Jan Víteček

Arginine-Based Inhibitors of Nitric Oxide Synthase: Therapeutic Potential and Challenges

In the past three decades, nitric oxide has been well established as an important bioactive molecule implicated in regulation of cardiovascular, nervous, and immune systems. Therefore, it is not surprising that much effort has been made to find specific inhibitors of nitric oxide synthases (NOS), the enzymes responsible for production of nitric oxide. Among the many NOS inhibitors developed to date, inhibitors based on derivatives and analogues of arginine are of special interest, as this category includes a relatively high number of compounds with good potential for experimental as well as clinical application. Though this group of inhibitors covers early nonspecific compounds, modern drug design strategies such as biochemical screening and computer-aided drug design have provided NOS-isoform-specific inhibitors. With an emphasis on major advances in this field, a comprehensive list of inhibitors based on their structural characteristics is discussed in this paper. We provide a summary of their biochemical properties as well as their observed effects both in vitro and in vivo. Furthermore, we focus in particular on their pharmacology and use in recent clinical studies. The potential of newly designed specific NOS inhibitors developed by means of modern drug development strategies is highlighted.

Glutathione deficiency of the Arabidopsis mutant pad2-1 affects oxidative stress-related events, defense gene expression, and the hypersensitive response.

The Arabidopsis (Arabidopsis thaliana) phytoalexin-deficient mutant pad2-1 displays enhanced susceptibility to a broad range of pathogens and herbivorous insects that correlates with deficiencies in the production of camalexin, indole glucosinolates, and salicylic acid (SA). The pad2-1 mutation is localized in the GLUTAMATE-CYSTEINE LIGASE (GCL) gene encoding the first enzyme of glutathione biosynthesis. While pad2-1 glutathione deficiency is not caused by a decrease in GCL transcripts, analysis of GCL protein level revealed that pad2-1 plants contained only 48% of the wild-type protein amount. In contrast to the wild type, the oxidized form of GCL was dominant in pad2-1, suggesting a distinct redox environment. This finding was corroborated by the expression of GRX1-roGFP2, showing that the cytosolic glutathione redox potential was significantly less negative in pad2-1. Analysis of oxidative stress-related gene expression showed a higher transcript accumulation in pad2-1 of GLUTATHIONE REDUCTASE, GLUTATHIONE-S-TRANSFERASE, and RESPIRATORY BURST OXIDASE HOMOLOG D in response to the oomycete Phytophthora brassicae. Interestingly, oligogalacturonide elicitation in pad2-1 revealed a lower plasma membrane depolarization that was found to act upstream of an impaired hydrogen peroxide production. This impaired hydrogen peroxide production was also observed during pathogen infection and correlated with a reduced hypersensitive response in pad2-1. In addition, a lack of pathogen-triggered expression of the ISOCHORISMATE SYNTHASE1 gene, coding for the SA-biosynthetic enzyme isochorismate synthase, was identified as the cause of the SA deficiency in pad2-1. Together, our results indicate that the pad2-1 mutation is related to a decrease in GCL protein and that the resulting glutathione deficiency negatively affects important processes of disease resistance.

The potentiation of myeloperoxidase activity by the glycosaminoglycan-dependent binding of myeloperoxidase to proteins of the extracellular matrix.

BACKGROUND Myeloperoxidase (MPO) is an abundant hemoprotein expressed by neutrophil granulocytes that is recognized to play an important role in the development of vascular diseases. Upon degranulation from circulating neutrophil granulocytes, MPO binds to the surface of endothelial cells in an electrostatic-dependent manner and undergoes transcytotic migration to the underlying extracellular matrix (ECM). However, the mechanisms governing the binding of MPO to subendothelial ECM proteins, and whether this binding modulates its enzymatic functions are not well understood. METHODS We investigated MPO binding to ECM derived from aortic endothelial cells, aortic smooth muscle cells, and fibroblasts, and to purified ECM proteins, and the modulation of these associations by glycosaminoglycans. The oxidizing and chlorinating potential of MPO upon binding to ECM proteins was tested. RESULTS MPO binds to the ECM proteins collagen IV and fibronectin, and this association is enhanced by the pre-incubation of these proteins with glycosaminoglycans. Correspondingly, an excess of glycosaminoglycans in solution during incubation inhibits the binding of MPO to collagen IV and fibronectin. These observations were confirmed with cell-derived ECM. The oxidizing and chlorinating potential of MPO was preserved upon binding to collagen IV and fibronectin; even the potentiation of MPO activity in the presence of collagen IV and fibronectin was observed. CONCLUSIONS Collectively, the data reveal that MPO binds to ECM proteins on the basis of electrostatic interactions, and MPO chlorinating and oxidizing activity is potentiated upon association with these proteins. GENERAL SIGNIFICANCE Our findings provide new insights into the molecular mechanisms underlying the interaction of MPO with ECM proteins.

Simple non-invasive analysis of embryonic stem cell-derived cardiomyocytes beating in vitro.

The analysis of digital video output enables the non-invasive screening of various active biological processes. For the monitoring and computing of the beating parameters of cardiomyocytes in vitro, CB Analyser (cardiomyocyte beating analyser) software was developed. This software is based on image analysis of the video recording of beating cardiomyocytes. CB Analyser was tested using cardiomyocytes derived from mouse embryonic stem cells at different stages of cardiomyogenesis. We observed that during differentiation (from day 18), the beat peak width decreased, which corresponded to the increased speed of an individual pulse. However, the beating frequency did not change. Further, the effects of epinephrine modulating mature cardiomyocyte functions were tested to validate the CB Analyser analysis. In conclusion, data show that CB Analyser is a useful tool for evaluating the functions of both developing and mature cardiomyocytes under various conditions in vitro.

A simple microviscometric approach based on Brownian motion tracking

Viscosity-an integral property of a liquid-is traditionally determined by mechanical instruments. The most pronounced disadvantage of such an approach is the requirement of a large sample volume, which poses a serious obstacle, particularly in biology and biophysics when working with limited samples. Scaling down the required volume by means of microviscometry based on tracking the Brownian motion of particles can provide a reasonable alternative. In this paper, we report a simple microviscometric approach which can be conducted with common laboratory equipment. The core of this approach consists in a freely available standalone script to process particle trajectory data based on a Newtonian model. In our study, this setup allowed the sample to be scaled down to 10 μl. The utility of the approach was demonstrated using model solutions of glycerine, hyaluronate, and mouse blood plasma. Therefore, this microviscometric approach based on a newly developed freely available script can be suggested for determination of the viscosity of small biological samples (e.g., body fluids).

Printing inks of electroactive polymer PEDOT:PSS: The study of biocompatibility, stability, and electrical properties: Electroactive Polymer PEDOT:PSS

Biocompatibility tests and a study of the electrical properties of thin films prepared from six electroactive polymer ink formulations based on poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) were performed. The aim was to find a suitable formulation of PEDOT:PSS and conditions for preparing thin films in order to construct printed bioelectronic devices for biomedical applications. The stability and electrical properties of such films were tested on organic electrochemical transistor (OECT)-based sensor platforms and their biocompatibility was evaluated in assays with 3T3 fibroblasts and murine cardiomyocytes. It was found that the thin films prepared from inks without an additive or any thin film post-treatment provide limited conductivity and stability for use in biomedical applications. These properties were greatly improved by using ethylene glycol and thermal annealing. Addition or post-treatment by ethylene glycol in combination with thermal annealing provided thin films with electrical resistance and a stability sufficient to be used in sensing of animal cell physiology. These films coated with collagen IV showed good biocompatibility in the assay with 3T3 fibroblasts when compared to standard cell culture plastics. Selected films were then used in assays with murine cardiomyocytes. We observed that these cells were able to attach to the PEDOT:PSS films and form an active sensor element. Spontaneously beating clusters were formed, indicating a good physiological status for the cardiomyocyte cells. These results open the door to construction of cheap printed electronic devices for biointerfacing in biomedical applications.

MPO (Myeloperoxidase) Reduces Endothelial Glycocalyx Thickness Dependent on Its Cationic Charge

Objective— The leukocyte heme-enzyme MPO (myeloperoxidase) exerts proinflammatory effects on the vascular system primarily linked to its catalytic properties. Recent studies have shown that MPO, depending on its cationic charge, mediates neutrophil recruitment and activation. Here, we further investigated MPO’s extracatalytic properties and its effect on endothelial glycocalyx (EG) integrity. Approach and Results— In vivo staining of murine cremaster muscle vessels with Alcian Blue 8GX provided evidence of an MPO-dependent decrease in anionic charge of the EG. MPO binding to the glycocalyx was further characterized using Chinese hamster ovary cells and its glycosaminoglycan mutants—pgsA-745 (mutant Chinese hamster ovary cells lacking heparan sulfate and chondroitin sulfate glycosaminoglycan) and pgsD-677 (mutant Chinese hamster ovary cells lacking heparan sulfate glycosaminoglycan), which revealed heparan sulfate as the main mediator of MPO binding. Further, EG integrity was assessed in terms of thickness using intravital microscopy of murine cremaster muscle. A significant reduction in EG thickness was observed on infusion of catalytically active MPO, as well as mutant inactive MPO and cationic polymer polylysine. Similar effects were also observed in wild-type mice after a local inflammatory stimulus but not in MPO-knockout mice. The reduction in EG thickness was reversed after removal of vessel-bound MPO, suggesting a possible physical collapse of the EG. Last, experiments with in vivo neutrophil depletion revealed that MPO also induced neutrophil-mediated shedding of the EG core protein, Sdc1 (syndecan-1). Conclusions— These findings provide evidence that MPO, via ionic interaction with heparan sulfate side chains, can cause neutrophil-dependent Sdc1 shedding and collapse of the EG structure.