Collagens are the most abundant proteins in mammals and form an extracellular matrix (ECM) with other components as the structural support of muscle, skin, corneas and blood vessels etc. Other than providing structura...Collagens are the most abundant proteins in mammals and form an extracellular matrix (ECM) with other components as the structural support of muscle, skin, corneas and blood vessels etc. Other than providing structural support, the ECM exhibits active communication with cells and influences many cellular processes including migration, wound healing, differentiation and cancer metastasis. Though collagen proteins contain highly repetitive primary sequences and defined tertiary structures, more and more studies have shown that many short peptides/motifs within collagen proteins play key roles in various biological processes. These short sequences are effective within triple helical structures or independently as stand-alone molecules resulting from proteolytic degradation. Besides endogenous ECM-derived peptides, many more functional peptides have been produced by tissue processing, chemical synthesis, and recombinant protein production. In this review, we summarize different peptides/motifs identified in collagen and other ECM proteins and discuss their potential for medical, personal care, and cosmetics applications.展开更多
Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.M...Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.展开更多
Our previous studies have reported that activation of the NLRP3(NOD-,LRR-and pyrin domain-containing protein 3)-inflammasome complex in ethanol-treated astrocytes and chronic alcohol-fed mice could be associated with ...Our previous studies have reported that activation of the NLRP3(NOD-,LRR-and pyrin domain-containing protein 3)-inflammasome complex in ethanol-treated astrocytes and chronic alcohol-fed mice could be associated with neuroinflammation and brain damage.Mesenchymal stem cell-derived extracellular vesicles(MSC-EVs)have been shown to restore the neuroinflammatory response,along with myelin and synaptic structural alterations in the prefrontal cortex,and alleviate cognitive and memory dysfunctions induced by binge-like ethanol treatment in adolescent mice.Considering the therapeutic role of the molecules contained in mesenchymal stem cell-derived extracellular vesicles,the present study analyzed whether the administration of mesenchymal stem cell-derived extracellular vesicles isolated from adipose tissue,which inhibited the activation of the NLRP3 inflammasome,was capable of reducing hippocampal neuroinflammation in adolescent mice treated with binge drinking.We demonstrated that the administration of mesenchymal stem cell-derived extracellular vesicles ameliorated the activation of the hippocampal NLRP3 inflammasome complex and other NLRs inflammasomes(e.g.,pyrin domain-containing 1,caspase recruitment domain-containing 4,and absent in melanoma 2,as well as the alterations in inflammatory genes(interleukin-1β,interleukin-18,inducible nitric oxide synthase,nuclear factor-kappa B,monocyte chemoattractant protein-1,and C–X3–C motif chemokine ligand 1)and miRNAs(miR-21a-5p,miR-146a-5p,and miR-141-5p)induced by binge-like ethanol treatment in adolescent mice.Bioinformatic analysis further revealed the involvement of miR-21a-5p and miR-146a-5p with inflammatory target genes and NOD-like receptor signaling pathways.Taken together,these findings provide novel evidence of the therapeutic potential of MSC-derived EVs to ameliorate the hippocampal neuroinflammatory response associated with NLRP3 inflammasome activation induced by binge drinking in adolescence.展开更多
The purpose of this study was to investigate changes of β 3 integrins and extra cellular matrix proteins including fibronectin (FN), laminin (LN) and collagen type Ⅳ (CL typeⅣ) on the endometrium of secret...The purpose of this study was to investigate changes of β 3 integrins and extra cellular matrix proteins including fibronectin (FN), laminin (LN) and collagen type Ⅳ (CL typeⅣ) on the endometrium of secretory phase from 31 fertile women (fertility group)and 34 women with unexplained infertility (infertility group) by a histochemical method.The results were as follows:In glandular epithelium, β 3 integrin appeared in the mid secretory phase and continued to late secretory phase in the fertility group, but was not expressed during the secretory phase in the infertility group. Extracellular matrix proteins from the fertility group were expressed more strongly in mid secretory phase than that in the early secretory phase, and were weakest in the late secretory phase. Compared with the fertility group, the levels of extracellular matrix proteins in the infertility group were elevated in the secretory phase. In conclusion: our current study demonstrate that β 3 integrin and extracellular matrix proteins are expressed at different levels in the endometrium during the menstrual cycle. They are involved in endometrial changes during the menstrual cycle and during the implantation of the blastocyst. Their unusual expression result in the failure of implantation.展开更多
Four batch experiments of hydrolysis and acidification were carried out to investigate the distributions of proteins (PN) and polysaccharides (PS) in the sludge, the PN/PS ratio, the particle sizes, and their rela...Four batch experiments of hydrolysis and acidification were carried out to investigate the distributions of proteins (PN) and polysaccharides (PS) in the sludge, the PN/PS ratio, the particle sizes, and their relationship with sludge dewaterability (as determined by capillary suction time, CST). The sludge flocs were stratified through centrifugation- and ultrasound-based method into four fractions: (1) slime, (2) loosely bound extracellular polymeric substances (LB-EPS), (3) tightly bound EPS (TB-EPS), and (4) pellet. The results showed that PN was mainly partitioned in the pellet (80.7%) and TB-EPS (9.6%) fractions, while PS distributed evenly in the four fractions. During hydrolysis and acidification, PN was transferred from the pellet and TB-EPS fractions to the slime fraction, but PS had no significant transfer trends. The mean particle sizes of the sludge flocs decreased with hydrolysis and acidification. The pH had a more significant influence on the dewaterability of sludge flocs than temperature. Sludge dewaterability during hydrolysis and acidification processes greatly deteriorated from 9.7 s at raw sludge to 340-450 s under alkaline conditions. However, it was just slightly increased under acidic conditions. Further investigation suggested that CST was affected by soluble PN, soluble PN/PS, and particle sizes of sludge flocs, but was affected slightly by total PN, PS, or PN/PS in the whole sludge flocs and other fractions (except slime).展开更多
After onset of myocardial infarction(MI),the left ventricle(LV) undergoes a continuum of molecular,cellular,and extracellular responses that result in LV wall thinning,dilatation,and dysfunction.These dynamic changes ...After onset of myocardial infarction(MI),the left ventricle(LV) undergoes a continuum of molecular,cellular,and extracellular responses that result in LV wall thinning,dilatation,and dysfunction.These dynamic changes in LV shape,size,and function are termed cardiac remodeling.If the cardiac healing after MI does not proceed properly,it could lead to cardiac rupture or maladaptive cardiac remodeling,such as further LV dilatation and dysfunction,and ultimately death.Although the precise molecular mechanisms in this cardiac healing process have not been fully elucidated,this process is strictly coordinated by the interaction of cells with their surrounding extracellular matrix(ECM) proteins.The components of ECM include basic structural proteins such as collagen,elastin and specialized proteins such as fibronectin,proteoglycans and matricellular proteins.Matricellular proteins are a class of non-structural and secreted proteins that probably exert regulatory functions through direct binding to cell surface receptors,other matrix proteins,and soluble extracellular factors such as growth factors and cytokines.This small group of proteins,which includesosteopontin,thrombospondin-1/2,tenascin,periostin,and secreted protein,acidic and rich in cysteine,shows a low level of expression in normal adult tissue,but is markedly upregulated during wound healing and tissue remodeling,including MI.In this review,we focus on the regulatory functions of matricellular proteins during cardiac tissue healing and remodeling after MI.展开更多
Mammalian cells express two isoforms of Cu- and Zn-containing superoxide dismutases(SODs), CuZn-SOD and extracellular SOD(EC-SOD), involved in the defense system against reactive oxygen species(ROS). The two SOD...Mammalian cells express two isoforms of Cu- and Zn-containing superoxide dismutases(SODs), CuZn-SOD and extracellular SOD(EC-SOD), involved in the defense system against reactive oxygen species(ROS). The two SODs have structurally homologous centre domain with distinct N- and C-terminuses, resulting in the different characteristics of the structure and function of the two molecules. We generated a hybrid SOD molecule(namely hy- SOD) via replacing the N- and C-terminuses of hCuZnSOD with the counterparts of hEC-SOD. The hySOD was expressed in host Pichia pastoris and the purified protein was a dimer with a molecular weight of about 34000. A series of activity analyses indicates that the hySOD is similar to hEC-SOD in heat-stability, and has the activity of protecting the host cell against heat shock and oxidative stress. Our results show evidence for the study on the compound activity of multiple SOD molecules, and may be important for understanding the relationship between structure and function of hEC-SOD and hCuZnSOD.展开更多
Populus euphratica Olive is the only tree species that can grow in the saline land and also survive cold winters in northwest China, and it plays a very important role in stabilizing the vulnerable ecosystem there. A ...Populus euphratica Olive is the only tree species that can grow in the saline land and also survive cold winters in northwest China, and it plays a very important role in stabilizing the vulnerable ecosystem there. A cell suspension culture was initiated from callus derived from plantlets of Populus euphratica. Cold acclimation was induced (LT50 of 17.5 ℃) in cell suspension at 45 ℃ in the dark for 30 days and the freezing tolerance increased from LT50 of 12.5 ℃ in nonacclimated cells to LT50 of 17.5 ℃ in cold-acclimated cells. Microvacuolation, cytoplasmic augmentation and accumulation of starch granules were observed in cells that were cold-acclimated by exposure to low temperatures. Several qualitative and quantitative changes in proteins were noted during cold acclimation. Antibodies to carrot extracellular (apoplastic) 36 kD antifreeze protein did not cross react on immunoelectroblots with extracellular proteins in cell suspension culture medium of Populus euphratica, indicating no common epitopes in the carrot 36 kD antifreeze protein and P. euphratica extracellular proteins. The relationship of these changes to cold acclimation in Populus euphratica cell cultures was discussed.展开更多
BACKGROUND Extracellular vesicles(EVs)derived from hypoxia-preconditioned(HP)mesenchymal stem cells(MSCs)have better cardioprotective effects against myocardial infarction(MI)in the early stage than EVs isolated from ...BACKGROUND Extracellular vesicles(EVs)derived from hypoxia-preconditioned(HP)mesenchymal stem cells(MSCs)have better cardioprotective effects against myocardial infarction(MI)in the early stage than EVs isolated from normoxic(NC)-MSCs.However,the cardioprotective mechanisms of HP-EVs are not fully understood.AIM To explore the cardioprotective mechanism of EVs derived from HP MSCs.METHODS We evaluated the cardioprotective effects of HP-EVs or NC-EVs from mouse adipose-derived MSCs(ADSCs)following hypoxia in vitro or MI in vivo,in order to improve the survival of cardiomyocytes(CMs)and restore cardiac function.The degree of CM apoptosis in each group was assessed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling and Annexin V/PI assays.MicroRNA(miRNA)sequencing was used to investigate the functional RNA diversity between HP-EVs and NC-EVs from mouse ADSCs.The molecular mechanism of EVs in mediating thioredoxin-interacting protein(TXNIP)was verified by the dual-luciferase reporter assay.Co-immunoprecipitation,western blotting,and immunofluorescence were performed to determine if TXNIP is involved in hypoxia-inducible factor-1 alpha(HIF-1α)ubiquitination and degradation via the chromosomal region maintenance-1(CRM-1)-dependent nuclear transport pathway.RESULTS HP-EVs derived from MSCs reduced both infarct size(necrosis area)and apoptotic degree to a greater extent than NC-EVs from CMs subjected to hypoxia in vitro and mice with MI in vivo.Sequencing of EV-associated miRNAs showed the upregulation of 10 miRNAs predicted to bind TXNIP,an oxidative stress-associated protein.We showed miRNA224-5p,the most upregulated miRNA in HP-EVs,directly combined the 3’untranslated region of TXNIP and demonstrated its critical protective role against hypoxia-mediated CM injury.Our results demonstrated that MI triggered TXNIP-mediated HIF-1αubiquitination and degradation in the CRM-1-mediated nuclear transport pathway in CMs,which led to aggravated injury and hypoxia tolerance in CMs in the early stage of MI.CONCLUSION The anti-apoptotic effects of HP-EVs in alleviating MI and the hypoxic conditions of CMs until reperfusion therapy may partly result from EV miR-224-5p targeting TXNIP.展开更多
AIM:To study the expression and phosphorylation of extracellular signal-regulated kinase(ERK)1 and ERK2 in multidrug resistant(MDR)hepatocellular carcinoma(HCC)cells.METHODS:MDR HCC cell lines,HepG2/adriamycin(ADM)and...AIM:To study the expression and phosphorylation of extracellular signal-regulated kinase(ERK)1 and ERK2 in multidrug resistant(MDR)hepatocellular carcinoma(HCC)cells.METHODS:MDR HCC cell lines,HepG2/adriamycin(ADM)and SMMC7721/ADM,were developed by exposing parental cells to stepwise increasing concentrations of ADM.MTT assay was used to determine drug sensitivity.Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein(P-gp)and multidrug resistant protein 1(MRP1)expression levels.ERK1 and ERK2 mRNA expression levels were measured by quantitative real-time PCR(QRTPCR).Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot.SMMC7721/ADM were resistant not only to ADM,but also to multiple anticancer drugs.The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells(8.92%±0.22%vs 0.88%±0.05%,P<0.001)and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells(7.37%±0.26%vs 1.74%±0.25%,P<0.001).However,the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells.In addition,the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase.QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells.Compared with the expression of parental cells,ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells.However,ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells.Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.CONCLUSION:ERK1 and ERK2 activities are downregulated in P-gp-mediated MDR HCC cells.ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells.展开更多
Objective To investigate the molecular mechanism of nectin-like molecule 1(NECL1) inhibiting the migration and invasion of U251 glioma cells.Methods We infected U251 glioma cells with adeno-nectin-like molecule 1(Ad-N...Objective To investigate the molecular mechanism of nectin-like molecule 1(NECL1) inhibiting the migration and invasion of U251 glioma cells.Methods We infected U251 glioma cells with adeno-nectin-like molecule 1(Ad-NECL1) or empty adenovirus(Ad).Transwell and wound healing assays were performed to observe the migration of U251 cells incubated with the cell supernatant from Ad-NECL1 or Ad infected U251 cells.DNA microarray was applied to screen the gene expression profile after the restoration of NECL1 in U251 glioma cell lines.The differential expression of osteopontin(OPN),a gene related to migration and invasion,was further analyzed with semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR),Western blot,and immunohistochemistry.Results The restoration of NECL1 inhibited migration of U251 cells significantly(P<0.05).Altogether 195 genes were found differentially expressed by microarray,in which 175 were up-regulated and 20 down-regulated,including 9 extracellular matrix proteins involved in the migration of cells.Both mRNA and protein expressions of OPN,the most markedly reduced extracellular matrix protein,were found decreased in U251 cells after restoration of NECL1.Immunohistochemical assay also detected an increase of OPN in glioma tissues,related with the progressing of malignant grade.Conclusion A link might exist between NECL1 and the extracellular matrix protein OPN in inhibiting the migration and invasion of U251 glioma cells.展开更多
Adipose mesenchymal stem cells(ADSCs)have protective effects against glutamate-induced excitotoxicity,but ADSCs are limited in use for treatment of optic nerve injury.Studies have shown that the extracellular vesicles...Adipose mesenchymal stem cells(ADSCs)have protective effects against glutamate-induced excitotoxicity,but ADSCs are limited in use for treatment of optic nerve injury.Studies have shown that the extracellular vesicles(EVs)secreted by ADSCs(ADSC-EVs)not only have the function of ADSCs,but also have unique advantages including non-immunogenicity,low probability of abnormal growth,and easy access to target cells.In the present study,we showed that intravitreal injection of ADSC-EVs substantially reduced glutamate-induced damage to retinal morphology and electroretinography.In addition,R28 cell pretreatment with ADSC-EVs before injury inhibited glutamate-induced overload of intracellular calcium,downregulation ofα-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor(AMPAR)subunit GluA2,and phosphorylation of GluA2 and protein kinase C alpha in vitro.A protein kinase C alpha agonist,12-O-tetradecanoylphorbol 13-acetate,inhibited the neuroprotective effects of ADSC-EVs on glutamate-induced R28 cells.These findings suggest that ADSCEVs ameliorate glutamate-induced excitotoxicity in the retina through inhibiting protein kinase C alpha activation.展开更多
Alzheimer's disease is a severe, highly disabling neurodegenerative disease, clinically characterized by a progressive decline in cognitive functions, and is the most common form of dementia in the elderly. For de...Alzheimer's disease is a severe, highly disabling neurodegenerative disease, clinically characterized by a progressive decline in cognitive functions, and is the most common form of dementia in the elderly. For decades, the search for disease-modifying therapies has focused on the two main Alzheimer's disease histopathological hallmarks, seeking to prevent, mitigate, or clear the formation of extracellular aggregates of β-amyloid peptide and intracellular neurofibrillary tangles of tau protein, although without clinical success. Mesenchymal stem cell-based therapy has emerged as a promising alternative for the treatment of Alzheimer's disease, especially because it also targets other crucial players in the pathogenesis of the disease, such as neuroinflammation, synaptic dysfunction/loss, oxidative stress, and impaired neurogenesis. Herein, we review current knowledge of the therapeutic potential of mesenchymal stem cells and their extracellular vesicles for Alzheimer's disease, discussing the most recent findings in both preclinical and clinical trials as well as how advanced technologies have helped to overcome some limitations and contributed to stimulate the development of more effective treatments.展开更多
Effect of sludge retention time( SRT) on the removal of potential and polysaccharides in an anaerobic / anoxic / aerobic( AAO) process was investigated. The Lowry method and anthrone method were used to detect protein...Effect of sludge retention time( SRT) on the removal of potential and polysaccharides in an anaerobic / anoxic / aerobic( AAO) process was investigated. The Lowry method and anthrone method were used to detect proteins and polysaccharides. Total removal efficiency of proteins at SRT of 10 to 25 d in the AAO system was higher than 90%. Polysaccharides removal efficiencies were above 80% when SRT was increased from 15 to 25 d,whereas only 81% of polysaccharides was removed at SRT of 10 d. The biodegradation part of proteins and polysaccharides increased from87. 40% to 93% and from 74. 22% to 86. 94% with increasing SRTs.The ratios of polysaccharides and proteins in extracellular polymer substances( EPSs) were around 1. 5-3 in different SRTs. As SRT increasing,polysaccharides and proteins discharged with residual sludge decreased gradually. The amount of EPSs decreased with increasing SRTs.展开更多
Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB le...Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.展开更多
Protein quality control involves many processes that jointly act to regulate the expression, localization, turnover, and degradation of proteins, and has been highlighted in recent studies as critical to the different...Protein quality control involves many processes that jointly act to regulate the expression, localization, turnover, and degradation of proteins, and has been highlighted in recent studies as critical to the differentiation of stem cells during regeneration. The roles of constitutively secreted extracellular chaperones in neuronal injury and disease are poorly understood. Extracellular chaperones are multifunctional proteins expressed by many cell types, including those of the nervous system, known to facilitate protein quality control processes. These molecules exert pleiotropic effects and have been implicated as playing important protective roles in a variety of stress conditions, including tissue damage, infections, and local tissue inflammation. This article aims to provide a critical review of what is currently known about the functions of extracellular chaperones in neuronal repair and regeneration and highlight future directions for this important research area. We review what is known of four constitutively secreted extracellular chaperones directly implicated in processes of neuronal damage and repair, including transthyretin, clusterin, α2-macroglobulin, and neuroserpin, and propose that investigation into the effects of these and other extracellular chaperones on neuronal repair and regeneration has the potential to yield valuable new therapies.展开更多
文摘Collagens are the most abundant proteins in mammals and form an extracellular matrix (ECM) with other components as the structural support of muscle, skin, corneas and blood vessels etc. Other than providing structural support, the ECM exhibits active communication with cells and influences many cellular processes including migration, wound healing, differentiation and cancer metastasis. Though collagen proteins contain highly repetitive primary sequences and defined tertiary structures, more and more studies have shown that many short peptides/motifs within collagen proteins play key roles in various biological processes. These short sequences are effective within triple helical structures or independently as stand-alone molecules resulting from proteolytic degradation. Besides endogenous ECM-derived peptides, many more functional peptides have been produced by tissue processing, chemical synthesis, and recombinant protein production. In this review, we summarize different peptides/motifs identified in collagen and other ECM proteins and discuss their potential for medical, personal care, and cosmetics applications.
基金Nanjing Science and Technology Plan Project(No.ZX20200009)Jiangsu Province Postgraduate Research and Practice Innovation Program(No.SJCX22-0895)。
文摘Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.
基金Project(50674101)supported by the National Natural Science Foundation of ChinaProject(2004CB619201)supported by the National Basic Research Program of ChinaProject(50621063)supported by the Chinese Science Foundation for Distinguished Group
基金supported by grants from the Spanish Ministry of Health-PNSD(2019-I039 and 2023-I024)(to MP)FEDER/Ministerio de Ciencia e Innovación-Agencia Estatal de Investigación PID2021-1243590B-I100(to VMM)+2 种基金GVA(CIAICO/2021/203)(to MP)the Primary Addiction Care Research Network(RD21/0009/0005)(to MP)a predoctoral fellowship from the Generalitat Valenciana(ACIF/2021/338)(to CPC).
文摘Our previous studies have reported that activation of the NLRP3(NOD-,LRR-and pyrin domain-containing protein 3)-inflammasome complex in ethanol-treated astrocytes and chronic alcohol-fed mice could be associated with neuroinflammation and brain damage.Mesenchymal stem cell-derived extracellular vesicles(MSC-EVs)have been shown to restore the neuroinflammatory response,along with myelin and synaptic structural alterations in the prefrontal cortex,and alleviate cognitive and memory dysfunctions induced by binge-like ethanol treatment in adolescent mice.Considering the therapeutic role of the molecules contained in mesenchymal stem cell-derived extracellular vesicles,the present study analyzed whether the administration of mesenchymal stem cell-derived extracellular vesicles isolated from adipose tissue,which inhibited the activation of the NLRP3 inflammasome,was capable of reducing hippocampal neuroinflammation in adolescent mice treated with binge drinking.We demonstrated that the administration of mesenchymal stem cell-derived extracellular vesicles ameliorated the activation of the hippocampal NLRP3 inflammasome complex and other NLRs inflammasomes(e.g.,pyrin domain-containing 1,caspase recruitment domain-containing 4,and absent in melanoma 2,as well as the alterations in inflammatory genes(interleukin-1β,interleukin-18,inducible nitric oxide synthase,nuclear factor-kappa B,monocyte chemoattractant protein-1,and C–X3–C motif chemokine ligand 1)and miRNAs(miR-21a-5p,miR-146a-5p,and miR-141-5p)induced by binge-like ethanol treatment in adolescent mice.Bioinformatic analysis further revealed the involvement of miR-21a-5p and miR-146a-5p with inflammatory target genes and NOD-like receptor signaling pathways.Taken together,these findings provide novel evidence of the therapeutic potential of MSC-derived EVs to ameliorate the hippocampal neuroinflammatory response associated with NLRP3 inflammasome activation induced by binge drinking in adolescence.
文摘The purpose of this study was to investigate changes of β 3 integrins and extra cellular matrix proteins including fibronectin (FN), laminin (LN) and collagen type Ⅳ (CL typeⅣ) on the endometrium of secretory phase from 31 fertile women (fertility group)and 34 women with unexplained infertility (infertility group) by a histochemical method.The results were as follows:In glandular epithelium, β 3 integrin appeared in the mid secretory phase and continued to late secretory phase in the fertility group, but was not expressed during the secretory phase in the infertility group. Extracellular matrix proteins from the fertility group were expressed more strongly in mid secretory phase than that in the early secretory phase, and were weakest in the late secretory phase. Compared with the fertility group, the levels of extracellular matrix proteins in the infertility group were elevated in the secretory phase. In conclusion: our current study demonstrate that β 3 integrin and extracellular matrix proteins are expressed at different levels in the endometrium during the menstrual cycle. They are involved in endometrial changes during the menstrual cycle and during the implantation of the blastocyst. Their unusual expression result in the failure of implantation.
基金supported by the National Hi-Tech Research and Development Program (863) of China (No. 2006AA06Z384).
文摘Four batch experiments of hydrolysis and acidification were carried out to investigate the distributions of proteins (PN) and polysaccharides (PS) in the sludge, the PN/PS ratio, the particle sizes, and their relationship with sludge dewaterability (as determined by capillary suction time, CST). The sludge flocs were stratified through centrifugation- and ultrasound-based method into four fractions: (1) slime, (2) loosely bound extracellular polymeric substances (LB-EPS), (3) tightly bound EPS (TB-EPS), and (4) pellet. The results showed that PN was mainly partitioned in the pellet (80.7%) and TB-EPS (9.6%) fractions, while PS distributed evenly in the four fractions. During hydrolysis and acidification, PN was transferred from the pellet and TB-EPS fractions to the slime fraction, but PS had no significant transfer trends. The mean particle sizes of the sludge flocs decreased with hydrolysis and acidification. The pH had a more significant influence on the dewaterability of sludge flocs than temperature. Sludge dewaterability during hydrolysis and acidification processes greatly deteriorated from 9.7 s at raw sludge to 340-450 s under alkaline conditions. However, it was just slightly increased under acidic conditions. Further investigation suggested that CST was affected by soluble PN, soluble PN/PS, and particle sizes of sludge flocs, but was affected slightly by total PN, PS, or PN/PS in the whole sludge flocs and other fractions (except slime).
文摘After onset of myocardial infarction(MI),the left ventricle(LV) undergoes a continuum of molecular,cellular,and extracellular responses that result in LV wall thinning,dilatation,and dysfunction.These dynamic changes in LV shape,size,and function are termed cardiac remodeling.If the cardiac healing after MI does not proceed properly,it could lead to cardiac rupture or maladaptive cardiac remodeling,such as further LV dilatation and dysfunction,and ultimately death.Although the precise molecular mechanisms in this cardiac healing process have not been fully elucidated,this process is strictly coordinated by the interaction of cells with their surrounding extracellular matrix(ECM) proteins.The components of ECM include basic structural proteins such as collagen,elastin and specialized proteins such as fibronectin,proteoglycans and matricellular proteins.Matricellular proteins are a class of non-structural and secreted proteins that probably exert regulatory functions through direct binding to cell surface receptors,other matrix proteins,and soluble extracellular factors such as growth factors and cytokines.This small group of proteins,which includesosteopontin,thrombospondin-1/2,tenascin,periostin,and secreted protein,acidic and rich in cysteine,shows a low level of expression in normal adult tissue,but is markedly upregulated during wound healing and tissue remodeling,including MI.In this review,we focus on the regulatory functions of matricellular proteins during cardiac tissue healing and remodeling after MI.
文摘Mammalian cells express two isoforms of Cu- and Zn-containing superoxide dismutases(SODs), CuZn-SOD and extracellular SOD(EC-SOD), involved in the defense system against reactive oxygen species(ROS). The two SODs have structurally homologous centre domain with distinct N- and C-terminuses, resulting in the different characteristics of the structure and function of the two molecules. We generated a hybrid SOD molecule(namely hy- SOD) via replacing the N- and C-terminuses of hCuZnSOD with the counterparts of hEC-SOD. The hySOD was expressed in host Pichia pastoris and the purified protein was a dimer with a molecular weight of about 34000. A series of activity analyses indicates that the hySOD is similar to hEC-SOD in heat-stability, and has the activity of protecting the host cell against heat shock and oxidative stress. Our results show evidence for the study on the compound activity of multiple SOD molecules, and may be important for understanding the relationship between structure and function of hEC-SOD and hCuZnSOD.
基金the National Natural Science Foundation of China (Grant No. 30271067)Fok Ying Tung Education Foundation (71030)+1 种基金 Key Teachers Foundation of the Educational Ministry of China and the State Key Basic Research and Development Plan of China (G199901600
文摘Populus euphratica Olive is the only tree species that can grow in the saline land and also survive cold winters in northwest China, and it plays a very important role in stabilizing the vulnerable ecosystem there. A cell suspension culture was initiated from callus derived from plantlets of Populus euphratica. Cold acclimation was induced (LT50 of 17.5 ℃) in cell suspension at 45 ℃ in the dark for 30 days and the freezing tolerance increased from LT50 of 12.5 ℃ in nonacclimated cells to LT50 of 17.5 ℃ in cold-acclimated cells. Microvacuolation, cytoplasmic augmentation and accumulation of starch granules were observed in cells that were cold-acclimated by exposure to low temperatures. Several qualitative and quantitative changes in proteins were noted during cold acclimation. Antibodies to carrot extracellular (apoplastic) 36 kD antifreeze protein did not cross react on immunoelectroblots with extracellular proteins in cell suspension culture medium of Populus euphratica, indicating no common epitopes in the carrot 36 kD antifreeze protein and P. euphratica extracellular proteins. The relationship of these changes to cold acclimation in Populus euphratica cell cultures was discussed.
基金Supported by National Natural Science Foundation of China,No. 81870264 and No. 81470546the Shanghai Committee of Science and Technology,No. 18411950500+1 种基金the Major Disease Joint Project of Shanghai Health System,No. 2014ZYJB0501Talent Cultivation Project of The Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,No. JC202005
文摘BACKGROUND Extracellular vesicles(EVs)derived from hypoxia-preconditioned(HP)mesenchymal stem cells(MSCs)have better cardioprotective effects against myocardial infarction(MI)in the early stage than EVs isolated from normoxic(NC)-MSCs.However,the cardioprotective mechanisms of HP-EVs are not fully understood.AIM To explore the cardioprotective mechanism of EVs derived from HP MSCs.METHODS We evaluated the cardioprotective effects of HP-EVs or NC-EVs from mouse adipose-derived MSCs(ADSCs)following hypoxia in vitro or MI in vivo,in order to improve the survival of cardiomyocytes(CMs)and restore cardiac function.The degree of CM apoptosis in each group was assessed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling and Annexin V/PI assays.MicroRNA(miRNA)sequencing was used to investigate the functional RNA diversity between HP-EVs and NC-EVs from mouse ADSCs.The molecular mechanism of EVs in mediating thioredoxin-interacting protein(TXNIP)was verified by the dual-luciferase reporter assay.Co-immunoprecipitation,western blotting,and immunofluorescence were performed to determine if TXNIP is involved in hypoxia-inducible factor-1 alpha(HIF-1α)ubiquitination and degradation via the chromosomal region maintenance-1(CRM-1)-dependent nuclear transport pathway.RESULTS HP-EVs derived from MSCs reduced both infarct size(necrosis area)and apoptotic degree to a greater extent than NC-EVs from CMs subjected to hypoxia in vitro and mice with MI in vivo.Sequencing of EV-associated miRNAs showed the upregulation of 10 miRNAs predicted to bind TXNIP,an oxidative stress-associated protein.We showed miRNA224-5p,the most upregulated miRNA in HP-EVs,directly combined the 3’untranslated region of TXNIP and demonstrated its critical protective role against hypoxia-mediated CM injury.Our results demonstrated that MI triggered TXNIP-mediated HIF-1αubiquitination and degradation in the CRM-1-mediated nuclear transport pathway in CMs,which led to aggravated injury and hypoxia tolerance in CMs in the early stage of MI.CONCLUSION The anti-apoptotic effects of HP-EVs in alleviating MI and the hypoxic conditions of CMs until reperfusion therapy may partly result from EV miR-224-5p targeting TXNIP.
基金Supported by Innovation Fund of Fujian Province,No.2007-CXB-7Key Science and Technology Project of Xiamen,No.3502Z20077045
文摘AIM:To study the expression and phosphorylation of extracellular signal-regulated kinase(ERK)1 and ERK2 in multidrug resistant(MDR)hepatocellular carcinoma(HCC)cells.METHODS:MDR HCC cell lines,HepG2/adriamycin(ADM)and SMMC7721/ADM,were developed by exposing parental cells to stepwise increasing concentrations of ADM.MTT assay was used to determine drug sensitivity.Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein(P-gp)and multidrug resistant protein 1(MRP1)expression levels.ERK1 and ERK2 mRNA expression levels were measured by quantitative real-time PCR(QRTPCR).Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot.SMMC7721/ADM were resistant not only to ADM,but also to multiple anticancer drugs.The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells(8.92%±0.22%vs 0.88%±0.05%,P<0.001)and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells(7.37%±0.26%vs 1.74%±0.25%,P<0.001).However,the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells.In addition,the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase.QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells.Compared with the expression of parental cells,ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells.However,ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells.Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.CONCLUSION:ERK1 and ERK2 activities are downregulated in P-gp-mediated MDR HCC cells.ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells.
基金Supported by National Natural Science Foundation of China (30421003,30828004)
文摘Objective To investigate the molecular mechanism of nectin-like molecule 1(NECL1) inhibiting the migration and invasion of U251 glioma cells.Methods We infected U251 glioma cells with adeno-nectin-like molecule 1(Ad-NECL1) or empty adenovirus(Ad).Transwell and wound healing assays were performed to observe the migration of U251 cells incubated with the cell supernatant from Ad-NECL1 or Ad infected U251 cells.DNA microarray was applied to screen the gene expression profile after the restoration of NECL1 in U251 glioma cell lines.The differential expression of osteopontin(OPN),a gene related to migration and invasion,was further analyzed with semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR),Western blot,and immunohistochemistry.Results The restoration of NECL1 inhibited migration of U251 cells significantly(P<0.05).Altogether 195 genes were found differentially expressed by microarray,in which 175 were up-regulated and 20 down-regulated,including 9 extracellular matrix proteins involved in the migration of cells.Both mRNA and protein expressions of OPN,the most markedly reduced extracellular matrix protein,were found decreased in U251 cells after restoration of NECL1.Immunohistochemical assay also detected an increase of OPN in glioma tissues,related with the progressing of malignant grade.Conclusion A link might exist between NECL1 and the extracellular matrix protein OPN in inhibiting the migration and invasion of U251 glioma cells.
基金supported by the National Key R&D Program of China,No.2016YFC1201800(to JFH)the Key Research and Development Program of Hunan Province,Nos.2018SK2090(to JFH),2022SK2079(to JFH)+2 种基金the Natural Science Foundation of Hu nan Province,No.2021JJ30891(to DC)the Human Resource Bank Program of Hunan Province,No.2020TP3003(to JFH)the School-Enterprise Joint Program of Central South University,No.2021XQLH092(to TQD)。
文摘Adipose mesenchymal stem cells(ADSCs)have protective effects against glutamate-induced excitotoxicity,but ADSCs are limited in use for treatment of optic nerve injury.Studies have shown that the extracellular vesicles(EVs)secreted by ADSCs(ADSC-EVs)not only have the function of ADSCs,but also have unique advantages including non-immunogenicity,low probability of abnormal growth,and easy access to target cells.In the present study,we showed that intravitreal injection of ADSC-EVs substantially reduced glutamate-induced damage to retinal morphology and electroretinography.In addition,R28 cell pretreatment with ADSC-EVs before injury inhibited glutamate-induced overload of intracellular calcium,downregulation ofα-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor(AMPAR)subunit GluA2,and phosphorylation of GluA2 and protein kinase C alpha in vitro.A protein kinase C alpha agonist,12-O-tetradecanoylphorbol 13-acetate,inhibited the neuroprotective effects of ADSC-EVs on glutamate-induced R28 cells.These findings suggest that ADSCEVs ameliorate glutamate-induced excitotoxicity in the retina through inhibiting protein kinase C alpha activation.
基金supported by gran ts and fellowships from the Deportomento de Ciência e Tecnologia (DECIT-MS) do Ministério da SaúdeConselho Nacionol de Desenvolvimento Científico e Tecnológico (CNPq)+2 种基金Instituto Nacional de Ciência e Tecnologia em Medicina RegenerotivaFundacao de AmporoàPesquisa do Estado do Rio de Janeiro(FAPERJ)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)(all to RMO)。
文摘Alzheimer's disease is a severe, highly disabling neurodegenerative disease, clinically characterized by a progressive decline in cognitive functions, and is the most common form of dementia in the elderly. For decades, the search for disease-modifying therapies has focused on the two main Alzheimer's disease histopathological hallmarks, seeking to prevent, mitigate, or clear the formation of extracellular aggregates of β-amyloid peptide and intracellular neurofibrillary tangles of tau protein, although without clinical success. Mesenchymal stem cell-based therapy has emerged as a promising alternative for the treatment of Alzheimer's disease, especially because it also targets other crucial players in the pathogenesis of the disease, such as neuroinflammation, synaptic dysfunction/loss, oxidative stress, and impaired neurogenesis. Herein, we review current knowledge of the therapeutic potential of mesenchymal stem cells and their extracellular vesicles for Alzheimer's disease, discussing the most recent findings in both preclinical and clinical trials as well as how advanced technologies have helped to overcome some limitations and contributed to stimulate the development of more effective treatments.
基金National Natural Science Foundations of China(Nos.21477018,21007010)Hunan Province Ministry of Transportation Scientific Research Projects,China(Nos.200908,201105)Ministry of Transport Science and Technology Program,China(No.2010353343290)
文摘Effect of sludge retention time( SRT) on the removal of potential and polysaccharides in an anaerobic / anoxic / aerobic( AAO) process was investigated. The Lowry method and anthrone method were used to detect proteins and polysaccharides. Total removal efficiency of proteins at SRT of 10 to 25 d in the AAO system was higher than 90%. Polysaccharides removal efficiencies were above 80% when SRT was increased from 15 to 25 d,whereas only 81% of polysaccharides was removed at SRT of 10 d. The biodegradation part of proteins and polysaccharides increased from87. 40% to 93% and from 74. 22% to 86. 94% with increasing SRTs.The ratios of polysaccharides and proteins in extracellular polymer substances( EPSs) were around 1. 5-3 in different SRTs. As SRT increasing,polysaccharides and proteins discharged with residual sludge decreased gradually. The amount of EPSs decreased with increasing SRTs.
基金supported by Research Start-up Funding of Shenzhen Traditional Chinese Medicine Hospital,No.2021-07(to FB)Sanming Project of Medicine in Shenzhen,No.SZZYSM 202111011(to XDQ and FB)+1 种基金Key Discipline Established by Zhejiang Province,Jiaxing City Jointly-Pain Medicine,No.2019-ss-ttyx(to LSX)Jiaxing Key Laboratory of Neurology and Pain Medicine,No.[2014]81(to LSX)。
文摘Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.
文摘Protein quality control involves many processes that jointly act to regulate the expression, localization, turnover, and degradation of proteins, and has been highlighted in recent studies as critical to the differentiation of stem cells during regeneration. The roles of constitutively secreted extracellular chaperones in neuronal injury and disease are poorly understood. Extracellular chaperones are multifunctional proteins expressed by many cell types, including those of the nervous system, known to facilitate protein quality control processes. These molecules exert pleiotropic effects and have been implicated as playing important protective roles in a variety of stress conditions, including tissue damage, infections, and local tissue inflammation. This article aims to provide a critical review of what is currently known about the functions of extracellular chaperones in neuronal repair and regeneration and highlight future directions for this important research area. We review what is known of four constitutively secreted extracellular chaperones directly implicated in processes of neuronal damage and repair, including transthyretin, clusterin, α2-macroglobulin, and neuroserpin, and propose that investigation into the effects of these and other extracellular chaperones on neuronal repair and regeneration has the potential to yield valuable new therapies.