Membrane ATPase Activity Research Articles

Exomer Is Part of a Hub Where Polarized Secretion and Ionic Stress Connect.

Plasma membrane and membranous organelles contribute to the physiology of the Eukaryotic cell by participating in vesicle trafficking and the maintenance of ion homeostasis. Exomer is a protein complex that facilitates vesicle transport from the trans-Golgi network to the plasma membrane, and its absence leads to the retention of a set of selected cargoes in this organelle. However, this retention does not explain all phenotypes observed in exomer mutants. The Schizosaccharomyces pombe exomer is composed of Cfr1 and Bch1, and cfr1Δ and bch1Δ were sensitive to high concentrations of potassium salts but not sorbitol, which showed sensitivity to ionic but not osmotic stress. Additionally, the activity of the plasma membrane ATPase was higher in exomer mutants than in the wild-type, pointing to membrane hyperpolarization, which caused an increase in intracellular K+ content and mild sensitivity to Na+, Ca2+, and the aminoglycoside antibiotic hygromycin B. Moreover, in response to K+ shock, the intracellular Ca2+ level of cfr1Δ cells increased significantly more than in the wild-type, likely due to the larger Ca2+ spikes in the mutant. Microscopy analyses showed a defective endosomal morphology in the mutants. This was accompanied by an increase in the intracellular pools of the K+ exporting P-type ATPase Cta3 and the plasma membrane Transient Receptor Potential (TRP)-like Ca2+ channel Pkd2, which were partially diverted from the trans-Golgi network to the prevacuolar endosome. Despite this, most Cta3 and Pkd2 were delivered to the plasma membrane at the cell growing sites, showing that their transport from the trans-Golgi network to the cell surface occurred in the absence of exomer. Nevertheless, shortly after gene expression in the presence of KCl, the polarized distribution of Cta3 and Pkd2 in the plasma membrane was disturbed in the mutants. Finally, the use of fluorescent probes suggested that the distribution and dynamics of association of some lipids to the plasma membrane in the presence of KCl were altered in the mutants. Thus, exomer participation in the response to K+ stress was multifaceted. These results supported the notion that exomer plays a general role in protein sorting at the trans-Golgi network and in polarized secretion, which is not always related to a function as a selective cargo adaptor.

Escherichia coli Dcu C4-dicarboxylate transporters dependent proton and potassium fluxes and FOF1-ATPase activity during glucose fermentation at pH 7.5

During fermentation in Escherichia coli succinate is transported via Dcu transporters, encoded dcuA, dcuB, dcuC and dcuD although the role of DcuD protein has not been elucidated yet. It has been shown contribution of Dcu transporters in the N,N’-dicyclohexylcarbodiimide (DCCD) sensitive proton and potassium transport through the cytoplasmic membrane and membrane-associated ATPase activity. Total H± efflux was decreased ~ 40% while K± uptake was absent in dcuD mutant. DCCD-sensitive H± flux was absent in dcuD nevertheless it was increased ~ 3 fold in dcuACB. K± uptake in dcuACB was stimulated ~ 30% compared to wild type but in DCCD assays K± ions were effluxed with the rate of 0.15 mmol/min per 109 cells/ml. In dcuACB mutant membrane potential (ΔΨ) was ~ 30 mV higher than in wild type. dcuD gene expression was increased in the dcuACB mutant respect to wild type at pH 7.5 (~120%), suggesting that an increment of DcuD activity compensates the lack of DcuA, DcuC and DcuB carriers. It can be concluded that active DcuD is important for H± efflux via the FOF1-ATPase and K± uptake at pH 7.5. In addition, DcuA, DcuB and DcuC transporters are crucial for regulating DCCD-sensitive K± transport and ΔΨ in E. coli.

Vacuolar Membrane ATPase Activity 21 Predicts a Favorable Outcome and Acts as a Suppressor in Colorectal Cancer.

Extracellular and/or intracellular manipulation of pH in tumor may have noticeable potential in cancer treatment. Although the assembly factor genes of V0 domain of the V-ATPase complex are required for intracellular pH homeostasis, their significance in colorectal cancer (CRC) remains largely unknown. Here, we used bioinformatics to identify the candidates from known assembly factor genes of the V0 domain, which were further evaluated by immunohistochemistry (IHC) in CRC and adjacent normal specimens from 661 patients. Univariate and multivariate Cox analyses were used to evaluate factors contributing to prognosis. The effects of variations in the expression of VMA21 on tumor growth were assessed in vitro and in vivo. Of five known assembly factors, only VMA21 showed differential expression between CRC and adjacent normal tissues at both mRNA and protein levels. Patients with high VMA21 expression had higher differentiation grade and longer disease-specific survival (DSS) at stages I–III disease. High VMA21 expression in tumors was also an independent predictor of DSS (hazard ratio, 0.345; 95% confidence interval, 0.123–0.976), with covariates included TNM stage and differentiation grade. VMA21 overexpression decreased CRC growth, whereas VMA21 knockdown increased CRC growth in vitro and in vivo. VMA21 expression suppresses CRC growth and predicts a favorable DSS in patients with stage I-III disease.

Trans-2-enoyl-CoA reductase limits Ca2+ accumulation in the endoplasmic reticulum by inhibiting the Ca2+ pump SERCA2b

The endoplasmic reticulum (ER) contains various enzymes that metabolize fatty acids (FAs). Given that FAs are the components of membranes, FA metabolic enzymes might be associated with regulation of ER membrane functions. However, it remains unclear whether there is the interplay between FA metabolic enzymes and ER membrane proteins. Trans-2-enoyl-CoA reductase (TER) is an FA reductase present in the ER membrane and catalyzes the last step in the FA elongation cycle and sphingosine degradation pathway. Here we identify sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b), an ER Ca2+ pump responsible for Ca2+ accumulation in the ER, as a TER-binding protein by affinity purification from HEK293 cell lysates. We show that TER directly binds to SERCA2b by in vitro assays using recombinant proteins. Thapsigargin, a specific SERCA inhibitor, inhibits this binding. TER binds to SERCA2b through its conserved C-terminal region. TER overexpression suppresses SERCA2b ATPase activity in microsomal membranes of HEK293 cells. Depletion of TER increases Ca2+ storage in the ER and accelerates SERCA2b-dependent Ca2+ uptake to the ER after ligand-induced Ca2+ release. Moreover, depletion of TER reduces the Ca2+-dependent nuclear translocation of nuclear factor of activated T cells 4. These results demonstrate that TER is a negative regulator of SERCA2b, implying the direct linkage of FA metabolism and Ca2+ accumulation in the ER.

Cell-penetrating peptidomimetics derived from cathelicidin CAP-18 showed inhibition of Na+ /K+, Cu(I) and Zn(II)ATPases in mycobacterial plasma membrane

Aims & objectives: Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), which is one of the ten most deadly diseases and the first cause of death by bacterial origin around the world. One of the causes that increase the problem are the M. tuberculosis resistant strains to conventional antibiotics, multi-drug resistant tuberculosis (MDR-TB) and extensively-drug resistant tuberculosis (XDR-TB); so it is necessary to develop new drugs with different mechanisms of action. Methods: In this study, the effect on the mycobacterial cell membrane ATPase activity of CAP-18 derived peptide with unnatural amino acids was assessed. Using bioinformatic tools, the amino acid sequence of CAP-18 with the best features of helical structure and antibacterial activity was determined; the corresponding peptide had changes for D-Lys and N-methyl-Gly in the sequence, generating the peptides CAP18-BD and CAP18-CD and the peptoid CAP18-BP. Results: In vitro assesament of the peptides and peptidomimetics were tested in plasma membrame from Mycobacterium smegmatis mc2 155. The inorganic phosphate released (Pi) was quantified during the ATP hydrolysis reaction by the action of ATPases, using the Fiske and Subarrow method modified by Cariani et al., in the presence and absence of the peptidic sequences to be evaluated, where the basal ATPase activity in the absence corresponds to zero percent inhibition of these enzymes, and 100% of the residual ATPase activity, understood as the enzymatic activity present with or without peptide treatment. Conclusions: The results showed inhibition of basal ATPase activity and Na+ /K+, Cu(I), Zn (II) pumps in the plasma membrane, which is associated with its mechanism of action, which suggests that CAP18-BD, CAP18-CD and CAP18-BP can be considered as a potential antimycobacterial compounds targeting cell membrane ATPases.

Induction of mitochondrial-mediated apoptosis by methanol extract of Cajanus cajan

Induction of mitochondrial-mediated apoptosis by methanol extract of Cajanus cajanAlao O Oa,b and Olorunsogo O.Oa aDepartment of Biochemistry, University of Ibadan, Nigeria bDepartment of Biochemistry, Lead City University, Ibadan, Nigeria. *All correspondence: olajualao@yahoo.co.uk +2348034728112Background: Attention has recently focused on mitochondrial-mediated apoptosis as a signaling pathway that is disrupted during carcinogenesis. The chemopreventive effects of dietary phytochemicals can be explored in order to combat conditions emanating from dysregulation of mitochondria-mediated apoptosis. Cajanus cajan is a medicinal plant commonly used in Nigeria for the traditional treatment of many diseases such as jaundice, measles dysentery and tumours. This study aims to investigate the effects of Cajanus cajan on the opening of the mitochondrial membrane permeability transition(MMPT) pore, ATPase activities, liver enzyme activities and immunohistochemical expression of apoptotic biomarkers in rat liver. Methods: Eighteen male Wistar rats (150±2.00 g) were assigned into three groups of six animals each and orally administered corn oil (control), 100 and 200 mg/kg MECC once daily for thirty consecutive days. Mitochondrial membrane permeability transition and ATPase activity were determined spectrophotometrically. Caspase 3, caspase 9 and cytochrome C release were determined by immunohistochemistry.Results: In vitro, at 10 and 30 µg/mL pore opening was not observed , however at 50 and 70 µg/mL MECC induced pore opening by 7 and 13 folds. Similarly, enhancement of ATPase activities was also observed. In the in vivo studies; the MECC induced opening at 200 mg/kg bwt.There was a significant increase in the activities of AST, ALT and ALP and caspase 3, caspase 9 and cytochrome C were activated Conclusion: Methanol extract of Cajanus cajan induced mitochondrial -mediated apoptosis in normal rat liver . Hence Cajanus cajan can be used in the management of conditions associated with dysregulation of apoptosis.Keywords: Mitochondria membrane permeability transition pore : apoptosis and Cajanus cajan

Morin treatment for acute ethanol exposure in rats

ABSTRACT Ethanol intoxication increases oxidative stress and pro-inflammatory proteins, which cause neurodegeneration. Morin is a natural flavonoid obtained from plants of the Moraceae family that exhibits antioxidant, anti-inflammatory, hepatoprotective, anticancer and cardioprotective properties. We investigated the neuroprotective effect of morin on ethanol intoxicated rats. Rats exposed to ethanol exhibit increased cholesterol, triglycerides, phospholipids, free fatty acids, and lipid oxidative byproducts, and decreased the activity of antioxidant enzymes and membrane ATPase. We found that ethanol increased activation of microglia and astrocytes in the brain. Administration of morin to rats exposed to ethanol significantly decreased lipid oxidative byproducts, enhanced antioxidant enzymes, normalized lipid levels and decreased microglia and astrocyte activation. Morin exhibits neuroprotective properties against ethanol intoxication by increasing the antioxidant defense mechanism and decreasing the inflammatory response caused by neuroglia and astrocytes.

Direct micro-electric stimulation alters phenanthrene-degrading metabolic activities of Pseudomonas sp. strain DGYH-12 in modified bioelectrochemical system

Bioelectrochemical systems (BESs) have great potential for treating wastewater containing polycyclic aromatic hydrocarbons (PAHs); however, detailed data on cell physiological activities in PAH biodegradation pathways stimulated by BESs are still lacking. In this paper, a novel BES device was assembled to promote the growth of Pseudomonas sp. DGYH-12 in phenanthrene (PHE) degradation. The results showed that in the micro-electric field (0.2 V), cell growth rate and PHE degradation efficiency were 22% and 27.2% higher than biological control without electric stimulation (BC), respectively. The extracellular polymeric substance (EPS) concentration in BES (39.38 mg L−1) was higher than control (33.36 mg L−1); moreover, the membrane permeability and ATPase activities were also enhanced and there existing phthalic acid and salicylic acid metabolic pathways in the strain. The degradation genes nahAc, pcaH, and xylE expression levels were upregulated by micro-electric stimulation. This is the first study to analyze the physiological and metabolic effect of micro-electric stimulation on a PHE-degrading strain in detail and systematically.

ATPASE ERYTHROCYTE TRANSPORT SYSTEMS IN HUMAN BLOOD

Aim. The article deals with establishing the features of ATPase erythrocytes in the athletes of the first and second categories and non-athletes from different age groups. Material and methods. We studied the capillary blood of track-and-field athletes and healthy non-athletes of the same age and anthropometric characteristics (n = 60). Blood analysis was performed with the help of the Dixion hematological analyzer (Russia). The activity of erythrocyte membrane ATPases was studied according to K.S. Keeton. Results. The study revealed significant (p < 0.05) differences in certain (HGB and RBC) hematological blood indicators in participants from different age groups depending on their physical fitness. Age differences between groups are not significant (p > 0.05). The results obtained demonstrate the significantly (p < 0.001) higher values of ATPase activity in the athletes of the first and second categories from various age groups. The increase in Mg2+, Na+, K+-, Mg2+- and Na+, K+-ATPases activity in athletes is connected with more intensive metabolism in this group because transport ATPases participate in the energy supply of training and competition loads. Age-related differences (p < 0.05) in the activity of erythrocyte membrane ATPases in both groups can be possibly connected with the conformation features of erythrocyte membrane protein. The two-factor dispersion analysis revealed that age significantly (Р < 0.001) determined Mg2+, Na+, K+-, Mg2+- and Na+, K+-ATPases erythrocyte membrane activity at 89.15, 87.46, and 81.40 % respectively; physical activity at 96.19, 95.45 and 93.80 % respectively. Conclusion. General physical fitness and age can be considered as the factors determining the activity of erythrocyte membrane ATPases.

A 4-cyano-3-methylisoquinoline inhibitor of Plasmodium falciparum growth targets the sodium efflux pump PfATP4

We developed a novel series of antimalarial compounds based on a 4-cyano-3-methylisoquinoline. Our lead compound MB14 achieved modest inhibition of the growth in vitro of the human malaria parasite, Plasmodium falciparum. To identify its biological target we selected for parasites resistant to MB14. Genome sequencing revealed that all resistant parasites bore a single point S374R mutation in the sodium (Na+) efflux transporter PfATP4. There are many compounds known to inhibit PfATP4 and some are under preclinical development. MB14 was shown to inhibit Na+ dependent ATPase activity in parasite membranes, consistent with the compound targeting PfATP4 directly. PfATP4 inhibitors cause swelling and lysis of infected erythrocytes, attributed to the accumulation of Na+ inside the intracellular parasites and the resultant parasite swelling. We show here that inhibitor-induced lysis of infected erythrocytes is dependent upon the parasite protein RhopH2, a component of the new permeability pathways that are induced by the parasite in the erythrocyte membrane. These pathways mediate the influx of Na+ into the infected erythrocyte and their suppression via RhopH2 knockdown limits the accumulation of Na+ within the parasite hence protecting the infected erythrocyte from lysis. This study reveals a role for the parasite-induced new permeability pathways in the mechanism of action of PfATP4 inhibitors.

Cellular expression and function of naturally occurring variants of the human ABCG2 multidrug transporter

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics; thus the relatively frequent polymorphic and mutant ABCG2 variants in the population may significantly alter disease conditions and pharmacological effects. Low-level or non-functional ABCG2 expression may increase individual drug toxicity, reduce cancer drug resistance, and result in hyperuricemia and gout. In the present work we have studied the cellular expression, trafficking, and function of nine naturally occurring polymorphic and mutant variants of ABCG2. A comprehensive analysis of the membrane localization, transport, and ATPase activity, as well as retention and degradation in intracellular compartments was performed. Among the examined variants, R147W and R383C showed expression and/or protein folding defects, indicating that they could indeed contribute to ABCG2 functional deficiency. These studies and the applied methods should significantly promote the exploration of the medical effects of these personal variants, promote potential therapies, and help to elucidate the specific role of the affected regions in the folding and function of the ABCG2 protein.

Study on the protective effect of ligustrazine on the transporting function of hepatocellular mitochondria membrane in the septic rats

To investigate the protective effect of ligustrazine on the transporting function of hepatocellular mitochondria membrane in the rats with sepsis-induced acute liver injury (SALI). The Sprague-Dawley (SD) rats were randomly divided into sham operation group, SALI group [established by cecal ligation and puncture (CLP)], ligustrazine treatment group (injection of ligustrazine 60 mg/kg through tail vein after CLP) and ligustrazine preventive group (7 days before CLP, ligustrazine was injected daily through tail vein for 60 mg/kg), and there were 12 rats in each group. Abdominal aorta blood and liver were harvested at 10 hours after operation. The content of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and mitochondrial aspartate aminotransferase (m-AST) were determined by enzyme coupling rate method. The content of ATP was detected by colorimetric and chemical fluorescein method. The activity of mitochondrial ATPase was detected by phosphorus quantification. The expressions of mitochondrial membrane aquaporin 8 (AQP8) and carnitine palmitoyl transferase (CPT) were detected by Western Blot. Compared with sham operation group, the levels of serum ALT, AST and m-AST were significantly increased in SALI group, ligustrazine treatment group and ligustrazine preventive group, and the content of ATP was reduced, the activity of mitochondrial membrane ATPase, the expressions of AQP8 and CPT-1A were significantly decreased. Compared with SALI group, the levels of serum ALT, AST and m-AST were significantly decreased in ligustrazine treatment and ligustrazine preventive groups [ALT (U/L): 123.8±32.8, 105.0±44.5 vs. 233.0±110.1; AST (U/L): 427.0±117.9, 303.9±110.3 vs. 742.6±441.4; m-AST (U/L): 239.6±64.9, 168.2±60.0 vs. 412.8±252.6; all P < 0.01], the content of ATP were significantly increased (nmol/mg: 29.5±10.3, 34.6±11.2 vs. 19.3±8.8, both P < 0.01), the activity of ATPase in hepatocellular mitochondrial membrane were significantly increased [Na+-K+-ATPase (U/mg): 3.91±0.30, 3.97±0.35 vs. 2.87±0.82; Mg2+-ATPase (U/mg): 3.75±0.38, 3.88±0.35 vs. 2.64±1.06; Ca2+-ATPase (U/mg): 3.15±0.58, 2.98±0.31 vs. 1.75±1.25; Ca2+-Mg2+-ATPase (U/mg): 3.82±0.31, 3.91±0.42 vs. 2.57±1.01, all P < 0.01], the expressions of AQP8 and CPT-1A were significantly increased [percentage increase from sham operation group (100%), AQP8/COX-IV: (79.12±7.79)%, (88.40±9.22)% vs. (62.08±11.91)%; CPT-1A/COX-IV: (87.92±10.06)%, (84.91±17.48)% vs. (72.11±7.82)%, all P < 0.01]. The levels of serum AST and m-AST in ligustrazine preventive group were significant lower than those in ligustrazine treatment group [AST (U/L): 303.9±110.3 vs. 427.0±117.9; m-AST (U/L): 168.2±60.0 vs. 239.6±64.9, both P < 0.05]. There was no significant difference in the expression of CPT-2 in mitochondrial membrane between the four groups. Ligustrazine could play a protective role on the mitochondrial membrane function of transporting water, ion and fat in the rats with SALI. The preventive function of ligustrazine is better than the treatment effect of the rats with sepsis.

Biochemical characterization and chemical inhibition of PfATP4-associated Na+-ATPase activity in Plasmodium falciparum membranes

The antimalarial activity of chemically diverse compounds, including the clinical candidate cipargamin, has been linked to the ATPase PfATP4 in the malaria-causing parasite Plasmodium falciparum The characterization of PfATP4 has been hampered by the inability thus far to achieve its functional expression in a heterologous system. Here, we optimized a membrane ATPase assay to probe the function of PfATP4 and its chemical sensitivity. We found that cipargamin inhibited the Na+-dependent ATPase activity present in P. falciparum membranes from WT parasites and that its potency was reduced in cipargamin-resistant PfATP4-mutant parasites. The cipargamin-sensitive fraction of membrane ATPase activity was inhibited by all 28 of the compounds in the "Malaria Box" shown previously to disrupt ion regulation in P. falciparum in a cipargamin-like manner. This is consistent with PfATP4 being the direct target of these compounds. Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na+ transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K+] or [Ca2+]. With an apparent Km for ATP of 0.2 mm and an apparent Km for Na+ of 16-17 mm, the protein is predicted to operate at below its half-maximal rate under normal physiological conditions, allowing the rate of Na+ efflux to increase in response to an increase in cytosolic [Na+]. In membranes from a cipargamin-resistant PfATP4-mutant line, the apparent Km for Na+ is slightly elevated. Our study provides new insights into the biochemical properties and chemical sensitivity of an important new antimalarial drug target.

Diverse antimalarials from whole-cell phenotypic screens disrupt malaria parasite ion and volume homeostasis

Four hundred structurally diverse drug-like compounds comprising the Medicines for Malaria Venture’s ‘Pathogen Box’ were screened for their effect on a range of physiological parameters in asexual blood-stage malaria (Plasmodium falciparum) parasites. Eleven of these compounds were found to perturb parasite Na+, pH and volume in a manner consistent with inhibition of the putative Na+ efflux P-type ATPase PfATP4. All eleven compounds fell within the subset of 125 compounds included in the Pathogen Box on the basis of their having been identified as potent inhibitors of the growth of asexual blood-stage P. falciparum parasites. All eleven compounds inhibited the Na+-dependent ATPase activity of parasite membranes and showed reduced efficacy against parasites carrying mutations in PfATP4. This study increases the number of chemically diverse structures known to show a ‘PfATP4-associated’ phenotype, and adds to emerging evidence that a high proportion (7–9%) of the structurally diverse antimalarial compounds identified in whole cell phenotypic screens share the same mechanism of action, exerting their antimalarial effect via an interaction with PfATP4.

Active proton efflux, nutrient retention and boron-bridging of pectin are related to greater tolerance of proton toxicity in the roots of two Erica species

Background and aimsTolerance to soil acidity was studied in two species of Ericaceae that grow in mine-contaminated soils (S Portugal, SW Spain) to find out if there are interspecific variations in H+ tolerance which might be related to their particular location. MethodsTolerance to H+ toxicity was tested in nutrient solutions using seeds collected in SW Spain. Plant growth and nutrient contents in leaves, stems and roots were determined. Viability tests and proton exchange were studied in roots exposed, short-term, to acidic conditions. Membrane ATPase activity and the cell-wall pectic polysaccharide domain rhamnogalacturonan-II (RG-II) were analysed to find out interspecific differences. ResultsVariation in survival, growth and mineral composition was found between species. The H+-tolerant species (Erica andevalensis) showed greater concentration of nutrients than E. australis. Very low pH (pH 2) produced a significant loss of root nutrients (K, P, Mg) in the sensitive species. Root ATPase activity was slightly higher in the tolerant species with a correspondingly greater H+ efflux capacity. In both species, the great majority of the RG-II domains were in their boron-bridged dimeric form. However, shifting to a medium of pH 2 caused some of the boron bridges to break in the sensitive species. ConclusionsVariation in elements linked to the cell wall-membrane complex and the stability of their components (RG-II, H+-ATPases) are crucial for acid stress tolerance. Thus, by maintaining root cell structure, active proton efflux avoided toxic H+ build-up in the cytoplasm and supported greater nutrient acquisition in H+-tolerant species.

Response of Membrane Fatty Acid Composition and ATPase Activity in Brassica rapa L. to Temperature in North China

Response of Membrane Fatty Acid Composition and ATPase Activity in <i>Brassica rapa</i> L. to Temperature in North China

Вивчення рівня активності мембранної AТР-ази бактерій C. perfringens та критеріїв впливу на їхні метаб

Вивчення рівня активності мембранної AТР-ази бактерій C. perfringens та критеріїв впливу на їхні метаб

Biochemical markers of safety of nano-particles of metals on the model of isolated subcultural fractions of eukaryotes

Unique sizes and a high level of bioavailability allow nanoparticles of metals (NPMe) to come into direct contact with biological systems, with infectious agents, toxins, as well as with different chemical compounds and separate cell structures (proteins, lipids, nucleic acids). Other biological effects, including less toxicity than in microscopic substances, require attention to be paid to the study of the potential risk of using nanoparticles of each type in a particular way, therefore scientific support is absolutely necessary in this direction. It is believed that the cytotoxicity of nanomaterials is due to genomic and mutagenic effects, but the mechanical forces of interaction of NPMе with cells, obviously, will change not only cytological but also their metabolic reactions. Therefore, the purpose of this research was to determine the biochemical markers of safety (potential toxicity) of NPMe (Au, Ag, Cu, Fe, Co, GFCo, Zn, MnO2) on the model of isolated membrane and cytosolic fractions of eukaryotic test cells of CHO-K1 and U937 lines. Under conditions of preincubation of experimental samples of NPMe at a final concentration of 1 μg/cm3 by the metal with preparations of subcellular fractions of CHO-K1 and U937 (in the final amount of protein 150–200 μg/cm3) for 3 minutes at 37 ± 1 ºС, there was determined the magnitude of membrane ATP-ase and cytosolic LDH-ase activity compared to intact cells ("control"). According to the results of the research, colloidal dispersions of NPAg average size ~30 nm, NPFe ~100 nm, NPCu ~70 nm, and NPMnO2 ~50 nm are safe and biocompatible by their membranotropic effect on subcellular fractions of eukaryotic test cells, as evidenced by an increase in the level of membrane ATPase and cytosolic LDHase of test-cells CHO-K1, and the experimental samples NPCo, NPGFCo and NPZn average size of ~100 nm are membrane-toxic, that is, dangerous. By the nature of the changes in the enzymatic activity of the test cells U937, the discrete dimensions of the membranotropic action of NPAu have been demonstrated: nanoparticles of size ~10 nm caused the inhibition of the membrane Na+,K+-ATPase, and the size of ~30 nm and ~45 nm – its induction; nanoparticles of size ~10, ~20 and ~30 nm induced cytosolic LDHase and the size of ~45 nm – its inhibition relative to the control level of enzymes, so NPAu ~10 and ~45 nm can be considered membrane toxic, and size ~30 nm – safe and biocompatible for eukaryotic cells. Based on the hypothesis about the involvement of metabolism-dependent mechanisms of contact interaction of colloidal dispersions of experimental samples of NPMe with cells through membranotropic properties, the study of their potential danger or biocompatibility in further research can be carried out by determining the intensity of oxidation of the main structural components of biomembranes of cells – lipids and proteins and indicators of their AO-regulation.

Effects of proteasome inhibitor MG-132 on the parasite Schistosoma mansoni.

Proteasome is a proteolytic complex responsible for intracellular protein turnover in eukaryotes, archaea and in some actinobacteria species. Previous work has demonstrated that in Schistosoma mansoni parasites, the proteasome inhibitor MG-132 affects parasite development. However, the molecular targets affected by MG-132 in S. mansoni are not entirely known. Here, we used expression microarrays to measure the genome-wide changes in gene expression of S. mansoni adult worms exposed in vitro to MG-132, followed by in silico functional analyses of the affected genes using Ingenuity Pathway Analysis (IPA). Scanning electron microscopy was used to document changes in the parasites’ tegument. We identified 1,919 genes with a statistically significant (q-value ≤ 0.025) differential expression in parasites treated for 24 h with MG-132, when compared with control. Of these, a total of 1,130 genes were up-regulated and 790 genes were down-regulated. A functional gene interaction network comprised of MG-132 and its target genes, known from the literature to be affected by the compound in humans, was identified here as affected by MG-132. While MG-132 activated the expression of the 26S proteasome genes, it also decreased the expression of 19S chaperones assembly, 20S proteasome maturation, ubiquitin-like NEDD8 and its partner cullin-3 ubiquitin ligase genes. Interestingly, genes that encode proteins related to potassium ion binding, integral membrane component, ATPase and potassium channel activities were significantly down-regulated, whereas genes encoding proteins related to actin binding and microtubule motor activity were significantly up-regulated. MG-132 caused important changes in the worm tegument; peeling, outbreaks and swelling in the tegument tubercles could be observed, which is consistent with interference on the ionic homeostasis in S. mansoni. Finally, we showed the down-regulation of Bax pro-apoptotic gene, as well as up-regulation of two apoptosis inhibitor genes, IAP1 and BRE1, and in contrast, down-regulation of Apaf-1 apoptotic activator, thus suggesting that apoptosis is deregulated in S. mansoni exposed to MG-132. A considerable insight has been gained concerning the potential of MG-132 as a gene expression modulator, and overall the data suggest that the proteasome might be an important molecular target for the design of new drugs against schistosomiasis.

Erythrocyte membrane ATPase activity in Sickle cell crisis

Background: Sickle cell disease is a genetic disease caused by a point mutation in the beta globin gene on chromosome 11 leading to the substitution of valine in place of glutamic acid in beta subunit of globin molecule. The vaso-occlusive crisis, or sickle cell crisis, is a common painful complication of sickle cell disease in adolescents and adults. Many studies have suggested the alteration in intracellular concentration of various ions associated with sickle cell disease.Aims and Objectives: This study aims to evaluate the activity of red blood cell membrane ATPase activity of sickle cell crisis patients in comparison to sickle cell patients in steady state.Material and Methods: Twenty patients of sickle cell crisis were included in the study. Twenty sickle cell patients in steady state were also registered who served as controls. The RBC Membrane ATPase activity were estimated and expressed as μmol of Pi/μg of protein/hour.Results: The activity of membrane ATPase were found to be significantly high in patients of sickle cell crisis in comparison to steady state ones.Conclusion: Membrane ATPases play a crucial role in the pathophysiology of sickle cell crisis. This study can be a based upon for future studies to understand the pathophysiology of sickle cell crisis and development of diagnostic and treatment modalities for the same.Asian Journal of Medical Sciences Vol.8(5) 2017 27-30