Molecular Basis for the Involvement of Mammalian Serum Albumin in the AGE/RAGE Axis: A Comprehensive Computational Study.

In mammals, glycated serum albumin (gSA) contributes to the pathogenesis of many metabolic diseases by activating the receptors (RAGE) for advanced glycation end products (AGEs). Many aspects of the gSA-RAGE interaction remain unknown. The purpose of the present paper was to study the interaction of glycated human albumin (gHSA) with RAGE using molecular modeling methods. Ten models of gHSA modified with different lysine residues to carboxymethyl-lysines were prepared. Complexes of gHSA-RAGE were obtained by the macromolecular docking method with subsequent molecular dynamics simulation (MD). According to the MD, the RAGE complexes with gHSA glycated at Lys233, Lys64, Lys525, Lys262 and Lys378 are the strongest. Three-dimensional models of the RAGE dimers with gHSA were proposed. Additional computational experiments showed that the binding of fatty acids (FAs) to HSA does not affect the ability of Lys525 (the most reactive lysine) to be glycated. In contrast, modification of Lys525 reduces the affinity of albumin for FA. The interspecies differences in the molecular structure of albumin that may affect the mechanism of the gSA-RAGE interaction were discussed. The obtained results will help us to learn more about the molecular basis for the involvement of serum albumin in the AGE/RAGE axis and improve the methodology for studying cellular signaling pathways involving RAGE.

Immunological Profile and Markers of Endothelial Dysfunction in Elderly Patients with Cognitive Impairments.

The process of aging is accompanied by a dynamic restructuring of the immune response, a phenomenon known as immunosenescence. Further, damage to the endothelium can be both a cause and a consequence of many diseases, especially in elderly people. The purpose of this study was to carry out immunological and biochemical profiling of elderly people with acute ischemic stroke (AIS), chronic cerebral circulation insufficiency (CCCI), prediabetes or newly diagnosed type II diabetes mellitus (DM), and subcortical ischemic vascular dementia (SIVD). Socio-demographic, lifestyle, and cognitive data were obtained. Biochemical, hematological, and immunological analyses were carried out, and extracellular vesicles (EVs) with endothelial CD markers were assessed. The greatest number of significant deviations from conditionally healthy donors (HDs) of the same age were registered in the SIVD group, a total of 20, of which 12 were specific and six were non-specific but with maximal differences (as compared to the other three groups) from the HDs group. The non-specific deviations were for the MOCA (Montreal Cognitive Impairment Scale), the MMSE (Mini Mental State Examination) and life satisfaction self-assessment scores, a decrease of albumin levels, and ADAMTS13 (a Disintegrin and Metalloproteinase with a Thrombospondin Type 1 motif, member 13) activity, and an increase of the VWF (von Willebrand factor) level. Considering the significant changes in immunological parameters (mostly Th17-like cells) and endothelial CD markers (CD144 and CD34), vascular repair was impaired to the greatest extent in the DM group. The AIS patients showed 12 significant deviations from the HD controls, including three specific to this group. These were high NEFAs (non-esterified fatty acids) and CD31 and CD147 markers of EVs. The lowest number of deviations were registered in the CCCI group, nine in total. There were significant changes from the HD controls with no specifics to this group, and just one non-specific with a maximal difference from the control parameters, which was α1-AGP (alpha 1 acid glycoprotein, orosomucoid). Besides the DM patients, impairments of vascular repair were also registered in the CCCI and AIS patients, with a complete absence of such in patients with dementia (SIVD group). On the other hand, microvascular damage seemed to be maximal in the latter group, considering the biochemical indicators VWF and ADAMTS13. In the DM patients, a maximum immune response was registered, mainly with Th17-like cells. In the CCCI group, the reaction was not as pronounced compared to other groups of patients, which may indicate the initial stages and/or compensatory nature of organic changes (remodeling). At the same time, immunological and biochemical deviations in SIVD patients indicated a persistent remodeling in microvessels, chronic inflammation, and a significant decrease in the anabolic function of the liver and other tissues. The data obtained support two interrelated assumptions. Taking into account the primary biochemical factors that trigger the pathological processes associated with vascular pathology and related diseases, the first assumption is that purine degradation in skeletal muscle may be a major factor in the production of uric acid, followed by its production by non-muscle cells, the main of which are endothelial cells. Another assumption is that therapeutic factors that increase the levels of endothelial progenitor cells may have a therapeutic effect in reducing the risk of cerebrovascular disease and related neurodegenerative diseases.

Searching for New Biomarkers to Assess COVID-19 Patients: A Pilot Study.

During the initial diagnosis of urgent medical conditions, which include acute infectious diseases, it is important to assess the severity of the patient's clinical state as quickly as possible. Unlike individual biochemical or physiological indicators, derived indices make it possible to better characterize a complex syndrome as a set of symptoms, and therefore quickly take a set of adequate measures. Recently, we reported on novel diagnostic indices containing butyrylcholinesterase (BChE) activity, which is decreased in COVID-19 patients. Also, in these patients, the secretion of von Willebrand factor (vWF) increases, which leads to thrombosis in the microvascular bed. The objective of this study was the determination of the concentration and activity of vWF in patients with COVID-19, and the search for new diagnostic indices. One of the main objectives was to compare the prognostic values of some individual and newly derived indices. Patients with COVID-19 were retrospectively divided into two groups: survivors (n = 77) and deceased (n = 24). According to clinical symptoms and computed tomography (CT) results, the course of disease was predominantly moderate in severity. The first blood sample (first point) was taken upon admission to the hospital, the second sample (second point)-within 4-6 days after admission. Along with the standard spectrum of biochemical indicators, BChE activity (BChEa or BChEb for acetylthiocholin or butyrylthiocholin, respectively), malondialdehyde (MDA), and vWF analysis (its antigen level, AGFW, and its activity, ActWF) were determined and new diagnostic indices were derived. The pooled sensitivity, specificity, and area under the receiver operating curve (AUC), as well as Likelihood ratio (LR) and Odds ratio (OR) were calculated. The level of vWF antigen in the deceased group was 1.5-fold higher than the level in the group of survivors. Indices that include vWF antigen levels are superior to indices using vWF activity. It was found that the index [Urea] × [AGWF] × 1000/(BChEb × [ALB]) had the best discriminatory power to predict COVID-19 mortality (AUC = 0.91 [0.83, 1.00], p < 0.0001; OR = 72.0 [7.5, 689], p = 0.0002). In addition, [Urea] × 1000/(BChEb × [ALB]) was a good predictor of mortality (AUC = 0.95 [0.89, 1.00], p < 0.0001; OR = 31.5 [3.4, 293], p = 0.0024). The index [Urea] × [AGWF] × 1000/(BChEb × [ALB]) was the best predictor of mortality associated with COVID-19 infection, followed by [Urea] × 1000/(BChEb × [ALB]). After validation in a subsequent cohort, these two indices could be recommended for diagnostic laboratories.

Comprehensive clinical assays for molecular diagnostics of gliomas: the current state and future prospects.

Glioma is one of the most intractable types of cancer, due to delayed diagnosis at advanced stages. The clinical symptoms of glioma are unclear and due to a variety of glioma subtypes, available low-invasive testing is not effective enough to be introduced into routine medical laboratory practice. Therefore, recent advances in the clinical diagnosis of glioma have focused on liquid biopsy approaches that utilize a wide range of techniques such as next-generation sequencing (NGS), droplet-digital polymerase chain reaction (ddPCR), and quantitative PCR (qPCR). Among all techniques, NGS is the most advantageous diagnostic method. Despite the rapid cheapening of NGS experiments, the cost of such diagnostics remains high. Moreover, high-throughput diagnostics are not appropriate for molecular profiling of gliomas since patients with gliomas exhibit only a few diagnostic markers. In this review, we highlighted all available assays for glioma diagnosing for main pathogenic glioma DNA sequence alterations. In the present study, we reviewed the possibility of integrating routine molecular methods into the diagnosis of gliomas. We state that the development of an affordable assay covering all glioma genetic aberrations could enable early detection and improve patient outcomes. Moreover, the development of such molecular diagnostic kits could potentially be a good alternative to expensive NGS-based approaches.

Structure-Dependent Mechanism of Organophosphate Release from Albumin and Butyrylcholinesterase Adducts When Exposed to Fluoride Ion: A Comprehensive In Silico Study.

The most favorable targets for retrospectively determining human exposure to organophosphorus pesticides, insecticides, retardants, and other industrial organophosphates (OPs) are adducts of OPs with blood plasma butyrylcholinesterase (BChE) and human serum albumin (HSA). One of the methods for determining OP exposure is the reactivation of modified BChE using a concentrated solution of KF in an acidic medium. It is known that under the action of fluoride ion, OPs or their fluoroanhydrides can be released not only from BChE adducts but also from the adducts with albumin; however, the contribution of albumin to the total pool of released OPs after plasma treatment with KF has not yet been studied. The efficiency of OP release can be affected by many factors associated with the experimental technique, but first, the structure of the adduct must be taken into account. We report a comparative analysis of the structure and conformation of organophosphorus adducts on HSA and BChE using molecular modeling methods and the mechanism of OP release after fluoride ion exposure. The conformational analysis of the organophosphorus adducts on HSA and BChE was performed, and the interaction of fluoride ions with modified proteins was studied by molecular dynamics simulation. The geometric and energy characteristics of the studied adducts and their complexes with fluoride ion were calculated using molecular mechanics and semiempirical approaches. The structural features of modified HSA and BChE that can affect the efficiency of OP release after fluoride ion exposure were revealed. Using the proposed approach, the expediency of using KF for establishing exposure to different OPs, depending on their structure, can be assessed.

Serum Albumin in Health and Disease: From Comparative Biochemistry to Translational Medicine.

Being one of the main proteins in the human body and many animal species, albumin plays a decisive role in the transport of various ions, electrically neutral molecules and in maintaining the colloid osmotic pressure of the blood [...].

Synergistic Interaction of 5-HT1B and 5-HT2B Receptors in Cytoplasmic Ca2+ Regulation in Human Umbilical Vein Endothelial Cells: Possible Involvement in Pathologies.

The aim of this work was to explore the involvement of 5-HT1B and 5-HT2B receptors (5-HT1BR and 5-HT2BR) in the regulation of free cytoplasmic calcium concentration ([Ca2+]i) in human umbilical vein endothelial cells (HUVEC). We have shown by quantitative PCR analysis, that 5-HT1BR and 5-HT2BR mRNAs levels are almost equal in HUVEC. Immunofluorescent staining demonstrated, that 5-HT1BR and 5-HT2BR are expressed both in plasma membrane and inside the cells. Intracellular 5-HT1BR are localized mainly in the nuclear region, whereas 5-HT2BR receptors are almost evenly distributed in HUVEC. 5-HT, 5-HT1BR agonist CGS12066B, or 5-HT2BR agonist BW723C86 added to HUVEC caused a slight increase in [Ca2+]i, which was much lower than that of histamine, ATP, or SFLLRN, an agonist of protease-activated receptors (PAR1). However, activation of 5-HT1BR with CGS12066B followed by activation of 5-HT2BR with BW723C86 manifested a synergism of response, since several-fold higher rise in [Ca2+]i occurred. CGS12066B caused more than a 5-fold increase in [Ca2+]i rise in HUVEC in response to 5-HT. This 5-HT induced [Ca2+]i rise was abolished by 5-HT2BR antagonist RS127445, indicating that extracellular 5-HT acts through 5-HT2BR. Synergistic [Ca2+]i rise in response to activation of 5-HT1BR and 5-HT2BR persisted in a calcium-free medium. It was suppressed by the phospholipase C inhibitor U73122 and was not inhibited by the ryanodine and NAADP receptors antagonists dantrolene and NED-19. [Ca2+]i measurements in single cells demonstrated that activation of 5-HT2BR alone by BW723C86 caused single asynchronous [Ca2+]i oscillations in 19.8 ± 4.2% (n = 3) of HUVEC that occur with a long delay (66.1 ± 4.3 s, n = 71). On the contrary, histamine causes a simultaneous and almost immediate increase in [Ca2+]i in all the cells. Pre-activation of 5-HT1BR by CGS12066B led to a 3-4 fold increase in the number of HUVEC responding to BW723C86, to synchronization of their responses with a delay shortening, and to the bursts of [Ca2+]i oscillations in addition to single oscillations. In conclusion, to get a full rise of [Ca2+]i in HUVEC in response to 5-HT, simultaneous activation of 5-HT1BR and 5-HT2BR is required. 5-HT causes an increase in [Ca2+]i via 5-HT2BR while 5-HT1BR could be activated by the membrane-permeable agonist CGS12066B. We hypothesized that CGS12066B acts via intracellular 5-HT1BR inaccessible to extracellular 5-HT. Intracellular 5-HT1BR might be activated by 5-HT which could be accumulated in EC under certain pathological conditions.

Anticholinesterase and Serotoninergic Evaluation of Benzimidazole-Carboxamides as Potential Multifunctional Agents for the Treatment of Alzheimer's Disease.

The etiology and pathogenesis of Alzheimer's disease are multifactorial, so one of the treatment strategies is the development of the drugs that affect several targets associated with the pathogenesis of the disease. Within this roadmap, we investigated the interaction of several substituted 1,3-dihydro-2-oxo-1H-benzimidazol-2-ones with their potential molecular targets: cholinesterases (ChE) and three types of the Gs-protein-coupled serotonin receptors (5-HTR) 5-HT6, 5-HT4 and 5-HT7 (5-HT4R, 5-HT6R and 5-HT7R, respectively). A microplate modification of the Ellman method was used for the biochemical analysis of the inhibitory ability of the drugs towards ChE. Molecular modeling methods, such as molecular docking and molecular dynamics (MD) simulation in water and the lipid bilayer, were used to study the interaction of the compounds with ChE and 5-HTR. In vitro experiments showed that the tested compounds had moderate anticholinesterase activity. With the help of molecular modeling methods, the mechanism of interaction of the tested compounds with ChE was investigated, the binding sites were described and the structural features of the drugs that determine the strength of their anticholinesterase activity were revealed. Primary in silico evaluation showed that benzimidazole-carboxamides effectively bind to 5-HT4R and 5-HT7R. The pool of the obtained data allows us to choose N-[2-(diethylamino)ethyl]-2-oxo-3-(tert-butyl)-2,3-dihydro-1H-benzimidazole-1-carboxamide hydrochloride (compound 13) as the most promising for further experimental development.

Albumin Is a Component of the Esterase Status of Human Blood Plasma.

The esterase status of blood plasma can claim to be one of the universal markers of various diseases; therefore, it deserves attention when searching for markers of the severity of COVID-19 and other infectious and non-infectious pathologies. When analyzing the esterase status of blood plasma, the esterase activity of serum albumin, which is the major protein in the blood of mammals, should not be ignored. The purpose of this study is to expand understanding of the esterase status of blood plasma and to evaluate the relationship of the esterase status, which includes information on the amount and enzymatic activity of human serum albumin (HSA), with other biochemical parameters of human blood, using the example of surviving and deceased patients with confirmed COVID-19. In experiments in vitro and in silico, the activity of human plasma and pure HSA towards various substrates was studied, and the effect of various inhibitors on this activity was tested. Then, a comparative analysis of the esterase status and a number of basic biochemical parameters of the blood plasma of healthy subjects and patients with confirmed COVID-19 was performed. Statistically significant differences have been found in esterase status and biochemical indices (including albumin levels) between healthy subjects and patients with COVID-19, as well as between surviving and deceased patients. Additional evidence has been obtained for the importance of albumin as a diagnostic marker. Of particular interest is a new index, [Urea] × [MDA] × 1000/(BChEb × [ALB]), which in the group of deceased patients was 10 times higher than in the group of survivors and 26 times higher than the value in the group of apparently healthy elderly subjects.

κ- and λ-Carrageenans from Marine Alga Chondrus armatus Exhibit Anticancer In Vitro Activity in Human Gastrointestinal Cancers Models.

The carrageenans isolated from red algae demonstrated a variety of activities from antiviral and immunomodulatory to antitumor. The diverse structure and sulfation profile of carrageenans provide a great landscape for drug development. In this study, we isolated, purified and structurally characterized κo- and λo- oligosaccharides from the marine algae Chondrus armatus. We further examined the tumor suppressive activity of both carrageenans in gastrointestinal cancer models. Thus, using MTT assay, we could demonstrate a pronounced antiproliferative effect of the carrageenans in KYSE-30 and FLO-1 as well as HCT-116 and RKO cell lines with IC50 184~405 µg/mL, while both compounds were less active in non-cancer epithelial cells RPE-1. This effect was stipulated by the inhibition of cell cycle progression in the cancer cells. Specifically, flow cytometry revealed an S phase delay in FLO-1 and HCT-116 cells under κo-carrageenan treatment, while KYSE-30 demonstrated a pronounced G2/M cell cycle delay. In line with this, western blotting revealed a reduction of cell cycle markers CDK2 and E2F2. Interestingly, κo-carrageenan inhibited cell cycle progression of RKO cells in G1 phase. Finally, isolated κo- and λo- carrageenans induced apoptosis on adenocarcinomas, specifically with high apoptosis induction in RKO cells. Overall, our data underline the potential of κo- and λo- carrageenans for colon and esophageal carcinoma drug development.

Molecular and Cellular Interactions in Pathogenesis of Sporadic Parkinson Disease.

An increasing number of the population all around the world suffer from age-associated neurodegenerative diseases including Parkinson's disease (PD). This disorder presents different signs of genetic, epigenetic and environmental origin, and molecular, cellular and intracellular dysfunction. At the molecular level, α-synuclein (αSyn) was identified as the principal molecule constituting the Lewy bodies (LB). The gut microbiota participates in the pathogenesis of PD and may contribute to the loss of dopaminergic neurons through mitochondrial dysfunction. The most important pathogenetic link is an imbalance of Ca2+ ions, which is associated with redox imbalance in the cells and increased generation of reactive oxygen species (ROS). In this review, genetic, epigenetic and environmental factors that cause these disorders and their cause-and-effect relationships are considered. As a constituent of environmental factors, the example of organophosphates (OPs) is also reviewed. The role of endothelial damage in the pathogenesis of PD is discussed, and a 'triple hit hypothesis' is proposed as a modification of Braak's dual hit one. In the absence of effective therapies for neurodegenerative diseases, more and more evidence is emerging about the positive impact of nutritional structure and healthy lifestyle on the state of blood vessels and the risk of developing these diseases.

Molecular Mechanisms of Acute Organophosphate Nephrotoxicity.

Organophosphates (OPs) are toxic chemicals produced by an esterification process and some other routes. They are the main components of herbicides, pesticides, and insecticides and are also widely used in the production of plastics and solvents. Acute or chronic exposure to OPs can manifest in various levels of toxicity to humans, animals, plants, and insects. OPs containing insecticides were widely used in many countries during the 20th century, and some of them continue to be used today. In particular, 36 OPs have been registered in the USA, and all of them have the potential to cause acute and sub-acute toxicity. Renal damage and impairment of kidney function after exposure to OPs, accompanied by the development of clinical manifestations of poisoning back in the early 1990s of the last century, was considered a rare manifestation of their toxicity. However, since the beginning of the 21st century, nephrotoxicity of OPs as a manifestation of delayed toxicity is the subject of greater attention of researchers. In this article, we present a modern view on the molecular pathophysiological mechanisms of acute nephrotoxicity of organophosphate compounds.

Novel assay to measure chromosome instability identifies Punica granatum extract that elevates CIN and has a potential for tumor- suppressing therapies.

Human artificial chromosomes (HACs) have provided a useful tool to study kinetochore structure and function, gene delivery, and gene expression. The HAC propagates and segregates properly in the cells. Recently, we have developed an experimental high-throughput imaging (HTI) HAC-based assay that allows the identification of genes whose depletion leads to chromosome instability (CIN). The HAC carries a GFP transgene that facilitates quantitative measurement of CIN. The loss of HAC/GFP may be measured by flow cytometry or fluorescence scanning microscope. Therefore, CIN rate can be measured by counting the proportion of fluorescent cells. Here, the HAC/GFP-based assay has been adapted to screen anticancer compounds for possible induction or elevation of CIN. We analyzed 24 cytotoxic plant extracts. Punica granatum leaf extract (PLE) indeed sharply increases CIN rate in HT1080 fibrosarcoma cells. PLE treatment leads to cell cycle arrest, reduction of mitotic index, and the increased numbers of micronuclei (MNi) and nucleoplasmic bridges (NPBs). PLE-mediated increased CIN correlates with the induction of double-stranded breaks (DSBs). We infer that the PLE extract contains a component(s) that elevate CIN, making it a candidate for further study as a potential cancer treatment. The data also provide a proof of principle for the utility of the HAC/GFP-based system in screening for natural products and other compounds that elevate CIN in cancer cells.

A Novel C1q Domain-Containing Protein Isolated from the Mollusk Modiolus kurilensis Recognizing Glycans Enriched with Acidic Galactans and Mannans.

C1q domain-containing (C1qDC) proteins are a group of biopolymers involved in immune response as pattern recognition receptors (PRRs) in a lectin-like manner. A new protein MkC1qDC from the hemolymph plasma of Modiolus kurilensis bivalve mollusk widespread in the Northwest Pacific was purified. The isolation procedure included ammonium sulfate precipitation followed by affinity chromatography on pectin-Sepharose. The full-length MkC1qDC sequence was assembled using de novo mass-spectrometry peptide sequencing complemented with N-terminal Edman's degradation, and included 176 amino acid residues with molecular mass of 19 kDa displaying high homology to bivalve C1qDC proteins. MkC1qDC demonstrated antibacterial properties against Gram-negative and Gram-positive strains. MkC1qDC binds to a number of saccharides in Ca2+-dependent manner which characterized by structural meta-similarity in acidic group enrichment of galactose and mannose derivatives incorporated in diversified molecular species of glycans. Alginate, κ-carrageenan, fucoidan, and pectin were found to be highly effective inhibitors of MkC1qDC activity. Yeast mannan, lipopolysaccharide (LPS), peptidoglycan (PGN) and mucin showed an inhibitory effect at concentrations three orders of magnitude greater than for the most effective saccharides. MkC1qDC localized to the mussel hemal system and interstitial compartment. Intriguingly, MkC1qDC was found to suppress proliferation of human adenocarcinoma HeLa cells in a dose-dependent manner, indicating to the biomedical potential of MkC1qDC protein.

Nephrotoxic Effects of Paraoxon in Three Rat Models of Acute Intoxication.

The delayed effects of acute intoxication by organophosphates (OPs) are poorly understood, and the various experimental animal models often do not take into account species characteristics. The principal biochemical feature of rodents is the presence of carboxylesterase in blood plasma, which is a target for OPs and can greatly distort their specific effects. The present study was designed to investigate the nephrotoxic effects of paraoxon (O,O-diethyl O-(4-nitrophenyl) phosphate, POX) using three models of acute poisoning in outbred Wistar rats. In the first model (M1, POX2x group), POX was administered twice at doses 110 µg/kg and 130 µg/kg subcutaneously, with an interval of 1 h. In the second model (M2, CBPOX group), 1 h prior to POX poisoning at a dose of 130 µg/kg subcutaneously, carboxylesterase activity was pre-inhibited by administration of specific inhibitor cresylbenzodioxaphosphorin oxide (CBDP, 3.3 mg/kg intraperitoneally). In the third model (M3), POX was administered subcutaneously just once at doses of LD16 (241 µg/kg), LD50 (250 µg/kg), and LD84 (259 µg/kg). Animal observation and sampling were performed 1, 3, and 7 days after the exposure. Endogenous creatinine clearance (ECC) decreased in 24 h in the POX2x group (p = 0.011). Glucosuria was observed in rats 24 h after exposure to POX in both M1 and M2 models. After 3 days, an increase in urinary excretion of chondroitin sulfate (CS, p = 0.024) and calbindin (p = 0.006) was observed in rats of the CBPOX group. Morphometric analysis revealed a number of differences most significant for rats in the CBPOX group. Furthermore, there was an increase in the area of the renal corpuscles (p = 0.0006), an increase in the diameter of the lumen of the proximal convoluted tubules (PCT, p = 0.0006), and narrowing of the diameter of the distal tubules (p = 0.001). After 7 days, the diameter of the PCT lumen was still increased in the nephrons of the CBPOX group (p = 0.0009). In the M3 model, histopathological and ultrastructural changes in the kidneys were revealed after the exposure to POX at doses of LD50 and LD84. Over a period from 24 h to 3 days, a significant (p = 0.018) expansion of Bowman's capsule was observed in the kidneys of rats of both the LD50 and LD84 groups. In the epithelium of the proximal tubules, stretching of the basal labyrinth, pycnotic nuclei, and desquamation of microvilli on the apical surface were revealed. In the epithelium of the distal tubules, partial swelling and destruction of mitochondria and pycnotic nuclei was observed, and nuclei were displaced towards the apical surface of cells. After 7 days of the exposure to POX, an increase in the thickness of the glomerular basement membrane (GBM) was observed in the LD50 and LD84 groups (p = 0.019 and 0.026, respectively). Moreover, signs of damage to tubular epithelial cells persisted with blockage of the tubule lumen by cellular detritus and local destruction of the surface of apical cells. Comparison of results from the three models demonstrates that the nephrotoxic effects of POX, evaluated at 1 and 3 days, appear regardless of prior inhibition of carboxylesterase activity.

Esterase Activity of Serum Albumin Studied by 1H NMR Spectroscopy and Molecular Modelling.

Serum albumin possesses esterase and pseudo-esterase activities towards a number of endogenous and exogenous substrates, but the mechanism of interaction of various esters and other compounds with albumin is still unclear. In the present study, proton nuclear magnetic resonance (1H NMR) has been applied to the study of true esterase activity of albumin, using the example of bovine serum albumin (BSA) and p-nitrophenyl acetate (NPA). The site of BSA esterase activity was then determined using molecular modelling methods. According to the data obtained, the accumulation of acetate in the presence of BSA in the reaction mixture is much more intense as compared with the spontaneous hydrolysis of NPA, which indicates true esterase activity of albumin towards NPA. Similar results were obtained for p-nitophenyl propionate (NPP) as substrate. The rate of acetate and propionate release confirms the assumption that there is a site of true esterase activity in the albumin molecule, which is different from the site of the pseudo-esterase activity Sudlow II. The results of molecular modelling of BSA and NPA interaction make it possible to postulate that Sudlow site I is the site of true esterase activity of albumin.

Serum Albumin in Health and Disease: Esterase, Antioxidant, Transporting and Signaling Properties.

Being one of the main proteins in the human body and many animal species, albumin plays a decisive role in the transport of various ions-electrically neutral and charged molecules-and in maintaining the colloidal osmotic pressure of the blood. Albumin is able to bind to almost all known drugs, as well as many nutraceuticals and toxic substances, largely determining their pharmaco- and toxicokinetics. Albumin of humans and respective representatives in cattle and rodents have their own structural features that determine species differences in functional properties. However, albumin is not only passive, but also an active participant of pharmacokinetic and toxicokinetic processes, possessing a number of enzymatic activities. Numerous experiments have shown esterase or pseudoesterase activity of albumin towards a number of endogeneous and exogeneous esters. Due to the free thiol group of Cys34, albumin can serve as a trap for reactive oxygen and nitrogen species, thus participating in redox processes. Glycated albumin makes a significant contribution to the pathogenesis of diabetes and other diseases. The interaction of albumin with blood cells, blood vessels and tissue cells outside the vascular bed is of great importance. Interactions with endothelial glycocalyx and vascular endothelial cells largely determine the integrative role of albumin. This review considers the esterase, antioxidant, transporting and signaling properties of albumin, as well as its structural and functional modifications and their significance in the pathogenesis of certain diseases.

The Use of Fluorescently Labeled ARC1779 Aptamer for Assessing the Effect of H2O2 on von Willebrand Factor Exocytosis.

Here, we propose a new approach for quantitative estimation of von Willebrand factor (vWF) exposed on the surface of endothelial cells (ECs) using the ARC1779 aptamer that interacts with the vWF A1 domain. To visualize complex formation between vWF and the aptamer, the latter was conjugated with the Cy5 fluorescent label. Cultured human umbilical vein endothelial cells (HUVEC) were stained with the ARC1779-Cy5 conjugate and imaged with a fluorescence microscope. The images were analyzed with the CellProfiler software. vWF released from the Weibel-Palade bodies was observed as bright dot-like structures of round and irregular shape, the number of which increased several times after HUVEC exposure to histamine or thrombin. Staining with ARC1779-Cy5 also revealed long filamentous vWF structures on the surface of activated HUVEC. vWF secretion by ECs is activated by the second messengers cAMP and Ca2+. There is evidence that hydrogen peroxide also acts as a second messenger in ECs. In addition, exogenous H2O2 formed in leukocytes can enter ECs. The aim of our study was to determine the effect of H2O2 on the vWF exposure at the surface of HUVEC using the proposed method. It was shown that hydrogen peroxide at concentration 100 µM, which is lower than the cytotoxicity threshold of H2O2 for cultured HUVEC, increased several times the number of dot-like structures and total amount of vWF exposed on plasma membrane of HUVEC, which suggest that H2O2 acts as a mediator that activates exocytosis of Weibel-Palade bodies and vWF secretion in the vascular endothelium during inflammation and upon elevated generation of endogenous reactive oxygen species in ECs.

Low-Dose Ammonium Preconditioning Enhances Endurance in Submaximal Physical Exercises.

Preconditioning is often used in medicine to protect organs from ischemic damage and in athletes to enhance the performances. We tested whether low-dose ammonium preconditioning (AMP) could have a beneficial effect on physical exercises (PE). We used Cardiopulmonary Exercise Testing (CPET) on a treadmill to investigate the effects of low-dose AMP on the physical exercise capacity of professional track and field athletes and tested twenty-five athletes. Because of the individual differences between athletes, we performed a preliminary treadmill test (Pre-test) and, according to the results, the athletes were randomly allocated into the AMP and control (placebo, PL) group based on the similarity of the total distance covered on a treadmill. In the AMP group, the covered distance increased (11.3 ± 3.6%, p < 0.02) compared to Pre-test. Similarly, AMP significantly increased O2 uptake volume-VO2 (4.6 ± 2.3%, p < 0.03) and pulmonary CO2 output-VCO2 (8.7 ± 2.8%, p < 0.01). Further, the basic blood parameters (pH, pO2, and lactate) shift was lower despite the greater physical exercise progress in the AMP group compared to Pre-test, whereas in the placebo group there were no differences between Pre-test and Load-test. Importantly, the AMP significantly increased red blood cell count (6.8 ± 2.0%, p < 0.01) and hemoglobin concentration (5.3 ± 1.9%, p < 0.01), which might explain the beneficial effects in physical exercise progress. For the first time, we showed that low-dose AMP had clear beneficial effects on submaximal PE.

The Universal Soldier: Enzymatic and Non-Enzymatic Antioxidant Functions of Serum Albumin.

As a carrier of many biologically active compounds, blood is exposed to oxidants to a greater extent than the intracellular environment. Serum albumin plays a key role in antioxidant defence under both normal and oxidative stress conditions. This review evaluates data published in the literature and from our own research on the mechanisms of the enzymatic and non-enzymatic activities of albumin that determine its participation in redox modulation of plasma and intercellular fluid. For the first time, the results of numerous clinical, biochemical, spectroscopic and computational experiments devoted to the study of allosteric modulation of the functional properties of the protein associated with its participation in antioxidant defence are analysed. It has been concluded that it is fundamentally possible to regulate the antioxidant properties of albumin with various ligands, and the binding and/or enzymatic features of the protein by changing its redox status. The perspectives for using the antioxidant properties of albumin in practice are discussed.