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PubMed1993

Dopaminergic lateral efferent innervation of the guinea-pig cochlea: immunoelectron microscopy of catecholamine-synthesizing enzymes and effect of 6-hydroxydopamine.

We have used an immunocytochemical approach to gain further data supporting a possible neurotransmitter or neuromodulator function for dopamine at the level of efferent (olivocochlear) innervations of the guinea-pig cochlea. Immunofluorescence screening was first done on cochleas two or seven days after infusion with the neurotoxin 6-hydroxydopamine. Two days after neurotoxin perfusion, the intensity of the tyrosine hydroxylase-like immunoreactivity was decreased in the inner and tunnel spiral bundles of the organ of Corti (and in known noradrenergic sympathetic fibers outside this organ), with respect to the control contralateral cochleas. In cochleas screened seven days after 6-hydroxydopamine infusion, no tyrosine hydroxylase-like immunoreactivity could be found in the organ of Corti. Only occasional faint fluorescence could be detected in sympathetic fibers. In another set of experiments, a pre-embedding immunoperoxidase technique was used to localize tyrosine hydroxylase and aromatic amino acid decarboxylase, another catecholamine-synthesizing enzyme, at the ultrastructural level. With both types of antibody, the same kind of results were observed. Immunoreactivities were only seen in vesiculated fibers within the inner and tunnel spiral bundles, thus are likely in lateral efferent varicosities. These immunostained fibers accounted for approximately half of the efferent profiles in the inner spiral bundle. Within this bundle, the immunoreactive fibers established axodendritic synapses with the radial afferent processes of type I neurons which contacted the inner hair cells. In no case was immunoreactivity to either enzyme observed in the outer hair cell region, at the level of medial efferent terminals. The synaptic localization of tyrosine hydroxylase- and aromatic amino acid decarboxylase-like immunoreactivities in the lateral efferent varicosities of the inner spiral bundle, as well as the effect of 6-hydroxydopamine on the tyrosine hydroxylase-like immunoreactivity in this same bundle, further support the hypothesis that a catecholamine could act as a lateral efferent neurotransmitter or neuromodulator. Based on previous data reporting a lack of dopamine-beta-hydroxylase-like immunoreactivity within the organ of Corti, and the effectiveness of a D2 agonist on the cochlear compound action potential of the auditory nerve, this catecholamine could well be dopamine.

Animals↗
PubMedDate not supplied

Immunoelectron microscopy and immunofluorescence antigenic mapping: diagnostic applications.

In selected situations, direct and indirect IEM may provide important diagnostic information for the clinician, as pertains to the diagnosis and therefore prognosis in a given patient. As discussed, however, all of the more conventional approaches should be pursued before consideration is given to the performance of IEM, knowing its relative cost and technical difficulty. In contrast, we consider immunofluorescence antigenic mapping to be the diagnostic method of choice for the determination of the type of inherited EB present in affected individuals. When additional ultrastructural or morphometric data is required, then TEM can be performed as an adjunct study once it is determined by mapping technique that additional useful information may be derived.

Antigens↗
PubMed1994

Immunoelectron microscopy reveals significant granule fusion upon stimulation of electropermeabilized human neutrophils.

Although electropermeabilization has become an important tool for studying the signal requirements of exocytosis, relatively little is known about the morphological changes accompanying this response in electropermeabilized cells. In this study, we determined that electropermeabilization of human neutrophils by itself caused only minor changes in the morphology as determined by transmission electron microscopy. The structure of the plasma membrane did not show detectable changes, whereas the cytoplasm was more electron lucent as compared to intact cells. Activation of intact neutrophils with formyl-methionyl-leucyl-phenylalanine (FMLP), in the presence of cytochalasin-B, caused the development of invaginations of the plasma membrane. In contrast, activation of electropermeabilized cells with 1 microM Ca2+ and/or 50 microM GTP-gamma-S caused the development of vacuoles that did not seem to be in contact (or had previously been in contact) with the extracellular environment. However, fusion of azurophilic and specific granules with these vacuoles clearly had taken place. The response characteristics of this fusion induced by Ca2+ and GTP-gamma-S were quite similar to those of the direct fusion of granules with the plasma membrane. We conclude that in electropermeabilized human neutrophils, two processes involving granule fusion can be distinguished. First, a direct fusion of granules with the plasma membrane. Secondly, the fusion of granules leading to the formation of vacuoles, not in contact with the extracellular space.

Calcium↗
PubMed1992

Localization of the cholesterol oxidase in Rhodococcus erythropolis IMET 7185 studied by immunoelectron microscopy.

Rhodococcus erythropolis IMET 7185 produces an inducible cholesterol oxidase (COD) which can easily be extracted by treatment of cells with 0.1% Triton X-100. The yield of the enzyme was 3.3 U/g wet wt from induced cells, which is about 5 times more than from non-induced cells. A study of the location of COD on intact cells and on ultrathin sections by means of immunogold electron microscopy revealed the following distribution, which corresponds with the biochemical results: (1) COD, which is extractable by the detergent, was localized in a distance up to 80 nm above the cell surface. It belongs to a surface layer, which only became visible after lysine/glutaraldehyde treatment and staining with ruthenium red, indicating a high carbohydrate content. (2) Non-extractable COD was found on the cell surface in a shorter distance to the cell wall as well as within the cell wall, in the cytoplasmic membrane and in the peripheral cytoplasm. In the latter clusters of gold particles on some places suggest the presence of larger amounts of insoluble enzyme.

Blotting, Western↗
PubMedDate not supplied

Improved technique for immunoelectron microscopy. How to prepare epoxy resin to obtain approximately the same immunogold labeling for epoxy sections as for acrylic sections without any etching.

The purpose of this study was to improve the immunogold labeling of epoxy sections and to increase our knowledge of the mechanism for how antigens become immunolabeled on resin sections. Tissues from pancreas, thyroid and fibrin clots were embedded in an epoxy resin and LR-White. The epoxy mixture was composed and treated in different ways, especially with respect to altered amounts of accelerator (DMP-30). Immunogold labeling was performed with anti-glucagon, anti-thyroglobulin and anti-fibrinogen respectively. By increasing the amount of DMP-30 in the infiltration steps and/or embedding step, we observed a significant rise in the immunogold labeling. For the largest proteins the labeling was up to 8 times more intense than the labeling achieve with epoxy sections produced by 'normal' amount of accelerator in the embedding mixture and without accelerator in the infiltration mixture. For the smallest protein, glucagon, the differences were almost absent. The labeling of thyroglobulin and fibrinogen on the high accelerator epoxy sections was up to 70% of the labeling of LR-White sections, while conventional epoxy sections showed a labeling of 5-10% of that obtained with acrylic labeling. The cutting qualities of the high-accelerator blocks were similar to that of conventional epoxy embedding. The ultrastructure of the sections from the high-accelerator epoxy blocks were good, and the contrast was improved when tannic acid was used as enhancer. Our theory to explain the improved labeling is that the antigens are less tightly incorporated in the polymer network when the concentration of the accelerator is increased. The method outlined significantly improves the detectability of antigens on epoxy sections, which is the embedding resin routinely used in many laboratories.

Acrylic Resins↗
PubMed1991

Immunoelectron microscopy studies with a monoclonal antibody directed against a receptor recognition site epitope in human alpha 2-macroglobulin.

Alpha 2-Macroglobulin (alpha 2M) is a plasma proteinase inhibitor that binds up to 2 mole of proteinase per mole of inhibitor. Proteinase binding or reaction with small primary amines causes a major conformational change in alpha 2M. As a result of this conformational change, a new epitope recognized by monoclonal antibody 7H11D6 is exposed. The association of alpha 2M-proteinase or alpha 2M-methylamine with alpha 2M cellular receptors is prevented by 7H11D6. In this investigation, the binding of 7H11D6 to alpha 2M was studied by electron microscopy. 7H11D6 bound to alpha 2M-methylamine and alpha 2M-trypsin but not to native alpha 2M. The structure of alpha 2M after conformational change resembled the letter "H." 7H11D6 epitopes were identified near the apices of the four arms in the alpha 2M "H" structure. 7H11D6 that was adducted to colloidal gold (7HAu) retained the specificity of the free antibody (binding to alpha 2M-trypsin but not to native alpha 2M). alpha 2M conformational change intermediates prepared by sequential reaction with a protein crosslinker and trypsin also bound 7HAu. These results suggest that a complete alpha 2M conformational change is not necessary for 7H11D6 epitope exposure and may not be required for receptor recognition. 7HAu was used to isolate a preparation consisting primarily of binary alpha 2M-trypsin (1 mole trypsin per mole alpha 2M instead of 2). Structures resembling the letter "H" were most common; however, each field showed some atypical molecules with arms that were compacted instead of thin and elongated.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1996

Nonuniform elasticity of titin in cardiac myocytes: a study using immunoelectron microscopy and cellular mechanics.

Titin (also known as connectin) is a muscle-specific giant protein found inside the sarcomere, spanning from the Z-line to the M-line. The I-band segment of titin is considered to function as a molecular spring that develops tension when sarcomeres are stretched (passive tension). Recent studies on skeletal muscle indicate that it is not the entire I-band segment of titin that behaves as a spring; some sections are inelastic and do not take part in the development of passive tension. To better understand the mechanism of passive tension development in the heart, where passive tension plays an essential role in the pumping function, we investigated titin's elastic segment in cardiac myocytes using structural and mechanical techniques. Single cardiac myocytes were stretched by various amounts and then immunolabeled and processed for electron microscopy in the stretched state. Monoclonal antibodies that recognize different titin epitopes were used, and the locations of the titin epitopes in the sarcomere were studied as a function of sarcomere length. We found that only a small region of the I-band segment of titin is elastic; its contour length is estimated at approximately 75 nm, which is only approximately 40% of the total I-band segment of titin. Passive tension measurements indicated that the fundamental determinant of how much passive tension the heart develops is the strain of titin's elastic segment. Furthermore, we found evidence that in sarcomeres that are slack (length, approximately 1.85 microns) the elastic titin segment is highly folded on top of itself. Based on the data, we propose a two-stage mechanism of passive tension development in the heart, in which, between sarcomere lengths of approximately 1.85 microns and approximately 2.0 microns, titin's elastic segment straightens and, at lengths longer than approximately 2.0 microns, the molecular domains that make up titin's elastic segment unravel. Sarcomere shortening to lengths below slack (approximately 1.85 microns) also results in straightening of the elastic titin segment, giving rise to a force that opposes shortening and that tends to bring sarcomeres back to their slack length.

Animals↗
PubMed1999

Immunoelectron microscopy of AMPA receptor subunits reveals three types of putative glutamatergic synapse in the rat vestibular end organs.

To characterize the synapses between hair cells and afferent nerve endings in the rat vestibular end organs, the ultrastructural localization of AMPA receptor subunits (GluR1-4) was examined by postembedding immunogold cytochemistry. Immunoreactivities for GluR2/3 and GluR4 were associated with the synapses between type I hair cells and the surrounding chaliceal nerve endings and with the bouton type nerve endings contacting type II hair cells. There was no detectable immunoreactivity for GluR1. A third type of immunoreactive synapse was found between the outer face of chalices and type II hair cells. While the linear densities of gold particles (particles per micrometer postsynaptic specialization) of bouton type endings and chaliceal nerve endings were the same, the former type of ending showed larger postsynaptic specializations and, hence, a higher number of receptor molecules. These data indicate that there are three types of putative glutamatergic synapse in the vestibular end organ.

Animals↗
PubMed1998

Desmin myopathy involving cardiac, skeletal, and vascular smooth muscle: report of a case with immunoelectron microscopy.

Desmin myopathy is a rare idiopathic disorder characterized by abnormal aggregates of desmin-type intermediate filaments, which affects cardiac and skeletal muscle, and rarely the intestinal smooth muscle. We report a 42-year-old woman with atrial fibrillation and progressive restrictive cardiomyopathy. Left ventricular biopsy, cardiac explant, and subsequent autopsy study of skeletal muscle revealed cytoplasmic granulo-filamentous inclusions that were continuous with Z-lines and were immunoreactive for desmin filaments both at the light immunohistochemical and electron microscopic level. In addition, we report the presence of characteristic inclusions within the smooth muscle of intramural coronary blood vessels. This is the first description of desmin inclusions within vascular smooth muscle, and underscores the systemic nature of this rare myopathy.

Adult↗
PubMed1993

LR Gold and LR White embedding of lung tissue for immunoelectron microscopy.

The immunogold technique in conjunction with LR Gold or LR White embedding was used to localize different antigens in normal rat and mini pig lung tissues. Lycopersicon esculentum and Maclura pomifera lectins and diverse cytoskeletal antigens such as cytokeratins, beta-tubulin, smooth muscle-actin, tropomyosin and villin were labelled well. Factor VIII-related antigen was found in Weibel-Palade bodies of endothelial cells. A 10 kDa Clara cell protein was localized in Clara cells. The morphological preservation of LR Gold-embedded tissue was superior to that of LR White-embedded tissues, but significant differences in antigenicity were not seen.

Acrylic Resins↗
PubMed1999

Demonstration of new sites of expression of the CCK-B/gastrin receptor in pancreatic acinar AR42J cells using immunoelectron microscopy.

The CCK-B/gastrin receptor has been characterised in both normal and tumour tissues. Endocytosis of the CCK-B/gastrin receptor has recently been demonstrated and this has similarly been described for other peptide receptors. In addition, ligand and ligand-receptor translocation to the nucleus has been demonstrated for other peptides. The aim of this study was to identify the sites of expression of the CCK-B/gastrin receptor in the known CCK-B/gastrin receptor bearing pancreatic acinar AR42J cells. The specificity of the CCK-B/gastrin receptor antibody (alpha-CCKBR-Ser antibody) was demonstrated by inhibition ELISA studies, radioligand inhibition studies and immunofluorescence binding studies on AR42J cells. Western blotting and immunogold electron microscopy techniques were used to identify the receptor in AR42J cell preparations. The affinity purified alpha-CCKBR-Ser antibody was shown to be specific for the CCK-B/gastrin receptor. The receptor was expressed on the cell membrane, in the cytoplasm and within the nucleus. Isoforms of the receptor protein identified in extra-nuclear and nuclear extracts ranged in molecular weight from 58 to 66 kDa. We conclude that the CCK-B/gastrin receptor is not only expressed on the cell membrane, but also in the cytoplasm and nucleus of AR42J pancreatic acinar cells.

Animals↗
PubMed2002

GABAergic innervation of corticotropin-releasing hormone (CRH)-secreting parvocellular neurons and its plasticity as demonstrated by quantitative immunoelectron microscopy.

GABA has been identified as an important neurotransmitter in stress-related circuitry mediating inhibitory effects on neurosecretory neurons that comprise the central limb of the hypothalamo-pituitary-adrenocortical axis. Using combinations of pre-embedding immunostaining and postembedding immunogold methods at the ultrastructural level, direct synaptic contacts were revealed between GABA-containing terminals and neurosecretory cells that were immunoreactive for corticotropin-releasing hormone (CRH) in the hypothalamic paraventricular nucleus (PVN). The vast majority of axo-dendritic GABA synapses was symmetric (inhibitory) type, and 46% of all synaptic boutons in the medial parvocellular subdivision of the PVN were immunoreactive to GABA. Using the disector method, an unbiased stereological method on serial ultrathin sections, the total calculated number of synaptic contacts within the medial parvocellular subdivision of the PVN was 55.4 x 10(6)/mm(3). On CRH-positive profiles 20.1 x 10(6) GABAergic synaptic boutons were detected per mm(3) in control, colchicine-treated rats. In the medial parvocellular subdivision, 79% of GABAergic boutons terminated on CRH neurons. Following adrenalectomy, which increases the synthetic and secretory activities of CRH neurons, the number of GABAergic synapses that terminate on CRH-positive profiles was increased by 55%. GABA-containing boutons appeared to be swollen, while the contact surfaces of cellular membranes between GABAergic boutons and CRH-positive profiles were shorter in adrenalectomized animals than in controls. Our data provide ultrastructural evidence for direct inhibitory GABAergic control of stress-related CRH neurons and suggest a pivotal role of GABA-containing inputs in the functional plasticity of parvocellular neurosecretory neurons seen in response to adrenalectomy.

Adrenalectomy↗