Biologia Serbica

IX Conference of Serbian Biochemical Society "Diversity in Biochemistry" Belgrade, 14-16 November 2019

Marija Gavrović-Jankulović — Thu, 27 Feb 2020 00:00:00 +0100

Detoxification of tabun-exposed mice by an acetylcholinesterase mutant using a novel pyridinium aldoxime

Zrinka Kovarik, Nikolina Maček Hrvat, Suzana Žunec, Maja Katalinić — Thu, 27 Feb 2020 00:00:00 +0100

Nerve agents, such as tabun, are covalent inhibitors of acetylcholinesterase (AChE), an essential enzyme in neurotransmission whose inhibition may lead to death. Currently used therapy, consisting of an anticholinergic drug and an oxime as the reactivator of inhibited AChE is particularly ineffective in cases of tabun exposure. Thus, an optimal re-activator is still needed. Click-chemistry, utilizing Cu (I)-catalyzed azide-alkyne cycloaddition of a library of small molecule building blocks, has made possible the rapid synthesis of a variety of new oximes. Among the new oximes tested in recent studies as re-activators of tabun-inhibited choline binding site AChE mutants, oxime 5B, a lengthened alkylchain congener of the standard oxime 2-PAM, as appeared to be a candidate for tabun ex vivo scavenging when paired with the Y337A mutant of AChE. Herein, we pursued the antidotal in vivo detoxification of tabun-exposed mice by assembling oxime-assisted catalytic scavenging using the mutant combined with oxime 5B. Although the antidotal treatment requires optimization, our findings offer a platform for further development of more potent means of counteracting tabun and related phosphoramidate exposure.

The role of proprotein convertase subtilisin/kexin type 9 in atherosclerosis

Jelena Vekić, Dragana Bojanin, Vesna Spasojević-Kalimanovska — Thu, 27 Feb 2020 00:00:00 +0100

Dyslipidemia is one of the predominant causes of atherosclerosis and cardiovascular disease (CVD) development. Accordingly, lifestyles approaches and therapeutic targeting of low-density lipoprotein (LDL)-cholesterol remain the main strategies for CVD prevention and treatment. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory serin-protease with important roles in lipoprotein metabolism. In particular, PCSK9 promotes degradation of hepatic LDL-receptors, leading to reduced clearance of LDL particles and increased plasma LDL-cholesterol levels. To date, a large body of evidence from experimental, genetic and clinical studies indicates that PCSK9 is implicated in the development of atherosclerosis. As this research is moving forward, additional roles of PCSK9 beyond cholesterol metabolism and atherosclerosis are being discovered. In the present paper, we will discuss our current knowledge of the role of PCSK9 in atherosclerosis and its associations with cardiometabolic risk factors, and provide a brief overview of the recent achievements in pharmacological inhibition of PCSK9-mediated LDL-receptor degradation toward LDL-cholesterol reduction and prevention of CVD development.

How FEBS supports Young Scientists´ Careers

Irene Díaz-Moreno, Vlastimil Kulda, Anna Jagusiak — Thu, 27 Feb 2020 00:00:00 +0100

The Federation of European Biochemical Societies (FEBS) brings together molecular life scientists in Europe and neighbouring regions. There are many activities of FEBS targeted to young scientists with the aim of promoting their careers. This article provides a brief overview of such FEBS initiatives.

Proteins that sense cellular environments – examples and implications

Snezana Djordjevic — Thu, 27 Feb 2020 00:00:00 +0100

The first step in the process of signal perception and transduction involves interaction between a stimulus and the specific protein that has the capacity to recognise the stimulus and to translate the interaction to the physical manifestation of a signal. The physical manifestation of the signal involves what is commonly referred to as a ‘protein conformational change’ that results in a change in a conformational equilibrium of the proteins that perceive the stimuli. Reflecting on our work, here I describe two specific examples of stimuli perception and signal transduction mechanisms, one relating to the protein AioX that is found in a prokaryotic organism adapted to living under conditions of arsenic contamination, and the second example involving neuropilins - transmembrane proteins of significance for human health.

A journey along the TIM23 complex, the major protein translocase of the mitochondrial inner membrane

Umut Günsel, Dejana Mokranjac — Thu, 27 Feb 2020 00:00:00 +0100

Proper structure and function of mitochondria depends on translocation of over a thousand different mitochondrial proteins from the cytosol. Mitochondrial proteins carry a number of different targeting signals and are translocated into the mitochondria by a number of different protein translocases. Here, we provide an overview of mitochondrial protein translocation pathways, and focus on the TIM23 complex, the major protein translocase of the mitochondrial inner membrane.

Routine and novel methods for isolation of extracellular vesicles

Milica Popovic — Thu, 27 Feb 2020 00:00:00 +0100

Extracellular vesicles (EV) play an important role in many physiological and pathological processes. Three main classes of EV are recognized, based on their biogenesis: exosomes, microvesicles and apoptotic bodies. Exosomes are extracellular-vesicles of 30 to 150 nm found in many bodily fluids (blood, urine, milk, cerebrospinal fluid, etc.). Due to their cellular origin and role in physiological and pathological processes, exosomes present in body fluids are considered a unique source of non-invasive and clinically relevant biomarkers. Analysis of exosomes can provide insight into the state of the parent-cell from which they originated. However, there is great heterogeneity in the methodologies used for exosome purification affecting the results of downstream analysis. The most commonly used methods for purification are based on ultracentrifugation (UC), ultrafiltration (UF) and precipitation. However, these are hard to standardize, leading to confounding and misleading results during downstream analyses, especially when highly-sensitive techniques such as mass spectrometry are used. Furthermore, loss of certain fractions or damage of EVs can lead to loss in obtained protein and RNA profile. Consequently, there is an emerging need to obtain consensus protocols for exosome isolation and identification of specific sub-populations. This manuscript will critically review the most commonly used techniques for EV purification such as UC, UF, size-exclusion, precipitation and immunoaffinity (IA) methods. We will also review the use of nano-antibodies for the development of novel IA protocols and identification of new EV biomarkers.

Molecular adaptation to high temperatures: pernisine from the archaeon Aeropyrum pernix K1

Kevin Hartman, Miha Bahun, Marko Šnajder, Nataša Poklar Ulrih — Thu, 27 Feb 2020 00:00:00 +0100

Pernisine is a subtilisin-like protease from the hyperthermophilic archaeon Aeropyrum pernix. Due to its high thermal stability and its activity in the presence of denaturants, pernisine represents a promising enzyme for use in various industrial applications. Another potentially applicable characteristic of this protease is its ability to degrade infectious prion aggregates. Production of pernisine in A. pernix does not provide sufficient yield for its commercial use, and alternative production strategies are hence needed. This review summarizes the biochemical and biophysical characteristics of pernisine and progress that has been made toward production of recombinant pernisine using Escherichia coli and Streptomyces rimosus as expression systems.

Gonadotropin-releasing hormone regulated transcription of gonadotropin subunit genes

Marija M. Janjic, Ana Milosevic, Ivana Bjelobaba — Thu, 27 Feb 2020 00:00:00 +0100

Two gonadotropins, luteinizing hormone and follicle-stimulating hormone, are synthetized and secreted by anterior pituitary gonadotropes and act on the gonads, controlling gametogenesis and sex hormone production. These hormones are glycoprotein polypeptides, composed of specific beta subunits and a common, alpha subunit. Both transcription and secretion of gonadotropins are regulated by gonadotropin-releasing hormone (GnRH), which is produced by a small number of hypothalamic neurons within the preoptic area and mediobasal hypothalamus. GnRH is released and reaches the pituitary in pulses, a pattern of secretion that is crucial for proper reproductive functions. This mini review covers mechanisms of transcriptional control of gonadotropin subunit genes by GnRH, predominantly focusing on in vivo experiments with mice and rats and in vitro experiments using primary pituitary cell cultures and immortalized pituitary cell lines derived from these species. We also provide an overview of the promoter regions of gonadotropin genes and major transcription factors involved in GnRH-driven expression of gonadotropin subunit genes.

An overview of the biological activities of less known wild onions (genus Allium sect. Codonoprasum)

Thu, 27 Feb 2020 00:00:00 +0100

Cultivated forms of Allium species, such as onion, garlic, leek and shalot, are widely used in the human diet and are also respected as medicinal plants. In contrast, wild onions, esspecially species from Codonoprasum, are less well known and remain understudied, even though they are present in local diets and traditional medicines. Therefore, in recent years, the chemical composition and biological activities of several species of onions from sect. Codonoprasum have been intensively studied in order to estimate their potential for applications in medicine. Headspace GC–MS analyses revealed that dimethyl-disulfide is by far the most dominant volatile sulfur compound in fresh bulbs of species from Codonoprasum section. LC-MS/MS analysis of phenolic compounds showed that these species are rich in phenolics, particularly in quercetin glycosides and kaempferol 3-O-glucoside. Allium flavum expressed the highest antioxidant activity in common in vitro assays and high anti-inflammatory activity in human platelets. Allium flavum and A. melanantherum exhibited high levels of antigenotoxic effects by activation of cellular antioxidant defence mechanisms. Allium flavum and A. carinatum drastically improved the activity of doxorubicin (Dox) against cancer cells. In zebrafish models, A. flavum and A. carinatum expressed high anti-angiogenic activity and protective effects against Dox-caused cardiac dysfunction and neutropenia. The results of these rewieved studies are indicative of the multiple beneficial pharmacological activities of wild Allium species.

The role of molecular markers of angiogenesis in disease prediction in breast cancer patients

D Cvetković, A Cvetkovic, S Ninković, M Milutinovic, S Mitrović, S Marković — Thu, 27 Feb 2020 00:00:00 +0100

Breast cancer is the most common malignant tumor in women around the world. It is a disease of complex etiology, characteristics and response to therapy. Oncology therapy is the most expensive form of treatment and represents a significant fraction of the overall health budget of both developed and transition countries. Progress in molecular biology has had a major impact on the development of a more personalized approach. Our study clearly indicates that cancer causes changes in both cancerous and peritumoral tissue that are detectable on the molecular but not the pathohistological level. Therefore, analysis not only of cancer but also of the peritumoral tissue is very valuable, because often the changes that are very significant in the prognostic sense occur predominantly in the microenvironment of the carcinoma. Markers of angiogenesis in cancer and peritumor tissue such as MMP-9 concentration, expression of VEGF-A, CXCL-12, HIF-1 and iNOS genes can serve as reliable predictors of disease outcome in patients with breast cancer, which can give useful suggestions for the choice of treatment. Using modern methods of molecular biology, a group of patients with an increased risk of metastases and recurrence can be identified, providing important information to guide decision making with respect to further treatments.

Metallomics and mass spectrometry for drug development: ICP-MS and MALDI TOF MS for assessment of protein-drug interactions

Romana Masnikosa, Ana Rilak-Simović, Suzana Veličković — Thu, 27 Feb 2020 00:00:00 +0100

Ever since the discovery of the anticancer drug cisplatin, the field of inorganic medicinal chemistry has been expanding. A plethora of metal-based compounds (Pt, Ru, Au, Pd, Cu) containing organic or inorganic ligands (NH₃, Cl, DMSO, η⁶-arene, imidazole, indazole, terpyridine, phosphoadamantane) has been developed with the aim to find new and improved anticancer therapeutics, which would have less side-effects, and to overcome tumour resistance. The medicinal inorganic chemistry community must determine: how these compounds enter living cells, how they distribute inside cells, what are their intracellular targets, how they behave in circulation (free vs protein-bound fraction), what are their target tissues, and what biomolecules are responsible for their transport to biological targets. All of these questions have been recently addressed with the aid of different mass spectrometry (MS) techniques. Inductively coupled plasma MS is a highly sensitive method for determining the metal content in biological samples. Matrix-assisted laser desorption/ionisation and electrospray MS are used to characterise adducts between metallodrugs and proteins. MS methods provide information on binding kinetics, binding parameters, the nature of the adducts and target sequences in proteins. This review deals with the most interesting results obtained by the most influential mass spectrometrists working in the field of inorganic biochemistry.

How to use educational technology to make education better - Not just different or entertaining!

Ferhan G. Sağin — Thu, 27 Feb 2020 00:00:00 +0100

Educational Technologies (EdTech) have the potential to improve learning and teaching both in- and outside of the classroom. EdTech tools can potentially make the education process more meaningful, more engaging, more interesting, more personalized and more flexible. There may be many reasons (changes in school policy, pressures from authorities, observing models of efficient use, etc.) and facilitating factors (support from the school, collaborations among faculty, etc.) driving implementation of EdTech, but there are also barriers (lack of technological tools, etc.) and drawbacks (technological competence, established teaching practices, etc.). In practice, educators are also faced with challenges in designing or integrating EdTech in a pedagogically meaningful way. Most of the time, educators try to effectively implement EdTech in their curriculum. However, we have seen numerous examples of EdTech implementations without a clear aim, vision, assessment and/or positive learning outcomes. This article will start with a definition of EdTech, delineating what it is not and what it is, and continue with a discussion of ‘why’ should EdTech be integrated, and ‘how’ to choose and to implement components of EdTech. Finally, we provide some take home messages and tips on successful integration of EdTech.

Longitudinal distribution of apoplastic antioxidative components in maize root

Jelena Dragišić Maksimović, Miloš Mojović, Željko Vučinić, Vuk Maksimović — Thu, 27 Feb 2020 00:00:00 +0100

The apoplast is a liquid- and gas-filled extracellular continuum which includes cell wall polymer networks and the external surface of the plasma membrane. Apoplastic constituents, such as various organic molecules, enzymes and proteins play major roles in a wide range of physiological processes. In order to investigate apoplastic fluid, two isolation procedures were compared and critically evaluated: infiltration and/or centrifugation techniques to obtain Apoplastic Washing Fluid and filter paper strips which, based on our results, enable collection of experimental data from intact plants. Different components of the antioxidative system (enzymes, phenolics, sugars, organic acids) present in apoplastic fluid were analyzed using different techniques. Three classes of non-enzymatic compounds (organic acids, sugars and phenolics) have been identified and quantified by HPLC. Detection of hydroxyl radicals was performed by EPR methods using spin-trap DEPMPO, which is capable of forming different spin-adducts with hydroxyl and superoxide anion radicals. Spectrophotometrically estimated total protein concentrations, peroxidase and superoxide dismutase specific activities, as well as their different isoforms, were visually confirmed by isoelectric focusing. These high sensitivity techniques (HPLC-ECD, EPR), as well as electrophoresis, in combination with the filter strip method provided us with the tools necessary to study the components of the antioxidative system in the apoplast of developing plant organs and their spatial-temporal changes. Such an experimental setup provided a powerful non-invasive analytical tool for studying metabolic processes occurring in the apoplast and local changes in small regions of intact root tissue.