scholarly journals Parathyroid hormone receptor in intact embryonic chicken bone: characterization and cellular localization.

1982 ◽  
Vol 94 (2) ◽  
pp. 379-386 ◽  
Author(s):  
C M Silve ◽  
G T Hradek ◽  
A L Jones ◽  
C D Arnaud
Keyword(s):  
Parathyroid Hormone ◽  
Cellular Localization ◽  
Adenosine Monophosphate ◽  
Biologically Active ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Parathyroid Hormone Receptor ◽  
Specific Localization ◽  
Grain Distribution

The specific localization and the characterization of the parathyroid hormone (PTH) receptor in bone have been studied using 18-d embryonic chick calvariae and biologically active, electrolytically labeled [125I] bovine PTH(1-34). Binding was initiated by adding [125I]-bPTH(1-34) to bisected calvariae at 30 degrees C. Steady state binding was achieved at 90 min at which time 10 mg drg wt of calvaria specifically bound 17% of the added [125I]bPTH(1-34). Nonspecific binding in the presence of 244 nM unlabeled bPTH(1-34) was less than 2%. Insulin, glucagon, and calcitonin (1 microgram/ml) did not compete for PTH binding sites. Half-maximal inhibition of binding was achieved at concentrations of unlabeled bPTH(1-34) or bPTH(1-84) of about 10 nM. The range of concentration (2-100 nM) over which bPTH(1-34) and bPTH(1-84) stimulated cyclic 3'5'adenosine monophosphate (cAMP) production was similar to that which inhibited the binding of [125I]bPTH(1-34). Light microscope autoradiograms showed that grains were concentrated over cells (osteoblasts and progenitor cells) at the external surface of the calvariae and in trabeculae. In the presence of excess unlabeled PTH, labeling of control autoradiograms was reduced to near background levels. No labeling of osteocytes or osteoclasts was observed. At the electron microscopic level, grains were localized primarily over cell membranes. A quantitative analysis of grain distribution suggested that cellular internalization of PTH occurred.

The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

1973 ◽  
Vol 31 ◽  
pp. 574-575
Author(s):  
J. T. Stasny ◽  
R. C. Burns ◽  
R. W. F. Hardy
Keyword(s):  
Cellular Localization ◽  
Direct Evidence ◽  
Azotobacter Vinelandii ◽  
Intracellular Localization ◽  
Protein Component ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Fe Protein ◽  
Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

scholarly journals A Novel Karyoskeletal Protein: Characterization of Protein NO145, the Major Component of Nucleolar Cortical Skeleton inXenopus Oocytes

2001 ◽  
Vol 12 (12) ◽  
pp. 3904-3918 ◽  
Author(s):  
Sandra Kneissel ◽  
Werner W. Franke ◽  
Joseph G. Gall ◽  
Hans Heid ◽  
Sonja Reidenbach ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Protein Characterization ◽  
Electron Microscopic Level ◽  
Expression Library ◽  
Electron Microscopic ◽  
Egg Formation ◽  
Complex Protein ◽  
Calculated Mass ◽  
Synaptonemal Complex Protein

The nucleolus is a ubiquitous, mostly spheroidal nuclear structure of all protein-synthesizing cells, with a well-defined functional compartmentalization. Although a number of nonribosomal proteins involved in ribosome formation have been identified, the elements responsible for the shape and internal architecture of nucleoli are still largely unknown. Here, we report the molecular characterization of a novel protein, NO145, which is a major and specific component of a nucleolar cortical skeleton resistant to high salt buffers. The amino acid sequence of this polypeptide with a SDS-PAGE mobility corresponding to M r 145,000 has been deduced from a cDNA clone isolated from a Xenopus laevis ovary expression library and defines a polypeptide of 977 amino acids with a calculated mass of 111 kDa, with partial sequence homology to a synaptonemal complex protein, SCP2. Antibodies specific for this protein have allowed its recognition in immunoblots of karyoskeleton-containing fractions of oocytes from differentXenopus species and have revealed its presence in all stages of oogenesis, followed by a specific and rapid degradation during egg formation. Immunolocalization studies at the light and electron microscopic level have shown that protein NO145 is exclusively located in a cage-like cortical structure around the entire nucleolus, consisting of a meshwork of patches and filaments that dissociates upon reduction of divalent cations. We propose that protein NO145 contributes to the assembly of a karyoskeletal structure specific for the nucleolar cortex of the extrachromosomal nucleoli ofXenopus oocytes, and we discuss the possibility that a similar structure is present in other cells and species.

Structural characterization of the parathyroid hormone receptor domains determinant for ligand binding

2007 ◽  
Vol 35 (4) ◽  
pp. 721-723 ◽  
Author(s):  
D.F. Mierke ◽  
L. Mao ◽  
M. Pellegrini ◽  
A. Piserchio ◽  
J. Plati ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
G Protein Coupled Receptors ◽  
Peptide Ligands ◽  
Receptor Complex ◽  
Receptor Model ◽  
Receptor System ◽  
Parathyroid Hormone Receptor ◽  
Contact Points ◽  
G Protein Coupled

Over the years, the association of peptide ligands to Family B GPCRs (G-protein coupled receptors) has been characterized by a number of experimental and theoretical techniques. For the PTH (parathyroid hormone) ligand–receptor system, important insight has been provided by photoaffinity labelling experiments and the elucidation of direct contact points between ligand and receptor. Our research has focused on the structural elucidation of the receptor domains shown to be involved in the binding of PTH. Employing a combination of carefully designed receptor domains, solution-state NMR carried out in the presence of membrane mimetics and extensive computer simulations, we have obtained a well-resolved model of the ligand–receptor complex for PTH. Here, we review the development of this model and highlight some inherent limitations of the methods employed and their consequences on interpretation of the ligand–receptor model.

scholarly journals An approach to postembedding staining of protein (immunoglobulin) antigen embedded in plastic: prerequisites and limitations.

1980 ◽  
Vol 28 (10) ◽  
pp. 1041-1049 ◽  
Author(s):  
H Takamiya ◽  
S Batsford ◽  
A Vogt
Keyword(s):  
Biopsy Specimen ◽  
Picric Acid ◽  
Renal Tissue ◽  
Microscopic Level ◽  
Tissue Structure ◽  
Electron Microscopic Level ◽  
Light Microscopic Level ◽  
Electron Microscopic ◽  
Specific Localization ◽  
Ultrathin Sections

A method is described for performing postembedding staining of protein (immunoglobulin) antigen embedded in styrene-methacrylate resin. Fixation of specimens in a combination of 4% paraformaldehyde and 0.2% picric acid and washing in buffer containing 7% sucrose, followed by abrupt dehydration with absolute acetone in the cold preserved the antigenicity, although in a masked form. The masked antigenicity could be reexposed by treatment with nonspecific protease. Staining with fluorescent-, peroxidase-, or ferritin-labeled antibodies on semi- and ultrathin sections resulted in specific localization of the antigen. We applied this technique to the localization of rabbit immunoglobulin in specimens of renal tissue obtained from rats with anti-glomerular basement membrane nephritis; we also localized human IgG in a renal biopsy specimen. The prerequisites for recovery of antigenicity are such that preservation of tissue structure at the light microscopic level is good, but relatively poor at the electron microscopic level.

Correlative immunolocalization with high-resolution light microscopy and Electron Microscopy using London Resin Gold embedded tissue

1995 ◽  
Vol 53 ◽  
pp. 702-703
Author(s):  
M. L. Grove ◽  
B. A. Evans ◽  
D. N. Misra ◽  
J. Zhao ◽  
D. H. Alpers ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Polyclonal Antibodies ◽  
Intracellular Localization ◽  
Intrinsic Factor ◽  
Gastric Epithelium ◽  
Electron Microscopic Level ◽  
Immunoperoxidase Staining ◽  
Rat Tissues ◽  
Electron Microscopic ◽  
Thin Sections

Immunoelectron microscopy specimens are often embedded in hydrophilic resins, like London Resin Gold(LRG), which permit antibody staining without etching (or deplasticizing) the sections. This characteristic of hydrophilic resins also allows for immunohistochemistry at the light level on semi thin sections (0.5μm - 1μm). The ability to do immunohistochemistry at both the light and electron microscopic level on the same tissue block allows focused ultrastructural study. Immunohistochemistry on semi-thin sections displays cellular localization of macromolecules, permitting more specificity in the selection of areas for studying intracellular localization ultrastructurally. We have developed a method for immunoperoxidase staining of LRG embedded tissues, utilizing anti-human polyclonal antibodies directed against intrinsic factor (Fig. 1). Intrinsic factor (IF), a cobalamin binding protein, is known to be produced in the stomach, pancreas and salivary glands of most mammals. We are interested in distribution of IF in gastric epithelium, small intestine (ileum) and supporting tissues in both gastrointestinal tract sites. Previously, we have described the cellular localization of IF in human and rat tissues.

scholarly journals Preparation and Purification of Tritiated Phytohemagglutinin and Studies of Cellular Localization in Human Leukocyte Cultures

Blood ◽  
1970 ◽  
Vol 35 (1) ◽  
pp. 44-55 ◽  
Author(s):  
ROBERT A. CONARD ◽  
CHARLES F. DEMOISE
Keyword(s):  
Cellular Localization ◽  
Mononuclear Cells ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Human Leukocyte ◽  
Chemical Extraction ◽  
Mitochondrial Activity ◽  
Electron Microscopic ◽  
Grain Distribution ◽  
Rna And Dna

Abstract A tritiated bean extract was obtained from bean plants (Phaseolus vulgaris) grown hydroponically in nutrient media containing various concentrations of tritium water. Purification was accomplished by ammonium sulfate precipitation and column chromatography using DEAE, CM-52 cellulose columns, and finally by Sephadex G-100 gel filtration. The purified product showed greatly increased mitogenic activity and electrophoretically showed a single labeled protein band on electrophoresis. Radioactivity of the purified PHA varied between 0.1-0.5 µCi/mg. which, though not as high as desirable, was sufficient for autoradiographic and subcellular fractionation studies in human leukocyte cultures. Within a few hours cytoplasmic localization of the label was noted in most of the cells in culture, including neutrophils and larger mononuclear cells. Breakdown of many of these cells during the first 24 hr. resulted in labeled amorphous basophilic staining masses of cell particles. Though agglutination of cells was commonly seen, the lack of label in red cells or surface label of leukocytes was notable. Blast forms which appeared by the second day showed prominent labeling which appeared to be largely cytoplasmic as evidenced by grain distribution. Several reasons were discussed which made it seem likely that the mitogenic molecule was represented in the cell label. Assay of subcellular fractions showed the major portion of radioactivity in the mitochondrial fraction. Specific activity, however, was too low to permit electron microscopic localization of label in individual organelles. Lack of label of RNA and DNA proteins was demonstrated by cellular distribution of the label and chemical extraction of RNA and DNA. Interesting speculation arises as to whether the PHA may stimulate mitochondrial activity and induce RNA synthesis and blastogenesis. Further experiments are in progress to obtain a purer PHA with higher specific radioactivity in order to more precisely define the site and mechanism of action of the mitogen.

scholarly journals Identification of immunoreactive sites in bovine parathyroid cells to antibodies raised against the NH2-terminal sequence of parathyroid hormone.

1979 ◽  
Vol 27 (8) ◽  
pp. 1209-1214 ◽  
Author(s):  
W Limacher ◽  
P Wild ◽  
E Manser ◽  
H Lutz
Keyword(s):  
Parathyroid Hormone ◽  
Protein A ◽  
Electron Microscopic Level ◽  
Enzyme Linked Immunosorbent Assay ◽  
Staphylococcal Protein A ◽  
Terminal Sequence ◽  
Electron Microscopic ◽  
Secretion Granules ◽  
Parenchymal Cells ◽  
Bovine Parathyroid Hormone

The NH2-terminal sequence of bovine parathyroid hormone (1-84) was localized with different immunocytochemical methods on the light and electron microscopic level in bovine parathyroid glands and in isolated bovine parathyroid parenchymal cells. The peroxidase labeled staphylococcal protein A and the peroxidase anti-peroxidase method were found to be advantageous for light and electron microscopic localization, respectively. Reaction product was found light microscopically in the cytoplasma of the parenchymal cells and electron microscopically largely over the secretion granules of the parenchymal cells. The immunoreactive sites were subsequently identified to represent only intact parathyroid hormone (1-84) by gel electrophoresis derived enzyme linked immunosorbent assay.

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