Liver cancer is a prevalent malignant cancer,ranking third in terms of mortality rate.Metastasis and recurrence primarily contribute to the high mortality rate of liver cancer.Hepatocellular carcinoma(HCC)has low expr...Liver cancer is a prevalent malignant cancer,ranking third in terms of mortality rate.Metastasis and recurrence primarily contribute to the high mortality rate of liver cancer.Hepatocellular carcinoma(HCC)has low expression of focal adhesion kinase(FAK),which increases the risk of metastasis and recurrence.Nevertheless,the efficacy of FAK phosphorylation inhibitors is currently limited.Thus,investigating the mechanisms by which FAK affects HCC metastasis to develop targeted therapies for FAK may present a novel strategy to inhibit HCC metastasis.This study examined the correlation between FAK expression and the prognosis of HCC.Additionally,we explored the impact of FAK degradation on HCC metastasis through wound healing experiments,transwell invasion experiments,and a xenograft tumor model.The expression of proteins related to epithelial-mesenchymal transition(EMT)was measured to elucidate the underlying mechanisms.The results showed that FAK PROTAC can degrade FAK,inhibit the migration and invasion of HCC cells in vitro,and notably decrease the lung metastasis of HCC in vivo.Increased expression of E-cadherin and decreased expression of vimentin indicated that EMT was inhibited.Consequently,degradation of FAK through FAK PROTAC effectively suppressed liver cancer metastasis,holding significant clinical implications for treating liver cancer and developing innovative anti-neoplastic drugs.展开更多
Background:Increasing understanding on the functions of amino acids (AA) has led to new commercial applications and expansion of the worldwide markets.However,the current technologies rely heavily on non-food grade mi...Background:Increasing understanding on the functions of amino acids (AA) has led to new commercial applications and expansion of the worldwide markets.However,the current technologies rely heavily on non-food grade microorganism and chemical synthesis for the production of AA.Several studies reported that lactic acid bacteria (LAB) have the capability of producing AA owing to their well-established proteolytic system and amino acid biosynthesis genes.Hence,the objectives of this study were to explore the extracellular proteolytic activity of LAB isolated from various Malaysian fermented foods and their potential to produce AA extracellularly as feed supplements.Results:All the studied LAB isolates were versatile extracellular protease producers,whereby extracellular protease activities were detected from acidic to alkaline pH (pH 5,pH 6.5,pH 8) using qualitative and quantitative proteolytic assays.The highest proteolytic activity at pH 5 (15.76 U/mg) and pH 8 (19.42 U/mg) was achieved by Lactobacillus plantarum RG14,while Lactobacillus plantarum RS5 exhibited the highest proteolytic activity of 17.22 U/mg at pH 6.5.As for the results of AA production conducted in de Man,Rogosa and Sharpe medium and analysed by high pressure liquid chromatography system,all LAB isolates were capable of producing an array of AA.Generally,Pediococcus sp.showed greater ability for AA production as compared to Lactobacillus sp.Moreover,the studied LAB were able to produce a few major feed supplement AA such as methionine,lysine,threonine and tryptophan.P.pentosaceus TL-3 recorded the highest methionine and threonine productivity of 3.72 mg/L/h and 5.58 mg/L/h respectively.However,L.plantarum I-UL4 demonstrated a lysine productivity of 1.24 mg/L/h,while P.acidilactici TP-6 achieved up to 1.73 mg/L/h of tryptophan productivity.Conclusion:All the 17 studied LAB isolates possessed versatile extracellular proteolytic system and have vast capability of producing various amino acids including a few major feed supplement AA such as methionine,lysine,threonine and tryptophan.Despite AA production was strain dependent,the studied LAB isolates possessed vast potential and can be exploited further as a bio-agent or an alternative amino acids and bioactive peptide producers.展开更多
The effect of a proteolytic starter culture isolated from Nanx Wudl,on microbiological,biochemical and organoleptic attributes of dry fermented sausages was investigated during processing.Based on preliminary screenin...The effect of a proteolytic starter culture isolated from Nanx Wudl,on microbiological,biochemical and organoleptic attributes of dry fermented sausages was investigated during processing.Based on preliminary screening,the combination of Staphylococcus xylosus SX16 and Lactobacillus plantarum CMRC6,showing excellent proteolytic activity in vitro,was selected as the multi-strain starter(starter LS).For comparison,the single-strain starter culture of L.plantarum CMRC6(starter LB)and non-inoculated control were also tested.During fermentation,lactic acid bacteria and staphylococci dominated the microbiota and suppressed the Enterobacteriaceae growth in LS-inoculated sausages.The addition of LS starter accelerated acidification and proteolysis during ripening,improving the contents of total free amino acids and several essential free amino acids(Phe,Ile and Leu).Volatile compounds analysis revealed that LS-fermented sausage showed the highest abundance of 3-methyl-1-butanol,probably due to the inoculated S.xylosus.The inoculation of LS starter improved the sensory properties of sausages,especially the flavor attribute.Therefore,S.xylosus SX16 and L.plantarum CMRC6 are promising candidates for inclusion as multi-strain starters in the manufacture of gourmet fermented dry sausage.展开更多
Afifella marina strain ME (KC205142), a purple non-sulfur bacterium was isolated from mangrove habitats of Sabah. The effects of light intensities and photoperiods on proteolytic activity in Afifella marina strain ME ...Afifella marina strain ME (KC205142), a purple non-sulfur bacterium was isolated from mangrove habitats of Sabah. The effects of light intensities and photoperiods on proteolytic activity in Afifella marina strain ME (KC205142) were investigated. Secretion of proteolytic enzymes in Afifella marina was preliminarily assessed by skim milk agarose media. Subsequently, light intensities, such as, dark, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 and 5000 lux were used to evaluate the effects on proteolytic activity in Afifella marina strain ME under anaerobic condition. After that, the effect of photoperiods on proteolytic activity was monitored under anaerobic light condition (3000 lux) at 0 h (0L/24D), 6 h (6L/18D), 12 h (12L/12D), 18 h (18L/6D) and 24 h (24L/0D) of photoperiod. The highest proteolytic activity of 74.67 U was recorded at 3000 lux illumination light intensity. The proteolytic activity in bacterium Afifella marina strain ME was positively associated with the dry cell weight. The proteolytic activity of 72.67 U in bacterium Afifella marina strain ME at 18 h (18L/6D) photoperiod is not significantly different (p > 0.05) from proteolytic activity of 74.67 U recorded at continuous light (24L/0D) condition. Light intensity of 3000 lux, culture period of 48 h and a photoperiod of 18 h (18L/ 6D) were the optimum parameters for proteolytic activity in bacterium Afifella marina strain ME.展开更多
In this study,seven coal-based activated carbons(ACs)were adopted to remove trimethylamine(TMA)in an aqueous solution as environmentally friendly and harmless adsorbents.The results showed that columnar AC(CAC)had a c...In this study,seven coal-based activated carbons(ACs)were adopted to remove trimethylamine(TMA)in an aqueous solution as environmentally friendly and harmless adsorbents.The results showed that columnar AC(CAC)had a clear scale and honeycomb structures with few fragments and micropores,contributing to superior TMA removal capacity compared to granular AC(GAC)(71.67%for 6.0 mm CAC and 69.92%for 40–60 mesh GAC).In addition,the process of adsorption was accompanied by desorption,and the recommended absorbed time was 120–180 min.The short time to achieve equilibrium indicated that adsorption was kinetically controlled,and pseudo-second-order kinetics was more appropriate than pseudo-first-order kinetics in explaining the adsorption mechanism in both water and oyster enzymatic hydrolysate.The intraparticle diffusion model presented that the adsorption processes could be divided into three steps for GAC and two steps for CAC.The adsorption processes were consistent with the Freundlich model,indicating the existence of physisorption and chemisorption as multilayer adsorption.The results indicated that AC,especially CAC,has great potential for TMA elimination in aquatic product processing.展开更多
Objective:To investigate the effect of MUC2 antisense oligodeoxynucleotide (ASODN) on cell proliferation, adhesion and proteolytic enzyme inhuman gastric carcinoma cell line (SGC7901). Methods: Phosphorothioate ...Objective:To investigate the effect of MUC2 antisense oligodeoxynucleotide (ASODN) on cell proliferation, adhesion and proteolytic enzyme inhuman gastric carcinoma cell line (SGC7901). Methods: Phosphorothioate MUC2 ASODN was synthesized and packaged by lipofectin, and then transfected to SGC7901 cells. The expression of MUC2 mRNA and protein after transfection was detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical method respectively, and the effect of MUC2 ASODN on cell proliferation, adhesion and proteolytic enzyme was determined by flow cytometry(FCM), MTT method, Rose Bengal and immunohistochemical method. Results: Compared with the blank control group, ASODN efficiently downregulated the expression of MUC2 mRNA and protein in SGC7901 cells 48h after transfection(P〈0.01). Various concentrations of ASODN could significantly inhibit the growth of SGC7901, and the inhibition peaked at the 48th hour after transfection(P〈0.05). The apoptosis rate of the experimental group was about 4.38%, and the percentage of S-phase cells rose while G0/G1-phase cells fell because most of them were blocked at S-phase. In addition, cells treated with MUC2 ASODN showed lower adhesion ability with matrix and endothelial cells than control cells in vitro(P〈0.01). By immunohistochemical method, the upregulation of E-cadherin proteins and the downregulation of MMP2 and cathepsinD proteins were also observed(P〈0.05). Conclusion: MUC2 ASODN could efficiently inhibit SGC7901 cell proliferation, reduce cell adhesion ability and downregulate the expression levels of proteolytic enzyme in vitro.展开更多
In this study, two bacilli strains namely S2-3 and S4-5, isolated from Terasi, a traditional fermented seafood product of Indonesia, were studied in terms of their phenotypic and genotypic properties. Both strains are...In this study, two bacilli strains namely S2-3 and S4-5, isolated from Terasi, a traditional fermented seafood product of Indonesia, were studied in terms of their phenotypic and genotypic properties. Both strains are of great interests due to their high proteolytic activity. Initially, they were subjected to morphological determination and a series of biochemical tests. These bacteria were Gram-positive, endospore-forming bacilli. Based on 16S rRNA gene sequence analysis, the identities of the strains S2-3 and S4-5 were confirmed as Bacillus thuringiensis and B. subtilis, respectively. Additionally, the two strains were also evaluated for their antibiogram profiles. It was found that they were susceptible to chloramphenicol, erythromycin, kanamycin, tetracycline and vancomycin and resistant to ampicillin and intermediately susceptible to bacitracin.展开更多
Summary of main observation and conclusion The SpyTag/SpyCatcher reaction is a powerful tool for bioconjugation, but it leaves a complex of considerable size after ligation. To facilitate removal of the catalytic frag...Summary of main observation and conclusion The SpyTag/SpyCatcher reaction is a powerful tool for bioconjugation, but it leaves a complex of considerable size after ligation. To facilitate removal of the catalytic fragment, proteolytic recog nit ion sites (such as DDDDK, AVLQ, and WELQ) were directly engineered into the first or second loop of SpyCatcher at locations after the reactive lysine to give a set of cleavable SpyCatcher variants. Among them, SpyCatcherDDDDK exhibits excellent reactivity with SpyTag and could still be cleaved proteolytically by enterokinase after ligation. Notably, SpyCatcherDDDDK is disordered in solution and forms an ordered complex upon reaction with SpyTag with a second order rate constant of 99.2 ± 0.1M^-1·S^-1 which is comparable to, if not faster than, most click reactions. The results demonstrate the high sequence plasticity of SpyCatcher and suggest that covalent bond formation may confer robustness on the folded structure against extensive mutation. These variants add to the expanding toolbox of genetically-encoded peptide-protein chemistry with diverse features.展开更多
Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion,growth,proliferation and differentiation.Recently,covalent and selective functionalization of glass and titanium surfaces with an...Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion,growth,proliferation and differentiation.Recently,covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide(HVP)mapped on[351e359]sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays,and to promote osseointegration in in vivo studies.For the first time to our knowledge,in this study we investigated the resistance of adhesion sequences to proteolytic digestion:HVP was completely cleaved after 5 h.In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence.A retro-inverted peptide D-2HVP,composed of D amino acids,was completely stable in serum-containing medium.In addition,glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium.Interestingly,D-2HVP increased expression of IBSP,VTN and SPP1 genes as compared to HVP functionalized surfaces.Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces.Therefore,the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.展开更多
In a number of renal disease tubular epithelial cells often display hypertrophy rather than hyperplasia. This hypertrophy, characterized by an increase in protein conten and cell size, as well as an accumulation of ex...In a number of renal disease tubular epithelial cells often display hypertrophy rather than hyperplasia. This hypertrophy, characterized by an increase in protein conten and cell size, as well as an accumulation of extracellular matrix, is a key process which may lead subsequently to tubulointerstitial fibrosis and end-stage renal failure.展开更多
The integral membrane,Kunitz-type serine protease inhibitors HAI-1 and HAI-2,can suppress the proteolytic activity of the type 2 transmembrane serine protease matriptase with high specificity and potency.High levels o...The integral membrane,Kunitz-type serine protease inhibitors HAI-1 and HAI-2,can suppress the proteolytic activity of the type 2 transmembrane serine protease matriptase with high specificity and potency.High levels of extracellular matriptase proteolytic activity have,however,been observed in some neoplastic B-cells with high levels of endogenous HAI-2,indicating that HAI-2 may be an ineffective matriptase inhibitor at the cellular level.The different effectiveness of the HAIs in the control of extracellular matriptase proteolytic activity is examined here.Upon inducing matriptase zymogen activation in the HAI Teton Daudi Burkitt lymphoma cells,which naturally express matriptase with very low levels of HAI-2 and no HAI-1,nascent active matriptase was rapidly inhibited or shed as an enzymatically active enzyme.With increasing HAI-1 expression,cellular matriptase-HAI-1 complex increased,and extracellular active matriptase decreased proportionally.Increasing HAI-2 expression,however,resulted in cellular matriptase-HAI-2 complex levels reaching a plateau,while extracellular active matriptase remained high.In contrast to this differential effect,both HAI-1 and HAI-2,even at very low levels,were shown to promote the expression and cell-surface translocation of endogenous matriptase.The difference in the suppression of extracellular active matriptase by the two closely related serine protease inhibitors could result from the primarily cell surface expression of HAI-1 compared to the mainly intracellular localization of HAI-2.The HAIs,therefore,resemble one another with respect to promoting matriptase expression and surface translocation but differ in their effectiveness in the control of extracellular matriptase enzymatic activity.展开更多
In the last decades,the role of the prion protein(PrP) in neurodegenerative diseases has been intensively investigated,initially in prion diseases of humans(e.g., Creutzfeldt-J akob disease) and animals(e.g.,scrapie i...In the last decades,the role of the prion protein(PrP) in neurodegenerative diseases has been intensively investigated,initially in prion diseases of humans(e.g., Creutzfeldt-J akob disease) and animals(e.g.,scrapie in sheep,chronic wasting disease in deer and elk,or "mad cow disease" in cattle).Templated misfolding of physiological cellular prion protein(PrPC) into an aggregation-prone isoform(termed PrP "Scrapie"(PrPSc)),self-re plication and spreading of the latter inside the brain and to peripheral tissues,and the associated formation of infectious proteopathic seeds(termed "prions")are among the essential pathogenic mechanisms underlying this group of fatal and transmissible spongiform encephalopathies.Late r,key roles of the correctly folded PrPCwere identified in more common human brain diseases(such as Alzheimer s disease or Parkinson’s disease) associated with the misfolding and/or accumulation of other proteins(such as amyloid-β,tau or α-synuclein,respectively).PrPChas also been linked with n euro protective and regenerative functions,for instance in hypoxic/ischemic conditions such as stroke.However,despite a mixed "bouquet" of suggested functions,our understanding of pathological and,especially,physiological roles played by PrPCin the brain and beyond is ce rtainly incomplete.Interactions with various other proteins at the cell surfa ce or within intracellular compartments may account for the functional diversity linked with PrPC.Moreover,conserved endogenous proteolytic processing of PrPCgenerates seve ral defined PrPCfragments,possibly holding intrinsic functions in physiological and pathological conditions,thus making the "true and complete biology" of this protein more complicated to be elucidated.Here,we focus on one of those released PrPCfragments,namely shed PrP(sPrP),generated by a membrane-proximate ADAM10-mediated cleavage event at the cell surfa ce.Similar to other soluble PrP fragments(such as the N1 fragment representing PrP’s released N-terminal tail upon the major α-cleavage event)or expe rimentally employed recombinant PrP,sPrP is being suggested to act n euro protective in Alzheimer’s disease and other protein misfolding diseases.Seve ral lines of evidence on extracellular PrPC(fragments) suggest that induction of PrPCrelease co uld be a future therapeutic option in various brain disorders.Our recent identification of a substrate-specific approach to stimulate the shedding by ADAM 10,based on ligands binding to cell surface PrPC,may further set the stage for research into this direction.展开更多
Cells of Candida albicans(C.albicans) can invade humans and may lead to mucosal and skin infections or to deep-seated my coses of almost all inner organs,especially in immunocompromised patients.In this context,both t...Cells of Candida albicans(C.albicans) can invade humans and may lead to mucosal and skin infections or to deep-seated my coses of almost all inner organs,especially in immunocompromised patients.In this context,both the host immune status and the ability of C.albicans to modulate the expression of its virulence factors are relevant aspects that drive the candidal susceptibility or resistance;in this last case,culminating in the establishment of successful infection knownas candidiasis.C.albicans possesses a potent arma-mentarium consisting of several virulence moleculesthat help the fungal cells to escape of the host immuneresponses.There is no doubt that the secretion of aspartyl-type proteases,designated as Saps,are one of the major virulence attributes produced by C.albicans cells,since these hydrolytic enzymes participate in a wide range of fungal physiological processes as well as in different facets of the fungal-host interactions.For these reasons,Saps clearly hold promise as new potential drug targets.Corroborating this hypothesis,the introduction of new anti-human immunodeficiency virus drugs of the as party l protease inhibitor-type(HIV PIs) have emerged as new agents for the inhibition of Saps.The introduction of HIV PIs has revolutionized the treatment of HIV disease,reducing opportunistic infections,especially candidiasis.The attenuation of candidal infections in HIV-infected individuals might not solely have resulted from improved immunological status,but also as a result of direct inhibition of C.albicans Saps.In this article,we review updates on the beneficial effects of HIV PIs against the human fungal pathogen C.albicans,focusing on the effects of these compounds on Sap activity,growth behavior,morphological architecture,cellular differentiation,fungal adhesion to animal cells and abiotic materials,modulation of virulence factors,experimental candidiasis infection,and their synergistic actions with classical antifungal agents.展开更多
Recently we have shown the presence of catalytically active IgGs, capable to cleave histone H1 and bovine myelin basic protein (MBP), in blood serum of SLE patients. Here we present data that demonstrate the correlati...Recently we have shown the presence of catalytically active IgGs, capable to cleave histone H1 and bovine myelin basic protein (MBP), in blood serum of SLE patients. Here we present data that demonstrate the correlation between a) proteolytic activity towards histone H1 and MBP of IgG-antibodies from blood serum of SLE patients and b) disease severity level in these patients. IgGs were isolated from blood serum by chromatography on protein G-sepharose. Commercial preparations of bovine myelin basic proteins (MBP) and calf thymus histone H1 were used as substrates. Analysis of the proteolytic activity showed that 16 of 38 lgG-preparations (42,1%) obtained from blood serum of SLE patients were capable of cleaving both histone H1 and MBP with different efficiency. It was revealed that the presence in blood serum of lgGs possessing proteolytic activity towards both histone H1 and bMBP closely correlates with manifestation of the disease severity in SLE patients.展开更多
Objective:To study the biological activities of the tissue extract of Cantharus tranquebaricus(C.tranquebaricus).Methods:Crude extract of gastropod was tested for inhibition of bacterial growth.Antibacterial assay was...Objective:To study the biological activities of the tissue extract of Cantharus tranquebaricus(C.tranquebaricus).Methods:Crude extract of gastropod was tested for inhibition of bacterial growth.Antibacterial assay was carried out by disc diffusion method and the activity was measured accordingly based on the inhibition zone around the disc impregnated with gastropod extract.Molecular weight of the extract was determined by using SDS-PAGE.Plasma coagulation,Fibrin plate assay and substrate SDS-PAGE were used to determine the effect of sample on plasma coagulation,fibrin(ogen)olytic and proteolytic;activity.Results:The maximum inhibition zone(10 mm)was observed against Vibrio cholera(V.cholera)and minimum inhibition zone(2 mm)was noticed against Proteus mirablis(P.mirablis).The molecular weight was determined as 47-106kDa.The tissue extract shows proteolytic activity above 48 kDa.SDS-PAGE analysis of fibrinogen after incubation with the tissue extract showed fibrinogenolytic activity.In plasma coagulation assay C.tranquebaricus tissue extract showed procoagulant property and it coagulated chicken plasma within 150 s,while control took 5 min to clot.The 9 HU hemolytic units were found against chicken blood and also exhibit high level of brine shrimp lethality.Conclusions:This study suggests that C.tranquebaricus could be used as potential source for isolating bioactive compounds,since it is explored first time and found with promising results.展开更多
Adeno-associated virus(AAV)is an essential instrument in the neuroscientist’s toolkit,which allows delivery of DNA to provide labeling with fluorescent proteins or genetic instructions to regulate gene expression.In ...Adeno-associated virus(AAV)is an essential instrument in the neuroscientist’s toolkit,which allows delivery of DNA to provide labeling with fluorescent proteins or genetic instructions to regulate gene expression.In the field of neural regeneration,the transduction of neurons enables the observation and regulation of axon growth and regeneration,and in the future will likely be a mechanism for delivering molecular therapies to promote sprouting and regeneration after central nervous system injury.Traditional formulations of AAV preparations permit efficient viral transduction under physiologic conditions,but an improved understanding of the mechanistic limitations of AAV transduction may facilitate production of more resilient AAV strains for investigative and therapeutic purposes.We studied AAV transduction in the context of prior exposure of AAV serotype 8(AAV8)to environmental pH within the range encountered during endosomal endocytosis(pH 7.4 to pH 4.4),during which low pH-triggered structural and autoproteolytic changes to the viral capsid are believed to be necessary for endosome escape and virus uncoating.Due to the fundamental nature of these processes,we hypothesized that premature exposure of AAV8 particles to acidic pH would decrease viral transduction of HT1080 cells in vitro,as measured by fluorescent reporter gene expression using high-content imaging analysis.We found that increasingly acidic incubation conditions were associated with concomitant reductions in transduction efficiency,and that quantitative levels of reporter gene expression in transduced cells were similarly decreased.The biggest decrease in transduction occurred between pH 7.4 and pH 6.4,suggesting the possible co-occurrence of a pH-associated event and viral inactivation within that range.Taken together,these findings indicate that exposure of AAV8 to acidic pH for as little as 1 hour is deleterious to transduction ability.Future studies are necessary to understand the pH-associated causative mechanisms involved.This study was approved by the University of Miami Institutional Animal Care and Use Committee,USA(Protocol#18-108-LF)on July 12,2018.展开更多
A kind of macroporous bead carrier of copolymer containing monomer units of N-aminoethyl acrylamide and vinylalcohol was synthesized, i.e. the MR-AA carrier. Papain was immobilized on the carrier using glutaraldehyde ...A kind of macroporous bead carrier of copolymer containing monomer units of N-aminoethyl acrylamide and vinylalcohol was synthesized, i.e. the MR-AA carrier. Papain was immobilized on the carrier using glutaraldehyde as the couplingagent. The enzymatic activity of the immobilized papain was compared with free papain using casein as a substrate, and theeffects of glutaraldehyde concentration, pH, temperature, time and papain amount added on the activity recovery were alsoinvestigated. The results show that the MR-AA carrier contains reactive primary amine groups, hydrophilic amido links andhydroxyl groups, as well as macroporous structures based on its matrix (MR-AV matrix), furthermore, the activity recoveryof papain in the immobilization could reach 48%/~58%. In comparison with free papain, the resulting immobilized papainexhibits a remarkable thermostability and better reusability.展开更多
The Synechocystis sp. PCC 6803 genome harbours a Deg gene family consisting of three members, htrA (degP, slr1204), hhoA (degQ, sll1679) and hhoB (degS, sll1427). This work provided biochemical characterization ...The Synechocystis sp. PCC 6803 genome harbours a Deg gene family consisting of three members, htrA (degP, slr1204), hhoA (degQ, sll1679) and hhoB (degS, sll1427). This work provided biochemical characterization of HhoA, HtrA and HhoB from Synechocystis sp. PCC 6803. Firstly mature HhoA, HhoB and HtrA from Synechocystis sp. PCC 6803 were cloned and expressed as soluble recombinant his-tagged fusion protein in Escherichia coli. Then the proteolytic activity of HhoA, HhoB and HtrA was tested using casein, bovine serum albumin, five recombinant chromoproteins and cyanobacterial phycocyanin as substrates in vitro. The experimental results showed that HhoA and HtrA had proteolytic activity on casein, five recombinant chromoproteins and cyanobacterial phycocyanin. No proteolytic activity of HhoB was found using all substrates in vitro, indicating functional difference among Deg proteases from Synechocystis sp. PCC 6803. Therefore, the results indicated the biochemical properties of HhoA and HtrA on hydrolysis of proteins and phycobiliproteins in vitro, which implicated that they were proteases possibly involved in phycobiliprotein degradation in vivo.展开更多
Crude enzyme extracts were prepared from leaves and stems of Linn. (Fabaceae) from Cameroon under optimized conditions. Proteolytic enzymes were precipitated with ammonium sulfate at 35% (w/v) saturation and assayed f...Crude enzyme extracts were prepared from leaves and stems of Linn. (Fabaceae) from Cameroon under optimized conditions. Proteolytic enzymes were precipitated with ammonium sulfate at 35% (w/v) saturation and assayed for enzyme activity. The effects of temperature, pH, incubation time and substrate specificity were studied. SDS-PAGE was used to determine molecular weight of precipitated protease. Results indicated that proteolytic activity of crude extract was 35.20 U/ml compared to 51.03 U/ml of partial purified extract. The optimum enzyme activity was found to be at 40°C, while 50% of activity was maintained at 60°C after 60 min incubation. Partial purified crude extract exhibited two optimum pH (2.75 and 9.0). The highest enzyme activity towards Bovine Serum Albumine (25.9 U/ml) was noted. SDS-PAGE gels exhibited molecular weight between 40 - 60 KDa. This result confirms that partial purified extract of A. precatorius contains proteases and could be a promising source for proteolytic enzyme extraction.展开更多
Brain-derived neurotrophic factor (BDNF) is a neurotrophin that elicits neuronal survival and differentiation, synaptic transmission, and the modulation of synaptic plasticity. The biological actions of BDNF are media...Brain-derived neurotrophic factor (BDNF) is a neurotrophin that elicits neuronal survival and differentiation, synaptic transmission, and the modulation of synaptic plasticity. The biological actions of BDNF are mediated via two distinct receptors: the high-affinity tropomyosin-related kinase B (TrkB) receptor and the low-affinity p75 neurotrophin receptor (p75NTR). Recent findings regarding the actions and mechanisms of BDNF are reviewed here. Activity-dependent synaptic plasticity, as exemplified by long-term potentiation (LTP) and long-term depression (LTD), underlies the cellular mechanism of learning and memory. An accumulating body of evidence shows that BDNF modulates synaptic plasticity. This function requires extracellular neurotrophin release, synaptic activity-dependent local protein synthesis. In addition, a precursor of BDNF, proBDNF, is emerging as a new ligand with biological activities that are distinct from those of BDNF. The proteolytic cleavage of proBDNF is also proposed as a mechanism that determines the direction of BDNF actions. This review discusses the post-translational processing of proBDNF, the modulatory roles of the human BDNF polymorphism Val66Met, recent reports of the novel mechanisms of BDNF expression, and clinical reports showing the roles of BDNF in the blood. Taken together, these data provide new insights into the biological roles of BDNF and its related molecules in the central nervous system.展开更多
基金supported by the National Natural Science Foundation of China Fund Project(82272956).
文摘Liver cancer is a prevalent malignant cancer,ranking third in terms of mortality rate.Metastasis and recurrence primarily contribute to the high mortality rate of liver cancer.Hepatocellular carcinoma(HCC)has low expression of focal adhesion kinase(FAK),which increases the risk of metastasis and recurrence.Nevertheless,the efficacy of FAK phosphorylation inhibitors is currently limited.Thus,investigating the mechanisms by which FAK affects HCC metastasis to develop targeted therapies for FAK may present a novel strategy to inhibit HCC metastasis.This study examined the correlation between FAK expression and the prognosis of HCC.Additionally,we explored the impact of FAK degradation on HCC metastasis through wound healing experiments,transwell invasion experiments,and a xenograft tumor model.The expression of proteins related to epithelial-mesenchymal transition(EMT)was measured to elucidate the underlying mechanisms.The results showed that FAK PROTAC can degrade FAK,inhibit the migration and invasion of HCC cells in vitro,and notably decrease the lung metastasis of HCC in vivo.Increased expression of E-cadherin and decreased expression of vimentin indicated that EMT was inhibited.Consequently,degradation of FAK through FAK PROTAC effectively suppressed liver cancer metastasis,holding significant clinical implications for treating liver cancer and developing innovative anti-neoplastic drugs.
基金The Long-Term Research Grant(LRGS)of the Ministry of Education of Malaysia supported this work
文摘Background:Increasing understanding on the functions of amino acids (AA) has led to new commercial applications and expansion of the worldwide markets.However,the current technologies rely heavily on non-food grade microorganism and chemical synthesis for the production of AA.Several studies reported that lactic acid bacteria (LAB) have the capability of producing AA owing to their well-established proteolytic system and amino acid biosynthesis genes.Hence,the objectives of this study were to explore the extracellular proteolytic activity of LAB isolated from various Malaysian fermented foods and their potential to produce AA extracellularly as feed supplements.Results:All the studied LAB isolates were versatile extracellular protease producers,whereby extracellular protease activities were detected from acidic to alkaline pH (pH 5,pH 6.5,pH 8) using qualitative and quantitative proteolytic assays.The highest proteolytic activity at pH 5 (15.76 U/mg) and pH 8 (19.42 U/mg) was achieved by Lactobacillus plantarum RG14,while Lactobacillus plantarum RS5 exhibited the highest proteolytic activity of 17.22 U/mg at pH 6.5.As for the results of AA production conducted in de Man,Rogosa and Sharpe medium and analysed by high pressure liquid chromatography system,all LAB isolates were capable of producing an array of AA.Generally,Pediococcus sp.showed greater ability for AA production as compared to Lactobacillus sp.Moreover,the studied LAB were able to produce a few major feed supplement AA such as methionine,lysine,threonine and tryptophan.P.pentosaceus TL-3 recorded the highest methionine and threonine productivity of 3.72 mg/L/h and 5.58 mg/L/h respectively.However,L.plantarum I-UL4 demonstrated a lysine productivity of 1.24 mg/L/h,while P.acidilactici TP-6 achieved up to 1.73 mg/L/h of tryptophan productivity.Conclusion:All the 17 studied LAB isolates possessed versatile extracellular proteolytic system and have vast capability of producing various amino acids including a few major feed supplement AA such as methionine,lysine,threonine and tryptophan.Despite AA production was strain dependent,the studied LAB isolates possessed vast potential and can be exploited further as a bio-agent or an alternative amino acids and bioactive peptide producers.
基金the National Key R&D Program of China(grant no.2018YFD0400404).
文摘The effect of a proteolytic starter culture isolated from Nanx Wudl,on microbiological,biochemical and organoleptic attributes of dry fermented sausages was investigated during processing.Based on preliminary screening,the combination of Staphylococcus xylosus SX16 and Lactobacillus plantarum CMRC6,showing excellent proteolytic activity in vitro,was selected as the multi-strain starter(starter LS).For comparison,the single-strain starter culture of L.plantarum CMRC6(starter LB)and non-inoculated control were also tested.During fermentation,lactic acid bacteria and staphylococci dominated the microbiota and suppressed the Enterobacteriaceae growth in LS-inoculated sausages.The addition of LS starter accelerated acidification and proteolysis during ripening,improving the contents of total free amino acids and several essential free amino acids(Phe,Ile and Leu).Volatile compounds analysis revealed that LS-fermented sausage showed the highest abundance of 3-methyl-1-butanol,probably due to the inoculated S.xylosus.The inoculation of LS starter improved the sensory properties of sausages,especially the flavor attribute.Therefore,S.xylosus SX16 and L.plantarum CMRC6 are promising candidates for inclusion as multi-strain starters in the manufacture of gourmet fermented dry sausage.
文摘Afifella marina strain ME (KC205142), a purple non-sulfur bacterium was isolated from mangrove habitats of Sabah. The effects of light intensities and photoperiods on proteolytic activity in Afifella marina strain ME (KC205142) were investigated. Secretion of proteolytic enzymes in Afifella marina was preliminarily assessed by skim milk agarose media. Subsequently, light intensities, such as, dark, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 and 5000 lux were used to evaluate the effects on proteolytic activity in Afifella marina strain ME under anaerobic condition. After that, the effect of photoperiods on proteolytic activity was monitored under anaerobic light condition (3000 lux) at 0 h (0L/24D), 6 h (6L/18D), 12 h (12L/12D), 18 h (18L/6D) and 24 h (24L/0D) of photoperiod. The highest proteolytic activity of 74.67 U was recorded at 3000 lux illumination light intensity. The proteolytic activity in bacterium Afifella marina strain ME was positively associated with the dry cell weight. The proteolytic activity of 72.67 U in bacterium Afifella marina strain ME at 18 h (18L/6D) photoperiod is not significantly different (p > 0.05) from proteolytic activity of 74.67 U recorded at continuous light (24L/0D) condition. Light intensity of 3000 lux, culture period of 48 h and a photoperiod of 18 h (18L/ 6D) were the optimum parameters for proteolytic activity in bacterium Afifella marina strain ME.
基金the National Key R&D Program of China(No.2018YFC0311202)the Key-Area Research and Development Program of Guang-dong Province(No.2020B1111030004)+4 种基金the Science and Technology Program of Guangzhou,China(Nos.201804010364 and 201804010321)the Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory(Guangzhou)(No.GML2019ZD0406)the National Key R&D Program of China(No.2018YFC0311202)the Natural Science Foun-dation of Guangdong Province,China(Nos.2018A030313088,2018A030313626)the Academician Work-station Foundation for Young Scientists of Chinese Aca-demy of Sciences Guangzhou Branch(No.20180313).
文摘In this study,seven coal-based activated carbons(ACs)were adopted to remove trimethylamine(TMA)in an aqueous solution as environmentally friendly and harmless adsorbents.The results showed that columnar AC(CAC)had a clear scale and honeycomb structures with few fragments and micropores,contributing to superior TMA removal capacity compared to granular AC(GAC)(71.67%for 6.0 mm CAC and 69.92%for 40–60 mesh GAC).In addition,the process of adsorption was accompanied by desorption,and the recommended absorbed time was 120–180 min.The short time to achieve equilibrium indicated that adsorption was kinetically controlled,and pseudo-second-order kinetics was more appropriate than pseudo-first-order kinetics in explaining the adsorption mechanism in both water and oyster enzymatic hydrolysate.The intraparticle diffusion model presented that the adsorption processes could be divided into three steps for GAC and two steps for CAC.The adsorption processes were consistent with the Freundlich model,indicating the existence of physisorption and chemisorption as multilayer adsorption.The results indicated that AC,especially CAC,has great potential for TMA elimination in aquatic product processing.
基金This work was supported by the Science Research Foundation of the Health Bureau of Chongqing Municipality(No.2000-48)
文摘Objective:To investigate the effect of MUC2 antisense oligodeoxynucleotide (ASODN) on cell proliferation, adhesion and proteolytic enzyme inhuman gastric carcinoma cell line (SGC7901). Methods: Phosphorothioate MUC2 ASODN was synthesized and packaged by lipofectin, and then transfected to SGC7901 cells. The expression of MUC2 mRNA and protein after transfection was detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical method respectively, and the effect of MUC2 ASODN on cell proliferation, adhesion and proteolytic enzyme was determined by flow cytometry(FCM), MTT method, Rose Bengal and immunohistochemical method. Results: Compared with the blank control group, ASODN efficiently downregulated the expression of MUC2 mRNA and protein in SGC7901 cells 48h after transfection(P〈0.01). Various concentrations of ASODN could significantly inhibit the growth of SGC7901, and the inhibition peaked at the 48th hour after transfection(P〈0.05). The apoptosis rate of the experimental group was about 4.38%, and the percentage of S-phase cells rose while G0/G1-phase cells fell because most of them were blocked at S-phase. In addition, cells treated with MUC2 ASODN showed lower adhesion ability with matrix and endothelial cells than control cells in vitro(P〈0.01). By immunohistochemical method, the upregulation of E-cadherin proteins and the downregulation of MMP2 and cathepsinD proteins were also observed(P〈0.05). Conclusion: MUC2 ASODN could efficiently inhibit SGC7901 cell proliferation, reduce cell adhesion ability and downregulate the expression levels of proteolytic enzyme in vitro.
基金Supported by Mae Fah Luang University(MFU)(57101010027)
文摘In this study, two bacilli strains namely S2-3 and S4-5, isolated from Terasi, a traditional fermented seafood product of Indonesia, were studied in terms of their phenotypic and genotypic properties. Both strains are of great interests due to their high proteolytic activity. Initially, they were subjected to morphological determination and a series of biochemical tests. These bacteria were Gram-positive, endospore-forming bacilli. Based on 16S rRNA gene sequence analysis, the identities of the strains S2-3 and S4-5 were confirmed as Bacillus thuringiensis and B. subtilis, respectively. Additionally, the two strains were also evaluated for their antibiogram profiles. It was found that they were susceptible to chloramphenicol, erythromycin, kanamycin, tetracycline and vancomycin and resistant to ampicillin and intermediately susceptible to bacitracin.
基金the financial support from the National Natural Science Foundation of China (Grants 21474003, 91427304)1000 Plan (Youth), the Open Project of State Key Laboratory of Supramolecular Structure and Materials of Jilin University (Grant No. sklssm201834), and the Interdisciplinary Medicine Seed Fund of Peking University (Grant No. BMU2018MC001).
文摘Summary of main observation and conclusion The SpyTag/SpyCatcher reaction is a powerful tool for bioconjugation, but it leaves a complex of considerable size after ligation. To facilitate removal of the catalytic fragment, proteolytic recog nit ion sites (such as DDDDK, AVLQ, and WELQ) were directly engineered into the first or second loop of SpyCatcher at locations after the reactive lysine to give a set of cleavable SpyCatcher variants. Among them, SpyCatcherDDDDK exhibits excellent reactivity with SpyTag and could still be cleaved proteolytically by enterokinase after ligation. Notably, SpyCatcherDDDDK is disordered in solution and forms an ordered complex upon reaction with SpyTag with a second order rate constant of 99.2 ± 0.1M^-1·S^-1 which is comparable to, if not faster than, most click reactions. The results demonstrate the high sequence plasticity of SpyCatcher and suggest that covalent bond formation may confer robustness on the folded structure against extensive mutation. These variants add to the expanding toolbox of genetically-encoded peptide-protein chemistry with diverse features.
文摘Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion,growth,proliferation and differentiation.Recently,covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide(HVP)mapped on[351e359]sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays,and to promote osseointegration in in vivo studies.For the first time to our knowledge,in this study we investigated the resistance of adhesion sequences to proteolytic digestion:HVP was completely cleaved after 5 h.In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence.A retro-inverted peptide D-2HVP,composed of D amino acids,was completely stable in serum-containing medium.In addition,glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium.Interestingly,D-2HVP increased expression of IBSP,VTN and SPP1 genes as compared to HVP functionalized surfaces.Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces.Therefore,the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.
文摘In a number of renal disease tubular epithelial cells often display hypertrophy rather than hyperplasia. This hypertrophy, characterized by an increase in protein conten and cell size, as well as an accumulation of extracellular matrix, is a key process which may lead subsequently to tubulointerstitial fibrosis and end-stage renal failure.
基金This study was supported by Cancer Institute Grant R01 CA 123223(to MDJ and CYL)Grant(MAB-108-079)from the Ministry of Defense Medical Affairs Bureau+2 种基金Grants(CMNDMC10705,CMNDMC10813)from Chi-Mei Medical Center(to J.-K.Wang)Grant(TSGH-E-109213-003)from Tri-Service General Hospital(to Y.-Y.Wu)Grants(108-2311-B-016-001-,109-2320-B-016-004-)from Ministry of Science and Technology(to Y.-L.Chiu).
文摘The integral membrane,Kunitz-type serine protease inhibitors HAI-1 and HAI-2,can suppress the proteolytic activity of the type 2 transmembrane serine protease matriptase with high specificity and potency.High levels of extracellular matriptase proteolytic activity have,however,been observed in some neoplastic B-cells with high levels of endogenous HAI-2,indicating that HAI-2 may be an ineffective matriptase inhibitor at the cellular level.The different effectiveness of the HAIs in the control of extracellular matriptase proteolytic activity is examined here.Upon inducing matriptase zymogen activation in the HAI Teton Daudi Burkitt lymphoma cells,which naturally express matriptase with very low levels of HAI-2 and no HAI-1,nascent active matriptase was rapidly inhibited or shed as an enzymatically active enzyme.With increasing HAI-1 expression,cellular matriptase-HAI-1 complex increased,and extracellular active matriptase decreased proportionally.Increasing HAI-2 expression,however,resulted in cellular matriptase-HAI-2 complex levels reaching a plateau,while extracellular active matriptase remained high.In contrast to this differential effect,both HAI-1 and HAI-2,even at very low levels,were shown to promote the expression and cell-surface translocation of endogenous matriptase.The difference in the suppression of extracellular active matriptase by the two closely related serine protease inhibitors could result from the primarily cell surface expression of HAI-1 compared to the mainly intracellular localization of HAI-2.The HAIs,therefore,resemble one another with respect to promoting matriptase expression and surface translocation but differ in their effectiveness in the control of extracellular matriptase enzymatic activity.
基金supported by funding from the Creutzfeldt-Jakob Disease FoundationInc.(USA)+4 种基金the Alzheimer Forschung Initiative (AFI e.V.,Germany)the Werner-Otto-Stiftung (Hamburg,Germany)(all to HCA)the China Scholarship Council (to FS)European Union’s Horizon 2020 Research and Innovation Program under the Marie Sklodowska-Curie Grant Agreement N°101030402 (to AMA)Deutsche Forschungsgemeinschaft (DFG) Collaborative Research Center (CRC) 877"Proteolysis as a regulatory event in pathophysiology"(to MG)。
文摘In the last decades,the role of the prion protein(PrP) in neurodegenerative diseases has been intensively investigated,initially in prion diseases of humans(e.g., Creutzfeldt-J akob disease) and animals(e.g.,scrapie in sheep,chronic wasting disease in deer and elk,or "mad cow disease" in cattle).Templated misfolding of physiological cellular prion protein(PrPC) into an aggregation-prone isoform(termed PrP "Scrapie"(PrPSc)),self-re plication and spreading of the latter inside the brain and to peripheral tissues,and the associated formation of infectious proteopathic seeds(termed "prions")are among the essential pathogenic mechanisms underlying this group of fatal and transmissible spongiform encephalopathies.Late r,key roles of the correctly folded PrPCwere identified in more common human brain diseases(such as Alzheimer s disease or Parkinson’s disease) associated with the misfolding and/or accumulation of other proteins(such as amyloid-β,tau or α-synuclein,respectively).PrPChas also been linked with n euro protective and regenerative functions,for instance in hypoxic/ischemic conditions such as stroke.However,despite a mixed "bouquet" of suggested functions,our understanding of pathological and,especially,physiological roles played by PrPCin the brain and beyond is ce rtainly incomplete.Interactions with various other proteins at the cell surfa ce or within intracellular compartments may account for the functional diversity linked with PrPC.Moreover,conserved endogenous proteolytic processing of PrPCgenerates seve ral defined PrPCfragments,possibly holding intrinsic functions in physiological and pathological conditions,thus making the "true and complete biology" of this protein more complicated to be elucidated.Here,we focus on one of those released PrPCfragments,namely shed PrP(sPrP),generated by a membrane-proximate ADAM10-mediated cleavage event at the cell surfa ce.Similar to other soluble PrP fragments(such as the N1 fragment representing PrP’s released N-terminal tail upon the major α-cleavage event)or expe rimentally employed recombinant PrP,sPrP is being suggested to act n euro protective in Alzheimer’s disease and other protein misfolding diseases.Seve ral lines of evidence on extracellular PrPC(fragments) suggest that induction of PrPCrelease co uld be a future therapeutic option in various brain disorders.Our recent identification of a substrate-specific approach to stimulate the shedding by ADAM 10,based on ligands binding to cell surface PrPC,may further set the stage for research into this direction.
基金Supported by Grants from the Brazilian Agencies:CNPq(Conselho Nacional de Desenvolvimento Científico e Tecnológico)CAPES(Coordenao de Aperfeioamento Pessoalde Nível Superior)+1 种基金FAPERJ(Fundao Carlos Chagas Filhode Amparoà Pesquisa do Estado do Rio de Janeiro)supported by a CNPq fellowship
文摘Cells of Candida albicans(C.albicans) can invade humans and may lead to mucosal and skin infections or to deep-seated my coses of almost all inner organs,especially in immunocompromised patients.In this context,both the host immune status and the ability of C.albicans to modulate the expression of its virulence factors are relevant aspects that drive the candidal susceptibility or resistance;in this last case,culminating in the establishment of successful infection knownas candidiasis.C.albicans possesses a potent arma-mentarium consisting of several virulence moleculesthat help the fungal cells to escape of the host immuneresponses.There is no doubt that the secretion of aspartyl-type proteases,designated as Saps,are one of the major virulence attributes produced by C.albicans cells,since these hydrolytic enzymes participate in a wide range of fungal physiological processes as well as in different facets of the fungal-host interactions.For these reasons,Saps clearly hold promise as new potential drug targets.Corroborating this hypothesis,the introduction of new anti-human immunodeficiency virus drugs of the as party l protease inhibitor-type(HIV PIs) have emerged as new agents for the inhibition of Saps.The introduction of HIV PIs has revolutionized the treatment of HIV disease,reducing opportunistic infections,especially candidiasis.The attenuation of candidal infections in HIV-infected individuals might not solely have resulted from improved immunological status,but also as a result of direct inhibition of C.albicans Saps.In this article,we review updates on the beneficial effects of HIV PIs against the human fungal pathogen C.albicans,focusing on the effects of these compounds on Sap activity,growth behavior,morphological architecture,cellular differentiation,fungal adhesion to animal cells and abiotic materials,modulation of virulence factors,experimental candidiasis infection,and their synergistic actions with classical antifungal agents.
文摘Recently we have shown the presence of catalytically active IgGs, capable to cleave histone H1 and bovine myelin basic protein (MBP), in blood serum of SLE patients. Here we present data that demonstrate the correlation between a) proteolytic activity towards histone H1 and MBP of IgG-antibodies from blood serum of SLE patients and b) disease severity level in these patients. IgGs were isolated from blood serum by chromatography on protein G-sepharose. Commercial preparations of bovine myelin basic proteins (MBP) and calf thymus histone H1 were used as substrates. Analysis of the proteolytic activity showed that 16 of 38 lgG-preparations (42,1%) obtained from blood serum of SLE patients were capable of cleaving both histone H1 and MBP with different efficiency. It was revealed that the presence in blood serum of lgGs possessing proteolytic activity towards both histone H1 and bMBP closely correlates with manifestation of the disease severity in SLE patients.
基金grateful to UGC[G4/9493]and MoES[G4/12319]for providing financial support
文摘Objective:To study the biological activities of the tissue extract of Cantharus tranquebaricus(C.tranquebaricus).Methods:Crude extract of gastropod was tested for inhibition of bacterial growth.Antibacterial assay was carried out by disc diffusion method and the activity was measured accordingly based on the inhibition zone around the disc impregnated with gastropod extract.Molecular weight of the extract was determined by using SDS-PAGE.Plasma coagulation,Fibrin plate assay and substrate SDS-PAGE were used to determine the effect of sample on plasma coagulation,fibrin(ogen)olytic and proteolytic;activity.Results:The maximum inhibition zone(10 mm)was observed against Vibrio cholera(V.cholera)and minimum inhibition zone(2 mm)was noticed against Proteus mirablis(P.mirablis).The molecular weight was determined as 47-106kDa.The tissue extract shows proteolytic activity above 48 kDa.SDS-PAGE analysis of fibrinogen after incubation with the tissue extract showed fibrinogenolytic activity.In plasma coagulation assay C.tranquebaricus tissue extract showed procoagulant property and it coagulated chicken plasma within 150 s,while control took 5 min to clot.The 9 HU hemolytic units were found against chicken blood and also exhibit high level of brine shrimp lethality.Conclusions:This study suggests that C.tranquebaricus could be used as potential source for isolating bioactive compounds,since it is explored first time and found with promising results.
基金This work was supported by grants to JLB and VPL from the National Institutes of Health(NS100531)the Craig H.Neilsen Foundation(598684)the Miami Project to Cure Paralysis.VPL holds the Walter G.Ross Distinguished Chair in Developmental Neuroscience.
文摘Adeno-associated virus(AAV)is an essential instrument in the neuroscientist’s toolkit,which allows delivery of DNA to provide labeling with fluorescent proteins or genetic instructions to regulate gene expression.In the field of neural regeneration,the transduction of neurons enables the observation and regulation of axon growth and regeneration,and in the future will likely be a mechanism for delivering molecular therapies to promote sprouting and regeneration after central nervous system injury.Traditional formulations of AAV preparations permit efficient viral transduction under physiologic conditions,but an improved understanding of the mechanistic limitations of AAV transduction may facilitate production of more resilient AAV strains for investigative and therapeutic purposes.We studied AAV transduction in the context of prior exposure of AAV serotype 8(AAV8)to environmental pH within the range encountered during endosomal endocytosis(pH 7.4 to pH 4.4),during which low pH-triggered structural and autoproteolytic changes to the viral capsid are believed to be necessary for endosome escape and virus uncoating.Due to the fundamental nature of these processes,we hypothesized that premature exposure of AAV8 particles to acidic pH would decrease viral transduction of HT1080 cells in vitro,as measured by fluorescent reporter gene expression using high-content imaging analysis.We found that increasingly acidic incubation conditions were associated with concomitant reductions in transduction efficiency,and that quantitative levels of reporter gene expression in transduced cells were similarly decreased.The biggest decrease in transduction occurred between pH 7.4 and pH 6.4,suggesting the possible co-occurrence of a pH-associated event and viral inactivation within that range.Taken together,these findings indicate that exposure of AAV8 to acidic pH for as little as 1 hour is deleterious to transduction ability.Future studies are necessary to understand the pH-associated causative mechanisms involved.This study was approved by the University of Miami Institutional Animal Care and Use Committee,USA(Protocol#18-108-LF)on July 12,2018.
基金This work was supported by the Lanzhou University Foundation for Middle-aged & Yonger Specialists (LD810718).
文摘A kind of macroporous bead carrier of copolymer containing monomer units of N-aminoethyl acrylamide and vinylalcohol was synthesized, i.e. the MR-AA carrier. Papain was immobilized on the carrier using glutaraldehyde as the couplingagent. The enzymatic activity of the immobilized papain was compared with free papain using casein as a substrate, and theeffects of glutaraldehyde concentration, pH, temperature, time and papain amount added on the activity recovery were alsoinvestigated. The results show that the MR-AA carrier contains reactive primary amine groups, hydrophilic amido links andhydroxyl groups, as well as macroporous structures based on its matrix (MR-AV matrix), furthermore, the activity recoveryof papain in the immobilization could reach 48%/~58%. In comparison with free papain, the resulting immobilized papainexhibits a remarkable thermostability and better reusability.
基金Funded by the National Natural Science Foundation of China (Nos.30870541,30870519)
文摘The Synechocystis sp. PCC 6803 genome harbours a Deg gene family consisting of three members, htrA (degP, slr1204), hhoA (degQ, sll1679) and hhoB (degS, sll1427). This work provided biochemical characterization of HhoA, HtrA and HhoB from Synechocystis sp. PCC 6803. Firstly mature HhoA, HhoB and HtrA from Synechocystis sp. PCC 6803 were cloned and expressed as soluble recombinant his-tagged fusion protein in Escherichia coli. Then the proteolytic activity of HhoA, HhoB and HtrA was tested using casein, bovine serum albumin, five recombinant chromoproteins and cyanobacterial phycocyanin as substrates in vitro. The experimental results showed that HhoA and HtrA had proteolytic activity on casein, five recombinant chromoproteins and cyanobacterial phycocyanin. No proteolytic activity of HhoB was found using all substrates in vitro, indicating functional difference among Deg proteases from Synechocystis sp. PCC 6803. Therefore, the results indicated the biochemical properties of HhoA and HtrA on hydrolysis of proteins and phycobiliproteins in vitro, which implicated that they were proteases possibly involved in phycobiliprotein degradation in vivo.
文摘Crude enzyme extracts were prepared from leaves and stems of Linn. (Fabaceae) from Cameroon under optimized conditions. Proteolytic enzymes were precipitated with ammonium sulfate at 35% (w/v) saturation and assayed for enzyme activity. The effects of temperature, pH, incubation time and substrate specificity were studied. SDS-PAGE was used to determine molecular weight of precipitated protease. Results indicated that proteolytic activity of crude extract was 35.20 U/ml compared to 51.03 U/ml of partial purified extract. The optimum enzyme activity was found to be at 40°C, while 50% of activity was maintained at 60°C after 60 min incubation. Partial purified crude extract exhibited two optimum pH (2.75 and 9.0). The highest enzyme activity towards Bovine Serum Albumine (25.9 U/ml) was noted. SDS-PAGE gels exhibited molecular weight between 40 - 60 KDa. This result confirms that partial purified extract of A. precatorius contains proteases and could be a promising source for proteolytic enzyme extraction.
文摘Brain-derived neurotrophic factor (BDNF) is a neurotrophin that elicits neuronal survival and differentiation, synaptic transmission, and the modulation of synaptic plasticity. The biological actions of BDNF are mediated via two distinct receptors: the high-affinity tropomyosin-related kinase B (TrkB) receptor and the low-affinity p75 neurotrophin receptor (p75NTR). Recent findings regarding the actions and mechanisms of BDNF are reviewed here. Activity-dependent synaptic plasticity, as exemplified by long-term potentiation (LTP) and long-term depression (LTD), underlies the cellular mechanism of learning and memory. An accumulating body of evidence shows that BDNF modulates synaptic plasticity. This function requires extracellular neurotrophin release, synaptic activity-dependent local protein synthesis. In addition, a precursor of BDNF, proBDNF, is emerging as a new ligand with biological activities that are distinct from those of BDNF. The proteolytic cleavage of proBDNF is also proposed as a mechanism that determines the direction of BDNF actions. This review discusses the post-translational processing of proBDNF, the modulatory roles of the human BDNF polymorphism Val66Met, recent reports of the novel mechanisms of BDNF expression, and clinical reports showing the roles of BDNF in the blood. Taken together, these data provide new insights into the biological roles of BDNF and its related molecules in the central nervous system.