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PubMed1993

Detection of DNA in the nucleoids of chloroplasts and mitochondria in Euglena gracilis by immunoelectron microscopy.

DNA in the nucleoids of chloroplasts and of mitochondria in Euglena gracilis was detected with anti-DNA antibodies by immunoelectron microscopy. After treatment with the antibodies, DNA in these organelles combined with gold particles that had been coated with anti-IgM antibodies such that it was possible to trace the outlines of the nucleoids. Nucleoids in chloroplasts appeared to be composed of twisted threads 50-70 nm in diameter. The twisted threads were entangled to form thicker nodes of 100-200 nm diameter. Most nucleoids in mitochondria were spherical or ovoid, 70-130 nm in diameter. Nucleoids both in chloroplasts and in mitochondria contained cores with which DNA threads were in tight contact. The structure of the nucleoids was very different from those previously observed by conventional electron microscopy.

Animals↗

PubMed1987

Mitochondrial myopathy involving ubiquinol-cytochrome c oxidoreductase (complex III) identified by immunoelectron microscopy.

The distribution of respiratory chain complexes in bovine heart and human muscle mitochondria has been explored by immunoelectron microscopy with antibodies made against bovine heart mitochondrial proteins in conjunction with protein A-colloidal gold (12-nm particles). The antibodies used were made against NADH-coenzyme Q reductase (complex I), ubiquinol cytochrome c oxidoreductase (complex III), cytochrome c oxidase, core proteins isolated from complex III and the non-heme iron protein of complex III. Labeling of bovine heart tissue with any of these antibodies gave gold particles randomly distributed along the mitochondrial inner membrane. The labeling of muscle tissue from a patient with a mitochondrial myopathy localized by biochemical analysis to complex III was quantitated and compared with the labeling of human control muscle tissue. Complex I and cytochrome c oxidase antibodies reacted to the same level in myopathic and normal muscle samples. Antibodies to complex III or its components reacted very poorly to the patient's tissue but strongly to control muscle samples. Immunoelectron microscopy using respiratory chain antibodies appears to be a promising approach to the diagnosis and characterization of mitochondrial myopathies when only limited amounts of tissue are available for study.

Electron Transport↗

PubMed1996

Dual over-expression pattern of membrane-type metalloproteinase-1 in cancer and stromal cells in human gastrointestinal carcinoma revealed by in situ hybridization and immunoelectron microscopy.

Membrane-type metalloproteinase-I (MTI-MMP) is a transmembrane metalloproteinase, which activates pro-gelatinase A. There has been disagreement as to whether the cell types expressing MTI-MMP are cancer cells or stromal fibroblasts. Using human gastrointestinal carcinomas, the present study disclosed the tissue localization of MTI-MMP mRNA by in situ hybridization and ultrastructural localization of its protein by immunoelectron microscopy. In normal colon and stomach tissues, MTI-MMP mRNA and protein were negative or faintly positive both in epithelial cells and in stromal fibroblasts, except in the fundic gland of the stomach, which showed the positivity for MTI-MMP. In contrast, gastrointestinal cancer tissue showed over-expression of MTI-MMP mRNA and protein both in cancer cells and in stromal cells (fibroblasts). Stromal fibroblasts also expressed mRNA for gelatinase A and type-I procollagen. Double immunohistochemistry revealed that macrophages were also positive for MTI-MMP. Immunoelectron microscopy showed that MTI-MMP was localized along the plasma membrane of cancer cells and macrophages and in rough endoplasmic reticulum of fibroblasts. The present study reveals a dual over-expression pattern of MTI-MMP both in cancer cells and in stromal fibroblasts; the expression in cancer cells may be related to the invasive growth, whereas that in fibroblasts may be related to the tissue remodeling process caused by invasive growth of cancer cells.

Carcinoma↗

PubMed1993

Topology of Na,K-ATPase alpha subunit epitopes analyzed with oligopeptide-specific antibodies and double-labeling immunoelectron microscopy.

Using four oligopeptide-specific polyclonal antibodies, we mapped the alpha subunit of Na,K-ATPase by double-labeling immunoelectron microscopy combined with negative staining. The results show that the epitopes of the N-terminus (Gly1-His13), C-terminus (Ile1002-Tyr1016) and Leu815-Gln828 are located on the same face of crystallized Na,K-ATPase membranes from pig kidney, whereas the epitope Asn889-Gln903 is present on the opposite side. The present study demonstrates the cytoplasmic location of C-terminus and that Leu815-Gln828 is exposed on the cytoplasmic and Asn889-Gln903 on the extracellular side. The results are consistent with an eight- or ten-segment model, and support the existence of an M5/M6 loop and the presence of one transmembrane segment between Leu815-Gln828 and Asn889-Gln903.

Amino Acid Sequence↗

PubMed1995

Ultrastructural effects of pressure stress to the nucleus in Saccharomyces cerevisiae: a study by immunoelectron microscopy using frozen thin sections.

The effects of hydrostatic pressure on subcellular structures, particularly the nucleus, of Saccharomyces cerevisiae were investigated by immunoelectron microscopy. Cells were treated with hydrostatic pressure from 0.1 to 400 MPa for 10 min at room temperature. Frozen thin sections of the cells revealed that spindle pole bodies disappeared at 100 MPa. At 150 MPa, the deposition of gold particles for anti alpha-tubulin was noticed in the nucleus, although the filamentous structure of microtubules was lost. At 200 MPa, fewer gold particles were scattered in the nucleus and the nuclear membrane in several portions was also observed to be open at 300 MPa. These results show that elements of the nuclear division apparatus were susceptible to pressure stress, particularly spindle pole bodies and microtubules. The damage to spindle pole bodies, microtubules, and nuclear membrane caused by pressure stress was followed by the inhibition of nuclear division. After the release of pressure, the spindle pole bodies and microtubules of pressurized cells at below 200 MPa regained their normal appearance at 24 h.

Cell Division↗

PubMed1997

Immunoelectron microscopy on epoxy sections without deplasticizing to detect glomerular immunoglobulin and complement deposits in renal diseases.

Twenty renal biopsies were studied by immunoelectron microscopy (IEM) after embedding in epoxy resin. Immunogold labeling for immunoglobulins and complement C3 was performed on the epoxy sections, which were not subjected to any kind of etching or deplasticizing prior to the immunolabeling. The concentration of accelerator, DMP-30 (Tri (Dimethyl Amino Methyl) Phenol), was increased in the infiltration and embedding steps far beyond the values normally used to make immunolabeling of these antigens possible on epoxy sections. The sections were stained with tannic acid accompanied by uranyl acetate and lead citrate. Immunofluorescence (IF) for light microscopy was carried out on frozen sections of parallel tissue samples. Some cases with IgA-nephritis demonstrated a higher sensitivity for IEM than IF, in the sense that smaller amounts of antigen were detectable with IEM. Ultrastructural preservation with this method was approximately the same as that usually seen on epoxy-embedded material. By combining excellent immunolabeling with nearly optimal ultrastructural morphology in one procedure, this method is useful particularly in situations where the material available is limited, such as in studies of renal biopsies. As far as we know, this is the first time that immunoglobulins have been satisfactorily immunolabeled on epoxy sections without etching or deplasticizing.

Complement C3c↗

PubMed1992

Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy.

The membrane compartments responsible for Golgi functions in wild-type Saccharomyces cerevisiae were identified and characterized by immunoelectron microscopy. Using improved fixation methods, Golgi compartments were identified by labeling with antibodies specific for alpha 1-6 mannose linkages, the Sec7 protein, or the Ypt1 protein. The compartments labeled by each of these antibodies appear as disk-like structures that are apparently surrounded by small vesicles. Yeast Golgi typically are seen as single, isolated cisternae, generally not arranged into parallel stacks. The location of the Golgi structures was monitored by immunoelectron microscopy through the yeast cell cycle. Several Golgi compartments, apparently randomly distributed, were always observed in mother cells. During the initiation of new daughter cells, additional Golgi structures cluster just below the site of bud emergence. These Golgi enter daughter cells at an early stage, raising the possibility that much of the bud's growth might be due to secretory vesicles formed as well as consumed entirely within the daughter. During cytokinesis, the Golgi compartments are concentrated near the site of cell wall synthesis. Clustering of Golgi both at the site of bud formation and at the cell septum suggests that these organelles might be directed toward sites of rapid cell surface growth.

Biological Transport↗

PubMed1997

Detection of intracellular interleukin-8 in human mast cells: flow cytometry as a guide for immunoelectron microscopy.

The chemokine interleukin-8 (IL-8) mediates infiltration and adhesion of neutrophils during inflammatory processes. We have previously shown that this cytokine can be produced and released by normal and leukemic human mast cells (HMC-1 cells). To assess whether and to what extent this cytokine is stored intracellularly, we investigated production and localization of IL-8 at the single-cell level by combined use of flow cytometry (FACS) and immunoelectron microscopy. Conditions necessary for optimal fixation and permeabilization of HMC-1 cells were determined by measuring changes in cell-specific light scatter parameters and by estimating cellular uptake of propidiumiodide (PI). In this way, we were able to detect IL-8 with a monoclonal antibody in stimulated cells that were microwave-fixed with a combination of paraformaldehyde (4%) and glutaraldehyde (0.1%), followed by permeabilization with saponin (0.025%). FACS analysis revealed time-dependent synthesis of IL-8 with at most 50% positively stained cells at 8-12 hr after stimulation. For pre-embedding immunogold electron microscopy, cells were treated according to the protocol established by flow cytometry. IL-8 was found to be located in specific cytoplasmic, electron-dense granules of stimulated HMC-1 cells. These results confirm and extend our previous findings by demonstrating IL-8 expression in HMC-1 cells at the single-cell level. In addition, we propose that quantitative FACS can be reliably used in a timesaving manner to establish appropriate conditions for pre-embedding immunoelectron microscopy of intracellular antigens.

Antibodies, Monoclonal↗

PubMed1993

Localization of gap junction proteins, connexins 32 and 26, in rat and guinea pig liver as revealed by quick-freeze, deep-etch immunoelectron microscopy.

By use of site-specific antibodies against synthetic oligopeptides, we examined the localizations of the gap junction proteins connexin 32 (Cx32) and connexin 26 (Cx26) in rat and guinea pig liver. Double-labeling immunofluorescence microscopy revealed that in guinea pig liver both proteins were spread throughout the liver lobules and seemed to localize together within the same gap junction plaque. In rat liver, co-localization of both Cx32 and Cx26 in the same plaques was also suggested in periportal zones. Quick-freeze, deep-etch immunoelectron microscopy showed that immunolabeling of isolated guinea pig liver gap junction plaques with either Cx32 or Cx26 antiserum yielded complete and dense antibody decoration of the cytoplasmic surface of the plaques. In isolated rat liver plaques, the cytoplasmic surfaces were densely decorated with Cx32 antiserum, whereas Cx26 labeling yielded diffuse decoration with variable intensity of the plaques. In both species we did not observe any focal or patchy clusters of the labeling in any plaques examined. Double-labeling immunoelectron microscopy confirmed that both Cx32 and Cx26 are co-localized in the same gap junction plaques. These results suggest that in hepatocytes expressing both Cx32 and Cx26, both types of gap junction proteins are not segregated but intermingle randomly within the same plaques.

Amino Acid Sequence↗

PubMed1993

Ultrastructural changes of extracellular matrices in diabetic nephropathy revealed by high resolution scanning and immunoelectron microscopy.

BACKGROUND: Diabetic nephropathy is invariably associated with proteinuria. EXPERIMENTAL DESIGN: To delineate the mechanism(s) of proteinuria in diabetic nephropathy, ultrastructural changes of the glomerular basement membranes (GBMs) were studied by high resolution scanning and immunoelectron microscopy. Acellular glomeruli from diabetic and age-matched control human subjects were prepared by detergent method and subjected to conductive staining, the technique in which the tissues are impregnated with metals rather than surface-coated with metallic alloys for visualization by electron microscopy. Subsequent to conductive staining, the tissues were examined by in-lens field emission scanning electron microscopy. RESULTS: Thirty glomeruli, each from the control and diabetic groups, were examined by scanning microscopy. In diabetic GBMs, a loose meshwork structure consisting of numerous pores of approximately 8 nm in diameter and distinct strands was observed. In contrast, meshwork structure was not readily discernible in controls and a few pores were observed. Five glomerular capillary loops, each from control and diabetic groups, were examined by immunoelectron microscopy. In controls, heparan sulfate-proteoglycan was localized in the lamina rara interna and externa, and type-IV collagen was distributed throughout the whole width of the GBM. In diabetic GBMs, a relative loss of staining of heparan sulfate-proteoglycan, both in the lamina rara interna and externa of the GBM, was observed. Type IV collagen was distributed in all layers of the thickened GBM, and the absolute number of the immunogold particles was increased. However, immunogold particle density of type IV collagen per unit area was decreased as compared with the control. CONCLUSIONS: These ultrastructural and immunoelectron microscopic changes in the GBM may explain the loss of charge as well as size selectivities of the glomerulus, as observed in diabetic nephropathy associated with proteinuria.

Aged↗

PubMed1990

Structural studies of the contractile tail sheath protein of bacteriophage T4. 2. Structural analyses of the tail sheath protein, Gp18, by limited proteolysis, immunoblotting, and immunoelectron microscopy.

The molecular structure of the T4 phage tail sheath protein, gp18, was studied by limited proteolysis, immunoblotting, and immunoelectron microscopy. Gp18 is extremely resistant to proteolysis in the assembled form of either extended or contracted sheaths, but it is readily cleaved by proteases in the monomeric form, giving rise to stable protease-resistant fragments. Limited proteolysis with trypsin gave rise to a trypsin-resistant fragment, Ala82-Lys316, with a molecular weight of 27K. Chymotrypsin- and thermolysin-resistant fragments were also mapped close to the trypsin-resistant region. The time course of trypsin digestion of the monomeric gp18 as monitored by SDS-polyacrylamide gel electrophoresis and immunoblotting of the gel revealed that the polypeptide chain consisting of 658 amino acid residues is sequentially cleaved at several positions from the C terminus. The N-terminal portion, Thr1-Arg81, was then removed to form the trypsin-resistant fragment. Immunoelectron microscopy revealed that the polyclonal antibodies against the trypsin-resistant fragment bound to the tail sheath. This supported the idea that at least part of the protease-resistant region of gp18 constitutes the protruding part of the sheath protein as previously revealed with three-dimensional image reconstruction from electron micrographs by Amos and Klug [Amos, L. A., & Klug, A. (1975) J. Mol. Biol. 99, 51-73].

Amino Acid Sequence↗

PubMed2000

Immunoelectron microscopy of PCNA as an efficient marker for studying replication times and sites during pollen development.

Here we report for the first time the ultrastructural localization of DNA replication sites in the nucleus of plant cells and the timing of replication through the pollen developmental programme by proliferating cell nuclear antigen (PCNA) immunogold labelling. Replication sites were identified by labelling with anti-PCNA antibodies in fibrils of the interchromatin region close to the condensed chromatin, defining a perichromatin subdomain in the interchromatin space where DNA replication takes place. The same nuclear structures are decorated by anti-BrdU (5-bromo-2'-deoxyuridine) immunogold after short pulses of BrdU labelling. Double immunogold labelling for PCNA and DNA show colocalization on these perichromatin structures. PCNA immunoelectron microscopy also allows correlation of replicative activity with the dynamics of chromatin condensation. DNA replication was also monitored at different phases during pollen development by PCNA immunoelectron microscopy, revealing two peaks of DNA synthesis, at the beginning (early tetrad), and the end (late vacuolate), of microspore interphase. High-resolution autoradiography after [3H]thymidine incorporation also showed high replicative activity at the same two periods of microspore interphase. In the bicellular pollen grain, PCNA immunogold labelling revealed that DNA replication in the generative cell starts at an intermediate stage of pollen maturation, whereas the vegetative nucleus does not replicate and is arrested in G1. The use of anti-PCNA antibodies at the ultrastructural level is an easier, faster and more feasible method than the detection of in vivo-incorporated nucleotides, especially in plant systems with long cell cycles. PCNA immunogold labelling is, therefore, proposed as an efficient marker for mapping the sites and timing of replication at the electron microscopy level.

Autoradiography↗

PubMed1990

Immunoelectron microscopy on the topographical distribution of the poliovirus receptor.

The topographical distribution of the poliovirus receptor on the cell surface was demonstrated by immunoelectron microscopy using monoclonal antibodies and immunogold markers. The receptor appeared in small clusters, which were randomly distributed over the cell surface and along cellular processes. The distribution pattern of the clusters corresponded to that of absorbed and immunogold-labelled poliovirus particles and suggests a multivalent organization of poliovirus binding sites. Freeze-fracturing and ultrathin sectioning did not reveal any specific ultrastructures within the plasma membrane at labelled receptor areas. Incubation of native cells with anti-receptor antibodies did not remove the receptor molecule from the cell surface nor did it induce ultrastructural alterations within the plasma membrane. The antibody-receptor complexes exhibited lateral mobility within the plasma membrane and were able to aggregate into large immune complexes after incubation with a second ligand.

Animals↗

PubMed2006

Evaluation of neoplastic human mast cells by tryptase-immunoelectron microscopy.

AIMS: To analyse and characterize the ultrastructural morphology of normal tissue mast cells (MC) and neoplastic bone marrow MC. METHODS: We have examined the ultrastructure and cytomorphological features of MC derived from cord blood cells, neoplastic bone marrow MC in patients with systemic mastocytosis (SM, n = 4), myelomastocytic leukaemia (MML, n = 2), mast cell leukaemia (MCL, n = 2) and tryptase-positive acute myeloid leukaemia (AML, n = 4). RESULTS: Based on their ultrastructure and morphology, four distinct cell types could be delineated: (i) mature well-granulated tissue MC exhibiting a round central nucleus; (ii) atypical MC type I with oval nuclei, hypogranulated cytoplasm, and prominent surface projections; (iii) immature atypical MC with bi- or polylobed nuclei (atypical MC type II = promastocytes); and (iv) metachromatic blasts. Type I atypical MC were detected in a patient with indolent SM, whereas type II MC and metachromatic blasts were primarily found in MML, MCL and tryptase-positive AML. In all samples examined, the identity of MC could be reconfirmed by immunoelectron microscopy, irrespective of the stage of cell maturation or the disease variant, all types of MC contained tryptase in their cytoplasmic granules. CONCLUSION: Immunoelectron microscopy may be a helpful approach in confirming the identity of neoplastic MC in myeloid neoplasms.

Adult↗

PubMed1997

Localization of germination-specific spore-lytic enzymes in Clostridium perfringens S40 spores detected by immunoelectron microscopy.

The localization of germination-specific spore-lytic enzymes, an amidase and a muramidase, in Clostridium perfringens S40 spores was examined by immunoelectron microscopy with respective antisera raised against the enzymes and a colloidal gold-immunoglobulin G complex. For both antisera, immunogold particles were visualized on the outside of the cortex of dormant spores, and they were not detected in germinated spores and decoated spores.

Antibodies, Fungal↗

PubMed1997

Localization by immunoelectron microscopy of antigens of Chlamydia psittaci suitable for diagnosis or vaccine development.

Two different antigens of serotype 1 Chlamydia psittaci were localized using three immunoelectron microscopy techniques: non-embedding, pre-embedding and post-embedding. The antigens had previously been described as being of potential use in diagnosis (80-90 kDa protein region) and vaccine development (110 kDa protein). The results show a direct relationship between the protective capacity of the antigens and their surface localization on the elementary bodies, which are the infectious form of Chlamydia. The 80-90 kDa protein region is located on the surface of reticulate bodies but not of elementary bodies, where it was located periplasmically, while the 110 kDa protein occurs on the surface of both elementary and reticulate bodies.

Animals↗

PubMed1991

A study of protein A-gold resolution for immunoelectron microscopy.

For the purpose of investigating a topographical correlation between antigen molecules and protein A-gold(PAG) particles which localized as an immunocytochemical probe, the simplest model on a localization pattern of antigen molecules, which were arranged two-dimensionally on a plane surface of the resin, was used. Ultrathin sections of a G-actin layer, which was adsorbed on epoxy resin and was re-embedded subsequently in JB-4 resin, was stained indirectly with rabbit anti-actin antibody and subsequently by PAG. From this immunoelectron microscopy, a histogram (relative frequency, denoted by y vs. relative length, denoted by x) was obtained using a computer-assisted method. For this histogram, a fitting curve was calculated by a least squares optimization and three parameters (H, U, and W) of the curve which could be useful for a study on the topographical organization of antigen molecules were estimated. Parameter H (maximum y of the curve) would reflect the maximum amount of epitopes at x = U. Half width W, which is the width of the curve at y = H/2, would reflect a breath of epitope masses. This fitting curve was separated into two overlapping curves whose Ws were different from each other. The one constituent curve of which value W was smaller than the other was regarded as a unit curve and the other constituent curve could be resolved into many unit curves whose W values are the same. From these unit curves, the resolution power of the immunoelectron microscopy, using a post-embedding procedure of ultrathin sections, was estimated as 58-66 A degrees.

Actins↗

PubMed1992

Phorbol ester- and calcium-induced reorganization of 180-kDa bullous pemphigoid antigen on the ventral surface of cultured human keratinocytes as studied by immunofluorescence and immunoelectron microscopy.

The hemidesmosome is an adhesion structure of the epidermal-dermal junction in keratinocytes. When keratinocytes migrate laterally or upward to differentiate, they must control the formation and disintegration of the hemidesmosomes. When keratinocytes are cultured in low-calcium (below 0.1 mM) medium, all cells behave like basal cells, adhere to the culture dish, and proliferate without differentiation. The calcium addition induces the differentiation. A bullous pemphigoid antigen, 180-kDa BPA, has been shown to be a component of the hemidesmosome. Using a monoclonal antibody to the 180-kDa BPA and a human squamous cell carcinoma cell line (DJM-1 cells), the fate of hemidesmosomes was studied after the addition of calcium to low-calcium-grown cells and 12-tetradecanoylphorbol-13-acetate (TPA) to high-calcium (1.87 mM) grown cells by immunofluorescence and immunoelectron microscopy. The antigen was distributed evenly as fine dots on the entire ventral surface of low-calcium cells, whereas they formed a peculiar, concentric ring or arch arrangement on the ventral surface of high-calcium cells. Immunoelectron microscopy revealed the deposits of gold particles at sites on the membrane surface, where some filamentous or electron-dense materials were associated, although the complete structure of hemidesmosomes was not formed. They deposited directly onto the membrane surface in low-calcium cells and with a distance of 20-50 nm from the membrane surface in high-calcium cells. The calcium addition caused a profound reduction of the 180-kDa BPA-positive area for 30 to 120 min and then formed the high-calcium-ring pattern after 4 to 6 h. A similar calcium response was seen in normal human keratinocytes. TPA (16 nM) treatment caused disintegration of the ring pattern in high-calcium DJM-1 cells. This was inhibited with a protein kinase C (PKC) inhibitor. H7 (20 microM). These results suggest that the hemidesmosome is a dynamic structure and PKC can be one of the major factors in controlling the hemidesmosome, since it is known that the low-high calcium shift induces a calcium influx and a PKC activation, and TPA activates PKC in keratinocytes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗