The aim of the present study was to determine the MICs using broth microdilution method and their combination effect by （minimum inhibitory concentrations） of selected antibiotic agents determination of FICindex （f...The aim of the present study was to determine the MICs using broth microdilution method and their combination effect by （minimum inhibitory concentrations） of selected antibiotic agents determination of FICindex （fractional inhibitory concentrations）. Lysozyme Anodonta cygnea was more active than Lysozyme Uniopictorum, especially against Gram-negative bacterias. Combination of Macropen and Lysozyme Unio pictorum has a synergistic effect, while a separation of these antibacterial agents was not active against some of the strains of microorganisms.展开更多
The purpose of this research is to investigate the effects of the variously sulfated chitosans on lysozyme activity and structure. It was shown that the specific enzymatic activity of lysozyme remained almost similar ...The purpose of this research is to investigate the effects of the variously sulfated chitosans on lysozyme activity and structure. It was shown that the specific enzymatic activity of lysozyme remained almost similar to the native protein after being bound to 6-O-sulfated chitosan （6S-chitosan） and 3,6-O-sulfated chitosan （3,6S-chitosan）, but decreased greatly after being bound to 2-N-6-O-sulfated chitosan （2,6S-chitosan）. Meanwhile, among these sulfated chitosans, 2,6S-chitosan induced the greatest conformational change in lysozyme as indicated by the fluorescence spectra. These findings demonstrated that when sulfated chitosans of different structures bind to lysozyme, lysozyme undergoes conformational change of different magnitudes, which results in corresponding levels of lysozyme activity. Further study on the interaction of sulfated chitosans with lysozyme by surface plasmon resonance （SPR） suggested that their affinities might be determined by their molecular structures.展开更多
The aggregation interaction between reduced-denatured egg white lysozymes during refolding procedure in urea solution was studied by means of reducing and non-reducing protein electrophoreses. Results of non-reducing ...The aggregation interaction between reduced-denatured egg white lysozymes during refolding procedure in urea solution was studied by means of reducing and non-reducing protein electrophoreses. Results of non-reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis （SDS-PAGE） of the supernatant and aggregate precipitate formed in refolding process show that except being refolded to native egg white lysozymes, the reduced-denatured lysozymes can also form the aggregates with molecular weights （MW） being separately about 30.0 and 35.0 kD, while the reducing SDS-PAGE and the refolding results in the presence of sodium dodecyl sulphate show that these aggregates are formed chiefly through the misconnection of disulfide bonds between the reduced-denatured lysozymes, and the aggregate precipitates are formed through the non-covalent interactions between the aggregates with molecular weight being about 30.0 kD. From the results of electrophoresis and size-exclusion chromatographic analyses, it can be inferred that the aggregates with molecular weights being about 30.0 and 35.0 kD are bi-molecular and tri-molecular egg white lysozyme aggregates, respectively. And finally, a suggested refolding mechanism of reduced-denatured egg white lysozymes in urea solution was presented.展开更多
Lysozyme(EC3.2.1.17)plays an important role in the immune response;as a nonspecific immune factor,it can resist causative agents.Lysozyme can be divided into c-type and g-type in fish.In a previous study,through genom...Lysozyme(EC3.2.1.17)plays an important role in the immune response;as a nonspecific immune factor,it can resist causative agents.Lysozyme can be divided into c-type and g-type in fish.In a previous study,through genome-wide association analysis,the g-type lysozyme gene,which is named NaLyg in yellow drum(Nibea albiflora),was found to be a key candidate gene for disease resistance in response to Vibrio harveyi infection.The cDNA of NaLyg was 1025 bp,including four exons and three introns,and its open reading frame(ORF)had a full-length of 582 bp,encoding 193 amino acids.NaLyg was found to be conserved during evolution through bioinformatic analyses.The NaLyg protein possessed a sugar binding domain and three catalytic sites,including Glu71,Asp84 and Asp101.Quantitative qRT-PCR results confirmed that NaLyg gene mRNA was visibly increased after V.harveyi infection.The NaLyg protein purified by prokaryotic expression killed some gram-negative bacterial pathogens by inducing cell wall destruction,including V.harveyi,Aeromonas hydrophila and Edwardsiella tarda.Moreover,the NaLyg protein killed two gram-positive bacteria,Bacillus subtilis and Staphylococcus aureus.Taken together,the experimental results suggested that the NaLyg protein of N.albiflora played an important role in fighting bacterial infections.展开更多
A new robust bio-inspired route by using lysozyme aqueous solution for surface modification on 1,3,5,7-tetranitro-1,3,5,7-tetrazocane(HMX)was described in this paper.HMX crystals were coated by in situ phase transitio...A new robust bio-inspired route by using lysozyme aqueous solution for surface modification on 1,3,5,7-tetranitro-1,3,5,7-tetrazocane(HMX)was described in this paper.HMX crystals were coated by in situ phase transition of lysozyme(PTL)molecules.The HMX decorated by PTL was characterized by SEM,XRD,FTIR and XPS,demonstrating a dense core-shell coating layer.The coverage of lysozyme on HMX crystal was calculated by the ratio of sulfur content.The surface coverage increased from 60.5% to 93.5% when the content of PTL was changed from 0.5 wt% to 2.0 wt%,indicating efficient coating.The thermal stability of HMX was investigated by in situ XRD and DSC.The thermal phase transition temperature of HMX(β to δ phase)was delayed by 42℃ with 2.0 wt% PTL coating,which prevented HMX from thermal damage and sensitivity by the effect of PTL coating.After heating at 215℃,large cracks appeared in the naked HMX crystal,while the PTL coated HMX still maintained intact,with the impact energy of HMX dropped dramatically from 5 J to 2 J.However,the impact energy of HMX with 1.0 wt% and 2.0 wt% coating content(HMX@PTL-1.0 and HMX@PTL-2.0)was unchanged(5 J).Present results potentially enable large-scale fabrication of polymorphic energetic materials with outstanding thermal stability by novel lysozyme coating.展开更多
Hydrophobic Val derivative Schiff base copper(II) complexes and dipeptide (AlaAla, GlyGly) derivative Schiff base copper(II) complexes were introduced into egg white lysozyme. X-ray crystal structure analysis revealed...Hydrophobic Val derivative Schiff base copper(II) complexes and dipeptide (AlaAla, GlyGly) derivative Schiff base copper(II) complexes were introduced into egg white lysozyme. X-ray crystal structure analysis revealed amino acid derivative Schiff base copper(II) complexes were obtained. Herein we discuss primarily on the binding mode of copper(II) of the complexes obtained with egg white lysozyme. The electron density of copper(II) ions was confirmed by X-ray crystal structure analysis. The Val derivative Schiff base copper(II) complex was weakly bound at Arg114 of egg white lysozyme. In other copper(II) complexes, binding of copper(II) ions with dissociated ligands to various residues was observed. The binding sites of copper(II) ions were compared with computational scientific predictions.展开更多
G-protein coupled receptors （GPCRs） class C represent a distant group among the large family of GPCRs. This class includes the receptors for the main neurotransmitters, glutamate and gamma-aminobutyric acid （GABA）...G-protein coupled receptors （GPCRs） class C represent a distant group among the large family of GPCRs. This class includes the receptors for the main neurotransmitters, glutamate and gamma-aminobutyric acid （GABA）, and the receptors for Ca2＋, some taste and pheromone molecules, as well as some orphan receptors. Like any other GPCRs, these receptors possess a heptahelical domain （HD） involved in heterotrimeric G-protein activation, but most of them also have a large extracellular domain （VFT） responsible for agonist recognition and binding. These receptors are dimers, either homo or heterodimers. Then whereas have mGluRs is homodimers, GABAB receptor was the first heteromeric G-protein coupled receptor （GPCR） identified. Indeed, both GB1 and GB2 subunits appear necessary to get a functional GABAB receptor. We have demonstrated that the interactions be- tween VFT domain of both GB1 and GB2 were important for receptor activation. We have also shown the dynamic movement of trans-membrane of mGluRs within dimers. Then we have found that the GABAB receptor induced acti- vation of ERK1/2/CREB and protected neurons from apoptosis by trans-activating IGF-1R. We have also demon- strated that GABAB receptor activation has been modulated by the dynamic protein-protein interactions between re- ceptors and its downstream signal proteins such as FAK1 and Rap l. Finally, we have performed the HTS screening and found the first negative allosteric modulator for GABAB receptors.展开更多
Aim To induce and express the T4 lysozyme in Pichia pastoris and test the antibacterial activity of the protein. Methods T4 lysozyme gene was inserted into expression vector pPIC9K of Pichia pastoris with the fusion a...Aim To induce and express the T4 lysozyme in Pichia pastoris and test the antibacterial activity of the protein. Methods T4 lysozyme gene was inserted into expression vector pPIC9K of Pichia pastoris with the fusion at N terminal. The recombinant plasmid was digested by Sal I and then introduced into prepared GS115 competent cells by electroporation. Positive clone and multiple inserts were screened. The secreted proteins in the supernatants were tested. In the agar holes diffusion assay, our expressed protein showed significant antibacterial circles. Results T4 lysozyme protein inhibited the growth of staphylococcus aureus and streptococcus Pneumoniae. There was no difference in the bactericidal activity and the amount of protein expression between the single and multiple copies. The antibacterial activity of expressed protein remained the same during the heat stability test. Conclusion T4 lysozyme was successfully induced and expressed in Pichia pastoris. There is no relationship between copy number and expression. T4 lysozyme protein is heat stable.展开更多
To develop a gene therapy strategy for treating bovine mastitis, a new mammary-specific vector containing human lysozyme (hLYZ) cDNA and kanamycin resistance gene was constructed for intramammary expression and clinic...To develop a gene therapy strategy for treating bovine mastitis, a new mammary-specific vector containing human lysozyme (hLYZ) cDNA and kanamycin resistance gene was constructed for intramammary expression and clinical studies. After one time acupuncture or intracisternal infusion of healthy cows with 400 μg of the p215C3LYZ vector, over 2.0 μg/ml of rhLYZ could be detected by enzymatic assay for about 3 weeks in the milk samples. Western blotting showed that rhLYZ secreted into milk samples from the vector-injected cows had molecular weight similar to that of the natural hLYZ in human colostrums. Twenty days after the primary injection, the quarters were re-injected with the same vector by quarter acupuncture and even higher concentrations of rhLYZ could be detected. Indirect competitive ELISA of milk samples showed that the vector injection did not induce detectable humoral immune response against hLYZ. Clinical studies showed that twice acupuncture of quarters with the p215C3LYZ vector had overt therapeutic effect on clinical and subclinical mastitis previously treated with antibiotics, including disappearance of clinical symptoms and relatively high microbiological cure rates. These data provide a solid rationale for using the vector to develop gene therapy for treating bovine mastitis.展开更多
文摘The aim of the present study was to determine the MICs using broth microdilution method and their combination effect by （minimum inhibitory concentrations） of selected antibiotic agents determination of FICindex （fractional inhibitory concentrations）. Lysozyme Anodonta cygnea was more active than Lysozyme Uniopictorum, especially against Gram-negative bacterias. Combination of Macropen and Lysozyme Unio pictorum has a synergistic effect, while a separation of these antibacterial agents was not active against some of the strains of microorganisms.
基金supported by the National Natural Science Foundation of China(Nos.20920102035,20974086, and 21074083)the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)
文摘The purpose of this research is to investigate the effects of the variously sulfated chitosans on lysozyme activity and structure. It was shown that the specific enzymatic activity of lysozyme remained almost similar to the native protein after being bound to 6-O-sulfated chitosan （6S-chitosan） and 3,6-O-sulfated chitosan （3,6S-chitosan）, but decreased greatly after being bound to 2-N-6-O-sulfated chitosan （2,6S-chitosan）. Meanwhile, among these sulfated chitosans, 2,6S-chitosan induced the greatest conformational change in lysozyme as indicated by the fluorescence spectra. These findings demonstrated that when sulfated chitosans of different structures bind to lysozyme, lysozyme undergoes conformational change of different magnitudes, which results in corresponding levels of lysozyme activity. Further study on the interaction of sulfated chitosans with lysozyme by surface plasmon resonance （SPR） suggested that their affinities might be determined by their molecular structures.
基金Project supported by the Natural Science Foundation of Shaanxi Province [No. 2001K10-G3-（3）].
文摘The aggregation interaction between reduced-denatured egg white lysozymes during refolding procedure in urea solution was studied by means of reducing and non-reducing protein electrophoreses. Results of non-reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis （SDS-PAGE） of the supernatant and aggregate precipitate formed in refolding process show that except being refolded to native egg white lysozymes, the reduced-denatured lysozymes can also form the aggregates with molecular weights （MW） being separately about 30.0 and 35.0 kD, while the reducing SDS-PAGE and the refolding results in the presence of sodium dodecyl sulphate show that these aggregates are formed chiefly through the misconnection of disulfide bonds between the reduced-denatured lysozymes, and the aggregate precipitates are formed through the non-covalent interactions between the aggregates with molecular weight being about 30.0 kD. From the results of electrophoresis and size-exclusion chromatographic analyses, it can be inferred that the aggregates with molecular weights being about 30.0 and 35.0 kD are bi-molecular and tri-molecular egg white lysozyme aggregates, respectively. And finally, a suggested refolding mechanism of reduced-denatured egg white lysozymes in urea solution was presented.
基金supported by the National Natural Science Foundation of China(No.32072969)the National Key R&D Program of China(No.2022YFD2401002)+1 种基金the Natural Science Foundation of Fujian Province(No.2022 J01325)the Open Research Fund Program of Fujian Provincial Key Laboratory of Marine Fishery Resources and Eco-Environment(No.Z822280).
文摘Lysozyme(EC3.2.1.17)plays an important role in the immune response;as a nonspecific immune factor,it can resist causative agents.Lysozyme can be divided into c-type and g-type in fish.In a previous study,through genome-wide association analysis,the g-type lysozyme gene,which is named NaLyg in yellow drum(Nibea albiflora),was found to be a key candidate gene for disease resistance in response to Vibrio harveyi infection.The cDNA of NaLyg was 1025 bp,including four exons and three introns,and its open reading frame(ORF)had a full-length of 582 bp,encoding 193 amino acids.NaLyg was found to be conserved during evolution through bioinformatic analyses.The NaLyg protein possessed a sugar binding domain and three catalytic sites,including Glu71,Asp84 and Asp101.Quantitative qRT-PCR results confirmed that NaLyg gene mRNA was visibly increased after V.harveyi infection.The NaLyg protein purified by prokaryotic expression killed some gram-negative bacterial pathogens by inducing cell wall destruction,including V.harveyi,Aeromonas hydrophila and Edwardsiella tarda.Moreover,the NaLyg protein killed two gram-positive bacteria,Bacillus subtilis and Staphylococcus aureus.Taken together,the experimental results suggested that the NaLyg protein of N.albiflora played an important role in fighting bacterial infections.
基金the China National Nature Science Foundation(Grant No.12102404)。
文摘A new robust bio-inspired route by using lysozyme aqueous solution for surface modification on 1,3,5,7-tetranitro-1,3,5,7-tetrazocane(HMX)was described in this paper.HMX crystals were coated by in situ phase transition of lysozyme(PTL)molecules.The HMX decorated by PTL was characterized by SEM,XRD,FTIR and XPS,demonstrating a dense core-shell coating layer.The coverage of lysozyme on HMX crystal was calculated by the ratio of sulfur content.The surface coverage increased from 60.5% to 93.5% when the content of PTL was changed from 0.5 wt% to 2.0 wt%,indicating efficient coating.The thermal stability of HMX was investigated by in situ XRD and DSC.The thermal phase transition temperature of HMX(β to δ phase)was delayed by 42℃ with 2.0 wt% PTL coating,which prevented HMX from thermal damage and sensitivity by the effect of PTL coating.After heating at 215℃,large cracks appeared in the naked HMX crystal,while the PTL coated HMX still maintained intact,with the impact energy of HMX dropped dramatically from 5 J to 2 J.However,the impact energy of HMX with 1.0 wt% and 2.0 wt% coating content(HMX@PTL-1.0 and HMX@PTL-2.0)was unchanged(5 J).Present results potentially enable large-scale fabrication of polymorphic energetic materials with outstanding thermal stability by novel lysozyme coating.
文摘Hydrophobic Val derivative Schiff base copper(II) complexes and dipeptide (AlaAla, GlyGly) derivative Schiff base copper(II) complexes were introduced into egg white lysozyme. X-ray crystal structure analysis revealed amino acid derivative Schiff base copper(II) complexes were obtained. Herein we discuss primarily on the binding mode of copper(II) of the complexes obtained with egg white lysozyme. The electron density of copper(II) ions was confirmed by X-ray crystal structure analysis. The Val derivative Schiff base copper(II) complex was weakly bound at Arg114 of egg white lysozyme. In other copper(II) complexes, binding of copper(II) ions with dissociated ligands to various residues was observed. The binding sites of copper(II) ions were compared with computational scientific predictions.
文摘G-protein coupled receptors （GPCRs） class C represent a distant group among the large family of GPCRs. This class includes the receptors for the main neurotransmitters, glutamate and gamma-aminobutyric acid （GABA）, and the receptors for Ca2＋, some taste and pheromone molecules, as well as some orphan receptors. Like any other GPCRs, these receptors possess a heptahelical domain （HD） involved in heterotrimeric G-protein activation, but most of them also have a large extracellular domain （VFT） responsible for agonist recognition and binding. These receptors are dimers, either homo or heterodimers. Then whereas have mGluRs is homodimers, GABAB receptor was the first heteromeric G-protein coupled receptor （GPCR） identified. Indeed, both GB1 and GB2 subunits appear necessary to get a functional GABAB receptor. We have demonstrated that the interactions be- tween VFT domain of both GB1 and GB2 were important for receptor activation. We have also shown the dynamic movement of trans-membrane of mGluRs within dimers. Then we have found that the GABAB receptor induced acti- vation of ERK1/2/CREB and protected neurons from apoptosis by trans-activating IGF-1R. We have also demon- strated that GABAB receptor activation has been modulated by the dynamic protein-protein interactions between re- ceptors and its downstream signal proteins such as FAK1 and Rap l. Finally, we have performed the HTS screening and found the first negative allosteric modulator for GABAB receptors.
文摘Aim To induce and express the T4 lysozyme in Pichia pastoris and test the antibacterial activity of the protein. Methods T4 lysozyme gene was inserted into expression vector pPIC9K of Pichia pastoris with the fusion at N terminal. The recombinant plasmid was digested by Sal I and then introduced into prepared GS115 competent cells by electroporation. Positive clone and multiple inserts were screened. The secreted proteins in the supernatants were tested. In the agar holes diffusion assay, our expressed protein showed significant antibacterial circles. Results T4 lysozyme protein inhibited the growth of staphylococcus aureus and streptococcus Pneumoniae. There was no difference in the bactericidal activity and the amount of protein expression between the single and multiple copies. The antibacterial activity of expressed protein remained the same during the heat stability test. Conclusion T4 lysozyme was successfully induced and expressed in Pichia pastoris. There is no relationship between copy number and expression. T4 lysozyme protein is heat stable.
基金Project (Nos. BJ2001315 and BE2004611) supported by the De-partment of Science and Technology of Jiangsu Province, China
文摘To develop a gene therapy strategy for treating bovine mastitis, a new mammary-specific vector containing human lysozyme (hLYZ) cDNA and kanamycin resistance gene was constructed for intramammary expression and clinical studies. After one time acupuncture or intracisternal infusion of healthy cows with 400 μg of the p215C3LYZ vector, over 2.0 μg/ml of rhLYZ could be detected by enzymatic assay for about 3 weeks in the milk samples. Western blotting showed that rhLYZ secreted into milk samples from the vector-injected cows had molecular weight similar to that of the natural hLYZ in human colostrums. Twenty days after the primary injection, the quarters were re-injected with the same vector by quarter acupuncture and even higher concentrations of rhLYZ could be detected. Indirect competitive ELISA of milk samples showed that the vector injection did not induce detectable humoral immune response against hLYZ. Clinical studies showed that twice acupuncture of quarters with the p215C3LYZ vector had overt therapeutic effect on clinical and subclinical mastitis previously treated with antibiotics, including disappearance of clinical symptoms and relatively high microbiological cure rates. These data provide a solid rationale for using the vector to develop gene therapy for treating bovine mastitis.
