Biochemical analysis of mutations in P450 oxidoreductase

2006 ◽  
Vol 34 (6) ◽  
pp. 1186-1191 ◽  
Author(s):  
A.V. Pandey
Keyword(s):  
Drug Metabolism ◽  
Biochemical Analysis ◽  
Three Dimensional ◽  
Polymorphic Form ◽  
Extensive Study ◽  
Expression Vectors ◽  
Missense Mutations ◽  
Three Dimensional Model ◽  
Biochemical Basis ◽  
Initial Report

All microsomal P450s require POR (cytochrome P450 reductase) for catalytic activity. Most of the clinically used drugs are metabolized by a small number of P450s and polymorphisms in the cytochrome P450s are known to cause changes in drug metabolism. We have recently found a number of POR missense mutations in the patients with disordered steroidogenesis. Our initial report described five missense mutations (A284P, R454H, V489E, C566Y and V605F) identified in four patients. We built bacterial expression vectors for each POR variant, purified the membranes expressing normal or variant POR and characterized their activities with cytochrome c and P450c17 assays. We have recently completed an extensive study of the range of POR mutations and characterized the mutants/polymorphisms A112V, T139A, M260V, Y456H, A500V, G536R, L562P, R613X, V628I and F643del from sequencing of patient DNA. We also studied POR variants Y179D, P225L, R313W, G410S and G501R that were available in databases or the published literature. We analysed the mutations with a three-dimensional model of human POR that was based on an essentially similar rat POR with known crystal structure. The missense mutations found in patients with disordered steroidogenesis mapped to functionally important domains of POR and the apparent polymorphisms mapped to less crucial regions. Since a variation in POR can alter the activity of all microsomal P450s, it can also affect the drug metabolism even with a normal P450. Understanding the genetic and biochemical basis of POR-mediated drug metabolism will provide valuable information about possible differences in P450-mediated reactions among the individuals carrying a variant or polymorphic form of POR.

scholarly journals Four novel mutations in deficiency of coagulation factor XIII: consequences to expression and structure of the A-subunit

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 141-151 ◽  
Author(s):  
H Mikkola ◽  
VC Yee ◽  
M Syrjala ◽  
R Seitz ◽  
R Egbring ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Nonsense Mutation ◽  
Factor Xiii ◽  
Three Dimensional ◽  
Factor Xiiia ◽  
Missense Mutations ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Core Domain ◽  
A Subunit

Abstract The characterization of naturally occurring mutations is one way to approach functionally significant domains of polypeptides. About 10 mutations have been reported in factor XIII (FXIII) A-subunit deficiency, but very little is known about the effects of the mutations on the expression or the structure of this enzyme. In this study, the recent crystallization of FXIII A-subunit and determination of the three-dimensional model were used for the first time to pursue the structural consequences of mutations in the A-subunit. The molecular analysis of four families from Sweden, Germany, and Denmark revealed four previously unreported point mutations. Three of the mutations were missense mutations, Arg326-->Gln, Arg252-->Ile, and Leu498-->Pro, and one was a nonsense mutation, a deletion of thymidine in codon for Phe8 resulting in early frameshift and premature termination of the polypeptide chain. In the case of the nonsense mutation, delT Phe8, the steady-state mRNA level of FXIII A-subunit was reduced, as quantitated by reverse transcriptase-polymerase chain reaction and solid-phase minisequencing. In contrast, none of the missense mutations affected mRNA levels, indicating the possible translation of the mutant polypeptides. However, by enzyme-linked immunosorbent analysis and immunofluorescence, all the patients demonstrated a complete lack of detectable factor XIIIA antigen in their platelets. In the structural analysis, we included the mutations described in this work and the Met242-->Thr mutation reported earlier by us. Interestingly, in the three-dimensional model, all four missense mutations are localized in the evolutionarily conserved catalytic core domain. The substitutions are at least 15 A away from the catalytic cleft and do not affect any of the residues known to be directly involved in the enzymatic reaction. The structural analyses suggest that the mutations are most likely interfering with proper folding and stability of the protein, which is in agreement with the observed absence of detectable FXIIIA antigen. Arg326, Arg252, and Met242 are all buried within the molecule. The Arg326-->Gln and Arg252-->Ile mutations are substitutions of smaller, neutral amino acids for large, charged residues. They disrupt the electrostatic balance and hydrogen-bonding interactions in structurally significant areas. The Met242-->Thr mutation is located in the same region of the core domain as the Arg252-->Ile site and is expected to have a destabilizing effect due to an introduction of a smaller, polar residue in a tightly packed hydrophobic pocket. The substitution of proline for Leu498 is predicted to cause unfavorable interatomic contacts and a disruption of the alpha-helix mainchain hydrogen-bonding pattern; it is likely to form a kink in the helix next to the dimer interface and is expected to impair proper dimerization of the A-subunits. In the case of all four missense mutations studied, the knowledge achieved from the three-dimensional model of crystallized FXIII A-subunit provides essential information about the structural significance of the specific residues and aids in understanding the biologic consequences of the mutations observed at the cellular level.

scholarly journals Four novel mutations in deficiency of coagulation factor XIII: consequences to expression and structure of the A-subunit

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 141-151 ◽  
Author(s):  
H Mikkola ◽  
VC Yee ◽  
M Syrjala ◽  
R Seitz ◽  
R Egbring ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Nonsense Mutation ◽  
Factor Xiii ◽  
Three Dimensional ◽  
Factor Xiiia ◽  
Missense Mutations ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Core Domain ◽  
A Subunit

The characterization of naturally occurring mutations is one way to approach functionally significant domains of polypeptides. About 10 mutations have been reported in factor XIII (FXIII) A-subunit deficiency, but very little is known about the effects of the mutations on the expression or the structure of this enzyme. In this study, the recent crystallization of FXIII A-subunit and determination of the three-dimensional model were used for the first time to pursue the structural consequences of mutations in the A-subunit. The molecular analysis of four families from Sweden, Germany, and Denmark revealed four previously unreported point mutations. Three of the mutations were missense mutations, Arg326-->Gln, Arg252-->Ile, and Leu498-->Pro, and one was a nonsense mutation, a deletion of thymidine in codon for Phe8 resulting in early frameshift and premature termination of the polypeptide chain. In the case of the nonsense mutation, delT Phe8, the steady-state mRNA level of FXIII A-subunit was reduced, as quantitated by reverse transcriptase-polymerase chain reaction and solid-phase minisequencing. In contrast, none of the missense mutations affected mRNA levels, indicating the possible translation of the mutant polypeptides. However, by enzyme-linked immunosorbent analysis and immunofluorescence, all the patients demonstrated a complete lack of detectable factor XIIIA antigen in their platelets. In the structural analysis, we included the mutations described in this work and the Met242-->Thr mutation reported earlier by us. Interestingly, in the three-dimensional model, all four missense mutations are localized in the evolutionarily conserved catalytic core domain. The substitutions are at least 15 A away from the catalytic cleft and do not affect any of the residues known to be directly involved in the enzymatic reaction. The structural analyses suggest that the mutations are most likely interfering with proper folding and stability of the protein, which is in agreement with the observed absence of detectable FXIIIA antigen. Arg326, Arg252, and Met242 are all buried within the molecule. The Arg326-->Gln and Arg252-->Ile mutations are substitutions of smaller, neutral amino acids for large, charged residues. They disrupt the electrostatic balance and hydrogen-bonding interactions in structurally significant areas. The Met242-->Thr mutation is located in the same region of the core domain as the Arg252-->Ile site and is expected to have a destabilizing effect due to an introduction of a smaller, polar residue in a tightly packed hydrophobic pocket. The substitution of proline for Leu498 is predicted to cause unfavorable interatomic contacts and a disruption of the alpha-helix mainchain hydrogen-bonding pattern; it is likely to form a kink in the helix next to the dimer interface and is expected to impair proper dimerization of the A-subunits. In the case of all four missense mutations studied, the knowledge achieved from the three-dimensional model of crystallized FXIII A-subunit provides essential information about the structural significance of the specific residues and aids in understanding the biologic consequences of the mutations observed at the cellular level.

Methods for making stereoslides and -prints, with freeze-etched olfactory epithelium as example

1984 ◽  
Vol 42 ◽  
pp. 704-705
Author(s):  
Bert Ph. M. Menco ◽  
Ido F. Menco ◽  
Frans L.T. Verdonk
Keyword(s):  
Electron Microscope ◽  
Olfactory Epithelium ◽  
Three Dimensional ◽  
Supporting Cell ◽  
Structural Features ◽  
Extensive Study ◽  
Sprague Dawley ◽  
Freezing Method ◽  
Respiratory Epithelia ◽  
Three Dimensional Presentation

Previously we presented an extensive study of the distributions of intramembranous particles of structures in apical surfaces of nasal olfactory and respiratory epithelia of the Sprague-Dawley rat. For the same structures these distributions were compared in samples which were i) chemically fixed and cryo-protected with glycerol before cryo-fixation, after excision, and ii)ultra-rapidly frozen by means of the slam-freezing method. Since a three-dimensional presentation markedly improves visualization of structural features micrographs were presented as stereopairs. Two exposures were made by tiling the sample stage of the electron microscope 6° in either direction with an eucentric goniometer. The negatives (Agfa Pan 25 Professional) were reversed with Kodak Technical Pan Film 2415 developed in D76 1:1. The prints were made from these reversed negatives. As an example tight-junctional features of an olfactory supporting cell in a region where this cell conjoined with two other cells are presented (Fig. 1).

Three-Dimensional Model for Virtual Surgical Simulation, during Complex Skull Base Surgery and Aneurysm Surgery

Skull Base ◽  
2008 ◽  
Vol 18 (S 01) ◽  
Author(s):  
Akio Morita ◽  
Toshikazu Kimura ◽  
Shigeo Sora ◽  
Kengo Nishimura ◽  
Hisayuki Sugiyama ◽  
...  
Keyword(s):  
Skull Base ◽  
Surgical Simulation ◽  
Skull Base Surgery ◽  
Three Dimensional ◽  
Aneurysm Surgery ◽  
Dimensional Model ◽  
Three Dimensional Model

Digitalization system of ancient architecture decoration art based on neural network and image features

2020 ◽  
pp. 1-12
Author(s):  
Wu Xin ◽  
Qiu Daping
Keyword(s):  
Neural Network ◽  
Construction Industry ◽  
Three Dimensional ◽  
Performance Testing ◽  
Image Features ◽  
Three Dimensional Model ◽  
Performance Effect ◽  
Data Process ◽  
And Performance ◽  
Construction Mode

The inheritance and innovation of ancient architecture decoration art is an important way for the development of the construction industry. The data process of traditional ancient architecture decoration art is relatively backward, which leads to the obvious distortion of the digitalization of ancient architecture decoration art. In order to improve the digital effect of ancient architecture decoration art, based on neural network, this paper combines the image features to construct a neural network-based ancient architecture decoration art data system model, and graphically expresses the static construction mode and dynamic construction process of the architecture group. Based on this, three-dimensional model reconstruction and scene simulation experiments of architecture groups are realized. In order to verify the performance effect of the system proposed in this paper, it is verified through simulation and performance testing, and data visualization is performed through statistical methods. The result of the study shows that the digitalization effect of the ancient architecture decoration art proposed in this paper is good.

Phonetic iconicity in the evaluative morphology of a sample of Indo-European, Niger-Congo and Austronesian languages

2010 ◽  
Vol 3 (2) ◽  
pp. 156-180 ◽  
Author(s):  
Renáta Gregová ◽  
Lívia Körtvélyessy ◽  
Július Zimmermann
Keyword(s):  
Three Dimensional ◽  
Sound Symbolism ◽  
Three Dimensional Model ◽  
Austronesian Languages ◽  
Place Of Articulation ◽  
European Languages ◽  
Phonetic Symbolism ◽  
The Individual ◽  
Core Vocabulary

Universals Archive (Universal #1926) indicates a universal tendency for sound symbolism in reference to the expression of diminutives and augmentatives. The research ( Štekauer et al. 2009 ) carried out on European languages has not proved the tendency at all. Therefore, our research was extended to cover three language families – Indo-European, Niger-Congo and Austronesian. A three-step analysis examining different aspects of phonetic symbolism was carried out on a core vocabulary of 35 lexical items. A research sample was selected out of 60 languages. The evaluative markers were analyzed according to both phonetic classification of vowels and consonants and Ultan's and Niewenhuis' conclusions on the dominance of palatal and post-alveolar consonants in diminutive markers. Finally, the data obtained in our sample languages was evaluated by means of a three-dimensional model illustrating the place of articulation of the individual segments.

Sign in / Sign up

Export Citation Format

Share Document