Mechanisms of regulation of PFKFB expression in pancreatic and gastric cancer cells

Enzymes 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and -4 (PFKFB-3 and PFKFB-4) play a significant role in the regulation of glycolysis in cancer cells as well as its proliferation and survival. The expression of these mRNAs is increased in malignant tumors and strongly induced in different cancer cell lines by hypoxia inducible factor (HIF) through active HIF binding sites in promoter region of PFKFB-4 and PFKFB-3 genes. Moreover, the expression and hypoxia responsibility of PFKFB-4 and PFKFB-3 was also shown for pancreatic (Panc1, PSN-1, and MIA PaCa-2) as well as gastric (MKN45 and NUGC3) cancer cells. At the same time, their basal expression level and hypoxia responsiveness vary in the different cells studied: the highest level of PFKFB-4 protein expression was found in NUGC3 gastric cancer cell line and lowest in Panc1 cells, with a stronger response to hypoxia in the pancreatic cancer cell line. Overexpression of different PFKFB in pancreatic and gastric cancer cells under hypoxic condition is correlated with enhanced expression of vascular endothelial growth factor (VEGF) and Glut1 mRNA as well as with increased level of HIF-1α protein. Increased expression of different PFKFB genes was also demonstrated in gastric, lung, breast, and colon cancers as compared to corresponding non-malignant tissue counterparts from the same patients, being more robust in the breast and lung tumors. Moreover, induction of PFKFB-4 mRNA expression in the breast and lung cancers is stronger than PFKFB-3 mRNA. The levels of both PFKFB-4 and PFKFB-3 proteins in non-malignant gastric and colon tissues were more pronounced than in the non-malignant breast and lung tissues. It is interesting to note that Panc1 and PSN-1 cells transfected with dominant/negative PFKFB-3 (dnPFKFB-3) showed a lower level of endogenous PFKFB-3, PFKFB-4, and VEGF mRNA expressions as well as a decreased proliferation rate of these cells. Moreover, a similar effect had dnPFKFB-4. In conclusion, there is strong evidence that PFKFB-4 and PFKFB-3 isoenzymes are induced under hypoxia in pancreatic and other cancer cell lines, are overexpressed in gastric, colon, lung, and breast malignant tumors and undergo changes in their metabolism that contribute to the proliferation and survival of cancer cells. Thus, targeting these PFKFB may therefore present new therapeutic opportunities.

Effect of hypoxia and glutamine or glucose deprivation on the expression of retinoblastoma and retinoblastoma-related genes in ERN1 knockdown glioma U87 cell line

The expression of retinoblastoma and several retinoblastoma-related genes was studied in glioma cell line U87 and its subline with knockdown of ERN1 (endoplasmic reticulum—nuclei-1), the main endoplasmic reticulum stress sensing and signaling enzyme. It was shown that a blockade of the ERN1 enzyme function increases the expression levels of retinoblastoma, retinoblastoma-like 1 and most retinoblastoma related genes: EID1, JARID1B, E2F1, E2F3, RBAP48 and CTIP, does not change RNF40 and RBAP46 and decreases KDM5A. We have also demonstrated that hypoxia reduces the expression levels of retinoblastoma, EID1, and E2F1 in ERN1-deficient glioma cells only. At the same time, the expression levels of retinoblastoma-like 1, E2F3, RBAP46, RBAP48 and CTIP decrease, while JARID1B and RBBP2 increase in both types of cells in hypoxic conditions, but the expression is much stronger in cells with suppressed function of ERN1. The expression level of JARID1B and KDM-5A mRNA is also enhanced in glutamine deprivation condition in both tested cell types, moreover, this effect is amplified by the blockade of the ERN1 enzyme function. The expression levels of retinoblastoma, EID1, RBAP48, and E2F3 are decreased in glutamine deprivation condition only in ERN1-deficient glioma cells, but RBL1, CTIP, RBAP46, and E2F1—in both tested cell types with more significant effect in ERN1-deficient cells. Glucose deprivation condition leads to a decrease of expression levels of retinoblastoma, RBL1, E2F3, RBAP46, and RBAP48 in both used cell types and of EID1 and E2F1 only in glioma cells with suppressed function of signaling enzyme ERN1. Thus, expression levels of retinoblastoma and most retinoblastoma-related genes are increased under a blockade of ERN1 enzyme function and significantly changed in hypoxia, glucose or glutamine deprivation conditions both in control U87 cells and ERN1-deficient cells, but inhibition of the unfolded protein response sensor ERN1 predominantly enhances these effects. Moreover, it is possible that the induction of the expression of retinoblastoma and most retinoblastoma-related genes after knockdown of ERN1 plays an important role in suppression of glioma proliferation.

Effect of Hypoxia on the Expression of a Subset of Proliferation Related Genes in IRE1 Knockdown U87 Glioma Cells

We have studied the expression of a subset of genes encoding important tumor growth related factors in U87 glioma cells with IRE1 (inositol requiring enzyme-1) knockdown as well as their hypoxic regulation. It was shown that the expression levels of activating transcription factor 6 (ATF6), clusterin (CLU), adhesion G protein-coupled receptor E5 (ADGRE5), transglutaminase?2, C polypeptide (TGM2), leukemia inhibitory factor (LIF), phosphoserine aminotransferase 1 (PSAT1), glyoxalase I (GLO1) and tetraspanin 13 (TSPAN13) are significantly down-regulated in glioma cells with the knockdown of IRE1 signaling enzyme. It was also shown that in glioma cells subjected to hypoxia, the expression levels of PSAT1, TSPAN13, EIF2AK3, and TGM2 genes were up-regulated, whereas the expression of ATF6 gene was down-regulated. At the same time, the expression levels of LIF, CLU, and ADGRE5 genes did not change in response to hypoxic treatment.?Furthermore, inhibition of IRE1, a key effector of an unfolded protein response pathway, modified the effect of hypoxia on the expression of most studied genes. Present study demonstrates that IRE1 knockdown down-regulated the expression of most studied genes and modified their hypoxic regulation and that these changes possibly contributed to the suppression of glioma growth in cells without IRE1 signaling enzyme function.

The Content of Selenium and Other Ten Chemical Elements in Boletus Edulis from Bulgaria and Inhibition of Lipid Peroxidation

The goal of this study was to determine the content of such biological active metals as Se, Hg, AI, Cu, Zn, As, Cd, Pb, Mg, Ca and Fe in Boletus Edulis mushrooms and to study the effect of mushrooms as inhibitors of blood serum copper-initiated lipid peroxidation. The metals content was determined by ICP-OES technique and blood lipids peroxidation in vitro was assessed by thiobarbituric acid-reactive substances measurement. The dependency between quality and content of the determined biological active metals has been traced. Samples were analyzed of wild growing mushrooms Boletus Edulis from two mountain regions in Bulgaria. On the average the content of Se in Boletus Edulis was found to be 25 mg/kg dried mushroom, this content being higher in tubules than in fleshy part. We found that Boletus Edulis mushrooms inhibited lipid peroxidation in the concentration dependent manner. The effective concentration of Boletus Edulis is in 5 times lower compared to the concentration of Cantharellus Cibarius resulting in similar lipid peroxidation inhibition. This effect can be explained by 56 times higher content of Se and by 1.5 and 3 times lower content of such initiators of lipid peroxidation as Cu and Fe in Boletus Edulis compared to Cantharellus Cibarius. A system with a source of infrared radiation heating, developed by authors, was used for the mushroom mineralization. We conclude that Boletus Edulis is an effective inhibitor of blood lipid peroxidation and in 5 times stronger rather than Cantharellus Cibarius.

Protective Effect of Ubiquinone and Precursors of ItsSynthesis on Mitochondrial Respiratory Chain andActivity of Matrix Metalloproteinases in Animal Tissuesunder Effect of Doxorubicin

Mitochondrial dysfunction, oxidative stress, and their regulation are important fields of study in modem clinical research.Exogenous CoQ is an efficient therapeutic agent, yet its application has leads to continued suppression of endogenous CoQ synthesis,which limits CoQ applicability. Our aim was to study the state of mitochondrial electron transport chain components, CoQ contentand redox state, superoxide anion radicals and NO production rates, and active MMP-2 and MMP-9 content in rat liver and heartunder treatment with Doxorubicin, CoQ10, and complex preparation of modulators and precursors of CoQ biosynthesis （EPMcomplex）. The results demonstrate that treatment with EPM complex and CoQ10 in addition to Doxorubicin administration exertsprotective effect on liver and heart mitochondria, evidenced by restoration of electron transport in respiratory chain, which isexpressed as decreased nitrile complexes formation with Fe-S-proteins and increased ubisemiquinone content. The protective effectsof EPM complex on mitochondrial electron transport chain under Doxorubicin administration is on par with those of CoQ10, anddecreased MMP2 and MMP9 activities signify lessened extracellular matrix destruction. These results demonstrate the viability ofapproaches to correct adverse effects of Doxorubicin by treatment with CoQ10 and e complex of precursors and modulators of itsbiosynthesis.

Mesenchymal stem cells as a potential therapeutic tool to cure cognitive impairment caused by neuroinflammation

An established contribution of neuroinflammation to multiple brain pathologies has raised the requirement for therapeutic strategies to overcome it in order to prevent age-and disease-dependent cognitive decline.Mesenchymal stem cells(MSCs)produce multiple growth and neurotrophic factors and seem to evade immune rejection due to low expression of major histocompatibility complex class I molecules.Therefore,MSCs are widely used in experiments and clinical trials of regenerative medicine.This review summarizes recent data concerning the optimization of MSC use for therapeutic purposes with the emphasis on the achievements of the last 2 years.Specific attention is paid to extracellular vesicles secreted by MSCs and to the role ofα7 nicotinic acetylcholine receptors.The reviewed data demonstrate that MSCs have a significant therapeutic potential in treating neuroinflammation-related cognitive disfunctions including age-related neurodegenerative diseases.The novel data demonstrate that maximal therapeutic effect is being achieved when MSCs penetrate the brain and produce their stimulating factors in situ.Consequently,therapeutic application using MSCs should include measures to facilitate their homing to the brain,support the survival in the brain microenvironment,and stimulate the production of neurotrophic and anti-inflammatory factors.These measures include but are not limited to genetic modification of MSCs and pre-conditioning before transplantation.

Blood coagulation factors and platelet response to drug-induced hepatitis and hepatosis in rats

Background:Knowing the variability of blood coagulation responses to liver damage of different origins can provide a key to curing liver tissues or to mitigating treatment side effects.The aim of the present work was to compare the changes in the main components of hemostasis under experimental drug-induced hepatosis and hepatitis in rats.Methods:We modeled diclofenac-induced hepatitis and tetracycline-induced hepa-tosis.Hemostasis response was gauged by measuring fibrinogen,factor X,protein C(PC),and prothrombin in plasma.The decarboxylated form of prothrombin was de-tected by measuring prothrombin index and ecamulin index.Platelet reactivity was studied using aggregometry.Results:Both hepatitis and hepatosis decreased the synthesis of fibrinogen,factor X,and prothrombin.However,protein carboxylation was not disrupted in hepatosis but was much impaired in hepatitis.PC decreased in both models as a consequence of its consumption possibly during inflammatory response.Platelet aggregation rate was lower in hepatosis but higher in hepatitis.Conclusions:Our findings imply the need for a thorough monitoring of the hemostasis system in liver diseases to avoid possible thrombotic complications.Its state indicates the disorder's rate and character.

The vascular endothelial growth factor genes expression in glioma U87 cells is dependent from ERN1 signaling enzyme function

The expression of different vascular endothelial growth factor (VEGF) genes was studied in glioma U87 cells with endoplasmic reticulum–nuclei-1 (ERN1) loss of function and its regulation by hypoxia and glutamine or glucose deprivation conditions as model of ischemia. The blockade of function of the ERN1 enzyme, which is a major sensor of endoplasmic reticulum stress, leads to a decrease of the VEGFA, VEGFB and VEGFC mRNA expression level. The level of VEGFA proteins also decreases at this experimental condition in the cytosolic fraction, but increases in the nuclear fraction. Hypoxia does not affect VEGFC and increases the expression level of VEGFA and VEGFB mRNA in both used cell types, however, the change was much less profound in cells with suppressed function of ERN1. The expression level of VEGFC mRNA decreases in both used cell types in glutamine deprivation condition, however, the change was more profound in control glioma cells. At the same time, the expression level of VEGFA mRNA increases and VEGFB—decreases in gluta-mine deprivation condition in control glioma cells only. Exposure of glioma cells to glucose deprivation condition increases VEGFB mRNA expression level in both used cell types;however, VEGFA—in control glioma cells only and VEGFC—in cells with ERN1 signaling enzyme loss of function only. Thus, the results of this study clearly demonstrated the down-regulation of the expression of all three VEGF genes in glioma cells with ERN1 loss of function which correlates to the suppressed angiogenesis and proliferation rate of these cells. Moreover, the effect of hy-poxia and glutamine or glucose deprivation condition on the expression level of all VEGF genes is different and mainly depends on ERN1 signaling enzyme function.

Meizothrombin Preparation and Its Role in Fibrin Formation and Platelet Aggregation

Meizothrombin (MT) is one of prothrombin derivatives which appears in haemostasis activation area. However, its role in haemostasis regulation isn’t clear. We studied the role of MT in fibrin formation, platelet activation and aggregation. A new effective method of obtaining MT from native human prothrombin was developed using immobilised prothrombin activator from Echis multisquamatis venom. The protein was stable and electrophoretically pure. Platelet-rich plasma for aggregation study and gel-sieved platelets for flow-cytometry were separated from blood of healthy donors. It was shown that MT transformed fibrinogen to fibrin and activated clotting factor XIII. MT didn’t activate gel-sieved intact platelets, but in platelet-rich plasma, increased platelet aggregation induced by ADP, collagen and adrenalin.

Gibbs-Donnan Potential as a Tool for Membrane Vesicles Polarization

It has been theoretically predicted that under conditions leading to Gibbs-Donnan equilibrium in case when size of one compartment is very different from another (as in system “membrane vesicle/liposomes—incubation medium”) stable transmembrane potential can be formed, which value is sufficient to fit requirement of real transmembrane potential. Four partial cases were considered with different location and charge of impermeable ion and it was concluded that locations of impermeable ions in medium provide stable transmembrane potential with sufficient value of 60 - 70 mV. Potential-sensitive probe, such as DiOC6(3) and oxonol VI, were used to confirm the calculated potential. According to the change in fluorescence level and emission/excitation shift, a stable and relatively high transmembrane potential can be formed if salt of impermeable ion is located in incubation medium. Impermeable cations and anions may be used to create positive and negative transmembrane potential respectively.

The Expression of <i>TIMP</i>1, <i>TIMP</i>2, <i>VCAN</i>, <i>SPARC</i>, <i>CLEC</i>3<i>B</i>and <i>E</i>2<i>F</i>1 in Subcutaneous Adipose Tissue of Obese Males and Glucose Intolerance

We investigated the expression of TIMP1, TIMP2, SPARC, VCAN, and CLEC3B genes, encoded matricellular proteins with pleiotropic functions, and glucose intolerance in obese male subjects with normal and impaired glucose tolerance. The purpose of this study was to examine the association between the gene expressions and glucose intolerance in obesity. The results indicate that obesity leads to significant increase of TIMP1, TIMP2, E2F1 and CLEC3B gene expressions in subcutaneous adipose tissue, especially TIMP2 gene. However, more significant increase of the expression of TIMP1 and TIMP2 was found in adipose tissue of obese patients with glucose intolerance. No significant changes were found in the expression of VCAN and SPARC genes in adipose tissue of obese subjects with normal glucose tolerance but increased in the group of obese subjects with glucose intolerance. At the same time, the E2F1 and CLEC3B gene expressions were decreased in adipose tissue of obese patients with glucose intolerance. Results of this study provide evidence that changes in the expression of genes encoded TIMP1, TIMP2, VCAN, SPARC, E2F1 and CLEC3B in subcutaneous adipose tissue of obese individuals associate with glucose intolerance.

Green synthesis of biocompatible Gd^(3+)-doped ultrasmall carbon-based nanohybrids from coffee wastes

A cheap method allowing fabrication of biocompatible,ultra-small(2-10 nm)and fluorescent(λ_(em)=425-500 nm)nanohybrids(NHs)from coffee wastes is reported.The gadolinium-doped nanohybrids(GDNHs)or gadolinium-free carbon dots(GFCDs)can be synthesized in a domestic microwave oven according to green synthesis principles.Hydrodynamic sizes,chemical composition,impact on proton magnetic resonance relaxation time and optical properties of the GDNHs and GFCDs were studied in details and compared.In particular,doping of the NHs with Gd^(3+)ions,up to 1.87%w/w of gadolinium per particles’weight,will allow their application for magnetic resonance imaging(MRI).Furthermore,cell culture tests on human adenocarcinomic alveolar basal epithelial cells line(A549)have shown high biocompatibility of the GDNHs and in a wide concentration range 100-1000μg/ml.

Essential variables for air quality estimation

Within this survey we describe the conceptual architecture of the infrastructure to measure PM2.5/PM10 concentration in the atmosphere over the Kyiv city using modern monitoring instruments.We define the requirements for information tools and network for informing Kyiv city community on the state of PM pollutions that will be created.This infrastructure will provide long-term PM2.5/PM10 observations that could be included in the AirBase network.The comprehensive review of in-situ and satellite measurements of PM2.5/PM10 is provided as well as the description current state-of-the-art for Air Quality monitoring with intelligent sensors and systems in Ukraine as-awhole and in Kyiv in particular.It is proposed to apply the concept of essential variables(EVs)used in Earth Observation to identify the variables that should be measured in priority when designing,deploying and maintaining observation systems.In this study we use and validate the global air quality products from Copernicus Atmosphere Monitoring Service obtained from modeling by GEOS-Chem model and other sources.The influence of PM and aerosols on a human health is estimated in terms of possible diseases and dangerous concentrations.