scholarly journals Xylanase Attachment to the Cell Wall of the Hyperthermophilic Bacterium Thermotoga maritima

2007 ◽  
Vol 190 (4) ◽  
pp. 1350-1358 ◽  
Author(s):  
Wolfgang Liebl ◽  
Christoph Winterhalter ◽  
Wolfgang Baumeister ◽  
Martin Armbrecht ◽  
Michael Valdez
Keyword(s):  
Outer Membrane ◽  
Signal Peptide ◽  
Cellular Localization ◽  
Culture Supernatant ◽  
Thermotoga Maritima ◽  
Surrounding Medium ◽  
Hydrophobic Core ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Amino Terminal

ABSTRACT The cellular localization and processing of the endo-xylanases (1,4-β-d-xylan-xylanohydrolase; EC 3.2.1.8) of the hyperthermophile Thermotoga maritima were investigated, in particular with respect to the unusual outer membrane (“toga”) of this gram-negative bacterium. XynB (40 kDa) was detected in the periplasmic fraction of T. maritima cells and in the culture supernatant. XynA (120 kDa) was partially released to the surrounding medium, but most XynA remained cell associated. Immunogold labeling of thin sections revealed that cell-bound XynA was localized mainly in the outer membranes of T. maritima cells. Amino-terminal sequencing of purified membrane-bound XynA revealed processing of the signal peptide after the eighth residue, thereby leaving the hydrophobic core of the signal peptide attached to the enzyme. This mode of processing is reminiscent of type IV prepilin signal peptide cleavage. Removal of the entire XynA signal peptide was necessary for release from the cell because enzyme purified from the culture supernatant lacked 44 residues at the N terminus, including the hydrophobic part of the signal peptide. We conclude that toga association of XynA is mediated by residues 9 to 44 of the signal peptide. The biochemical and electron microscopic localization studies together with the amino-terminal processing data indicate that XynA is held at the cell surface of T. maritima via a hydrophobic peptide anchor, which is highly unusual for an outer membrane protein.

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.

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 Polypeptides of the chloroplast envelope membranes as visualized by immunochemical techniques.

1986 ◽  
Vol 34 (5) ◽  
pp. 577-583 ◽  
Author(s):  
J van Berkel ◽  
M Steup ◽  
W Völker ◽  
H Robenek ◽  
U I Flügge
Keyword(s):  
Outer Membrane ◽  
Spinacia Oleracea ◽  
Leaf Tissue ◽  
Fluorescein Isothiocyanate ◽  
Chloroplast Envelope ◽  
Envelope Membrane ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Microscopic Evaluation ◽  
Freeze Etching

The polypeptides of relative molecular masses (Mr) 22,000, 29,000, and 36,000 represent three major constituents of the chloroplast envelope of spinach (Spinacia oleracea L.) leaves. The Mr 22,000 polypeptide has been localized in the outer membrane, whereas the two other peptides have been attributed to the inner envelope membrane (Joyard et al., 1983). The Mr 29,000 polypeptide has been identified as the "phosphate translocator" (Flügge and Heldt, 1979). In this investigation, we studied the three envelope polypeptides by means of immunocytochemistry. Using indirect immunofluorescence, all three polypeptides were visualized in cryostat sections of formaldehyde-fixed leaf tissue. They were found in both palisade and spongy parenchyma cells and in guard cells, as indicated by a strong fluorescence in the chloroplast periphery. In contrast, fluorescein isothiocyanate or protein A-gold labeling of isolated fixed chloroplasts resulted only in visualization of the Mr 22,000 polypeptide, a constituent of the outer membrane. We further studied the morphological distribution and frequency of this peptide by electron microscopic evaluation of platinum-carbon replicas after freeze-etching or label-fracture and of ultra-thin sections. By use of these three methods, the polypeptide was found to be randomly distributed in the outer envelope membrane and easily accessible to the immunomarker. Average marker density, as obtained by freeze-etching and label-fracture, was approximately 130 gold particles per square micron.

scholarly journals Critical Regions of secM That Control Its Translation and Secretion and Promote Secretion-Specific secA Regulation

2002 ◽  
Vol 184 (9) ◽  
pp. 2360-2369 ◽  
Author(s):  
Shameema Sarker ◽  
Donald Oliver
Keyword(s):  
Signal Peptide ◽  
Mutational Analysis ◽  
Regulatory System ◽  
Specific Form ◽  
Hydrophobic Core ◽  
Translational Initiation ◽  
Amino Terminal ◽  
Direct Support ◽  
Peptide Level ◽  
Critical Regions

ABSTRACT SecA is an essential ATP-driven motor protein that binds to presecretory or membrane proteins and the translocon and promotes the translocation or membrane integration of these proteins. secA is subject to a protein secretion-specific form of regulation, whereby its translation is elevated during secretion-limiting conditions. A novel mechanism that promotes this regulation involves translational pausing within the gene upstream of secA, secM. The secM translational pause prevents formation of an RNA helix that normally blocks secA translational initiation. The duration of this pause is controlled by the rate of secretion of nascent SecM, which in turn depends on its signal peptide and a functional translocon. We characterized the atypical secM signal peptide and found that mutations within the amino-terminal region specifically affect the secM translational pause and secA regulation, while mutations in the hydrophobic core region affect SecM secretion as well as translational pausing and secA regulation. In addition, mutational analysis of the 3′ end of secM allowed us to identify a conserved region that is required to promote the translational pause that appears to be operative at the peptide level. Together, our results provide direct support for the secM translational pause model of secA regulation, and they pinpoint key sequences within secM that promote this important regulatory system.

scholarly journals Recombinant Porphyromonas gingivalis FimA preproprotein expressed in Escherichia coli is lipidated and the mature or processed recombinant FimA protein forms a short filament in vitro

2010 ◽  
Vol 56 (11) ◽  
pp. 959-967 ◽  
Author(s):  
Mikio Shoji ◽  
Atsutoshi Yoshimura ◽  
Hidenobu Yoshioka ◽  
Akemi Takade ◽  
Yasuko Takuma ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Outer Membrane ◽  
Porphyromonas Gingivalis ◽  
Signal Peptide ◽  
Periodontal Diseases ◽  
Microscopic Analysis ◽  
Base Substitution ◽  
Electron Microscopic

The gram-negative anaerobic bacterium Porphyromonas gingivalis is an etiologically important pathogen for chronic periodontal diseases in adults. Our previous study suggested that the major structural components of both Fim and Mfa fimbriae in this organism are secreted through their lipidated precursors. In this study, we constructed Escherichia coli strains expressing various fimA genes with or without the 5′-terminal DNA region encoding the signal peptide, and we determined whether lipidation of recombinant FimA proteins occurred in E. coli. Lipidation occurred for a recombinant protein from the fimA gene with the 5′-terminal DNA region encoding the signal peptide but not for a recombinant protein from the fimA gene without the signal-peptide-encoding region, as revealed by [3H]palmitic acid labeling experiments. A TLR2-dependent signaling response was induced by the recombinant protein from the fimA gene with the signal-peptide-encoding region but not by a recombinant protein from the fimA gene with the signal-peptide-encoding region that had a base substitution causing an amino acid substitution (C19A). Electron microscopic analysis revealed that recombinant FimA (A-47 – W-383) protein was autopolymerized to form filamentous structures of about 80 nm in length in vitro. The results suggest that FimA protein, a major subunit of Fim fimbriae, is transported to the outer membrane by the lipoprotein sorting system, and a mature or processed FimA protein on the outer membrane is autopolymerized to form Fim fimbriae.

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.

The High Resolution Appearance of Biological Substrates

1969 ◽  
Vol 27 ◽  
pp. 416-417
Author(s):  
Glen B. Haydon
Keyword(s):  
High Resolution ◽  
Phase Image ◽  
Biological Structure ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Resolution Electron ◽  
Discrete Structures ◽  
Large Phase ◽  
Biological Substrates ◽  
High Resolution Electron Micrographs

High resolution electron microscopic study of negatively stained macromolecules and thin sections of tissue embedded in a variety of media are difficult to interpret because of the superimposed phase image granularity. Although all of the information concerning the biological structure of interest may be present in a defocused electron micrograph, the high contrast of large phase image granules produced by the substrate makes it impossible to distinguish the phase ‘points’ from discrete structures of the same dimensions. Theory predicts the findings; however, it does not allow an appreciation of the actual appearance of the image under various conditions. Therefore, though perhaps trivial, training of the cheapest computer produced by mass labor has been undertaken in order to learn to appreciate the factors which affect the appearance of the background in high resolution electron micrographs.

Determination of the phase structure of polymerblends by electron microscopy

1978 ◽  
Vol 36 (1) ◽  
pp. 492-493
Author(s):  
Dr. G. Kaemof
Keyword(s):  
Screw Extruder ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Single Screw Extruder ◽  
Transmission Electron ◽  
The Matrix ◽  
Twin Screw ◽  
Special Case ◽  
Microscopic Investigations

A mixture of polycarbonate (PC) and styrene-acrylonitrile-copolymer (SAN) represents a very good example for the efficiency of electron microscopic investigations concerning the determination of optimum production procedures for high grade product properties.The following parameters have been varied:components of charge (PC : SAN 50 : 50, 60 : 40, 70 : 30), kind of compounding machine (single screw extruder, twin screw extruder, discontinuous kneader), mass-temperature (lowest and highest possible temperature).The transmission electron microscopic investigations (TEM) were carried out on ultra thin sections, the PC-phase of which was selectively etched by triethylamine.The phase transition (matrix to disperse phase) does not occur - as might be expected - at a PC to SAN ratio of 50 : 50, but at a ratio of 65 : 35. Our results show that the matrix is preferably formed by the components with the lower melting viscosity (in this special case SAN), even at concentrations of less than 50 %.

An Electron Microscopic Study of the Acute Effects of Hypothalamic Releasing Factors on the Anterior Pituitary Gland

1968 ◽  
Vol 26 ◽  
pp. 232-233
Author(s):  
P.W. Coates ◽  
E.A. Ashby ◽  
L. Krulich ◽  
A. Dhariwal ◽  
S. McCann
Keyword(s):  
Anterior Pituitary ◽  
Microscopic Investigation ◽  
Uranyl Acetate ◽  
Electron Microscopic Investigation ◽  
Electron Microscopic ◽  
Tissue Samples ◽  
Thin Sections ◽  
Dense Membrane ◽  
Membrane Bound ◽  
Releasing Factors

The morphologic effects on somatotrophs of crude sheep hypothalamic extract prepared from stalk-median eminence were studied by electron microscopy in conjunction with concurrently run bioassays performed on the same tissue samples taken from young adult male Sherman rats.Groups were divided into uninjected controls and injected experimentals sacrificed at 5', 15', and 30' after injection. Half of each anterior pituitary was prepared for electron microscopic investigation, the other half for bioassay. Fixation using collidine buffered osmium tetroxide was followed by dehydration and embedment in Maraglas. Uranyl acetate and lead citrate were used as stains. Thin sections were examined in a Philips EM 200.Somatotrophs from uninjected controls appeared as described in the literature (Fig. 1). In addition to other components, these cells contained moderate numbers of spherical, electron-dense, membrane-bound granules approximately 350 millicrons in diameter.

Toxic effects of inhaled DMMP on the kidneys of Fischer-344 rats

1987 ◽  
Vol 45 ◽  
pp. 880-881
Author(s):  
D.R. Mattie ◽  
C.J. Hixson
Keyword(s):  
Left Kidney ◽  
Inhalation Exposure ◽  
Electron Microscopic Examination ◽  
Exposure Period ◽  
Male Rats ◽  
Continuous Exposure ◽  
Fischer 344 Rats ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Fischer 344

Dimethylmethylphosphonate (DMMP) is a simple organophosphate used industrially as a flame retardant and to lower viscosity in polyester and epoxy resins. The military considered the use of DMMP as a nerve gas simulant. Since military use of DMMP involved exposure by inhalation, there was a need for a subchronic inhalation exposure to DMMP to fully investigate its toxic potential.Male Fischer-344 rats were exposed to 25 ppm or 250 ppm DMMP vapor on a continuous basis for 90 days. An equal number of control rats were sham-exposed. Following the 90-day continuous exposure period, 15 male rats were sacrificed from each group. Two rats from each group had the left kidney perfused for electron microscopic examination. The kidneys were perfused from a height of 150 cm water with 1% glutaraldehyde in Sorensen's 0.1M phosphate buffer pH 7.2. An additional kidney was taken from a rat in each group and fixed by immersion in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer pH 7.4. A portion of the 9 kidneys collected for electron microscopy were processed into Epon 812. Thin sections, stained with uranyl acetate and lead citrate, were examined with a JEOL 100B Transmission Electron Microscope. Microvilli height was measured on photographs of the cells of proximal tubules. This data, along with morphologic features of the cells, allows the proximal convoluted tubules (PCT) to be identified as being S1, S2, or S3 segment PCT.

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