scholarly journals Functional characterization of canine wild type glucocorticoid receptor and an insertional mutation in a dog

2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Kosei Yamanaka ◽  
Masaru Okuda ◽  
Takuya Mizuno
Keyword(s):  
Glucocorticoid Receptor ◽  
Cushing Syndrome ◽  
Functional Characterization ◽  
Wild Type ◽  
Key Factors ◽  
Truncated Form ◽  
Glucocorticoid Sensitivity ◽  
Almost All

Abstract Background Glucocorticoids, among the most widely utilized drugs in veterinary medicine, are employed to treat a wide variety of diseases; however, their use often induces adverse events in dogs. The efficacy of glucocorticoids usually depends on dosage, although differences in sensitivity to glucocorticoids in individual animals have been reported. Glucocorticoids bind to the cytoplasmic glucocorticoid receptor (GR), which is expressed in almost all cells. These receptors are key factors in determining individual sensitivity to glucocorticoids. This study examined individual differences in glucocorticoid sensitivity in dogs, focusing on reactivity of the GR to prednisolone. Results We first molecularly cloned the GR gene from a healthy dog. We discovered a mutant GR in a dog suspected to have iatrogenic Cushing syndrome. The mutant GR had extra nucleotides between exons 6 and 7, resulting in a truncated form of GR that was 98 amino acids shorter than the wild-type dog GR. The truncated GR exhibited very low reactivity to prednisolone, irrespective of concentration. Conclusions We have identified the truncated form of canine GR in a dog with iatrogenic Cushing syndrome. This truncated form showed the very less sensitivity to glucocorticoid in vitro, unfortunately, we could not elucidate its clinical significance. However, our data is a first report about the function of canine GR, and will facilitate the analysis of canine glucocorticoid sensitivity.

scholarly journals Functional characterization of the rod visual pigment of the echidna (Tachyglossus aculeatus), a basal mammal

2012 ◽  
Vol 29 (4-5) ◽  
pp. 211-217 ◽  
Author(s):  
CONSTANZE BICKELMANN ◽  
JAMES M. MORROW ◽  
JOHANNES MÜLLER ◽  
BELINDA S.W. CHANG
Keyword(s):  
Half Life ◽  
Functional Characterization ◽  
Egg Laying ◽  
Sensory Transduction ◽  
Wild Type ◽  
Functional Variation ◽  
Bovine Rhodopsin ◽  
Tachyglossus Aculeatus

AbstractMonotremes are the most basal egg-laying mammals comprised of two extant genera, which are largely nocturnal. Visual pigments, the first step in the sensory transduction cascade in photoreceptors of the eye, have been examined in a variety of vertebrates, but little work has been done to study the rhodopsin of monotremes. We isolated the rhodopsin gene of the nocturnal short-beaked echidna (Tachyglossus aculeatus) and expressed and functionally characterized the protein in vitro. Three mutants were also expressed and characterized: N83D, an important site for spectral tuning and metarhodopsin kinetics, and two sites with amino acids unique to the echidna (T158A and F169A). The λmax of echidna rhodopsin (497.9 ± 1.1 nm) did not vary significantly in either T158A (498.0 ± 1.3 nm) or F169A (499.4 ± 0.1 nm) but was redshifted in N83D (503.8 ± 1.5 nm). Unlike other mammalian rhodopsins, echidna rhodopsin did react when exposed to hydroxylamine, although not as fast as cone opsins. The retinal release rate of light-activated echidna rhodopsin, as measured by fluorescence spectroscopy, had a half-life of 9.5 ± 2.6 min−1, which is significantly shorter than that of bovine rhodopsin. The half-life of the N83D mutant was 5.1 ± 0.1 min−1, even shorter than wild type. Our results show that with respect to hydroxylamine sensitivity and retinal release, the wild-type echidna rhodopsin displays major differences to all previously characterized mammalian rhodopsins and appears more similar to other nonmammalian vertebrate rhodopsins such as chicken and anole. However, our N83D mutagenesis results suggest that this site may mediate adaptation in the echidna to dim light environments, possibly via increased stability of light-activated intermediates. This study is the first characterization of a rhodopsin from a most basal mammal and indicates that there might be more functional variation in mammalian rhodopsins than previously assumed.

scholarly journals 2066 Functional characterization of mutant BRCA1

2018 ◽  
Vol 2 (S1) ◽  
pp. 13-13
Author(s):  
John Barrows ◽  
David Long
Keyword(s):  
Functional Characterization ◽  
Coiled Coil ◽  
Wild Type ◽  
Coiled Coil Region ◽  
Egg Extracts ◽  
Study Population ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

OBJECTIVES/SPECIFIC AIMS: The objective of this work is to determine the mechanistic consequences of BRCA1 mutants in inter-strand crosslink (ICL) repair. METHODS/STUDY POPULATION: Our lab uses Xenopus egg extracts to study ICL repair. These extracts can be depleted of endogenous BRCA1 by immunoprecipitation. The goal of this work is to rescue endogenous depletion with in vitro translated, wild type BRCA1. Once achieved, we can supplement the depleted extract with BRCA1 mutants to access their function in ICL repair. RESULTS/ANTICIPATED RESULTS: We hypothesize that the BRCT and RING domain mutations will abrogate ICL repair, while mutations in the coiled coil region will not affect repair. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings will have an immense impact on the understanding of BRCA1 domains. Importantly these results will spur personalized therapy of BRCA1 mutants by showing which domains are sensitive to cross-linking agents.

scholarly journals Functional characterization of Ost3p. Loss of the 34-kD subunit of the Saccharomyces cerevisiae oligosaccharyltransferase results in biased underglycosylation of acceptor substrates.

1995 ◽  
Vol 130 (3) ◽  
pp. 567-577 ◽  
Author(s):  
D Karaoglu ◽  
D J Kelleher ◽  
R Gilmore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Characterization ◽  
Null Mutant ◽  
Wild Type ◽  
Membrane Glycoproteins ◽  
En Bloc ◽  
Oligosaccharide Moiety

Within the lumen of the rough endoplasmic reticulum, oligosaccharyltransferase catalyzes the en bloc transfer of a high mannose oligosaccharide moiety from the lipid-linked oligosaccharide donor to asparagine acceptor sites in nascent polypeptides. The Saccharomyces cerevisiae oligosaccharyltransferase was purified as a heteroligomeric complex consisting of six subunits (alpha-zeta) having apparent molecular masses of 64 kD (Ost1p), 45 kD (Wbp1p), 34 kD, 30 kD (Swp1p), 16 kD, and 9 kD. Here we report a structural and functional characterization of Ost3p which corresponds to the 34-kD gamma-subunit of the oligosaccharyltransferase. Unlike Ost1p, Wbp1p, and Swp1p, expression of Ost3p is not essential for viability of yeast. Instead, ost3 null mutant yeast grow at wild-type rates on solid or in liquid media irrespective of culture temperature. Nonetheless, detergent extracts prepared from ost3 null mutant membranes are twofold less active than extracts prepared from wild-type membranes in an in vitro oligosaccharyltransferase assay. Furthermore, loss of Ost3p is accompanied by significant underglycosylation of soluble and membrane-bound glycoproteins in vivo. Compared to the previously characterized ost1-1 mutant in the oligosaccharyltransferase, and the alg5 mutant in the oligosaccharide assembly pathway, ost3 null mutant yeast appear to be selectively impaired in the glycosylation of several membrane glycoproteins. The latter observation suggests that Ost3p may enhance oligosaccharide transfer in vivo to a subset of acceptor substrates.

scholarly journals Functional characterization of wild-type and 24 CYP2D6 allelic variants on gefitinib metabolism in vitro

2017 ◽  
Vol Volume 11 ◽  
pp. 1283-1290 ◽  
Author(s):  
Ping Fang ◽  
Xiang Zheng ◽  
Jiayang He ◽  
Honglei Ge ◽  
Pengfei Tang ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Wild Type ◽  
Allelic Variants

Functional characterization of the H+/K+ ATPase in wild type and gastrin knock-out mice

2007 ◽  
Vol 45 (05) ◽  
Author(s):  
A Schnur ◽  
P Hegyi ◽  
V Venglovecz ◽  
Z Rakonczay ◽  
I Ignáth ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Wild Type ◽  
Knock Out ◽  
Knock Out Mice

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals NFB-01. FUNCTIONAL CHARACTERIZATION OF ATRX LOSS IN NF1-ASSOCIATED GLIOMA AND MPNST

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii417-iii418
Author(s):  
Ming Yuan ◽  
Karlyne Reilly ◽  
Christine Pratilas ◽  
Christopher Heaphy ◽  
Fausto Rodriguez
Keyword(s):  
Cell Lines ◽  
Functional Characterization ◽  
Tert Promoter ◽  
Aggressive Tumors ◽  
Sirna Knockdown ◽  
Nih 3T3 ◽  
Vitro Effect ◽  
Murine Glioma

Abstract To identify the biologic relevance of ATRX loss in NF1-associated gliomagenesis, we studied the effects of Atrx loss using four previously characterized Nf1+/-Trp53+/- murine glioma lines. Lines 130G#3 and 158D#8 (corresponding to grade IV and III gliomas, respectively) displayed preserved ATRX protein expression compared to NIH-3T3 cells. We studied the effects of Atrx knockdown in these two lines in the presence and absence of the TERT inhibitor, BIRBR1532. Using a telomere-specific FISH assay, we identified increased signal intensity after Atrx knockdown, only in the presence of the TERT inhibitor. These features are reminiscent of ALT, although there were no significant alterations in cell growth. Next, we studied the effect of ATRX loss in MPNST lines ST88-14, NF90-8, STS-26T. These cell lines all expressed ATRX and DAXX. However, STS-26T contained a TERT promoter mutation and ST88-14 had a known SNP in the TERT promoter, while NF90-8 had no alterations. ATRX siRNA knockdown showed no significant effects in cell proliferation or apoptosis. However, ATRX knockdown resulted in rare ultra-bright foci, indicative of ALT. Next, we studied the in vitro effect of the ATR inhibitor VE-821 in MPNST cell lines. Only NF90-8 (lacking TERT alterations) demonstrated a decrease in growth after ATRX knockdown and VE-821 treatment. However, ATRX knockdown alone did not affect sensitivity to carboplatin. Our findings further support a role for ATRX loss with subsequent ALT activation in a biologic subset of NF1-associated malignancies, thereby opening an opportunity for therapeutic targeting of these aggressive tumors using specific classes of drugs.

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 2372-2382
Author(s):  
K M Arndt ◽  
S L Ricupero ◽  
D M Eisenmann ◽  
F Winston
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Repeat ◽  
Single Amino Acid ◽  
Wild Type ◽  
Dna Binding Specificity ◽  
Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

Functional Characterization of GABAA Receptor Ligands In Vitro

2004 ◽  
pp. 85-94
Author(s):  
Bjarke Ebert ◽  
Sally Anne Thompson ◽  
Signe Í. Stórustovu ◽  
Keith A. Wafford
Keyword(s):  
Gabaa Receptor ◽  
Functional Characterization ◽  
Receptor Ligands
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