Separation of the enantiomers of coumarinic anticoagulant drugs by capillary electrophoresis using maltodextrins as chiral modifiers

The separation and quantitation of coumarinic anticoagulant drug enantiomers were achieved by direct chiral capillary electrophoresis using complex maltooligosaccharide mixtures as stereoselective electrolyte modifiers. Chiral separations were characterized by a high selectivity and efficiency, enabling enantiomeric excess determinations. In addition, preliminary results indicate the applicability of the method for the determination of individual enantiomers in biological samples. So the method can be used to perform stereoselective pharmacokinetic studies. © 1994 Wiley-Liss, Inc.     

Fipronil recognition by the FA1 site of human serum albumin

Fipronil is a broad‐spectrum pesticide widely used in agriculture, horticulture, and forestry. Because fipronil can cause a variety of toxic effects in animals and humans, its use is authorized as a pesticide in veterinary medicinal products for pets, but not for the treatment of livestock animals whose products are intended for consumption. Recently, however, the presence of fipronil residues has been detected in the eggs and meat of layer hens from farms located in different European countries. Given the relevance of fipronil toxicity for human health, it is important to gain information concerning its fate in the human body, including its binding mode to human serum albumin (HSA), the most abundant protein in plasma. Here, the inhibition of heme‐Fe(III) binding to the fatty acid site 1 (FA1) of HSA by fipronil is reported. Docking simulations support functional data, indicating that the FA1 site is the preferential cleft for fipronil recognition by HSA. The affinity of fipronil for HSA (K_f = 1.9 × 10^?6 M, at pH 7.3, and 20.0°C) may be relevant in vivo. Indeed, HSA could play a pivotal role in fipronil transport and scavenging, thus reducing the pesticide‐free plasmatic levels, with consequent reduced systemic toxicity. In turn, fipronil binding to the FA1 site of HSA could impair the recognition of endogenous and exogenous molecules.     

单管PCR-Pyrosequencing快速检测华法林代谢酶基因多态性方法的建立

文章旨在建立一种单管、快速及高通量的华法林药物代谢酶相关基因多态性的检测方法。通过抽取人外周血DNA,应用带有生物素标记的扩增引物,经PCR扩增并制备焦磷酸测序单链模板,于PyroMark ID焦磷酸测序仪上进行焦磷酸测序,以Sanger测序法测序结果为对照,观察分析的准确性。结果显示,华法林药物代谢酶的3个相关基因多态性(CYP2C9*2、CYP2C9*3、VKORC1(-1693))于单管中可被同时检测,一次可获得96份DNA的华法林药物代谢相关多态性位点检测结果。经与Sanger测序方法比较,符合率为100%。结果表明本方法可准确、高通量、快速检测华法林药物代谢酶相关基因多态性,与单管检测一个位点的焦磷酸测序方法相比,能有效降低检测成本,节省检测时间。该方法在个性化医疗上有较大的推广应用价值,也可以将该平台运用于其他疾病相关基因多态性检测。     

Thematic Review Series: Fat-Soluble Vitamins: Vitamin K: Recent trends in the metabolism and cell biology of vitamin K with special reference to vitamin K cycling and MK-4 biosynthesis

In contrast to other fat-soluble vitamins, dietary vitamin K is rapidly lost to the body resulting in comparatively low tissue stores. Deficiency is kept at bay by the ubiquity of vitamin K in the diet, synthesis by gut microflora in some species, and relatively low vitamin K cofactor requirements for γ-glutamyl carboxylation. However, as shown by fatal neonatal bleeding in mice that lack vitamin K epoxide reductase (VKOR), the low requirements are dependent on the ability of animals to regenerate vitamin K from its epoxide metabolite via the vitamin K cycle. The identification of the genes encoding VKOR and its paralog VKOR-like 1 (VKORL1) has accelerated understanding of the enzymology of this salvage pathway. In parallel, a novel human enzyme that participates in the cellular conversion of phylloquinone to menaquinone (MK)-4 was identified as UbiA prenyltransferase-containing domain 1 (UBIAD1). Recent studies suggest that side-chain cleavage of oral phylloquinone occurs in the intestine, and that menadione is a circulating precursor of tissue MK-4. The mechanisms and functions of vitamin K recycling and MK-4 synthesis have dominated advances made in vitamin K biochemistry over the last five years and, after a brief overview of general metabolism, are the main focuses of this review.     

Five recombinant fragments of human serum albumin-tools for the characterization of the warfarin binding site

Human serum albumin (HSA) interacts with a vast array of chemically diverse ligands at specific binding sites. To pinpoint the essential structural elements for the formation of the warfarin binding site on human serum albumin, a defined set of five recombinant proteins comprising combinations of domains and/or subdomains of the N-terminal part were prepared and characterized by biochemical standard procedures, tryptophanyl fluorescence, and circular dichroic measurements, indicating well-preserved secondary and tertiary structures. Affinity constants for binding to warfarin were estimated by fluorescence titration experiments and found to be highest for HSA-DOM I-II and HSA, followed by HSA-DOM IB-II, HSA-DOM II, and HSA-DOM I-IIA. In addition, ultraviolet difference spectroscopy and induced circular dichroism experiments were carried out to get an in depth understanding of the binding mechanism of warfarin to the fragments as stand-alone proteins. This systematic study indicates that the primary warfarin binding site is centered in subdomain IIA with indispensable structural contributions of subdomain IIB and domain I, while domain III is not involved in this binding site, underlining the great potential that lies in the use of combinations of recombinant fragments for the study and accurate localization of ligand binding sites on HSA.     

Prenatal exposure to environmental factors and congenital limb defects

Limb congenital defects afflict approximately 0.6:1000 live births. In addition to genetic factors, prenatal exposure to drugs and environmental toxicants, represents a major contributing factor to limb defects. Examples of well‐recognized limb teratogenic agents include thalidomide, warfarin, valproic acid, misoprostol, and phenytoin. While the mechanism by which these agents cause dymorphogenesis is increasingly clear, prediction of the limb teratogenicity of many thousands of as yet uncharacterized environmental factors (pollutants) remains inexact. This is limited by the insufficiencies of currently available models. Specifically, in vivo approaches using guideline animal models have inherently deficient predictive power due to genomic and anatomic differences that complicate mechanistic comparisons. On the other hand, in vitro two‐dimensional (2D) cell cultures, while accessible for cellular and molecular experimentation, do not reflect the three‐dimensional (3D) morphogenetic events in vivo nor systemic influences. More robust and accessible models based on human cells that accurately replicate specific processes of embryonic limb development are needed to enhance limb teratogenesis prediction and to permit mechanistic analysis of the adverse outcome pathways. Recent advances in elucidating mechanisms of normal development will aid in the development of process‐specific 3D cell cultures within specialized bioreactors to support multicellular microtissues or organoid constructs that will lead to increased understanding of cell functions, cell‐to‐cell signaling, pathway networks, and mechanisms of toxicity. The promise is prompting researchers to look to such 3D microphysiological systems to help sort out complex and often subtle interactions relevant to developmental malformations that would not be evident by standard 2D cell culture testing. Birth Defects Research (Part C) 108:243–273, 2016. © 2016 Wiley Periodicals, Inc.     

Association between polymorphisms of VKORC1 and CYP2C9 genes with warfarin maintenance dose in a group of warfarin users in Birjand city,Iran

Warfarin is the cardinal anticoagulant drug prescribed around the world. Due to stochastic bleeding in patients, it is essential to adjust the dose for every individual. The aim of the present study was to evaluate the frequency of CYP2C9 and VKORC1 gene polymorphisms and their association with warfarin maintenance dose in a sample of cardiovascular patients in Birjand, South-Khorasan province of Iran. Patients with a history of cardiovascular disorders who take warfarin daily were selected. CYP2C9 and VKORC1 gene polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism in all participants. A total of 114 patients (mean age: 52.7 ± 14.9 years, M/F ratio: 0.76) participated in this study. Regarding CYP2C9 gene polymorphisms, the most frequent genotype was 1*/1* (80.4% in females and 62.5% in males). The frequency of 1*/2* and 2*/2* variants was 13% and 6.5% in females and 25% and 12.5% in males, respectively. The frequency of VKORC1 gene (1639 G > A), was 31.5%, 39.5%, and 29% for GG, GA, and AA in males, respectively. Besides, the mentioned genotype frequencies for females were 50%, 40.5%, and 9.5%, respectively. Moreover, there was a statistically significant correlation between VKORC1 gene −1639 G > A variant and warfarin maintenance dose (P < 0.001) but not for CYP2C9 variants. The results of the current study confirmed that the mutant variants of CYP2C9 are not frequent and do not have any impact on warfarin dose. In the case of VKORC1, the mutant allele (A) showed a positive correlation with warfarin dose adjustment.     

Analysis of sequence,structure of GAPDH of Leishmania donovani and its interactions

Drug resistance acquired by Leishmania donovani (Ldv) is a major problem in the treatment and control of visceral leishmaniasis (VL). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a major glycolytic enzyme has been targeted as is found in other protozoan which cause diseases like sleeping sickness. GAPDH gene of Ldv (AG83 strain) was amplified, sequenced, and modeled on the basis of crystal structure of Leishmania mexicana. The model of the Ldv GAPDH exhibited NAD-binding domain with Rossmann folding. Virtual screening of different experimentally proved compounds with the crystal and the modeled structures of GAPDH of Leishmania strains revealed diverse binding affinities of different compounds. Comparison of binding affinities (based on different programs) of compounds revealed that discovery studio v2.5 (Ligandfit) was able to predict the most hit compounds, the best hit compounds against GAPDH of Leishmania strains are hydrazine, vetrazine, and benzyl carbazate. It is predicted that patients suffering from both VL and cardiac disorders (atrial fibrillation) may benefit if they are treated with warfarin in conjunction with first-line antileishmanial therapies such as miltefosine and AmBisome.     

Structural and functional insights into human vitamin K epoxide reductase and vitamin K epoxide reductase-like1

Abstract

Human vitamin K epoxide reductase (hVKOR) is a small integral membrane protein involved in recycling vitamin K. hVKOR produces vitamin K hydroquinone, a crucial cofactor for γ-glutamyl carboxylation of vitamin K dependent proteins, which are necessary for blood coagulation. Because of this, hVKOR is the target of a common anticoagulant, warfarin. Spurred by the identification of the hVKOR gene less than a decade ago, there have been a number of new insights related to this protein. Nonetheless, there are a number of key issues that have not been resolved; such as where warfarin binds hVKOR, or if human VKOR shares the topology of the structurally characterized but distantly related prokaryotic VKOR. The pharmacogenetics and single nucleotide polymorphisms of hVKOR used in personalized medicine strategies for warfarin dosing should be carefully considered to inform the debate. The biochemical and cell biological evidence suggests that hVKOR has a distinct fold from its ancestral protein, though the controversy will likely remain until structural studies of hVKOR are accomplished. Resolving these issues should impact development of new anticoagulants. The paralogous human protein, VKOR-like1 (VKORL1) was recently shown to also participate in vitamin K recycling. VKORL1 was also recently characterized and assigned a functional role as a housekeeping protein involved in redox homeostasis and oxidative stress with a potential role in cancer regulation. As the physiological interplay between these two human paralogs emerge, the impacts could be significant in a number of diverse fields from coagulation to cancer.