bluuebunny/crossref_metadata_2025 huggingface dataset:
The key role of the redox status in regulation of metabolism in photosynthesizing organisms.
Authors: Kornas Andrzej , Kuźniak Elzbieta , Slesak Ireneusz , Miszalski Zbigniew
Published: March 2019

The redox status of the cell is described by the ratio of reduced to non-reduced compounds. Redox reactions which determine the redox state are an essential feature of all living beings on Earth. However, the first life forms evolved under strongly anoxic conditions of the young Earth, and the redox status probably was based on iron and sulphur compounds. Nowadays, redox reactions in cells have developed in strict connection to molecular oxygen and its derivatives i.e. reactive oxygen species (ROS). Oxygen has started to accumulate on the Earth due to oxygenic photosynthesis. All aspects of aerobic life involve ROS, reactive nitrogen species (RNS), antioxidants and redox regulation. Many different redox-active compounds are involved in the complex of redox processes, including pyridine nucleotides, thioredoxins, glutaredoxins and other thiol/disulphide-containing proteins. Redox regulation is integrated with the redox-reactions in photosynthesis and respiration to achieve an overall energy balance and to maintain a reduced state necessary for the biosynthetic pathways that are reductive in nature. It underlies the physiological and developmental flexibility in plant response to environmental signals.
A preliminary assessment of singlet oxygen scavenging, cytotoxic and genotoxic properties of Geranium macrorrhizum extracts.
Authors: Venskutonis Petras Rimantas , Dedonyte Veronika , Lazutka Juozas , Slapsyte Grazina , Maroziene Audrone , Nemeikaite-Ceniene Ausra , Cenas Narimantas , Miliauskas Giedrius
Published: March 2019

Strong radical-scavenging activity of Geranium macrorrhizum extracts isolated by using various solvent systems has been reported previously. This study aimed at expanding the knowledge on the bioactivities of antioxidatively active G. macrorrhizum butanol fraction, which was isolated from ethanolic extract (EB), and water fraction, which was isolated from water extract (WW) by measuring their singlet oxygen scavenging properties, as well as preliminary assessment of cytotoxicity and genotoxicity toward mammalian cells. The cytotoxicity (necrosis induction) of the extracts in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was partly prevented by antioxidants and stimulated by the prooxidant BCNU (N,N'-bis(2-chloroethyl)-N-nitrosourea). This indicates that the cytotoxicity of G. macrorrhizum extracts is at least partly attributed to their prooxidant action, presumably due to the formation of quinoidal products of their (auto)oxidation. The latter was evidenced by the nature of the peroxidase-catalyzed oxidation products, which supported DT-diaphorase-catalyzed oxidation of NADPH and participated in conjugation reactions with reduced glutathione. The genotoxic properties were studied using chromosome aberration (CA) and sister chromatid exchange (SCE) tests in human lymphocytes in vitro and Drosophila melanogaster somatic mutation and recombination test (SMART) in vivo. In the CA test, only the highest doses of both fractions significantly increased chromosome aberration frequency. In the SCE test, both fractions induced SCEs in a clear dose-dependent manner. G. macrorrhizum extracts were not genotoxic in the SMART test in vivo. Our data indicate that in spite of the possible beneficial (antioxidant) effects of Geranium extracts, the possibilities of their use as ingredients of functional foods and/or food supplements should be further examined due to their cyto- and genotoxic effects resulting mainly from the action of quercetin-derived components abundant in the extracts.
Cytotoxicity of β-carotene cleavage products and its prevention by antioxidants.
Authors: Alija Avdulla J , Bresgen Nikolaus , Bojaxhi Ekramije , Vogl Cornelia , Siems Werner , Eckl Peter M
Published: March 2019

When we investigated the genotoxicity of beta-carotene cleavage products (CPs) in primary rat hepatocytes stimulated to proliferate, we observed dose-dependent increases of chromosomal aberrations, sister chromatid exchanges and micronuclei. In contrast to other genotoxic substances, however, this increased genotoxicity was not accompanied by increased cytotoxicity. As a consequence we observed metaphases showing massive chromosomal damage, indicating inhibition of apoptosis by CPs enabling these cells to proceed in the cell cycle. Since proliferative stimulation by growth factors may support this effect, the in vitro toxicological effects of CPs were studied on proliferatively quiescent primary rat hepatocytes. A significant increase of both apoptosis and necrosis was found. Supplementation with antioxidants did not significantly lower the level of apoptosis, while the level of necrosis was significantly reduced by Trolox and N-acetylcysteine at all concentrations tested as well as ascorbic acid (50 microM) and a combination of Trolox (50 microM) and ascorbic acid (50 microM). These observations indicate that a) the cytotoxic potential in combination with the genotoxic potential of CPs may promote the initiation of cells due to compensatory cell division in exposed tissues and may aggravate inflammatory processes under chronic exposure, and b) the applied antioxidants may protect from cytotoxicity primarily via the detoxification of aldehydic beta-carotene cleavage products.
Reduction of oxidative stress in adjuvant arthritis. Comparison of efficacy of two pyridoindoles: stobadine dipalmitate and SMe1.2HCl.
Authors: Ponist Silvester , Mihalova Danica , Jancinova Viera , Snirc Vladimír , Ondrejickova Olga , Mascia Cinzia , Poli Giuseppe , Stancikova Maria , Nosal Radomir , Bauerová Katarína
Published: March 2019

The aim of this study was to evaluate the therapeutic potential of oxidative stress (OS) reduction by using pyridoindole (PI) antioxidants in adjuvant arthritis (AA). The substances tested were stobadine dipalmitate (STB) and SMe1. AA was used as animal model. The experiments included healthy animals, control arthritic animals and arthritic animals with administration of PI in the oral daily dose of 15 mg/kg b.m during 28 experimental days. The rats were sacrificed on day 28. Clinical and biochemical parameters were determined. The effect of PI administration was evaluated on the basis of the following parameters: (a) arthritis (volume of hind paws - HPW, change of animal body mass - CBM), (b) OS (chemiluminescence of whole blood - CWB, levels of thiobarbituric acid reacting substance - TBARS and of HNE- and MDA-protein adducts in plasma and activity of gamma-glutamyltransferase (GGT) in hind paw joint homogenates). The PI studied significantly increased the CBM of animals and corrected the HPW. STB also significantly decreased the activity of GGT in joint homogenates. SMe1 was more effective in decreasing plasmatic TBARS levels, but STB was more effective in reducing plasmatic HNE- and MDA-protein adducts. The assay for HNE- and MDA-adducts in plasma as a function of time was applied for the first time in AA. STB markedly decreased spontaneous and PMA-stimulated CWB and reduced neutrophil count. In summary, STB was more effective than SMe1 in reducing OS in AA. Our results showed that the reduction of OS in arthritis also corrected the clinical manifestations of the disease.
Evaluation of antioxidant activity of medicinal plants containing polyphenol compounds. Comparison of two extraction systems.
Authors: Kratchanova Maria , Denev Petko , Ciz Milan , Lojek Antonin , Mihailov Atanas
Published: March 2019

This study investigates the influence of extraction system on the extractability of polyphenol compounds and antioxidant activity of various medicinal plants. Oxygen radical absorbance capacity (ORAC) and total polyphenol content of 25 Bulgarian medicinal plants subjected to water or 80 % acetone extractions were investigated and compared. The type of extragent significantly influenced the efficiency of the polyphenol extraction and the antioxidant activity. In all cases ORAC results and total polyphenol content were higher for acetone extraction than for water extraction. The acetone extract of peppermint had the highest ORAC value - 2917 micromol Trolox equivalent (TE)/g dry weight (DW) and polyphenol content - 20216 mg/100 g DW. For water extraction thyme exhibited the highest ORAC antioxidant activity - 1434 micromol TE/g DW. There was a significant linear correlation between the concentration of total polyphenols and ORAC in the investigated medicinal plants. It can be concluded that the solvent used affects significantly the polyphenol content and the antioxidant activity of the extract and therefore it is recommended to use more than one extraction system for better assessment of the antioxidant activity of natural products. Several of the investigated herbs contain substantial amounts of free radical scavengers and can serve as a potential source of natural antioxidants for medicinal and commercial uses.
Suppressor of cytokine signaling and accelerated atherosclerosis in kidney disease.
Authors: Wesoly Joanna , Sikorski Krzysztof , Lee Chien-Kuo , Bluyssen Hans A R
Published: March 2019

The prevalence of cardiovascular disease in patients with renal failure is extremely high and accounts for a large part of the morbidity and mortality. Inflammation participates importantly in host defense against infectious agents and injury, but also contributes to the pathophysiology of many diseases, including cardiovascular atherosclerosis, which is a main problem in patients with renal failure. Recruitment of blood leukocytes to the injured vascular endothelium characterizes the initiation and progression of atherosclerosis and involves many inflammatory mediators, modulated by cells of both innate and adaptive immunity. Excessive inflammatory and immune responses, communicated by these different cell types, are driven by inflammatory cytokines that promote associated tissue damage if cytokine signaling pathways remain unregulated. Thus, pathways capable of suppressing proinflammatory cytokine signaling hold the potential to limit life-threatening cardiovascular events caused by atherogenesis. Suppressor of cytokine signaling (SOCS) are a family of intracellular proteins, several of which have emerged as key physiological regulators of cytokine-mediated homeostasis, including innate and adaptive immunity. Accumulating evidence supports the idea that dysregulation of cytokine signaling by differential SOCS expression is involved in the pathogenesis of various inflammatory, and immunological diseases, including atherosclerosis. Based on recent observations, in which SOCS expression levels are profoundly altered in kidney disease, we discuss the possibilities of SOCS as new intracellular markers of inflammation as well as their potential atherogenic properties in renal failure related cardiovascular disease.
Personal remarks on the future of protein crystallography and structural biology.
Authors: Jaskolski Mariusz
Published: March 2019

Protein crystallography, the main experimental method of structural biology, has undergone in the recent past three revolutionary changes leading to its unexpected renaissance. They were connected with (i) the introduction of synchrotron radiation sources for X-ray diffraction experiments, (ii) implementation of Se-Met multiwavelength anomalous diffraction (MAD) for phasing, and (iii) initiation of structural genomics (SG) programs. It can be foreseen that in the next 10-15 years protein crystallography will continue to be in this revolutionary phase. We can expect not only an avalanche of protein crystal structures from SG centers, but also attacking of more demanding projects, such as the structure of membrane proteins and of very large macromolecular complexes. On the technological front, the introduction of X-ray radiation from free-electron lasers will revolutionize the experimental possibilities, making feasible even the imaging of single molecules and of intact biological cells.
Microbial transformation of citral by Penicillium sp..
Authors: Esmaeili Akbar , Tavassoli Afsaneh
Published: March 2019

Thymol is present in the essential oils from herbs and spices, such as thyme. It is produced by these plant species as a chemical defense against phytopathogenic microorganisms. Therefore, this compound has attracted great attention in food industry, i.e., it has been used as a natural preservative in foods such as cheese to prevent fungal growth. Previous studies concerning the biotransformation of nerol by Penicillium sp. and microbial transformation of citral by sporulated surface cultures method (SSCM) of Penicillium digitatum have been reported. The objective of this research was to study the pathway involved during biotransformation of citral by Penicillium sp. using two methods. The culture preparation was done using different microbial methods and incubation periods to obtain Penicillium for citral biotransformation. The biotransformation products were identified by gas chromatography (GC) and gas chromatography/mass spectroscopy (GC/MS). A comparison of the two methods showed that SSCM was more effective, its major products were thymol (21.5 %), geranial (18.6 %) and nerol (13.7 %). LM produced only one compound — thymol — with a low efficiency.
DNAzyme as an efficient tool to modulate invasiveness of human carcinoma cells.
Authors: Wiktorska Magdalena , Papiewska-Pająk Izabela , Okruszek Andrzej , Sacewicz-Hofman Izabela , Niewiarowska Jolanta
Published: March 2019

In this study we evaluated efficiency of DNAzymes to modulate motility of cancer cells, an important factor in the progression and metastasis of cancers. For this purpose we targeted β1 integrins that are predominant adhesive receptors in various carcinoma cell lines (CX1.1, HT29, LOVO, LS180, PC-3). To evaluate invasiveness of cancer cells, we used a transwell migration assay that allowed analyzing chemotactic migration of colon carcinoma cell lines across an ECM-coated membrane. Their adhesive properties were also characterized by the analysis of adhesion to fibronectin, laminin and collagen. In addition, the expression of major integrin subunits, selected intact β1 integrins, and other adhesive receptors (ICAM, E-selectin, uPAR) was analyzed by flow cytometry. Inhibition of β1 integrin expression by DNAzyme to β1 mRNA almost abolished the invasiveness of the CX1.1, HT29, LS180, LOVO and PC-3 cells in vitro. These data show that DNAzymes to β1 integrin subunit can be used to inhibit invasiveness of carcinoma cells.
Distribution of apolipoprotein ai-containing lipoprotein subclasses in plasma of normolipidemic subjects.
Authors: Atmeh Ragheb F , Kasasbeh Amani Z , Abu Odeh Mohammad R
Published: March 2019

The distribution of apoA-I among apoA-I-containing lipoprotein (AI-Lp) subclasses in plasma was studied by immunoblotting utilizing agarose gel matrix incorporating anti-apoA-I as the transfer medium. Nine AI-Lp subclasses were detected in the plasma of normolipidemics, with relative molecular masses ranging from 70,000 to ≥ 354,000 and diameters from 7.12 to ≥ 11.6 nm. The mass distribution of AI-Lp subclasses was significantly different between males and females, and some subclasses increased gradually with age while others decreased. There was a significant strong positive correlation between subclass 1 (M(r) 70,000–75,000) and subclass 3 (M(r) 105,000–126,000) in all subjects and age groups. Analysis of similar AI-Lp or HDL subclasses reported in the literature showed variability in the sizes reported by various workers. This stresses the need for a unified classification of such subclasses, and this work contributes to this direction. The quantitative nature of the method used in this work compared with the semiquantitative approaches used earlier makes it a better method for the study of the quantitative changes of the subclasses in various physiological and pathological states. The method helps to generate ideas for in vitro and in vivo studies of apoA-I exchange among subclasses and in vivo kinetic studies. Conclusion. Plasma level of the AILp subclasses varied quantitatively with age and gender, and strong correlations were detected between some subclasses. This work contributes to a better classification of AI-Lp subclasses according to their size. Comparison of the method used here with the methods reported in the literature revealed its advantages.
Isolation and functional expression of a novel lipase gene isolated directly from oil-contaminated soil.
Authors: Zuo Kaijing , Zhang Lida , Yao Hongyan , Wang Jin
Published: March 2019

A lipase gene SR1 encoding an extracellular lipase was isolated from oil-contaminated soil and expressed in Escherichia coli. The gene contained a 1845-bp reading frame and encoded a 615-amino-acid lipase protein. The mature part of the lipase was expressed with an N-terminal histidine tag in E. coli BL21, purified and characterized biochemically. The results showed that the purified lipase combines the properties of Pseudomonas chlororaphis and other Serratia lipases characterized so far. Its optimum pH and temperature for hydrolysis activity was pH 5.5-8.0 and 37°C respectively. The enzyme showed high preference for short chain substrates (556.3±2.8 U/µg for C10 fatty acid oil) and surprisingly it also displayed high activity for long-chain fatty acid. The deduced lipase SR1 protein is probably from Serratia, and is organized as a prepro-protein and belongs to the GXSXG lipase family.
Pain and mobility improvement and MDA plasma levels in degenerative osteoarthritis, low back pain, and rheumatoid arthritis after infrared A-irradiation.
Authors: Siems Werner , Bresgen Nikolaus , Brenke Rainer , Siems Renate , Kitzing Manfred , Harting Heike , Eckl Peter M
Published: March 2019

Infrared (IR)-A irradiation can be useful in back and musculoskeletal pain therapy. In this study joint and vertebral column pain and mobility were measured during two weeks of IR-A irradiation treatment of patients suffering from degenerative osteoarthritis of hip and knee, low back pain, or rheumatoid arthritis. Additionally, before and after IR-A treatment MDA serum levels were measured to check if MDA variations accompany changes in pain intensity and mobility. Two-hundred and seven patients were divided into verum groups getting IR-irradiation, placebo groups getting visible, but not IR irradiation, and groups getting no irradiation. In osteoarthritis significant pain reduction according to Visual Analogue Scale and mobility improvements occurred in the verum group. Even though beneficial mean value changes occurred in the placebo group, the improvements in the placebo and No Irradiation groups were without statistical significance. In low back pain, pain and mobility improvements (by 35-40%) in the verum group were found, too. A delayed (2nd week) mobility improvement in rheumatoid arthritis was seen. However, pain relief was seen immediately. In patients suffering from low back pain or rheumatoid arthritis, the pain and mobility improvements were accompanied by significant changes of MDA serum levels. However, MDA appears not a sensitive biofactor for changes of the pain intensity in degenerative osteoarthritis. Nevertheless, unaffected or lowered MDA levels during intensive IR-A therapy argue against previous reports on free radical formation upon infrared. In conclusion, rapid beneficial effects of IR-A towards musculoskeletal pain and joint mobility loss were demonstrated.
Effects of high sucrose diet, gemfibrozil, and their combination on plasma paraoxonase 1 activity and lipid levels in rats.
Authors: Macan Marija , Vrkić Nada , Vrdoljak Ana Lucić , Radić Božica , Bradamante Vlasta
Published: March 2019

We investigated the influence of high sucrose diet (HSD) after 3 or 5 weeks of administration on paraoxonase 1 (PON1) activity in plasma of normolipidemic rats and the relationship between serum PON1 activity, triacylglycerides (TGs), HDL and total cholesterol vs. the control group of rats fed normal, control diet (CD). Because the data about the influence of gemfibrozil (GEM) on PON1 activity are controversial, we also investigated its effects (administration in the 4th and 5th week in rats on HSD and CD) on plasma PON1 activity and lipid levels in normolipidemic rats, and in rats with hypertriglyceridemia caused by HSD. Our results obtained in rats on HSD show a significant increase of plasma TGs levels by 47% (P
Cytokines of the Th1 and Th2 type in sera of rheumatoid arthritis patients; correlations with anti-Hsp40 immune response and diagnostic markers.
Authors: Tukaj Stefan , Kotlarz Agnieszka , Jóźwik Agnieszka , Smoleńska Zaneta , Bryl Ewa , Witkowski Jacek M , Lipińska Barbara
Published: March 2019

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease which affects approximately 1% of the population worldwide. Recent research on the role of heat shock proteins (Hsps) in RA development indicates that they may have pro- or anti-inflammatory effect, most probably via modulating cytokine secretion. We investigated type Th1 (INFγ, TNFα, IL-2) and type Th2 (IL-10, IL-6, IL-4) cytokine levels in sera of RA patients and healthy controls, using flow cytometric bead array assay, and searched for correlations between the cytokine levels and serum antibodies against bacterial (DnaJ) and human (Hdj1, Hdj2 and Hdj3) Hsp40 proteins, as well as clinical and laboratory parameters. The levels of all cytokines studied were significantly increased in RA patients; the highest increase relative to healthy controls (7-fold) was observed for IL-6 and its levels correlated positively with the antibodies directed to DnaJ and to the C-terminal domain of Hdj2, and with diagnostic parameters (DAS 28, Steinbrocker RTG criteria, ARA/7, ESR, TEN, SW and GH). INFγ levels correlated negatively with DAS 28, ESR, TEN and SW. No correlations were found for TNFα, IL-2 or IL-4. Our results support the hypothesis of Hsp40 involvement in RA as well as indicate that IL-6 serum level is a good marker of the RA activity.
Combined methotrexate and coenzyme Q₁₀ therapy in adjuvant-induced arthritis evaluated using parameters of inflammation and oxidative stress.
Authors: Bauerova Katarina , Paulovicova Ema , Mihalova Danica , Drafi Frantisek , Strosova Miriam , Mascia Cinzia , Biasi Fiorella , Rovensky Jozef , Kucharska Jarmila , Gvozdjakova Anna , Ponist Silvester
Published: March 2019

Rheumatoid arthritis is a common severe joint disease that affects all age groups, it is thus of great importance to develop new strategies for its treatment. The aim of the present study was to examine the combined effect of coenzyme Q₁₀ (CoQ₁₀) and methotrexate (MTX) on the progression of adjuvant-induced arthritis in rats. Adjuvant arthritis (AA) was induced by a single intradermal injection of heat-inactivated Mycobacterium butyricum in incomplete Freund's adjuvant. The experiments included healthy animals, arthritic animals not treated, arthritic animals treated with CoQ₁₀, with methotrexate, and with a combination of CoQ₁₀ and methotrexate. The two latter groups received a daily oral dose of 20 mg/kg b.w. of CoQ₁₀, either alone or with methotrexate in an oral dose of 0.3 mg/kg b.w. twice a week. We found that CoQ₁₀ potentiated both the antiarthritic (decrease of hind paw volume) and the antioxidant effect of methotrexate on the level of oxidation of proteins (suppression of protein carbonyl level in plasma) as well as lipoperoxidation (suppression of levels of HNE-adducts and MDA-adducts to plasma proteins). The same effect was observed for plasmatic levels of CoQ₉ and IL-1α, and partially also for γ-glutamyltransferase activity assessed in joints and spleen. Moreover, the combination therapy improved the functionality of peripheral blood neutrophils in AA, with a balancing effect on the immunosuppression caused by MTX monotherapy. In summary, combined administration of CoQ₁₀ and methotrexate suppressed arthritic progression in rats more effectively than did MTX alone. This finding may help improve treatment of rheumatoid arthritis.
Depletion of intracellular glutathione and increased lipid peroxidation mediate cytotoxicity of hematite nanoparticles in MRC-5 cells.
Authors: Radu Mihaela , Munteanu Maria Cristina , Petrache Sorina , Serban Andreea Iren , Dinu Diana , Hermenean Anca , Sima Cornelia , Dinischiotu Anca
Published: March 2019

Particles generated from numerous anthropogenic and/or natural sources, such as crystalline α-Fe₂O₃ nanoparticles, have the potential to damage lung cells. In our study we investigated the effects of these nanoparticles (12.5 µg/ml) on lipid peroxidation and the antioxidative system in MRC-5 lung fibroblast cells following exposure for 24, 48 or 72h. Exposure to α-Fe₂O₃ nanoparticles increased lipid peroxidation by 81%, 189% and 110% after 24, 48 and 72h, respectively. Conversely, the reduced glutathione concentration decreased by 23.2% and 51.4% after 48 and 72h of treatment, respectively. In addition, an augmentation of the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione transferase and glutathione reductase within the interval between 48-72h was noticed. Taking into account that the reduced glutathione level decreased and the malondialdehyde level, a lipid peroxidation product, remained highly increased up to 72h of exposure, it would appear that the MRC-5 antioxidant defense mechanisms did not efficiently counteract the oxidative stress induced by exposure to hematite nanoparticles.
Fluorometric assay of oleate-activated phospholipase D isoenzyme in membranes of rat nervous tissue and human platelets.
Authors: Krzystanek Marek , Trzeciak Henryk I , Krzystanek Ewa , Małecki Andrzej
Published: March 2019

Phospholipase D plays a key role in the biosynthesis of phosphatidic acid, a second messenger involved in essential cellular processes. Oleate-activated phospholipase D was the first mammalian phospholipase D isoform to be discovered but is the least known. The study was aimed to test a fluorometric method of assessment of oleate-activated phospholipase D activity in different biological materials. The brain cortex of male Wistar rats, cultured rat brain astrocytes, and human platelets were processed to yield plasmatic membranes for experiments. To assess phospholipase D activity the modified fluorometric method was used. Previously, the method was used only to determine H₂O₂. In this enzyme-coupled assay phospholipase D activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine. First, phospholipase D cleaves exogenous phosphatidylcholine to yield choline and phosphatidic acid. Second, choline is oxidized by choline oxidase to betaine and H₂O₂. Finally, in the presence of horseradish peroxidase, H₂O₂ reacts with 10-acetyl-3,7-dihydroxyphenoxazine to generate the highly fluorescent product, resorufin. The concentration of resorufin was measured using excitation and emission at 560 nm and 590 nm, respectively. The proposed optimal parameters of the tested assay are 25 µg of rat brain cortex protein, 50 µg of rat brain astrocyte protein, and 50 µg of human platelet protein in a reaction volume of 200 µL, and 2 min enzymatic reaction at 37°C. The fluorometric method may be applied to assay phospholipase D in different biological materials.
Conjugated linoleic acids regulate triacylglycerol and cholesterol concentrations in macrophages/foam cells by the modulation of CD36 expression.
Authors: Stachowska Ewa , Baskiewicz Magdalena , Marchlewicz Mariola , Czupryńska Katarzyna , Kaczmarczyk Mariusz , Wiszniewska Barbara , Machaliński Bogusław , Chlubek Dariusz
Published: March 2019

Atherosclerosis is an inflammatory disease characterised by the accumulation of lipids and their metabolites in the artery wall. During inflammation circulating LDL are taken up by macrophages through two major scavenger receptors: CD36 and scavenger receptor A (SRA). Fatty acids that are common in food, e.g. linoleic acid and n-3 unsaturated fatty acids can modulate expression of CD36 on the macrophage surface. Conjugated linoleic acid isomers (CLA) that originate from the human diet, have demonstrated antiatherogenic properties in several experiments. Animal study evidenced that CLA could induce resolution of plaque by activation of peroxisome proliferator activated receptors and down-regulation of pro-inflammatory genes. Less unequivocal results were obtained in human studies (on the CLA effects on the inflammatory process). Therefore in this study we investigated the influence of CLA on CD36 expression and lipid accumulation in human macrophages. Macrophages were incubated with 30 μM cis-9,trans-11 CLA, trans-10,cis-12 CLA or linoleic acid for 48 h. After that, expression of CD36 as well as accumulation of lipids were measured by flow cytometry, microscopy and a spectroscopic method. We demonstrate that both cis-9,trans-11 C 18 : 2 CLA and linoleic acid slightly elevated expression of CD36, whereas second isomer — trans-10,cis-12 CLA — did not. Nevertheless, only trans-10,cis-12 CLA triggered delipidation of macrophages, that is decreased triacylglycerols concentration. Also in human adipocytes, trans-10,cis-12 CLA causes cell delipidation by reduction of PPAR receptor expression. We propose a similar mechanism for human macrophages/foam cells.
Impact of diabetes-associated lipoproteins on oxygen consumption and mitochondrial enzymes in porcine aortic endothelial cells.
Authors: Xie Xueping , Chowdhury Subir Roy , Sangle Ganesh , Shen Garry X
Published: March 2019

Impairments in mitochondrial function have been proposed to play an important role in the pathogenesis of diabetes. Atherosclerotic coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Mitochondrial dysfunction and increased production of reactive oxygen species (ROS) are associated with diabetes and CAD. Elevated levels of glycated low density lipoproteins (glyLDL) and oxidized LDL (oxLDL) were detected in patients with diabetes. Our previous studies demonstrated that oxLDL and glyLDL increased the generation of ROS and altered the activities of antioxidant enzymes in vascular endothelial cells (EC). The present study examined the effects of glyLDL and oxLDL on mitochondrial respiration, membrane potential and the activities and proteins of key enzymes in mitochondrial electron transport chain (mETC) in cultured porcine aortic EC (PAEC). The results demonstrated that glyLDL or oxLDL significantly reduced oxygen consumption in Complex I, II/III and IV of mETC in PAEC compared to LDL or vehicle control using oxygraphy. Incubation with glyLDL or oxLDL significantly reduced mitochondrial membrane potential, the activities of mitochondrial ETC enzymes - NADH dehydrogenase (Complex I), succinate cytochrome c reductase (Complex II + III), ubiquinol cytochrome c reductase (Complex III), and cytochrome c oxidase (Complex IV) in PAEC compared to LDL or control. Treatment with oxLDL or glyLDL reduced the abundance of subunits of Complex I, ND1 and ND6 in PAEC. However, the effects of oxLDL on mitochondrial activity and proteins were not significantly different from glyLDL. The findings suggest that the glyLDL or oxLDL impairs mitochondrial respiration, as a result from the reduction of the abundance of several key enzymes in mitochondria of vascular EC, which potentially may lead to oxidative stress in vascular EC, and the development of diabetic vascular complications.
Metabolic control analysis of integrated energy metabolism in permeabilized cardiomyocytes - experimental study.
Authors: Tepp Kersti , Timohhina Natalja , Chekulayev Vladimir , Shevchuk Igor , Kaambre Tuuli , Saks Valdur
Published: March 2019

The main focus of this research was to apply Metabolic Control Analysis to quantitative investigation of the regulation of respiration by components of the Mitochondrial Interactosome (MI, a supercomplex consisting of ATP Synthasome, mitochondrial creatine kinase (MtCK), voltage dependent anion channel (VDAC), and tubulin) in permeabilized cardiomyocytes. Flux control coefficients (FCC) were measured using two protocols: 1) with direct ADP activation, and 2) with MtCK activation by creatine (Cr) in the presence of ATP and pyruvate kinase-phosphoenolpyruvate system. The results show that the metabolic control is much stronger in the latter case: the sum of the measured FCC is 2.7 versus 0.74 (ADP activation). This is consistent with previous data showing recycling of ADP and ATP inside the MI due to the functional coupling between MtCK and ANT and limited permeability of VDAC for these compounds, PCr being the major energy carrier between the mitochondria and ATPases. In physiological conditions, when the MI is activated, the key sites of regulation of respiration in mitochondria are MtCK (FCC = 0.93), adenine nucleotide translocase ANT (FCC = 0.95) and CoQ cytochrome c oxidoreductase (FCC = 0.4). These results show clearly that under the physiological conditions the energy transfer from mitochondria to the cytoplasm is regulated by the MI supercomplex and is very sensitive to metabolic signals.
Methotrexate binding causes structural and functional changes in lung cystatin.
Authors: Khan Mohd Shahnawaz , Priyadarshini Medha , Sumbul Sadia , Bano Bilqees
Published: March 2019

Regulation of cysteine proteinases and their inhibitors is of utmost importance in diseases like lung cancer, chronic inflammatory conditions such as asthma, emphysema, and idiopathic pulmonary fibrosis. Protease-antiprotease imbalance accelerates disease progression. In the present study, the effect of antineoplastic and antirheumatic drug methotrexate (MTX) on lung cystatin (a cysteine protease inhibitor) was studied to explore drug induced changes in functional and structural integrity of the protein. The basic binding interaction was studied by UV-absorption, FT-IR and fluorescence spectroscopy. The quenching of protein fluorescence confirmed the binding of MTX with goat lung cystatin (GLC-I). Stern-Volmer analysis of MTX-GLC-I system at different temperatures indicates the presence of static component in the quenching mechanism. The thermodynamic parameters ΔH⁰ and ΔS⁰ were -3.8 kJ/mol and 94.97 J•mol⁻¹•K⁻¹, respectively, indicating that both hydrogen bonds and hydrophobic interactions played a major role in the binding of MTX to GLC-I. Methotrexate (7 µM) caused complete inactivation of lung cystatin after 6 hours. The results of FT-IR spectroscopy reflect perturbation of the goat lung cystatin on interaction with MTX. Methotrexate induced loss of function change in the inhibitor could provide a rationale for the off target tissue injury caused by the drug and for the design of agents against such an injury.
Comparative proteomic analysis of Bombyx mori hemolymph and fat body after calorie restriction.
Authors: Chen Huiqing , Li Yijia , Chen Keping , Yao Qin , Li Guohui , Wang Lin
Published: March 2019

Calorie restriction (CR) is known to extend life span from yeast to mammals. To gain an insight into the effects of CR on growth and development of the silkworm Bombyx mori at protein level, we employed comparative proteomic approach to investigate proteomic differences of hemolymph and fat body of the silkworm larvae subjected to CR. Thirty-nine differentially expressed proteins were identified by MALDI TOF/TOF MS. Among them, 19 were from the hemolymph and 20 from the fat body. The hemolymph of the CR group contained two down-regulated and 17 up-regulated proteins, whereas the fat body contained 15 down-regulated and five up-regulated ones. These proteins belonged to those functioning in immune system, in signal transduction and apoptosis, in regulation of growth and development, and in energy metabolism. Our results suggest that CR can alter the expression of proteins related to the above four aspects, implying that these proteins may regulate life span of the silkworm through CR.
Raised plasma insulin level and homeostasis model assessment (HOMA) score in cerebral malaria: evidence for insulin resistance and marker of virulence.
Authors: Eltahir Elrashid M , El Ghazali Gehad , A-Elgadir Thoraya M E , A-Elbasit Ishraga E , Elbashir Mustafa I , Giha Hayder A
Published: March 2019

To study the glycaemic profile of patients with severe malaria (SM).
For this purpose, 110 SM patients were recruited. Pre-treatment random blood glucose and plasma insulin were measured in a subset of donors. An ex-vivo experiment was developed for estimation of glucose consumption by parasitized erythrocytes.
Hyperglycaemia was frequent in SM but more commonly associated with cerebral malaria (CM), while hyperinsulinaemia was recognized in severe-malarial-hypotension (median, 25 %-75 %, 188.2, 93.8-336.8 pmol/L). The plasma insulin level was positively correlated with age (CC = 0.457, p < 0.001) and negatively with parasitaemia (CC = -0.368, p = 0.045). Importantly, fatal-CM was associated with hyperglycaemia (12.22, 6.5-14.6 mmol/L), hyperinsulinaemia (141.0, 54.0-186.8 pmol/L) and elevated homeostasis model assessment (HOMA) values. However, there was a trend of higher glucose consumption by parasites in CM compared with that in uncomplicated malaria (UM).
Hyperglycaemia, hyperinsulinaemia and elevated HOMA are evidence for insulin resistance and possibly pancreatic B-cell dysfunction in fatal-CM.
Identification of novel putative regulators of the major apoptotic nuclease DNA Fragmentation Factor.
Authors: Hanus Jakub , Kalinowska-Herok Magdalena , Widlak Piotr
Published: March 2019

Yeast two- and three-hybrid systems were used to screen cDNA libraries from HeLa cells and human brain tissue to identify novel protein partners of DNA Fragmentation Factor, the major apoptotic nuclease. The two-hybrid system revealed the DFF45 inhibitory subunit of the nuclease as the only identified partner of the DFF40 catalytic subunit. Similar analysis revealed several protein candidates that potentially interact with the DFF45 subunit: FBXO28, FOSL1, PGK1, PCNT, FHL1 and GFAP. Recombinant GFAP protected DFF45 against cleavage with caspase-3 and prevented activation of the DFF nuclease in vitro. In addition, three-hybrid system results revealed a putative novel protein partner of the DFF40-DFF45 heterodimer. The candidate cDNA contained two open reading frames that mapped to an intron of the GBF1 gene. Products of the candidate cDNA derived from a cell-free transcription/translation system inhibited DNA cleavage by recombinant caspase-activated DFF. This putative partner of DFF may have functional importance in regulating the apoptotic response because its RNAi silencing facilitated cleavage of the DFF45 inhibitor subunit and affected chromatin fragmentation in HeLa cells undergoing apoptosis. This hypothetical protein, named DRIG based on an acronym specifying its genomic location, could be a novel factor involved in regulation of DFF40 apoptotic nuclease.
Expression, purification and functional characterization of recombinant human acyl-CoA-binding protein (ACBP) from erythroid cells.
Authors: Augoff Katarzyna , Kolondra Adam , Chorzalska Anna , Lach Agnieszka , Grabowski Krzysztof , Sikorski Aleksander F
Published: March 2019

Fatty acyl-CoA esters are extremely important in cellular homeostasis. They are intermediates in both lipid metabolism and post-translational protein modifications. Among these modification events, protein palmitoylation seems to be unique by its reversibility which allows dynamic regulation of the protein hydrophobicity. The recent discovery of an enzyme family that catalyze protein palmitoylation has increased the understanding of the enzymology of the covalent attachment of fatty acids to proteins. Despite that, the molecular mechanism of supplying acyl-CoA esters to this reaction is yet to be established. Acyl-coenzyme A-binding proteins are known to bind long-chain acyl-CoA esters with very high affinity. Therefore, they play a significant role in intracellular acyl-CoA transport and pool formation. The purpose of this work is to explore the potential of one of the acyl-CoA-binding proteins to participate in the protein palmitoylation. In this study, a recombinant form of ACBP derived from human erythroid cells was expressed in E. coli, purified, and functionally characterized. We demonstrate that recombinant hACBP effectively binds palmitoyl-CoA in vitro, undergoing a shift from a monomeric to a dimeric state, and that this ligand-binding ability is involved in erythrocytic membrane phosphatidylcholine (PC) remodeling but not in protein acylation.
Effect of tunicamycin on the biogenesis of hepatitis C virus glycoproteins.
Authors: Reszka Natalia , Krol Ewelina , Patel Arvind H , Szewczyk Boguslaw
Published: March 2019

Hepatitis C virus (HCV) infects humans, with a prevalence around 3% of population, causing acute and chronic hepatitis and hepatocellular carcinoma. We studied the effect of inhibition of glycosylation on the assembly of the HCV particle. HCV possesses two envelope glycoproteins E1 and E2 that are highly modified by N-glycans. These glycan residues are crucial for viral entry and maturation of the progeny. Here, we examined the influence of inhibition of N-glycosylation on expression of E1 and E2. Since the propagation of HCV in cell culture is limited, we used a recombinant baculovirus producing viral-like particles in insect cells. Our data showed that blocking of N-glycan transfer to the nascent polypeptide chain with the antibiotic tunicamycin resulted in the loss of E1 and E2. We also found that a dose of tunicamycin that did not influence the cell viability significantly reduced the E2 level in infected cells. The results indicate that blocking of glycosylation at an early step efficiently reduces the assembly of HCV virions. Thus, we suggest that derivatives of tunicamycin that preferentially block glycosylation of viral proteins may become potential therapeutic agents against HCV.
Atorvastatin improves tubular status in non-diabetic patients with chronic kidney disease - placebo controlled, randomized, cross-over study.
Authors: Renke Marcin , Tylicki Leszek , Rutkowski Przemysław , Neuwelt Alexander , Larczyński Wojciech , Ziętkiewicz Marcin , Aleksandrowicz Ewa , Lysiak-Szydłowska Wiesława , Rutkowski Bolesław
Published: March 2019

There is evidence that dyslipidemia is associated with chronic kidney disease (CKD) and it has been implicated in the progression of renal damage. Optimal management of dyslipidemia should therefore lead to renal benefits. A number of experimental models demonstrate a beneficial effect of statins in ameliorating renal damage. However, the exact mechanism by which statins protect against renal damage remains unclear.
In a placebo-controlled, randomized, cross-over study we evaluated the influence of atorvastatin (ATO) 40 mg/day added to the renin-angiotensin-aldosterone systeme (RAAS) blockade on proteinuria and surrogate biomarkers of tubular damage or injury in 14 non-diabetic patients with proteinuria (0.4-1.8 g per 24 h) with normal or declined kidney function (eGFR 55-153 ml/min). In the eight-week run-in period, therapy using angiotensin converting enzyme inhibitors (ACEI) and/or angiotensin II subtype 1 receptor antagonists (ARB) was adjusted to achieve a blood pressure below 130/80 mm Hg. Next, patients were randomly assigned to one of two treatment sequences: ATO/washout/placebo or placebo/washout/ATO. Clinical evaluation and laboratory tests were performed at the randomization point and after each period of the study. The primary end point of this study was a change in proteinuria measured as 24-h urine protein excretion (DPE). Secondary end points included urine N-acetyl-β-D-glucosaminidase (NAG) and α1-microglobulin (α1m) excretion.
The ATO therapy significantly reduced urine excretion of α₁m (p=0.033) and NAG (p=0.038) as compared to placebo. There were no differences in proteinuria, blood pressure, eGFR and serum creatinine between the ATO and placebo groups.
Atorvastatin treatment is safe and improves biomarkers of tubular damage or injury in non-diabetic patients with CKD.
Comparison of the utility of five commercial kits for extraction of DNA from Aspergillus fumigatus spores.
Authors: Nawrot Urszula , Wlodarczyk Katarzyna , Wrobel Magdalena , Wasik Anita , Dobosz Tadeusz
Published: March 2019

The aim of this study was to compare the efficiency of DNA extraction from water as well as from blood samples spiked with A. fumigatus spores, using selected commercial kits. Extraction of DNA according to manufacturer's protocols was preceded by blood cells lysis and disruption of fungal cells by enzymatic digestion or bead beating. The efficiency of DNA extraction was measured by PCR using Aspergillus-specific primers and SYBR Green I dye or TaqMan probes targeting 28S rRNA gene. All methods allowed the detection of Aspergillus at the lowest tested density of water suspensions of spores (10¹ cells/ml). The highest DNA yield was obtained using the ZR Fungal/Bacterial DNA kit, YeastStar Genomic DNA kit, and QIAamp DNA Mini kit with mechanical cell disruption. The ZR Fungal/Bacterial DNA and YeastStar kits showed the highest sensitivity in examination of blood samples spiked with Aspergillus (100 % for the detection of 10² spores and 75 % for 10¹ spores). Recently, the enzymatic method ceased to be recommended for examination of blood samples for Aspergillus, thus ZR Fungal/Bacterial DNA kit and QIAamp DNA Mini kit with mechanical cell disruption could be used for extraction of Aspergillus DNA from clinical samples.
Adult-type hypolactasia and lactose malabsorption in Poland.
Authors: Mądry Edyta , Lisowska Aleksandra , Kwiecień Jarosław , Marciniak Ryszard , Korzon-Burakowska Anna , Drzymała-Czyż Sławomira , Mojs Ewa , Walkowiak Jarosław
Published: March 2019

The available data on the incidence of lactose malabsorption are contradictory. Potential bias in random selection is a major drawback of studies performed to-date. Moreover, molecular analysis of polymorphism -13910 C>T upstream of the lactase (LCT) gene (NM_005915.4:c.1917+326C>T; rs4988235) has not been reported in those studies. Therefore, in this study we aimed to assess genetic predisposition and clinical manifestation of adult-type hypolactasia (ATH).
In two-hundred randomly chosen healthy subjects (HS) aged from 18 to 20 years, the presence of -13910 C>T polymorphic variants upstream of the LCT gene was assessed. In a subgroup of subjects with genotype predisposing to ATH, hydrogen-methane breath test (BT) with lactose loading was conducted to determine the current state of lactase activity. In addition, clinical symptoms typical for lactose malabsorption were assessed using the questionnaire method.
Sixty-three out of 200 (31.5 %) HS had -13910 C/C genotype. Thus, genetically determined lactase persistence is expected in the remaining 137 (68.5 %) subjects. Thirteen out of 53 (24.5 %) HS having -13910 C/C genotype were proved to be lactose intolerant. Recalculating the data for the entire studied population it implies the incidence of lactose malabsorption in 7.7 % of subjects. Only three out of 13 (23.1 %) subjects with abnormal BT results, reported clinical symptoms related to lactose consumption.
Significantly lower than previously reported incidence of clinically detectable lactose malabsorption in young healthy adults in Poland has been documented. The -13910 C/C genotype upstream of the LCT gene indicates a predisposition to ATH, but definitely does not define the current ability to tolerate lactose.