scholarly journals Partial Characterization of a Vicilin-Like Glycoprotein from Seeds of Flowering Tobacco (Nicotiana sylvestris)

2009 ◽  
Vol 2009 ◽  
pp. 1-12 ◽  
Author(s):  
Jared Q. Gerlach ◽  
Veer P. Bhavanandan ◽  
Paul A. Haynes ◽  
Lokesh Joshi
Keyword(s):  
Compositional Analysis ◽  
Nicotiana Sylvestris ◽  
Proteolytic Processing ◽  
Protein Subunits ◽  
Lectin Staining ◽  
Disulphide Bonds ◽  
Sds Page ◽  
Solanaceae Family

A vicilin-like glycoprotein from the seeds of Nicotiana sylvestris, flowering tobacco, has been identified using nanoLC/ESI-MS/MS. Sequences from a fragment of protein demonstrated homology with vicilins from other members of the Solanaceae family, notably potato (Solanum demissum). Reducing and nonreducing SDS-PAGE analyses of the identified protein indicated that fragments resulting from in situ proteolytic processing are joined by intrachain disulphide bonds. Staining with Con A lectin was specifically inhibited by mannose suggested the presence of -linked glycosylation which was confirmed by carbohydrate compositional analysis of PVDF-bound protein subunits. HPAEC-PAD analysis of the monosaccharides released from the glycoprotein by acid hydrolysis revealed glucosamine and mannose. -acetylglucosamine termination of attached oligosaccharides was further verified by inhibitable WGA lectin staining. Immunostaining of PVDF-bound N. sylvestris proteins with antibodies against G. max total protein demonstrated cross-staining at masses corresponding to fragments from the proteolytically processed protein subunits.

scholarly journals Characterization of MAX.3 antigen, a glycoprotein expressed on mature macrophages, dendritic cells and blood platelets: identity with CD84

2000 ◽  
Vol 346 (3) ◽  
pp. 729-736 ◽  
Author(s):  
Stefan W. KRAUSE ◽  
Michael REHLI ◽  
Sven HEINZ ◽  
Reinhard EBNER ◽  
Reinhard ANDREESEN
Keyword(s):  
Dendritic Cells ◽  
Blood Platelets ◽  
Cell Types ◽  
Variable Expression ◽  
Surface Molecules ◽  
Sds Page ◽  
Molecular Masses ◽  
Antigen A

MAX.3 is a monoclonal antibody that preferentially reacts with mature macrophages (MAC), monocyte-derived dendritic cells, megakaryocytes and platelets. In this study, we describe the characterization, purification and identification of the MAX.3 antigen. Immunoprecipitation and SDS/PAGE revealed different molecular masses of MAX.3 antigen in MAC (60-90 kDa) and platelets (58-64 kDa), whereas a similar size (45 kDa) was observed in both cell types after digestion with N-glycosidase F. Lectin affinity and sequential treatment with different glycosidases suggests complex type glycosylation of MAX.3 antigen in MAC and hybrid type glycosylation in platelets. Amino acid sequencing led to the identification of a corresponding cDNA clone and showed its identity to the sequence of the CD84 antigen, a member of the CD2 family of cell surface molecules. MAX.3/CD84 was further studied by immunohistochemistry and a variable expression was found on tissue MAC, confirming this antigen to be mainly a marker for MAC in situ.

Excreted/secreted products of developing Taenia saginata metacestodes

Parasitology ◽  
1988 ◽  
Vol 97 (3) ◽  
pp. 477-487 ◽  
Author(s):  
G. W. P. Joshua ◽  
L. J. S. Harrison ◽  
M. M. H. Sewell
Keyword(s):  
Taenia Saginata ◽  
Cellular Infiltrate ◽  
Diagnostic Assays ◽  
Antigenic Characterization ◽  
Sds Page ◽  
Host Parasite Relationship ◽  
Parasite Relationship ◽  
Host Parasite

SummaryExcretions and secretions (ES) and somatic components of 4, 8, 12 and 16-week-old Taenia saginata metacestodes were biosynthetically radio-isotope labelled by incubating the larvae in the presence of [35S]methionine. Despite their small size, 4-week-old metacestodes produced as much isotope-labelled ES/parasite as older metacestodes, indicating a proportionately greater metabolic activity of the parasite at this age. In situ the 4-week-old metacestodes were surrounded by a marked granulomatous cellular infiltrate which had largely resolved around 8-week-old metacestodes. Examination of the isotope-labelled ES by SDS-PAGE revealed distinct age-specific components from 4- and 12-week-old metacestodes and other ES components which were produced by all the ages of metacestodes examined. In comparison the labelled somatic components were conserved. Antigenic characterization of the ES by immunoprecipitation against a panel of clinically defined bovine sera combined with SDS-PAGE analysis, identified some highly immunogenic parasite products and others which did not elicit an antibody response demonstrable by immunoprecipitation. These components are of interest in relation to the host/parasite relationship, to the construction of diagnostic assays for the detection of T. saginata cysticercosis, and to the immunity that cattle develop against this parasite.

scholarly journals Isolation, Production, Purification, Assay and Characterization of Insulin From Goat (Capra Hircus) Pancreas By RDT Using Saccharomyces Cerevisae Vector

2020 ◽  
pp. 107-110
Keyword(s):  
Recombinant Dna ◽  
Polypeptide Chain ◽  
Capra Hircus ◽  
Β Cell ◽  
Recombinant Dna Technology ◽  
Disulphide Bonds ◽  
Recombinant Insulin ◽  
Sds Page ◽  
A Chain

Insulin is a polypeptide hormone secreted by the β-cell of Islet’s of langerhan’s of the pancreas and cosists of two polypeptide chain-A and chain- B. It is linked by two inter chain disulphide bonds (A7- B7 and A20-B19) and also has an intra-chain disulphide bond in the A-Chain (A6-A11). For production of Insulin from goat pancreas saccharomyces cerevisae using as a vector. In this study Recombinant DNA technology, chromatography, Elecrophoresis, Spectrophotometer, SDS-PAGE, zymography etc techaniques were used. Humulin was tanken as a marker. It was found, goat insulin showed good result. 5.8 – 6.5 KDa recombinant insulin was calculated. It did not show antigenic proterties.

scholarly journals Homologous expression and characterization of gassericin T and gassericin S, a novel class IIb bacteriocin produced byLactobacillus gasseriLA327

2018 ◽  
Author(s):  
Genki Kasuga ◽  
Masaru Tanaka ◽  
Yuki Harada ◽  
Hiroshi Nagashima ◽  
Taisei Yamato ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Synergistic Activity ◽  
Bacteriocin Activity ◽  
Homologous Expression ◽  
Sds Page ◽  
Class Iib ◽  
Active Class

AbstractLactobacillus gasseriLA327 isolated from the large intestine tissue in humans is a bacteriocinogenic strain and is predicted to produce two kinds of class IIb bacteriocins, i.e. gassericin T (GT) and acidocin LF221A (Acd LF221A). In this study, DNA sequencing of the genes for GT and Acd LF221A onLb. gasseriLA327 revealed that the amino acid sequences for GT completely corresponded with those ofgatexcept for GatK (histidine kinase). However, those for the Acd LF221A had analogues which differed in at least one amino acid residue to be a putative class IIb bacteriocin designated as gassericin S (GS). By deletion test of GT structural genes (gatAX), the LA327 strain retained the bacteriocin activity, and the LA327 mutant strain lacking the ABC-type transporter gene (gatT) completely lost the bacteriocin activity. This indicates that LA327 strain is a GS producer, and GS production is performed viagatwith the inclusion ofgatT. Homologous expression using deletion mutants for GS and GT containing each single peptide elucidated that GS (GasAX) and GT (GatAX) showed synergistic activity as class IIb bacteriocins, respectively, and no synergistic activity was observed between each peptide of GS and GT. The molecular mass of GS was estimated to be theoretical ca. 5,400 Da byin situactivity assay after SDS-PAGE, clarifying that GS was actually expressed as an active class IIb bacteriocin. Furthermore, stability of GS expressed against pH, heat and protease was determined.ImportanceWe determined the complete DNA sequence for GS, a novel class IIb bacteriocin ofLb. gasseri, and succeeded to express GS as active bacteriocins. Our results clarified the interaction of each class IIb component peptide for GT in addition to GS via construction of homologous mutants which were not dependent on the purification. These data may demonstrate the characteristics of class IIb bacteriocins forLb. gasseri.

scholarly journals Characterization of N-ethylmaleimide-sensitive thiol groups required for the GTP-dependent fusion of endoplasmic reticulum membranes

1995 ◽  
Vol 312 (1) ◽  
pp. 23-30 ◽  
Author(s):  
A V Sokoloff ◽  
T Whalley ◽  
J Zimmerberg
Keyword(s):  
Endoplasmic Reticulum ◽  
Sodium Periodate ◽  
Potent Inhibitor ◽  
Immunoblot Analysis ◽  
Fusion Activity ◽  
Thiol Groups ◽  
Chemical Probes ◽  
Disulphide Bonds

The GTP-dependent fusion activity of endoplasmic reticulum membranes is thought to be required for the structural maintenance and post-mitotic regeneration of the endoplasmic reticulum. This fusion is sensitive to the thiol-alkylating agent N-ethylmaleimide. In many intracellular fusion events N-ethylmaleimide-sensitivity is associated with a homotrimeric ATPase called N-ethylmaleimide-sensitive fusion protein or NSF. The addition of cytosol containing NSF is known to restore fusion activity to N-ethylmaleimide-treated membranes. We found that the inhibition of fusion of rat liver endoplasmic reticulum membranes (microsomes) by N-ethylmaleimide was not reversed by the addition of untreated cytosol. Fusion was also unaffected by treatment with a buffer known to remove NSF from membranes. Accordingly, no membrane-associated NSF was detected by immunoblot analysis. These data suggest that microsome fusion requires an N-ethylmaleimide-sensitive component distinct from NSF. This component was tightly associated with the membranes, so we used a number of chemical probes to characterize it in situ. Its thiol groups did not appear to be part of a GTP-binding site. They showed relatively low reactivity with sodium periodate, which induces the formation of disulphide bonds between proximate thiol groups. The thiols were not protected against N-ethylmaleimide by Zn2+, a potent inhibitor of fusion which is known to efficiently co-ordinate thiol groups. To characterize the topology of the fusion-related thiol groups we used bulky thiol-specific reagents prepared by conjugating BSA or 10 kDa aminodextran to the bifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. The inhibition of fusion by these reagents indicated that these thiols are highly exposed on the membranes. This exposure might be important for the function of these groups during GTP-triggered fusion.

The Use of Advanced Analytical Techniques for Characterization of the Chemical, Physical, and Crystallographic Nature of a Surface

1973 ◽  
Vol 31 ◽  
pp. 132-133 ◽  
Author(s):  
R. E. Herfert
Keyword(s):  
Surface Characterization ◽  
Analytical Techniques ◽  
X Ray Diffraction ◽  
Depth Of Penetration ◽  
X Ray ◽  
The Past ◽  
Transmission Electron ◽  
Scanning Electron

Studies of the nature of a surface, either metallic or nonmetallic, in the past, have been limited to the instrumentation available for these measurements. In the past, optical microscopy, replica transmission electron microscopy, electron or X-ray diffraction and optical or X-ray spectroscopy have provided the means of surface characterization. Actually, some of these techniques are not purely surface; the depth of penetration may be a few thousands of an inch. Within the last five years, instrumentation has been made available which now makes it practical for use to study the outer few 100A of layers and characterize it completely from a chemical, physical, and crystallographic standpoint. The scanning electron microscope (SEM) provides a means of viewing the surface of a material in situ to magnifications as high as 250,000X.

A very rapid in situ Kikuchi technique to determine relative crystal misorientation

1988 ◽  
Vol 46 ◽  
pp. 830-831
Author(s):  
J. I. Bennetch
Keyword(s):  
Electron Beam ◽  
Analytical Techniques ◽  
Superplastic Forming ◽  
The Other ◽  
Kikuchi Pattern ◽  
Kikuchi Line ◽  
Line Analysis

In a recent study of the superplastic forming (SPF) behavior of certain Al-Li-X alloys, the relative misorientation between adjacent (sub)grains proved to be an important parameter. It is well established that the most accurate way to determine misorientation across boundaries is by Kikuchi line analysis. However, the SPF study required the characterization of a large number of (sub)grains in each sample to be statistically meaningful, a very time-consuming task even for comparatively rapid Kikuchi analytical techniques.In order to circumvent this problem, an alternate, even more rapid in-situ Kikuchi technique was devised, eliminating the need for the developing of negatives and any subsequent measurements on photographic plates. All that is required is a double tilt low backlash goniometer capable of tilting ± 45° in one axis and ± 30° in the other axis. The procedure is as follows. While viewing the microscope screen, one merely tilts the specimen until a standard recognizable reference Kikuchi pattern is centered, making sure, at the same time, that the focused electron beam remains on the (sub)grain in question.

Coherent electron nanodiffraction from clean silver nano particles in a UHV STEM

1993 ◽  
Vol 51 ◽  
pp. 1058-1059
Author(s):  
J. Liu ◽  
M. Pan ◽  
G. E. Spinnler
Keyword(s):  
Supported Catalysts ◽  
Imaging Techniques ◽  
Nano Particles ◽  
Metal Particles ◽  
Shape Structure ◽  
Dynamical Simulations ◽  
Small Metal Particles ◽  
Extract Information

Small metal particles have peculiar chemical and physical properties as compared to bulk materials. They are especially important in catalysis since metal particles are common constituents of supported catalysts. The structural characterization of small particles is of primary importance for the understanding of structure-catalytic activity relationships. The shape and size of metal particles larger than approximately 5 nm in diameter can be determined by several imaging techniques. It is difficult, however, to deduce the shape of smaller metal particles. Coherent electron nanodiffraction (CEND) patterns from nano particles contain information about the particle size, shape, structure and defects etc. As part of an on-going program of STEM characterization of supported catalysts we report some preliminary results of CEND study of Ag nano particles, deposited in situ in a UHV STEM instrument, and compare the experimental results with full dynamical simulations in order to extract information about the shape of Ag nano particles.

Characterization of the glycoconjugates of boar testis and epididymis

Reproduction ◽  
2000 ◽  
pp. 325-335 ◽  
Author(s):  
A Calvo ◽  
LM Pastor ◽  
S Bonet ◽  
E Pinart ◽  
M Ventura
Keyword(s):  
Ricinus Communis ◽  
Lectin Histochemistry ◽  
Apical Part ◽  
Seminiferous Tubules ◽  
In Situ Characterization ◽  
Intense Staining ◽  
Terminal Galactose ◽  
Boar Spermatozoa

Lectin histochemistry was used to perform in situ characterization of the glycoconjugates present in boar testis and epididymis. Thirteen horseradish peroxidase- or digoxigenin-labelled lectins were used in samples obtained from healthy fertile boars. The acrosomes of the spermatids were stained intensely by lectins with affinity for galactose and N-acetyl-galactosamine residues, these being soybean, peanut and Ricinus communis agglutinins. Sertoli cells were stained selectively by Maackia ammurensis agglutinin. The lamina propria of seminiferous tubules showed the most intense staining with fucose-binding lectins. The Golgi area and the apical part of the principal cells of the epididymis were stained intensely with many lectins and their distribution was similar in the three zones of the epididymis. On the basis of lectin affinity, both testis and epididymis appear to have N- and O-linked glycoconjugates. Spermatozoa from different epididymal regions showed different expression of terminal galactose and N-acetyl-galactosamine. Sialic acid (specifically alpha2,3 neuraminic-5 acid) was probably incorporated into spermatozoa along the extratesticular ducts. These findings indicate that the development and maturation of boar spermatozoa are accompanied by changes in glycoconjugates. As some lectins stain cellular or extracellular compartments specifically, these lectins could be useful markers in histopathological evaluation of diseases of boar testis and epididymis.

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