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1.
The binding of rabbit anti-human IgG labeled with 125I, shellfish glycogen or ferritin to human IgG attached to the surface of rabbit RBC with chromic chloride was studied. Maximum binding was noted with 125I labeled antibody. Slightly but consistently less binding was found with shellfish glycogen labeled antibody. The binding of ferritin labeled antibody was strikingly reduced--usually one-third or less of that found with 125I labeled antibody alone. This suggests that under the conditions of these experiments, the attachment of large labels to antibody molecules results in reduced antibody binding to surface antigen. Steric hindrance is probably at least in part responsible for this reduced binding. 相似文献
2.
Measurement of serum ferritin by a 2-site immunoradiometric assay 总被引:18,自引:0,他引:18
A 2-site immunoradiometric assay (2-site IRMA) for human serum ferritin is carried out by reaction of the ferritin solution with a solid-phase anti(human ferritin), followed by a second reaction in which the insoluble product is incubated with purified, radioactively labeled anti(human ferritin). Unreacted labeled antibodies remain in solution and are washed away. As the amount of ferritin increases, the radioactivity in the solid-phase increases. Factors affecting the assay were evaluated including (a) concentration and stability of solid-phase antibody, (b) variation in temperature, reaction times, and reagent concentrations, (c) stability and storage of antibody coated tubes, (d) effect of tube washing cycles, (e) effect of serum proteins and anticoagulants, (f) organ specificity of ferritin.A paradoxical fall in dose-response was seen at high dose. Statistical analysis, dose interpolation, and automatic data processing were carried out by a generalization of the logit/log method and computer programs used in conventional radioimmunoassay. The dependence of the variance on the position on the dose-response curve is different than that seen in radioimmunoassay systems and is reflected in a greater effective assay range. 2-Site IRMA may also have advantages in reagent stability, specificity, antigen protection, and suitability for automation. The properties of 2-site IRMA are closely related to the usual IRMA assay system, but 2-site IRMA is more economical in antigen, is unlikely to be subject to deleterious allosteric reactions, and has a lower zero dose-response (0.5–2% of the total radioactivity in the system). 相似文献
3.
ULTRASTRUCTURAL LOCALIZATION OF INTRACELLULAR ANTIGEN USING ENZYME-LABELED ANTIBODY FRAGMENTS 总被引:7,自引:1,他引:6
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The efficiency of small enzyme-labeled tracers for the demonstration of intracellular antigen was investigated in tissues fixed with picric acid-formaldehyde. The influence of fixation on the immunological activity was tested in vitro by radial immunodiffusion. The experimental model consisted of newborn pig jejunum after absorption of ferritin from the intestinal lumen. Ferritin was located after 1 hr in vacuoles scattered in the cytoplasm of the absorptive cells and represented an easily recognizable intracellular antigen. After immunohistochemical treatments with antiferritin preparations, the distribution of labeling enzyme reaction product was examined by morphometry. The ratio of the labeled volume to the total volume of vacuoles containing ferritin indicated the degree of specific labeling of the antigen. In both direct and indirect methods, the degree of labeling was low when enzyme-labeled immunoglobulin G was the tracer. With antigen binding fragments (Fab), the labeling was significantly increased. In the indirect method, the degree of labeling was influenced by the first-step reagents. Onlywhen the serum titer was optimum was a high degree of labeling obtained. With antigen binding fragments or papain-digested serum the effect of the titer was negligible and maximum labeling was achieved. In both methods, with peroxidase as the labeling enzyme, a diffuse nonspecific deposition of reaction product was observed. This could be avoided by using cytochrome c instead. 相似文献
4.
J. H. W. M. Rombout A. A. van den Berg C. T. G. A. van den Berg P. Witte E. Egberts 《Journal of fish biology》1989,35(2):179-186
Mucosal and systemic (serum) immune responses were studied after oral, anal or intramuscular (i.m.) immunization with particulate ( Vibrio anguillarum ) or soluble (ferritin) antigen. Antigen specific antibodies were found by ELISA in skin mucus after repeated oral or anal administration of bacteria, but not after immunization with ferritin. Daily feeding with bacteria did not give detectable antibodies in serum, while regular oral administration of ferritin resulted in an increase of specific antibodies during the first 3 weeks. From that time immunosuppression was observed, as the antibody titre decreased despite the continued ferritin feeding. Immunosuppression was also found after a second anal intubation or i.m. injection with ferritin, independent of the route of priming (i.m. or anal). On the contrary, a second anal intubation of bacteria resulted in a secondary serum response. These results combined with those reported in Parts I and II of the study indicate an important immunological role for the second gut segment. Because mucosal as well as serum responses can be obtained by anal immunization with bacteria, the significance for oral vaccination is discussed. 相似文献
5.
Daniel Radoux Ernst Heinen Cécile Kinet-Denoël Elisabeth Tihange Léon Simar 《Cell and tissue research》1984,235(2):267-274
Summary Horse-spleen ferritin or bovine serum albumin conjugated to colloidal gold (BSA-gold) were injected subcutaneously in preimmunized mice. In draining lymph nodes both antigens were located in macrophages or between the cytoplasmic processes of follicular dendritic cells (FDC). Some of the antigens remained trapped on FDC until day 31 after injection. Simultaneous injection of both antigens showed that they were located between the infoldings of the same FDC. These cells are thus able to retain at least two different antigens on their surface. The peculiar arrangement of ferritin between the cytoplasmic infoldings suggests that this antigen is fixed on both cell membranes by specific antibodies. The trapped immune complexes could thus stabilize the FDC membrane system.The antigen retention requires the presence of specific antibodies since BSA-gold or ferritin injected without preimmunization were not found between FDC processes. Nonantigenic materials, such as colloidal gold or carbon particles, are not trapped by FDC, except when injected in large amounts.The antigens were trapped on the surface of FDC, however unfrequently in close contact with lymphocytes. FDC might protect lymphocytes against an excess of immune complexes and act as regulators of contacts between lymphocytes and immune complexes.Abbreviations BSA
bovine serum albumin
- BSA-gold
BSA conjugated to colloidal gold particles
- FDC
follicular dendritic cells 相似文献
6.
Neurons in the somatic pretectum receive input from the dorsal column nuclei (DCN) and project to a comparable "somatic" portion of the dorsal accessory nucleus of the inferior olive (DAO). This somatic DAO is reciprocally connected with the anterior interpositus nucleus of the cerebellum. One question that arises is whether this circuitry is further controlled by an output specifically from the anterior interpositus nucleus to the somatic pretectum. Wheatgerm agglutinin conjugated to horseradish peroxidase was injected into various parts of the cat pretectum. Injection sites were interpreted as including the somatic pretectum if neurons in the DCN were retrogradely labeled and if anterograde terminal labeling occurred in somatic DAO. The locations of retrogradely labeled neurons within the deep cerebellar nuclei were then compared in cases in which the injection sites included or excluded the somatic pretectum. In all cases in which the injection site included the somatic pretectum, retrogradely labeled neurons were observed in the anterior interpositus nucleus as well as in the lateral cerebellar nuclei. In some of these cases, neurons in the posterior interpositus and medial nuclei were also labeled. In contrast, in cases in which the pretectal injection site was located outside or at the border of the somatic pretectum, retrogradely labeled neurons were observed only in the lateral, posterior interpositus, and medial nuclei. Thus, the somatic pretectum appears to receive input primarily from neurons in the anterior interpositus nucleus, along with some input from neurons in the lateral nucleus. These results provide additional evidence for a pathway through the DCN in which sequentially processed somatic information has access to and is modulated by cerebellar circuitry. The existence of such a pathway supports the conclusion that neurons in the DCN convey somatic information important not only for cutaneous, kinesthestic, and other bodily sensations, but also for the control of movement. 相似文献
7.
Antibodies against a lysosomal membrane antigen recognize a prelysosomal compartment involved in the endocytic pathway in cultured prolactin cells 总被引:8,自引:5,他引:3
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Antibodies against a lysosomal membrane antigen (A-Ly-M) have recently been obtained and characterized (Reggio, H., D. Bainton, E. Harms, E. Coudrier, and D. Louvard, 1984, J. Cell Biol., 99:1511-1526). They recognize a 100,000-mol-wt antigen immunologically related to a purified [H+,K+]ATPase from pig gastric mucosa. In the present study, we have localized this antigen during adsorptive endocytosis in rat prolactin cells in culture using cationized ferritin (CF) as a tracer. CF was rapidly internalized (after 5 min) in coated pits and vesicles that were labeled by antibodies against clathrin. The tracer was then delivered (after 15 min) to vacuoles and multivesicular bodies. These structures were labeled with A-Ly-M. These organelles were devoid of acid phosphatase activity. At later stages (after 30 min) CF was observed within larger structures that were strongly stained by A-Ly-M and displayed a strong acid phosphatase activity. These findings clearly indicate that A-Ly-M react with prelysosomal and lysosomal compartments involved in the endocytic pathway in cultured prolactin cells. The membrane of these structures therefore contains antigenic determinant(s) related to the 100,000-mol-wt polypeptide. Our results suggest that the prelysosomal structure stained by A-Ly-M may represent in GH3 cells the acidic prelysosomal compartment recently described in the early steps of endocytosis in other cell types (Tycko, B., and F. R. Maxfield, 1982, Cell, 28:643-651). 相似文献
8.
Stage-specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies 总被引:5,自引:0,他引:5
We have identified three germ cell surface carbohydrate antigens that exhibit a common, stage-specific pattern of expression during spermatogenesis in the mouse. IgM-class monoclonal antibodies designated "J1," "C6," and "A5" were absorbed by adult testis, but not by any adult somatic tissue tested. In indirect immunofluorescence assays using collagenase-dissociated prepuberal and adult testicular cells, these antibodies labeled the surfaces of early and late pachytene spermatocytes and round spermatids. Gonocytes from fetal and neonatal testes were not labeled. In paraffin sections of prepuberal and adult testes, sialidase treatment exposed antigens recognized by antibodies C6 and A5 on preleptotene, leptotene, and zygotene spermatocytes located near the perimeter of seminiferous tubules. The determinants recognized by antibodies J1, C6, and A5 were characterized partially using a sugar hapten inhibition assay. The binding of J1 to adult testicular cells was inhibited specifically by N-acetylglucosamine and the binding of both C6 and A5 was inhibited by N-acetyllactosamine. The glycoconjugates recognized by J1, C6, and A5 eluted from gel filtration columns with an apparent molecular weight greater than 1 X 10(6) and were sensitive to endo-beta-galactosidase (keratanase) treatment. The apparent high molecular weight of these glycoconjugates was confirmed by immunolabeling Western blots of testis extracts separated by SDS-polyacrylamide gel electrophoresis. The results suggest that polylactosamine (keratan) glycoconjugates of high molecular weight are associated with the plasma membranes of meiotic and haploid male germ cells. The effects of sialidase on antibody labeling patterns suggest that changes in cell surface sialylation accompany the transition of early meiotic germ cells to pachytene spermatocytes during spermatogenesis. 相似文献
9.
Ultrastructural visualization of anionic sites on mycoplasma membranes by polycationic ferritin. 总被引:7,自引:3,他引:4
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Anionic sites on mycoplasma membranes were visualized in the electron microscope by a polycationized ferritin derivative. The technique of thin sectioning was used. Staining prior to fixation led to clustering of ferritin granules on the mycoplasma cell surface. On glutaraldehyde-fixed Mycoplasma mycoides subsp. capri, M. gallisepticum, M. pneumoniae, and Acholeplasma laidlawii, the anionic sites were uniformly distributed over the entire membrane surface. M. hominis did not bind the polycationic ferritin label. Chemical and enzymatic treatments of the mycoplasmas indicated that the anionic sites may be lipid phosphate groups. Isolated M. mycoides subsp. capri membranes were labeled exclusively on only one membrane surface, presumably the outer one. Liposomes prepared from diphosphatidylglycerol and phosphatidylcholine were also labeled by the polycationic ferritin. 相似文献
10.
Evidence for conservation of ferritin sequences among plants and animals and for a transit peptide in soybean 总被引:13,自引:0,他引:13
M Ragland J F Briat J Gagnon J P Laulhere O Massenet E C Theil 《The Journal of biological chemistry》1990,265(30):18339-18344
Ferritin is a large multisubunit protein that stores iron in plants, animals, and bacteria. In animals, the protein is mainly cytoplasmic and is highly conserved, while in plants ferritin is found in chloroplasts and other plastids. Ferritin is synthesized in plants as a larger precursor of the mature subunit. There is no sequence information for ferritin from plants, except an NH2-terminal peptide of 35 residues which shows little similarity to any known ferritin sequences or transit peptides (Laulhere, J. P., Laboure, A. M., and Briat, J. F. (1989) J. Biol. Chem. 264, 3629-3635). To understand the genetic origin and the location of ferritin synthesis in plant cells, as well as the structure of ferritin from plants, we have sequenced both CNBr peptides from pea seed ferritin and nucleotides of a soybean hypocotyl ferritin cDNA, identified using a frog ferritin cDNA as a probe. Comparison of pea and soybean sequences showed an identity of 89%. Alignment of the plant ferritin sequences with animal ferritins showed 55-65% sequence identity in the common regions. However, a peptide of 28 amino acids extended the NH2 terminus of the plant ferritins. Furthermore, the cDNA encoded additional amino acids which appear to be a transit peptide. None of the sequences in soybean ferritin were found in the tobacco chloroplast genome, suggesting, as does the transit peptide, a nuclear location of ferritin gene(s) in plants. Plant ferritin mRNA is 400-500 nucleotides longer than animal ferritin mRNAs, a difference accounted for in part by the extra peptides encoded. The size of soybean ferritin mRNA was constant in different tissues but expression varied in different tissues (leaf greater than hypocotyl). Thus, higher plants and animal ferritins display sequence homology and differential tissue expression. An ancient, common progenitor apparently gave rise to contemporary eukaryotic ferritins after specific modifications, e.g. transport to plasmids. 相似文献
11.
Dr. Richard M. Pino 《Cell and tissue research》1986,243(1):157-164
Summary The location and chemical composition of anionic sites on the endothelium of the choriocapillaris was investigated with cationic ferritin and enzyme digestion techniques. Cationic ferritin administered intravenously initially labeled essentially all fenestral diaphragms. Within 30 min after injection, no diaphrams remained labeled, but they could be relabeled by a second cationic ferritin injection. Following perfusion of cationic ferritin, the entire luminal front of the endothelium was labeled: the plasmalemma and fenestral, vesicle, and channel diaphragms. Perfusion of neuraminidase or chondroitinase did not affect subsequent cationic ferritin binding. In contrast, heparitinase removed anionic sites on all structures except fenestral diaphragms. Cationic ferritin did not mark the endothelium following heparinase digestion. All sites were cleaved with pronase E. These results indicate that heparin is the anionic moiety on fenestral diaphragms while the glycocalcyces of the plasmalemma and vesicle and channel diaphragms are rich in a heparan sulfate proteoglycan. Furthermore, since the heparan sulfate localized to these structures was digested by both heparinase and heparitinase, it is in a form similar to heparin. These findings demonstrate that the endothelium of the choriocapillaris bears cell-surface anionic components that are different than those described for fenestrated endothelia lining other vascular beds.Supported by NIH EY 03776 相似文献
12.
13.
Both the J5 and BA-3 monoclonal antibodies are considered to be specific for epitopes on the common acute lymphoblastic leukemia antigen (CALLA). Flow-cytometric analyses of three cell lines and one normal bone marrow sample using these antibodies as CALLA markers demonstrated that J5-labeled cells were always brighter than those labeled with BA-3, and that the ratio of their fluorescence intensities varied widely in the different systems. Furthermore, one of the lines, RPMI 8226, while positive for J5, appeared to be negative when labeled with BA-3, except for a slight displacement of the fluorescence distribution relative to the control. A possible explanation for the observed results is that the BA-3 binding epitope or epitopes on CALLA may vary in their number and/or accessibility to the antibody. These observations suggest that the use of a single monoclonal antibody to detect a cell surface antigen may be misleading, particularly when a negative result is obtained. 相似文献
14.
An aberrant retinal pathway and visual centers in Xenopus tadpoles share a common cell surface molecule, A5 antigen 总被引:1,自引:0,他引:1
A monoclonal antibody A5 (MAb-A5), which was raised against Xenopus tadpole tectal cells, recognizes a cell surface-related protein molecule (A5 antigen) expressed on the visual centers of Xenopus tadpoles (S. Takagi, T. Tsuji, T. Amagai, T. Takamatsu, and H. Fujisawa, 1987, Dev. Biol. 122, 90-100). The present immunohistochemistry using MAb-A5 indicated that, in addition to the visual centers, A5 antigen was expressed on the general somatic sensory tract in the medulla and spinal cord of Xenopus tadpoles. As the general somatic sensory tract has been shown to be a pathway for ectopically transplanted retinal axons (M. Constantine-Paton and R. R. Capranica, 1976, J. Comp. Neurol. 170, 17-32; M. J. Katz and R. J. Lasek, 1979, J. Comp. Neurol. 183, 817-832), we examined whether retinal axons transplanted close to the spinal cord or medulla preferentially grow into the A5 antigen-positive general somatic sensory tract. We performed eye transplantation at embryonic stages and detected precise locations and trajectories of transplanted retinal axons within the medulla and spinal cord in tadpoles after filling retinal axons with horseradish peroxidase (HRP). HRP histochemistry in combination with MAb-A5 immunohistochemistry indicated that almost all HRP-filled transplanted retinal axons joined the A5 antigen-positive general somatic sensory tract. These findings suggest the involvement of A5 antigen in specific cell-cell recognition between retinal axons and their targets. 相似文献
15.
Morphogenesis of Type 2 Parainfluenza Virus Examined by Light and Electron Microscopy 总被引:9,自引:12,他引:9
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The development of type 2 parainfluenza virus in HeLa and stable human amnion cells was examined by use of antisera labeled with fluorescein and ferritin. Serum containing antibody predominantly to soluble viral antigen gave specific fluorescence which was first detectable in small cytoplasmic foci 8 to 10 hr after initiation of infection. By 20 to 24 hr, when the production of infective virus and hemagglutinin was maximal, large perinuclear aggregates of fluorescence were observed which corresponded in distribution and time of appearance to the eosinophilic inclusions seen in similar preparations stained with azure eosin. The inclusions, examined by electron microscopy, were composed of fibrils, presumably viral ribonucleoprotein, which specifically bound the antibody labeled with ferritin. With antiserum to concentrated virus, on the other hand, specific fluorescence was most marked at the surface of infected cells. Foci of fluorescence at the surface represented segments of membrane which had become differentiated morphologically and antigenically to resemble the viral envelope. These were the sites where mature virions appeared. The latter exhibited marked pleomorphism; in some instances, particles were formed which lacked recognizable internal fibrils but which possessed an enclosing membrane bearing viral antigen. Filamentous forms showing an organized internal structure were also observed at the cell surface, but were never encountered in negatively stained preparations. No clear relationship between these filaments and the spherical or oval forms could be established. In negatively stained preparations, nucleocapsid released by rupture of viral particles was similar in appearance to that reported for other paramyxoviruses. It seems probable that this component has a helical configuration. 相似文献
16.
Nymalm Y Kravchuk Z Salminen T Chumanevich AA Dubnovitsky AP Kankare J Pentikäinen O Lehtonen J Arosio P Martsev S Johnson MS 《Journal of structural biology》2002,138(3):171-186
The antiferritin variable light domain (VL) dimer binds human spleen ferritin ( approximately 85% L subunits) but with approximately 50-fold lower affinity, K(a)=4 x 10(7) x M(-1), than the parent F11 antibody (K(a)=2.1 x 10(9) x M(-1)). The VL dimer does not recognize either rL (100% L subunits) or rH (100% H subunits) human ferritin, whereas the parent antibody recognizes rL-ferritin. To help explain the differences in ferritin binding affinities and specificities, the crystal structure of the VL domain (2.8A resolution) was determined by molecular replacement and models of the antiferritin VL-VH dimer were made on the basis of antilysozyme antibody D1.3. The domain interface is smaller in the VL dimer but a larger number of interdomain hydrogen bonds may prevent rearrangement on antigen binding. The antigen binding surface of the VL dimer is flatter, lacking a negatively charged pocket found in the VL-VH models, contributed by the CDR3 loop of the VH domain. Loop CDR2 (VL dimer) is located away from the antigen binding site, while the corresponding loop of the VH domain would be located within the antigen binding site. Together these differences lead to 50-fold lower binding affinity in the VL dimer and to more restricted specificity than is seen for the parent antibody. 相似文献
17.
This research documents in situ wastewater biofilm formation, structure, and physiochemical properties as revealed by scanning and transmission electron microscopy. Cationized ferritin was used to label anionic sites of the biofilm glycocalyx for viewing in thin section. Wastewater biofilm formation paralleled the processes involved in marine biofilm formation. Scanning electron microscopy revealed a dramatic increase in cell colonization and growth over a 144-h period. Constituents included a variety of actively dividing morphological types. Many of the colonizing bacteria were flagellated. Filaments were seen after primary colonization of the surface. Transmission electron microscopy revealed a dominant gram-negative cell wall structure in the biofilm constituents. At least three types of glycocalyces were observed. The predominant glycocalyx possessed interstices and was densely labeled with cationized ferritin. Two of the glycocalyces appeared to mediate biofilm adhesion to the substratum. The results suggest that the predominant glycocalyx of this thin wastewater biofilm serves, in part, to: (i) enclose the bacteria in a matrix and anchor the biofilm to the substratum and (ii) provide an extensive surface area with polyanionic properties. 相似文献
18.
Electron microscopic examination of wastewater biofilm formation and structural components. 总被引:11,自引:7,他引:4
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This research documents in situ wastewater biofilm formation, structure, and physiochemical properties as revealed by scanning and transmission electron microscopy. Cationized ferritin was used to label anionic sites of the biofilm glycocalyx for viewing in thin section. Wastewater biofilm formation paralleled the processes involved in marine biofilm formation. Scanning electron microscopy revealed a dramatic increase in cell colonization and growth over a 144-h period. Constituents included a variety of actively dividing morphological types. Many of the colonizing bacteria were flagellated. Filaments were seen after primary colonization of the surface. Transmission electron microscopy revealed a dominant gram-negative cell wall structure in the biofilm constituents. At least three types of glycocalyces were observed. The predominant glycocalyx possessed interstices and was densely labeled with cationized ferritin. Two of the glycocalyces appeared to mediate biofilm adhesion to the substratum. The results suggest that the predominant glycocalyx of this thin wastewater biofilm serves, in part, to: (i) enclose the bacteria in a matrix and anchor the biofilm to the substratum and (ii) provide an extensive surface area with polyanionic properties. 相似文献
19.
Ascorbic acid inhibits lysosomal autophagy of ferritin 总被引:2,自引:0,他引:2
K R Bridges 《The Journal of biological chemistry》1987,262(30):14773-14778
Ascorbic acid retards ferritin degradation in K562 erythroleukemia cells leading to an increase in the availability of cellular iron (Bridges, K. R., and Hoffman, K. E. (1986) J. Biol. Chem. 261, 14273-14277). To explore the mechanism of this effect, the influence of ascorbate on subcellular ferritin distribution was examined. Cellular ferritin was pulse-labeled with 59Fe for 2 h, after which the cells were hypotonically lysed and fractionated on an 8% Percoll density gradient. Immediately after the labeling, all of the ferritin was in the cytoplasmic fractions at the top of the gradient. When the labeling was followed by a 24-h period of growth, a portion of the ferritin shifted to the lysosome-associated fractions at the bottom of the gradient, consistent with lysosomal autophagy of cytoplasmic ferritin. When ascorbate was added to the culture medium during the 24-h incubation, the magnitude of the shift was reduced. This process was also examined by size-fractionation of the contents of labeled cells using a Sepharose CL-6B column. Immediately after labeling, ferritin emerged from the column in two peaks, indicating the existence of both ferritin monomer and aggregates within the cytoplasm. After a 24-h period of growth, the monomer peak disappeared, while a new ferritin peak coincident with lysosomes emerged again, indicative of lysosomal autophagy of ferritin. In cells cultured with ascorbate for 24-h, there was a marked attenuation of the shift of ferritin to the lysosomal fractions. The monomer peak disappeared, as in the controls, but there was instead, an accumulation of ferritin as cytoplasmic aggregates. The total ferritin content of the ascorbate-treated cells was increased by 4-fold over that of the control. These experiments indicate that ascorbate blocks the degradation of cytoplasmic ferritin by reducing lysosomal autophagy of the protein. The access to the cell of the potentially toxic iron stored within the ferritin molecule is thereby increased. 相似文献
20.
K L Goldenthal K Hedman J W Chen J T August P Vihko I Pastan M C Willingham 《The journal of histochemistry and cytochemistry》1988,36(4):391-400
We have used electron microscopic immunocytochemistry to compare the distribution of LAMP-1, a marker for lysosomal membranes, with the intracellular localization of alpha 2-macroglobulin (alpha 2-M) and transferrin at various time points after their endocytosis into cultured NIH 3T3 cells. The purposes of this study were (a) to determine how soon endocytic ligands reach lysosomal organelles, (b) to examine whether the intermediate endocytic vesicles gained lysosomal markers gradually or in a precipitous, discrete event, and (c) to examine the relationship, if any, between the pathway of recycling ligands and lysosomes. At early time points (0-5 min) after initiation of endocytosis, most structures containing alpha 2-M labeled with colloidal gold (receptosomes) were not labeled by anti-LAMP-1 detected using ferritin bridge or peroxidase immunocytochemistry. At late time points (greater than or equal to 15 min), the structures containing alpha 2-M (lysosomes) were strongly labeled by anti-LAMP-1. In contrast, transferrin that was directly labeled with ferritin was mostly located in LAMP-1-negative structures at all time points studied. The proportion of alpha 2-M-gold containing vesicles strongly labeled for LAMP-1 roughly paralleled the proportion of alpha 2-M-gold-containing structures positive for cytochemically detectable acid phosphatase. Our data indicate that ligands such as transferrin that are internalized through coated pits and receptosomes, but not delivered to lysosomes, do not traverse a lysosomal organelle compartment as marked by LAMP-1 content. Ligands such as alpha 2-M that are destined for lysosomal delivery reach a LAMP-1-positive organelle compartment only after they traverse LAMP-1-negative, non-lysosomal vesicles previously described as receptosomes. 相似文献