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.

scholarly journals Chemical probes to potently and selectively inhibit endocannabinoid cellular reuptake

2017 ◽  
Vol 114 (25) ◽  
pp. E5006-E5015 ◽  
Author(s):  
Andrea Chicca ◽  
Simon Nicolussi ◽  
Ruben Bartholomäus ◽  
Martina Blunder ◽  
Alejandro Aparisi Rey ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Cannabinoid Receptor ◽  
Facilitated Diffusion ◽  
Chemical Probes ◽  
Analgesic Effects ◽  
Complex Process ◽  
Arachidonoyl Glycerol ◽  
The Brain

The extracellular effects of the endocannabinoids anandamide and 2-arachidonoyl glycerol are terminated by enzymatic hydrolysis after crossing cellular membranes by facilitated diffusion. The lack of potent and selective inhibitors for endocannabinoid transport has prevented the molecular characterization of this process, thus hindering its biochemical investigation and pharmacological exploitation. Here, we report the design, chemical synthesis, and biological profiling of natural product-derivedN-substituted 2,4-dodecadienamides as a selective endocannabinoid uptake inhibitor. The highly potent (IC50= 10 nM) inhibitorN-(3,4-dimethoxyphenyl)ethyl amide (WOBE437) exerted pronounced cannabinoid receptor-dependent anxiolytic, antiinflammatory, and analgesic effects in mice by increasing endocannabinoid levels. A tailored WOBE437-derived diazirine-containing photoaffinity probe (RX-055) irreversibly blocked membrane transport of both endocannabinoids, providing mechanistic insights into this complex process. Moreover, RX-055 exerted site-specific anxiolytic effects on in situ photoactivation in the brain. This study describes suitable inhibitors to target endocannabinoid membrane trafficking and uncovers an alternative endocannabinoid pharmacology.

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 Synthesis and characterization of a cysteine xyloglucan conjugate as mucoadhesive polymer

2013 ◽  
Vol 49 (2) ◽  
pp. 285-292 ◽  
Author(s):  
Mangesh Bhalekar ◽  
Savita Sonawane ◽  
Shamkant Shimpi
Keyword(s):  
Rheological Properties ◽  
Covalent Attachment ◽  
Synthesis And Characterization ◽  
Thiol Groups ◽  
Chemically Modified ◽  
Mucoadhesive Polymer ◽  
Disulphide Bonds ◽  
Group Content

The aim of this study was to improve the mucoadhesive potential of xyloglucan polymer by the covalent attachment of cysteine as thiol moiety. The parent polymer xyloglucan was chemically modified by introducing sulphydryl bearing compound L-cysteine HCl. Different batches of xyloglucan-cysteine conjugates were prepared at varying reaction pH (2-6) and evaluated for optimum thiol incorporation, disulphide group content, swelling behavior, rheological properties and mucoadhesive properties. The obtained conjugates characterized in vitro by quantification of immobilized thiol groups; showed maximum thiol incorporation on xyloglucan (7.67 ± 0.14 %) at pH 5. The disulphide group content was found maximum (2.83 ± 0.12) at pH 6. The water uptake at end of 4 h was 5.0 for xyloglucan and was found to decrease in thiolated derivatives with increase in thiolation. Mucoadhesion studies revealed that mucoadhesion of xyloglucan-cysteine conjugate increased more than twice compared to the unmodified polymer. The viscosity of thiomer was more than that of xyloglucan because of formation of disulphide bonds.

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.

Spectro-mechanical Characterization of Aromaticity and Maturity of Kerogens in Oil Shale at 6 nm Spatial Resolution

2018 ◽  
Author(s):  
Devon Jakob ◽  
Le Wang ◽  
Haomin Wang ◽  
Xiaoji Xu
Keyword(s):  
Spatial Resolution ◽  
Oil Shale ◽  
Infrared Imaging ◽  
Mechanical Characterization ◽  
Optical Diffraction ◽  
Chemical Compositions ◽  
Valuable Insight ◽  
Eagle Ford Shale

<p>In situ measurements of the chemical compositions and mechanical properties of kerogen help understand the formation, transformation, and utilization of organic matter in the oil shale at the nanoscale. However, the optical diffraction limit prevents attainment of nanoscale resolution using conventional spectroscopy and microscopy. Here, we utilize peak force infrared (PFIR) microscopy for multimodal characterization of kerogen in oil shale. The PFIR provides correlative infrared imaging, mechanical mapping, and broadband infrared spectroscopy capability with 6 nm spatial resolution. We observed nanoscale heterogeneity in the chemical composition, aromaticity, and maturity of the kerogens from oil shales from Eagle Ford shale play in Texas. The kerogen aromaticity positively correlates with the local mechanical moduli of the surrounding inorganic matrix, manifesting the Le Chatelier’s principle. In situ spectro-mechanical characterization of oil shale will yield valuable insight for geochemical and geomechanical modeling on the origin and transformation of kerogen in the oil shale.</p>

Sign in / Sign up

Export Citation Format

Share Document