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1.
Various strategies were evaluated for their ability to minimize the rate of breakdown of endogenous membrane lipids during cell fractionation studies with potato tubers. Buffering the homogenates at pH 7.5 to 8.0 resulted in significantly lower rates of phosphatidylcholine (PC) hydrolysis than were observed at lower pH values. Several potential inhibitors were added to homogenates to evaluate their ability to inhibit membrane lipid hydrolysis. The addition of bovine serum albumin (1% w/v) inhibited the rate of PC hydrolysis by 50%. The addition of low concentrations (25–100 μM) dibucaine (nupercaine) inhibited PC hydrolysis, but at higher concentrations (1-2 mM) it caused a 5- to 6-fold stimulation. Because dibucaine is a calmodulin antagonist, two other calmodulin antagonists (trifluoperazine and chlorpromazine) were tested and found to exhibit similar patterns of inhibition and stimulation. Similarly, the addition of low concentrations of deoxycholate also inhibited PC hydrolysis and high concentrations stimulated it. These results indicate that the hydrophobic properties of deoxycholate, dibucaine, and other calmodülin antagonists may explain their unusual effects on the rates of PC hydrolysis in potato tuber homogenates. Although the addition of exogenous calcium increased the rate of PC hydrolysis, the addition of calmodulin (bovine brain) had no effect. Other experiments revealed that the addition of 1% bovine serum albumin improved the yield and stability of mitochondrial and microsomal fractions from potato tubers. In constrast, the addition of 100 μM dibucaine caused deleterious effects.  相似文献   

2.
The influence of arginine on autolysis and proteolysis was studied. Arginine at the concentration of 0.5 and 1.0 microM/ml was added to the incubation mixture. Proteolytic processes were studied in the acid, neutral and alkaline media (pH 4.5; 7.4; 8.5). Autolysis was determined by incubation of the brain and liver homogenates and proteolysis by the use of bovine serum albumin as a substrate. Autolytic and proteolytic activities were calculated as an increase of Folin positive compounds or amino nitrogen in the samples. It was established that the influence in vitro of arginine on the proteolytic processes depended on pH, type of the peptide-hydrolases, to a lesser extent, on the arginine concentration and did not depend on the tissue type. Arginine displayed its regulative action in the brain and liver by the same way. The addition of arginine had an effect on autolysis and proteolysis in the neutral and alkaline media. Determination of autolytic and proteolytic activities by Folin positive compounds has shown that arginine addition into the samples decreased autolysis and proteolysis. At the same time determination of autolysis and proteolysis by amino nitrogen in the presence of arginine has shown that autolytic and proteolytic activities increased.  相似文献   

3.
Avian anterior (ALD) and posterior (PLD) latissimus dorsi muscle homogenates were tested for autolysis of proteins at pH values for which muscle proteases have been described (pH 4.0, 7.0, and 8.5). The action of MgCl2 and CaCl2 at physiological concentrations on these autolytic activities was measured to determine if changes in ion concentration could initiate proteolysis. At pH 7.0, it was found that alterations in CaCl2 concentrations within physiological limits in the presence of a MgCl2 concentration below physiological levels could activate or inhibit autolysis. Changes in magnesium concentration also could activate autolysis. This calcium effect was greatest for the PLD muscle. The possible role of calcium compartmental changes in initiation of contractile protein breakdown following denervation is discussed.  相似文献   

4.
AIM: To determine the autolytic phenotype of five species in the Bacillus cereus group. METHODS AND RESULTS: The autolytic rate of 96 strains belonging to five species in the B. cereus group was examined under starvation conditions at pH 6, 6.5 and 8.5 in different buffers. The autolytic rate was strain-dependent with a wide variability at pH 6, but higher and more uniform at pH 6.5. At pH 8.5, and respect to the extent of autolysis at pH 6.5, it was relatively low for most of the strains with the lowest values between 13 and 52% in Bacillus mycoides and Bacillus pseudomycoides. Peptidoglycan hydrolase patterns evaluated by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cells of Bacillus thuringiensis ssp. tolworthi HD125 as an indicator, revealed complex profiles with lytic bands of about 90, 63, 46, 41, 38, 32, 28 and 25 kDa in B. cereus, B. thuringiensis and Bacillus weihenstephanensis. Bacillus mycoides and B. pseudomycoides had simpler profiles with lytic bands of 63, 46 and 38 kDa. Changes in the autolytic pattern were observed for cells harvested at the stationary phase of growth (72 h) showing an increase in the intensity of the 25 kDa band in the case of B. cereus, B. thuringiensis and B. weihenstephanensis, while no changes were observed for B. mycoides. Using Micrococcus lysodeicticus and Listeria monocytogenes as indicators lytic activity was retained by proteins of 63, 46, 38, 32 and 25 kDa and a new one of about 20 kDa in B. mycoides. Growth in the different media did not affect the autolytic pattern. NaCl abolished the activity of all the peptidoglycan hydrolases except for those of B. mycoides and B. weihenstephanensis. Lytic activity was retained in the presence of MgCl(2), MnCl(2) and EDTA and increased at basic pH. CONCLUSIONS: Bacillus cereus/B. thuringiensis/B. weihenstephanensis showed a high extent of autolysis around neutral pH, even though they presented relatively complex autolysin profiles at alkaline pH. Bacillus mycoides/B. pseudomycoides had a higher extent of autolysis at acidic pH and a simpler autolysin pattern. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the autolytic phenotype expand the phenotypic characterization of the different species in the B. cereus group.  相似文献   

5.
Induction and control of the autolytic system of Escherichia coli.   总被引:17,自引:16,他引:1       下载免费PDF全文
Various methods of inducing autolysis of Escherichia coli cells were investigated, some being described here for the first time. For the autolysis of growing cells only induction methods interfering with the biosynthesis of peptidoglycan were taken into consideration, whereas with harvested cells autolysis was induced by rapid osmotic or EDTA shock treatments. The highest rates of autolysis were observed after induction by moenomycin, EDTA, or cephaloridine. The different autolyses examined shared certain common properties. In particular, regardless of the induction method used, more or less extensive peptidoglycan degradation was observed, and 10(-2) M Mg2+ efficiently inhibited the autolytic process. However, for other properties a distinction was made between methods used for growing cells and those used for harvested cells. Autolysis of growing cells required RNA, protein, and fatty acid synthesis. No such requirements were observed with shock-induced autolysis performed with harvested cells. Thus, the effects of Mg2+, rifampicin, chloramphenicol, and cerulenin clearly suggest that distinct factors are involved in the control of the autolytic system of E. Coli. Uncoupling agents such as sodium azide, 2,4-dinitrophenol, and carbonyl-cyanide-m-chlorophenyl hydrazone used at their usual inhibiting concentration had no effect on the cephaloridine or shock-induced autolysis.  相似文献   

6.
AIMS: To evaluate the autolytic phenotype of Bacillus thuringiensis. METHODS AND RESULTS: The autolytic rate of 87 strains belonging to different subsp. of B. thuringiensis was examined at pH 6, 6.5 and 8.5 in different buffers under starvation conditions. At pH 6 the extent of autolysis (average in the strain collection 38.3 +/- 21.1) was strain-dependent with wide variability, while at pH 6.5 and 8.5 (averages 72.0 +/- 9.0 and 63.1 +/- 8.2, respectively) it was much more uniform with only a few strains showing low autolytic rates. Forty-one per cent of the strains showed high resistance (>/=80%) to mutanolysin, a commercial muramidase from Streptomyces. The peptidoglycan hydrolase pattern was evaluated by renaturing SDS-PAGE using cells of B. thuringiensis subsp. tolworthi HD125 as indicator. The strain collection showed seven major lytic bands of about 90, 63, 46, 38, 32, 28 and 25 kDa, and in the stationary growth phase (72 h) there was a more intense 25 kDa band in the autolytic pattern. Using Micrococcus lysodeicticus and Listeria monocytogenes as the indicators lytic activity was retained, as seen by the bands of 63, 46, 38, 32 and 25 kDa. Growth in the different media did not affect the autolytic pattern. NaCl abolished the activity of all the peptidoglycan hydrolases in the gel, but in the presence of KCl, MgCl(2), MnCl(2) and EDTA some activity was retained. At basic pH the lytic activity increased. CONCLUSIONS: The autolytic phenotype of B. thuringiensis was found to be strain-dependent, and different proteins exibited peptidoglycan hydrolase activity, particularly at alkaline pH. Several of these proteins retained lytic activity against other bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterisation of the autolytic phenotype of B. thuringiensis should expand the prospects of using this species in bacterial bio-control and field applications.  相似文献   

7.
The molecular mechanism of the autolysis of rat alpha-chymotrypsin B was investigated. In addition to the two already known autolytic sites, Tyr146 and Asn147, a new site formed by Phe114 was identified. The former two sites and the latter one are located in the autolysis and the interdomain loops, respectively. By eliminating these sites by site-directed mutagenesis, their involvement in the autolysis and autolytic inactivation processes was studied. Mutants Phe114-->Ile and Tyr146-->His/Asn147-->Ser, that had the same enzymatic activity and molecular stability as the wild-type enzyme, displayed altered routes of autolytic degradation. The Phe114-->Ile mutant also exhibited a significantly slower autolytic inactivation (its half-life was 27-fold longer in the absence and sixfold longer in the presence of Ca2+ ions) that obeyed a first order kinetics instead of the second order displayed by wild-type chymotrypsin inactivation. The comparison of autolysis and autolytic inactivation data showed that: (a) the preferential cleavage of sites followed the order of Tyr146-Asn147 --> Phe114 --> other sites; (b) the cleavage rates at sites Phe114 and Tyr146-Asn147 were independent from each other; and (c) the hydrolysis of the Phe114-Ser115 bond was the rate determining step in autolytic inactivation. Thus, it is the cleavage of the interdomain loop and not of the autolysis or other loops that determines the half-life of chymotrypsin activity.  相似文献   

8.
The Coomassie brilliant blue G-250 dye-binding technique of M. M. Bradford (1976, Anal. Biochem. 72, 248-254) for protein quantification has been used to measure autolytic protein breakdown in cell-free extracts of Euglena gracilis. Specific autolysis rates were calculated from the difference between initial and actual absorbances at different incubation times of Coomassie brilliant blue-stained protein. They were found to depend linearly on time and initial protein concentration. Calibration against another method of protein determination is necessary due to different color yields with various protein mixtures. The high sensitivity and reproducibility of this method permit determination of specific autolysis rates below 0.1% mg-1 h-1 over a pH range between 3 and 8, without protein precipitation or pH adjustment, and in the presence of high amounts of amino acids and/or small peptides. Results obtained by this method are comparable to those of other autolysis measurements and to proteolytic activity determination by azocaseinolysis. Proteolytic autolysis has been observed in both the soluble and the particulate fractions of E. gracilis cell-free extracts, but displays different pH optima and specific activities in these fractions, as is also the case for azocaseinolysis. The method described is easy to perform, inexpensive, time saving, and should be applicable to other biological systems as well.  相似文献   

9.
The addition of several different antibiotics to growing cultures of Streptococcus faecalis, ATCC 9790, was found to inhibit autolysis of cells in sodium phosphate buffer. When added to exponential-phase cultures, mitomycin C (0.4 mug/ml) or phenethyl alcohol (3 mg/ml) inhibited deoxyribonucleic acid synthesis, but did not appreciably affect the rate of cellular autolysis. Addition of chloramphenicol (10 mug/ml), tetracycline (0.5 mug/ml), puromycin (25 mug/ml), or 5-azacytidine (5 mug/ml) to exponential-phase cultures inhibited protein synthesis and profoundly decreased the rate of cellular autolysis. Actinomycin D (0.075 mug/ml) and rifampin (0.01 mug/ml), both inhibitors of ribonucleic acid (RNA) synthesis, also reduced the rate of cellular autolysis. However, the inhibitory effect of actinomycin D and rifampin on cellular autolysis was more closely correlated with their concomitant secondary inhibition of protein synthesis than with the more severe inhibition of RNA synthesis. The dose-dependent inhibition of protein synthesis by 5-azacytidine was quickly diluted out of a growing culture. Reversal of inhibition was accompanied by a disproportionately rapid increase in the ability of cells to autolyze. Thus, inhibition of the ability of cells to autolyze can be most closely related to inhibition of protein synthesis. Furthermore, the rapidity of the response of cellular autolysis to inhibitors of protein synthesis suggests that regulation is exerted at the level of autolytic enzyme activity and not enzyme synthesis.  相似文献   

10.
The effect of physiological conditions on autolysis and autolytic activity in various strains ofStaphylococcus aureus was determined. The rate of whole cell autolysis ofS. aureus was growth phase dependent and a maximum rate was observed in early stationary phase cultures. However, the autolysins extracted by the freeze-thaw method (cell-wall bound autolytic activity) did not show any significant increase in activity. The addition of NaCl to the growth medium enhanced the rate of autolysis with the highest rate being displayed by cultures grown in 1.5 M NaCl. However, lower autolytic activity was found in the freeze-thaw extracts of cultures grown at higher concentrations of NaCl. The rate of autolysis of cultures grown at 30°C was higher than cultures grown at 37 or 43°C. Thus, the rate of autolysis seems to be independent of the bacterial growth rate. Cultures grown in slightly acidic conditions showed a faster rate of autolysis compared to cultures grown under alkaline conditions. SDS-polyacrylamide gel containing 0.2% crude cell-wall ofS. aureus did not show any obvious correlation with the appearance of any particular lytic band in the zymogram to autolytic activity or rate of autolysis of cultures grown under various environmental conditions. A nonhemolytic phenotype, mutations in the accessory gene regulator, and lysogeny (phages ø11, ø12, ø13) had no obvious effect either on the rate of autolysis or on the pattern of lytic bands in the zymograms.  相似文献   

11.
Potato leaves contain high levels of lipolytic acyl hydrolase activity which degrades phospholipids and galactolipids during homogenization and organelle isolation. Four calmodulin antagonists (dibucaine, tetracaine, trifluoperazine a and chlorpromazine) were found to inhibit the rate of hydrolysis of endogenous membrane lipids in homogenates of potato leaves. In contrast, the addition of calcium and purified calmodulin stimulated the rate of hydrolysis. These results indicate that a lipolytic acyl hydrolase activity in potato leaves appears to be mediated either directly or indirectly by calcium and calmodulin.  相似文献   

12.
Abstract By a comparison of the rate øX174 gene E product (gpE)-induced autolysis of Escherichia coli RM4101 and its autolysis deficient mutant strains RK232, RK238 and RK316, it was shown that gpE-induced autolysis differs from autolysis induced by EDTA or moenomycin. Subclones of these strains which could no longer be lysed by gpE can be lysed by EDTA shock treatment or moenomycin at almost normal rates. GpE seems to induce only partially the activity of the autolytic system of E. coli.  相似文献   

13.
Seven strains of viridans streptococci of the species Streptococcus sanguis, S. mutans and S. mitis were investigated for autolysis. The effect of pH, salt concentration and temperature on the autolytic process was studied in Na2HPO4/NaH2PO4 buffer. Whole cells and walls of all strains autolysed most rapidly at pH values above 7. Autolysis of whole cells of S. sanguis and one strain of S. mitis (ATCC15909) was maximal in 0-05 TO 0-2 M buffer, while the two S. mutans strains and S. mitis ATCC15912 showed maximal autolysis in 0-5 and 1-0 M buffers. Cultures harvested in the stationary phase of growth possessed only slightly decreased autolytic activity compared with those from the exponential phase. Whole cells autolysed more rapidly at 37 degrees C Than at 45 degrees C and 10 degrees C. Autolysis of isolated walls of three strains of S. mitis (ATCC903, ATCC15909 and ATCC15912) was maximal at pH 7-0 AND 7-5 and in 1-0 M buffers. Streptococcus mitis ATCC15909 also showed maximal lysis in 0-01 M and 0-5 M buffers. An endopeptidase action of the autolytic system of S. mitis ATCC15912 was indicated by the progressive release of soluble amino groups during autolysis of the walls. No release of reducing groups was observed. Several free amino acids were released during autolysis of these walls, alanine, lysine and glutamic acid being in greatest quanitity.  相似文献   

14.
AIMS: To evaluate the autolytic phenotype of Pediococcus acidilactici and P. pentosaceus, the peptidoglycan hydrolases content and the effect of pediocin AcH/PA-1 and autolysins on cell lysis. METHODS AND RESULTS: The autolytic phenotype of Pediococcus strains was evaluated under starvation conditions in potassium phosphate buffer. The strains tested showed an extent of autolysis ranging between 40 and 90% after 48 h of starvation at 37 degrees C. Peptidoglycan hydrolase content was evaluated by renaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using cells of Micrococcus lysodeikticus as a target for the enzymatic activity and a major activity band migrating at about 116 kDa was detected. Additional secondary lytic bands migrating in a range of molecular weight between 45 and 110 kDa were also detected. The lytic activity, evaluated in the presence of different chemicals, was retained in 15 mM CaCl2 and in a range of pH between 5 and 9.5 but was strongly reduced in presence of 8% NaCl and in the presence of protease inhibitors. The substrate specificity of peptidoglycan hydrolases of Pediococcus strains was evaluated in renaturing SDS-PAGE incorporating cells of different bacterial species. Lytic activity was detected against cells of Lactococcus lactis subsp. lactis, L. delbrueckii subsp. bulgaricus, Lactobacillus helveticus and Listeria monocytogenes. The interaction between pediocin AcH/PA-1 and autolysis was evaluated and a relevant effect of bacteriocin in cell-induced lysis was observed. CONCLUSIONS: The autolytic phenotype is widely distributed among P. acidilactici and P. pentosaceus and the rate of autolysis is high in the majority of the analysed strains. Several autolytic bands, detected by renaturing SDS-PAGE, retained their activity against several lactic acid bacteria and L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of the autolytic phenotype of Pediococcus acidilactici and P. pentosaceus strains should expand the knowledge of their role in fermentation processes where these species occur as primary or secondary bacterial population.  相似文献   

15.
The autolytic N-acetylmuramidase present in Lactobacillus acidophilus strain 63 AM Gasser has an optimal pH between 5 and 6 when lysing intact cells or isolated cell walls. Cellular lysis at pH 5 is two to four times more rapid in citrate buffer of 0.01 M and 0.5 M or higher than in 0.1 M acetate buffer. It seems that sulfhydryl groups are required for both cell and wall autolysis. Heavy metal ions and p-chloro-mercuribenzoate, at low concentrations, are powerful inhibitors. Ethylenediaminetetraacetic acid stimulates cellular but not wall autolysis in acetate buffer to the level obtained in citrate buffer. The possible involvement of sulfhydryl groups in a mechanism of control of cellular autolytic activity is discussed. The autolytic enzyme, although unstable in solution at 37 C, can be extracted from walls by the use of solutions of bovine serum albumin (100 mug/ml) in 0.01 N NaOH. Soluble enzyme extracted from walls rebinds on to sodium decylsulfate-treated walls, but three times as much of the wall material is required to completely re-adsorb the activity.  相似文献   

16.
A comparative study was performed of the processes of autolytic degradation of the cells of Saccharomyces cerevisiae and Schizosaccharomyces pombe under conditions simulating the phase of cell death in microbial cultures: (1) during autolysis induced by oleic acid, which is the chemical analogue of factors d2 (autolysis autoinducer), (2) under the effect of extracellular microbial proteinases (enzymatic lysis), and (3) under concomitant effect of the enzymes of the endogenous autolytic complex and exogenous proteinases (heterolysis). Regulatory mechanisms controlling the rate and profundity of autolysis were elucidated, relying on the stabilization of hydrolytic enzymes and enhancement of their activity in their complexes with a chemical analogue of microbial autoregulatory factors d1, which belong to alkylhydroxybenzenes and fulfil functions of chemical chaperons. The changes in the activity of proteinases and enzymes of the autolytic complex were shown to be dependent on the concentration of the analogue at the moment of complex formation.  相似文献   

17.
Cell walls isolated from ripening tomato ( Lycopersicon esculentum Mill. cv. Rutgers) fruit released pectic polymers when incubated under conditions that allow activity of wall-bound polygalacturonase (EC 3.2.1.15). Autolysis was optimally stimulated by 150–300 m M NaCl at either pH 2.5 or 4.5. This stimulation was negated by exposure to pH 6.5 or higher and by pretreatment of walls with boiling 80% ethanol. Five m M CaCl2 did not affect autolysis at pH 2.5, but significantly inhibited at pH 4.5 or higher. Inclusion of 1 M NaCl at selected steps in the extraction scheme did not inhibit subsequent autolysis of isolated walls. Exposure of isolated walls to 1 M NaCl at pH 2.5–8.5 also did not inhibit autolytic activity compared to walls that received no ionic treatment. These data support the concept that cell wall hydrolysis during tomato fruit softening is regulated by pH, Ca2+ levels and ionic strength of the apoplast.  相似文献   

18.
A comparative study was performed of the processes of autolytic degradation of the cells of Saccharomyces cerevisiae and Schizosaccharomyces pombe under conditions simulating the phase of cell death in microbial cultures: (1) during autolysis induced by oleic acid, which is the chemical analogue of factors d2 (autolysis autoinducer), (2) under the effect of extracellular microbial proteinases (enzymatic lysis), and (3) under the concomitant effect of the enzymes of the endogenous autolytic complex and exogenous proteinases (heterolysis). Regulatory mechanisms controlling the rate and profundity of autolysis were elucidated, relying on the stabilization of hydrolytic enzymes and enhancement of their activity in their complexes with a chemical analogue of microbial autoregulatory factors d1, which belong to alkylhydroxybenzenes and fulfill functions of chemical chaperones. The changes in the activity of proteinases and enzymes of the autolytic complex were shown to be dependent on the concentration of the analogue at the moment of complex formation.  相似文献   

19.
The mycelium of Mucor rouxii reached a 50% degree of lysis after 50 days incubation, and was then stable with the incubation time. The pH of the medium was 4.3 when autolysis began, rising to pH 7.6 after 6 days of autolysis and remaining there for the duration of the experiment. Maximum degradation of mycelium occurs during the first days of autolysis. Glucosamine is present in the culture liquid during all the autolytic process. Enzymes implicated in the degradation of chitosan and chitin were studied in the culture fluid during autolysis. An exochitosanase activity was detected after a day of autolysis, and its activity increased during 20 days of autolysis and afterwards remained constant until the end of the process. An endochitosanase activity was detected in the culture fluid from the beginning of the autolysis, having its maximum activity after 34 days of incubation. Both activities show an optimum pH of 5.5, but the pH range of activity for endochitosanase was broader than for exochitosanase. Both activities were not inhibited by 0.5 mM glucosamine. Activities of the enzymes B-N-acetylglucosaminidase and chitinase were not found. The chitosan content in the cell walls decreased with the incubation time. In these cell walls the chitin content experienced an increase at the beginning of the autolysis, decreasing afterwards. The enzymatic complex obtained from autolyzed cultures of M. rouxii hydrolyzed 2-day-old cell walls of this fungus. The hydrolysis was 21% after 24 h of incubation, liberating glucose and glucosamine. As a consequence protoplasts from M. rouxii germinated spores were obtained with its own lytic enzymes in adequate osmotic conditions. The involvement of chitosanases in the autolysis of this fungus have been studied.  相似文献   

20.
Abstract The impacts of potato psyllid (Bactericera cockerelli) feeding on potato foliage on the free amino acids (FAAs) composition in potato leaf and tubers were determined under greenhouse conditions. The free amino acids in plant extracts were separated by high‐performance liquid chromatography, and in both leaf and tuber samples, at least 17 FAAs were detected. Psyllid feeding significantly changed the levels of several FAAs in both leaf and tuber samples. The concentration of leucine increased 1.5‐fold, whereas that of serine and proline increased 2‐ and 3‐fold, respectively. In contrast, the concentrations of glutamic acid, aspartic acid and lyscine were significantly reduced by 42.0%, 52.1% and 27.5%, respectively. There were also significant changes in the levels of FAAs in the Zebra chip (ZC) infected tubers compared with the healthy tubers, and the levels of six of the FAAs increased, and the levels of nine of the FAAs decreased. The results from this study indicate that potato psyllid causes major changes in free amino acid composition of plant tissues, and this change in plant metabolism may contribute to the plant stress as indicated by increased levels of proline in the leaves and hence promoting the development of plant diseases such as ZC disease.  相似文献   

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