A monoclonal antibody study of protein distribution in the membrane skeleton of the ciliate Pseudomicrothorax

1992 ◽  
Vol 103 (4) ◽  
pp. 1117-1125 ◽  
Author(s):  
S. Curtenaz ◽  
R.K. Peck
Keyword(s):  
Monoclonal Antibody ◽  
Cell Shape ◽  
Membrane Skeleton ◽  
The Other ◽  
Regional Differentiation ◽  
Protein Distribution ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Western Blots

The membrane skeleton, or epiplasm, of the ciliated protozoon Pseudomicrothorax dubius is a chemically and structurally complex layer. It is responsible for the cell shape and the positioning of some cortical organelles. One may expect that its possible morphogenetic role can be achieved only via a regional differentiation of the protein distribution in the epiplasm. We have tried to demonstrate such differentiation by preparing an epiplasm extract, which consists predominantly of concanavalin A (ConA)-positive glycoproteins. This fraction, either untreated or deglycosylated, was used to raise monoclonal antibodies (mAbs), whose specificity was tested on western blots of either untreated or deglycosylated epiplasm. The recognized polypeptides were then localized in situ by fluorescence and electron microscopic immunocytochemistry. Six mAbs are presented here. Four of them are directed against ConA-positive glycoproteins and show a localization of the latter on the outer surface of the epiplasm. The two others are directed against other epiplasmic polypeptides: one is specific for a common epitope shared by most of the epiplasmic proteins, but not by the glycoproteins, and labels the entire membrane skeleton, whereas the other recognizes three minor polypeptides, which seem localized to the inner part of the epiplasm.

scholarly journals Localized endocrine conversion of ventricular cardiocytes in ventricular aneurysm.

1990 ◽  
Vol 38 (11) ◽  
pp. 1659-1668 ◽  
Author(s):  
J Gu ◽  
L B McGrath
Keyword(s):  
Messenger Rna ◽  
Secretory Granules ◽  
Scar Tissue ◽  
Border Zone ◽  
Ventricular Aneurysm ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Low Concentrations ◽  
Fibrous Scar Tissue

Atrial natriuretic peptide (ANP) is synthesized and stored in the atria of the heart, but not or at very low concentrations in the ventricles. We investigated the occurrence of ANP and its messenger RNA (mRNA) in human ventricular aneurysm where the cardiocytes were physically over-stretched. The techniques of light and electron microscopic immunocytochemistry, and RNA-RNA tissue in situ hybridization were employed. A large amount of ANP immunoreactivity was found in the cytoplasm of the cardiocytes in and around the aneurysm, but not in fibrous scar tissue or in the normal ventricles. Immunoelectron microscopy localized the immunoreactivity mainly to specific secretory granules in the cytoplasm of the cardiocytes. ANP mRNA was also detected in these cardiocytes. The abundance of both was much higher than that found in the hypertrophic ventricles of other types. The highest concentration of ANP immunoreactivity and of ANP mRNA was found in the cardiocytes located at the border zone. The quantities of both ANP and its mRNA decreased in cardiocytes more distant from the lesion. Our findings suggest that human ventricular cardiocytes in and around aneurysm can convert to produce large amounts of the endocrine peptide ANP. This ventricular endocrine conversion was localized and was probably caused by physical over-stretch of the cardiocytes.

scholarly journals Metagenomic Analysis Reveals Unexpected Subgenomic Diversity of Magnetotactic Bacteria within the PhylumNitrospirae

2010 ◽  
Vol 77 (1) ◽  
pp. 323-326 ◽  
Author(s):  
Wei Lin ◽  
Christian Jogler ◽  
Dirk Schüler ◽  
Yongxin Pan
Keyword(s):  
Sequence Analysis ◽  
Genome Organization ◽  
Cell Shape ◽  
Magnetotactic Bacteria ◽  
Comparative Sequence Analysis ◽  
The Other ◽  
Metagenomic Analysis ◽  
Beijing China ◽  
Metagenomic Approach

ABSTRACTA targeted metagenomic approach was applied to investigate magnetotactic bacteria (MTB) within the phylumNitrospiraein Lake Miyun near Beijing, China. Five fosmids containing rRNA operons were identified. Comparative sequence analysis of a total of 172 kb provided new insights into their genome organization and revealed unexpected subgenomic diversity of uncultivated MTB in the phylumNitrospirae. In addition, affiliation of two novel MTB with the phylumNitrospiraewas verified by fluorescencein situhybridization. One of them was morphologically similar to “CandidatusMagnetobacterium bavaricum,” but the other differed substantially in cell shape and magnetosome organization from all previously described “Ca.Magnetobacterium bavaricum”-like bacteria.

Regional differentiation of the membrane skeleton in Tetrahymena

1987 ◽  
Vol 87 (3) ◽  
pp. 457-463
Author(s):  
N.E. Williams ◽  
J.E. Honts ◽  
R.F. Jaeckel-Williams
Keyword(s):  
Cell Surface ◽  
Cell Shape ◽  
Surface Membrane ◽  
Tetrahymena Pyriformis ◽  
Membrane Skeleton ◽  
Regional Differentiation ◽  
Basal Bodies ◽  
Structural Elements ◽  
Molecular Weights ◽  
Triton X

Antisera have been raised in rabbits against three high molecular weight proteins that are present in Triton X-100-insoluble residues of Tetrahymena pyriformis GL cells. These proteins, called A, B and C, have apparent molecular weights of 235, 135 and 125 (X 10(3)), respectively, in SDS-polyacrylamide gels. The antisera obtained are specific for these proteins, as shown by immunoblotting. Immunolocalization studies are reported that suggest that these proteins are present throughout the epiplasmic layer beneath the cell surface (membrane skeleton). Images obtained with the fluorescence microscope, however, suggest that the membrane skeleton is modified in discrete zones: (1) around somatic basal bodies, (2) within the oral apparatus, (3) in the cytoproct, (4) in contractile vacuole pores, (5) in the fission zone in late division, and (6) at the mating junction in conjugating cells. These regions may represent areas of increased rigidity at the cell surface. The transition from pliable to rigid epiplasm in spatially delimited areas is apparently a recurring theme in cortical morphogenesis in Tetrahymena. Together, the two types of epiplasm probably allow for extensive changes in cell shape while preserving essential relationships between structural elements within the cortex.

scholarly journals Light and electron microscopic immunolocalization of rat submandibular gland mucin glycoprotein and glutamine/glutamic acid-rich proteins.

1989 ◽  
Vol 37 (4) ◽  
pp. 515-528 ◽  
Author(s):  
J E Moreira ◽  
L A Tabak ◽  
G S Bedi ◽  
D J Culp ◽  
A R Hand
Keyword(s):  
Monoclonal Antibody ◽  
Acinar Cells ◽  
Electron Microscopic ◽  
Western Blots ◽  
Gold Particles ◽  
Blood Group A ◽  
Mucin Glycoprotein ◽  
Group A ◽  
Secretory Products ◽  
Rat Submandibular Gland

We studied the subcellular localization of two major secretory products of adult rat submandibular gland (RSMG), blood group A-reactive mucin glycoprotein and glutamine/glutamic acid-rich protein (GRP), by light and electron microscopic immunocytochemistry. The structure of the major neutral oligosaccharide of the mucin was shown to be: GalNAc alpha 1,3(Fuc alpha 1,2)Gal beta 1,3GalNAc. A mouse monoclonal antibody (1F9) with specificity for blood group A determinants was prepared against the mucin. The antibody recognized a single band of approximately 114 KD on Western blots of RSMG extract. A previously characterized monoclonal antibody (59) against GRP (Mirels et al.: J Biol Chem 262: 7289, 1987) reacted with a doublet of 45-50 KD on Western blots of extraparotid saliva. Immunofluorescence and immunoperoxidase staining of cryostat sections of RSMG with anti-mucin antibodies and anti-GRP antibodies revealed reactivity in acinar cells of the gland. No specific labeling was seen in duct cells of RSMG or in mucous acinar cells of the adjacent sublingual gland. Post-embedding immunogold labeling of thin sections of glutaraldehyde-fixed RSMG with anti-mucin showed strong labeling of the Golgi apparatus and secretory granules of acinar cells. Gold particles were seen mainly over electron-lucent areas of the granules. No labeling occurred over the endoplasmic reticulum. The labeling pattern with the anti-GRP antibodies was similar, except that both electron-dense and -lucent areas of the granules were labeled, and the endoplasmic reticulum was reactive. Double labeling with two different sizes of gold particles showed that both mucin and GRP co-localized in the same granules. Pre-absorption of the antibodies with their respective antigens eliminated immunolabeling of the acinar cells. These antibodies will be useful in studies of cell differentiation in RSMG and of synthesis, processing, and packaging of RSMG secretory products.

Immunocytochemistry. A Review of Methodology for Electron Microscopic Applicaiton

1974 ◽  
Vol 32 ◽  
pp. 328-329
Author(s):  
Joseph E. Mazurkiewicz
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Electron Microscopic ◽  
Biological Interest ◽  
Investigative Approach ◽  
Immunologic Activity ◽  
Dense Label

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

Artifact-free electron microscopic immunocytochemistry on rat brain tissue

1991 ◽  
Vol 49 ◽  
pp. 272-273
Author(s):  
Veronika Burmeister ◽  
N. Ludvig ◽  
P.C. Jobe
Keyword(s):  
Microscopic Examination ◽  
Free Electron ◽  
Electron Microscopic Examination ◽  
Ultrastructural Localization ◽  
Rabbit Serum ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Phosphate Buffered Saline ◽  
Rat Brain Tissue ◽  
Immune Rabbit

Electron microscopic immunocytochemistry provides an important tool to determine the ultrastructural distribution of various molecules in both normal and pathologic tissues. However, the specific immunostaining may be obscured by artifactual immunoreaction product, misleading the investigator. Previous observations show that shortening the incubation period with the primary antibody from the generally used 12-24 hours to 1 hour substantially reduces the artifactual immunostaining. We now extend this finding by the demonstration of artifact-free ultrastructural localization of the Ca2/calmodulindependent cyclic nucleotide phosphodiesterase (CaM-dependent PDE) immunoreactivity in brain.Anesthetized rats were perfused transcardially with phosphate-buffered saline followed by a fixative containing paraformaldehyde (4%) and glutaraldehyde (0.25%) in PBS. The brains were removed, and 40μm sections were cut with a vibratome. The sections were processed for immunocytochemistry as described by Ludvig et al. Both non-immune rabbit serum and specific CaM-dependent PDE antibodies were used. In both experiments incubations were at one hour and overnight. The immunostained sections were processed for electron microscopic examination.

Sign in / Sign up

Export Citation Format

Share Document