共查询到20条相似文献,搜索用时 31 毫秒
1.
S Marleau B Fruteau de Laclos A B Sanchez P E Poubelle P Borgeat 《Journal of immunology (Baltimore, Md. : 1950)》1999,163(6):3449-3458
Studies were undertaken to define the role of 5-lipoxygenase (5-LO) products and, in particular, of leukotriene (LT) B4 in the polymorphonuclear leukocyte (PMN) emigration process using a rabbit model of dermal inflammation. Our results show that i.v. administration to rabbits of MK-0591, a compound that inhibits LT biosynthesis in blood and tissues when administered in vivo, significantly reduced 51Cr-labeled PMN accumulation in response to intradermally injected chemotactic agonists, including IL-8, FMLP, C5a, and LTB4 itself. In addition, pretreatment of the labeled PMN with MK-0591 ex vivo before their injection in recipient animals was equally effective in reducing 51Cr-labeled PMN emigration to dermal inflammatory sites. These results support a role for de novo synthesis of 5-LO metabolites by PMN for their chemotactic response to inflammatory mediators. Other studies demonstrated that elevated intravascular concentration of LTB4 interferes with PMN extravasation inasmuch as a continuous i.v. infusion of LTB4, in the range of 5-300 ng/min/kg, dose-dependently inhibited extravascular PMN accumulation to acute inflammatory skin sites elicited by the chemoattractants LTB4, FMLP, C5a, and IL-8 and by TNF-alpha, IL-1beta, and LPS; such phenomena may constitute a natural protective mechanism from massive tissue invasion by activated PMN in specific pathologic conditions such as ischemia (and reperfusion). These studies demonstrate additional functions of 5-LO products in the regulation of PMN trafficking, distinct from the well-characterized chemotactic activity of LTB4 present in the extravascular compartment. 相似文献
2.
J G Bender D E Van Epps D E Chenoweth 《Journal of immunology (Baltimore, Md. : 1950)》1987,139(9):3028-3033
The fluoresceinated chemotactic factors, C5a, formyl-methionyl-leucyl-phenylalanyl-lysine (FMLPL), and casein were used in conjunction with flow cytometry to examine chemotactic factor receptor expression on polymorphonuclear leukocytes (PMN) activated with phorbol myristate acetate (PMA), C5a, or formyl-methionyl-leucyl-phenylalanine. Activation with PMA resulted in a dose-dependent increase in binding of fluorescein-labeled (FL)-casein and (FL-FMLPL) over the range of PMA concentrations from 0.5 to 50 ng/ml. In contrast, activation of PMN with PMA resulted in a dose-dependent decrease in FL-C5a binding, and activation with concentrations above 5 ng/ml resulted in a complete loss of binding. This loss of binding was not caused by inactivation of the ligand or prevented by the addition of superoxide dismutase and catalase or protease inhibitors. Furthermore, incubation of PMN with supernatants from PMN stimulated to degranulate did not reduce the availability of C5a receptors. This pattern of increased FMLPL and casein binding with decreased C5a binding was also observed with cytochalasin B-pretreated PMN that were stimulated with chemotactic factors. Parallel studies of superoxide anion generation demonstrated that PMA-treated PMN were still responsive to formyl-methionyl-leucyl-phenylalanine, but not to C5a. These data demonstrate that the activation of PMN up-regulates formyl peptide and casein receptors whereas C5a receptors are down-regulated under similar conditions. 相似文献
3.
Although prior studies with mAb have defined an endogenous chymotrypsin-like protease in the neutrophil (polymorphonuclear leukocyte (PMN)) membrane that is associated with initiation of superoxide response to inflammatory stimuli, it is not known whether extracellular proteases (in the inflammatory milieu) can also influence PMN activation. This study examined the ability of four neutral proteases: cathepsin G, elastase, chymotrypsin, and trypsin, to modify PMN superoxide response to FMLP, PMA, and arachidonate. In response to 1 microM FMLP, PMN treated with cathepsin G, chymotrypsin, or elastase showed 64%, 60%, and 32% increases, respectively, in superoxide generation when compared with control, untreated cells (p less than 0.05 for each). These increments were dependent on intact enzymatic function of the proteases, were greatest when enzyme and stimulus were added concurrently, and persisted after PMN were washed free of enzyme. Enhancement of superoxide response was not stimulus specific; in response to 10 ng/ml PMA, cells treated with cathepsin G showed a 84%, and elastase a 57%, increase in superoxide generation (p less than 0.05 for both) with a marked reduction in the time required for onset of this response. For cell activation with 80 microM arachidonate, treatment with elastase produced a 180% increase in superoxide production (p less than 0.025). Neutrophils incubated with trypsin demonstrated significant decreases in superoxide response to PMA (-34%, p less than 0.05) and arachidonate (-39%, p less than 0.01). The enzymes themselves were not stimuli for superoxide production nor were they scavengers for superoxide in cellfree system. We conclude that local release of the PMN primary-granule neutral proteases, cathepsin G, and elastase within inflammatory sites can augment neutrophil effector function by up-regulating oxidative response to defined inflammatory stimuli. This autocrine/paracrine function may provide a significant increase in antimicrobial activity, but may also enhance the potential for host tissue injury. 相似文献
4.
H D Perez O Ohtani D Banda R Ong K Fukuyama I M Goldstein 《Journal of immunology (Baltimore, Md. : 1950)》1983,131(1):397-402
The thiol proteinase cathepsin H, isolated and purified from rat liver lysosomes, provokes acute inflammation characterized by the accumulation of polymorphonuclear leukocytes (PMN) when injected intracutaneously into newborn rats. We have examined the possibility that the accumulation of PMN at skin sites injected with cathepsin H is due, in part, to generation locally of C-derived chemotactic factors. We have found that cathepsin H acts in a concentration- and time-dependent fashion in whole human (and rat) EDTA-plasma to generate C5-derived peptides with chemotactic activity for PMN. Chemotactic activity was not generated in EDTA-plasma by either heat-inactivated cathepsin H or by a combination of active enzyme and a thiol proteinase inhibitor isolated from rat epidermis. Cathepsin H also acted in a concentration- and time-dependent fashion on isolated (functionally pure) human C5 to yield chemotactic activity for PMN as well as PMN lysosomal enzyme-releasing activity. Whereas 10 ng/ml cathepsin H generated significant chemotactic activity from isolated C5 (1000 CH50 U/ml), 7 to 10 micrograms/ml were required to generate chemotactic activity in whole EDTA-plasma. Cathepsin H not only was capable of generating biologically active, C5-derived peptides, but also was capable of degrading these peptides. Incubation of either whole EDTA-plasma or isolated C5 with high concentrations of cathepsin H (e.g., 25 micrograms/ml and 100 ng/ml, respectively) caused the rapid appearance of chemotactic activity followed by an equally rapid disappearance. PMN accumulated more rapidly in the skin of newborn rats injected with cathepsin H-treated C5 than in the skin of animals injected with cathepsin H alone. These data suggest that generation by cathepsin H of C-derived chemotactic activity contributes to the ability of this enzyme to induce dermal inflammation. 相似文献
5.
T J Sayers T A Wiltrout C A Bull A C Denn A M Pilaro B Lokesh 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(5):1670-1677
The i.p. injection of mice with highly purified recombinant human rIL-1 alpha or beta resulted in the rapid influx of a large number of polymorphonuclear neutrophils (PMN) into the peritoneal cavity. Significant increases in the number of PMN were induced by doses of IL-1 which ranged from 0.005 to 5 ng/injection. Interestingly the dose response for PMN influx was bell-shaped because 50 ng of IL-1 did not result in a significant increase in peritoneal PMN. IL-1 induced PMN infiltration was detectable by 1 h with peak levels of PMN obtained by about 2 h, followed by a subsequent decline by 24 h. Other cytokines, IL-2, IFN-gamma, IFN alpha beta, granulocyte-CSF, granulocyte-macrophage-CSF, IL-3, TNF-alpha, and TNF-beta were compared to IL-1 for their ability to induce a PMN influx into the peritoneum. Only TNF-alpha or TNF-beta (lymphotoxin) were able to induce a significant influx of PMN within 2 h. However, based on total protein administered, about 100 times more TNF than IL-1 was required to produce a comparable PMN infiltration. Intraperitoneal injection of inhibitors of the cyclooxygenase or lipoxygenase pathways did not inhibit the IL-1-induced influx of PMN. Also, neither IL-1 nor TNF triggered an increase in PG or leukotriene release from peritoneal cells in vitro. Furthermore, direct peritoneal injection of leukotriene B4, a potent PMN chemoattractant in vitro, did not induce any significant increase in PMN in the peritoneal cavity indicating that chemotactic activity alone is insufficient for inducing peritoneal infiltration. These results suggest that the local production of very low levels of IL-1 in vivo would be sufficient to initiate a sequence of events that results in a rapid accumulation of PMN. Because IL-1 was not chemotactic for PMN in vitro, our data suggest that IL-1 induces production of factors that are chemotactic for PMN. Alternatively, IL-1 may act on other stages of the complex sequence of events that regulates the emigration of PMN into tissue sites in vivo. The synergy apparent in PMN influx when suboptimal concentrations of IL-1 and TNF were injected suggests that the local production of very low concentrations of these cytokines in situ could play a critical role in the emigration of PMN during infection. 相似文献
6.
A thermostable and dialyzable peptide, released from rabbit IgG by rabbit neutrophil neutral thiol protease, exhibited a distinct chemotactic activity for rat lymphocytes; it was assumed to be derived from the Fc fragment (but not from the Fab fragment) by the enzyme. This substance seemed to be effective for adherent cells (B cells) from rat spleen, but not for nonadherent cells (T cells). The chemotactic peptide was purified by gel filtration on Sephadex G-50 and G-15 and then by high-voltage paper electrophoresis. As previously described, the IgG residue after release of dialyzable peptide(s) exhibited chemotactic activity for neutrophils but not for macrophages. 相似文献
7.
Heat-stable proteases from psychrotrophic pseudomonads: comparison of immunological properties. 总被引:2,自引:3,他引:2
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A heat-stable extracellular protease from Pseudomonas fluorescens was purified by chromatography on a DEAE-cellulose column and gel filtration on a Sephadex G100 column. The homogeneous enzyme preparation was used to prepare antiserum in rabbits. The rabbit antiserum was used to study the antigenic relatedness of proteases from 19 psychrotrophic pseudomonads isolated from raw milk. The inhibition of the proteases by the antiserum and the gel precipitin reactions revealed similar antigenic determinants in proteases from different isolates. Rabbit antiserum to the purified protease gave precipitin bands with antigens (proteases) from 10 different isolates. However, the same antiserum did not inhibit the protease activity in cell extracts of isolates T10, T13, and T24. By determining serological cross-reactions, proteases from psychrotrophic pseudomonads were shown to be different from one another. 相似文献
8.
A heat-stable extracellular protease from Pseudomonas fluorescens was purified by chromatography on a DEAE-cellulose column and gel filtration on a Sephadex G100 column. The homogeneous enzyme preparation was used to prepare antiserum in rabbits. The rabbit antiserum was used to study the antigenic relatedness of proteases from 19 psychrotrophic pseudomonads isolated from raw milk. The inhibition of the proteases by the antiserum and the gel precipitin reactions revealed similar antigenic determinants in proteases from different isolates. Rabbit antiserum to the purified protease gave precipitin bands with antigens (proteases) from 10 different isolates. However, the same antiserum did not inhibit the protease activity in cell extracts of isolates T10, T13, and T24. By determining serological cross-reactions, proteases from psychrotrophic pseudomonads were shown to be different from one another. 相似文献
9.
Calf thymus histone as a substrate for neutral and acid proteases of leucocyte lysosomes and other proteolytic enzymes 总被引:2,自引:0,他引:2
The use of calf thymus histone as a substrate has revealed a previously unknown neutral protease, optimally active at pH 7.2–7.3, in rabbit PMN lysosomes. The Sakaguchi reaction for the colorimetric determination of arginine has been modified, allowing a slow, linear development of color measurable at 500 nm over a period of up to 4 hr at 0–2°C. Specificity for arginine was shown since no other amino acid tested gave any color in the reaction. This new method has been used to measure arginine-reactive hydrolysis products released from histone by PMN lysosomes at neutral pH. Release of tyrosine, measured by the Folin method, was also used as an indicator of hydrolytic activity. Histone proved to be a useful substrate for the acid cathepsins of PMN, comparing favorably with hemoglobin, commonly used to measure such activity. Crystalline trypsin and chymotrypsin also hydrolyzed histone. The kinetics of arginine release by these enzymes over a period of 24 hr resembled those of neutral protease from PMN lysosomes. 相似文献
10.
Neutrophil-mediated maturation of chemerin: a link between innate and adaptive immunity 总被引:9,自引:0,他引:9
Wittamer V Bondue B Guillabert A Vassart G Parmentier M Communi D 《Journal of immunology (Baltimore, Md. : 1950)》2005,175(1):487-493
Dendritic cells and macrophages are professional APCs that play a central role in initiating immune responses, linking innate and adaptive immunity. Chemerin is a novel chemoattractant factor that specifically attracts APCs through its receptor ChemR23. Interestingly, chemerin is secreted as a precursor of low biological activity, prochemerin, which upon proteolytic removal of a C-terminal peptide, is converted into a potent and highly specific agonist of its receptor. Given the fact that APCs are often preceded by polymorphonuclear cells (PMN) in inflammatory infiltrates, we hypothesized that PMN could mediate chemerin generation. We demonstrate here that human degranulated PMNs release proteases that efficiently convert prochemerin into active chemerin. The use of specific protease inhibitors allowed us to identify the neutrophil serine proteases cathepsin G and elastase as responsible for this process. Mass spectrometry analysis of processed prochemerin showed that each protease generates specifically a distinct form of active chemerin, differing in their C terminus and initially identified in human inflammatory fluids. These findings strongly suggest that bioactive chemerin generation takes place during the early stages of inflammation, underscoring the functional contribution of chemerin as a bridge between innate and adaptive immunity. 相似文献
11.
During the recruitment of human polymorphonuclear neutrophils (PMN) to sites of inflammation, leukocyte adhesion molecules of the beta2 integrin (CD11/CD18) family mediate firm adhesion of these cells to the endothelial cell monolayer lining the vessel wall. This process is a prerequisite for shape change and spreading of PMN on the endothelium which eventually allows PMN emigration into the extravascular space. In order to elucidate the molecular mechanisms which mediate this sequence of events, intracellular protein tyrosine signaling was studied subsequent to beta2 integrin-mediated ligand binding. Using western blotting technique, beta2 integrin-mediated adhesion was found to induce tyrosine phosphorylation of different proteins. The effect was absent in PMN derived from CD18-deficient mice which lack any beta2 integrin expression on the cell surface demonstrating the specificity of the observed response. Inhibition of beta2 integrin-mediated tyrosine signaling by herbimycin A almost completely inhibited adhesion, shape change, and subsequent spreading of PMN. Herbimycin A also diminished chemotactic migration of these cells in response to the soluble mediator N-formyl-Met-Leu-Phe (fMLP). In contrast, treatment of PMN with cytochalasin D had no substantial effect on beta2 integrin-mediated signaling or adhesion but inhibited shape change, spreading, and chemotactic migration of PMN. This suggests that the signaling capacity exerted by beta2 integrins upon ligand binding was independent of an intact cytoskeleton. Moreover, the beta2 integrin-mediated activation of intracellular signal transduction pathways was critical for firm adhesion of PMN, the prerequisite subsequent shape change and spreading, which allows emigration of PMN into the extravascular space. 相似文献
12.
The generation of chemotactic activity in sera of germ-free newborn piglets by interaction with rough strain ofEscherichia coli 总被引:1,自引:0,他引:1
I. Miller 《Folia microbiologica》1969,14(6):560-565
The interaction of sera of newborn precolostral piglets with rough strain ofEscherichia coli or its endotoxin leads to formation of a factor which is chemotactic for rabbit polymorphonuclear neutrophil leucocytesin vitro (as tested using the Boyden's diffusion two-compartment chamber). Smooth strain ofEscherichia coli does not induce chemotaxin formation. The generation of chemotactic factor can be prevented by heating of the serum, addition of EDTA or yeast phosphomannan. The generation of the chemotactic activity is explained by the fixation of piglet complement system which is activated by rough bacteria even in the absence of detectable antibodies in newborn sera. 相似文献
13.
Numanami H Koyama S Sato E Haniuda M Nelson DK Hoyt JC Freels JL Habib MP Robbins RA 《American journal of physiology. Lung cellular and molecular physiology》2003,284(5):L882-L890
Chemotactic chemokines can be released from lung fibroblasts in response to interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. An imbalance between proteases and antiproteases has been observed at inflammatory sites, and, therefore, protease inhibitors might modulate fibroblast release of chemotactic cytokines. To test this hypothesis, serine protease inhibitors (FK-706, alpha(1)-antitrypsin, or N(alpha)-p-tosyl-L-lysine chloromethyl ketone) were evaluated for their capacity to attenuate the release of neutrophil chemotactic activity (NCA) or monocyte chemotactic activity (MCA) from human fetal lung fibroblasts (HFL-1). Similarly, the release of the chemoattractants IL-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, macrophage colony-stimulating factor, and granulocyte/macrophage colony-stimulating factor, from HFL-1, were evaluated in response to IL-1beta and TNF-alpha. NCA, MCA, and chemotactic cytokines were attenuated by FK-706. However, matrix metalloproteinase inhibitors were without effect, and cysteine protease inhibitors only slightly attenuated chemotactic or cytokine release. These data suggest that IL-1beta and TNF-alpha may stimulate lung fibroblasts to release NCA and MCA by a protease-dependent mechanism and that serine protease inhibitors may attenuate the release. 相似文献
14.
K Sveen 《Acta pathologica et microbiologica Scandinavica. Section B, Microbiology》1979,87(5):285-290
Purified lipopolysaccharide (LPS) from Veillonella incubated in normal rabbit serum was tested for chemotactic activity on rabbit polymorphonuclear leukocytes (PMNs) in modified Boyden chambers. In doses above those giving optimal response (over-optimal dose), a decrease of the PMN migration activity was found. This decrease also correlated well with an increase in the migration inhibition of the PMNs as demonstrated with the capillary tube assay. The PMN chemotactic factor isolated from LPS-induced inflammatory exudate (LPS-CF) in rabbits, produced both a decrease in chemotactic response and a migration inhibition of PMNs in over-optimal doses. This inhibitory effect was not due to cytotoxicity, proved by the trypan blue exclusion test. Also, a reduced locomotion of PMNs first preincubated with chemoattractants and then reactivated, was shown when the same PMNs were restimulated to migration using the same chemoattractants. This was interpreted as a deactivation of the cells. A cross-deactivation was demonstrated between LPS-CF and casein. The results from the experiments reported show that the Boyden chamber may be used to disciminate directional chemotaxis and migration inhibition. It may also be concluded from the study that the reduced migration activity of PMNs at over-optimal doses of chemoattractants is not due to cytotoxicity, but most probably is caused by a deactivation of the cells. 相似文献
15.
Emigration and accumulation of PMN-leukocytes induced by endotoxin, interleukin 1 and other chemotactic substances 总被引:1,自引:0,他引:1
H Z Movat M I Cybulsky I G Colditz 《Folia histochemica et cytobiologica / Polish Academy of Sciences, Polish Histochemical and Cytochemical Society》1986,24(2):75-88
This publication describes polymorphonuclear leukocyte (PMN) emigration and accumulation, which is prerequisite for their defensive function in infected tissues. The extravasated PMNs can kill microorganisms, but in this process they also release proteolytic enzymes and other cell constituents which can alter and even injure the tissues, primarily the microcirculation. In the first part of the paper in vivo quantitation of the acute inflammatory reaction is described with emphasis on PMN emigration and accumulation. With 51Cr-labeled PMNs the kinetics of their emigration induced by a number of chemotaxins and chemotaxinigens was found to be similar, peaking in 1-4 hour old lesions and returning to baseline values thereafter. The most potent substance tested was endotoxin, which induced a PMN influx at a molar concentration a least 3 orders of magnitude lower than the other substances tested, implying the these substances are not the primary endogenous mediators of endotoxin induced inflammation. Next we describe an observation which shed considerable light on the mechanisms underlying PMN emigration. When a chemotaxin or endotoxin was injected intradermally and after varying periods of time reinjected into the same site, the PMN influx into those sites was diminished, compared to sites not previously injected, i. e. injected for the first time. This tachyphylaxis or diminished responsiveness was attributed to a downregulation of receptors, presumably on endothelial cells, coupled to a facilitatory mechanism. Other mechanism proposed to terminate emigration of PMNs during inflammatory reaction were unlikely, based on our experimental findings. Endotoxin is not chemotactic in vitro but it induces PMN emigration when injected intradermally. Hence the third part of the publication deals with PMN emigration induced by interleukin 1 and its significance for endotoxin-induced inflammation. IL 1 is the only chemotaxin which induces PMN accumulation at a concentration comparable to that of endotoxin and considerably lower than the other chemotaxins. There was cross tachyphylaxis between endotoxin and IL 1 and vice versa. The PMN influx into IL 1 sites injected 6 hours earlier with IL 1 or with endotoxin was diminished compared to IL 1 sites injected into normal skin. Sites injected first with IL 1 and then with a low dose of endotoxin also exhibited cross tachyphylaxis. FMLP or LTB4 injected into sites pretreated with endotoxin did not exhibit cross tachyphylaxis, i. e. the PMN influx was similar to sites injected for the first time with these chemotaxins.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
16.
Mucor pusillus was grown in different media for a period of 92 h, and the media were investigated for both milk-clotting and protease activities. It was observed that the ratio of extracellular milk-clotting activity to protease activity was the highest for 3% corn steep liquor containing 1% glucose as the source of carbon. Variation of both milk-clotting and protease activities was studied during the growth of the organism in the medium stated above. Separation of protease was carried out by ion-exchange chromatography at pH 8.0. Fractions collected were assayed for both activities simultaneously. The findings suggested that, instead of only one major acid protease, as reported by previous workers, two major acid proteases were produced. One of them had significant rennin-like activity, and the other lacked it. The former could be assumed to be the enzyme reported and studied by previous workers. The existence of two proteases was further confirmed by the appearance of two protease activity bands on polyacrylamide gels after electrophoresis. An attempt was made to separate the rennin-like enzyme from nonspecific protease activity by ammonium sulfate fractionation followed by ion-exchange chromatography at pH 6.0. The results indicated that the nonspecific protease activity due to the enzyme that lacked rennin action was substantially removed by the ammonium sulfate fractionation. 相似文献
17.
H Ogawa S L Kunkel J C Fantone P A Ward 《Virchows Archiv. B, Cell pathology including molecular pathology》1981,38(2):149-157
Recently our laboratory has shown that neutrophils contain enzymatic activity within their lysosomal granules which will generate chemotactic activity for neutrophils and tumor cells from the fifth component of complement (C5). We have now expanded this initial observation and have demonstrated that eosinophils can release enzymatic activity from their lysosomal granules upon stimulation with immune complexes or opsoninized zymosan, but not with C5a or synthetic chemotactic peptides. Furthermore, the enzymatic activity released from the eosinophil lysosomal granules can cleave C5 into eosinophil-specific chemotactic activity. The generation of the eosinophil chemotactic activities from C5 is blocked by prior treatment of the eosinophil preparations with a number of protease inhibitors. The eosinophil-derived C5 cleaving activity possesses a pH optimum of 7.2, thus suggesting the enzymatic activity is a neutral protease. The demonstration that enzyme activities derived from eosinophils have the ability to generate eosinophil chemotactic factor(s) from C5 may explain why eosinophils are the predominant inflammatory cell in both nasal polyps and in the nasopharynx and bronchi of patients with allergic conditions such as hay fever and asthma. 相似文献
18.
《Process Biochemistry》2007,42(11):1492-1497
This study is aimed at developing a two-step process (fermentation plus enzymatic hydrolysis) for protease and peptone production, using a bioethanol industry by-product – corn distiller's dried grains with solubles (CDDGS) – as the sole carbon/nitrogen and protein source, respectively.Bacillus licheniformis was used for protease production. CDDGS concentration is the main parameter controlling protease generation, only low substrate concentration (below 2%, w/v) induces sporulation followed by enzyme excretion.The enzymatic peptone production process was implemented using the B. licheniformis fermentation broth (proteases) generated in the first step as hydrolytic tool, and CDDGS as a protein source.The protein present in CDDGS is solubilized yielding a peptone (protein concentration >80%), mainly composed of peptides and oligopeptides, soluble at practically all pH values. Both products, proteases and peptones, could be of great potential in industrial processes and in nutrition and food science. 相似文献
19.
Jake Zondagh Alison Williams Ikechukwu Achilonu Heini W. Dirr Yasien Sayed 《The protein journal》2018,37(4):369-379
In recent years, various strategies have been used to overexpress and purify HIV-1 protease because it is an essential drug target in anti-retroviral therapy. Obtaining sufficient quantities of the enzyme, however, remains challenging. Overexpression of large quantities is prevented due to the enzyme’s autolytic nature and its inherent cytotoxicity in Escherichia coli cells. Here, we describe a novel HIV-1 protease purification method using a thioredoxin–hexahistidine fusion system for the wild-type and two variant proteases. The fusion proteases were overexpressed in E. coli and recovered by immobilised metal ion affinity chromatography. The proteases were cleaved from the fusion constructs using thrombin. When compared to the standard overexpression and purification protocol in use in our laboratory, the expression of the fusion-derived wild-type protease was increased from 0.83 to 2.5 mg/l of culture medium. The expression levels of the two variant proteases ranged from 1.5 to 2 mg/l of culture medium. The fusion wild-type and variant proteases were inactive before the cleavage of the thioredoxin–hexahistidine fusion tag as no enzymatic activity was observed. The proteases were, however, active after cleavage of the tag. The novel thioredoxin–hexahistidine fusion system, therefore, enables the successful overexpression and purification of catalytically active HIV-1 proteases. 相似文献
20.
Human hepatocytes metabolizing ethanol generate a non-polar chemotactic factor for human neutrophils 总被引:2,自引:0,他引:2
F J Roll D M Bissell H D Perez 《Biochemical and biophysical research communications》1986,137(2):688-694
When human hepatocytes were incubated with low concentrations of ethanol they general chemotactic activity for human neutrophils. Generation of chemotactic activity was dependent upon duration of incubation and concentration of ethanol used. Production of chemotactic activity by ethanol-treated hepatocytes was inhibited completely in the presence of the alcohol dehydrogenase inhibitor 4-methylpyrazole. PMN isolated from rats, in contrast, do not respond chemotactically to the factor released by homologous cells. Preliminary studies indicated that the chemotactic factor is non-polar in nature (perhaps related to leukotriene B4). These results indicate that human hepatocytes, when exposed to ethanol, generate chemotactic factor(s) for human PMN. The occurrence of this phenomenon may explain, in part, the PMN infiltrates observed in human liver during the course of acute alcoholic hepatitis. 相似文献