scholarly journals Mechanism of Binding of Prothioconazole toMycosphaerella graminicolaCYP51 Differs from That of Other Azole Antifungals

2010 ◽  
Vol 77 (4) ◽  
pp. 1460-1465 ◽  
Author(s):  
Josie E. Parker ◽  
Andrew G. S. Warrilow ◽  
Hans J. Cools ◽  
Claire M. Martel ◽  
W. David Nes ◽  
...  
Keyword(s):  
Scientific Literature ◽  
Competitive Inhibitor ◽  
Sterol Composition ◽  
Specific Information ◽  
Type Ii ◽  
Wild Type ◽  
Binding Studies ◽  
Difference Spectra ◽  
Mechanism Of Inhibition ◽  
Noncompetitive Inhibitors

ABSTRACTProthioconazole is one of the most important commercially available demethylase inhibitors (DMIs) used to treatMycosphaerella graminicolainfection of wheat, but specific information regarding its mode of action is not available in the scientific literature. Treatment of wild-typeM. graminicola(strain IPO323) with 5 μg of epoxiconazole, tebuconazole, triadimenol, or prothioconazole ml−1resulted in inhibition ofM. graminicolaCYP51 (MgCYP51), as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol in azole-treated cells. Successful expression of MgCYP51 inEscherichia colienabled us to conduct spectrophotometric assays using purified 62-kDa MgCYP51 protein. Antifungal-binding studies revealed that epoxiconazole, tebuconazole, and triadimenol all bound tightly to MgCYP51, producing strong type II difference spectra (peak at 423 to 429 nm and trough at 406 to 409 nm) indicative of the formation of classical low-spin sixth-ligand complexes. Interaction of prothioconazole with MgCYP51 exhibited a novel spectrum with a peak and trough observed at 410 nm and 428 nm, respectively, indicating a different mechanism of inhibition. Prothioconazole bound to MgCYP51 with 840-fold less affinity than epoxiconazole and, unlike epoxiconazole, tebuconazole, and triadimenol, which are noncompetitive inhibitors, prothioconazole was found to be a competitive inhibitor of substrate binding. This represents the first study to validate the effect of prothioconazole on the sterol composition ofM. graminicolaand the first on the successful heterologous expression of active MgCYP51 protein. The binding affinity studies documented here provide novel insights into the interaction of MgCYP51 with DMIs, especially for the new triazolinethione derivative prothioconazole.

Two Different UGT1A1 Mutations causing Crigler–Najjar Syndrome types I and II in an Iranian Family

2015 ◽  
Vol 24 (4) ◽  
pp. 523-526 ◽  
Author(s):  
Yoshihiro Maruo ◽  
Mahdiyeh Behnam ◽  
Shinichi Ikushiro ◽  
Sayuri Nakahara ◽  
Narges Nouri ◽  
...  
Keyword(s):  
Serum Bilirubin ◽  
Total Serum Bilirubin ◽  
Total Serum ◽  
Compound Heterozygous ◽  
Type I ◽  
Syndrome Type ◽  
Type Ii ◽  
Wild Type ◽  
Iranian Family ◽  
Udp Glucuronosyltransferase

Background: Crigler–Najjar syndrome type I (CN-1) and type II (CN-2) are rare hereditary unconjugated hyperbilirubinemia disorders. However, there have been no reports regarding the co-existence of CN-1 and CN-2 in one family. We experienced a case of an Iranian family that included members with either CN-1 or CN-2. Genetic analysis revealed a mutation in the bilirubin UDP-glucuronosyltransferase (UGT1A1) gene that resulted in residual enzymatic activity.Case report: The female proband developed severe hyperbilirubinemia [total serum bilirubin concentration (TB) = 34.8 mg/dL] with bilirubin encephalopathy (kernicterus) and died after liver transplantation. Her family history included a cousin with kernicterus (TB = 30.0 mg/dL) diagnosed as CN-1. Her great grandfather (TB unknown) and uncle (TB = 23.0 mg/dL) developed jaundice, but without any treatment, they remained healthy as CN-2. Results: The affected cousin was homozygous for a novel frameshift mutation (c.381insGG, p.C127WfsX23). The affected uncle was compound heterozygous for p.C127WfsX23 and p.V225G linked with A(TA)7TAA. p.V225G-UGT1A1 reduced glucuronidation activity to 60% of wild-type. Thus, linkage of A(TA)7TAA and p.V225G might reduce UGT1A1 activity to 18%–36 % of the wild-type. Conclusion: Genetic and in vitro expression analyses are useful for accurate genetic counseling for a family with a history of both CN-1 and CN-2. Abbreviations: CN-1: Crigler–Najjar syndrome type I; CN-2: Crigler–Najjar syndrome type II; GS: Gilbert syndrome; UGT1A1: bilirubin UDP-glucuronosyltransferase; WT: Wild type; TB: total serum bilirubin.

Estrogen Receptor-Independent Catechol Estrogen Binding Activity:  Protein Binding Studies in Wild-Type, Estrogen Receptor-α KO, and Aromatase KO Mice Tissues†

Biochemistry ◽  
2004 ◽  
Vol 43 (21) ◽  
pp. 6698-6708 ◽  
Author(s):  
Brian J. Philips ◽  
Pete J. Ansell ◽  
Leslie G. Newton ◽  
Nobuhiro Harada ◽  
Shin-Ichiro Honda ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Protein Binding ◽  
Estrogen Receptor Α ◽  
Binding Activity ◽  
Wild Type ◽  
Binding Studies ◽  
Catechol Estrogen ◽  
Estrogen Binding

scholarly journals Transcription of rDNA insertions in bobbed mutants of Drosophila melanogaster

1986 ◽  
Vol 48 (3) ◽  
pp. 167-174 ◽  
Author(s):  
Régine Terracol
Keyword(s):  
Drosophila Melanogaster ◽  
28S Rrna ◽  
Type Ii ◽  
Wild Type

SummaryIn Drosophila melanogaster a large number of the genes coding for 18S and 28S rRNA are interrupted in the 28S region by insertions of two types. Ribosomal insertion transcripts were compared in wild-type and bobbed strains. We found that the level of insertion transcripts increased in bobbed mutants after deletion of 50% of INS− genes, and inversely decreased in revertants when more than 50% of wild-type levels. Among type II insertion transcripts we found a predominant 3·5 kb RNA, precisely of the most frequent insertion size. No primary insertion transcript has been found, although it could be undetected if very fast splicing leads to mature 28S occurs.

scholarly journals Evidence for HrpXo-Dependent Expression of Type II Secretory Proteins in Xanthomonas oryzae pv. oryzae

2004 ◽  
Vol 186 (5) ◽  
pp. 1374-1380 ◽  
Author(s):  
Ayako Furutani ◽  
Seiji Tsuge ◽  
Kouhei Ohnishi ◽  
Yasufumi Hikichi ◽  
Takashi Oku ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Gene ◽  
Secretion System ◽  
Xanthomonas Oryzae ◽  
Nucleotide Sequence Analysis ◽  
Secretory Proteins ◽  
Type Ii ◽  
Wild Type ◽  
Type Iii

ABSTRACT Xanthomonas oryzae pv. oryzae is a causal agent of bacterial leaf blight of rice. Recently, an efficient hrp-inducing medium, XOM2, was established for this bacterium. In this medium, more than 10 proteins were secreted from the wild-type strain of X. oryzae pv. oryzae. Many of these proteins disappeared or decreased in amount in culture on XOM2 when incubated with the strain that has a mutation in the hrp regulatory gene. Interestingly, the secretory protein profile of a mutant lacking a type III secretion system (TTSS), components of which are encoded by hrp genes, was similar to that of the wild-type strain except that a few proteins had disappeared. This finding suggests that many HrpXo-dependent secretory proteins are secreted via systems other than the TTSS. By isolating mutant strains lacking a type II secretion system, we examined this hypothesis. As expected, many of the HrpXo-dependent secretory proteins disappeared or decreased when the mutant was cultured in XOM2. By determining the N-terminal amino acid sequence, we identified one of the type II secretory proteins as a cysteine protease homolog, CysP2. Nucleotide sequence analysis revealed that cysP2 has an imperfect plant-inducible-promoter box, a consensus sequence which HrpXo regulons possess in the promoter region, and a deduced signal peptide sequence at the N terminus. By reverse transcription-PCR analysis and examination of the expression of CysP2 by using a plasmid harboring a cysP2::gus fusion gene, HrpXo-dependent expression of CysP2 was confirmed. Here, we reveal that the hrp regulatory gene hrpXo is also involved in the expression of not only hrp genes and type III secretory proteins but also some type II secretory proteins.

A mutant androgen receptor from patients with Reifenstein syndrome: identification of the function of a conserved alanine residue in the D box of steroid receptors

1993 ◽  
Vol 13 (12) ◽  
pp. 7850-7858
Author(s):  
F Kaspar ◽  
H Klocker ◽  
A Denninger ◽  
A C Cato
Keyword(s):  
Androgen Receptor ◽  
Dna Binding ◽  
Steroid Receptors ◽  
Regulatory Elements ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Binding Studies ◽  
Mutant Receptor ◽  
Alanine Residue

Reifenstein syndrome is an eponymic term that describes partial androgen-insensitive disorders. Androgen receptor isolated from five patients with this syndrome contains a specific mutation in the DNA binding domain of the receptor. This mutation converts an alanine to a threonine at position 596 next to the zinc catenation site at the second finger. The threonine 596 mutant receptor mediated normal androgen response at promoters with closely positioned multiple regulatory elements for the androgen receptor and other transcription factors. Promoters with single isolated androgen response elements were not transactivated by the mutant receptor. In in vitro receptor-DNA binding studies, interaction with DNA by the mutant receptor was achieved only in the presence of an anti-androgen receptor antibody. Exchanging alanine 596 in the wild-type androgen receptor with serine or valine produced mutants with properties indistinguishable from those of the naturally occurring threonine 596 mutant receptor. These results indicate that an alanine residue at position 596 contributes important structural and functional activities to the androgen receptor. In the androgen receptor from the patients with Reifenstein syndrome, in which this alanine is converted to a threonine, wild-type receptor properties can be restored by exchanging an additional threonine at position 602 to an alanine. An alanine residue at position 596 or 602 in the DNA binding domain of the androgen receptor is therefore important for the full function of this receptor. In all steroid receptors that bind the core sequence AGAACANNNTGTTCT, an alanine residue is also present at a position equivalent to alanine 596 in the androgen receptor.

scholarly journals Efficient endosomal localization of major histocompatibility complex class II-invariant chain complexes requires multimerization of the invariant chain targeting sequence.

1995 ◽  
Vol 129 (5) ◽  
pp. 1217-1228 ◽  
Author(s):  
L S Arneson ◽  
J Miller
Keyword(s):  
Cell Surface ◽  
Class Ii ◽  
Cell Surface Expression ◽  
Invariant Chain ◽  
Antigenic Peptides ◽  
Type Ii ◽  
Wild Type ◽  
Chain Complexes ◽  
Cytosolic Domain ◽  
Localization Signal

During biosynthesis, MHC class II-invariant chain complexes are transported into endosomal compartments where invariant chain (Ii) is degraded and class II encounters antigenic peptides. One of the signals that determines this intracellular transport route has been localized to the cytosolic domain of Ii. Deletion of this signal disrupts endosomal targeting and results in the stable expression of class II-Ii complexes at the surface. In this paper we have examined the role of Ii trimerization on the generation of this endosomal localization signal. In L cell transfectants expressing class II and both wild type Ii and a truncated form of Ii that lacks this endosomal localization signal, Ii was found to form multimers which could contain both wild type and truncated Ii. The multimers were not large aggregates but were found to be discrete complexes, probably the nine molecule class II-Ii complex that has been observed in human B cells. The co-expression of truncated Ii allowed for cell surface expression of a subset of wild type Ii. This surface-expressed wild type Ii associated with truncated Ii in multimers at a 2:1 ratio, indicating that these trimers contain two truncated and one wild type Ii molecule. These data suggest a division in trafficking of Ii trimers: if two wild type Ii molecules are present, the complex is transported to and rapidly degraded in endosomes, whereas the presence of only one wild type Ii results in trafficking and expression of the heterotrimer on the cell surface. Following surface arrival, complexes containing only a single wild type Ii molecule are internalized more rapidly and have a shorter half-life than complexes containing only truncated Ii molecules. These data suggest that although a single Ii cytosolic domain can function as a plasma membrane internalization signal, multimerization of Ii is required for efficient Golgi complex to endosome targeting of class II-Ii complexes.

Role of common cytokine receptor γ chain (γc)– and Jak3-dependent signaling in the proliferation and survival of murine mast cells

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2172-2180 ◽  
Author(s):  
Kotaro Suzuki ◽  
Hiroshi Nakajima ◽  
Norihiko Watanabe ◽  
Shin-ichiro Kagami ◽  
Akira Suto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Cytokine Receptor ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Wild Type ◽  
Peritoneal Mast Cells ◽  
The Common

Abstract The regulatory roles of the common cytokine receptor γ chain (γc)– and Jak3-dependent signaling in the proliferation and survival of mast cells were determined using γc-deficient (γc−) and Jak3-deficient (Jak3−) mice. Although the mast cells in γc− and Jak3− mice were morphologically indistinguishable from those in wild-type mice, the number of peritoneal mast cells was decreased in γc− and Jak3− mice as compared with that in wild-type mice. Among γc-related cytokines, interleukin (IL)-4 and IL-9, but not IL-2, IL-7, or IL-15, enhanced the proliferation and survival of bone marrow–derived mast cells (BMMCs) from wild-type mice. However, the effects of IL-4 and IL-9 were absent in BMMCs from γc− and Jak3−mice. In addition, IL-4Rα, γc, and Jak3, but not IL-2Rβ or IL-7Rα, were expressed in BMMCs. In contrast, IL-13 did not significantly induce the proliferation and survival of BMMCs even from wild-type mice, and IL-13Rα1 was not expressed in BMMCs. Furthermore, IL-4 phosphorylated the 65-kd isoform of Stat6 in BMMCs from wild-type mice but not from γc− and Jak3− mice. These results indicate that γc- and Jak3-dependent signaling is essential for IL-4– and IL-9–induced proliferation and survival of murine mast cells, that the effects of IL-4 are mediated by type I IL-4R and that type II IL-4R is absent on mast cells, and that IL-4 phosphorylates the 65-kd isoform of Stat6 in mast cells in a γc- and Jak3-dependent manner.

scholarly journals Characterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron

1990 ◽  
Vol 272 (3) ◽  
pp. 805-811 ◽  
Author(s):  
K Ramotar ◽  
B Boyd ◽  
G Tyrrell ◽  
J Gariepy ◽  
C Lingwood ◽  
...  
Keyword(s):  
Culture Medium ◽  
Polymyxin B ◽  
Exchange Chromatography ◽  
Structural Studies ◽  
Wild Type ◽  
Binding Studies ◽  
B Subunit ◽  
Tac Promoter ◽  
Glycolipid Receptor

The cistron encoding the B subunit of Escherichia coli Shiga-like toxin I (SLT-I) was cloned under control of the tac promoter in the expression vector pKK223-3 and the SLT-I B subunit was expressed constitutively in a wild-type background and inducibly in a lacIq background. The B subunit was located in the periplasmic space, and less than 10% was found in the culture medium after 24 h incubation. Polymyxin B extracts contained as much as 160 micrograms of B subunit/ml of culture. B subunit was purified to homogeneity by ion-exchange chromatography followed by chromatofocusing. Cross-linking analysis of purified native B subunit showed that it exists as a pentamer. In gels containing 0.1% SDS the native protein dissociated into monomers. B subunit was found to have the same glycolipid-receptor-specificity as SLT-I holotoxin. Competitive binding studies showed that B subunit and holotoxin had the same affinity for the globotriosylceramide receptor. We conclude that this recombinant plasmid is a convenient source of large amounts of purified SLT-I B subunit, which could be used for biophysical and structural studies or as a natural toxoid.

Sign in / Sign up

Export Citation Format

Share Document