Immunogold labeling of the bacterial surface components of shigella flexneri 2a

1995 ◽  
Vol 53 ◽  
pp. 1052-1053
Author(s):  
D.H. Chen ◽  
G.H. Wang ◽  
H.Q. Zhang ◽  
H. Li
Keyword(s):  
Outer Surface ◽  
Bacterial Cell ◽  
Shigella Flexneri ◽  
Electron Microscopic Examination ◽  
Immunogold Labeling ◽  
Electron Microscopic ◽  
Bacterial Surface ◽  
Cell Wall Binding ◽  
Shigella Flexneri 2A ◽  
Pure Cultures

Some bacterial outer membrane components are efficient protective antigens against Shigella spp. Electron microscopic immunogold labeling provides a possibility for investigating the surface exposure of antigens (1). The aim of the present study was to localise surface antigens of Shigella flexneri 2a by using an electron microscopic immunogold labeling technique.Seven monoclonal antibodies (MAbs: 2A3, 3A6,2E6,1G8,1Al and 4F1) used in this study were produced as described previously (2). Pure cultures of Shigella flaxneri 2a were grown in broth, harvested by centrifugation, prefixed and then immunolabelled by MAbs and anti-mouse IgG conjugated to colloidal gold particles (GAMG15 and GAMG5) respectively. Ultrathin sections of labelled bacteial cells were cut and viewed in a transmission electron microscope (Philips EM 400T).Electron microscopic examination of the sectioned organisms showed that gold labeling was confined to the outer surface of the bacterial cell wall. Binding of the MAb on whole bacterial cell indicated the antigens recognized by MAbs 3A6, 2E6, 4F1 and 2A3 were exposed on the outer surface of cell membrane (Fig 1, a-c), and those by MAbs 1A1 and 1G8 were not.

scholarly journals Genetic Characterization and Immunogenicity of Coli Surface Antigen 4 from Enterotoxigenic Escherichia coli when It Is Expressed in a Shigella Live-Vector Strain

2003 ◽  
Vol 71 (3) ◽  
pp. 1352-1360 ◽  
Author(s):  
Zeev Altboum ◽  
Myron M. Levine ◽  
James E. Galen ◽  
Eileen M. Barry
Keyword(s):  
Escherichia Coli ◽  
Shigella Flexneri ◽  
Regulatory Protein ◽  
Electron Microscopic Examination ◽  
Amino Acid Sequences ◽  
Enterotoxigenic Escherichia Coli ◽  
Electron Microscopic ◽  
Bacterial Surface ◽  
Fimbrial Subunit ◽  
Mucosal Antibody

ABSTRACT The genes that encode the enterotoxigenic Escherichia coli (ETEC) CS4 fimbriae, csaA, -B, -C, -E, and -D′, were isolated from strain E11881A. The csa operon encodes a 17-kDa major fimbrial subunit (CsaB), a 40-kDa tip-associated protein (CsaE), a 27-kDa chaperone-like protein (CsaA), a 97-kDa usher-like protein (CsaC), and a deleted regulatory protein (CsaD′). The predicted amino acid sequences of the CS4 proteins are highly homologous to structural and assembly proteins of other ETEC fimbriae, including CS1 and CS2, and to CFA/I in particular. The csaA, -B, -C, -E operon was cloned on a stabilized plasmid downstream from an osomotically regulated ompC promoter. pGA2-CS4 directs production of CS4 fimbriae in both E. coli DH5α and Shigella flexneri 2a vaccine strain CVD 1204, as detected by Western blot analysis and bacterial agglutination with anti-CS4 immune sera. Electron-microscopic examination of Shigella expressing CS4 confirmed the presence of fimbriae on the bacterial surface. Guinea pigs immunized with CVD 1204(pGA2-CS4) showed serum and mucosal antibody responses to both the Shigella vector and the ETEC fimbria CS4. Among the seven most prevalent fimbrial antigens of human ETEC, CS4 is the last to be cloned and sequenced. These findings pave the way for CS4 to be included in multivalent ETEC vaccines, including an attenuated Shigella live-vector-based ETEC vaccine.

scholarly journals Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.

1980 ◽  
Vol 85 (3) ◽  
pp. 890-902 ◽  
Author(s):  
K D McCarthy ◽  
J de Vellis
Keyword(s):  
Electron Microscopic Examination ◽  
Brain Extract ◽  
Cell Protein ◽  
Glycerol Phosphate ◽  
Electron Microscopic ◽  
Morphological Effect ◽  
Oligodendroglial Cell ◽  
Process Formation ◽  
Cyclic Amp Accumulation ◽  
Pure Cultures

A novel method has been developed for the preparation of nearly pure separate cultures of astrocytes and oligodendrocytes. The method is based on (a) the absence of viable neurons in cultures prepared from postnatal rat cerebra, (b) the stratification of astrocytes and oligodendrocytes in culture, and (c) the selective detachment of the overlying oligodendrocytes when exposed to sheer forces generated by shaking the cultures on an orbital shaker for 15--18 h at 37 degrees C. Preparations appear greater than 98% pure and contain approximately 1-2 x 10(7) viable cells (20--40 mg of cell protein). Three methods were used to characterize these two culture t ypes. First, electron microscopic examination was used to identify the cells in each preparation (mixed and separated cultures of astrocytes and oligodendrocytes) and to assess the purity of each preparation. Second, two oligodendroglial cell markers, 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37) and glycerol phosphate dehydrogenase (EC 1.1.1.8) were monitored. Third, the regulation of cyclic AMP accumulation in each culture type was examined. In addition to these studies, we examined the influence of brain extract and dibutyryl cAMP on the gross morphology and ultrastructure of each preparation. These agents induced astroglial process formation without any apparent morphological effect on oligodendrocytes. Collectively, the results indicate that essentially pure cultures of astrocytes and of oligodendrocytes can be prepared and maintained. These preparations should significantly aid in efforts to examine the biochemistry, physiology, and pharmacology of these two major classes of central nervous system cells.

scholarly journals Establishment of Unipolar Localization of IcsA in Shigella flexneri 2a Is Not Dependent on Virulence Plasmid Determinants

1999 ◽  
Vol 67 (1) ◽  
pp. 350-356 ◽  
Author(s):  
Robin C. Sandlin ◽  
Anthony T. Maurelli
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Bacterial Cell ◽  
Shigella Flexneri ◽  
Virulence Plasmid ◽  
Wild Type ◽  
Common Pathway ◽  
Plasmid Copy ◽  
E Coli ◽  
Shigella Flexneri 2A

ABSTRACT Unipolar localization of IcsA on the surface of Shigella flexneri is required for efficient formation of actin tails and protrusions in infected eucaryotic cells. Lipopolysaccharide (LPS) mutations have been demonstrated to affect either the establishment or the maintenance of IcsA in a unipolar location, although the mechanism is unknown. In order to analyze the contribution of virulence plasmid determinants on the unipolar localization of IcsA, we examined the localization of IcsA expressed from a cloned plasmid copy in two different genetic backgrounds. The localization of IcsA was first examined in a virulence plasmid-cured derivative of the wild-typeS. flexneri 2a isolate 2457T. This approach examined the contribution of virulence plasmid-borne factors, including the previously identified virulence plasmid-borne protease that is responsible for cleaving IcsA in the outer membrane and releasing the 95-kDa secreted form from the cell surface. IcsA localization in a related but nonpathogenic Escherichia coli strain expressing LPS of the O8 serotype was also examined. IcsA surface presentation in both of these genetic backgrounds continued to be unipolar, demonstrating that virulence plasmid-borne determinants are not responsible for unipolar localization of IcsA. The unipolar localization of IcsA in the E. coli background suggests that a common pathway that allows IcsA to be spatially restricted to one pole on the bacterial cell surface exists in Shigellaand E. coli.

Electron microscopic studies on ultrathin frozen sections of lactating mouse mammary gland

1978 ◽  
Vol 36 (2) ◽  
pp. 46-47
Author(s):  
Jan Zarzycki ◽  
Joseph Szroeder
Keyword(s):  
Mammary Gland ◽  
Protein Denaturation ◽  
Chemical Constituents ◽  
Freeze Substitution ◽  
Electron Microscopic Examination ◽  
Mouse Mammary Gland ◽  
Electron Microscopic ◽  
Preparation Methods ◽  
Microscopic Studies ◽  
Ultrathin Frozen Sections

The mammary gland ultrastructure in various functional states is the object of our investigations. The material prepared for electron microscopic examination by the conventional chemical methods has several limitations, the most important are the protein denaturation processes and the loss of large amounts of chemical constituents from the cells. In relevance to this,one can't be sure about a degree the observed images are adequate to the realy ultrastructure of a living cell. To avoid the disadvantages of the chemical preparation methods,some autors worked out alternative physical methods based on tissue freezing / freeze-drying, freeze-substitution, freeze-eatching techniqs/; actually the technique of cryoultraraicrotomy,i,e.cutting ultrathin sections from deep frozen specimens is assented as a complete alternative method. According to the limitations of the routine plastic embbeding methods we were interested to analize the mammary gland ultrastructure during lactation by the cryoultramicrotomy method.

A scanning electron microscopic study on dissolving process of cholesterol gallstones by chenodeoxycholic acid (CDCA)

1978 ◽  
Vol 36 (2) ◽  
pp. 522-523
Author(s):  
T. Kanetaka ◽  
M. Cho ◽  
S. Kawamura ◽  
T. Sado ◽  
K. Hara
Keyword(s):  
Electron Microscope ◽  
Chenodeoxycholic Acid ◽  
Outer Surface ◽  
Electron Microscopic ◽  
Cholesterol Gallstones ◽  
The Core ◽  
Scanning Electron Microscopic Study ◽  
Scanning Electron Microscopic ◽  
Acceleration Voltage ◽  
Scanning Electron

The authors have investigated the dissolution process of human cholesterol gallstones using a scanning electron microscope(SEM). This study was carried out by comparing control gallstones incubated in beagle bile with gallstones obtained from patients who were treated with chenodeoxycholic acid(CDCA).The cholesterol gallstones for this study were obtained from 14 patients. Three control patients were treated without CDCA and eleven patients were treated with CDCA 300-600 mg/day for periods ranging from four to twenty five months. It was confirmed through chemical analysis that these gallstones contained more than 80% cholesterol in both the outer surface and the core.The specimen were obtained from the outer surface and the core of the gallstones. Each specimen was attached to alminum sheet and coated with carbon to 100Å thickness. The SEM observation was made by Hitachi S-550 with 20 kV acceleration voltage and with 60-20, 000X magnification.

A Comparative Study of Fractographic Replicas

1974 ◽  
Vol 32 ◽  
pp. 566-567
Author(s):  
Loren Anderson ◽  
Pat Pizzo ◽  
Glen Haydon
Keyword(s):  
Electron Microscopy ◽  
Electron Microscopic Examination ◽  
Direct Examination ◽  
Electron Microscopic ◽  
Reliable Technique ◽  
Service Failures ◽  
Transmission Electron ◽  
Mode Of Failure ◽  
Scanning Electron Microscopic ◽  
Scanning Electron

Transmission electron microscopy of replicas has long been used to study the fracture surfaces of components which fail in service. Recently, the scanning electron microscope (SEM) has gained popularity because it allows direct examination of the fracture surface. However, the somewhat lower resolution of the SEM coupled with a restriction on the sample size has served to limit the use of this instrument in investigating in-service failures. It is the intent of this paper to show that scanning electron microscopic examination of conventional negative replicas can be a convenient and reliable technique for determining mode of failure.

Novel methods for demonstrating osseointegration of hydroxylapatite-coated titanium hip prostheses

1991 ◽  
Vol 49 ◽  
pp. 34-35 ◽  
Author(s):  
P. Frayssinet ◽  
J. Hanker ◽  
D. Hardy ◽  
B. Giammara
Keyword(s):  
Hip Prosthesis ◽  
Ethanol Concentration ◽  
Bone Matrix ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Hip Prostheses ◽  
Cellular Inclusions ◽  
Microscopic Studies ◽  
Diamond Saw ◽  
Plasma Sprayed

Prostheses implanted in hard tissues cannot be processed for electron microscopic examination or microanalysis in the same way as those in other tissues. For these reasons, we have developed methods allowing light and electron microscopic studies as well as microanalysis of the interface between bone and a metal biomaterial coated by plasma-sprayed hydroxylapatite(HA) ceramic.An HA-coated titanium hip prosthesis (Corail, Landos, France), which had been implanted for two years, was removed after death (unrelated to the orthopaedic problem). After fixation it was dehydrated in solutions of increasing ethanol concentration prior to embedment in polymethylmethacrylate(PMMA). Transverse femur sections were obtained with a diamond saw and the sections then carefully ground to a thickness of 200 microns. Plastic-embedded sections were stained for calcium with a silver methenamine modification of the von Kossa method for calcium staining and coated by carbon. They have been examined by back-scatter SEM on an ISI-SS60 operated at 25 KV. EDAX has been done on cellular inclusions and extracellular bone matrix.

Properties of Isolated Mitochondria of Agaricus Bisporus: Their Relationship to Dormancy and the Germination of Spores

1973 ◽  
Vol 31 ◽  
pp. 458-459
Author(s):  
K. S. McCarty ◽  
R. F. Weave ◽  
L. Kemper ◽  
F. S. Vogel
Keyword(s):  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Microscopic Examination ◽  
Agaricus Bisporus ◽  
Osmotic Shock ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Isolated Mitochondria ◽  
Dna Strands ◽  
Cytoplasmic Ribosomes

During the prodromal stages of sporulation in the Basidiomycete, Agaricus bisporus, mitochondria accumulate in the basidial cells, zygotes, in the gill tissues prior to entry of these mitochondria, together with two haploid nuclei and cytoplasmic ribosomes, into the exospores. The mitochondria contain prominent loci of DNA [Fig. 1]. A modified Kleinschmidt spread technique1 has been used to evaluate the DNA strands from purified whole mitochondria released by osmotic shock, mitochondrial DNA purified on CsCl gradients [density = 1.698 gms/cc], and DNA purified on ethidium bromide CsCl gradients. The DNA appeared as linear strands up to 25 u in length and circular forms 2.2-5.2 u in circumference. In specimens prepared by osmotic shock, many strands of DNA are apparently attached to membrane fragments [Fig. 2]. When mitochondria were ruptured in hypotonic sucrose and then fixed in glutaraldehyde, the ribosomes were released for electron microscopic examination.

Molecular Structure of the Outermost Cell Wall Component of Spirillum Serpens

1977 ◽  
Vol 35 ◽  
pp. 342-343
Author(s):  
Wah Chiu ◽  
David Grano
Keyword(s):  
Molecular Structure ◽  
Cell Wall ◽  
Outer Membrane ◽  
Gel Electrophoresis ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Cell Wall Component ◽  
Protein Components ◽  
Exposure Technique ◽  
Structural Aspects

The periodic structure external to the outer membrane of Spirillum serpens VHA has been isolated by similar procedures to those used by Buckmire and Murray (1). From SDS gel electrophoresis, we have found that the isolated fragments contain several protein components, and that the crystalline structure is composed of a glycoprotein component with a molecular weight of ∽ 140,000 daltons (2). Under an electron microscopic examination, we have visualized the hexagonally-packed glycoprotein subunits, as well as the bilayer profile of the outer membrane. In this paper, we will discuss some structural aspects of the crystalline glycoproteins, based on computer-reconstructed images of the external cell wall fragments.The specimens were prepared for electron microscopy in two ways: negatively stained with 1% PTA, and maintained in a frozen-hydrated state (3). The micrographs were taken with a JEM-100B electron microscope with a field emission gun. The minimum exposure technique was essential for imaging the frozen- hydrated specimens.

Electron Microscopic Examination of a Transplantable Rat Mammary Carcinoma

1968 ◽  
Vol 26 ◽  
pp. 20-21
Author(s):  
S. Shirahama ◽  
G. C. Engle ◽  
R. M. Dutcher
Keyword(s):  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Electron Microscopic Examination ◽  
Undifferentiated Carcinoma ◽  
Uranyl Acetate ◽  
Sprague Dawley ◽  
Electron Microscopic ◽  
North West ◽  
X Irradiation ◽  
Tissue Specimens

A transplantable carcinoma was established in North West Sprague Dawley (NWSD) rats by use of X-irradiation by Engle and Spencer. The tumor was passaged through 63 generations over a period of 32 months. The original tumor, an adenocarcinoma, changed into an undifferentiated carcinoma following the 19th transplant. The tumor grew well in NWSD rats of either sex at various ages. It was invariably fatal, causing death of the host within 15 to 35 days following transplantation.Tumor, thymus, spleen, and plasma from 7 rats receiving transplants of tumor at 3 to 9 weeks of age were examined with an electron microscope at intervals of 8, 15, 22 and 30 days after transplantation. Four normal control rats of the same age were also examined. The tissues were fixed in glutaraldehyde, postfixed in osmium tetroxide and embedded in Epon. The plasma was separated from heparanized blood and processed as previously described for the tissue specimens. Sections were stained with uranyl acetate followed by lead citrate and examined with an RCA EMU-3G electron microscope.

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