Inspired by the light-dependent signal transduction in nature, we herein report a fully synthetic receptor AZO with the capacity of transmembrane signaling, working by photo-induced change of molecular conformation. O...Inspired by the light-dependent signal transduction in nature, we herein report a fully synthetic receptor AZO with the capacity of transmembrane signaling, working by photo-induced change of molecular conformation. Our receptor has an anchoring group, a rigid and photoresponsive transmembrane unit and a precatalyst tailgroup. After doping in lipid membranes, AZO is membrane anchored and the extended trans-isomer enables the tailgroup to bind with intravesicular Zn^(2+), thereby achieving enzyme activation and triggering downstream events(ester hydrolysis). However, the shortened cis-isomer pulls the tailgroup into lipids, thereby preventing the complexation and all transduction processes. Upon alternative irradiation of ultraviolet(UV) and visible light, the transduction process can be reversible switch between“ON” and “OFF”, achieving light signal transduction. This study provides a new strategy for future design of artificial signal transduction receptors.展开更多
Infertility has become one of the most serious diseases worldwide,and 50% of this disease can be attributed to male-related factors.Spermatogenesis,by definition,is a complex process by which spermatogonial stem cells...Infertility has become one of the most serious diseases worldwide,and 50% of this disease can be attributed to male-related factors.Spermatogenesis,by definition,is a complex process by which spermatogonial stem cells(SSCs)self-renew to maintain stem cell population within the testes and differentiate into mature spermatids.It is of great significance to uncover gene regulation and signaling pathways that are involved in the fate determinations of SSCs with aims to better understand molecular mechanisms underlying human spermatogenesis and identify novel targets for gene therapy of male infertility.Significant achievement has recently been made in demonstrating the signaling molecules and pathways mediating the fate decisions of mammalian SSCs.In this review,we address key gene regulation and crucial signaling transduction pathways in controlling the self-renewal,differentiation,and apoptosis of SSCs,and we illustrate the networks of genes and signaling pathways in SSC fate determinations.We also highlight perspectives and future directions in SSC regulation by genes and their signaling pathways.This review could provide novel insights into the genetic regulation of normal and abnormal spermatogenesis and offer molecular targets to develop new approaches for gene therapy of male infertility.展开更多
Objective Melittin and its derivatives have been characterized to establish effective gene delivery systems.Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery.Nevertheles...Objective Melittin and its derivatives have been characterized to establish effective gene delivery systems.Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery.Nevertheless,little investigation has been conducted to explore its potential application in the context of viral vectors.Methods Various melittin-derived peptides were inserted into the loop VIII of the capsid proteins of recombinant adeno-associated virus vectors.These vectors carrying either gfp or fluc genes were subjected to qPCR assays and transduction assays of HEK293T cells to investigate the efficiency of vector production and gene delivery.In addition,the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice.Finally,the intricate details of the vector-mediated transduction mechanisms were revealed by specific pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle.Results A total of 76 melittin-related peptides were compiled from existing literature.Among them,cMA2,Melt13,p5RHH and aAR3 were found to significantly enhance the gene delivery efficiency of rAAV2 vectors.The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV.Mechanistically,bafilomycin A1,a vacuolar endosome acidification inhibitor,completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors.Most importantly,p5RHH-rAAV8 vectors also demonstrated increased hepatic transduction in vivo in C57BL/6 mice.Conclusion The incorporation of melittin analogues into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression.While further modifications remain an area of interest,our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery.展开更多
Precise targeting of specific regions within the central nervous system(CNS)is crucial for both scientific research and gene therapy in the context of brain diseases.Adeno-associated virus 13(AAV13)is known for its re...Precise targeting of specific regions within the central nervous system(CNS)is crucial for both scientific research and gene therapy in the context of brain diseases.Adeno-associated virus 13(AAV13)is known for its restricted diffusion range within the CNS,making it an ideal choice for precise labeling and administration within small brain regions.However,AAV13 mediates relatively low expression of target genes.Here,we introduced specifically engineered modifications to the AAV13 capsid protein to enhance its transduction efficiency.We first constructed AAV13-YF by mutating tyrosine to phenylalanine on the surface of the AAV13 capsid.We then inserted the 7m8 peptide,known to enhance cell transduction,into positions 587/588 and 585/586 of the AAV13 capsid,resulting in two distinct variants named AAV13-587-7m8 and AAV13-585-7m8,respectively.We found that AAV13-YF exhibited superior in vitro infectivity in HEK293T cells compared to AAV13,while AAV13-587-7m8 and AAV13-585-7m8 showed enhanced CNS infection capabilities in C57BL/6 mice,with AAV13-587-7m8 infection retaining a limited spread range.These modified AAV13 variants hold promising potential for applications in gene therapy and neuroscience research.展开更多
To cope with unpredictably environmental perturbations and sometimes stresses, plants have evolved with some mechanisms so that these developing stresses can be sensitively perceived and the physiology can be rapidl...To cope with unpredictably environmental perturbations and sometimes stresses, plants have evolved with some mechanisms so that these developing stresses can be sensitively perceived and the physiology can be rapidly regulated. Such perception and regulation can be a kind of feed_forward mechanism and may involve many biochemical and physiological processes and/or the expression of many genes. Although many dehydration_responsive genes have been identified, much fewer of their functions have been known. Such stress_ induced responses should include the initial perception of the dehydration signal, then the complicated signal transduction and cellular transmission until to the final gene activation or expression. As an important plant stress hormone abscisic acid (ABA) mediates many such responses. We believe that starting from the initial perception of dehydration to the gene expression leading to the stress_induced ABA biosynthesis is the most important stress signal transduction pathway among all the plant responses to stresses. Identification of the genes involved and understanding their roles during stress perception and physiological regulation shall be the most important and interesting research field in the coming years.展开更多
Abscisic acid (ABA) plays an important role in plant growth and developmental processes. Although some ABA signal molecules, such as cADPR, Ca2+, etc., have been reported, there. was no evidence proving the involvemen...Abscisic acid (ABA) plays an important role in plant growth and developmental processes. Although some ABA signal molecules, such as cADPR, Ca2+, etc., have been reported, there. was no evidence proving the involvement of cAMP in A-B-A, signal transduction. In this present study, the constructed gene ( rd29A-GUS) was transformed into Nicotiana tabacum, and calli was induced from the transgenic plant. The suspension cells obtained from the callus grew well and uniformly. Treatment of the suspension cells with ABA led to an increase in GUS activity, indicating that these transgenic suspension cells are useful for the study of ABA signaling. Addition of nicotinamide (cADPR inhibitor) or U-73122 (phospholiphase C inhibitor) could only partially inhibit the increase of GUS activity elicited by ABA. The inhibitory effect of nicotinamide was enhanced by application of K252a (inhibitor of protein kinase). Treatment of the suspension cells with 8-Br-cAMP, a membrane-permeable analogue of cAMP, could partially replace the effect of ABA. Furthermore, intracellular addition of IBMX (phosphodiesterase inhibitor) mimicked die effect of exogenous cAMP on the deduction of expression of rd29A promoter. These results suggested that cAMP was an important messenger in ABA signal transduction in tobacco suspension cell.展开更多
The immune responses of plants to foreign pathogens have developed relevant defense mechanisms, which formed complicated disease resistant signal transduction pathways. Salicylic acid（SA）, jasmonic acid（JA）/Ethyl...The immune responses of plants to foreign pathogens have developed relevant defense mechanisms, which formed complicated disease resistant signal transduction pathways. Salicylic acid（SA）, jasmonic acid（JA）/Ethylene（ET） and brassi- nosteroid （BR） could trigger the immune response to different pathogens in plants, making the plants show some resistance to the pathogens. The study on the trans- duction pathways of these three disease-resistant signals were introduced to provide some useful suggestions for the study on the transduction of disease-resistant sig- nals in plants.展开更多
The growth factor receptor-bound protein 2 （Grb2） -associated binder （Gab） proteins are intracellular scaffolding/ docking molecules,and participate in multiple signaling pathways,usually acting as the downstream ...The growth factor receptor-bound protein 2 （Grb2） -associated binder （Gab） proteins are intracellular scaffolding/ docking molecules,and participate in multiple signaling pathways,usually acting as the downstream effector of protein-tyrosine kinases （PTKs） -triggered signal transduction pathway.When phosphorylated by PTKs,Gab proteins can recruit several signaling molecules （p85,SHP2,and Crk） ,and subsequently activate multiple transmitting signals that are critical for cell growth,survival,differentiation and apoptosis.Recently,it has been reported that Gab2 polymorphism is associated with the increase in the risk of Alzheimer’s disease （AD） and is involved in the pathogenesis of AD.This review mainly focuses on the structure and function of Gab2 protein and its role in the pathogenesis of AD.展开更多
Advances of studies on the acupuncture and pain signal transduction mechanisms in complete Freud＇s adjuvant arthritis are reviewed from the three aspects, the first messenger of modulating pain signals and the relate...Advances of studies on the acupuncture and pain signal transduction mechanisms in complete Freud＇s adjuvant arthritis are reviewed from the three aspects, the first messenger of modulating pain signals and the related receptors, the second messenger of modulating pain signals and other factors possibly involved in modulation of pain signal transduction, etc. It is held that modulation of acupuncture for pain signals is a comprehensive course involved in multi-channels, multi-levels, multi-links, and in future, acupuncture analgesic mechanisms for Freud＇s adjuvant arthritis will be more deeply studied by use of more new techniques and new methods.展开更多
There are two degradation systems in mammalian cells, autophagy/lysosomal pathway and ubiquitin-proteasome pathway. Proteasome is consist of multiple protein subunits and plays important roles in degradation of short-...There are two degradation systems in mammalian cells, autophagy/lysosomal pathway and ubiquitin-proteasome pathway. Proteasome is consist of multiple protein subunits and plays important roles in degradation of short-lived cellular proteins. Recent studies reveal that proteasomal degradation system is also involved in signal transduction and regulation of various cellular functions. Dysfunction or dysregulation of proteasomal function may thus be an important pathogenic mechanism in certain neurological disorders. This paper reviews the biological functions of proteasome in signal transduction and its potential roles in neurodegenerative diseases.展开更多
AIM To compare the expression level of Fas gene and Bcl-2 gene in gastric cancer cells SGC7901 and gastric cancer MDR (multidrug resistant) cells SGC7901/VCR, to transduce Fas cDNA and Bcl-2 antisense nucleic acid int...AIM To compare the expression level of Fas gene and Bcl-2 gene in gastric cancer cells SGC7901 and gastric cancer MDR (multidrug resistant) cells SGC7901/VCR, to transduce Fas cDNA and Bcl-2 antisense nucleic acid into SGC7901/VCR cells respectively, and to observe the expression of two genes in transfectants and non-transfectants as well as their drug sensitivity.METHODS Eukaryotic expression vector pBK-Fas cDNA and pDOR-anti Bcl-2 were constructed and transfected into SGC7901/VCR cells by lipofectamine, respectively. Northern blot and Western blot were used to detect the expression of mRNA and protein in SGC7901/VCR and SGC7901 cells and transfectants, and drug sensitivity of transfectants for VCR, CDDP and 5-FU was analyzed with MTT assay.RESULTS After gene transfection, 80 for Fas and 120 for antisense Bcl-2 drug-resistant clones were selected from 2×105 cells, transfection rate being 0.04% and 0.06%. Two clones of SGC7901 Fas/VCR cells and SGC7901 anti Bcl-2/VCR cells were randomly selected for further incubation. Hybridization results showed that the expression level of Fas mRNA and protein in SGC7901/VCR cells was much lower, but that of Bcl-2 mRNA and protein was higher than that in SGC7901 cells. The expression of Fas mRNA and protein in SGC7901 Fas/VCR cells was higher, and of Bcl-2 mRNA and protein was lower in SGC7901 anti Bcl-2/VCR cells than that in non-transfectants. MTT assay showed that transfectants were more sensitive to VCR, CDDP, 5-FU than non-transfectants.CONCLUSION Bcl-2 gene displayed high expression while Fas gene had low expression in drug resistant gastric cancer cells. Expression of Bcl-2 protein was effectively blocked in SGC7901 anti Bcl-2/VCR cells by gene transfection. In contrast, the expression of Fas mRNA and protein in SGC7901 Fas/VCR cells increased. Fas gene and Bcl-2 antisense nucleic acid transfection sensitized drug resistant gastric cancer cells to chemotherapeutic drugs. These results suggest cell apoptosis plays an important role in the mechanism of MDR, and enhancing apoptosis might reverse MDR.展开更多
Objective: To study the effect of emodin on protein and gene expressions of the massagers in mobility signal transduction system of cholecyst smooth muscle cells in guinea pig with cholesterol calculus. Methods: The g...Objective: To study the effect of emodin on protein and gene expressions of the massagers in mobility signal transduction system of cholecyst smooth muscle cells in guinea pig with cholesterol calculus. Methods: The guinea pigs were randomly divided into 4 groups, such as control group, gall-stone(GS) group, emodin group and ursodesoxycholic acid(UA) group. Cholesterol calculus models were induced in guinea pigs of GS, emodin and UA groups of induced models by lithogenic diet, while emodin or UA were given to the corresponding group for 7 weeks. The histomorphological and ultrastructure change of gallbladder were detected by microscope and electron microscope, the content of plasma cholecystokinin(CCK) and [Ca^(2+)]i were analyzed successively by radioimmunoassay and flow cytometry. The protein and mR NA of Gsα, Giα and Cap in cholecyst cells were determined by western blotting and real time polymerase chain reaction(RT-PCR). Results: Emodin or UA can relieve pathogenic changes in epithelial cells and muscle cells in gallbladder of guinea pig with cholesterol calculus by microscope and transmission electron microscope. In the cholecyst cells of GS group, CCK levels in plasma and [Ca^(2+)]i decreased, the protein and m RNA of GS group were downregulated,the protein and m RNA of Gi and Cap were up-regulated. Emodin significantly decreased the formative rate of gallstone, improved the pathogenic change in epithelial cells and muscle cells, increased CCK levels in plasma and [Ca^(2+)]i in cholecyst cells, enhanced the protein and mR NA of Gs in cholecyst cells, reduced the protein and mR NA of Gi and Cap in cholecyst cells in guinea pig with cholesterol calculus. Conclusion: The dysfunction of gallbladder contraction gives rise to the disorders of mobility signal transduction system in cholecyst smooth muscle cells, including low content of plasma CCK and [Ca^(2+)]i in cholecyst cells, abnormal protein and mRNA of Gs, Gi and Cap. Emodin can enhance the contractibility of gallbladder and alleviate cholestasis by regulating plasma CCK levels, [Ca2+]i in cholecyst cells and the protein and mR NA of Gs, Gi and Cap.展开更多
The galactopoietic mechanism of Vaccaria segetalis is still unknown. Understanding dibutyl phthalate （DBP） separated from Vaccaria segetalis on the expression of lactation signal transduction genes of mammary gland ...The galactopoietic mechanism of Vaccaria segetalis is still unknown. Understanding dibutyl phthalate （DBP） separated from Vaccaria segetalis on the expression of lactation signal transduction genes of mammary gland epithelial cells, including prlr, erα, akt1, socs2, pparγ and elf5, will be helpful to reveal the molecular mechanism. Western blot and qRT- PCR were used to study the change of prlr, erα, akt, socs2, pparγ, and elf5 expression at mRNA and protein level. Co- localization expression of prolactin receptor （PRLR） and estrogen receptor α （ERα） was observed by immunofluorescence; the expression changes of miRNAs （21, 125b, 143, and 195） and the secretion of β-casein and lactose were detected by qRT-PCR and RP-HPLC. The results showed that Vaccaria segetalis active compound had similar fuctions as estrogen and/or prolactin （PRL） in dairy cow mammary gland epithelial cells （DCMECs）, increased the expressions of prlr, erα, akt1, and elf5 genes, while repressed pparγ expressions. DBP promoted socs2 mRNA expression, but its protein expressions were repressed. Furthermore, both DBP and PRL could repress the expressions of miRNA-125b, miRNA-143 and miRNA- 195 in DCMECs. DBP could repress the expression of miRNA-21, while the influence of PRL on miRNA-21 was not certain. DBP could promote the lactation ability of DCMECs by regulating the ER and PRLR cellular signal transduction pathway.展开更多
Objective:To study the mechanism of insulin resistance in the cholesterol gallstone formation from insulin signal transduction pathway so as to reveal the possible mechanism and the effective role of Albiflorin Granul...Objective:To study the mechanism of insulin resistance in the cholesterol gallstone formation from insulin signal transduction pathway so as to reveal the possible mechanism and the effective role of Albiflorin Granule on preventing the cholesterol gallstones.Methods:Serum triglycerides(TG),free fatty acid(FFA),and total cholesterol(TC) from different groups were measured and liver cells Ins R,PKB,IKK-β protein expression levels were detected by western blotting.Results:Albiflorin significantly decreased the cholesterol gallstone formation rate,increased glucose infusion rate in gallstone guinea pigs and improved insulin resistance.Compared with the normal group,insulin receptor and PKB protein expression in GS group were significantly reduced.IKK-β protein in the GS group increased significantly and Albiflorin could reduce IKK-β protein expression in guinea pig liver cells.Conclusions:The model of insulin resistance in cholesterol gallstone guinea pig was successfully established,which plays an important role in the cholesterol gallstone formation.All aspects of insulin signaling pathway are involved in gallstone formation.Albiflorin can regulate various aspects of insulin signal transduction pathway to prevent the formation of gallbladder.展开更多
The copper-binding, membrane-anchored, cellular prion protein （PrP~） has two constitutive cleavage sites producing distinct N- and C-terminal fragments （N1/C1 and N2/C2）. Using RK13 cells expressing either human P...The copper-binding, membrane-anchored, cellular prion protein （PrP~） has two constitutive cleavage sites producing distinct N- and C-terminal fragments （N1/C1 and N2/C2）. Using RK13 cells expressing either human PrPc, mouse PrPc or mouse PrP^C carrying the 3F4 epitope, this study explored the influence of the PrP^C primary sequence on endoproteolytic cleavage and one putative PrPc function, MAP kinase signal transduction, in response to exogenous copper with or without a perturbed membrane environment. PrPc primary sequence, especially that around the N1/C1 cleavage site, appeared to influence basal levels of proteolysis at this location and extracellular signal-regulat- ed kinase 1/2 （ERK1/2） phosphorylation, with increased processing demonstrating an inverse relationship with basal ERK1/2 activation. Human PrP^C showed increased N1/C1 cleavage in response to copper alone, accompanied by specific p38 and JNK/SAPK phosphorylation. Combined exposure to copper plus the cholesterol-sequestering antibiotic filipin resulted in a mouse PrP^C-specific substantial increase in signal protein phosphorylation, accompanied by an increase in N1/C1 cleavage. Mouse PrPc harboring the human N1/C1 cleavage site assumed more human-like profiles basally and in response to copper and altered membrane environments. Our results demonstrate that the PrPc pri- mary sequence around the N1/C1 cleavage site influences endoproteolytic processing at this location, which appears linked to MAP kinase signal transduction both basally and in response to copper. Further, the primary sequence appears to confer a mutual dependence of N1/C1 cleavage and membrane integrity on the fidelity of PrP^C-related signal transduction in response to exogenous stimuli.展开更多
Erythropoietin (EPO), a 34 kD glycopro-tein, is the principal growth factor regulating theproduction of circulating erythrocytes; EPO isessential for committed CFU - E erythroid pro-genitors to divide several times an...Erythropoietin (EPO), a 34 kD glycopro-tein, is the principal growth factor regulating theproduction of circulating erythrocytes; EPO isessential for committed CFU - E erythroid pro-genitors to divide several times and then to dif-ferentiate into erythrocytes. Like most receptorsfor hematopoietic growth factors, the erythro-poietin receptor (EPO - R) is a type I trans-membrane protein and a member of the cytokinereceptor superfamily. These receptors containfour conserved cysteines and a Trp - Ser - X -展开更多
Cold stress responses help insects to survive under low temperatures that would be lethal otherwise.This phenomenon might contribute to the invasion of some Bemisia tabaci cryptic species from subtropical areas to tem...Cold stress responses help insects to survive under low temperatures that would be lethal otherwise.This phenomenon might contribute to the invasion of some Bemisia tabaci cryptic species from subtropical areas to temperate regions.However,the molecular mechanisms regulating cold stress responses in whitefly are yet unclear.Mitogen-activated protein kinases（MAPKs）which including p38,ERK,and JNK,are well known for their roles in regulating metabolic responses to cold stress in many insects.In this study,we explored the possible roles of the MAPKs in response to low temperature stresses in the Mediterranean cryptic species（the Q-biotype）of the B.tabaci species complex.First,we cloned the p38 and ERK genes from the whitefly cDNA library.Next,we analyzed the activation of MAPKs during cold stress in the Mediterranean cryptic species by immuno-blotting.After cold stress,the level of phospho-p38 increased but no significant change was observed in the phosphorylation of ERK and JNK,thus suggesting that the p38 might be responsible for the defense response to low temperature stress.Furthermore,we demonstrated that：i）3 min chilling at 0°C was sufficient for the activation of p38 MAPK pathway in this whitefly;and ii）the amount of phosphorylated p38 increased significantly in the first 20 min of chilling,reversed by 60 min,and then returned to the original level by 120 min.Taken together,our results suggest that the p38 pathway is important during response to low temperature stress in the Mediterranean cryptic species of the B.tabaci species complex.展开更多
AIM: To investigate the role of TIP30 in apoptotic signal pathway in hepatoblastoma cells and to provide a basis for TIP30 as a gene therapy candidate in the regression of hepatoblastoma cells. METHODS: Apoptosis of h...AIM: To investigate the role of TIP30 in apoptotic signal pathway in hepatoblastoma cells and to provide a basis for TIP30 as a gene therapy candidate in the regression of hepatoblastoma cells. METHODS: Apoptosis of human hepatoblastoma cell lines HepG2 (p53 wild), Hep3B (p53 null) and PLC/RPF/5 (p53 mutant) infected with Ad-TIP30 (bearing a wild type human Tip30 gene) were analyzed and p53, Bax and Bcl-xl expression levels were compared among these cells. MTT assay, DNA fragmentation, in situ 3' end labeling of DNA, annexin-V FITC staining were used to detect cell death and apoptosis in cells at various time intervals subsequent to infection, and to determine whether TIP30 had an effect on the expression levels of some apoptosis-related gene products such as Bax, p53 and Bcl-xl. A similar time course experiment was performed by Western blotting. RESULTS: In MTT assay, the viability of HepG2 cells decreased significantly from 99.7% to 10% and displayed more massive cell death within 5-8 d than Hep3B and PLC/ RPF/5 cells, with their viability decreased from 97.8% to 44.3% and 98.1% to 50.4%, respectively. In annexin-V FITC assay, the percentage of apoptosis cells in HepG2 cells was two to three-fold higher than that in control cells (infected with Ad-GFP), two-fold higher than that in Hep3B cells and 1.4-fold higher than that in PLC/RPF/5 cells 36 h after infection, respectively. Moreover, in HepG2 cells, the p53 began to increase 6-8 h after infection, reaching a maximum level between 8 and 12 h after infection and then dropped. Bax showed a similar increase in the cells as p53 reached the maximum at 8-12 h and subsequently decreased. Interestingly, Bcl-xl protein levels were down regulated during 24 to 36 h after Ad-TIP30 infection. In contrast, ectopic expression of TIP30 in Hep3B and PLC/ RPF/5 cells had no effect on the regulation of Bax expression, but had an effect on Bcl-xl levels. In comparison with HepG2 cells, these data suggested that up-regulation of p53 levels by TIP30 might be a pre-requisite for Bax and Bax/Bcl-xl ratio increase. We hypothesied that TIP30 might regulate Bax gene partly through p53, which sensitizes cells to apoptosis by involving a p53 apoptosis signal transduction pathway. CONCLUSION: TIP30 plays an important role in predisposing hepatoblastoma cells to apoptosis through regulating expression levels of these genes. Ad-TIP30 carrying exogenous TIP30-anti-tumor genes may be regarded as a potential candidate for the treatment of hepatocellular carcinoma.展开更多
Drosophila dEAAT2, a member of the excitatory amino-acid transporter(EAAT) family, has been described as mediating the high-affinity transport of taurine, which is a free amino-acid abundant in both insects and mammal...Drosophila dEAAT2, a member of the excitatory amino-acid transporter(EAAT) family, has been described as mediating the high-affinity transport of taurine, which is a free amino-acid abundant in both insects and mammals. However, the role of taurine and its transporter in hearing is not clear. Here, we report that dEAAT2 is required for the larval startle response to sound stimuli. d EAAT2 was found to be enriched in the distal region of chordotonal neurons where sound transduction occurs. The Ca2+imaging and electrophysiological results showed that disrupted dEAAT2 expression significantly reduced the response of chordotonal neurons to sound.More importantly, expressing d EAAT2 in the chordotonal neurons rescued these mutant phenotypes. Taken together,these findings indicate a critical role for Drosophila dEAAT2 in sound transduction by chordotonal neurons.展开更多
AIM：To develop a new method to produce recombinant reprogramming proteins,c Myc,Klf4,Oct4,and Sox2,in soluble format with low cost for the generation of induced pluripotent stem cells（i PSCs）.METHODS：A short polyp...AIM：To develop a new method to produce recombinant reprogramming proteins,c Myc,Klf4,Oct4,and Sox2,in soluble format with low cost for the generation of induced pluripotent stem cells（i PSCs）.METHODS：A short polypeptide sequence derived from the HIV trans-activator of transcription protein（TAT） and the nucleus localization signal（NLS） polypeptide were fused to the N terminus of the reprogramming proteins and they were constructed into p Cold-SUMO vector which can extremely improve the solubility of recombinant proteins.Then these vector plasmids were transformed into E.coli BL21（DE3） Chaperone competent cells for amplification.The solubility of these recombinant proteins was determined by SDS-PAGE and Coomassie brilliant blue staining.The recombinant proteins were purified by NiNTA resin and identified by Western blot.The transduction of these proteins into HEK 293 T cells were evaluated by immunofluorescence staining.RESULTS：These four reprogramming proteins could be produced in soluble format in p Cold-SUMO expression vector system with the assistance of chaperone proteins in bacteria.The proteins were purified successfully with a purity of over 70% with a relative high transduction rate into 293 cells.CONCLUSION：The results in the present study indicate the four important reprogramming proteins,c Myc,Klf4,Oct4,and Sox2,can be produced in soluble format in bacteria with low cost.Our new method thus might be expected to greatly contribute to the future study of i PSCs.展开更多
基金supported by the National Natural Science Foundation of China (No. 22171085)Shanghai Frontier Science Research Base of Optogenetic Techniques for Cell Metabolism (Shanghai Municipal Education Commission, No. 2021 Sci & Tech 03–28)。
文摘Inspired by the light-dependent signal transduction in nature, we herein report a fully synthetic receptor AZO with the capacity of transmembrane signaling, working by photo-induced change of molecular conformation. Our receptor has an anchoring group, a rigid and photoresponsive transmembrane unit and a precatalyst tailgroup. After doping in lipid membranes, AZO is membrane anchored and the extended trans-isomer enables the tailgroup to bind with intravesicular Zn^(2+), thereby achieving enzyme activation and triggering downstream events(ester hydrolysis). However, the shortened cis-isomer pulls the tailgroup into lipids, thereby preventing the complexation and all transduction processes. Upon alternative irradiation of ultraviolet(UV) and visible light, the transduction process can be reversible switch between“ON” and “OFF”, achieving light signal transduction. This study provides a new strategy for future design of artificial signal transduction receptors.
基金supported by the grants from the National Nature Science Foundation of China(No.32170862)Major Scientific and Technological Projects for Collaborative Prevention and Control of Birth Defect in Hunan Province(No.2019SK1012)+1 种基金the Research Team for Reproduction Health and Translational Medicine of Hunan Normal University(No.2023JC101)Graduate Scientific Research Innovation Project of Hunan Province,China(No.CX2022520).
文摘Infertility has become one of the most serious diseases worldwide,and 50% of this disease can be attributed to male-related factors.Spermatogenesis,by definition,is a complex process by which spermatogonial stem cells(SSCs)self-renew to maintain stem cell population within the testes and differentiate into mature spermatids.It is of great significance to uncover gene regulation and signaling pathways that are involved in the fate determinations of SSCs with aims to better understand molecular mechanisms underlying human spermatogenesis and identify novel targets for gene therapy of male infertility.Significant achievement has recently been made in demonstrating the signaling molecules and pathways mediating the fate decisions of mammalian SSCs.In this review,we address key gene regulation and crucial signaling transduction pathways in controlling the self-renewal,differentiation,and apoptosis of SSCs,and we illustrate the networks of genes and signaling pathways in SSC fate determinations.We also highlight perspectives and future directions in SSC regulation by genes and their signaling pathways.This review could provide novel insights into the genetic regulation of normal and abnormal spermatogenesis and offer molecular targets to develop new approaches for gene therapy of male infertility.
基金sponsored by grants from the National Natural Science Foundation of China(No.82030117)the Wenzhou major scientific and technological innovation project(No.ZY2022001).
文摘Objective Melittin and its derivatives have been characterized to establish effective gene delivery systems.Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery.Nevertheless,little investigation has been conducted to explore its potential application in the context of viral vectors.Methods Various melittin-derived peptides were inserted into the loop VIII of the capsid proteins of recombinant adeno-associated virus vectors.These vectors carrying either gfp or fluc genes were subjected to qPCR assays and transduction assays of HEK293T cells to investigate the efficiency of vector production and gene delivery.In addition,the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice.Finally,the intricate details of the vector-mediated transduction mechanisms were revealed by specific pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle.Results A total of 76 melittin-related peptides were compiled from existing literature.Among them,cMA2,Melt13,p5RHH and aAR3 were found to significantly enhance the gene delivery efficiency of rAAV2 vectors.The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV.Mechanistically,bafilomycin A1,a vacuolar endosome acidification inhibitor,completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors.Most importantly,p5RHH-rAAV8 vectors also demonstrated increased hepatic transduction in vivo in C57BL/6 mice.Conclusion The incorporation of melittin analogues into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression.While further modifications remain an area of interest,our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery.
基金National Science and Technology Innovation 2030 Grant(2021ZD0201003)National Natural Science Foundation of China(31830035,31771156,21921004)+2 种基金Strategic Priority Research Program of the Chinese Academy of Sciences(XDB32030200)Shenzhen Key Laboratory of Viral Vectors for Biomedicine(ZDSYS20200811142401005)Key Laboratory of Quality Control Technology for Virus-Based Therapeutics,Guangdong Provincial Medical Products Administration(2022ZDZ13)。
文摘Precise targeting of specific regions within the central nervous system(CNS)is crucial for both scientific research and gene therapy in the context of brain diseases.Adeno-associated virus 13(AAV13)is known for its restricted diffusion range within the CNS,making it an ideal choice for precise labeling and administration within small brain regions.However,AAV13 mediates relatively low expression of target genes.Here,we introduced specifically engineered modifications to the AAV13 capsid protein to enhance its transduction efficiency.We first constructed AAV13-YF by mutating tyrosine to phenylalanine on the surface of the AAV13 capsid.We then inserted the 7m8 peptide,known to enhance cell transduction,into positions 587/588 and 585/586 of the AAV13 capsid,resulting in two distinct variants named AAV13-587-7m8 and AAV13-585-7m8,respectively.We found that AAV13-YF exhibited superior in vitro infectivity in HEK293T cells compared to AAV13,while AAV13-587-7m8 and AAV13-585-7m8 showed enhanced CNS infection capabilities in C57BL/6 mice,with AAV13-587-7m8 infection retaining a limited spread range.These modified AAV13 variants hold promising potential for applications in gene therapy and neuroscience research.
文摘To cope with unpredictably environmental perturbations and sometimes stresses, plants have evolved with some mechanisms so that these developing stresses can be sensitively perceived and the physiology can be rapidly regulated. Such perception and regulation can be a kind of feed_forward mechanism and may involve many biochemical and physiological processes and/or the expression of many genes. Although many dehydration_responsive genes have been identified, much fewer of their functions have been known. Such stress_ induced responses should include the initial perception of the dehydration signal, then the complicated signal transduction and cellular transmission until to the final gene activation or expression. As an important plant stress hormone abscisic acid (ABA) mediates many such responses. We believe that starting from the initial perception of dehydration to the gene expression leading to the stress_induced ABA biosynthesis is the most important stress signal transduction pathway among all the plant responses to stresses. Identification of the genes involved and understanding their roles during stress perception and physiological regulation shall be the most important and interesting research field in the coming years.
文摘Abscisic acid (ABA) plays an important role in plant growth and developmental processes. Although some ABA signal molecules, such as cADPR, Ca2+, etc., have been reported, there. was no evidence proving the involvement of cAMP in A-B-A, signal transduction. In this present study, the constructed gene ( rd29A-GUS) was transformed into Nicotiana tabacum, and calli was induced from the transgenic plant. The suspension cells obtained from the callus grew well and uniformly. Treatment of the suspension cells with ABA led to an increase in GUS activity, indicating that these transgenic suspension cells are useful for the study of ABA signaling. Addition of nicotinamide (cADPR inhibitor) or U-73122 (phospholiphase C inhibitor) could only partially inhibit the increase of GUS activity elicited by ABA. The inhibitory effect of nicotinamide was enhanced by application of K252a (inhibitor of protein kinase). Treatment of the suspension cells with 8-Br-cAMP, a membrane-permeable analogue of cAMP, could partially replace the effect of ABA. Furthermore, intracellular addition of IBMX (phosphodiesterase inhibitor) mimicked die effect of exogenous cAMP on the deduction of expression of rd29A promoter. These results suggested that cAMP was an important messenger in ABA signal transduction in tobacco suspension cell.
基金Supported by the National Natural Science Foundation of China(31360262)Zhuke Contract(2012HK209-38)the Innovation Capacity Platform Construction Project of Guizhou Science and Technology Department(2011018)~~
文摘The immune responses of plants to foreign pathogens have developed relevant defense mechanisms, which formed complicated disease resistant signal transduction pathways. Salicylic acid（SA）, jasmonic acid（JA）/Ethylene（ET） and brassi- nosteroid （BR） could trigger the immune response to different pathogens in plants, making the plants show some resistance to the pathogens. The study on the trans- duction pathways of these three disease-resistant signals were introduced to provide some useful suggestions for the study on the transduction of disease-resistant sig- nals in plants.
基金supported by the National Basic Research Development Program of China(No.2006CB500706)the National Natural Science Foundation of China(No.30700251,30872729,30971031)+1 种基金Shanghai Key Discipline Program(No.S30202)the Program for Out-standing Medical Academic Leader(No.LJ 06003)
文摘The growth factor receptor-bound protein 2 （Grb2） -associated binder （Gab） proteins are intracellular scaffolding/ docking molecules,and participate in multiple signaling pathways,usually acting as the downstream effector of protein-tyrosine kinases （PTKs） -triggered signal transduction pathway.When phosphorylated by PTKs,Gab proteins can recruit several signaling molecules （p85,SHP2,and Crk） ,and subsequently activate multiple transmitting signals that are critical for cell growth,survival,differentiation and apoptosis.Recently,it has been reported that Gab2 polymorphism is associated with the increase in the risk of Alzheimer’s disease （AD） and is involved in the pathogenesis of AD.This review mainly focuses on the structure and function of Gab2 protein and its role in the pathogenesis of AD.
基金Supported by Scientific Research Project Foundation of Shanghai City Science and Technology Committee:07dz19722-5
文摘Advances of studies on the acupuncture and pain signal transduction mechanisms in complete Freud＇s adjuvant arthritis are reviewed from the three aspects, the first messenger of modulating pain signals and the related receptors, the second messenger of modulating pain signals and other factors possibly involved in modulation of pain signal transduction, etc. It is held that modulation of acupuncture for pain signals is a comprehensive course involved in multi-channels, multi-levels, multi-links, and in future, acupuncture analgesic mechanisms for Freud＇s adjuvant arthritis will be more deeply studied by use of more new techniques and new methods.
基金This work was supported by the National Natural Science Foundation of China （No. 30470587, No. 30600197）.
文摘There are two degradation systems in mammalian cells, autophagy/lysosomal pathway and ubiquitin-proteasome pathway. Proteasome is consist of multiple protein subunits and plays important roles in degradation of short-lived cellular proteins. Recent studies reveal that proteasomal degradation system is also involved in signal transduction and regulation of various cellular functions. Dysfunction or dysregulation of proteasomal function may thus be an important pathogenic mechanism in certain neurological disorders. This paper reviews the biological functions of proteasome in signal transduction and its potential roles in neurodegenerative diseases.
文摘AIM To compare the expression level of Fas gene and Bcl-2 gene in gastric cancer cells SGC7901 and gastric cancer MDR (multidrug resistant) cells SGC7901/VCR, to transduce Fas cDNA and Bcl-2 antisense nucleic acid into SGC7901/VCR cells respectively, and to observe the expression of two genes in transfectants and non-transfectants as well as their drug sensitivity.METHODS Eukaryotic expression vector pBK-Fas cDNA and pDOR-anti Bcl-2 were constructed and transfected into SGC7901/VCR cells by lipofectamine, respectively. Northern blot and Western blot were used to detect the expression of mRNA and protein in SGC7901/VCR and SGC7901 cells and transfectants, and drug sensitivity of transfectants for VCR, CDDP and 5-FU was analyzed with MTT assay.RESULTS After gene transfection, 80 for Fas and 120 for antisense Bcl-2 drug-resistant clones were selected from 2×105 cells, transfection rate being 0.04% and 0.06%. Two clones of SGC7901 Fas/VCR cells and SGC7901 anti Bcl-2/VCR cells were randomly selected for further incubation. Hybridization results showed that the expression level of Fas mRNA and protein in SGC7901/VCR cells was much lower, but that of Bcl-2 mRNA and protein was higher than that in SGC7901 cells. The expression of Fas mRNA and protein in SGC7901 Fas/VCR cells was higher, and of Bcl-2 mRNA and protein was lower in SGC7901 anti Bcl-2/VCR cells than that in non-transfectants. MTT assay showed that transfectants were more sensitive to VCR, CDDP, 5-FU than non-transfectants.CONCLUSION Bcl-2 gene displayed high expression while Fas gene had low expression in drug resistant gastric cancer cells. Expression of Bcl-2 protein was effectively blocked in SGC7901 anti Bcl-2/VCR cells by gene transfection. In contrast, the expression of Fas mRNA and protein in SGC7901 Fas/VCR cells increased. Fas gene and Bcl-2 antisense nucleic acid transfection sensitized drug resistant gastric cancer cells to chemotherapeutic drugs. These results suggest cell apoptosis plays an important role in the mechanism of MDR, and enhancing apoptosis might reverse MDR.
基金supported by the National Science Foundation of China(30672698)
文摘Objective: To study the effect of emodin on protein and gene expressions of the massagers in mobility signal transduction system of cholecyst smooth muscle cells in guinea pig with cholesterol calculus. Methods: The guinea pigs were randomly divided into 4 groups, such as control group, gall-stone(GS) group, emodin group and ursodesoxycholic acid(UA) group. Cholesterol calculus models were induced in guinea pigs of GS, emodin and UA groups of induced models by lithogenic diet, while emodin or UA were given to the corresponding group for 7 weeks. The histomorphological and ultrastructure change of gallbladder were detected by microscope and electron microscope, the content of plasma cholecystokinin(CCK) and [Ca^(2+)]i were analyzed successively by radioimmunoassay and flow cytometry. The protein and mR NA of Gsα, Giα and Cap in cholecyst cells were determined by western blotting and real time polymerase chain reaction(RT-PCR). Results: Emodin or UA can relieve pathogenic changes in epithelial cells and muscle cells in gallbladder of guinea pig with cholesterol calculus by microscope and transmission electron microscope. In the cholecyst cells of GS group, CCK levels in plasma and [Ca^(2+)]i decreased, the protein and m RNA of GS group were downregulated,the protein and m RNA of Gi and Cap were up-regulated. Emodin significantly decreased the formative rate of gallstone, improved the pathogenic change in epithelial cells and muscle cells, increased CCK levels in plasma and [Ca^(2+)]i in cholecyst cells, enhanced the protein and mR NA of Gs in cholecyst cells, reduced the protein and mR NA of Gi and Cap in cholecyst cells in guinea pig with cholesterol calculus. Conclusion: The dysfunction of gallbladder contraction gives rise to the disorders of mobility signal transduction system in cholecyst smooth muscle cells, including low content of plasma CCK and [Ca^(2+)]i in cholecyst cells, abnormal protein and mRNA of Gs, Gi and Cap. Emodin can enhance the contractibility of gallbladder and alleviate cholestasis by regulating plasma CCK levels, [Ca2+]i in cholecyst cells and the protein and mR NA of Gs, Gi and Cap.
基金supported by the National High Tech-nologies R&D Program (863 Program) of China(2006AA10Z1A4)the Innovation Team Project of Northeast Agricultural University, China (LXT005-1-2)
文摘The galactopoietic mechanism of Vaccaria segetalis is still unknown. Understanding dibutyl phthalate （DBP） separated from Vaccaria segetalis on the expression of lactation signal transduction genes of mammary gland epithelial cells, including prlr, erα, akt1, socs2, pparγ and elf5, will be helpful to reveal the molecular mechanism. Western blot and qRT- PCR were used to study the change of prlr, erα, akt, socs2, pparγ, and elf5 expression at mRNA and protein level. Co- localization expression of prolactin receptor （PRLR） and estrogen receptor α （ERα） was observed by immunofluorescence; the expression changes of miRNAs （21, 125b, 143, and 195） and the secretion of β-casein and lactose were detected by qRT-PCR and RP-HPLC. The results showed that Vaccaria segetalis active compound had similar fuctions as estrogen and/or prolactin （PRL） in dairy cow mammary gland epithelial cells （DCMECs）, increased the expressions of prlr, erα, akt1, and elf5 genes, while repressed pparγ expressions. DBP promoted socs2 mRNA expression, but its protein expressions were repressed. Furthermore, both DBP and PRL could repress the expressions of miRNA-125b, miRNA-143 and miRNA- 195 in DCMECs. DBP could repress the expression of miRNA-21, while the influence of PRL on miRNA-21 was not certain. DBP could promote the lactation ability of DCMECs by regulating the ER and PRLR cellular signal transduction pathway.
基金supported by the National Science Foundation of China(30672698)
文摘Objective:To study the mechanism of insulin resistance in the cholesterol gallstone formation from insulin signal transduction pathway so as to reveal the possible mechanism and the effective role of Albiflorin Granule on preventing the cholesterol gallstones.Methods:Serum triglycerides(TG),free fatty acid(FFA),and total cholesterol(TC) from different groups were measured and liver cells Ins R,PKB,IKK-β protein expression levels were detected by western blotting.Results:Albiflorin significantly decreased the cholesterol gallstone formation rate,increased glucose infusion rate in gallstone guinea pigs and improved insulin resistance.Compared with the normal group,insulin receptor and PKB protein expression in GS group were significantly reduced.IKK-β protein in the GS group increased significantly and Albiflorin could reduce IKK-β protein expression in guinea pig liver cells.Conclusions:The model of insulin resistance in cholesterol gallstone guinea pig was successfully established,which plays an important role in the cholesterol gallstone formation.All aspects of insulin signaling pathway are involved in gallstone formation.Albiflorin can regulate various aspects of insulin signal transduction pathway to prevent the formation of gallbladder.
文摘The copper-binding, membrane-anchored, cellular prion protein （PrP~） has two constitutive cleavage sites producing distinct N- and C-terminal fragments （N1/C1 and N2/C2）. Using RK13 cells expressing either human PrPc, mouse PrPc or mouse PrP^C carrying the 3F4 epitope, this study explored the influence of the PrP^C primary sequence on endoproteolytic cleavage and one putative PrPc function, MAP kinase signal transduction, in response to exogenous copper with or without a perturbed membrane environment. PrPc primary sequence, especially that around the N1/C1 cleavage site, appeared to influence basal levels of proteolysis at this location and extracellular signal-regulat- ed kinase 1/2 （ERK1/2） phosphorylation, with increased processing demonstrating an inverse relationship with basal ERK1/2 activation. Human PrP^C showed increased N1/C1 cleavage in response to copper alone, accompanied by specific p38 and JNK/SAPK phosphorylation. Combined exposure to copper plus the cholesterol-sequestering antibiotic filipin resulted in a mouse PrP^C-specific substantial increase in signal protein phosphorylation, accompanied by an increase in N1/C1 cleavage. Mouse PrPc harboring the human N1/C1 cleavage site assumed more human-like profiles basally and in response to copper and altered membrane environments. Our results demonstrate that the PrPc pri- mary sequence around the N1/C1 cleavage site influences endoproteolytic processing at this location, which appears linked to MAP kinase signal transduction both basally and in response to copper. Further, the primary sequence appears to confer a mutual dependence of N1/C1 cleavage and membrane integrity on the fidelity of PrP^C-related signal transduction in response to exogenous stimuli.
文摘Erythropoietin (EPO), a 34 kD glycopro-tein, is the principal growth factor regulating theproduction of circulating erythrocytes; EPO isessential for committed CFU - E erythroid pro-genitors to divide several times and then to dif-ferentiate into erythrocytes. Like most receptorsfor hematopoietic growth factors, the erythro-poietin receptor (EPO - R) is a type I trans-membrane protein and a member of the cytokinereceptor superfamily. These receptors containfour conserved cysteines and a Trp - Ser - X -
基金supported by the National Natural Science Foundation of China(30730061)the National Basic Research Program of China(2009CB119203)
文摘Cold stress responses help insects to survive under low temperatures that would be lethal otherwise.This phenomenon might contribute to the invasion of some Bemisia tabaci cryptic species from subtropical areas to temperate regions.However,the molecular mechanisms regulating cold stress responses in whitefly are yet unclear.Mitogen-activated protein kinases（MAPKs）which including p38,ERK,and JNK,are well known for their roles in regulating metabolic responses to cold stress in many insects.In this study,we explored the possible roles of the MAPKs in response to low temperature stresses in the Mediterranean cryptic species（the Q-biotype）of the B.tabaci species complex.First,we cloned the p38 and ERK genes from the whitefly cDNA library.Next,we analyzed the activation of MAPKs during cold stress in the Mediterranean cryptic species by immuno-blotting.After cold stress,the level of phospho-p38 increased but no significant change was observed in the phosphorylation of ERK and JNK,thus suggesting that the p38 might be responsible for the defense response to low temperature stress.Furthermore,we demonstrated that：i）3 min chilling at 0°C was sufficient for the activation of p38 MAPK pathway in this whitefly;and ii）the amount of phosphorylated p38 increased significantly in the first 20 min of chilling,reversed by 60 min,and then returned to the original level by 120 min.Taken together,our results suggest that the p38 pathway is important during response to low temperature stress in the Mediterranean cryptic species of the B.tabaci species complex.
文摘AIM: To investigate the role of TIP30 in apoptotic signal pathway in hepatoblastoma cells and to provide a basis for TIP30 as a gene therapy candidate in the regression of hepatoblastoma cells. METHODS: Apoptosis of human hepatoblastoma cell lines HepG2 (p53 wild), Hep3B (p53 null) and PLC/RPF/5 (p53 mutant) infected with Ad-TIP30 (bearing a wild type human Tip30 gene) were analyzed and p53, Bax and Bcl-xl expression levels were compared among these cells. MTT assay, DNA fragmentation, in situ 3' end labeling of DNA, annexin-V FITC staining were used to detect cell death and apoptosis in cells at various time intervals subsequent to infection, and to determine whether TIP30 had an effect on the expression levels of some apoptosis-related gene products such as Bax, p53 and Bcl-xl. A similar time course experiment was performed by Western blotting. RESULTS: In MTT assay, the viability of HepG2 cells decreased significantly from 99.7% to 10% and displayed more massive cell death within 5-8 d than Hep3B and PLC/ RPF/5 cells, with their viability decreased from 97.8% to 44.3% and 98.1% to 50.4%, respectively. In annexin-V FITC assay, the percentage of apoptosis cells in HepG2 cells was two to three-fold higher than that in control cells (infected with Ad-GFP), two-fold higher than that in Hep3B cells and 1.4-fold higher than that in PLC/RPF/5 cells 36 h after infection, respectively. Moreover, in HepG2 cells, the p53 began to increase 6-8 h after infection, reaching a maximum level between 8 and 12 h after infection and then dropped. Bax showed a similar increase in the cells as p53 reached the maximum at 8-12 h and subsequently decreased. Interestingly, Bcl-xl protein levels were down regulated during 24 to 36 h after Ad-TIP30 infection. In contrast, ectopic expression of TIP30 in Hep3B and PLC/ RPF/5 cells had no effect on the regulation of Bax expression, but had an effect on Bcl-xl levels. In comparison with HepG2 cells, these data suggested that up-regulation of p53 levels by TIP30 might be a pre-requisite for Bax and Bax/Bcl-xl ratio increase. We hypothesied that TIP30 might regulate Bax gene partly through p53, which sensitizes cells to apoptosis by involving a p53 apoptosis signal transduction pathway. CONCLUSION: TIP30 plays an important role in predisposing hepatoblastoma cells to apoptosis through regulating expression levels of these genes. Ad-TIP30 carrying exogenous TIP30-anti-tumor genes may be regarded as a potential candidate for the treatment of hepatocellular carcinoma.
基金supported by funds from The Ministry of Science and Technology of China (2017YFA0103900 and 2016YFA0502800)The National Natural Science Foundation of China (31571083)+6 种基金supported by The National Natural Science Foundation of China (81470701)The National Natural Science Foundation of China (81771882)The Program for Professor of Special Appointment (Eastern Scholar of Shanghai TP2014008)The Shanghai Rising-Star Program (14QA1400800)a grant from the Young 1000 Talent Program of China to ZYThe Fundamental Research (Discipline Layout) Foundation from Shenzhen Committee of Science, Technology and Innovation (JCYJ20170817111912585) to FC
文摘Drosophila dEAAT2, a member of the excitatory amino-acid transporter(EAAT) family, has been described as mediating the high-affinity transport of taurine, which is a free amino-acid abundant in both insects and mammals. However, the role of taurine and its transporter in hearing is not clear. Here, we report that dEAAT2 is required for the larval startle response to sound stimuli. d EAAT2 was found to be enriched in the distal region of chordotonal neurons where sound transduction occurs. The Ca2+imaging and electrophysiological results showed that disrupted dEAAT2 expression significantly reduced the response of chordotonal neurons to sound.More importantly, expressing d EAAT2 in the chordotonal neurons rescued these mutant phenotypes. Taken together,these findings indicate a critical role for Drosophila dEAAT2 in sound transduction by chordotonal neurons.
基金Supported by the Foundation of Heilongjiang Provincial Foundation for Youths Project(No.QC2011C119)
文摘AIM：To develop a new method to produce recombinant reprogramming proteins,c Myc,Klf4,Oct4,and Sox2,in soluble format with low cost for the generation of induced pluripotent stem cells（i PSCs）.METHODS：A short polypeptide sequence derived from the HIV trans-activator of transcription protein（TAT） and the nucleus localization signal（NLS） polypeptide were fused to the N terminus of the reprogramming proteins and they were constructed into p Cold-SUMO vector which can extremely improve the solubility of recombinant proteins.Then these vector plasmids were transformed into E.coli BL21（DE3） Chaperone competent cells for amplification.The solubility of these recombinant proteins was determined by SDS-PAGE and Coomassie brilliant blue staining.The recombinant proteins were purified by NiNTA resin and identified by Western blot.The transduction of these proteins into HEK 293 T cells were evaluated by immunofluorescence staining.RESULTS：These four reprogramming proteins could be produced in soluble format in p Cold-SUMO expression vector system with the assistance of chaperone proteins in bacteria.The proteins were purified successfully with a purity of over 70% with a relative high transduction rate into 293 cells.CONCLUSION：The results in the present study indicate the four important reprogramming proteins,c Myc,Klf4,Oct4,and Sox2,can be produced in soluble format in bacteria with low cost.Our new method thus might be expected to greatly contribute to the future study of i PSCs.
