bluuebunny/crossref_metadata_2025_split huggingface dataset:
Distribution and movement of membrane‐associated platelet glycoproteins: Use of colloidal gold with correlative video‐enhanced light microscopy, low‐voltage high‐resolution scanning electron microscopy, and high‐voltage transmission electron microscopy
Authors: Albrecht Ralph M. , Goodman Steven L. , Simmons Scott R. 
Published: February 2005

AbstractScanning electron microscopy (SEM), especially low‐voltage (1 KeV) high‐resolution SEM, can be used in conjunction with stereo pair high‐voltage (1 MeV) transmission electron microscopy (HVEM) of whole spread cells or thick sections effectively to correlate surface structure with internal structure. Surface features such as microvilli, pits, pseudopodia, ruffles, attached virus, and other surface‐related morphologic characteristics can be identified using SEM, while underlying cytoskeletal structure and organelle organization can be viewed by HVEM of the same preparation. However, the need to “prepare” cells for electron microscopy precludes observation in the living state. The use of several types of video‐enhanced light microscopy (VLM) permits observation of living cells such that certain surface and internal features can be observed at a relatively high level of resolution or detection. Thus, changes in living cells can be followed, and at appropriate times the cells may be chemically fixed or rapidly frozen and prepared for ultrastructural examination by electron microscopy.We have utilized VLM in conjunction with SEM and HVEM to correlate changes in shape and surface structure with changes in the internal structure of platelets. In addition, we have found it advantageous to use colloidal gold‐labeling procedures, because these markers are detectable by all three forms of microscopy. Using this approach we have labeled platelet membrane GPIIb/IIIa, a receptor for RGD‐containing adhesive proteins, with gold‐fibrinogen or gold‐anti‐IIb/IIIa. The initial binding and subsequent movement of gold‐fibrinogen‐IIb/IIIa complexes in living platelets was followed by VLM. The movement of individual labels could be mapped. Subsequent observation by low‐voltage (1 KeV), high‐resolution SEM and HVEM permits visualization of the same individual receptors tracked by LM. The final position on the membrane or the position‐in‐transit when fixative was added was determined relative to surface ultrastructure (SEM) and internal, particularly cytoskeletal, ultrastructure (HVEM).
Localization of cellular regulatory proteins using postembedding immunogold labeling
Authors: Hand Arthur R. , Jungmann Richard A. 
Published: February 2005

AbstractCyclic AMP‐dependent protein kinase (cAPK) mediates the effects of catecholamines and hormones that cause elevation of intracellular cyclic AMP levels. The holoenzyme is a tetramer consisting of catalytic (C) and cyclic AMP‐binding regulatory (R) subunits. The type I and type II cAPK isoenzymes are defined by R subunits (RI and RI) of differing molecular weight, primary structure, and cyclic AMP‐binding properties. Postembedding immunogold labeling procedures and specific polyclonal and monoclonal antibodies to RI, RII, and C were used to study the subcellular distribution of cAPK sub‐units in several tissues. In the rat parotid gland, both RI and RII were present in the cytoplasm, nuclei, and secretory granules of the acinar cells, whereas secretory granules of intercalated and striated duct cells were poorly labeled. These results confirmed that the acinar secretory granules are the source of R subunits previously identified in saliva by specific photoaffinity labeling techniques. Zymogen granules of pancreatic acinar cells and secretory granules of seminal vesicle cells were labeled with antibody to RII. Pancreatic and seminal fluids were shown to contain cyclic AMP‐binding proteins. The granules of several endocrine cells (pituitary, pancreatic islet, intestinal) also labeled with RII antibody. Double labeling of ovarian granulosa cells showed that both RI and C were present in the nuclei and cytoplasm. The localization of cAPK subunits revealed by postembedding immunogold labeling is consistent with the postulated regulatory functions of these proteins in gene expression, cell proliferation, exocytosis, and various metabolic events The widespread occurrence of cAPK sub‐units in secretory granules and their release to the extracellular environment suggests that they play an important role in secretory cell function.
Localization of nucleic acid sequences by EM in situ hybridization using colloidal gold labels
Authors: Hamkalo Barbara A. , Narayanswami Sandya , Lundgren Karen 
Published: February 2005

AbstractIt is possible to combine hybridization to specimens on electron‐microscope grids of nucleic‐acid probes labelled nonisotopically with immunogold detection of hybrid sites to map the position of target sequences rapidly and precisely. The basic technique is described, and examples are provided to illustrate the types of questions which can be approached in the general area of higher‐order chromosome organization and function. A combination of two differentially labelled probes and two different‐sized gold particles permits the simultaneous detection of closely linked or interspersed sequences.
Ultrastructural localization of insulin and C‐peptide antigenic sites in rat pancreatic B cell obtained by applying the quantitative high‐resolution protein A–gold approach
Authors: Bendayan Moïse 
Published: February 2005

AbstractInsulin and C‐peptide antigenic sites have been revealed in rat pancreatic B cells by applying immunohistochemical and cytochemical techniques. Fluorescein and rhodamine stains at the light‐microscope level have detected both antigens in the same B cells. With the protein A–gold technique, labeling for both antigens was found in the cisternae of the rough endoplasmic reticulum, in those of the transitional elements, in all the cisternae of the Golgi apparatus except in the trans‐most one, in the smooth but not in the coated vesicles, in the immature and mature secretory granules, and in some lysosomal (multi‐granular) structures. The fixation procedure used yielded excellent Ultrastructural preservation which allowed for high resolution. The various control experiments demonstrated the high specificity of the results. Quantitative evaluations confirmed the qualitative observations in that they documented the specificity of the label and revealed the presence of an increasing gradient for both antigenic sites along the endoplasmic reticulum‐Golgi‐granule secretory pathway. The quantification also demonstrated various sites in which an increased labeling occurs: the rough endoplasmic reticulum, the smooth vesicles, the trans‐cisternae of the Golgi apparatus, and the immature and the mature secretory granules. The Golgi apparatus was composed of three different subcompartments distinguished by their concentration of label. These include the cisternae on the cis‐side, those on the trans‐side, and the trans‐most rigid cisternae. Since insulin and C‐peptide form the proinsulin chain, their antigenic sites were found in the same locations along the secretory pathway; differences in location appeared only in the secretory granules, where insulin was concentrated in the core, while C‐peptide was found in both the core and the halo of the granules. Furthermore, in the mature secretory granules displaying a crystalline core, insulin was restricted to the core, while C‐peptide was confined to the halo. These results are in accord with the biochemical data, which indicate that simultaneous localization of both antigenic sites in compartments upstream to the immature secretory granules reflects their presence in the form of proinsulin. However, upon dissociation of proinsulin into insulin and C‐peptide, both antigenic sites are segregated in different locations. The peptides appear to share parallel pathways and a fate which includes secretion through exocytosis or degradation by the lysosomal system.
Localization and quantification of hormones, ligands, and mRNA with affinity‐gold probes
Authors: Childs Gwen V. , Yamauchi Kohen , Unabia Geda 
Published: February 2005

AbstractThe application of immunogold techniques to localize pituitary hormones produces label that can be quantified and correlated with different secretory states. This report focuses on three major applications of the technology. In the first set of studies, immunogold labels for adrenocorticotropin (ACTH) or luteinizing hormone (LHβ) and follicle‐stimulating hormone (FSHβ) were applied to ultrathin sections of pituitaries from adrenalectomized rats or from rats in different stages of the estrous cycle. During the first week after adrenalectomy, ACTH cell area increased. The concentration of immunoperoxidase label (amount of label/area of the cortico‐tropes) decreased. Counts of gold markers showed that there were no changes in the concentration of antigens per granule. Three weeks after adrenalectomy, the amount of immunoperoxidase label increased along with the concentration of that label. The concentration of gold label for ACTH on granules also increased. All changes correlated well with increases in serum ACTH stimulated by adrenalectomy. In the studies of cycling rats, gonadotropes showed increases in the number of gold markers for LHβ or FSHβ per granule area just before an elevation in serum levels. There were also increases in the proportion of granules that contained only LHβ or FSHβ (monohormonal) before the rise in secretion. Thus, nonparallel release of gonadotropins might be attributed to changes in the ratio of gonadotropins packaged per granule. In the second series of studies, avidin‐gold labels were used to identify sites of binding of biotinylated ligands. These studies illustrate and quantify binding by biotinylated gonadotropin‐releasing hormone (GnRH) to ovarian or pituitary target cells. Triple‐labeling protocols (avidin‐peroxidase followed by immunogold) show that the target cells in the pituitary contain gonadotropins. In the third set of studies, avidin gold or avidin peroxidase was used to label sites of hybridization of a biotinylated cRNA probe to gonadotropin β subunit mRNA. The sites of hybridization appear on rough endoplasmic reticulum; however, further work is needed to improve cell ultrastructure and perserve antigens. Triple‐labeling protocols (avidin‐peroxidase followed by immunogold) show the feasibility of the technique as well as the need for further refinement. To summarize, these studies describe multiple applications of gold labels for the localization of antigens, ligands, and mRNA. The labels are sensitive for detection of antigens and ligands and easily quantified. Quantitative analyses show changes in concentration of gold label that correlate well with secretpry states.
Double‐labeling immunogold electron‐microscopic study of hormonal colocalization in nontumorous and adenomatous rat pituitaries
Authors: Losinski N. E. , Horvath E. , Kovacs K. 
Published: February 2005

AbstractAccording to the one cell, one hormone theory, the pituitary gland is composed of 5 cell types which secrete 6 hormones. Recent investigations indicate that this theory must be modified, as there are some bihormonal cells containing 2 hormones, i.e., mammosomatotrophs prolactin‐growth hormone (PRL‐GH). This study was undertaken in order to elucidate whether other adenohypophysial cells are capable of producing 2 hormones and to demonstrate the presence of cells coexpressing PRL‐GH, PRL‐thyrotropin (TSH), or TSH‐GH. Sixteen nontumorous and 16 adenomatous male and female Sprague‐Dawley and Long Evans rat pituitaries were removed immediately after the animals were killed and processed for transmission electron microscopy and the immunogold double‐labeling technique. Coexpression of PRL‐GH, PRL‐TSH, and TSH‐GH was found in both nontumorous and adenomatous pituitaries. Double labeling was present not only in the same cell cytoplasm but also in the same secretory granules. The question of whether these double‐labeled cells represent different cell populations, transitional cell types, or precursor cells requires further investigation.
Use of the protein A–gold immunocytochemical and enzyme‐gold cytochemical techniques in studies of vitellogenesis
Authors: Herbener George H. 
Published: February 2005

AbstractVitellogenesis in the frog hepatocyte was investigated by applying the protein A‐gold immunocytochemical and RNase‐gold cytochemical techniques in conjunction with morphometric and biochemical analyses. The morphometric studies demonstrated that the surface density of rough endoplasmic reticulum (RER) and nucleolar size increased more than fourfold and 1.25‐fold, respectively, while the nuclear size and the mitochondrial compartment size remained constant following estrogen treatment. Concurrently, liver RNA concentration increased 2.5‐fold while protein and DNA concentrations did not change. In addition, total plasma protein more than doubled, with vitellogenin accounting for 40% of the final volume. The secretory proteins vitellogenin and protein‐RcX (a nonvitellogenin, estrogen‐induced plasma protein of unknown function, found in the plasma of Rana catesbeiana) were detected immunocytochemically in the RER, Golgi apparatus, and secretory granules in hepatocytes only of estrogen‐treated frogs. Lysosomes also were labeled. These observations established that protein‐RcX was synthesized and secreted by the hepatocyte in parallel with vitellogenin and that both of these export proteins were confined to the secretory pathway and lysosomes. Quantitation of labeling density indicated that the concentration of vitellogenin increased as it progressed along the secretory vector. Albumin was detected immunocytochemically also within these same hepatocyte entities from both untreated and treated animals. In the untreated animals, albumin concentration also increased progressively along the secretory vector. A marked alteration of albumin processing was observed following estrogen treatment While albumin concentration in the RER was unchanged, its concentrations within the Golgi apparatus and secretory granules were lower than those observed in the RER or in counterpart compartments under control conditions. RNase‐gold cytochemistry for total RNA demonstrated a 1.5‐fold increase in labeling density over the nucleolus but no change in RER labeling following estrogen treatment. These labeling data, in combination with the morpho‐metric data, suggest an increase of approximately 80% in the total amount of RNA in the nucleolus and 430% in the RER in response to estrogen. This review thus illustrates the significant contributions which can be made by gold‐probe techniques, alone or in combination with morphometric and biochemical techniques, to investigations of the intracellular processing of secretory proteins.
Cytochemical studies of lipid metabolism: Immunogold probes for lipoprotein lipase and cholesterol
Authors: Blanchette‐Mackie E. Joan , Dwyer Nancy K. , Amende Linn A. 
Published: February 2005

AbstractIn this article, cytochemical methods are presented for the study of lipid metabolism both in normal cells and in mutant cells with genetic disorders characterized by abnormal lipid metabolism. The benefit of using an immunocytochemical approach to the study of lipase in tissues is discussed, and a review is presented of the results on immunolocalization of lipoprotein lipase in cardiac tissue of normal mice. Immunocytochemical techniques are applied to the study of lysosomal proliferation in hepatocytes from liver of mutant mice with a genetic defect responsible for the lack of hepatic lipase and lipoprotein lipase activity in these animals. Localization of lipids in tissues with structural techniques has been an area of great interest to our laboratory for many years. Attention is called to the development of a technique for the visualization of fatty acids as a function of their ionization state and the production of fatty‐acid myelin figures in membranes. Results on the use of filipin to detect unesterified cholesterol in membranes are reviewed. Filipin produces fluorescent filipin‐cholesterol complexes but also perturbs cell membranes. Application of this cytochemical probe, in combination with immunocytochemistry of lysosomes, produced useful information on defects in low‐density lipoprotein‐derived cholesterol translocation in mutant human fibroblasts. Initial results on the application of immunological techniques to the study of cholesterol in lipid model systems indicate a novel approach, which may be applicable to specialized cell systems. Recent advances in cryoultramicrotomy and development of immunoprobes present valuable opportunities for the structural assessment of lipids and lipases in cell organelles and cell membranes.
Comparison of detecting sensitivities of different sizes of gold particles with electron‐microscopic immunogold staining using atrial natriuretic peptide in rat atria as a model
Authors: Gu Jiang , D'Andrea Michael 
Published: February 2005

AbstractThe detecting sensitivities of different‐sized gold particles were compared in the localization of atrial natriuretic peptide (ANP) in rat atria. The secondary antibodies were goat antirabbit labeled with 5, 15, 30, or 40 nm colloidal gold diluted 1:2 to 1:100 in Tris buffer. The relative quantity of alpha‐ANP immunoreactivity in specific granules was determined by subtracting the number of gold particles in 1 μm2 nongranule area from that in 1 μm2 granule area measured with a computerized image analyzer. The optimal dilution that achieved the maximal contrast between specific and background label was influenced by the particle size. Optimal dilutions were 1:80, 1:30, 1:20, and 1:5 for 5, 15, 30, and 40 nm gold, respectively. At optimal dilutions, the maximal detecting sensitivity (MDS) was in inverse proportion to the gold particle size; however, this relationship is not entirely linear. The ratio among the MDSs of 5, 15, 30, and 40 nm gold particles was approximately 34:9:3:2. A double immunogold staining was performed to localize alpha‐ and beta‐ANPs with 15 and 5 nm gold, respectively. Both antigens were detected in the same granules. If the ratios established from the single staining data were used, the ratio between the alpha‐ and the beta‐ANP antigens in the same granules was approximately 2.8:1. The data obtained in this study provide a useful reference for applications of immunogold electron microscopy in a quantitative manner, particularly for double immunogold labeling.
Quantitative aspects of immunogold labeling in embedded and in nonembedded sections
Authors: Slot Jan W. , Posthuma George , Chang Lin‐Yi , Crapo James D. , Geuze Hans J. 
Published: February 2005

AbstractWe tried to control immunolabeling conditions so that information about antigen concentration could be achieved by quantifying labeling patterns. Working with immunogold labeling procedures in ultrathin cryosections, we observed that differential penetration of immunoreagents causes considerable differences in labeling efficiency between various cell structures. Therefore, in these nonembedded sections, labeling densities can only be used to measure variations in antigen concentration within one cell structure. After embedding the tissue in 30% polyacrylamide (PAA), differences in penetration were negated. The equalizing effect of PAA on the labeling efficiency enabled us to design a simple immunocytochemical method by which concentrations of a protein can be measured in situ at subcellular levels, provided that no variations in he protein's structural conformation occur that would affect its immunoreactivity.In spite of a higher sensitivity observed for Ig‐gold, we preferred to use protein A–gold in our system because of the low nonspecific labeling and the more precise antigen detection by the later immunomarker.
Structure, composition, and assembly of basement membrane
Authors: Leblond C. P. , Inoue S. 
Published: February 2005

AbstractBasement membranes are thin layers of matrix separating parenchymal cells from connective tissue. Their ultrastructure consists of a three‐dimensional network of irregular, fuzzy strands referred to as “cords”; the cord thickness averages 3–4 nm.Immunostaining reveals that the cords are composed of at least five substances: collagen IV, laminin, heparan sulfate proteoglycan, entactin, and fibronectin. Collagen IV has been identified as a filament of variable thickness persisting after the other components have been removed by plas‐min digestion or salt extraction. Heparan sulfate proteoglycan appears as sets of two parallel lines, referred to as “double tracks,” which run at the surface of the cords. Laminin is detected in the cords as diffuse material within which thin wavy lines may be distinguished. The entactin and fibronectin present within the cords have not been identified as visible structures.The ability of laminin, heparan sulfate proteoglycan, fibronectin, and entactin to bind to collagen IV has been demonstrated by visualization with rotary shadowing and/or biochemical studies. Incubation of three of these substances–collagen IV, laminin (with small entactin contamination), and proteoglycan–at 35°C for 1 hr resulted in a precipitate that was sectioned for electron microscopic examination and processed for gold im‐munolabeling for each of the three incubated substances. Three structures are present in the precipitate: (1) a lacework, exclusively composed of heparan sulfate proteoglycan in the form of two parallel lines, similar to double tracks; (2) semi‐solid, irregular accumulations, composed of the three initial substances distributed on a cord network; and (3) convoluted sheets, which are also composed of the three initial substances distributed on a cord network but which, in addition, have the uniform appearance and thickness of the lamina densa of basement membrane. Hence these sheets are closely similar to the main component of authentic basement membranes.
In vivo ciliogenesis in human fetal tracheal epithelium
Authors: Gaillard Dominique A. , Lallement Aude V. , Petit Annie F. , Puchelle Edith S. 
Published: February 2005

AbstractDevelopment of ciliated (CC) in the fetal human trachea was studied by light and electron microscopy in specimens obtained from 45 embryos or fetuses aged from 9 to 27 weeks of gestation (menstrual age). Four stages could be recognized during tracheal development. Up to 11 weeks (stage I), the trachea was covered with a columnar undifferentiated epithelium with abundant glycogen, apical microvilli, and primary cilia. From 12 to 18–19 weeks (stage II), centriolo‐genesis and secondary ciliogenesis were very active, and the percentage of CC and secretory cells (SC) progressively increased. From 20 to 22–23 weeks, the density of CC was higher but, in parallel, the percentage of SC decreased (stage III). Throughout this period, the different steps of ciliogenesis could be identified in the same field, and the ciliated borders consisted of ciliary shafts with a disorderly arrangement. Megacilia were identified. Some of the preciliated cells had both cilia and secretory granules in their apical cytoplasm. After 24 weeks (stage IV), the ciliated border was apparently mature, the rootlets lengthened, and the cilia were correctly orientated. Whatever the fetal age, the density of CC was significantly higher (P < .01) in the dorsal trachea compared to the ventral trachea. There are many similarities between animal and human ciliogenesis, but in human fetuses, most of the ciliary differentiation occurs early, during the first half of gestation. As demonstrated in experimental models, SC likely play a major role in genesis of CC during the fetal development of the human trachea.