scholarly journals In vitro uptake and processing of prezein and other maize preproteins by maize membranes.

1981 ◽  
Vol 90 (2) ◽  
pp. 427-434 ◽  
Author(s):  
F A Burr ◽  
B Burr
Keyword(s):  
Sodium Dodecyl ◽  
Protein Composition ◽  
Micrococcal Nuclease ◽  
Mrna Synthesis ◽  
Nucellar Tissue ◽  
A Cell ◽  
Synthesis And Processing ◽  
Zein Mrna ◽  
Low Molecular Weight Proteins

A cell-free, mRNA-dependent system has been developed for the translation and processing of zein preproteins. A rough endoplasmic reticulum (RER)-enriched fraction, isolated by sucrose density gradients, can be treated with micrococcal nuclease to destroy endogenous messages. When these membranes are added to a wheat germ protein-synthesizing system together with zein mRNA, synthesis and processing of the polypeptides to the mature products takes place. The RER fraction from the endosperm has a different protein composition than that prepared from either the shoot or nucellar tissue and processes prezein more efficiently. The cleavage of the preproteins appears to be a cotranslational step as the completed preprotein chains cannot be processed, although they can be taken up to a limited extent. This small uptake, or absorption, or unprocessed zein seems to be an artifact and may be related to the unusual solubility properties of zein. Finally a sodium dodecyl sulfate (SDS)-urea polyacrylamide gel system has been developed which is particularly suited for the separation of low molecular weight proteins (less than 10,000 daltons). Using this method, we examined the products of in vitro zein processing and detected no presequence polypeptides. This suggests that the zein cleavage proteinase is probably an exopeptidase.

scholarly journals Role of Protein O-Mannosyltransferase Pmt4 in the Morphogenesis and Virulence of Cryptococcus neoformans

2006 ◽  
Vol 6 (2) ◽  
pp. 222-234 ◽  
Author(s):  
Gillian M. Olson ◽  
Deborah S. Fox ◽  
Ping Wang ◽  
J. Andrew Alspaugh ◽  
Kent L. Buchanan
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Cell Separation ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Cell Wall Integrity ◽  
Abnormal Growth ◽  
Transmission Electron ◽  
A Cell ◽  
And Function

ABSTRACT Protein O mannosylation is initiated in the endoplasmic reticulum by protein O-mannosyltransferases (Pmt proteins) and plays an important role in the secretion, localization, and function of many proteins, as well as in cell wall integrity and morphogenesis in fungi. Three Pmt proteins, each belonging to one of the three respective Pmt subfamilies, are encoded in the genome of the human fungal pathogen Cryptococcus neoformans. Disruption of the C. neoformans PMT4 gene resulted in abnormal growth morphology and defective cell separation. Transmission electron microscopy revealed defective cell wall septum degradation during mother-daughter cell separation in the pmt4 mutant compared to wild-type cells. The pmt4 mutant also demonstrated sensitivity to elevated temperature, sodium dodecyl sulfate, and amphotericin B, suggesting cell wall defects. Further analysis of cell wall protein composition revealed a cell wall proteome defect in the pmt4 mutant, as well as a global decrease in protein mannosylation. Heterologous expression of C. neoformans PMT4 in a Saccharomyces cerevisiae pmt1pmt4 mutant strain functionally complemented the deficient Pmt activity. Furthermore, Pmt4 activity in C. neoformans was required for full virulence in two murine models of disseminated cryptococcal infection. Taken together, these results indicate a central role for Pmt4-mediated protein O mannosylation in growth, cell wall integrity, and virulence of C. neoformans.

scholarly journals Assembly of the Herpes Simplex Virus Capsid: Identification of Soluble Scaffold-Portal Complexes and Their Role in Formation of Portal-Containing Capsids

2003 ◽  
Vol 77 (18) ◽  
pp. 9862-9871 ◽  
Author(s):  
William W. Newcomb ◽  
Darrell R. Thomsen ◽  
Fred L. Homa ◽  
Jay C. Brown
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Scaffolding Protein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The herpes simplex virus type 1 (HSV-1) portal complex is a ring-shaped structure located at a single vertex in the viral capsid. Composed of 12 UL6 protein molecules, the portal functions as a channel through which DNA passes as it enters the capsid. The studies described here were undertaken to clarify how the portal becomes incorporated as the capsid is assembled. We tested the idea that an intact portal may be donated to the growing capsid by way of a complex with the major scaffolding protein, UL26.5. Soluble UL26.5-portal complexes were found to assemble when purified portals were mixed in vitro with UL26.5. The complexes, called scaffold-portal particles, were stable during purification by agarose gel electrophoresis or sucrose density gradient ultracentrifugation. Examination of the scaffold-portal particles by electron microscopy showed that they resemble the 50- to 60-nm-diameter “scaffold particles” formed from purified UL26.5. They differed, however, in that intact portals were observed on the surface. Analysis of the protein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that portals and UL26.5 combine in various proportions, with the highest observed UL6 content corresponding to two or three portals per scaffold particle. Association between the portal and UL26.5 was antagonized by WAY-150138, a small-molecule inhibitor of HSV-1 replication. Soluble scaffold-portal particles were found to function in an in vitro capsid assembly system that also contained the major capsid (VP5) and triplex (VP19C and VP23) proteins. Capsids that formed in this system had the structure and protein composition expected of mature HSV-1 capsids, including UL6, at a level corresponding to ∼1 portal complex per capsid. The results support the view that UL6 becomes incorporated into nascent HSV-1 capsids by way of a complex with UL26.5 and suggest further that UL6 may be introduced into the growing capsid as an intact portal.

scholarly journals Ciliostatic, hemagglutinating, and proteolytic activities in a cell extract of Mycoplasma pneumoniae

1980 ◽  
Vol 29 (3) ◽  
pp. 1111-1116
Author(s):  
D K Chandler ◽  
M F Barile
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Mycoplasma Pneumoniae ◽  
Sodium Dodecyl ◽  
Cell Extract ◽  
Ciliary Activity ◽  
Organ Cultures ◽  
Freeze Thaw ◽  
A Cell ◽  
Proteolytic Activities

An extract of Mycoplasma pneumoniae, prepared from glass-grown organisms by extraction with 2 M NaCl, followed by freeze-thaw, ultracentrifugation, dialysis, and lyophilization, yielded approximately 20% of the total mycoplasmal protein. The extract contained at least 20 protein bands on sodium dodecyl sulfate-polyacrylamide gels and 2 to 5% carbohydrate and inhibited 70 to 100% of the ciliary activity of hamster tracheal organ cultures (ciliostasis). The extent of ciliostasis was dependent on the concentration of the extract. The extract also produced hemagglutination of human O-positive erythrocytes and showed proteolytic activity with a synthetic tetrapeptide substrate, S-2222. These in vitro tissue-damaging activities may be associated with the virulence of the mycoplasmas and with the pathogenesis of M. pneumoniae disease.

scholarly journals Synthesis of phosphoenolpyruvate carboxykinase (guanosine triphosphate) by isolated liver polyribosomes

1974 ◽  
Vol 144 (2) ◽  
pp. 199-207 ◽  
Author(s):  
F J Ballard ◽  
M F Hopgood ◽  
L Reshef ◽  
S Tilghman ◽  
Richard W. Hanson
Keyword(s):  
Protein Synthesis ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Sodium Dodecyl ◽  
Enzyme Synthesis ◽  
Mrna Synthesis ◽  
Synthesis System ◽  
Isolated Liver ◽  
Protein Synthesis System ◽  
Specific Mrna

1. Phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was synthesized by postmitochondrial supernatants of rat liver in the presence of appropriate salts, an energy supply and [3H]leucine. Synthesis of enzyme released from polyribosomes was detected by immunoprecipitation with specific antibody followed by electrophoresis of the dissolved antibody–antigen precipitates on sodium dodecyl sulphate–polyacrylamide gels in the presence of a14C-labelled enzyme marker. 2. Enzyme synthesis in vitro occurs predominantly on free rather than bound polyribosomes. 3. Starved animals in which de-induction of phosphoenolpyruvate carboxykinase (GTP) had been initiated by re-feeding for 2h had a markedly decreased rate of enzyme synthesis, whether the measurements were made after injection of radioactive leucine into the intact animal or if synthesis was determined in vitro. 4. The low rate of enzyme synthesis by liver polyribosomes from re-fed animals was not due to the absence of soluble factors, nor could it be increased by the addition of cyclic AMP to the protein synthesis system. 5. Phosphoenolpyruvate carboxykinase (GTP) synthesis in vitro is diminished relative to total protein synthesis when the postmitochondrial supernatant is kept at 0°C for several hours before measurement of protein synthesis. Since this effect is blocked by heparin, it is probably caused by selective ribonuclease attack on enzyme mRNA. 6. De-induction of phosphoenolpyruvate carboxykinase (GTP) is tentatively explained as being due to a transcriptional block in specific mRNA synthesis, followed by rapid degradation of existing message.

scholarly journals Biosynthesis and processing of legumin-like storage proteins in Lupinus angustifolius (lupin)

1985 ◽  
Vol 232 (3) ◽  
pp. 673-679 ◽  
Author(s):  
E D Johnson ◽  
J Knight ◽  
K R Gayler
Keyword(s):  
Storage Proteins ◽  
Sodium Dodecyl ◽  
Subcellular Fractionation ◽  
Lupinus Angustifolius ◽  
Free Translation ◽  
Specific Stage ◽  
Synthesis And Processing ◽  
Membrane Bound

Synthesis, secretion and post-translational proteolysis of the storage proteins in cotyledons of Lupinus angustifolius L. (lupin) have been examined in vivo and in vitro by using a combination of pulse-chase experiments with [3H]- or [35S]-labelled amino acids, subcellular fractionation and cell-free translation from poly(A)+ (polyadenylylated) RNA or membrane-bound polyribosomes. Related polypeptides were identified by immunoprecipitation, separation on sodium dodecyl sulphate/polyacrylamide gels and fluorography. The synthesis and processing of two proteins were compared. Conglutin alpha, the 11 S protein, was found as a family of precursor polypeptides of Mr 68000-88000 when translated from poly(A)+ RNA under conditions where signal segments were not cleaved, and Mr 64000-85000 both when sequestered into the endoplasmic reticulum and when accumulated in the protein bodies. Pulse-chase labelling showed that cotyledons from early stages of development were completely incapable of further proteolysis of these precursors. Nevertheless, in the same juvenile cotyledons, the precursors of the minor storage protein conglutin gamma, two polypeptides with Mr 50000-51000, were proteolytically cleaved to mature subunits of Mr 32000 and 17000 within 2 h. Further cleavage of the precursors of conglutin alpha into families of mature subunits of Mr 21000-24000 and 42000-62000 was detected in more mature cotyledons. A model is proposed which suggests that the mature subunits are produced by a single proteolytic cleavage of each of the three major precursors of conglutin alpha and also suggests that a close similarity exists between these subunits and those of other legumin-like proteins. The enzyme responsible for this cleavage, which appears at a specific stage in the middle of cotyledonary development, seems to be an integral part of the programmed developmental sequence in these pods.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

Using FRET to Measure the Time it Takes for a Cell to Destroy a Virus

2019 ◽  
Author(s):  
Candace E. Benjamin ◽  
Zhuo Chen ◽  
Olivia Brohlin ◽  
Hamilton Lee ◽  
Stefanie Boyd ◽  
...  
Keyword(s):  
Cellular Uptake ◽  
Rhodamine B ◽  
Virus Like Particle ◽  
A Cell ◽  
Viral Nanoparticles ◽  
The Stability ◽  
Open Question ◽  
Viral Surface ◽  
Fret Pair

<div><div><div><p>The emergence of viral nanotechnology over the preceding two decades has created a number of intellectually captivating possible translational applications; however, the in vitro fate of the viral nanoparticles in cells remains an open question. Herein, we investigate the stability and lifetime of virus-like particle (VLP) Qβ - a representative and popular VLP for several applications - following cellular uptake. By exploiting the available functional handles on the viral surface, we have orthogonally installed the known FRET pair, FITC and Rhodamine B, to gain insight of the particle’s behavior in vitro. Based on these data, we believe VLPs undergo aggregation in addition to the anticipated proteolysis within a few hours of cellular uptake.</p></div></div></div>

Preincubation with Sn-complexes causes intensive intracellular retention of 99mTc in thyroid cells in vitro

2012 ◽  
Vol 51 (05) ◽  
pp. 179-185 ◽  
Author(s):  
M. Wendisch ◽  
D. Aurich ◽  
R. Runge ◽  
R. Freudenberg ◽  
J. Kotzerke ◽  
...  
Keyword(s):  
Cell Model ◽  
Low Energy ◽  
Thyroid Cells ◽  
Low Energy Electrons ◽  
A Cell ◽  
Vitro Model ◽  
Uptake Studies ◽  
Radiobiological Effects ◽  
Radioactivity Retention

SummaryTechnetium radiopharmaceuticals are well established in nuclear medicine. Besides its well-known gamma radiation, 99mTc emits an average of five Auger and internal conversion electrons per decay. The biological toxicity of these low-energy, high-LET (linear energy transfer) emissions is a controversial subject. One aim of this study was to estimate in a cell model how much 99mTc can be present in exposed cells and which radiobiological effects could be estimated in 99mTc-overloaded cells. Methods: Sodium iodine symporter (NIS)- positive thyroid cells were used. 99mTc-uptake studies were performed after preincubation with a non-radioactive (cold) stannous pyro - phosphate kit solution or as a standard 99mTc pyrophosphate kit preparation or with pure pertechnetate solution. Survival curves were analyzed from colony-forming assays. Results: Preincubation with stannous complexes causes irreversible intracellular radioactivity retention of 99mTc and is followed by further pertechnetate influx to an unexpectedly high 99mTc level. The uptake of 99mTc pertechnetate in NIS-positive cells can be modified using stannous pyrophosphate from 3–5% to >80%. The maximum possible cellular uptake of 99mTc was 90 Bq/cell. Compared with nearly pure extracellular irradiation from routine 99mTc complexes, cell survival was reduced by 3–4 orders of magnitude after preincubation with stannous pyrophosphate. Conclusions: Intra cellular 99mTc retention is related to reduced survival, which is most likely mediated by the emission of low-energy electrons. Our findings show that the described experiments constitute a simple and useful in vitro model for radiobiological investigations in a cell model.

Analysis of the Dynamics of the Electric Capacitance of a Cell Monolayer at the Late Stages of Caco-2 cell Differentiation

2019 ◽  
Vol 35 (6) ◽  
pp. 87-90
Author(s):  
S.V. Nikulin ◽  
V.A. Petrov ◽  
D.A. Sakharov
Keyword(s):  
Impedance Spectroscopy ◽  
Cell Monolayer ◽  
Microfluidic Chips ◽  
Russian Science ◽  
Electric Capacitance ◽  
A Cell ◽  
Static Conditions ◽  
Late Stages

The real-time monitoring of electric capacitance (impedance spectroscopy) allowed obtaining evidence that structures which look like intestinal villi can be formed during the cultivation under static conditions as well as during the cultivation in microfluidic chips. It was shown in this work via transcriptome analysis that the Hh signaling pathway is involved in the formation of villus-like structures in vitro, which was previously shown for their formation in vivo. impedance spectroscopy, intestine, villi, electric capacitance, Hh The study was funded by the Russian Science Foundation (Project 16-19-10597).

Proteomics-Based Characterization of the Effects of MMP14 on the Protein Content of Exosomes from Corneal Fibroblasts

2020 ◽  
Vol 27 (10) ◽  
pp. 979-988
Author(s):  
Kyu-Yeon Han ◽  
Jin-Hong Chang ◽  
Dimitri T. Azar
Keyword(s):  
Protein Content ◽  
Catalytic Domain ◽  
Protein Composition ◽  
Proteomics Analysis ◽  
Knock Out ◽  
Corneal Fibroblast ◽  
Flow Cytometric ◽  
Corneal Fibroblasts ◽  
Liquid Chromatography Tandem Mass

Background: Exosomes secreted by corneal fibroblasts contain matrix metalloproteinase (MMP) 14, which is known to influence pro-MMP2 accumulation on exosomes. Accordingly, we hypothesized that the enzymatic activity of MMP14 may alter the protein content of corneal fibroblast- secreted exosomes. Objective: The aim of this study was to investigate the effects of MMP14 on the composition and biological activity of corneal fibroblast-derived exosomes. Methods: Knock out of the catalytic domain (ΔExon4) of MMP14 in corneal fibroblasts was used to determine the effect of MMP14 expression on the characteristics of fibroblast-secreted exosomes. The amount of secreted proteins and their size distribution were measured using Nano Tracking Analysis. Proteins within exosomes from wild-type (WT) and ΔExon4-deficient fibroblasts were identified by liquid chromatography-tandem mass spectrometry (MS/MS) proteomics analysis. The proteolytic effects of MMP14 were evaluated in vitro via MS identification of eliminated proteins. The biological functions of MMP14-carrying exosomes were investigated via fusion to endothelial cells and flow cytometric assays. Results: Exosomes isolated from WT and ΔExon4-deficient fibroblasts exhibited similar size distributions and morphologies, although WT fibroblasts secreted a greater amount of exosomes. The protein content, however, was higher in ΔExon4-deficient fibroblast-derived exosomes than in WT fibroblast-derived exosomes. Proteomics analysis revealed that WT-derived exosomes included proteins that regulated cell migration, and ΔExon4 fibroblast-derived exosomes contained additional proteins that were cleaved by MMP14. Conclusion: Our findings suggest that MMP14 expression influences the protein composition of exosomes secreted by corneal fibroblasts, and through those biological components, MMP14 in corneal fibroblasts derived-exosomes may regulate corneal angiogenesis.

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