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1.
Several preparations of staphylococcal alpha-toxin and delta-lysin were studied in order to compare hemolytic activity with capacity to lyse bacterial protoplasts. delta-Lysin in relatively low concentration lysed protoplasts of Sarcina lutea, protoplasts of Streptococcus faecalis, and spheroplasts of Escherichia coli. Lysis of bacterial protoplasts by preparations of alpha-toxin appeared to be due to contamination of the preparations with delta-lysin. Data comparing the protoplast-lysing activity of various lytic agents are presented. 相似文献
2.
Crude and purified staphylococcal alpha-toxin were used to demonstrate that the platelet-damaging effect of crude alpha-toxin represents a fifth activity of the alpha-toxin molecule. The homogeneity of the purified toxin employed was demonstrated by ultracentrifugation, Ouchterlony, and immunoelectrophoretic methods. Continuous-flow electrophoretic migration studies demonstrated under a variety of conditions that the platelet-damaging and the alpha-hemolytic activities migrated as a unit. Fractionation studies with the use of Sephadex G-100, carboxymethyl cellulose, and diethylaminoethyl cellulose failed to separate these two activities. Further, when alpha-toxin of demonstrated purity and crude toxin were adjusted to the same hemolytic activity, they possessed the same platelet-damaging activity. In addition, heat-reactivation studies with crude alpha-toxin revealed that the platelet-damaging effect was inactivated and reactivated in parallel with alpha-hemolytic activity. Comparable studies with purified alpha-toxin showed parallel inactivation of both activities at 60 C. Additional heating at 100 C failed to reactivate either activity. Electron micrographs revealed that purified alpha-toxin produced distinct degenerative changes in rabbit platelets. These studies also provided definite evidence that purified alpha-toxin has a damaging effect on human platelets. Monovalent alpha-antisera prevented platelet damage. 相似文献
3.
The purification of staphylococcal β-hemolysin was accomplished by the successive use of three protein fractionation methods. The first method employed was a double precipitation with the use of ammonium sulfate at 65% saturation. The second phase of purification used Sephadex G-100 column fractionation. The third phase utilized either carboxymethyl cellulose or diethylaminoethyl cellulose fractionation. The last two fractionation methods both resulted in the separation of a relatively high concentration of cationic hot-cold lysin and a low concentration of anionic hot-cold lysin. Because of the low concentration of the anionic component, its purity could not be assessed. However, the purity of the cationic component was demonstrated by immunodiffusion, microimmunoelectrophoresis, and by disc polyacrylamide gel electrophoresis. In addition, antisera against purified cationic β-hemolysin yielded one line of precipitate when tested against the original crude β-hemolysin. The purified cationic β-hemolysin was stable in the lyophilized state. Crude β-hemolysin was dermonecrotic, whereas purified cationic β-hemolysin was not dermonecrotic even after Mg ++ activation. 相似文献
4.
The cadherins are a family of homophilic adhesion molecules that play a vital role in the formation of cellular junctions and in tissue morphogenesis. Members of the integrin family are also involved in cell to cell adhesion, but bind heterophilically to immunoglobulin superfamily molecules such as intracellular adhesion molecule (ICAM)–1, vascular cell adhesion molecule (VCAM)–1, or mucosal addressin cell adhesion molecule (MadCAM)–1. Recently, an interaction between epithelial (E-) cadherin and the mucosal lymphocyte integrin, α Eβ 7, has been proposed. Here, we demonstrate that a human E-cadherin–Fc fusion protein binds directly to soluble recombinant α Eβ 7, and to α Eβ 7 solubilized from intraepithelial T lymphocytes. Furthermore, intraepithelial lymphocytes or transfected JY′ cells expressing the α Eβ 7 integrin adhere strongly to purified E-cadherin–Fc coated on plastic, and the adhesion can be inhibited by antibodies to α Eβ 7 or E-cadherin. The binding of αEβ7 integrin to cadherins is selective since cell adhesion to P-cadherin–Fc through αEβ7 requires >100-fold more fusion protein than to E-cadherin–Fc. Although the structure of the αE-chain is unique among integrins, the avidity of αEβ7 for E-cadherin can be regulated by divalent cations or phorbol myristate acetate. Cross-linking of the T cell receptor complex on intraepithelial lymphocytes increases the avidity of αEβ7 for E-cadherin, and may provide a mechanism for the adherence and activation of lymphocytes within the epithelium in the presence of specific foreign antigen. Thus, despite its dissimilarity to known integrin ligands, the specific molecular interaction demonstrated here indicates that E-cadherin is a direct counter receptor for the αEβ7 integrin. 相似文献
5.
The interaction of the α5β1 integrin and its ligand, fibronectin (FN), plays a crucial role in the adhesion of cells to the extracellular matrix. An important intrinsic property of the α5β1/FN interaction is the dynamic response of the complex to a pulling force. We have carried out atomic force microscopy measurements of the interaction between α5β1 and a fibronectin fragment derived from the seventh through tenth type III repeats of FN (i.e., FN7-10) containing both the arg-gly-asp (RGD) sequence and the synergy site. Direct force measurements obtained from an experimental system consisting of an α5β1 expressing K562 cell attached to the atomic force microscopy cantilever and FN7-10 adsorbed on a substrate were used to determine the dynamic response of the α5β1/FN7-10 complex to a pulling force. The experiments were carried out over a three-orders-of-magnitude change in loading rate and under conditions that allowed for detection of individual α5β1/FN7-10 interactions. The dynamic rupture force of the α5β1/FN7-10 complex revealed two regimes of loading: a fast loading regime (>10,000 pN/s) and a slow loading regime (<10,000 pN/s) that characterize the inner and outer activation barriers of the complex, respectively. Activation by TS2/16 antibody increased both the frequency of adhesion and elevated the rupture force of the α5β1/wild type FN7-10 complex to higher values in the slow loading regime. In experiments carried out with a FN7-10 RGD deleted mutant, the force measurements revealed that both inner and outer activation barriers were suppressed by the mutation. Mutations to the synergy site of FN, however, suppressed only the outer barrier activation of the complex. For both the RGD and synergy deletions, the frequency of adhesion was less than that of the wild type FN7-10, but was increased by integrin activation. The rupture force of these mutants was only slightly less than that of the wild type, and was not increased by activation. These results suggest that integrin activation involved a cooperative interaction with both the RGD and synergy sites. 相似文献
6.
Purification of beta-hemolysin was achieved by ammonium sulfate precipitation, Sephadex G-100 gel filtration, carboxymethyl cellulose column chromatography, and density gradient electrophoresis. Active fractions eluted from carboxymethyl cellulose contained at least one nonhemolytic protein, and omission of this step was not detrimental to the purification process. Density gradient electrophoresis yielded approximately 1.6 mg of highly active purified beta-hemolysin per liter of culture supernatant liquid. Purified beta-hemolysin gave a single line on gel double diffusion and immunoelectrophoresis. A single symmetrical peak formed in the analytical ultracentrifuge, and the sedimentation coefficient was calculated to be 1.7S. The purified beta-hemolysin was stable at 4 C and could be lyophilized. Magnesium cations were required for full expression of beta-hemolytic activity. beta-Hemolysin was lethal for rabbits when injected intravenously in amounts between 40 and 160 mug. Crude beta-hemolysin was more stable than purified beta-hemolysin when heated at 60 C for 30 min. Purified beta-hemolysin lost almost all of its activity on subsequent heating at 100 C for 10 min. 相似文献
7.
α-Ionone, α-methylionone, and α-isomethylionone were converted by Aspergillus niger JTS 191. The individual bioconversion products from α-ionone were isolated and identified by spectrometry and organic synthesis. The major products were cis-3-hydroxy-α-ionone, trans-3-hydroxy-α-ionone, and 3-oxo-α-ionone. 2,3-Dehydro-α-ionone, 3,4-dehydro-β-ionone, and 1-(6,6-dimethyl-2-methylene-3-cyclohexenyl)-buten-3-one were also identified. Analogous bioconversion products from α-methylionone and α-isomethylionone were also identified. From results of gas-liquid chromatographic analysis during the fermentation, we propose a metabolic pathway for α-ionones and elucidation of stereochemical features of the bioconversion. 相似文献
8.
Concentrated preparations of staphylococcal delta-hemolysin were obtained by growing selected hemolytic colonies from the 146P strain of Staphylococcus aureus on dialysis membranes laid over Brain Liver Heart agar plates at 37 C for 20 hr under 10% CO(2) and harvesting the growth from five such membranes in 1.0 ml of deionized distilled water. Incubation in a humid environment facilitated this harvesting procedure. Incubation longer than 40 hr or incubation under CO(2) higher than 10 to 20% gave lower yields of delta-lysin. Addition of a sugar, fermented by the organisms, resulted in lower yields of delta-hemolysin. Agar, although separated from the growing cells by the dialysis membrane, did potentiate delta-hemolysin production. Addition of 0.1% agar to the inoculum further enhanced this potentiation. delta-Hemolysin produced in broth or semisolid cultures was excessively diluted with the media. Dialysis membranes prevented this dilution and thus yielded concentrated preparations of delta-hemolysin. 相似文献
9.
Sheep erythrocyte ghosts released water-soluble organic phosphorus when treated with purified beta-hemolysin. Phospholipid analysis demonstrated that sphingomyelin accounted for 53% of the phospholipids present in sheep erythrocytes. Purified beta-hemolysin showed phospholipase C activity when purified ox brain or sheep erythrocyte sphingomyelin was used as substrate. Such studies have also revealed that the disappearance of sphingomyelin from the reaction mixture was accompanied by a comparable increase in the concentration of phosphoryl choline. Thin-layer chromatography of phospholipids, extracted from sheep erythrocytes which had been exposed to beta-hemolysin, demonstrated that sphingomyelin was rapidly degraded. Activators of beta-hemolysin, such as Mg(++), enhanced the release of organic phosphorus from erythrocyte ghosts and from sphingomyelin. Inhibitors of beta-hemolysin, such as ethylenediaminetetraacetic acid, p-chloromercuribenzoate, and iodoacetamide, also inhibited the release of organic phosphorus from erythrocyte ghosts and from sphingomyelin. These studies strongly suggested that beta-hemolysin enzymatically degraded the sphingomyelin of the erythrocyte membrane. Such degradation probably resulted in the eventual lysis of the erythrocyte. 相似文献
11.
There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability. 相似文献
14.
A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg 2+, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration. 相似文献
15.
Intestinal intraepithelial lymphocytes (IELs) are distributed along the length of the intestine and are considered the frontline of immune surveillance. The precise molecular mechanisms, especially epigenetic regulation, of their development and function are poorly understood. The trimethylation of histone 3 at lysine 27 (H3K27Me3) is a kind of histone modifications and associated with gene repression. Kdm6b is an epigenetic enzyme responsible for the demethylation of H3K27Me3 and thus promotes gene expression. Here we identified Kdm6b as an important intracellular regulator of small intestinal IELs. Mice genetically deficient for Kdm6b showed greatly reduced numbers of TCRαβ +CD8αα + IELs. In the absence of Kdm6b, TCRαβ +CD8αα + IELs exhibited increased apoptosis, disturbed maturation and a compromised capability to lyse target cells. Both IL-15 and Kdm6b-mediated demethylation of histone 3 at lysine 27 are responsible for the maturation of TCRαβ +CD8αα + IELs through upregulating the expression of Gzmb and Fasl. In addition, Kdm6b also regulates the expression of the gut-homing molecule CCR9 by controlling H3K27Me3 level at its promoter. However, Kdm6b is dispensable for the reactivity of thymic precursors of TCRαβ +CD8αα + IELs (IELPs) to IL-15 and TGF-β. In conclusion, we showed that Kdm6b plays critical roles in the maturation and cytotoxic function of small intestinal TCRαβ +CD8αα + IELs.Subject terms: Epigenetics, Gene regulation, Immunological disorders, T cells 相似文献
17.
The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed via several microtubule nucleation pathways. Targeting Protein for XKlp2 (TPX2) stimulates the branching microtubule nucleation pathway, where new microtubules are nucleated from preexisting ones within mitotic or meiotic spindles. TPX2, like other spindle assembly factors, is sequestered by binding to nuclear importins-α/β until the onset of mitosis, yet the molecular nature of this regulation remains unclear. Here we demonstrate that TPX2 interacts with importins-α/β with nanomolar affinity in a 1:1:1 monodispersed trimer. We also identify a new nuclear localization sequence in TPX2 that contributes to its high-affinity interaction with importin-α. In addition, we establish that TPX2 interacts with importin-β via dispersed, weak interactions. We show that interactions of both importin-α and -β with TPX2 inhibit its ability to undergo phase separation, which was recently shown to enhance the kinetics of branching microtubule nucleation. In summary, our study informs how importins regulate TPX2 to facilitate spindle assembly, and provides novel insight into the functional regulation of protein phase separation. 相似文献
18.
A growing culture of Streptomyces lydicus converted biotin- 14C to α-dehydrobiotin- 14C. The conversion was demonstrated by isolating crystalline α-dehydrobiotin- 14C from fermentation liquors supplemented with biotin- 14C. The addition of pimelic acid- 14C to the growing culture did not produce any radioactive α-dehydrobiotin. α-Dehydrobiotin did not substitute for biotin in Lactobacillus plantarum or in Saccharomyces cerevisiae. Antimicrobial activity of α-dehydrobiotin was abolished by avidin. α-Dehydrobiotin appears to be different from several biotin vitamers described in the literature. It is concluded that α-dehydrobiotin is a product of biotin catabolism in S. lydicus. 相似文献
20.
The amyloid-β peptide (Aβ) can mediate cell attachment by binding to β1 integrins through an arg-his-asp sequence. We show here that the α5β1 integrin, a fibronectin receptor, is an efficient binder of Aβ, and mediates cell attachment to nonfibrillar Aβ. Cells engineered to express α5β1 internalized and degraded more added Aβ1-40 than did α5β1-negative control cells. Deposition of an insoluble Aβ1-40 matrix around the α5β1-expressing cells was reduced, and the cells showed less apoptosis than the control cells. Thus, the α5β1 integrin may protect against Aβ deposition and toxicity, which is a course of Alzheimer's disease lesions. 相似文献
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