scholarly journals Induction of an inactivation pathway for ecdysteroids in larvae of the cotton leafworm, Spodoptera littoralis

1994 ◽  
Vol 301 (1) ◽  
pp. 89-95 ◽  
Author(s):  
J H Chen ◽  
M Kabbouh ◽  
M J Fisher ◽  
H H Rees
Keyword(s):  
Spodoptera Littoralis ◽  
Actinomycin D ◽  
Hydroxylase Activity ◽  
Free System ◽  
Cotton Leafworm ◽  
Oxygenase Activity ◽  
Rna And Protein Synthesis ◽  
A Cell ◽  
Moulting Hormones ◽  
Aldehyde Derivative

Treatment of the last-instar larvae of the cotton leafworm (Spodoptera littoralis) with ecdysteroids (moulting hormones) results in the induction of an ecdysteroid-inactivation pathway. Administration of ecdysone, 20-hydroxyecdysone or an ecdysteroid agonist, RH 5849, leads to induction of an ecdysteroid 26-hydroxylase activity. This induction occurred in both early sixth-instar larvae and in older larvae which had been head-ligated to prevent the normal developmental increase in ecdysone 20-mono-oxygenase activity. The induction of 26-hydroxylase activity requires both RNA and protein synthesis, as demonstrated by experiments involving actinomycin D and cycloheximide. The 26-aldehyde derivative of ecdysone and ecdyson-26-oic acid were also formed from ecdysone in the RH 5849-induced systems. Formation of the aldehyde and the corresponding 26-oic acid (ecdysonoic acid) from 26-hydroxyecdysone was directly demonstrated in a cell-free system, thus establishing the following inactivation pathway: Ecdysteroid-->26-hydroxyecdysteroid-->ecdysteroid 26-aldehyde-->ecdysteroid 26-oic acid.

ADDITIONAL STUDIES ON THE MECHANISM OF ACTION OF ACTH

1965 ◽  
Vol 43 (7) ◽  
pp. 923-932 ◽  
Author(s):  
Kenneth W. McKerns
Keyword(s):  
Pentose Phosphate ◽  
Free System ◽  
Active Complex ◽  
Initial Event ◽  
Rna And Protein Synthesis ◽  
Lysine Residues ◽  
A Cell ◽  
Initial Activation ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Increased Activity

Additional studies are presented to support the concept that ACTH regulates the function of the adrenal cortex by activating adrenal glucose-6-phosphate dehydrogenase. Corticoid synthesis in a cell-free system of adrenal tissue, supplemented with glucose-6-phosphate, can be stimulated by synthetic 23 amino acid ACTH. ACTH is inactive if the lysine residues of the peptide chain are blocked. Estrogen competes with NADP+ binding to glucose-6-phosphate dehydrogenase and inhibits the ACTH stimulus to steroidogenesis. Puromycin and other protein inhibitors have no effect on the initial activation of glucose-6-phosphate dehydrogenase and steroidogenesis. With longer incubation times puromycin can completely inhibit steroidogenesis, possibly by poisoning microsomal or mitochondrial elements. The initial event in the ACTH effect would seem to be the formation of an active complex of ACTH with glucose-6-phosphate dehydrogenase. This increases the rate of metabolism of glucose-6-phosphate and the rate of reduction of NADP+ required for the hydroxylation reactions in corticoid synthesis. The increased activity of the pentose phosphate pathway with increased production of ribose sugars and ATP could increase DNA-directed RNA and protein synthesis as a secondary event.

scholarly journals Immunological analysis of developmental changes in ecdysone 20-mono-oxygenase expression in the cotton leafworm, Spodoptera littoralis

1994 ◽  
Vol 299 (3) ◽  
pp. 711-717 ◽  
Author(s):  
J H Chen ◽  
T Hara ◽  
M J Fisher ◽  
H H Rees
Keyword(s):  
Spodoptera Littoralis ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Close Correlation ◽  
Developmental Changes ◽  
Cotton Leafworm ◽  
Oxygenase Activity ◽  
Adrenodoxin Reductase ◽  
Cytochrome P 450

The developmental changes in ecdysone 20-mono-oxygenase during the sixth larval instar of the cotton leafworm, Spodoptera littoralis, were investigated. The specific activity of mitochondrial ecdysone 20-mono-oxygenase in the fat-body exhibited a distinct peak at 72 h, at which time the larvae stop feeding. Immunoblot analyses, using antibodies raised against components of vertebrate mitochondrial steroidogenic enzyme systems [anti-(cytochrome P-450scc), anti-(cytochrome P-450(11) beta), anti-adrenodoxin and anti-(adrenodoxin reductase) antibodies], revealed the presence of specific immunoreactive polypeptides in fat-body mitochondrial extracts. In addition, these antibodies effectively inhibited fat-body mitochondrial ecdysone 20-mono-oxygenase activity. This suggests that the S. littoralis steroid-hydroxylating system(s) may contain polypeptide components analogous to those present in vertebrates. A close correlation between developmental changes in mitochondrial ecdysone 20-mono-oxygenase activity and the abundance of polypeptides (approx. 66 kDa and 50 kDa) recognized by the anti-(cytochrome P-450(11) beta) antibody and a polypeptide (approx. 52 kDa) recognized by the anti-(adrenodoxin reductase) antibody were observed in both fat-body and midgut. These results suggest that developmental changes in the abundance of components of the ecdysone 20-mono-oxygenase system may play an important role in the developmental regulation of the enzyme expression and, hence, of 20-hydroxyecdysone titre.

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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