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71.
The plasma membrane of Listeria monocytogenes strain 42 was prepared by osmotic lysis of protoplasts with tris(hydroxymethyl)aminomethane (Tris) buffer, pH 8.2, containing MgCl2 and glucose, followed by washing with NaCl and MgCl2 in Tris buffer. Electron microscopy showed that the preparation was not contaminated with cytoplasmic material. The membrane preparation was composed of 55 to 60% protein, 1.5% ribonucleic acid, 0.1% deoxyribonucleic acid, 1.3 to 2.3% carbohydrate, 0.17 to 0.38% amino sugar, 0.2 to 0.4% rhamnose, 3.5 to 4.0% phosphorus, 10.5 to 12.0% nitrogen, and 30 to 35% lipid. Amino acid composition of the washed membrane showed some variation from that of the whole cells. Sulfur-containing amino acids were not present in the membrane hydrolysate. The membrane carbohydrate contained glucose, galactose, ribose, and arabinose. The membrane lipid was 80 to 85% phospholipid and 15 to 20% neutral lipid. The lipid contained 2.3 to 3.0% phosphorus, 2.5 to 3.0% carbohydrate, and a very small amount of nitrogen (0.2 to 0.3%). The phospholipid was of the phosphatidyl glycerol type. Electron micrographs of the washed membrane showed three layers. The outer and inner layers varied in thickness from 25 to 37 A and the middle layer from 20 to 25 A. The total thickness varied between 85 and 100 A. These preparations contained many vesicles which stained heavily with lead citrate. Some vesicles were also attached to the protoplast ghosts in the form of extrusions or intrusions, or both. Membrane preparations obtained by lysis of protoplasts in the absence of MgCl2 were fragmented and contained less lipid (20 to 22%) and ribonucleic acid (0.3 to 0.5%) than preparations prepared with MgCl2. 相似文献
72.
Fine Structure of Listeria monocytogenes in Relation to Protoplast Formation 总被引:19,自引:11,他引:8
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Among the eight strains of Listeria monocytogenes tested for lysozyme sensitivity, two were resistant to lysozyme but became sensitive after lipase pretreatment. Among the other six, one was very sensitive to lipase and another one was extremely susceptible to lysozyme. Stable protoplasts were formed from the lysozyme-resistant strain (42) by lipase and lysozyme treatment, which completely digested the cell wall. The cell wall (uranyl acetate-lead stained) was of a thick triple-layered profile, with the intermediate layer of low density. Lipase treatment for a short time (60 min) did not cause any alteration in structure, but prolonged treatment (180 min) caused extensive digestion of the plasma membrane and the cell wall, liberating cytoplasmic material. When the cells were treated with either lipase or lysozyme, a small number of protoplasts were extruded through the partly digested or weakened transverse cell wall, leaving an almost intact cell wall ghost. There were small vesicular structures in the interspace between cell wall and plasma membrane. Mesosomes of varied organization were prominent in electron micrographs, both in sections and in negatively stained preparations. These were largely everted during protoplasting in the form of tubules and as small peripheral buds; a few small vesicles also remained as intrusive structures, some of which were very unusual because they appeared to be enclosed by the inner layer of plasma membrane alone. Lysis of the protoplasts by dilution of the sucrose, while maintaining a constant ionic environment, liberated many small vesicular structures and fibrillar nuclear material. 相似文献
73.
74.
Influence of reagents reacting with metal, thiol and amino sites of catalytic activity and l-phenylalanine inhibition of rat intestinal alkaline phosphatase 总被引:7,自引:7,他引:0
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1. Studies on the inactivation of rat intestinal alkaline phosphatase by several metal-binding agents, namely EDTA, 8-hydroxyquinoline, pyridine-2,6-dicarboxylic acid, αα′-bipyridyl, o-phenanthroline and sodium cyanide, indicated the functional role of a metal, probably zinc, in the catalysis. The metal ligands lowered stereospecific uncompetitive inhibition of the enzyme by l-phenylalanine by an extent that paralleled the decline in enzyme activity. 2. The thiol reagents p-hydroxymercuribenzoate, iodoacetamide and iodine inactivated rat intestinal phosphatase. The enzyme could be protected from inactivation by either cysteine or substrate. The l-phenylalanine inhibition remained unchanged only in the presence of moderately inactivating concentrations of the thiol reagents. 3. Inactivation of the enzyme by the amino-group-blocking reagent, O-methylisourea, provided ample evidence for the participation in the catalysis of the -amino group of lysine. At the same time, l-phenylalanine inhibition remained unaltered even when the enzyme was strongly inactivated. This -amino-group-blocked enzyme exhibited no change in migration in starch gel, in contrast with enzyme treated with acetic anhydride, formaldehyde or succinic anhydride. The Michaelis constant of the enzyme was enhanced by such modifications, but the optimum pH remained the same. 4. d-Phenylalanine acted as a competitive or `co-operative' activator for intestinal alkaline phosphatase after it had been modified by acetylation. 相似文献
75.
76.
Kinetoplastid flagellates: surface-reactive carbohydrates detected by fluorescein-conjugated lectins
Membrane-associated carbohydrate residues of 3 isolates of Leishmania derived from etiological agents of visceral leishmaniasis (VL), postkala-azar dermal leishmaniasis (PKDL), and cutaneous leishmaniasis (CL), as well as 2 other nonpathogenic insect gut kinetoplastid flagellates, Bodo sp. and Herpetomonas sp., were characterized with the aid of 8 fluorescein-conjugated lectins. Four lectins, concanavalin A, Dolichos biflorus, phytohemagglutinin P, Ricinus communis agglutinin, bound to all kinetoplastid flagellates at different concentrations. All Leishmania promastigotes showed reactions with Ulex agglutinin. Although these lectins were bound to all kinetoplastids, the site and intensity of binding was different. All skin-dwelling Leishmania parasites, viz., Leishmania donovani of PKDL and Leishmania tropica of CL showed unique selectivity toward peanut agglutinin (PNA), soybean agglutinin, and wheatgerm agglutinin (WGA). More interestingly, Herpetomonas showed positive fluorescence with PNA and WGA, whereas Bodo was negative. The results demonstrated that no lectin could distinguish between the pathogenic and nonpathogenic status of kinetoplastid flagellates. Moreover, the antigenic (carbohydrate) profiles of Herpetomonas corresponded more closely to those of L. tropica, whereas Bodo shared some common lectin receptors with L. donovani of VL. 相似文献
77.
A chemically defined medium using commercially available alpha-MEM supplemented with hemin, HEPES, L-glutamine, D-glucose, folic acid, D-biotin and adenine supports the luxuriant growth and propagation of Leishmania donovani promastigotes. A peak parasite population of about 7.0 x 10(7)/ml at stationary phase and a population doubling time of 11.4 h for high-subpassage promastigotes were obtained. The medium was suitable for transformation of isolated amastigotes from infected hamster spleen. Promastigotes could be detected by culturing kala-azar patients' bone-marrow aspirate or spleen puncture material in this medium. Four out of six freshly transformed isolates gradually adapted and grew well in this medium. Macroscopic colonies appeared on agar plates prepared with the medium within 16-20 days after inoculation. The cloning efficiency was increased about five-fold by glycerol supplementation. 相似文献
78.
We previously reported that idiotype (Id)-loss, stable somatic variants of a B cell hybrid, 2C3E1, are generated both in vitro and in vivo, after interaction of the Id-positive tumor cells with autologous Id-specific effector T cells. The present investigation was undertaken to elucidate further the nature and functional characteristics of the effector T cells. We report here that the idiotype-specific cells mediating the generation of Id- tumor variants are Thy1+ L3T4+ Lyt-2- cells, which respond to specific idiotypic stimulation by secreting IL-2 in vitro. No IL-2 is secreted in response to unrelated Ig or an Id/Ig-2C3E1 tumor variant. Furthermore, the Id-specific T cells exert strong suppressive effects on the expression of 2C3E1 Ig and the effects can be reversed by blocking the L3T4+ T cells with monoclonal anti-L3T4 antibody in vitro during the initial 3 days of co-culture. After 4 days, the T cell-mediated suppression of the 2C3E1-Id is irreversible. In addition to the in vitro studies we have determined that the administration of anti-L3T4 mAb to mice just before priming with idiotype-bearing tumor cells also abrogates the suppressive effects of the idiotype primed spleen cells on Ig expression of 2C3E1. To study the Id-specific effector T cells in more detail we have generated functional Id-specific L3T4+ T cell lines. These T cell lines have been shown to recapitulate the generation of Id- tumor variants that we observed with Id-primed spleen cells. It is concluded that L3T4+, Id-specific Ts cells are responsible for the generation of somatic variants of the B cell hybrid 2C3E1 and that the induction or selection of these variants progresses from a reversible phase to an irreversible phase. 相似文献
79.
Summary To investigate whether anti-(carcinoembryonic antigen) monoclonal antibodies (mAb) react with single or repeated epitopes, sandwich radioimmunoassays in homologous and heterologous combinations were performed. Four mAb (I-27, I-47, II-17 and to some degree II-16) gave homologous binding while two mAb (I-38S1 and II-10) did not. Taken together with previous immunoprecipitation studies we conclude that all these mAb except II-10 react with repeated epitopes. The relative positions of the epitopes recognized by these mAb and of three additional mAb (II-6, II-7 and CB-CEA-1) were investigated using a plate antibody competition test with enzyme-labelled carcinoembryonic antigen (CEA). mAb I-38S1, II-6, II-7, II-10, II-16 and CB-CEA-1 were mutually cross-reactive, and were classified as belonging to one epitope group. mAb I-27 and I-47 fell outside this group and did not interfere with the binding of CEA conjugate to mAb II-17 either. They therefore represent a second epitope group. mAb II-17 showed no interference with the binding of CEA to any of the other mAb and must therefore represent a third epitope group. The slopes of the plate antibody competition curves were used for calculation of a correlation matrix, which in turn was used to depict the relative positions of the epitopes recognized by the mAb in the large group. 相似文献
80.
Eukaryotic initiation factor (eIF)-5, isolated from rabbit reticulocyte lysates, is a monomeric protein of Mr = 58,000-62,000. Immunochemical methods were employed to identify eIF-5 in crude cell lysates. Antisera against purified denatured eIF-5 were prepared in rabbits and characterized by immunoblotting and immunoprecipitation techniques using native and denatured eIF-5 as antigens. Monospecific antibodies to denatured eIF-5 were affinity-purified using eIF-5 blotted onto aminophenylthioether paper. Rabbit reticulocytes, HeLa cells and mouse L cells were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate. The denatured proteins were analyzed by polyacrylamide gel electrophoresis followed by immunoblotting with anti-eIF-5 antibodies. With each lysate, one major immunoreactive polypeptide was observed whose molecular weight corresponded to that of purified eIF-5 (Mr = 58,000-62,000). No degradation products or precursor forms of molecular weight higher than 62,000 were detected in any lysate. These results indicate that isolated eIF-5 is the same size as that found in crude lysates. Additional characterization of eIF-5 indicates that purified eIF-5 can be phosphorylated at serine residues in vitro by casein kinase II. Furthermore, in vitro phosphorylated eIF-5 retains full biological activity in catalyzing the joining of 60 S ribosomal subunits to a preformed 40 S ribosomal initiation complex to form an 80 S initiation complex. Based on its specific activity, we demonstrate that 1 pmol of rabbit reticulocyte eIF-5 mediates the formation of approximately 180 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions. 相似文献