scholarly journals Subcellular compartmentalization of maize storage proteins in Xenopus oocytes injected with zein messenger RNAs.

1981 ◽  
Vol 89 (2) ◽  
pp. 292-299 ◽  
Author(s):  
W J Hurkman ◽  
L D Smith ◽  
J Richter ◽  
B A Larkins
Keyword(s):  
Storage Proteins ◽  
Matrix Material ◽  
Membrane Vesicles ◽  
Subcellular Fractionation ◽  
Dense Matrix ◽  
Messenger Rnas ◽  
Material Density ◽  
Optimal Separation ◽  
High Concentrations ◽  
Subcellular Compartmentalization

Maize storage proteins synthesized in oocytes were compartmentalized in membrane vesicles because they were resistant to hydrolysis by protease, unless detergent was present. The site of storage protein deposition within the oocyte was determined by subcellular fractionation. Optimal separation of oocyte membranes and organelles was obtained when EDTA and high concentrations of NaCl were included in the homogenization and gradient buffers. Under these conditions, fractions in sucrose gradients containing a heterogeneous mixture of smooth membranes (presumably endoplasmic reticulum, Golgi apparatus, and plasma membrane, density = 1.10-1.12 g/cm3), mitochondria (densities = 1.14 and 1.16 g/cm3), yolk platelets (density = 1.21 g/cm3), and a dense matrix material (density = 1.22 g/cm3) could be separated. Some zein proteins were recovered in the mixed membrane fraction, but the majority occurred in vesicles sedimenting with yolk platelets and granular material at a density of approximately 1.22 g/cm3. When metrizamide was included in the gradient to increase the density, little of the dense matrix material was isolated, and vesicles containing zein proteins were separated from other oocyte components. These vesicles were similar to protein bodies in maize endosperm because they were of identical density and contained the same group of polypeptides.

SELECTION OF COLUMN AND OPERATING CONDITIONS FOR REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF PROTEINS IN CANADIAN WHEAT

1985 ◽  
Vol 65 (2) ◽  
pp. 285-298 ◽  
Author(s):  
J. E. KRUGER ◽  
B. A. MARCHYLO
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Storage Proteins ◽  
Sodium Dodecyl ◽  
Reversed Phase ◽  
Operating Conditions ◽  
Protein Patterns ◽  
Optimal Separation ◽  
Rp Hplc

Chromatographic conditions were optimized and three commercially available columns were evaluated for separation of alcohol-soluble storage proteins of Neepawa wheat using reversed-phase high-performance liquid chromatography (RP-HPLC). Optimal separation was achieved using an extracting solution of 50% 1-propanol, 1% acetic acid, and 4% dithiothreitol and an HPLC elution time of 105 min at a flow rate of 1.0 mL/min. HPLC columns evaluated (SynChropak RP-P, Ultrapore RPSC and Aquapore RP-300) varied in selectivity and resolution. The column providing the greatest versatility was Aquapore RP-300 available in cartridge form. Sodium dodecyl sulfate gradient-gel electrophoresis analysis of protein peaks resolved by RP-HPLC indicated that many of the eluted peaks contained more than one protein species. Chromatographic protein patterns obtained for Neepawa wheat grown at different locations and in different years were qualitatively the same.Key words: Protein, high-performance liquid chromatography, wheat

Effects of endogenous calcium transport inhibitor from heart muscle on the active calcium uptake and passive calcium release properties of sarcoplasmic reticulum

1989 ◽  
Vol 67 (9) ◽  
pp. 999-1006 ◽  
Author(s):  
Njanoor Narayanan ◽  
Philip Bedard ◽  
Trilochan S. Waraich
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Heart Muscle ◽  
Calcium Release ◽  
Membrane Vesicles ◽  
Transport Inhibitor ◽  
Low Concentrations ◽  
Active Calcium ◽  
High Concentrations ◽  
Stimulation Of

In the present study, the effects of the cytosolic Ca2+ transport inhibitor on ATP-dependent Ca2+ uptake by, and unidirectional passive Ca2+ release from, sarcoplassmic reticulum enriched membrane vesicles were examined in parallel experiments to determine whether inhibitor-mediated enhancement in Ca2+ efflux contributes to inhibition of net Ca2+ uptake. When assays were performed at pH 6.8 in the presence of oxalate, low concentrations (<100 μg/mL) of the inhibitor caused substantial inhibition of Ca2+ uptake by SR (28–50%). At this pH, low concentrations of the inhibitor did not cause enhancement of passive Ca2+ release from actively Ca2+-loaded sarcoplasmic reticulum. Under these conditions, high concentrations (>100 μg/mL) of the inhibitor caused stimulation of passive Ca2+ release but to a much lesser extent when compared with the extent of inhibition of active Ca2+ uptake (i.e., twofold greater inhibition of Ca2+ uptake than stimulation of Ca2+ release). When Ca2+ uptake and release assays were carried out at pH 7.4, the Ca2+ release promoting action of the inhibitor became more pronounced, such that the magnitude of enhancement in Ca2+ release at varying concentrations of the inhibitor (20–200 μg/mL) was not markedly different from the magnitude of inhibition of Ca2+ uptake. In the absence of oxalate in the assay medium, inhibition of Ca2+ uptake was observed at alkaline but not acidic pH. These findings imply that the inhibition of Ca2+ uptake observed at pH 6.8 is mainly due to decrease in the rate of active Ca2+ transport into the membrane vesicles rather than stimulation of passive Ca2+ efflux; at alkaline pH (pH 7.4), enhanced Ca2+ efflux contributes substantially, if not exclusively, to the decrease in Ca2+ uptake observed in the presence of the inhibitor. It is suggested that if the cytosolic inhibitor has actions similar to those observed in vitro in intact cardiac muscle, acid–base status of the intracellular fluid would be a major factor influencing the nature of its effects (inhibition of Ca2+ uptake or stimulation of Ca2+ release) on transmembrane Ca2+ fluxes across the sarcoplasmic reticulum.Key words: sarcoplasmic reticulum, Ca2+ uptake, Ca2+ release, endogenous inhibitor, heart muscle.

scholarly journals Rheological Behaviour of Highly Filled Materials for Injection Moulding and Additive Manufacturing: Effect of Particle Material and Loading

2020 ◽  
Vol 10 (22) ◽  
pp. 7993
Author(s):  
Marko Bek ◽  
Joamin Gonzalez-Gutierrez ◽  
Christian Kukla ◽  
Klementina Pušnik Črešnar ◽  
Boris Maroh ◽  
...  
Keyword(s):  
Matrix Material ◽  
Rheological Behaviour ◽  
Thermoplastic Polymer ◽  
Molten State ◽  
Binder System ◽  
Inorganic Fillers ◽  
High Concentrations ◽  
Shape And Size ◽  
Alloy Titanium ◽  
Number Of Particles

Within this paper, we are dealing with a mixture of thermoplastic polymer that is filled with inorganic fillers at high concentrations up to 60 vol.%. A high number of particles in the compound can substantially change the rheological behaviour of the composite and can lead to problems during processing in the molten state. The rheological behaviour of highly filled materials is complex and influenced by many interrelated factors. In the present investigation, we considered four different spherical materials: steel, aluminium alloy, titanium alloy and glass. Particles with similar particle size distribution were mixed with a binder system at different filling grades (30–60 vol.%). We showed that the rheological behaviour of highly filled materials is significantly dependent on the chemical interactions between the filler and matrix material. Moreover, it was shown that the changes of the particle shape and size during processing lead to unexpected rheological behaviour of composite materials as it was observed in the composites filled with glass beads that broke at high contents during processing.

scholarly journals A molecular pore spans the double membrane of the coronavirus replication organelle

Science ◽  
2020 ◽  
Vol 369 (6509) ◽  
pp. 1395-1398 ◽  
Author(s):  
Georg Wolff ◽  
Ronald W. A. L. Limpens ◽  
Jessika C. Zevenhoven-Dobbe ◽  
Ulrike Laugks ◽  
Shawn Zheng ◽  
...  
Keyword(s):  
Drug Target ◽  
Rna Synthesis ◽  
Transmembrane Protein ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Genome Replication ◽  
Messenger Rnas ◽  
Double Membrane ◽  
The Core ◽  
Cryo Electron Microscopy

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo–electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target.

The Influence of Internal Pore Pressure During Roll Forming of Structurally Porous Metals

1998 ◽  
Vol 521 ◽  
Author(s):  
D. M. Elzey ◽  
H. N. G. Wadley
Keyword(s):  
Pore Pressure ◽  
High Pressures ◽  
Hot Isostatic Pressing ◽  
Matrix Material ◽  
Porous Metal ◽  
Roll Forming ◽  
Dense Matrix ◽  
Porous Metals ◽  
Noticeable Effect ◽  
Internal Pore

ABSTRACTStructurally porous metal sandwich panels consisting of dense face sheets and porous cores of controlled relative density can be manufactured by trapping inert gas during hot isostatic pressing and modifying its distribution via subsequent thermo-mechanical forming. At high pressures, the internal gas is expected to influence the forming response. This paper describes a model for the roll forming of a porous metal panel and its use to explore the effects of internal pore pressure upon rolling response. It is shown that for gas pressures below about half the yield strength of the fully dense matrix material, there is essentially no influence on the forming response. Only in the case of very high initial pore pressures or at relative densities approaching full theoretical does a noticeable effect arise. In this case, a limiting upper density is attainable which depends on the specific rolling conditions and geometry.

Characteristics of calcium transport and binding by rat myometrium plasma membrane subfractions

1980 ◽  
Vol 239 (3) ◽  
pp. C66-C74 ◽  
Author(s):  
A. K. Grover ◽  
C. Y. Kwan ◽  
J. Crankshaw ◽  
D. J. Crankshaw ◽  
R. E. Garfield ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cytochrome C ◽  
Buoyant Density ◽  
Membrane Vesicles ◽  
Ionophore A23187 ◽  
Plasma Membrane Vesicles ◽  
Thin Sections ◽  
Cytochrome C Reductase ◽  
Ca Uptake ◽  
High Concentrations

A gradient has been designed to yield two subfractions of plasma membrane vesicles from rat myometrium, a low buoyant density (8-24% sucrose) fraction N1 richer in 5'-nucleotidase and a higher buoyant density (24-30% sucrose) fraction N2, instead of a previously described fraction F1. Both N1 and N2 had very low activities of NADPH-cytochrome c reductase and succinate-cytochrome c reductase. Electron micrographs of thin sections of N1 showed clear vesicles, whereas N2 consisted of vesicles with electron-dense bodies attached to them. These plasma membrane vesicles can actively take up Ca. The active uptake of Ca was potentiated by oxalate and phosphate and abolished by the Ca ionophore A23187. Dilution of actively loaded vesicles in isotonic media containing EGTA led to loss of a small proportion of the stored Ca instantaneously and the remainder more slowly in a biphasic manner. Dilution in hypotonic media with EGTA led to a release of a much larger proportion of the accumulated Ca. A23187 at high concentrations (10 microM) caused a release of all the sequestered Ca whether the active Ca uptake had been carried out in the presence or in the absence of oxalate. A23187, 0.5 microM, released all the sequestered Ca from the vesicles that were actively loaded in the absence of oxalate, but only 37% when the vesicles were actively loaded with Ca in the presence of oxalate. Comparison of the composite plasma membrane fraction F1 (8-30% sucrose) and the subfractions N1 and N2 showed that they had different capacities for Ca uptake in the presence and absence of ATP. An attempt has been made to analyze the active Ca-uptake data in terms of various Ca pools.

Parathyroid hormone-induced translocation of Na-H antiporters in rat proximal tubules

1989 ◽  
Vol 257 (4) ◽  
pp. C637-C645 ◽  
Author(s):  
C. B. Hensley ◽  
M. E. Bradley ◽  
A. K. Mircheff
Keyword(s):  
Parathyroid Hormone ◽  
Density Gradient ◽  
Brush Border ◽  
Gradient Centrifugation ◽  
Membrane Vesicles ◽  
Subcellular Fractionation ◽  
Proximal Tubules ◽  
Nylon Mesh ◽  
Sequence Of Events ◽  
Distinct Membrane

Parathyroid hormone (PTH) is believed to inhibit bicarbonate reabsorption by inhibiting Na-H antiport activity in proximal tubular brush-border membranes. The sequence of events triggered by PTH was investigated in a crude preparation of proximal tubules obtained by mechanical disruption and filtration through nylon mesh filters. Tubule samples were subjected to analytical subcellular fractionation after 2-, 5-, and 30-min treatments with 1 IU/ml PTH. These PTH-treatment intervals caused 54, 63, and 68% decreases in the Na-H antiport activity of a population of brush-border membrane vesicles that was resolved from a PTH-unresponsive brush-border population by density-gradient centrifugation. The rapid loss of Na-H antiport activity from the responsive population was accompanied by a transient increase in the Na-H antiport activity of a region of the density gradient, designated density window III, which was shown to contain two distinct membrane populations; these populations were both enriched in acid phosphatase activity, and one of them was also an important locus of galactosyltransferase activity. The increase in the Na-H antiport activity of window III accounted for 52% of the activity lost from the PTH-responsive population after 2 min, and for 43% of the activity lost after 5 min, but it was completely abolished after 25 more minutes in the presence of PTH. These observations suggest that PTH triggers a rapid translocation of Na-H antiporters from the microvillus membrane to a distinct membrane domain, where they are subsequently inactivated.

scholarly journals The product of HUM1, a novel yeast gene, is required for vacuolar Ca2+/H+ exchange and is related to mammalian Na+/Ca2+ exchangers.

1996 ◽  
Vol 16 (7) ◽  
pp. 3730-3741 ◽  
Author(s):  
T C Pozos ◽  
I Sekler ◽  
M S Cyert
Keyword(s):  
Mutant Strain ◽  
Critical Role ◽  
Ion Homeostasis ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Protein Product ◽  
Vacuolar Membrane ◽  
Yeast Cells ◽  
Vacuolar Acidification ◽  
High Concentrations

Calcineurin, or PP2B, plays a critical role in mediating Ca2+-dependent signaling in many cell types. In yeast cells, this highly conserved protein phosphatase regulates aspects of ion homeostasis and cell wall synthesis. We show that calcineurin mutants are sensitive to high concentrations of Mn2+ and identify two genes, CCC1 and HUM1, that, at high dosages, increase the Mn2+ tolerance of calcineurin mutants. CCC1 was previously identified by complementation of a Ca2+-sensitive (csg1) mutant. HUM1 (for "high copy number undoes manganese") is a novel gene whose predicted protein product shows similarity to mammalian Na+/Ca2+ exchangers. hum1 mutations confer Mn2+ sensitivity in some genetic backgrounds and exacerbate the Mn2+ sensitivity of calcineurin mutants. Furthermore, disruption of HUM1 in a calcineurin mutant strain results in a Ca2+-sensitive phenotype. We investigated the effect of disrupting HUM1 in other strains with defects in Ca2+ homeostasis. The Ca2+ sensitivity of pmc1 mutants, which lack a P-type ATPase presumed to transport Ca2+ into the vacuole, is exacerbated in a hum1 mutant strain background. Also, the Ca2+ content of hum1 pmc1 cells is less than that of pmc1 cells. In contrast, the Ca2+ sensitivity of vph1 mutants, which are specifically defective in vacuolar acidification, is not significantly altered by disruption of Hum1p function. These genetic interactions suggest that Hum1p may participate in vacuolar Ca2+/H+ exchange. Therefore, we prepared vacuolar membrane vesicles from wild-type and hum1 cells and compared their Ca2+ transport properties. Vacuolar membrane vesicles from hum1 mutants lack all Ca2+/H+ antiport activity, demonstrating that Hum1p catalyzes the exchange of Ca2+ for H+ across the yeast vacuolar membrane.

scholarly journals Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein–protein interactions

2014 ◽  
Vol 25 (20) ◽  
pp. 3178-3194 ◽  
Author(s):  
Georg H. H. Borner ◽  
Marco Y. Hein ◽  
Jennifer Hirst ◽  
James R. Edgar ◽  
Matthias Mann ◽  
...  
Keyword(s):  
Cluster Analysis ◽  
Protein Interactions ◽  
Large Scale ◽  
Protein Complexes ◽  
Membrane Vesicles ◽  
Subcellular Fractionation ◽  
Coated Vesicle ◽  
Universal Method ◽  
Diverse Range ◽  
Rapid Generation

We developed “fractionation profiling,” a method for rapid proteomic analysis of membrane vesicles and protein particles. The approach combines quantitative proteomics with subcellular fractionation to generate signature protein abundance distribution profiles. Functionally associated groups of proteins are revealed through cluster analysis. To validate the method, we first profiled >3500 proteins from HeLa cells and identified known clathrin-coated vesicle proteins with >90% accuracy. We then profiled >2400 proteins from Drosophila S2 cells, and we report the first comprehensive insect clathrin-coated vesicle proteome. Of importance, the cluster analysis extends to all profiled proteins and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes. We show that it also allows the detailed compositional characterization of complexes, including the delineation of subcomplexes and subunit stoichiometry. Our predictions are presented in an interactive database. Fractionation profiling is a universal method for defining the clathrin-coated vesicle proteome and may be adapted for the analysis of other types of vesicles and particles. In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps.

The unique stability of the marsupial sperm acrosomal membranes examined by unprotected freeze-thawing and treatment with the detergent Triton X-100

1993 ◽  
Vol 5 (1) ◽  
pp. 1 ◽  
Author(s):  
Y Sistina ◽  
M Lin ◽  
KE Mate ◽  
ES Robinson ◽  
JC Rodger
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Tammar Wallaby ◽  
Significant Loss ◽  
Monodelphis Domestica ◽  
Brushtail Possum ◽  
Plasma Membrane Vesicles ◽  
High Concentrations ◽  
Unique Stability

In this study of the unique stability of the marsupial acrosome, experiments were carried out on the acrosomes of spermatozoa of the tammar wallaby (Macropus eugenii), common brushtail possum (Trichosurus vulpecula) and grey short-tailed opossum (Monodelphis domestica). Light microscopy showed that 4% of opossum and 15% of possum and wallaby spermatozoa lost their acrosomes after freeze-thawing. Electron microscopy revealed that freeze-thawing also induced changes in the acrosomal matrix of some acrosome intact spermatozoa. In both possum and wallaby, freeze-thawing increased the number of spermatozoa with vesiculation of the acrosomal matrix. Freeze-thawing disrupted the plasma membrane of spermatozoa but the acrosomal membranes remained intact. Immediately on addition of high concentrations of TX-100 (0.02% and 0.04%) there was significant loss of acrosomes and motility in possum and wallaby spermatozoa. Lower concentrations of TX-100 (< or = 0.01%) did not affect motility for up to 30 min in all three species, and there was no significant loss of acrosomes. Although loss of acrosomes did not occur under mild detergent treatment, 56% of wallaby and 70% of possum spermatozoa had altered acrosomes after 30 min in 0.01% TX-100. Electron microscopy revealed that acrosomes were undergoing a vesiculation process similar to that seen after freeze-thawing. Often the plasma membrane of detergent-treated spermatozoa was disrupted and had formed plasma membrane vesicles. However, the acrosomal membranes remained intact despite major changes to the acrosomal matrix. The study confirmed the remarkable stability of the marsupial acrosome and suggested that this is probably based in the acrosomal membranes.

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