,per1,per2 and rorαmRNA levels obviously increased.bmal1 and cry1 mRNA expression hardly changed in the control and SD groups. Meanwhile,the protein levels of liver BMAL1,CLOCK,NPAS2,CRY1 and REV-ERBαwere significantly down-regulated and PER1,PER2 and RORαprotein levels were up-regulated in SD group compared with control group.Conclusion 72 h SD can result in abnormal expressions of several circadian clock genes in the rat liver at both transcriptional and translational levels.
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Objective To explore whether PERK is involved in the regulation of arsenite-induced autophagy.Methods Human hepatoma cells HepG2 were cultured and treated with arsenite.The expression level of autophagic hallmarks and the activation status of PERK were detected by Western blotting.The transactivation of p53 and the induction of its downstream target genes expression were also detected by Western blotting after knockdown of PERK expression.Transactivity of p53 was detected by dual luciferase reporter assay after knockdown of PERK expression.Results An increase in the LC3BII:I ratio,the induction of Beclin-1 expression and the degradation of p62 were readily observed in arsenite-treated HepG2 cells,but the effects were abolished after knockdown of PERK expression.Furthermore,phosphorylation of p53 at Ser15 and Ser392,transactivation of p53 and the induction of its downstream target gene DAPK1 expression were effectively inhibited under the same PERK knockdown conditions.Conclusion PERK regulates arsenite-induced autophagy by activating p53-dependent DAPK1 upregulation.
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Objective To investigate the effects of 72 h sleep deprivation (SD) on circadian clock gene expression in the rat spleen.Methods The rats were randomly divided into control group and SD group.An SD instrument was used to deprive the rats of sleep for 72 h.Then the lymphocytes from the spleen were obtained by Ficoll seperation medium before the expression of clock genes was detected by RT-PCR and Western blotting analysis respectively.Results Compared with the control group,the mRNA levels of bmal1,clock,per2 and rev-erbα strikingly decreased in the spleens of the SD group rats.However,npas2,per1,rorα and cry1 mRNA expression hardly changed in the control and SD group.Meanwhile,the protein levels of spleen BMAL1,NPAS2,CRY1 and RORα were significantly down-regulated and PER1 protein levels were up-regulated in SD group compared with control group.However REV-ERBα protein expression remained unchanged in the control and SD group.Conclusion 72 h SD can result in abnormal expressions of several circadian clock genes in lymphocytes of the spleen at both transcription and translation levels.
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Circadian rhythms are driven by the transcriptional and translational oscillator and post-transcriptional modification on clock genes (bmall,clock,cry and per) and clock control genes (rev-erbα,rorα,dbp,tefand hlf),which keeps the body's physiologies,behavior,and other life activities and shows roughly 24-hour oscillation.The circadian clock system consists of the central and peripheral clock system,rhythm input and output system.The input system receives and transfers the light signals to the central clock system that functions as the main pacemaker to generate and output the circadian signals to the peripheral organ,which works together with the local endogenous circadian system to maintain the body's physiological activities.A variety of endogenous and exogenous factors,such as light,temperature,time of food intake,nutrients and metabolic related factors,play important roles in regulating the circadian clock system and maintaining homeostasis of circadian rhythms.Here,we focuses on the regulating system and related regulating factors for the generation and maintenance of circadian rhythms.
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Objective To develop a rapid, accurate, visual, and portable detection method for adenovirus types B (AdvB) and E ( AdvE).Methods Universal primers were targeted on type-specific conserved regions to allow the simultaneous detection of both human Adv (HAdV) species.A detection method based on the combination of recombinase polymerase amplification ( RPA) and lateral flow dipstick ( LFD) was established the sensitivity and specificity evaluated , and throat swab specimens of 19 patients infected with AdvB and AdvE as well as 10 healthy volunteers were detected with this method.Results The detection limit of the method was 10 copies/μl Adv DNA, which was close to that of qPCR , and there were no cross-reactions with other species of Adv and unrelated virus .The detection could be finished within 15 to 20 min within the temperature range of 25 to 45℃.When applied to clinical samples , this method showed 100% sensitivity and specificity.Conclusion This detection assay is a sensitive , specific, rapid and simple method that eliminates the need for expensive equipment , trained personnel or laboratories .The characteristics of this system render it suitable for use in grass-roots healthcare departments , and the system is especially effective for field testing and on-site testing.
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The circadian rhythm generated by the endogenous circadian clock system depends on the feedback loop composed of a series of core clock genes (bmal1, clock, cry and per) and clock controlled genes (rev-erbα, rorα, dbp, tef and hlf) .The circadian rhythm is closely related to the body metabolism , which plays important roles in the metabolic process of the major nutrients , such as glucose and fat .Circadian rhythm disorders caused by exogenous factors will significantly increase the risk of metabolic syndrome .This paper focuses on the effect of circadian rhythms on metabolism and their roles in regulating metabolic syndrome .
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Objective To develop a rapid, accurate, visual, and portable detection method for adenovirus types B (AdvB) and E ( AdvE).Methods Universal primers were targeted on type-specific conserved regions to allow the simultaneous detection of both human Adv (HAdV) species.A detection method based on the combination of recombinase polymerase amplification ( RPA) and lateral flow dipstick ( LFD) was established the sensitivity and specificity evaluated , and throat swab specimens of 19 patients infected with AdvB and AdvE as well as 10 healthy volunteers were detected with this method.Results The detection limit of the method was 10 copies/μl Adv DNA, which was close to that of qPCR , and there were no cross-reactions with other species of Adv and unrelated virus .The detection could be finished within 15 to 20 min within the temperature range of 25 to 45℃.When applied to clinical samples , this method showed 100% sensitivity and specificity.Conclusion This detection assay is a sensitive , specific, rapid and simple method that eliminates the need for expensive equipment , trained personnel or laboratories .The characteristics of this system render it suitable for use in grass-roots healthcare departments , and the system is especially effective for field testing and on-site testing.
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The circadian rhythm generated by the endogenous circadian clock system depends on the feedback loop composed of a series of core clock genes (bmal1, clock, cry and per) and clock controlled genes (rev-erbα, rorα, dbp, tef and hlf) .The circadian rhythm is closely related to the body metabolism , which plays important roles in the metabolic process of the major nutrients , such as glucose and fat .Circadian rhythm disorders caused by exogenous factors will significantly increase the risk of metabolic syndrome .This paper focuses on the effect of circadian rhythms on metabolism and their roles in regulating metabolic syndrome .
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Objective To investigate the effect of 36 h continuous sleep deprivation(SD) on circadian clock gene expression in the rat liver and kidney and the alteration of urine biomarker levels.Methods Twelve rats were randomly divided into control group and SD group.An SD device was used to deprive the rats of sleep.After 36 h continuous SD, the abdominal cavity was exposed to obtain livers and kidneys, and RT-PCR and Western blotting were used to detect expression of clock genes.Then,the pelvic cavity was exposed to obtain urine, and the changes in bio-marker total bile acids(TBA) were tested with ELISA.Results Compared with the control group, the mRNA level of liver clock and bmal1 was obviously reduced in the SD-treated rats (P<0.05).However, no obvious change was found in the samples from the kidney.Sharp down-regulation of CLOCK and BMAL1 protein expression was also observed in the rat liver after SD treatment.Urine TBA content in SD treated rats was raised obviously (P<0.001), compared with control.Conclusion Thirty-six hours of continuous SD could result in deregulation of circadian clock gene and cholesterol metabolism disorder in the rat liver.TBA might be used as a noninvasive biomarker of liver injury under SD stress conditions.
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Circadian rhythms are endogenous 24 h variations found in virtually all physiological processes and behaviors, which are controlled by the transcriptional translational oscillator that consists of a series of core clock genes (bmal1, clock, cry and per) and clock controlled genes (rev-erbα, rorα, dbp, tef and hlf).Clock genes exist in immune organs, tissues and cells, leading to the immune cell function (migration and chemotaxis, phagocytosis, cytotoxicity and so on) and a variety of immune parameters (factor level of circulating immune cells and subsets of the relative and absolute number of cells) showing circadian rhythm changes, and playing an important role in maintaining immune homeostasis.In addition, some immune related diseases are closely correlated with circadian rhythms abnormalities.This paper will focus on the effect of circadian rhythms on immune functions and their roles in some immune related diseases.
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Objective To explore the role of the transcriptional factor activator protein (AP)-1 in mediating vascular endothelial growth factor ( VEGF) expression in human bronchial epithelial cells exposed to PM 2.5.Methods Beas-2B cells was treated with PM2.5.Luciferase assay was used to detect the activation status of AP-1 and transcription of VEGF in the Beas-2B cells.The induced activation of c-Jun, ATF2 and VEGF expression was tested by Western blotting assay.Results PM2.5 induced transactivation of the transcriptional factor AP-1, accompanied by phosphorylation of the AP-1 components, c-Jun and ATF2 in Beas-2B cells.Moreover, when AP-1 activation was inhibited by knocking down c-Jun or ATF2 expressions, induction of VEGF expression was partially attenuated in Beas-2B cells.Conclusion AP-1 is a critical transcriptional factor in mediating PM2.5-induced VEGF expression and inflammatory responses in human bronchial epithelial cells.
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Objective To explore the signal transduction mechanism of inhibitor kappa B kinase α( IKKα) , one of the catalytic subunits of IKK complex , for regulating p53 transactivation in the cellular ultraviolet radiation ( UVB) repsonse. Methods The transactivation of p53 was determined by dual-luciferase reporter gene analysis system while the expression and activation of IKKα, IKKβ, p53 and p38K was detected by Western blotting assay .Results UVB exposure induced activation and transactivation of p 53 in the wild type mouse fibroblasts ,but the effect was blocked by IKKa deficiency and recovered by reconstitution of IKKαexpression.Under the same conditions , IKKαregulated p38K activation, while inhibi-ting p38K activation down-regulated p53 transactivation under UVB exposure .Conclusion IKKαregulates UVB-induced phosphorylation and activation of p 53 in a p38K-dependent manner .
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Objective To investigate the molecular mechanism of vascular endothelial growth factor ( VEGF) expression in bronchial epithelial cells (Beas-2B)induced by particulate matter 2.5(PM2.5).Methods PM2.5 powder was dis-solved in DMEM medium and diluted into five concentrations , 0,12.5,25,50 and 100μg/ml, respectively.The double an-tibiotics ( streptomycin and penicillin ) and FBS were added into the solution to a 2% final concentration of serum system after being treated by ultrasound for 30 minutes.The cultured Beas-2B cells were then treated with different doses of PM2.5.Subsequently, nuclear factor-kappa B(NF-κB) transactivity and the transcriptional activation of vegf gene promot-er were tested by dual-luciferase reporter gene analysis system while phosphorylation of p 65 , expression levels of IκBαand VEGF were detected by Western blotting .Results PM2.5 induced up-regulation of VEGF expression in Beas-2B cells in a dose-dependent manner , accompanied by NF-κB transactivation at the highest level under 100 μg/ml of PM2.5 treatment. Moreover, PM2.5 induced degradation of the repressor protein IκBαand increase in the phosphorylation level of p 65 sub-unit in Beas-2B cells.Knockdown of NF-κB p65 expression significantly inhibited vegf gene promoter transcriptional activa-tion as well as VEGF protein expression in Beas-2B cells induced by PM2.5.Conclusion PM2.5 induces VEGF expres-sion via activation of NF-κB pathway in bronchial epithelial cells .
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The methods for searching traditional Chinese medical literature in English with subject headings in PubMed-MEDLINE database were discussed from the practical view, and seven useful searching methods with free words according to practical experiences were put forward in order to assist the domestic TCM researchers to utilize PubMed-MEDLINE database in searching overseas TCM literature in English.
Subject(s)
Information Storage and Retrieval , Methods , MEDLINE , Medicine, Chinese Traditional , Periodicals as Topic , PubMed , United StatesABSTRACT
In order to investigate the mechanism of stat3 nuclear import. positioned a characterized NLS of the SV40 large T antigen into Stat3-GFP, Dstat3-GFP respectively between the C-terminus of Stat3 and the N-terminus of GFP to create Stat3-NLS-GFP and Dstat3-NLS-GFP. With NLS-GFP as the positive control, Expression of the Stat3-NLS-GFP without IL-6 stimulation and Stat3-GFP with IL-6 stimulation resulted in a predominantly nuclear localization in 293T cell. Expression of Stat3-GFP and Dstat3-NLS-GFP without IL-6 stimulation resulted in predominantly cytoplasm localization in 293T cell. The results suggest that latent Stat3 is not anchored in the cytoplasm, and that nuclear localization in response to IL-6 is facilitated by gain of an NLS function.
Subject(s)
Active Transport, Cell Nucleus , Cell Nucleus , Metabolism , Nuclear Localization Signals , Plasmids , Metabolism , STAT3 Transcription Factor , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate apoptosis in XG-7, a human myeloma cell line, induced by IL-6 deprivation and the function of three anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-kappa(L), Mcl-1) in the apoptotic process.</p><p><b>METHODS</b>Apoptosis in XG-7 myeloma cells induced by IL-6 withdrawal was determined by flow cytometry with propidium iodide (PI) nuclear staining. Expressions of three Bcl-2 proteins in XG-7 cells were monitored by immunoblotting assay.</p><p><b>RESULTS</b>In the absence of IL-6 for a certain time, a significant percentage of apoptiotic XG-7 cells can be observed, as well as down-regulated expression of one of the three anti-apoptotic proteins (Mcl-1) in XG-7 cells. IL-6 re-stimulation in XG-7 cells following cytokine removal up-regulated the expression of Mcl-1 and inhibited cell apoptosis.</p><p><b>CONCLUSION</b>Mcl-1,instead of Bcl-2 and Bcl-kappa(L), plays an important role in IL-6 deprivation induced apoptosis in XG-7 human myeloma cells.</p>
Subject(s)
Humans , Apoptosis , Interleukin-6 , Physiology , Multiple Myeloma , Pathology , Myeloid Cell Leukemia Sequence 1 Protein , Neoplasm Proteins , Physiology , Proto-Oncogene Proteins c-bcl-2 , Tumor Cells, Cultured , bcl-X ProteinABSTRACT
<p><b>OBJECTIVE</b>To investigate the biological activity and molecular mechanism of interferon alpha (IFNalpha) on human myeloma cell line Sko-007.</p><p><b>METHODS</b>The effect of IFNalpha on the growth of Sko-007 cells was measured by MTT assay. Cells cycle distribution and the expression of two IL-6 receptor chains (IL-6R and gp130) on Sko-007 cell surface in the absence or presence of IFNalpha were monitored by FACS analysis. The activation state of protein kinase ERK, which is involved in Ras/MAPK signal transduction pathway mediating cell survival and proliferation, and the expression of anti-apoptotic Bcl-2 family proteins-Bcl-2, Bcl-x(L) and Mcl-1 in Sko-007 cells with or without IFNalpha were determined by immunoblot assay.</p><p><b>RESULT</b>IFNalpha arrested Sko-007 cell cycle progression. After stimulation with IFNalpha, an obvious increase in G(0)/G(1) phase (41.1%-->84.1%) and decrease in S phase (57.1%-->13.3%) of Sko-007 cell cycle distribution can be observed. Moreover, the proliferation of Sko-007 cells was dramatically inhibited in the presence of IFNalpha, with a maximal inhibitory rate up to 88%. In addition, the expression of gp130 on cell surface, the activation of protein kinase ERK and the expression of Bcl-2 and Bcl-x(L) were all down-regualted in IFNalpha-stimulated Sko-007 cells.</p><p><b>CONCLUSION</b>The inhibitory effect of IFNalpha on the proliferation of Sko-007 cells was mediated by gp130 down-regulation, degradation of Bcl-2 family anti-apoptotic proteins and inhibition of ERK activation.</p>
Subject(s)
Humans , Antigens, CD , Metabolism , Cell Cycle , Cell Division , Cytokine Receptor gp130 , Dose-Response Relationship, Drug , Down-Regulation , Enzyme Activation , G1 Phase , Immunoblotting , Interferon-alpha , Pharmacology , Membrane Glycoproteins , Metabolism , Mitogen-Activated Protein Kinases , Metabolism , Multiple Myeloma , Metabolism , Pathology , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Receptors, Interleukin-6 , Metabolism , Resting Phase, Cell Cycle , S Phase , Tumor Cells, Cultured , Metabolism , bcl-X ProteinABSTRACT
Objective To investigate the IL-6 signal transduction pathways and their regulation mechanism in a human myeloma cell line-U266. Methods Electrophoretic mobility shift assay (EMSA) was used to detect the activation of the transcription factors (TFs)-STAT3 and NF-IL-6 by IL-6. Cells were treated with chemical agents or transfected with the expression plasmids for the two TFs or the anti-sense oligonucleotide for protein kinase involved in one IL-6 signal transduction pathway. The change of the activation state of another IL-6 signal transduction pathway was also exhibited by EMSA. Results Two IL-6 signal transduction pathways (JAK/STAT and Ras/NF-IL-6) can be activated by IL-6 of different dose in U266 cells. When one of the two signal transduction pathways was up-regulated, the other one was down-regulated. Conclusion There is an antagonistic effect between the activation of two IL-6 signal transduction pathways in U266 cells.
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Objective:To investigate the mechanism of dexamethasone(DEX) induced apoptosis of human myeloma cell line Sko 007 and the inhibitory effect of IL 6 on apoptosis.Methods:Effect of DEX on the growth of Sko 007 cells was measured by MTT assay;Apoptosis of Sko 007 cells induced by DEX was determined by flow cytometry with propidium iodide(PI) staining of nuclei;The expression of three anti apoptotic Bcl 2 family proteins Bcl 2,Bcl X L and Mcl 1 in Sko 007 cells with or without DEX were determined by immunblot assay.Results:DEX inhibited the proliferation of Sko 007 cells and induced a significant cell apoptosis.In addition,degradation of Bcl 2 can be observed in Sko 007 cells in the presence of Dex.IL 6 stimulation prevented down regulation of Bcl 2 and the apoptosis of Sko 007 cells induced by DEX.Conclusion:IL 6 inhibits DEX induced apoptosis of Sko 007 cells through the specific regulation of Bcl 2.
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Objective: To investigate which step of IL-6 signal transduction pathways in myeloma cells can be taken as the acting target of the antagonists for IL-6. Methods: EMSA and immunoprecipitation were used to detect the activation of transcription factors(TFs)-STAT3, NF-IL-6 and protein kinase ERK in a myeloma cell line-Sko-007 by IL-6. then anti-sense expression plasmids and anti-sense ODN for these signal moleculars were constructed and designed,the effects of these anti-sense nucleic acids on IL-6 signal transduction in Sko-007 cells were analyzed by the same methods. Results: IL-6 signal transduction could be antagonized by these anti-sense nucleic acids at different extent.Conclusion: TFs or protein kinases in IL-6 signal trareduction pathways can be taken as the target for antagonists design.