scholarly journals Localization of sugar-binding sites in Staphylococcus aureus using gold-labeled neoglycoprotein.

1994 ◽  
Vol 42 (12) ◽  
pp. 1609-1613 ◽  
Author(s):  
H Morioka ◽  
A Suganuma ◽  
M Tachibana
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Bovine Serum Albumin ◽  
Binding Sites ◽  
Bacterial Strain ◽  
Wheat Germ ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Gold Particles ◽  
Sugar Binding

We studied post- and pre-embedding staining of sugar-binding sites on thin sections of Staphylococcus aureus with an electron microscopic neoglycoprotein-gold technique. Although gold particles of cellobiosyl bovine serum albumin (BSA)-glycosylated BSA-, lactosyl BSA-, and melibiosyl BSA-gold did not label, heavy labeling of N-acetylglucosaminide-BSA-gold was observed in both the cell wall and the cytoplasm on Spurr-embedded thin sections of S. aureus. Inhibition of labeling with wheat germ agglutinin-biotin and N-acetylglucosaminidase indicated that the labeling was due to N-acetylglucosamine. These data suggested that molecules that bind specifically with N-acetylglucosamine occur in the cell wall and cytoplasm of S. aureus. Pre-embedding staining revealed that these molecules are abundant at the surface of the cell wall and that the abundance differs depending on the bacterial strain. An N-acetylglucosamine-specific lectin-like substance, glucosaminidase, and toxins are proposed as candidates for molecules responsible for the labeling, and the possible functional significance of the findings is discussed briefly.

scholarly journals Polymyxin B binding sites in Escherichia coli as revealed by polymyxin B-gold labeling.

1987 ◽  
Vol 35 (2) ◽  
pp. 229-231 ◽  
Author(s):  
H Morioka ◽  
M Tachibana ◽  
M Machino ◽  
A Suganuma
Keyword(s):  
Escherichia Coli ◽  
Bovine Serum Albumin ◽  
Binding Sites ◽  
Colloidal Gold ◽  
Ultrastructural Localization ◽  
Polymyxin B ◽  
Thin Sections ◽  
Gold Particles ◽  
E Coli ◽  
Escherichia Coli Cells

A complex of polymyxin B, bovine serum albumin, and colloidal gold was prepared and used for the ultrastructural localization of polymyxin B binding sites on thin sections of Epon-embedded Escherichia coli cells. Gold particles were found on the outer membrane of E. coli, which is consistent with reported biochemical findings. We concluded that gold labeling with polymyxin B is useful in localizing the binding sites of polymyxin.

scholarly journals Aminoglycoside binding sites in Escherichia coli as revealed by neomycin-gold labeling.

1986 ◽  
Vol 34 (7) ◽  
pp. 909-912 ◽  
Author(s):  
H Morioka ◽  
M Tachibana ◽  
T Amagai ◽  
A Suganuma
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Bovine Serum Albumin ◽  
Binding Sites ◽  
Colloidal Gold ◽  
Cytoplasmic Membrane ◽  
Thin Sections ◽  
Gold Particles ◽  
E Coli ◽  
Cytochemical Technique

A cytochemical technique for demonstration of neomycin binding sites by electron microscopy was developed and applied to Escherichia coli. Neomycin was conjugated chemically with bovine serum albumin (BSA). Colloidal gold was coated with the conjugated neomycin-BSA. The neomycin-BSA-gold was applied to thin sections of Epon-embedded E. coli and examined. Gold particles were observed on the outer membrane and the cytoplasmic membrane of E. coli. It was probably the ribosomes that were being labeled in the cytoplasm. Different cytochemical controls, including a number of inhibition tests and the use of BSA-gold, proved the specificity of this cytochemical technique and provided the biochemical significance of the observations.

Immunocytochemical localization of p65 with one-nanometer gold particles following silver development

1989 ◽  
Vol 47 ◽  
pp. 1042-1043
Author(s):  
Richard W. Burry ◽  
Diane M. Hayes
Keyword(s):  
Plasma Membrane ◽  
Bovine Serum Albumin ◽  
Calf Serum ◽  
Specific Protein ◽  
Immunocytochemical Localization ◽  
Electron Microscopic ◽  
Gold Particles ◽  
Pass Through ◽  
Label Distribution ◽  
Phosphate Buffered Saline

Electron microscopic (EM) immunocytochemistry localization of the neuron specific protein p65 could show which organelles contain this antigen. Antibodies (Ab) labeled with horseradish peroxidase (HRP) followed by chromogen development show a broad diffuse label distribution within cells and restricting identification of organelles. Particulate label (e.g. 10 nm colloidal gold) is highly desirable but not practical because penetration into cells requires destroying the plasma membrane. We report pre-embedding immunocytochemistry with a particulate marker, 1 nm gold, that will pass through membranes treated with saponin, a mild detergent.Cell cultures of the rat cerebellum were fixed in buffered 4% paraformaldehyde and 0.1% glutaraldehyde (Glut.). The buffer for all incubations and rinses was phosphate buffered saline with: 1% calf serum, 0.2% saponin, 0.1% gelatin, 50 mM glycine 1 mg/ml bovine serum albumin, and (not in the HRP labeled cultures) 0.02% sodium azide. The monoclonal #48 to p65 was used with three label systems: HRP, 1 nm avidin gold with IntenSE M development, and 1 nm avidin gold with Danscher development.

THE MODE OF ACTION OF THE ANTIBIOTIC PAROMOMYCIN ON STAPHYLOCOCCUS AUREUS

1966 ◽  
Vol 12 (6) ◽  
pp. 1157-1165 ◽  
Author(s):  
A. von Seefried ◽  
D. C. Jordan
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Binding Sites ◽  
Bactericidal Action ◽  
Plate Method ◽  
Vitro Binding ◽  
Resistant Mutants ◽  
Intracellular Binding

Paromomycin (Humatin, Parke Davis & Co.), a broad-spectrum aminoglycosidic antibiotic, inhibits the incorporation of amino acids into the trypsinsoluble protein fraction of Staphylococcus aureus 257. Protein synthesis is inhibited immediately, but the synthesis of cell-wall mucopeptide and alcohol-soluble proteins and lipids is not affected for approximately 35 min after antibiotic addition to actively growing cells. Paromomycin, at the ribosomal level, prevents the attachment of amino acyl-s-RNA and causes accumulation of m-RNA.Divalent cations (Ca++ and Mg++) antagonize the bactericidal action of paromomycin and interfere with the in vivo binding of the antibiotic on both the cell surface and the intracellular binding sites. In vitro binding to free ribosomes can be prevented and reversed by both monovalent and divalent cations.Using a "cylinder-plate" method, involving the displacement of antibiotic from cellular fractions by 0.2 M MgCl2, the antibiotic can be recovered from the ribosomes, cytoplasm, and the cell wall of paromomycin-sensitive S. aureus cells, but is not found in any of these fractions isolated from paromomycin-resistant cells developed from the sensitive parent strain. The resistant mutants apparently have lost the ability to adsorb and transport the antibiotic into the cell.

scholarly journals Application of lectin--gold complexes for electron microscopic localization of glycoconjugates on thin sections.

1983 ◽  
Vol 31 (8) ◽  
pp. 987-999 ◽  
Author(s):  
J Roth
Keyword(s):  
Endoplasmic Reticulum ◽  
Concanavalin A ◽  
Binding Sites ◽  
Light And Electron Microscopy ◽  
Gold Complex ◽  
Electron Microscopic ◽  
Gold Complexes ◽  
Thin Sections ◽  
Renal Tubular Cells ◽  
Lowicryl K4m

A method is described for the electron microscopic detection of lectin-binding sites in different cellular compartments and extracellular structures that uses thin sections from resin-embedded tissues. Various lectins (Ricinus communis lectin I and II, peanut lectin, Lotus tetragonolobus lectin, Ulex europeus lectin I, Lens culinaris lectin, Helix pomatia lectin, and soybean lectin) were bound to particles of colloidal gold and used for direct staining of thin sections or glycoprotein--gold complexes were prepared and applied in an indirect technique (concanavalin A and horseradish peroxidase--gold complex; wheat germ lectin and ovomucoid--gold complex). The details for preparation of such complexes from 14 nm gold particles are reported. The conditions of tissue processing that gave satisfactory staining results and good fine structure preservation were mild aldehyde fixation without osmification and low temperature embedding with the hydrophilic resin Lowicryl K4M. None of the so-called etching procedures was necessary prior to labeling of Lowicryl K4M thin sections. Examples of the use of this approach for detection of glycoconjugates in the rough endoplasmic reticulum, Golgi apparatus, and mucin of intestinal goblet cells as well as plasma membrane and various intracellular structures of absorptive intestinal and renal tubular cells are shown. A comparison is made with preembedding staining results on Concanavalin A-binding site localization in rat liver which shows that problems of penetration common in such a technique are circumvented by the postembedding approach described here. Concanavalin A-binding sites were not only consistently found in nuclear envelope, rough and smooth endoplasmic reticulum, plasma membranes, and collagen fibers, but also in mitochondria, glycogen, ribosomes, and nucleus. These data and those of a previous investigation (Roth J, Cytochem 31:547, 1983) prove the applicability of this cytochemical technique for postembedding localization of glycoconjugates by light and electron microscopy.

scholarly journals Immunocytochemical localization of D-amino acid oxidase in the central clear matrix of rat kidney peroxisomes.

1986 ◽  
Vol 34 (12) ◽  
pp. 1709-1718 ◽  
Author(s):  
N Usuda ◽  
S Yokota ◽  
T Hashimoto ◽  
T Nagata
Keyword(s):  
Amino Acid ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Protein A ◽  
Immunoblot Analysis ◽  
Acid Oxidase ◽  
Electron Microscopic ◽  
Amino Acid Oxidase ◽  
Thin Sections ◽  
Gold Particles

Light and electron microscopic localizations of D-amino acid oxidase (DAO) in rat kidney was investigated using immunoenzyme and protein A-gold techniques. The enzyme was purified from rat kidney homogenate and its antibody was raised in rabbits. By Ouchterlony double-diffusion analysis and immunoblot analysis with anti-(rat kidney DAO) immunoglobulin, the antibody was confirmed to be monospecific. The tissue sections (200 micron thick) of fixed rat kidney were embedded in Epon or Lowicryl K4M. Semi-thin sections were stained for DAO by the immunoenzyme technique after removal of epoxy resin for LM, and ultra-thin sections of Lowicryl-embedded material were labeled for DAO by the protein A-gold technique for EM. By LM, fine cytoplasmic granules of proximal tubule were stained exclusively. Among three segments of proximal tubules, and S2 and S3 segments were heavily stained but the S1 segment only weakly so. By EM, gold particles indicating the antigenic sites for DAO were exclusively confined to peroxisomes. Within peroxisomes, the gold particles were localized in the central clear matrix but not in the peripheral tubular substructures. The results indicate that D-amino acid oxidase in rat kidney is present exclusively in peroxisomes in the proximal tubule and that within peroxisomes it is found only in central clear matrix and not in the peripheral tubular substructures.

scholarly journals Ultracytochemistry of calmodulin binding sites in myocardial cells by staining of frozen thin sections with colloidal gold-labeled calmodulin.

1989 ◽  
Vol 37 (2) ◽  
pp. 249-256 ◽  
Author(s):  
K Fujimoto ◽  
N Araki ◽  
K S Ogawa ◽  
S Kondo ◽  
T Kitaoka ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Sarcoplasmic Reticulum ◽  
Binding Sites ◽  
Colloidal Gold ◽  
Biologically Active ◽  
Myocardial Cells ◽  
Thin Sections ◽  
Gold Particles ◽  
Calmodulin Binding ◽  
Tissue Sections

Calmodulin (CaM) has been implicated as a multifunctional regulator of Ca2+ in the cytoplasm of cells. We have recently introduced biologically active colloidal gold-labeled CaM as a marker for identifying potential CaM binding sites (unoccupied by endogenous CaM at the time of fixation) by electron microscopy and have stained frozen thin sections of rat cardiac muscle with this conjugate. In the presence of Ca2+, gold particles indicating CaM binding sites were found localized on the sarcoplasmic reticulum, mitochondria, and gap junctions. Control tissue sections treated with EGTA or exposed to excess amounts of unlabeled native CaM before staining showed no binding. We believe that cytochemistry of potential CaM binding sites revealed by staining with labeled exogenous CaM is useful in correlating known biochemical reactions of CaM with particular cell activities.

scholarly journals Interaction of Azospirillum lipoferumwith Wheat Germ Agglutinin Stimulates Nitrogen Fixation

1999 ◽  
Vol 181 (13) ◽  
pp. 3949-3955 ◽  
Author(s):  
Eva Karpati ◽  
Peter Kiss ◽  
Tamas Ponyi ◽  
Istvan Fendrik ◽  
Miklos de Zamaroczy ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Gene Cluster ◽  
Binding Sites ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Side Chains ◽  
Receptor Complex ◽  
Sugar Binding ◽  
Signalling Cascade

ABSTRACT In vitro, the nitrogen fixation capability of A. lipoferum is efficiently increased in the presence of wheat germ agglutinin (WGA). A putative WGA-binding receptor, a 32-kDa protein, was detected in the cell capsule. The stimulatory effect requiredN-acetyl-d-glucosamine dimer (GlcNAcdi) terminated sugar side chains of the receptor and was dependent on the number of GlcNAcdi links involved in receptor-WGA interface. Binding to the primary sugar binding sites on WGA had a larger stimulatory effect than binding to the secondary sites. The WGA-receptor complex generated stimulus led to elevated transcription of the nifH and nifA genes and of the glnBA gene cluster but not of the glnA gene from its own promoter. There may well be a signalling cascade contributing to the regulation of nitrogen fixation.

scholarly journals Regional distribution of N-acetyl-D-galactosamine residues in the glycocalyx of glomerular podocytes.

1983 ◽  
Vol 96 (5) ◽  
pp. 1189-1196 ◽  
Author(s):  
J Roth ◽  
D Brown ◽  
L Orci
Keyword(s):  
Basement Membrane ◽  
Cell Surface ◽  
Binding Sites ◽  
Wheat Germ ◽  
Lectin Binding ◽  
Glomerular Cell ◽  
Thin Sections ◽  
Foot Process ◽  
Wheat Germ Lectin ◽  
Lectin Binding Sites

Helix pomatia lectin (HPL) bound to colloidal gold was used as a specific cytochemical probe for the localization of terminal nonreducing N-acetyl-D-galactosamine residues in thin sections of rat kidney. In the glomerulus, lectin-binding sites were associated only with the podocyte foot process bases and were not found on the free cell surface of podocytes or on any other glomerular components. Gold-particle label was often arranged in the form of clusters which extended from the foot process base to the lamina rare externa and lamina densa of the basement membrane. In contrast, wheat germ lectin (WGL)-binding sites (beta-[1 leads to 4] linked N-acetyl-D-glucosamine residues and N-acetylneuraminic acid residues) were found in all regions of the podocyte plasma membrane and on the cell surface of all other glomerular cell types. In addition, WGL-binding sites were present in all three layers of the glomerular basement membrane (GBM) as well as in the mesangial matrix. A quantitative evaluation of the pattern of labeling for HPL-binding sites together with the sugar specificity of this lectin suggest that a component of the glycocalyx is being detected rather than a basement membrane component. This was confirmed by the absence of H. pomatia lectin-binding sites in preparations of isolated GBM which retained, however, wheat germ lectin-binding sites. These data show that the glycocalyx of the foot process base is a highly specialized cell surface domain with respect to its carbohydrate composition.

Colloidal Gold Markers: Preparation and Cell Labeling for Transmission and Scanning Electron Microscopy

1985 ◽  
Vol 43 ◽  
pp. 530-533
Author(s):  
Robert S. Molday
Keyword(s):  
Cell Surface ◽  
Colloidal Gold ◽  
Critical Evaluation ◽  
Cell Labeling ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Gold Particles ◽  
Backscattered Electrons ◽  
Wide Range ◽  
Scanning Electron

Colloidal gold particles have become one of the most widely used markers to detect, localize and, in some cases, quantitate cell surface and intracellular antigens and receptors since their introduction as transmission electron microscopic (TFM) markers by Faulk and Taylor in 1971 and as scanning electron microscopic (SEM) markers by Horisberger et al. in 1975. This interest in colloidal gold markers for cell labeling is based on their versatile properties for detection under the electron microscope. Colloidal gold particles are highly electron-dense which enables them to be seen under the TEM in thin sections of heavily stained cells. They can be prepared in a wide range of highly uniform sizes for visualization at different magnifications and for multiple labeling studies. Under the SEM, gold particles emit a high quantity of secondary electrons, backscattered electrons and characteristic X-ray signals and as a result, with the appropriate detectors, they can be readily distinguished from cell surface structures having a similar morphological appearance. The successful application of colloidal gold particles as markers for TEM and SEM however requires (i) careful preparation and characterization of both the gold markers and the ligand (protein)-gold conjugates, (ii) utilization of specific labeling techniques employing necessary controls to confirm the specificity of labeling, and (iii) critical evaluation of results in relation to the conditions used in labeling. These aspects of gold labeling will be considered here. Additional information can be obtained from recent reviews dealing specifically with gold markers and more generally with cell labeling techniques.

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