Small interfering RNAs(siRNA)provide a novel and highly specific therapy due to their ability to effectively silence target genes,to date six siRNA therapeutics are approved for clinical use.Even so,some critical chal...Small interfering RNAs(siRNA)provide a novel and highly specific therapy due to their ability to effectively silence target genes,to date six siRNA therapeutics are approved for clinical use.Even so,some critical challenges remain to overcome in the therapeutic application of siRNAs,with delivery issues at the forefront.Among them,endo/lysosomal barrier is one of the important but often-neglected limitations hindering the delivery of siRNA therapeutics.In this review,we summarize the promising strategies that facilitate siRNAs overcoming endo/lysosomal barriers based on the cellular uptake and intracellular transport pathways,including promoting escape once endocytosis into the endo/lysosomes and bypassing lysosomes via endosome-Golgi-endoplasmic reticulum(ER)pathway or nonendocytosis pathway,and discuss the principal considerations and the future directions of promoting endo/lysosomal escape in the development of therapeutic siRNAs.展开更多
Cancer vaccines have garnered significant attention in cancer immunotherapy because they trigger tumor-specific immune responses.However,their effectiveness is hindered by inefficient antigen and adjuvant delivery at ...Cancer vaccines have garnered significant attention in cancer immunotherapy because they trigger tumor-specific immune responses.However,their effectiveness is hindered by inefficient antigen and adjuvant delivery at the subcellular level,which is essential to stimulate a robust CD8^(+)T cell response.This study presents PAR/OVA,a cancer nanovaccine developed by combining imiquimod(R837)-modified polyamidoamine dendrimers with the model protein antigen ovalbumin(OVA).Within the nanovaccine,R837 is an adjuvant for the Toll-like receptor 7(TLR7)pathway and a structural component that facilitates OVA loading.In dendritic cells(DCs),the high cytoplasmic glutathione levels triggered the breakdown of PAR/OVA,releasing OVA and R837,which induced DC activation and antigen cross-presentation.Furthermore,PAR/OVA vaccination showed a protective effect and effectively inhibited B16-OVA tumor progression,indicating its potential in cancer immunotherapy.Combining this vaccine with an immune checkpoint blockade enhanced antitumor efficacy by improving the ability of cytotoxic T lymphocytes to target cancer cells within the tumor microenvironment.These findings underscore the potential of this adjuvant/antigen-delivering nanovaccine in cancer immunotherapy.展开更多
Legume symbiotic nitrogen fixation(SNF)is suppressed by inorganic nitrogen(N)in the soil.High N inhibition of nitrogenase activity is associated with the deprivation of carbon allocation and metabolism in nodules.Howe...Legume symbiotic nitrogen fixation(SNF)is suppressed by inorganic nitrogen(N)in the soil.High N inhibition of nitrogenase activity is associated with the deprivation of carbon allocation and metabolism in nodules.However,the underlying molecular mechanisms remain unclear.Here,we identify Gm CIN1,which encodes a cytosolic invertase,as a gateway for the N-tuning of sucrose utilization in nodules.Gm CIN1 is enriched in mature soybean nodules,and its expression is regulated by nitrogen status.The knockout of Gm CIN1 using genome editing partially mimics the inhibitory effects of N on nitrogenase activity and sugar content and the impact of high N on nodule transcriptomes.This indicates that Gm CIN1 partially mediates the high N inhibition of nodule activity.Moreover,Ch IP-q PCR and EMSA reveal that SNAP1/2 transcription factors directly bind to the Gm CIN1 promoter.In addition,SNAP1/2 may be involved in the repression of Gm CIN1 expression in mature nodules at high N concentrations.Our findings provide insights into the involvement of the transcriptional tuning of carbon(C)metabolism genes by N-signaling modulators in the N-induced inhibition of nitrogenase activity.展开更多
The 1 195 bp 5′ flanking region of rice ( Oryza sativa L.) cytosolic fructose_1, 6_bisphosphatase (cyFBPase) can direct tissue, cell specific expression in transgenic rice. In order to identify sequence elements ...The 1 195 bp 5′ flanking region of rice ( Oryza sativa L.) cytosolic fructose_1, 6_bisphosphatase (cyFBPase) can direct tissue, cell specific expression in transgenic rice. In order to identify sequence elements responsible for the regulation of mesophyll_specific expression, the 5′ flanking regions of -1 195 bp, -1 102 bp, -768 bp, and -644 bp upstream of the translation initiation ATG codon were fused to the reporter gene encoding β_glucuronidase (GUS) and transferred to rice via particle bombardment. Analysis of the 5′ promoter deletions identified that a 93 bp fragment between -1 195 bp and -1 102 bp is essential for directing mesophyll specific expression. High constitutive expression of GUS reporter gene was found in the -768 deletion lines and another two deletion series. These results indicate the great potential utility of the promoter in rice biotechnology.展开更多
A genomic DNA fragment containing the 5'-upstream sequence and part of the open reading frame corresponding to the cytosolic fructose-1,6-bisphosphatase (cyFBPase) cDNA was isolated by Genome Walking. The 1 195 li...A genomic DNA fragment containing the 5'-upstream sequence and part of the open reading frame corresponding to the cytosolic fructose-1,6-bisphosphatase (cyFBPase) cDNA was isolated by Genome Walking. The 1 195 lip 5'-flanking region which started from the translation initiation ATG codon was fused to reporter gene encoding beta-glucuronidase (GUS) and stably transferred to rice via particle bombardment. Strong GUS activity was detected in leaves and leaf sheaths of transgenic rice, but not in culms and roots. Histochemical localization revealed that GUS expression was exclusively restricted to mesophyll cells in transgenic rice. Our results indicate that the 1 195 bp fragment contains all the cis-elements required for directing mesophyll-specific expression pattern in rice.展开更多
The effect of lanthanum （ Ⅲ ） （La^3 ＋ ） on cytosolic free calcium （ [ Ca^2 ＋ ] i ） in isolated rabbit mature osteoclasts was studied with the employment of fluo-3/AM as an intracellular calcium-sensitive fluo...The effect of lanthanum （ Ⅲ ） （La^3 ＋ ） on cytosolic free calcium （ [ Ca^2 ＋ ] i ） in isolated rabbit mature osteoclasts was studied with the employment of fluo-3/AM as an intracellular calcium-sensitive fluorescent probe by using a confocal laser scanning microscope. La^3＋ does not alter basal [Ca^2＋ ]i levels and cell spread area at the concentration of 1.00 × 10^- 8 mol· L ^- 1. However, La^3 ＋ at higher concentrations （ 1. 00 × 10^ - 5 and 1.00 × 10^- 7 mol· L^- 1 ） decreases [ Ca^2 ＋ ] i levels and cell spread area, and greater decreases are observed for the higher concentrations of La^3 ＋ . Since [Ca^2 ＋ ]i affects cytoskeleton and the adhesion properties of osteoclasts, our results seem to suggest that La^3 ＋ inhibit bone resorption by decreasing [Ca^2＋]i in rabbit mature osteoclasts.展开更多
AIM: To clarify whether Lysophosphatidic acid (LPA) activates the nuclear translocation of nuclear factor-κB (NF-κB) in pancreatic cancer. METHODS: Panc-1, a human pancreatic cancer cell line, was used throughout th...AIM: To clarify whether Lysophosphatidic acid (LPA) activates the nuclear translocation of nuclear factor-κB (NF-κB) in pancreatic cancer. METHODS: Panc-1, a human pancreatic cancer cell line, was used throughout the study. The expression of LPA receptors was confirmed by reverse-transcript polymerase chain reaction (RT-PCR). Cytosolic free calcium was measured by fluorescent calcium indicator fura-2, and the localization of NF-κB was visualized by immunofluorescent method with or without various agents, which effect cell signaling. RESULTS: Panc-1 expressed LPA receptors, LPA1, LPA2 and LPA3. LPA caused the elevation of cytosolic free calcium dose-dependently. LPA also caused the nuclear translocation of NF-κB. Cytosolic free calcium was attenuated by pertussis toxin (PTX) and U73122, an inhibitor of phospholipase C. The translocation of NF-κB was similarly attenuated by PTX and U73122, but phorbol ester, an activator of protein kinase C, alone did not translocate NF-κB. Furthermore, the translocation of NF-κB was completely blocked by Ca2+ chelator BAPTA-AM. Thapsigargin, an endoplasmic- reticulum Ca2+-ATPase pump inhibitor, also promoted the translocation of NF-κB. Staurosporine, a proteinkinase C inhibitor, attenuated translocation of NF-κB induced by LPA. CONCLUSION: These findings suggest that protein kinase C is activated endogenously in Panc-1, and protein kinase C is essential for activating NF-κB with cytosolic calcium and that LPA induces the nuclear translocation of NF-κB in Panc-1 by mobilizing cytosolic free calcium.展开更多
Responses to oligogalacturonic acid (OGA) were determined in transgenic Arabidopsis thaliana seedlings expressing the calcium reporter protein aequorin. OGA stimulated a rapid, substantial and transient increase in th...Responses to oligogalacturonic acid (OGA) were determined in transgenic Arabidopsis thaliana seedlings expressing the calcium reporter protein aequorin. OGA stimulated a rapid, substantial and transient increase in the concentration of cytosolic calcium ([Ca2+]cyt) that peaked after ca. 15 s. This increase was dose-dependent, saturating at ca. 50 μg Gal equiv/ml of OGA. OGA also stimulated a rapid generation of H2O2. A small, rapid increase in H2O2 content was followed by a much larger oxidative burst, with H2O2 content peaking after ca. 60 min and declining thereafter. Induction of the oxidative burst by OGA was also dose-dependent, with a maximum response again being achieved at ca. 50 μg Gal equiv/mL. Inhibitors of calcium fluxes inhibited both increases in [Ca2+]cyt and [H2O2], whereas inhibitors of NADPH oxidase blocked only the oxidative burst. OGA increased strongly the expression of the defence-related genes CHS,GST, PAL and PR-1. This induction was suppressed by inhibitors of calcium flux or NADPH oxidase, indicating that increases in both cytosolic calcium and H2O2 are required for OGA-induced gene expression.展开更多
Little information is available about the effects of exposure to pulsed microwaves on neuronal Ca^2＋ signaling under non-thermal conditions. In this study, rat pheochromocytoma （PC12） cells were exposed to pulsed m...Little information is available about the effects of exposure to pulsed microwaves on neuronal Ca^2＋ signaling under non-thermal conditions. In this study, rat pheochromocytoma （PC12） cells were exposed to pulsed microwaves for 6 min at a specific absorption rate （SAR） of 4 W/kg to assess possible real-time effects. During microwave exposure, free calcium dynamics in the cytosol, mitochondria, and nucleus of cells were monitored by time-lapse microfluorimetry using a genetically encoded calcium indicator （ratiometric-pericam, ratiometric-10ericam-mt,展开更多
Objective: To explore the effect of cytosolic phospholipase A2α(cPLA2α) on hepatocellular carcinoma(HCC) cell adhesion and the underlying mechanisms.Methods: Cell adhesion, detachment, and hanging-drop assays were u...Objective: To explore the effect of cytosolic phospholipase A2α(cPLA2α) on hepatocellular carcinoma(HCC) cell adhesion and the underlying mechanisms.Methods: Cell adhesion, detachment, and hanging-drop assays were utilized to examine the effect of cPLA2α on the cell-matrix and cell-cell adhesion. Downstream substrates and effectors of cPLA2α were screened via a phospho-antibody microarray.Associated signaling pathways were identified by the functional annotation tool DAVID. Candidate proteins were verified using Western blot and colocalization was investigated via immunofluorescence. Western blot and immunohistochemistry were used to detect protein expression in HCC tissues. Prognosis evaluation was conducted using Kaplan-Meier and Cox-proportional hazards regression analyses.Results: Our findings showed that cPLA2α knockdown decreases cell-matrix adhesion but increases cell-cell adhesion in HepG2 cells. Microarray analysis revealed that phosphorylation of multiple proteins at specific sites were regulated by cPLA2α. These phosphorylated proteins were involved in various biological processes. In addition, our results indicated that the focal adhesion pathway was highly enriched in the cPLA2α-relevant signaling pathway. Furthermore, cPLA2α was found to elevate phosphorylation levels of FAK and paxillin, two crucial components of focal adhesion. Moreover, localization of p-FAK to focal adhesions in the plasma membrane was significantly reduced with the downregulation of cPLA2α. Clinically, cPLA2α expression was positively correlated with p-FAK levels. Additionally, high expression of both cPLA2α and p-FAK predicted the worst prognoses for HCC patients.Conclusions: Our study indicated that cPLA2α may promote cell-matrix adhesion via the FAK/paxillin pathway, which partly explains the malignant cPLA2α phenotype seen in HCC.展开更多
Central nervous system(CNS)trauma,including traumatic brain injury and spinal cord injury,has a high rate of disability and mortality,and effective treatment is currently lacking.Previous studies have revealed that ne...Central nervous system(CNS)trauma,including traumatic brain injury and spinal cord injury,has a high rate of disability and mortality,and effective treatment is currently lacking.Previous studies have revealed that neural inflammation plays a vital role in CNS trauma.As the initial enzyme in neuroinflammation,cytosolic phospholipase A_(2)(cPLA2)can hydrolyze membranous phosphatides at the sn-2 position in a preferential way to release lysophospholipids andω3-polyunsaturated fatty acid dominated by arachidonic acid,thereby inducing secondary injuries.Although there is substantial fresh knowledge pertaining to cPLA2,in-depth comprehension of how cPLA2 participates in CNS trauma and the potential methods to amelio rate the clinical res ults after CNS trauma are still insufficient.The present review summarizes the latest understanding of how cPLA2 participates in CNS trauma,highlighting novel findings pertaining to how cPLA2 activation initiates the potential mechanisms specifically,neuroinflammation,lysosome membrane functions,and autophagy activity,that damage the CNS after trauma.Moreover,we focused on testing a variety of drugs capable of inhibiting cPLA2 or the upstream pathway,and we explored how those agents might be utilized as treatments to improve the results following CNS trauma.This review aimed to effectively understand the mechanism of cPLA2 activation and its role in the pathophysiological processes of CNS trauma and provide clarification and a new referential framework for future research.展开更多
Summary: The effects of 3, 4-Dihydroxyacetophenone (3, 4-DHAP) on cytosolic free calcium [Ca~2+ ]_i in pulmonary artery endothelia (PAECs) and smooth muscle cells (PASMCs) during acute hypoxia were studied. Porcine pu...Summary: The effects of 3, 4-Dihydroxyacetophenone (3, 4-DHAP) on cytosolic free calcium [Ca~2+ ]_i in pulmonary artery endothelia (PAECs) and smooth muscle cells (PASMCs) during acute hypoxia were studied. Porcine pulmonary artery endothelial and smooth muscle cells (PASMCs) were cultured primarily, and they were divided into 4 groups: groups incubated under normoxia or hypoxia and those with or without treatment with 3, 4-DHAP. The [Ca~2+ ]_i of both PAECs and PASMCs was measured by determining the fluorescence of fura 2 AM on spetrofluorometer. Our results showed that hypoxia caused significant elevation of [Ca~2+ ]_i, in both PAECs and PASMCs, 3, 4-DHAP could attenuate the hypoxic elevation of [Ca~2+ ]_i only in PASMCs but not in PAECs. It is concluded that 3, 4-DHAP decreases the hypoxic elevation of [Ca~2+ ]_i in PASMCs. This might contribute to its inhibitory effect on hypoxic pulmonary vasoconstriction.展开更多
BACKGROUND Aminoacyl tRNA synthetases/ligases(ARSs)are highly conserved enzymes involved in attaching amino acids to tRNA promoting protein synthesis.Although deficiencies of ARSs localized to the mitochondria classic...BACKGROUND Aminoacyl tRNA synthetases/ligases(ARSs)are highly conserved enzymes involved in attaching amino acids to tRNA promoting protein synthesis.Although deficiencies of ARSs localized to the mitochondria classically present with neuropathology,the clinical features of cytosolic ARS deficiencies are more variable.They have previously been associated with neonatal hepatitis,but never with early-onset inflammatory bowel disease.CASE SUMMARY A nine-year-old Bangladeshi boy presented with neonatal liver failure and deranged clotting,transaminitis and cholestasis.His parents were first cousins.Two older brothers and a sister were well.The patient suffered from loose stools from early infancy which became more troublesome and persistent from five years old with ten bloody motions a day.Repeated endoscopies showed persistent pancolitis,which was refractory to mesalazine,corticosteroids,azathioprine,sirolimus and anti-TNF(adalimumab)therapy,but has improved recently with subcutaneous methotrexate.Whole Genome Sequencing revealed a novel pathogenic missense variant(c.290A>G)in the cytosolic isoleucyl-tRNA synthetase gene,leading to an amino acid substitution(p.Asp97Gly).Pathogenic variants in other genes associated with inflammatory bowel disease(IBD)(ADAM17,EGFR,FOXP3,IL10RA,IL10RB,IL21R,NCF4,STAT3)were excluded.Cytokine assays demonstrated markedly elevated IL-2,IL-5,IL-13,IL-9 and IL-10 by the patient’s CD4+T-cells,while IL-17A,IL-17F,IFNβwere lower,and TNFαnot significantly different when compared to healthy controls.CONCLUSION This case report provides evidence that recessive mutations in cytosolic isoleucyltRNA synthetase are a novel monogenic cause of IBD,which should be considered,particularly in infants and children with a history of neonatal hepatitis and very early-onset IBD poorly responsive to treatment.展开更多
Vanishing white matter （VWM） disease - a disease of the cytosolic translation machinery： VWM is a recessive genet- ic neurodegenerative disease caused by mutations in any of the five genes encoding the subunits of ...Vanishing white matter （VWM） disease - a disease of the cytosolic translation machinery： VWM is a recessive genet- ic neurodegenerative disease caused by mutations in any of the five genes encoding the subunits of translation initiation factor 2B （eIF2B） （Leegwater et al., 2001; OMIM 306896）.展开更多
Elevated activities of cytosolic fructose-1,6-bisphosphatase(cyFBPase) and sedoheptulose-1,7-bisphosphatase(SBPase)are associated with higher yields in plants. In this study, the expression levels of the cyFBPase and ...Elevated activities of cytosolic fructose-1,6-bisphosphatase(cyFBPase) and sedoheptulose-1,7-bisphosphatase(SBPase)are associated with higher yields in plants. In this study, the expression levels of the cyFBPase and SBPase genes were increased by overexpressing rape(Brassica napus) cDNA in tobacco(Nicotiana tabacum) plants. The transgenic plants coexpressing cy FBPase and SBPase(TpFS), or expressing single cy FBPase(TpF) or SBPase(TpS) had 1.77-, 1.55-, 1.23-fold cyFBPase and 1.45-, 1.12-, 1.36-fold SBPase activities as compared to the wild-type(WT), respectively. Photosynthesis rates of TpF, TpS and TpFS increased 4, 20 and 25% compared with WT plants. The SBPase and cyFBPase positively regulated each other and functioned synergistically in transgenic tobacco plants. In addition, the sucrose contents of the three transgenic plants were higher than that of WT plants. The starch accumulation of the TpFS and TpS plants was improved by 53 and 37%, but slightly decreased in TpF plants. Moreover, the transgenic tobacco plants harbouring SBPase and/or cyFBPase genes showed improvements in their growth, biomass, dry weight, plant height, stem diameter, leaf size,flower number, and pod weight. In conclusion, co-expression of SBPase and cyFBPase may pave a new way for improving crop yield in agricultural applications.展开更多
Cytosolic phospholipase A2α （cPLA2α） catalyzes the release of arachidonic acid （AA） and lysophosphoglyceride from membrane phospholipids. Although the roles of AA and eicosanoids in cellular viability, the proce...Cytosolic phospholipase A2α （cPLA2α） catalyzes the release of arachidonic acid （AA） and lysophosphoglyceride from membrane phospholipids. Although the roles of AA and eicosanoids in cellular viability, the processes of inflammation and cancer cell development have been extensively studied, the function of cPLA2α in the processes of inflammation and cancer cell development is not clear. This review summarizes published evidences for the biochemical properties and regulatory mechanisms of cPLA2α. The potential for use of cPLA2α as a novel diagnostic target and predictive biomarker for tumors is also discussed.展开更多
Objectives To investigate the effects of β2-adrenergic antagonist on cytosolic Ca^2 ＋ （[Ca^2＋ ]i） in ventricular myocytes from infarcted rat heart. Methods A ligature was placed around left anterior descending co...Objectives To investigate the effects of β2-adrenergic antagonist on cytosolic Ca^2 ＋ （[Ca^2＋ ]i） in ventricular myocytes from infarcted rat heart. Methods A ligature was placed around left anterior descending coronary artery of rat hearts. Rats in the control group were sham-operated. Cardiomyocytes were dissociated at two, four, eight weeks after myocardial infarction （MI） and [Ca^2＋]i was measured via fura-2 fluorescence. The response of cardiomyocytes to isoproterenol in presence or absence of betal-adrenergic antagonist atenolol, beta2-adrenergic antagonist ICI118, 551 or non-selective β1, 2- adrenergic antagonists propranolol was examined. Results The followings were found that ICI 118, 551 had no significant effects on the rise of [Ca^2＋]i induced by isoproterenol in normal ventricular myocytes （P 〉 0.05）, ICI118, 551 only significantly attenuated the rise of [Ca^2＋]i induced by isoproterenol at four weeks and eight weeks after MI （24.5%±5.7% vs 57.8% ± 13.2%, P〈 0.01; 12.2%±7.9% vs 44.6%±11.3%, P〈 0.01）. Atenolol had suppressive effects only in the control group and the post-MI group of two weeks （P 〈 0.05）, and propranolol had suppressive effects in the control and all the three post-MI groups （P 〈 0.01）. Conclusions Beta2-adrenergic antagonist ICI118, 551 may exert negative effects on Ca^2＋ overload initiated by sympathetic stimulation after MI.展开更多
Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes...Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and patho- genicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicity. In this study, the latest molecular chaperone system associated with endoplasmic re- ticulum-associated protein degradation, the endoplasmic reticulum resident protein quality-control system and the ubiquitination proteasome system, is outlined. The molecular chaperone system directly correlates with the prion protein degradation pathway. Understanding the molecular mechanisms will help provide a fascinating avenue for further investigations on prion disease treatment and prion protein-induced neurodegenerative diseases.展开更多
Effects of procainamide (PA) on human platelet aggregation and the cytosolic free-Ca2+ concentration ([Ca2+]) were investigated in vitro. PA at doses of 8.5 , 34 and 136 μmol·L-1 could inhibit the human platelet...Effects of procainamide (PA) on human platelet aggregation and the cytosolic free-Ca2+ concentration ([Ca2+]) were investigated in vitro. PA at doses of 8.5 , 34 and 136 μmol·L-1 could inhibit the human platelet aggregation induced by 0. 5 μmol · L-1 A23187 with a good concentration -effect relationship. One min and maximal aggregation rates were also inhibited ( P<0. 01 ,vs control). The [Ca2+], was decreased in the presence of PA ,and the changes of [Ca2+], showed a significant linear correlation with 1 min or maximal aggregation rate (P<0.05). These results suggest that the mechanisms of PA inhibiting platelet aggregation relate to the decrease of [Ca2+].展开更多
Ferritin sequesters and stores iron and, consequently, protect cells against iron-mediated free radical damage. However, the mechanism of iron exit from the ferritin nanocage and
基金supported by National Natural Science Foundation of China(No.82173769)the National Key R&D Program of China(No.2021YFE0106900)+1 种基金Applied Basic Research Multiinvestment Foundation of Tianjin(No.21JCYBJC01540)the Science&Technology Development Fund of Tianjin Education Commission for Higher Education(No.2023ZD019)。
文摘Small interfering RNAs(siRNA)provide a novel and highly specific therapy due to their ability to effectively silence target genes,to date six siRNA therapeutics are approved for clinical use.Even so,some critical challenges remain to overcome in the therapeutic application of siRNAs,with delivery issues at the forefront.Among them,endo/lysosomal barrier is one of the important but often-neglected limitations hindering the delivery of siRNA therapeutics.In this review,we summarize the promising strategies that facilitate siRNAs overcoming endo/lysosomal barriers based on the cellular uptake and intracellular transport pathways,including promoting escape once endocytosis into the endo/lysosomes and bypassing lysosomes via endosome-Golgi-endoplasmic reticulum(ER)pathway or nonendocytosis pathway,and discuss the principal considerations and the future directions of promoting endo/lysosomal escape in the development of therapeutic siRNAs.
基金supported by the Guangdong Medical Science and Technology Research Fund Project(Nos.A2022124 and B2023148)the Guangdong Provincial Administration of Traditional Chinese Medicine Research Project(No.20231222)+3 种基金the National Natural Science Foundation of China(No.52403185)Guangdong Basic and Applied Basic Research Foundation(No.2023A1515011493)the Open Fund of Guangdong Provincial Key Laboratory of Spine and Spinal Cord Reconstruction(No.2023B121203001)the National Famous Old Traditional Chinese Medicine Experts Inheritance Studio Construction Program of the National Administration of TCM(No.(2022)75).
文摘Cancer vaccines have garnered significant attention in cancer immunotherapy because they trigger tumor-specific immune responses.However,their effectiveness is hindered by inefficient antigen and adjuvant delivery at the subcellular level,which is essential to stimulate a robust CD8^(+)T cell response.This study presents PAR/OVA,a cancer nanovaccine developed by combining imiquimod(R837)-modified polyamidoamine dendrimers with the model protein antigen ovalbumin(OVA).Within the nanovaccine,R837 is an adjuvant for the Toll-like receptor 7(TLR7)pathway and a structural component that facilitates OVA loading.In dendritic cells(DCs),the high cytoplasmic glutathione levels triggered the breakdown of PAR/OVA,releasing OVA and R837,which induced DC activation and antigen cross-presentation.Furthermore,PAR/OVA vaccination showed a protective effect and effectively inhibited B16-OVA tumor progression,indicating its potential in cancer immunotherapy.Combining this vaccine with an immune checkpoint blockade enhanced antitumor efficacy by improving the ability of cytotoxic T lymphocytes to target cancer cells within the tumor microenvironment.These findings underscore the potential of this adjuvant/antigen-delivering nanovaccine in cancer immunotherapy.
基金supported by funds from the National Natural Science Foundation of China(32072083)a Guangzhou Science Grant(2024A03J0010)awarded to Y.G。
文摘Legume symbiotic nitrogen fixation(SNF)is suppressed by inorganic nitrogen(N)in the soil.High N inhibition of nitrogenase activity is associated with the deprivation of carbon allocation and metabolism in nodules.However,the underlying molecular mechanisms remain unclear.Here,we identify Gm CIN1,which encodes a cytosolic invertase,as a gateway for the N-tuning of sucrose utilization in nodules.Gm CIN1 is enriched in mature soybean nodules,and its expression is regulated by nitrogen status.The knockout of Gm CIN1 using genome editing partially mimics the inhibitory effects of N on nitrogenase activity and sugar content and the impact of high N on nodule transcriptomes.This indicates that Gm CIN1 partially mediates the high N inhibition of nodule activity.Moreover,Ch IP-q PCR and EMSA reveal that SNAP1/2 transcription factors directly bind to the Gm CIN1 promoter.In addition,SNAP1/2 may be involved in the repression of Gm CIN1 expression in mature nodules at high N concentrations.Our findings provide insights into the involvement of the transcriptional tuning of carbon(C)metabolism genes by N-signaling modulators in the N-induced inhibition of nitrogenase activity.
文摘The 1 195 bp 5′ flanking region of rice ( Oryza sativa L.) cytosolic fructose_1, 6_bisphosphatase (cyFBPase) can direct tissue, cell specific expression in transgenic rice. In order to identify sequence elements responsible for the regulation of mesophyll_specific expression, the 5′ flanking regions of -1 195 bp, -1 102 bp, -768 bp, and -644 bp upstream of the translation initiation ATG codon were fused to the reporter gene encoding β_glucuronidase (GUS) and transferred to rice via particle bombardment. Analysis of the 5′ promoter deletions identified that a 93 bp fragment between -1 195 bp and -1 102 bp is essential for directing mesophyll specific expression. High constitutive expression of GUS reporter gene was found in the -768 deletion lines and another two deletion series. These results indicate the great potential utility of the promoter in rice biotechnology.
文摘A genomic DNA fragment containing the 5'-upstream sequence and part of the open reading frame corresponding to the cytosolic fructose-1,6-bisphosphatase (cyFBPase) cDNA was isolated by Genome Walking. The 1 195 lip 5'-flanking region which started from the translation initiation ATG codon was fused to reporter gene encoding beta-glucuronidase (GUS) and stably transferred to rice via particle bombardment. Strong GUS activity was detected in leaves and leaf sheaths of transgenic rice, but not in culms and roots. Histochemical localization revealed that GUS expression was exclusively restricted to mesophyll cells in transgenic rice. Our results indicate that the 1 195 bp fragment contains all the cis-elements required for directing mesophyll-specific expression pattern in rice.
文摘The effect of lanthanum （ Ⅲ ） （La^3 ＋ ） on cytosolic free calcium （ [ Ca^2 ＋ ] i ） in isolated rabbit mature osteoclasts was studied with the employment of fluo-3/AM as an intracellular calcium-sensitive fluorescent probe by using a confocal laser scanning microscope. La^3＋ does not alter basal [Ca^2＋ ]i levels and cell spread area at the concentration of 1.00 × 10^- 8 mol· L ^- 1. However, La^3 ＋ at higher concentrations （ 1. 00 × 10^ - 5 and 1.00 × 10^- 7 mol· L^- 1 ） decreases [ Ca^2 ＋ ] i levels and cell spread area, and greater decreases are observed for the higher concentrations of La^3 ＋ . Since [Ca^2 ＋ ]i affects cytoskeleton and the adhesion properties of osteoclasts, our results seem to suggest that La^3 ＋ inhibit bone resorption by decreasing [Ca^2＋]i in rabbit mature osteoclasts.
基金The Research Committee of Intractable Pancreatic Diseases, provided by the Ministry of Health, Labour, and Welfare, Japan, No. 50253448
文摘AIM: To clarify whether Lysophosphatidic acid (LPA) activates the nuclear translocation of nuclear factor-κB (NF-κB) in pancreatic cancer. METHODS: Panc-1, a human pancreatic cancer cell line, was used throughout the study. The expression of LPA receptors was confirmed by reverse-transcript polymerase chain reaction (RT-PCR). Cytosolic free calcium was measured by fluorescent calcium indicator fura-2, and the localization of NF-κB was visualized by immunofluorescent method with or without various agents, which effect cell signaling. RESULTS: Panc-1 expressed LPA receptors, LPA1, LPA2 and LPA3. LPA caused the elevation of cytosolic free calcium dose-dependently. LPA also caused the nuclear translocation of NF-κB. Cytosolic free calcium was attenuated by pertussis toxin (PTX) and U73122, an inhibitor of phospholipase C. The translocation of NF-κB was similarly attenuated by PTX and U73122, but phorbol ester, an activator of protein kinase C, alone did not translocate NF-κB. Furthermore, the translocation of NF-κB was completely blocked by Ca2+ chelator BAPTA-AM. Thapsigargin, an endoplasmic- reticulum Ca2+-ATPase pump inhibitor, also promoted the translocation of NF-κB. Staurosporine, a proteinkinase C inhibitor, attenuated translocation of NF-κB induced by LPA. CONCLUSION: These findings suggest that protein kinase C is activated endogenously in Panc-1, and protein kinase C is essential for activating NF-κB with cytosolic calcium and that LPA induces the nuclear translocation of NF-κB in Panc-1 by mobilizing cytosolic free calcium.
基金This work was supported by the National Natural Science Foundation of China(No.39870050)the Chinese Academy of Sciences(Grant No.KSCX2-SW-322).
文摘Responses to oligogalacturonic acid (OGA) were determined in transgenic Arabidopsis thaliana seedlings expressing the calcium reporter protein aequorin. OGA stimulated a rapid, substantial and transient increase in the concentration of cytosolic calcium ([Ca2+]cyt) that peaked after ca. 15 s. This increase was dose-dependent, saturating at ca. 50 μg Gal equiv/ml of OGA. OGA also stimulated a rapid generation of H2O2. A small, rapid increase in H2O2 content was followed by a much larger oxidative burst, with H2O2 content peaking after ca. 60 min and declining thereafter. Induction of the oxidative burst by OGA was also dose-dependent, with a maximum response again being achieved at ca. 50 μg Gal equiv/mL. Inhibitors of calcium fluxes inhibited both increases in [Ca2+]cyt and [H2O2], whereas inhibitors of NADPH oxidase blocked only the oxidative burst. OGA increased strongly the expression of the defence-related genes CHS,GST, PAL and PR-1. This induction was suppressed by inhibitors of calcium flux or NADPH oxidase, indicating that increases in both cytosolic calcium and H2O2 are required for OGA-induced gene expression.
基金supported by the National Natural Science Foundation of China[No.31400716 and No.61401497]
文摘Little information is available about the effects of exposure to pulsed microwaves on neuronal Ca^2＋ signaling under non-thermal conditions. In this study, rat pheochromocytoma （PC12） cells were exposed to pulsed microwaves for 6 min at a specific absorption rate （SAR） of 4 W/kg to assess possible real-time effects. During microwave exposure, free calcium dynamics in the cytosol, mitochondria, and nucleus of cells were monitored by time-lapse microfluorimetry using a genetically encoded calcium indicator （ratiometric-pericam, ratiometric-10ericam-mt,
基金This work was supported by grants from Key Project of Tianjin Natural Science Foundation (Grant No.18JCZDJC35200)NSFC-FRQS program (Grant No.81661128009)+1 种基金The Science & Technology Development Fund of Tianjin Education Commission for Higher Education (Grant No.2017KJ202)Scientific Research Foundation for Returned Scholars and Doctoral Program of Tianjin Medical University Cancer Institute and Hospital (Grant No.B1703)
文摘Objective: To explore the effect of cytosolic phospholipase A2α(cPLA2α) on hepatocellular carcinoma(HCC) cell adhesion and the underlying mechanisms.Methods: Cell adhesion, detachment, and hanging-drop assays were utilized to examine the effect of cPLA2α on the cell-matrix and cell-cell adhesion. Downstream substrates and effectors of cPLA2α were screened via a phospho-antibody microarray.Associated signaling pathways were identified by the functional annotation tool DAVID. Candidate proteins were verified using Western blot and colocalization was investigated via immunofluorescence. Western blot and immunohistochemistry were used to detect protein expression in HCC tissues. Prognosis evaluation was conducted using Kaplan-Meier and Cox-proportional hazards regression analyses.Results: Our findings showed that cPLA2α knockdown decreases cell-matrix adhesion but increases cell-cell adhesion in HepG2 cells. Microarray analysis revealed that phosphorylation of multiple proteins at specific sites were regulated by cPLA2α. These phosphorylated proteins were involved in various biological processes. In addition, our results indicated that the focal adhesion pathway was highly enriched in the cPLA2α-relevant signaling pathway. Furthermore, cPLA2α was found to elevate phosphorylation levels of FAK and paxillin, two crucial components of focal adhesion. Moreover, localization of p-FAK to focal adhesions in the plasma membrane was significantly reduced with the downregulation of cPLA2α. Clinically, cPLA2α expression was positively correlated with p-FAK levels. Additionally, high expression of both cPLA2α and p-FAK predicted the worst prognoses for HCC patients.Conclusions: Our study indicated that cPLA2α may promote cell-matrix adhesion via the FAK/paxillin pathway, which partly explains the malignant cPLA2α phenotype seen in HCC.
基金supported by the National Natural Science Foundation of China,No.82072192(to KLZ)Public Welfare Technology Research Project of Zhejiang Province,No.LGF20H150003(to KLZ)+1 种基金the Natural Science Foundation of Zhejiang Province,Nos.LY17H060009 and Y21H060050(both to WFN)Wenzhou Science and Technology Bureau Foundation,No.Y20210438(to KLZ)。
文摘Central nervous system(CNS)trauma,including traumatic brain injury and spinal cord injury,has a high rate of disability and mortality,and effective treatment is currently lacking.Previous studies have revealed that neural inflammation plays a vital role in CNS trauma.As the initial enzyme in neuroinflammation,cytosolic phospholipase A_(2)(cPLA2)can hydrolyze membranous phosphatides at the sn-2 position in a preferential way to release lysophospholipids andω3-polyunsaturated fatty acid dominated by arachidonic acid,thereby inducing secondary injuries.Although there is substantial fresh knowledge pertaining to cPLA2,in-depth comprehension of how cPLA2 participates in CNS trauma and the potential methods to amelio rate the clinical res ults after CNS trauma are still insufficient.The present review summarizes the latest understanding of how cPLA2 participates in CNS trauma,highlighting novel findings pertaining to how cPLA2 activation initiates the potential mechanisms specifically,neuroinflammation,lysosome membrane functions,and autophagy activity,that damage the CNS after trauma.Moreover,we focused on testing a variety of drugs capable of inhibiting cPLA2 or the upstream pathway,and we explored how those agents might be utilized as treatments to improve the results following CNS trauma.This review aimed to effectively understand the mechanism of cPLA2 activation and its role in the pathophysiological processes of CNS trauma and provide clarification and a new referential framework for future research.
文摘Summary: The effects of 3, 4-Dihydroxyacetophenone (3, 4-DHAP) on cytosolic free calcium [Ca~2+ ]_i in pulmonary artery endothelia (PAECs) and smooth muscle cells (PASMCs) during acute hypoxia were studied. Porcine pulmonary artery endothelial and smooth muscle cells (PASMCs) were cultured primarily, and they were divided into 4 groups: groups incubated under normoxia or hypoxia and those with or without treatment with 3, 4-DHAP. The [Ca~2+ ]_i of both PAECs and PASMCs was measured by determining the fluorescence of fura 2 AM on spetrofluorometer. Our results showed that hypoxia caused significant elevation of [Ca~2+ ]_i, in both PAECs and PASMCs, 3, 4-DHAP could attenuate the hypoxic elevation of [Ca~2+ ]_i only in PASMCs but not in PAECs. It is concluded that 3, 4-DHAP decreases the hypoxic elevation of [Ca~2+ ]_i in PASMCs. This might contribute to its inhibitory effect on hypoxic pulmonary vasoconstriction.
文摘BACKGROUND Aminoacyl tRNA synthetases/ligases(ARSs)are highly conserved enzymes involved in attaching amino acids to tRNA promoting protein synthesis.Although deficiencies of ARSs localized to the mitochondria classically present with neuropathology,the clinical features of cytosolic ARS deficiencies are more variable.They have previously been associated with neonatal hepatitis,but never with early-onset inflammatory bowel disease.CASE SUMMARY A nine-year-old Bangladeshi boy presented with neonatal liver failure and deranged clotting,transaminitis and cholestasis.His parents were first cousins.Two older brothers and a sister were well.The patient suffered from loose stools from early infancy which became more troublesome and persistent from five years old with ten bloody motions a day.Repeated endoscopies showed persistent pancolitis,which was refractory to mesalazine,corticosteroids,azathioprine,sirolimus and anti-TNF(adalimumab)therapy,but has improved recently with subcutaneous methotrexate.Whole Genome Sequencing revealed a novel pathogenic missense variant(c.290A>G)in the cytosolic isoleucyl-tRNA synthetase gene,leading to an amino acid substitution(p.Asp97Gly).Pathogenic variants in other genes associated with inflammatory bowel disease(IBD)(ADAM17,EGFR,FOXP3,IL10RA,IL10RB,IL21R,NCF4,STAT3)were excluded.Cytokine assays demonstrated markedly elevated IL-2,IL-5,IL-13,IL-9 and IL-10 by the patient’s CD4+T-cells,while IL-17A,IL-17F,IFNβwere lower,and TNFαnot significantly different when compared to healthy controls.CONCLUSION This case report provides evidence that recessive mutations in cytosolic isoleucyltRNA synthetase are a novel monogenic cause of IBD,which should be considered,particularly in infants and children with a history of neonatal hepatitis and very early-onset IBD poorly responsive to treatment.
基金funded by The Legacy Heritage Bio-Medical Program of the Israel Science Foundation(grant No.1629/13)
文摘Vanishing white matter （VWM） disease - a disease of the cytosolic translation machinery： VWM is a recessive genet- ic neurodegenerative disease caused by mutations in any of the five genes encoding the subunits of translation initiation factor 2B （eIF2B） （Leegwater et al., 2001; OMIM 306896）.
基金supported by the National Major Program on Transgenic Organisms from Ministry of Agriculture,China(2016ZX08005-004)。
文摘Elevated activities of cytosolic fructose-1,6-bisphosphatase(cyFBPase) and sedoheptulose-1,7-bisphosphatase(SBPase)are associated with higher yields in plants. In this study, the expression levels of the cyFBPase and SBPase genes were increased by overexpressing rape(Brassica napus) cDNA in tobacco(Nicotiana tabacum) plants. The transgenic plants coexpressing cy FBPase and SBPase(TpFS), or expressing single cy FBPase(TpF) or SBPase(TpS) had 1.77-, 1.55-, 1.23-fold cyFBPase and 1.45-, 1.12-, 1.36-fold SBPase activities as compared to the wild-type(WT), respectively. Photosynthesis rates of TpF, TpS and TpFS increased 4, 20 and 25% compared with WT plants. The SBPase and cyFBPase positively regulated each other and functioned synergistically in transgenic tobacco plants. In addition, the sucrose contents of the three transgenic plants were higher than that of WT plants. The starch accumulation of the TpFS and TpS plants was improved by 53 and 37%, but slightly decreased in TpF plants. Moreover, the transgenic tobacco plants harbouring SBPase and/or cyFBPase genes showed improvements in their growth, biomass, dry weight, plant height, stem diameter, leaf size,flower number, and pod weight. In conclusion, co-expression of SBPase and cyFBPase may pave a new way for improving crop yield in agricultural applications.
文摘Cytosolic phospholipase A2α （cPLA2α） catalyzes the release of arachidonic acid （AA） and lysophosphoglyceride from membrane phospholipids. Although the roles of AA and eicosanoids in cellular viability, the processes of inflammation and cancer cell development have been extensively studied, the function of cPLA2α in the processes of inflammation and cancer cell development is not clear. This review summarizes published evidences for the biochemical properties and regulatory mechanisms of cPLA2α. The potential for use of cPLA2α as a novel diagnostic target and predictive biomarker for tumors is also discussed.
文摘Objectives To investigate the effects of β2-adrenergic antagonist on cytosolic Ca^2 ＋ （[Ca^2＋ ]i） in ventricular myocytes from infarcted rat heart. Methods A ligature was placed around left anterior descending coronary artery of rat hearts. Rats in the control group were sham-operated. Cardiomyocytes were dissociated at two, four, eight weeks after myocardial infarction （MI） and [Ca^2＋]i was measured via fura-2 fluorescence. The response of cardiomyocytes to isoproterenol in presence or absence of betal-adrenergic antagonist atenolol, beta2-adrenergic antagonist ICI118, 551 or non-selective β1, 2- adrenergic antagonists propranolol was examined. Results The followings were found that ICI 118, 551 had no significant effects on the rise of [Ca^2＋]i induced by isoproterenol in normal ventricular myocytes （P 〉 0.05）, ICI118, 551 only significantly attenuated the rise of [Ca^2＋]i induced by isoproterenol at four weeks and eight weeks after MI （24.5%±5.7% vs 57.8% ± 13.2%, P〈 0.01; 12.2%±7.9% vs 44.6%±11.3%, P〈 0.01）. Atenolol had suppressive effects only in the control group and the post-MI group of two weeks （P 〈 0.05）, and propranolol had suppressive effects in the control and all the three post-MI groups （P 〈 0.01）. Conclusions Beta2-adrenergic antagonist ICI118, 551 may exert negative effects on Ca^2＋ overload initiated by sympathetic stimulation after MI.
基金supported by the National Natural Science Foundation of China,No.31001048
文摘Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and patho- genicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicity. In this study, the latest molecular chaperone system associated with endoplasmic re- ticulum-associated protein degradation, the endoplasmic reticulum resident protein quality-control system and the ubiquitination proteasome system, is outlined. The molecular chaperone system directly correlates with the prion protein degradation pathway. Understanding the molecular mechanisms will help provide a fascinating avenue for further investigations on prion disease treatment and prion protein-induced neurodegenerative diseases.
文摘Effects of procainamide (PA) on human platelet aggregation and the cytosolic free-Ca2+ concentration ([Ca2+]) were investigated in vitro. PA at doses of 8.5 , 34 and 136 μmol·L-1 could inhibit the human platelet aggregation induced by 0. 5 μmol · L-1 A23187 with a good concentration -effect relationship. One min and maximal aggregation rates were also inhibited ( P<0. 01 ,vs control). The [Ca2+], was decreased in the presence of PA ,and the changes of [Ca2+], showed a significant linear correlation with 1 min or maximal aggregation rate (P<0.05). These results suggest that the mechanisms of PA inhibiting platelet aggregation relate to the decrease of [Ca2+].
文摘Ferritin sequesters and stores iron and, consequently, protect cells against iron-mediated free radical damage. However, the mechanism of iron exit from the ferritin nanocage and
