scholarly journals Cell-Free Reconstitution of Microautophagic Vacuole Invagination and Vesicle Formation

2000 ◽  
Vol 151 (3) ◽  
pp. 529-538 ◽  
Author(s):  
Tanja Sattler ◽  
Andreas Mayer
Keyword(s):  
Structural Changes ◽  
Fluorescent Dyes ◽  
Vacuolar Membrane ◽  
Specific Gene ◽  
Temperature Dependent ◽  
Free System ◽  
Prolonged Incubation ◽  
A Cell ◽  
Vacuolar Membranes ◽  
Boundary Membrane

Many organelles change their shape in the course of the cell cycle or in response to environmental conditions. Lysosomes undergo drastic changes of shape during microautophagocytosis, which include the invagination of their boundary membrane and the subsequent scission of vesicles into the lumen of the organelle. The mechanism driving these structural changes is enigmatic. We have begun to analyze this process by reconstituting microautophagocytosis in a cell-free system. Isolated yeast vacuoles took up fluorescent dyes or reporter enzymes in a cytosol-, ATP-, and temperature-dependent fashion. During the uptake reaction, vacuolar membrane invaginations, called autophagic tubes, were observed. The reaction resulted in the transient formation of autophagic bodies in the vacuolar lumen, which were degraded upon prolonged incubation. Under starvation conditions, the system reproduced the induction of autophagocytosis and depended on specific gene products, which were identified in screens for mutants deficient in autophagocytosis. Microautophagic uptake depended on the activity of the vacuolar ATPase and was sensitive to GTPγS, indicating a requirement for GTPases and for the vacuolar membrane potential. However, microautophagocytosis was independent of known factors for vacuolar fusion and vesicular trafficking. Therefore, scission of the invaginated membrane must occur via a novel mechanism distinct from the homotypic fusion of vacuolar membranes.

Poly(A) shortening and degradation of the 3' A+U-rich sequences of human c-myc mRNA in a cell-free system

1988 ◽  
Vol 8 (4) ◽  
pp. 1697-1708
Author(s):  
G Brewer ◽  
J Ross
Keyword(s):  
Untranslated Region ◽  
Degradation Pathway ◽  
Second Step ◽  
Free System ◽  
Prolonged Incubation ◽  
Gamma Globin ◽  
Decay Products ◽  
Exon 1 ◽  
A Cell

The early steps in the degradation of human c-myc mRNA were investigated, using a previously described cell-free mRNA decay system. The first detectable step was poly(A) shortening, which generated a pool of oligoadenylated mRNA molecules. In contrast, the poly(A) of a stable mRNA, gamma globin, was not excised, even after prolonged incubation. The second step, degradation of oligoadenylated c-myc mRNA, generated decay products whose 3' termini were located within the A+U-rich portion of the 3' untranslated region. These products disappeared soon after they were formed, consistent with rapid degradation of the 3' region. In contrast, the 5' region, corresponding approximately to c-myc exon 1, was stable in vitro. The data indicate a sequential degradation pathway in which 3' region cleavages occur only after most or all of the poly(A) is removed. To account for rapid deadenylation, we suggest that the c-myc poly(A)-poly(A)-binding protein complex is readily dissociated, generating a protein-depleted poly(A) tract that is no longer resistant to nucleases.

INTERACTION OF 3H-OESTRADIOL WITH ANTERIOR PITUITARY NUCLEI IN A CELL-FREE SYSTEM

1972 ◽  
Vol 69 (2) ◽  
pp. 230-240 ◽  
Author(s):  
Jerry P. Friend ◽  
Wendell W. Leavitt
Keyword(s):  
Anterior Pituitary ◽  
Supernatant Fraction ◽  
Release Mechanism ◽  
Nuclear Fraction ◽  
Temperature Dependent ◽  
Free System ◽  
Cytoplasmic Component ◽  
C 23 ◽  
A Cell ◽  
Pituitary Homogenate

ABSTRACT The uptake and retention of 3H-oestradiol by the nuclear fraction of pituitary homogenate was dependent on factors present in the 800 g-supernatant fraction. The binding of 3H-oestradiol by the nuclear fraction was competitively inhibited by unlabelled oestradiol. The nuclear fraction retained different amounts of 3H-oestradiol following incubation at 4°C, 23°C and 37°C. Significant binding occurred at each temperature, and the rate of uptake was greatest at 37°C. Radioactivity was released rapidly from the nuclear fraction at 37°C, and this could be prevented by lowering the temperature. These studies revealed that there is a cytoplasmic component as well as a temperature-dependent release mechanism which controls oestradiol uptake and retention by the nuclear fraction. Evidence was obtained indicating that the cytoplasmic component is tissue specific.

scholarly journals Poly(A) shortening and degradation of the 3' A+U-rich sequences of human c-myc mRNA in a cell-free system.

1988 ◽  
Vol 8 (4) ◽  
pp. 1697-1708 ◽  
Author(s):  
G Brewer ◽  
J Ross
Keyword(s):  
Untranslated Region ◽  
Degradation Pathway ◽  
Second Step ◽  
Free System ◽  
Prolonged Incubation ◽  
Gamma Globin ◽  
Decay Products ◽  
Exon 1 ◽  
A Cell

The early steps in the degradation of human c-myc mRNA were investigated, using a previously described cell-free mRNA decay system. The first detectable step was poly(A) shortening, which generated a pool of oligoadenylated mRNA molecules. In contrast, the poly(A) of a stable mRNA, gamma globin, was not excised, even after prolonged incubation. The second step, degradation of oligoadenylated c-myc mRNA, generated decay products whose 3' termini were located within the A+U-rich portion of the 3' untranslated region. These products disappeared soon after they were formed, consistent with rapid degradation of the 3' region. In contrast, the 5' region, corresponding approximately to c-myc exon 1, was stable in vitro. The data indicate a sequential degradation pathway in which 3' region cleavages occur only after most or all of the poly(A) is removed. To account for rapid deadenylation, we suggest that the c-myc poly(A)-poly(A)-binding protein complex is readily dissociated, generating a protein-depleted poly(A) tract that is no longer resistant to nucleases.

Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

2019 ◽  
pp. 27-33
Author(s):  
YuE Kravchenko ◽  
SV Ivanov ◽  
DS Kravchenko ◽  
EI Frolova ◽  
SP Chumakov
Keyword(s):  
Phage Display ◽  
Selection Procedure ◽  
Free System ◽  
Phage Displays ◽  
High Affinity ◽  
Binding Domains ◽  
A Cell ◽  
Vhh Antibodies ◽  
Efficiency Of Selection ◽  
Selection Of

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

scholarly journals Effect of detergents on sterol synthesis in a cell-free system of yeast

1982 ◽  
Vol 23 (6) ◽  
pp. 803-810
Author(s):  
S Hata ◽  
T Nishino ◽  
N Ariga ◽  
H Katsuki
Keyword(s):  
Sterol Synthesis ◽  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals Reconstitution of Endosomal Proteolysis in a Cell-free System

1989 ◽  
Vol 264 (10) ◽  
pp. 5392-5399
Author(s):  
L S Mayorga ◽  
R Diaz ◽  
P D Stahl
Keyword(s):  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals Novel Method for Quantifying AhR-Ligand Binding Affinities Using Microscale Thermophoresis

Biosensors ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 60
Author(s):  
Anne Stinn ◽  
Jens Furkert ◽  
Stefan H. E. Kaufmann ◽  
Pedro Moura-Alves ◽  
Michael Kolbe
Keyword(s):  
Ligand Binding ◽  
Environmental Pollutants ◽  
Screening Strategy ◽  
Pharmacological Intervention ◽  
Binding Affinities ◽  
Free System ◽  
Microscale Thermophoresis ◽  
Aryl Hydrocarbon ◽  
Promising Target ◽  
A Cell

The aryl hydrocarbon receptor (AhR) is a highly conserved cellular sensor of a variety of environmental pollutants and dietary-, cell- and microbiota-derived metabolites with important roles in fundamental biological processes. Deregulation of the AhR pathway is implicated in several diseases, including autoimmune diseases and cancer, rendering AhR a promising target for drug development and host-directed therapy. The pharmacological intervention of AhR processes requires detailed information about the ligand binding properties to allow specific targeting of a particular signaling process without affecting the remaining. Here, we present a novel microscale thermophoresis-based approach to monitoring the binding of purified recombinant human AhR to its natural ligands in a cell-free system. This approach facilitates a precise identification and characterization of unknown AhR ligands and represents a screening strategy for the discovery of potential selective AhR modulators.

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