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PubMed1996

Twofold symmetry of human fibrinogen proved at the beta chain distal domains by monoclonal-immunoelectron microscopy and image analysis.

Using a murine antibody (F7) specific for the C-terminal domain of the beta chain of human fibrinogen combined with electron microscopy and image analysis, we show unequivocally that the epitopes for F7 are at the distal nodules of fibrinogen, equidistant from the center of the molecule and arranged not colinearly with the long axis of the molecule but at opposite sides of it, i.e., following twofold symmetry. Thus, given the monoclonality of the immunochemical probe used and the dimeric nature of the fibrinogen molecule, we can conclude that the distal domains of the two beta chains are arranged in the same manner as these epitopes and, therefore, that the fibrinogen molecule has twofold symmetry. This symmetry pattern found here for F7 is the same as that found recently for the platelet fibrinogen receptor binding sites [Weisel, J. W., Nagaswami, C., Vilaire, G., & Bennett, J. S. (1992) J. Biol. Chem. 23, 16637-16643], located almost certainly at the C-terminal end of the gamma chains, and gives further support to the most accurate model of fibrinogen available so far. We discuss the consequences of this symmetry pattern and of the molecular rigidity of fibrinogen in the actual models of fibrin polymerization and platelet aggregation and adhesion.

Amino Acid Sequence↗
PubMedDate not supplied

A novel embedment-free immunoelectron microscopy technique reveals association of apoptosis-regulating proteins with subcellular structures.

By employing two electron microscopy techniques, postembedding double- and triple immunocytochemical gold-labelling combined with embedment-free electron microscopy (EF-EM), we have detected previously unreported nuclear and cytoplasmic complexes between different proapoptatic proteins in a human cancer cell line COLO 205 stimulated to apoptosis by nimesulide, a specific cyclooxygenase-2 inhibitor. Experiments with the use of double- and triple immunolabelling visualized the colocalization of proapoptotic proteins such as Bax with Bid, Bax with Bid and voltage-dependent anion channel protein (VDAC-1), and Bax with Bid and caspase-8, on organellar membranes and within the nucleus. Application of this technique in combination with EF-EM technique augments our knowledge on the precise identification and relationship of subcellular structures containing Bax, Bid, VDAC-1 and caspase-8.

Adenocarcinoma↗
PubMed2002

Keratinocytes constitutively express the CD95 ligand molecule on the plasma membrane: an in situ immunoelectron microscopy study on ultracryosections of normal human skin.

BACKGROUND: Tissue homeostasis is mainly preserved by cytolytic functions. Cytolytic cells, when expressing the CD95 ligand (Fas-L) molecule on the cell membrane, are able to kill CD95 (Fas)-expressing target cells. Although cultured epidermal keratinocytes (KC) have been shown to express Fas-L, and normal skin has been shown to bear Fas-L mRNA, efforts so far to find possible constitutive Fas-L expression on the cell membrane by resting KC in normal human epidermis (i.e. in a functionally active location) have been inconclusive. OBJECTIVES: The aim of the present study was therefore to show the constitutive expression of Fas-L on the plasma membrane of KC. METHODS: Gold immunoelectronmicroscopy, a highly specific and sensitive immunodetection system, was performed in situ on skin sections obtained by ultracryomicrotomy, without previous embedding (i.e. in conditions strictly similar to the in vivo situation). RESULTS: Relatively few (51.55 +/- 28.61), 10-nm colloidal gold particles were observed at the cell surface of KC in the basal layer of the epidermis and an even smaller (P < 0.005) number of gold granules was detected in the KC of the spinous layer. CONCLUSIONS: Although scanty, the constitutive Fas-L expressed on the surface of KC can bind Fas expressed by possible occasional inflammatory cells entering the epidermis, and kill them, so preventing inflammation. Fas-L-expressing KC could moreover induce apoptosis of epidermal cells bearing viral or neoplastic antigens. Thus, the expression of Fas-L by KC may contribute to the preservation of epidermal homeostasis in vivo.

Apoptosis↗
PubMed2000

Localization of interleukin (IL) -4 but not IL-5 to human mast cell secretory granules by immunoelectron microscopy.

BACKGROUND: Human mast cells synthesize and secrete many cytokines of relevance to the pathogenesis of allergic diseases such as asthma and rhinitis. In particular, interleukin (IL) -4 and IL-5 are likely to play key roles in the development of the inflammatory response that characterizes these diseases. Immunohistochemical studies on human nasal and bronchial mucosal biopsies suggest that IL-4 and IL-5 may be stored preformed in mast cells. OBJECTIVE: To identify whether IL-4 and IL-5 are stored within mast cell secretory granules. METHODS: We used immunogold electron microscopic analysis on bronchial mucosa and lung parenchyma from resected lung specimens, and a nasal mucosal biopsy from a patient with active allergic rhinitis. Samples were fixed in 4% paraformaldehyde plus 0.5% glutaraldehyde and processed into Lowicryl K4M resin by the 'Progressive Lowering of Temperature' technique. Ultrathin sections were stained immunohistochemically by an indirect immunogold method. RESULTS: Immunoreactivity for IL-4, but not IL-5, was localized to the granules of mast cells in all tissue samples. IL-5 was localized to the matrix of eosinophil granules in these samples, but neither cytokine was detected in T cells. IL-4 immunoreactivity increased in the granules of mast cells 24 h after immunoglobulin (Ig) E-dependent activation (mean 17.5 +/- 1.4 gold particles per granule) compared with nonactivated mast cells (mean 6.8 +/- 0.8 gold particles per granule, P < 0.001), suggesting replenishment of stores by newly generated protein. Immunoreactive IL-5 remained undetectable in mast cells 24 h after activation, a time point at which they are known to secrete large quantities of this cytokine. CONCLUSION: Human mast cells store IL-4 within the matrix of their granules. Very few, if any, lung or nasal mast cells store IL-5. A store of preformed IL-4 within mast cell granules is likely to have an important influence during the initiation and maintenance of the allergic immunological response.

Cytoplasmic Granules↗
PubMed2003

Light chain crystal deposition as a manifestation of plasma cell dyscrasias: the role of immunoelectron microscopy.

Light chain crystal deposition disease is a rare and poorly characterized entity that can be confused with a number of different conditions, depending on where the disease process is manifested. The present study explored the role of ultrastructural immunogold labeling in the diagnosis of this condition. Seven cases of light chain crystal deposition (kappa light chain-related) are reported. Immunohistochemistry and immunofluorescence techniques play a rather limited role in the evaluation of these cases, as a result of the inability to detect monoclonal kappa light chains in association with the crystalline structures or high background staining. Ultrastructural labeling is the method of choice to fully characterize these cases. However, surgical pathologists must learn to recognize the findings associated with this condition to avoid misdiagnosis. If the diagnosis is at least suspected, then a complete hematologic workup may identify the underlying plasma cell dyscrasia. It must be emphasized that in some patients the plasma cell dyscrasia does not become clinically manifested until years after the diagnosis of light chain crystal deposition.

Adult↗
PubMedDate not supplied

Subcellular distribution of urokinase and urokinase receptor in human neutrophils determined by immunoelectron microscopy.

A high-affinity receptor for urokinase-type plasminogen activator (uPAR) has been identified on the plasma membrane of a number of different cell types, and has been shown to be important for plasminogen activation, cell adhesion, and possibly signal transduction. uPAR and uPA cosediment with secretory vesicles and specific granules by subcellular fractionation and translocate to the plasma membrane upon activation of neutrophils. Here the subcellular distribution of uPAR and uPA is studied by electron microscopy of neutrophils using immunogold double labeling for uPAR and uPA and a set of markers for well-defined subtypes of granules: matrix metalloproteinase type-9 (MMP-9) for gelatinase granules, lactoferrin (LF) for specific granules, and myeloperoxidase (MPO) and neutrophil elastase (NE) for primary granules. With this technique uPAR colocalizes with uPA in 71% of labeled granules. In granules containing uPAR the degree of coexpression with MMP-9, MPO and NE was 19, 66, and 74%, respectively. In granules labeled for uPA the corresponding overlap with MMP-9, MPO and NE was 24, 64, and 51%, respectively. Low levels of co-localization were found for uPAR and LF (7%) and for uPA and lactoferrin (5%). The results indicate that uPAR and uPA are present in gelatinase granules and primary granules, but rarely in specific granules. The demonstration of uPAR and uPA in primary granules is of particular interest, and may indicate that uPAR and uPA participate in the activation of latent hepatocyte growth factor of neutrophils.

Biomarkers↗
PubMedDate not supplied

Atypical amyloidosis: diagnostic challenges and the role of immunoelectron microscopy in diagnosis.

Renal AL-amyloidosis is usually lambda-light-chain-related, particularly lambda type 6. Kappa-light-chain-related renal amyloidosis is a rare entity. Relatively few cases have been reported in the literature. The pathologic findings in some of these cases may mimic other disease processes. It is important to recognize the different morphologic expressions of this entity in order to render the right diagnosis. The authors discuss one such case in which the initial pathologic impression was erroneous and, upon further studies, including ultrastructural immunogold labeling, the diagnosis of kappa-light-chain-related renal amyloidosis was established.

Aged↗
PubMed2003

Localization of the two constitutively expressed nitric oxide synthase isoforms (nNOS and eNOS) in the same cell types in the saccule maculae of the frog Rana pipiens by immunoelectron microscopy: evidence for a back-up system?

There is growing evidence for a nitric oxide/cyclic GMP pathway of signal transduction in the vestibular system. Recently, two isoforms of nitric oxide (NO) synthase (nNOS and eNOS) and NO itself have been identified at the light microscopic level in the vestibulocochlear system of mice using specific antibodies and a new fluorescence indicator. In order to acquire more information about signal transduction and tissue modulation in this neuroepithelium at the cellular and subcellular levels, ultrathin sections of London Resin White-embedded saccule maculae of the frog Rana pipiens were incubated with various concentrations of commercially available antibodies to nNOS and eNOS. The immunoreactivity was visualized by a gold-labelled secondary antibody and the amount of the immunoreactions per microm2 was quantified for the different cell types and subcellular regions. Significant eNOS immunoreactivity was identified in the hair bundles, cuticular plates and the rest of the cytoplasm of the hair cells as well as in different subcellular regions of the supporting cells. Gold-labelled anti-nNOS antibodies stained mainly stereovilli and cuticular structures of hair cells and supporting cells, whereas the number of the immunoreactions in the remaining cytoplasm of both cell types was near the background level. The spatial co-localization of the two NOS isotypes in the same cell regions of hair cells and supporting cells was confirmed in double-labelling experiments. The immunocytochemical findings are suggestive of a redundant system in which one NOS isoform can (partially) replace the other. The different subcellular localization of the NOS isoforms may allow for isoform specific regulation of NOS activity by different Ca2+ currents at the subcellular level, underlining the importance of NO-regulated processes in neuroepithelia of the inner ear.

Acoustic Maculae↗
PubMed2000

Immunoelectron microscopy in the age of molecular pathology.

The introduction of molecular biology-based diagnostic procedures in pathology has created substantial expectations in regard to screening, characterization, monitoring, and detection of predisposition to a variety of diseases, most notably malignant neoplasms. It should be emphasized, however, that molecular studies are only one component of the diagnostic process and that more traditional methods are still required in the evaluation of tumors and management of patients. The data obtained from the molecular biology-based studies must be always interpreted in conjunction with the clinical history, immunomorphologic findings, and other pertinent ancillary data. Routine evaluation of tissues using traditional light microscopy remains the backbone of pathologic evaluation. The additive role of molecular diagnostics often depends on how accurate the initial evaluation has been. Ancillary techniques such as immunohistochemistry and electron microscopy remain essential in properly characterizing diseased tissues and in speciation of tumors. Ultrastructural immunolabeling capitalizes on combining these two techniques and providing exquisite immunomorphologic evaluation. The extra time and effort required are more than compensated by the degree of sophistication that can be achieved when this diagnostic technique is utilized and the added expense is rather reasonable. The value of molecular biology-based diagnostics is potentially questionable if the tissue samples are not initially accurately characterized. The question that molecular diagnostics may be trying to answer may be the wrong one or the answer obtained may be interpreted incorrectly if the context of the clinicopathologic situation has not been clearly defined using traditional diagnostic techniques.

Humans↗
PubMed1994

Particle assembly and Vpr expression in human immunodeficiency virus type 1-infected cells demonstrated by immunoelectron microscopy.

The 96 amino acid viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) was detected during virus assembly in intracellular vacuoles and at the plasma membrane on peripheral blood mononuclear cells. In both immature and mature virus particles, Vpr was located immediately beneath the viral envelope, colocalizing with the core structural protein, Gag p24. Vpr was present in intracellular HIV-1 wild-type virions at 50% of the level found in extracellular HIV-1 particles. Cells infected with HIV-1 strains with C-terminal truncations of Vpr manifested a different pattern of Vpr expression. A mutant with an alteration of amino acids 79 to 85 exhibited a 23% reduction in total levels of Vpr expression, but a marked accumulation of Vpr in intracellular rather than extracellular virions. A mutant with the last 17 amino acids of Vpr deleted expressed only 10% of wild-type levels of Vpr. These observations indicate that Vpr is incorporated into virions from the cytoplasmic aspect of either the vacuolar or plasma membrane. Furthermore, the proportion of Vpr on intracellular compared to extracellular virions is affected by a specific locus within the protein.

Amino Acid Sequence↗
PubMed1995

Identification of hepatitis C virus by immunoelectron microscopy.

Sequencing of the hepatitis C virus (HCV) has provided a better understanding of the natural history, immunology, and epidemiology of this virus. However, the morphology of HCV has not been definitively characterized. In this study, through a sequence of concentration processes, virus-like particles were isolated from human serum and liver tissue, visualized by transmission electron microscopy and identified as hepatitis C virion by immunoelectron microscopy. Spherical flavi-like virus particles, approximately 70 nm in diameter, were observed in the fraction with 1.04-1.12 g ml-1 sucrose density and bound to immunogold particles with monoclonal antibodies (mAb) against hepatitis C. The nucleocapsid of the particles, which were 50 nm in diameter, appeared to be icosahedral in structure and surrounded by an envelope covered with surface projections. A 'tadpole' form of particles was also observed. The findings indicate that the low buoyant density in sucrose and the morphological features of the hepatitis C virion are consistent with the characteristics of flaviviruses and pestiviruses.

Animals↗
PubMed1990

Immunoelectron microscopy of human spermatozoa.

A number of immunocytochemical methods using various markers for electron microscopy have been developed in recent years. The immunogold technique has been especially effective in histotopochemical studies. The value of this technique for demonstrating sperm antigens results from the high electron density of gold, which makes it easily detectable under the electron microscope. The high resolution of the electron microscope permits precise localization of immunologic reactions in the sperm cell. Light microscopy findings can thus be elucidated. We tested a number of monoclonal antibodies that react with sperm antigens. Of three techniques for preparing the spermatozoa, the pre-embedding method and marking of cryoultra-microtome sections proved best.

Fluorescent Antibody Technique↗
PubMedDate not supplied

Localization of a Toxoplasma gondii rhoptry protein by immunoelectron microscopy during and after host cell penetration.

We immunolocalized a Toxoplasma gondii rhoptry protein (ROP1) before and after parasite host cell invasion of human fibroblasts and TG180 murine sarcoma cells by electron microscopy and immunogold labeling using either a monoclonal antibody (Tg49) or a monospecific rabbit antiserum (alpha 249). At all stages of parasite growth ROP1 was found within the body but rarely within the peduncle of rhoptries, even in those that appeared empty. Immediately after host cell invasion ROP1 was associated with the parasitophorous vacuole membrane. Within hours after invasion the amount of ROP1 immunodetectable on the parasitophorous vacuole membrane was markedly decreased. The localization of ROP1 suggests a role in the early establishment of infection in host cells, consistent with previous work that has indicated that monoclonal antibodies to ROP1 (including the one used in these studies) interfere with the phenomenon of penetration enhancement.

Animals↗
PubMedDate not supplied

Immunoelectron microscopy of Giardia lamblia cytoskeleton using antibody to acetylated alpha-tubulin.

Giardia lamblia trophozoites contain acetylated alpha-tubulin but lack detectable levels of tyrosinolated alpha-tubulin, as demonstrated in immunoblots with monoclonal antibodies specific for these tubulin forms. By immunofluorescence microscopy, acetylated alpha-tubulin is localized in axonemes, median bodies and in the adhesive disk. Post-embeddment immunogold labeling of thin sections of cells was used to evaluate acetylation at the level of individual microtubules by electron microscopy. Cells were fixed with glutaraldehyde and embedded in the acrylic resin LR Gold. Results indicate all microtubules in adhesive disk, axonemes, basal bodies, funis and the median bodies contain acetylated alpha-tubulin. Unlike immunofluorescence labeling, all microtubules of the adhesive disk and the funis could be gold labeled. No nonspecific labeling of the cytoplasm or of structures other than microtubules was observed. Acetylated microtubules in G. lamblia do not appear to be a subset of microtubules and acetylation appears uniform along the entire length of individual microtubules. Acetylation and the tyrosinolation state of microtubules in Giardia are discussed in the context of microtubule stability and crosslinked features of the cytoskeleton.

Acetylation↗
PubMed1994

Immunoelectron microscopy of antigens of Bordetella pertussis using monoclonal antibodies to agglutinogens 2 and 3, filamentous haemagglutinin, pertussis toxin, pertactin and adenylate cyclase toxin.

Immunogold electron microscopy and monoclonal antibodies (Mabs) were used to localize surface-related antigens of Bordetella pertussis. Unfixed organisms of B. pertussis strains which are included in the Danish whole-cell pertussis vaccine and fixed cells from a vial of vaccine were examined. Mabs to agglutinogens 2 and 3 labelled fimbria-like structures on both live and fixed cells in a serotype-specific manner. Mab against pertactin, a 69 kDa outer membrane protein, produced intense labelling of the surface of unfixed cells, whereas staining was reduced when fixed cells were examined. Mabs against filamentous haemagglutinin (FHA) stained aggregates of material between or adherent to both live and fixed cells. Negligible labelling of FHA on cell surfaces was observed. Mabs to pertussis toxin and adenylate cyclase toxin labelled loose-structured material which was adherent to or between cells, but neither of these toxin antigens was expressed on the surface of B. pertussis in Mab recognizable form. It is therefore suggested that these antigens are readily dispersed after exit from the outer membrane of B. pertussis.

Adenylate Cyclase Toxin↗