Dexamethasone-Induced Expression of Endothelial Mitogen-Activated Protein Kinase Phosphatase-1 Involves Activation of the Transcription Factors Activator Protein-1 and 3',5'-Cyclic Adenosine 5'-Monophosphate Response Element-Binding Protein and the Generation of Reactive Oxygen Species

We have recently identified the MAPK phosphatase (MKP)-1 as a novel mediator of the antiinflammatory properties of glucocorticoids (dexamethasone) in the human endothelium. However, nothing is as yet known about the signaling pathways responsible for the up-regulation of MKP-1 by dexamethasone in endothelial cells. Knowledge of the molecular basis of this new alternative way of glucocorticoid action could facilitate the identification of new antiinflammatory drug targets. Thus, the aim of our study was to elucidate the underlying molecular mechanisms. Using Western blot analysis, we found that dexamethasone rapidly activates ERK, c-jun Nterminal kinase (JNK), and p38 MAPK in human umbilical vein endothelial cells. By applying the kinase inhibitors PD98059 (MAPK kinase-1) and SP600125 (JNK), ERK and JNK were shown to be crucial for the induction of MKP-1. Using EMSA and a decoy oligonucleotide approach, the transcription factors activator protein-1 (activated by ERK and JNK) and cAMP response element-binding protein (activated by ERK) were found to be involved in the up-regulation of MKP-1 by dexamethasone. Interestingly, dexamethasone induces the generation of reactive oxygen species (measured by dihydrofluorescein assay), which participate in the signaling process by triggering JNK activation. Our work elucidates a novel alternative mechanism for transducing antiinflammatory effects of glucocorticoids in the human endothelium. Thus, our study adds valuable information to the efforts made to find new antiinflammatory principles utilized by glucocorticoids. This might help to gain new therapeutic options to limit glucocorticoid side effects and to overcome resistance

A single F153Sβ3 mutation causes constitutive integrin αIIbβ3 activation in a variant form of Glanzmann thrombasthenia

This report identifies a novel variant form of the inherited bleeding disorder Glanzmann thrombasthenia, exhibiting only mild bleeding in a physically active individual. The platelets cannot aggregate ex vivo with physiologic agonists of activation, although microfluidic analysis with whole blood displays moderate ex vivo platelet adhesion and aggregation consistent with mild bleeding. Immunocytometry shows reduced expression of αIIbβ3 on quiescent platelets that spontaneously bind/store fibrinogen, and activation-dependent antibodies (ligand-induced binding site-319.4 and PAC-1) report β3 extension suggesting an intrinsic activation phenotype. Genetic analysis reveals a single F153Sβ3 substitution within the βI-domain from a heterozygous T556C nucleotide substitution of ITGB3 exon 4 in conjunction with a previously reported IVS5(+1)G\u3eA splice site mutation with undetectable platelet messenger RNA accounting for hemizygous expression of S153β3. F153 is completely conserved among β3 of several species and all human β-integrin subunits suggesting that it may play a vital role in integrin structure/function. Mutagenesis of αIIb-F153Sβ3 also displays reduced levels of a constitutively activated αIIb-S153β3 on HEK293T cells. The overall structural analysis suggests that a bulky aromatic, nonpolar amino acid (F,W)153β3 is critical for maintaining the resting conformation of α2- and α1-helices of the βI-domain because small amino acid substitutions (S,A) facilitate an unhindered inward movement of the α2- and α1-helices of the βI-domain toward the constitutively active αIIbβ3 conformation, while a bulky aromatic, polar amino acid (Y) hinders such movements and restrains αIIbβ3 activation. The data collectively demonstrate that disruption of F153β3 can significantly alter normal integrin/platelet function, although reduced expression of αIIb-S153β3 may be compensated by a hyperactive conformation that promotes viable hemostasis

Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A

It is essential to improve therapies for controlling excessive bleeding in patients with haemorrhagic disorders. As activated blood platelets mediate the primary response to vascular injury, we hypothesize that storage of coagulation Factor VIII within platelets may provide a locally inducible treatment to maintain haemostasis for haemophilia A. Here we show that haematopoietic stem cell gene therapy can prevent the occurrence of severe bleeding episodes in dogs with haemophilia A for at least 2.5 years after transplantation. We employ a clinically relevant strategy based on a lentiviral vector encoding the ITGA2B gene promoter, which drives platelet-specific expression of human FVIII permitting storage and release of FVIII from activated platelets. One animal receives a hybrid molecule of FVIII fused to the von Willebrand Factor propeptide-D2 domain that traffics FVIII more effectively into α-granules. The absence of inhibitory antibodies to platelet-derived FVIII indicates that this approach may have benefit in patients who reject FVIII replacement therapies. Thus, platelet FVIII may provide effective long-term control of bleeding in patients with haemophilia A. Haemophilia is a genetic bleeding disorder associated with a deficiency in the coagulation factor VIII. Here, the authors use gene therapy to achieve stable overexpression of factor VIII in platelets of dogs with haemophilia A, preventing the occurrence of severe bleeding episodes for over 2.5 years

Pharmacogenetics of warfarin in a paediatric population: time in therapeutic range, initial and stable dosing and adverse effects

Warfarin is used in paediatric populations, but dosing algorithms incorporating pharmacogenetic data have not been developed for children. Previous studies have produced estimates of the effect of polymorphisms in Cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) on stable warfarin dosing, but data on time in therapeutic range, initial dosing and adverse effects are limited. Participants (n=97) were recruited, and routine clinical data and salivary DNA samples were collected from all participants and analysed for CYP2C9*2, *3 and VKORC1-1639 polymorphisms.VKORC1 -1639 was associated with a greater proportion of the first 6 months’ treatment time spent within the target International Normalised Ratio (INR) range, accounting for an additional 9.5% of the variance in the proportion of time. CYP2C9*2 was associated with a greater likelihood of INR values exceeding the target range during the initiation of treatment (odds ratio (OR; per additional copy) 4.18, 95% confidence interval (CI) 1.42, 12.34). CYP2C9*2 and VKORC1-1639 were associated with a lower dose requirement, and accounted for almost 12% of the variance in stable dose. VKORC1-1639 was associated with an increased likelihood of mild bleeding complications (OR (heterozygotes vs homozygotes) 4.53, 95% CI 1.59, 12.93). These data show novel associations between VKORC1-1639 and CYP2C9*2 and INR values in children taking warfarin, as well as replicating previous findings with regard to stable dose requirements. The development of pharmacogenomic dosing algorithms for children using warfarin has the potential to improve clinical care in this population

Kinetic constraints and features imposed by the immobilization of enzymes onto solid matrices: A key to advanced biotransformation

1045-1051The kinetics of immobilized enzymes can not be analyzed by means of the simple Michaelis-Menten concept, which generally fails to describe the immobilized state due to both its probable barriers, and because the active concentration of the enzyme approaches, or even exceeds this of its substrate(s). In such cases, the various experimental data are usually treated by complex rate equations comprising too many parameters acquiring different natures and meanings, depending on both the properties of the immobilization state and the experimental conditions; thus, more likely, only apparent values of the Michaelis-Menten kinetic parameters can be estimated experimentally. Likewise, immobilization is often a key method in optimizing the operational performance of enzymes, in both laboratory and industrial scale, and affects considerably the kinetics in non-aqueous and non-conventional media due to several issues as the structural changes of the enzyme molecule, the heterogeneity of the system, and the partial or total absence of water. In this work a theoretical approach is described on the formulation of simplified rate equations, reflecting also the actual mass balances of the reactants, in the case where esterification synthetic reactions are catalyzed by immobilized lipases, in either a non-aqueous organic solvent or in a non-solvent system

Flavin-containing monooxygenase 3 gene polymorphisms in Turkish population

Flavin-containing monooxygenases (FMOs) represent the second most important human monooxygenase system, after cytochrome P450s (CYPs) and catalyze the oxygenation of many chemicals containing nitrogen-, sulphur-, phosphorous-, selenium-and other nucleophilic heteroatoms. FMO3 is the prominent FMO form in adult human liver. For FMO3, both interindividual variability within a single ethnic group and variability between ethnic groups have been reported. In our study, three prevalent functional FMO3 variants (E158K, V257M, and E308G) were genotyped in healthy Turkish people by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) methods. The frequencies of alleles and haplotypes were compared with those obtained from different populations. It was found that FMO3 158K, 257M and 308G alleles, demonstrate impaired metabolism toward many FMO3 substrates, were observed frequently in Turkish population similar to the other populations. Also, the frequencies of haplotypes were determined based on individual allelic frequencies and it was observed that the most common haplotypes were haplotip EVE and KVE (E158K/V257M/E308G), which together accounted for 80% of all haplotypes. The obtained data from the present study could be useful for further studies assessing sensitivity to therapeutic drugs, environmental toxicants and common disease

Correction to: Molecular, biochemical and kinetic analysis of a novel, thermostable lipase (LipSm) from Stenotrophomonas maltophilia Psi‑1, the first member of a new bacterial lipase family (XIX)

We have recently (8th February 2018) published our article entitled “Molecular, biochemical and kinetic analysis of a novel, thermostable lipase (LipSm) from Stenotrophomonas maltophilia Psi-1, the first member of a new bacterial lipase family (XVIII)” [1]. While our manuscript was going through the final stages of publication, an article by Samoylova et al. [2] was published (12th January 2018) in the journal Extremophiles, entitled “Cloning, expression and characterization of the esterase estUT1 from Ureibacillus thermosphaericus which belongs to a new lipase family XVIII”. Since we could not have known of the work of Samoylova et al. [2] when we submitted our manuscript, and in order to avoid confusion in the scientific community, we propose to reclassify LipSm as the first characterized member of the new bacterial lipase family XIX. Therefore throughout our article [1] “lipase family XVIII” should read “lipase family XIX” (title included)