Plasma Membrane Enzyme Research Articles

Possible Association of Smokeless Tobacco Dependent Impairment in the Erythrocytes and Platelets Membranes of Human Male Volunteers: An Observation.

Background:Smokeless tobacco (SLT) acts as a modifier of erythrocyte and platelet membranes by disrupting antioxidant system with the concomitant increase in free radical production and induction of apoptosis. Methods:The SLT users was that individuals used gutkha and khaini products (Khaleja/mahak chaini brand respectively) habitually, at least >20 times per week consists of 50-60 g during the last 2-4 years. Results:The gutkha and khaini users found to be significantly increased levels of iNOS (Inducible nitric oxide synthase) enzyme in plasma, erythrocytes, and platelet membranes when compared to normal controls. The gutkha and khaini users exhibited that the significant increase in the levels of gene expression of apoptotic proteins (Bcl2-B cell lymphoma gene 2, Bax, caspases 8, caspase 10, and caspase 12), IL-6 (Interleukin-6), and decreased levels of TNF-α (Tumor necrosis factor-alpha) and decreased expression of caspase 12 of khaini users were observed from blood samples. The significant increase in the concentrations of peroxynitrites (ONOO-), nitric oxide (NO) (Nitrates and nitrites), malondialdehyde (MDA), cholesterol, and phospholipids were reported in the smokeless tobacco users of erythrocytes and platelets. The experimental subjects showed that the increased osmotic fragility and decreased membrane fluidity of erythrocytes and platelets in comparison with non-tobacco users. The normal subjects had been exposed that the proper functioning of antioxidant enzymes and decreased enzyme activities of antioxidants were reported by SLT users. Conclusion:The smokeless tobacco products are exerted chronic damage to membranes of erythrocytes and platelets and elevation of apoptosis in the prolonged periods of human male volunteers.

Evidences of plasma membrane-mediated ROS generation upon ELF exposure in neuroblastoma cells supported by a computational multiscale approach

BackgroundMolecular mechanisms of interaction between cells and extremely low frequency magnetic fields (ELF-MFs) still represent a matter of scientific debate. In this paper, to identify the possible primary source of oxidative stress induced by ELF-MF in SH-SY5Y human neuroblastoma cells, we estimated the induced electric field and current density at the cell level. MethodsWe followed a computational multiscale approach, estimating the local electric field and current density from the whole sample down to the single cell level. The procedure takes into account morphological modeling of SH-SY5Y cells, arranged in different topologies. Experimental validation has been carried out: neuroblastoma cells have been treated with Diphenyleneiodonium (DPI) -an inhibitor of the plasma membrane enzyme NADPH oxidase (Nox)- administered 24 h before exposure to 50 Hz (1 mT) MF. ResultsMacroscopic and microscopic dosimetric evaluations suggest that increased current densities are induced at the plasma membrane/extra-cellular medium interface; identifying the plasma membrane as the main site of the ELF-neuroblastoma cell interaction. The in vitro results provide an experimental proof that plasma membrane Nox exerts a key role in the redox imbalance elicited by ELF, as DPI treatment reverts the generation of reactive oxygen species induced by ELF exposure. General significanceMicroscopic current densities induced at the plasma membrane are likely to play an active physical role in eliciting ELF effects related to redox imbalance. Multiscale computational dosimetry, supported by an in vitro approach for validation, is proposed as the innovative and rigorous paradigm to unveil mechanisms underlying the complex ELF-MF interactions.

Antifungal Activity, Mode of Action, Docking Prediction and Anti-biofilm Effects of (+)-β-pinene Enantiomers against Candida spp.

The objective of this study was to investigate the effectiveness of (+)-β-pinene inhibition on Candida spp. growth, aiming at elucidation of the mechanism of action; to determine fungal cell enzyme binding activity (through molecular docking simulations) and its effects on biofilm reduction. Candida strains (n=25) from referenced and clinical origins, either susceptible or resistant to standard clinical antifungals, were tested for determination of Minimum Inhibitory Concentration (MIC); Minimum Fungicidal Concentration (MFC); and microbial death curves upon treatment with (+)-β-pinene; the effects of (+)-β-pinene on the cell wall (sorbitol assay), membrane ergosterol binding, and effects on biofilm were evaluated by microdilution techniques. We also evaluated the interactions between (+)-β-pinene and cell wall and membrane enzymes of interest. The MIC values of (+)-β-pinene ranged from <56.25 to 1800 µmol/L. The MIC of (+)-β-pinene did not increase when ergosterol was added to the medium, however it did increase in the presence of sorbitol, leading to a doubled MIC for C. tropicalis and C. krusei. The results of the molecular docking simulations indicated better interaction with delta-14-sterol reductase (-51 kcal/mol). (+)-β-pinene presents anti-biofilm activity against multiples species of Candida. (+)-β-pinene has antifungal activity and most likely acts through interference with the cell wall; through molecular interaction with Delta-14-sterol reductase and, to a lesser extent, with the 1,3-β- glucan synthase. This molecule was also found to effectively reduce Candida biofilm adhesion.

Responses of flocculated activated sludge to bimetallic Ag-Fe nanoparticles toxicity: Performance, activity enzymatic, and bacterial community shift.

Ever-increasing production and use of nanoparticles (NPs) have aroused overarching concerns for their toxic effects on the environment and human. In the present study, the toxic effects of Silver (Ag) and Iron (Fe) NPs on the performance of activated sludge were investigated under continuous aerobic/anoxic/anaerobic conditions in laboratory-scale sequencing batch reactors (SBRs).Activated sludge was exposed to various concentrations (5-100 mg/L) of Ag-Fe NPs for 60 days and its response was assessed through the enzymatic activity, COD, nitrogen (TN) and phosphorus (TP) removal, toxicity tests, as well as variations in bacterial community. Compared with the pristine control sample, the exposure to NPs suppressed TN and TP removal efficiencies. Indeed, the respiration rate and biomass concentration were significantly affected by the NPs. Although the simultaneous exposure to Ag-Fe NPs did affect the integrity of cell membrane (LDH) and key enzymes activities, the higher concentration induced an increased generation of reactive oxygen species (ROS). The metagenome analysis revealed a marked shift in the microbial community structure suggesting that both heterotrophic and autotrophic communities were affected by the presence of Ag-Fe NPs. Our results provide some evidence for compounded effects of NPs in their simultaneous presence, and generate new leads for future research efforts.

Lighting-Up Tumor for Assisting Resection via Spraying NIR Fluorescent Probe of γ-Glutamyltranspeptidas.

For the precision resection, development of near-infrared (NIR) fluorescent probe based on specificity identification tumor-associated enzyme for lighting-up the tumor area, is urgent in the field of diagnosis and treatment. Overexpression of γ-glutamyltranspeptidase, one of the cell-membrane enzymes, known as a biomarker is concerned with the growth and progression of ovarian, liver, colon and breast cancer compared to normal tissue. In this work, a remarkable enzyme-activated NIR fluorescent probe NIR-SN-GGT was proposed and synthesized including two moieties: a NIR dicyanoisophorone core as signal reporter unit; γ-glutamyl group as the specificity identification site. In the presence of γ-GGT, probe NIR-SN-GGT was transformed into NIR-SN-NH2, the recovery of Intramolecular Charge Transfer (ICT), liberating the NIR fluorescence signal, which was firstly employed to distinguish tumor tissue and normal tissues via simple “spraying” manner, greatly promoting the possibility of precise excision. Furthermore, combined with magnetic resonance imaging by T2 weight mode, tumor transplanted BABL/c mice could be also lit up for first time by NIR fluorescence probe having a large stokes, which demonstrated that probe NIR-SN-GGT would be a useful tool for assisting surgeon to diagnose and remove tumor in clinical practice.

Na+ , K+ -ATPase activity in children with autism spectrum disorder: Searching for the reason(s) of its decrease in blood cells.

Na+, K+‐ATPase (NKA) activity, which establishes the sodium and potassium gradient across the cell membrane and is instrumental in the propagation of the nerve impulses, is altered in a number of neurological and neuropsychiatric disorders, including autism spectrum disorders (ASD). In the present work, we examined a wide range of biochemical and cellular parameters in the attempt to understand the reason(s) for the severe decrease in NKA activity in erythrocytes of ASD children that we reported previously. NKA activity in leukocytes was found to be decreased independently from alteration in plasma membrane fluidity. The different subunits were evaluated for gene expression in leukocytes and for protein expression in erythrocytes: small differences in gene expression between ASD and typically developing children were not apparently paralleled by differences in protein expression. Moreover, no gross difference in erythrocyte plasma membrane oxidative modifications was detectable, although oxidative stress in blood samples from ASD children was confirmed by increased expression of NRF2 mRNA. Interestingly, gene expression of some NKA subunits correlated with clinical features. Excess inhibitory metals or ouabain‐like activities, which might account for NKA activity decrease, were ruled out. Plasma membrane cholesterol, but not phosphatidylcholine and phosphatidlserine, was slighty decreased in erythrocytes from ASD children. Although no compelling results were obtained, our data suggest that alteration in the erytrocyte lipid moiety or subtle oxidative modifications in NKA structure are likely candidates for the observed decrease in NKA activity. These findings are discussed in the light of the relevance of NKA in ASD. Autism Res 2018, 11: 1388–1403. © 2018 International Society for Autism Research, Wiley Periodicals, Inc.Lay SummaryThe activity of the cell membrane enzyme NKA, which is instrumental in the propagation of the nerve impulses, is severely decreased in erythrocytes from ASD children and in other brain disorders, yet no explanation has been provided for this observation. We strived to find a biological/biochemical cause of such alteration, but most queries went unsolved because of the complexity of NKA regulation. As NKA activity is altered in many brain disorders, we stress the relevance of studies aimed at understanding its regulation in ASD.

Calcium-engaged Mechanisms of Nongenomic Action of Neurosteroids.

Background: Neurosteroids form the unique group because of their dual mechanism of action. Classically, they bind to specific intracellular and/or nuclear receptors, and next modify genes transcription. Another mode of action is linked with the rapid effects induced at the plasma membrane level within seconds or milliseconds. The key molecules in neurotransmission are calcium ions, thereby we focus on the recent advances in understanding of complex signaling crosstalk between action of neurosteroids and calcium-engaged events.Methods: Short-time effects of neurosteroids action have been reviewed for GABAA receptor complex, glycine receptor, NMDA receptor, AMPA receptor, G protein-coupled receptors and sigma-1 receptor, as well as for several membrane ion channels and plasma membrane enzymes, based on available published research.Results: The physiological relevance of neurosteroids results from the fact that they can be synthesized and accumulated in the central nervous system, independently from peripheral sources. Fast action of neurosteroids is a prerequisite for genomic effects and these early events can significantly modify intracellular downstream signaling pathways. Since they may exert either positive or negative effects on calcium homeostasis, their role in monitoring of spatio-temporal Ca2+ dynamics, and subsequently, Ca2+-dependent physiological processes or initiation of pathological events, is evident.Conclusion: Neurosteroids and calcium appear to be the integrated elements of signaling systems in neuronal cells under physiological and pathological conditions. A better understanding of cellular and molecular mechanisms of nongenomic, calcium-engaged neurosteroids action could open new ways for therapeutic interventions aimed to restore neuronal function in many neurological and psychiatric diseases.

Soluble Amyloid Precursor Protein Alpha Interacts with alpha3-Na, K-ATPAse to Induce Axonal Outgrowth but Not Neuroprotection: Evidence for Distinct Mechanisms Underlying these Properties.

Amyloid precursor protein (APP) is cleaved not only to generate the amyloid peptide (Aß), involved in neurodegenerative processes, but can also be metabolized by alpha secretase to produce and release soluble N-terminal APP (sAPPα), which has many properties including the induction of axonal elongation and neuroprotection. The mechanisms underlying the properties of sAPPα are not known. Here, we used proteomic analysis of mouse cortico-hippocampal membranes to identify the neuronal specific alpha3 (α3)-subunit of the plasma membrane enzyme Na, K-ATPase (NKA) as a new binding partner of sAPPα. We showed that sAPPα recruits very rapidly clusters of α3-NKA at neuronal surface, and its binding triggers a cascade of events promoting sAPPα-induced axonal outgrowth. The binding of sAPPα with α3-NKA was not observed for sAPPα-induced Aß1-42 oligomers neuroprotection, neither the downstream events particularly the interaction of sAPPα with APP before endocytosis, ERK signaling, and the translocation of SET from the nucleus to the plasma membrane. These data suggest that the mechanisms of the axonal growth promoting and neuroprotective properties of sAPPα appear to be specific and independent. The signals at the cell surface specific to trigger these mechanisms require further study.

The absolute quantitative analysis of cingulate cortex metabolites in preschool children with autism spectrum disorder using proton MR spectroscopy

Objective To investigate the metabolite changes in the preschool children with autism spectrum disorder (ASD) using MR spectroscopy (MRS) and explore the associations between image findings and clinical variables, which may provide a noninvasive brain biochemical method for the early diagnosis and prevention of autism. Methods Twenty one cases of preschool ASD children (3-6 years old) and age-and sex-matched 20 preschool healthy controls underwent single voxel short (SVS) short TE (TE=30 ms) MRS. The absolute metabolite concentrations in the anterior cingulate cortex (ACC), anterior middle anterior cingulate cortex (aMCC) and posterior cingulate (PCC) were quantitatively analyzed using LCModel software. Two independent sample t tests were used for analysis. The relationships between metabolite concentrations and diagnostic and statistical manual of mental disorders (DSM-IV), childhood autism rating scale (CARS) and autism behavior checklist (ABC) were analyzed by Pearson correlation analysis. Results Compared to control subjects, ASD patients had significantly lower N-acetylaspartate (NAA) values (4.35±0.80, 6.34±0.82, 8.04±0.97 mmol/L respectively) in ACC, aMCC and PCC (t=2.640, P=0.012; t=2.182, P=0.035; t=3.343, P=0.002), had significantly lower choline (Cho) 1.32±0.22 mmol/L (t=2.905, P=0.006) and glutamine and glutamate complex (Glx) 10.02±0.88 mmol/L (t=2.090, P=0.043) in PCC. Cho, total creatine (tCr), myo-Inositol (MI) and Glx levels did not differ between groups in other aforementioned regions (P>0.05). Negative correlations between the NAA ualues in the PCC and CARS (r=-0.504, P=0.020) were detected, and no significant correlation among DSM-IV, CARS, ABC and other metabolite values (P>0.05). Conclusions The biochemical changes in the preschool children with ASD in cingulate reflect the neuronal loss, structural or functional damage and cell membrane enzyme metabolic dysfunctions, may reveal the pathological basis of ASD. These results may provide noninvasive and quantitative methods for the diagnosis and prognosis evaluation of ASD child. Key words: Autism spectrum disorder; Magnetic resonance spectroscopy; Cingulate cortex; Child

The role of brassinosteroids in the regulation of the plasma membrane H+-ATPase and NADPH oxidase under cadmium stress

The present research aim was to define the role of brassinosteroids (BRs) in plant adaptation to cadmium stress. We observed a stimulating effect of exogenous BR on the activity of two plasma membrane enzymes which play a key role in plants adaptation to cadmium stress, H+-ATPase (EC 3.6.3.14) and NADPH oxidase (EC 1.6.3.1). Using anti-phosphothreonine antibody we showed that modification of PM H+-ATPase activity under BR action could result from phosphorylation of the enzyme protein. Also the relative expression of genes encoding both PM H+-ATPase and NADPH oxidase was affected by BR. To confirm the role of BR in the cadmium stimulating effect on activity of both studied plasma membrane enzymes, an assay in the presence of a BR biosynthesis inhibitor (propiconazole) was performed. Moreover, as a tool in our work we used commercially available plant mutants unable to BR biosynthesis or with dysfunctional BR signaling pathway, to further confirm participation of BR in plant adaptation to heavy metal stress. Presented results demonstrate some elements of the brassinosteroid-induced pathway activated under cadmium stress, wherein H+-ATPase and NADPH oxidase are key factors.

Abstract 1270: Vitamin E related genetic variants and prostate cancer risk: A Mendelian randomization analysis

Abstract Alpha-tocopherol is a highly biologically active form of vitamin E that has been inconsistently associated with prostate cancer risk in cohorts and supplementation trials. In order to further elucidate the association using a Mendelian randomization approach that avoids sources of bias common in observational research, we examined genetic variants related to vitamin E status, including those from GWAS analyses of circulating vitamin E, in relation to prostate cancer risk in the Prostate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome (PRACTICAL) Consortium. The analysis included 38,868 cases and 25,210 controls from 35 participating cohorts and examined the association between 847 vitamin E-related single nucleotide polymorphisms (SNPs) in (or near) six genes and risk of prostate cancer. Logistical regression was used to estimate meta-analyzed case-control odds ratios (OR) and 95% confidence intervals (CI) for the per allele risk associations. Both genotyped and imputed data were examined, and results for the latter are presented based on the consistency between the two sets of findings. In addition to examining SNPs demonstrating nominal significance (alpha-error p&amp;lt;0.05), a Bonferroni threshold of &amp;lt;0.00006 was applied to adjust for multiple comparisons. We found 89 SNPs were nominally significantly associated with prostate cancer risk. This included SNPs involved in vitamin E transport (i.e., SEC14L2 and SCARB1) and metabolism (i.e., BUD13/ZNF259). The top three risk-associated SNPs (P &amp;lt;0.001) included rs141696823 (OR = 0.59, P=6.0x10-5) in CYP4F8 (cytochrome P450 family 4 subfamily F member 8) on 19p13.12 which functions as a prostaglandin hydroxylase in the seminal vesicles, and rs1915379 (OR = 1.04, P=0.0002) in CYP4F3 (cytochrome P450 family 4 subfamily F member 3) on 19p13.12 which oxidizes arachidonic acid and omega-hydroxylates tocopherol phytyl side chains, and has been directly associated with higher circulating alpha-tocopherol. The final SNP (r185614299, OR = 1.19, P=0.0004) is in NKAIN3 on 8q12.3, which encodes the Na+/K+ transporting ATPase Interacting Protein 3, a plasma membrane enzyme responsible for maintenance of intracellular electrolyte gradients. Vitamin E has been shown to reduce cell membrane lipid peroxidation, thereby promoting Na/K-ATPase activity. Other variants on this gene have previously been associated with serum alpha-tocopherol concentration in response to vitamin E supplementation in the ATBC Study. The present Mendelian randomization analysis suggests genetic variants related to vitamin E metabolism, its role as an antioxidant, and its function in inflammatory processes, may be associated with prostate cancer risk. Citation Format: Tracy M. Layne, Shakira M. Nelson, Stephanie J. Weinstein, Eric Karlins, Stephen J. Chanock, Demetrius Albanes, The PRACTICAL Consortium. Vitamin E related genetic variants and prostate cancer risk: A Mendelian randomization analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1270. doi:10.1158/1538-7445.AM2017-1270

Selective ligands for Na+/K+-ATPase α isoforms differentially and cooperatively regulate excitability of pyramidal neurons in distinct brain regions

Sodium-potassium ATPase (NaKA) is a plasma membrane enzyme responsible for influencing membrane physiology by direct electrogenic activity. It determines cellular excitability and synaptic neurotransmission, thus affecting learning and memory processes. A principle catalytic α subunit of NaKA has development-specific expression pattern. There are two α isoforms, α1 and α3, in adult brain neurons. Although NaKA is a housekeeping enzyme, the physiological differences between these two α isoforms in different brain regions have not been well explored. Endogenous cardiotonic steroids, including Marinobufagenin and Ouabain, control the cell homeostasis and cell functions via inhibiting NaKA. Here we employed selective inhibition of α1 and α3 NaKA isoforms by Marinobufagenin and Ouabain respectively, to measure the contribution of α subunits in cellular physiology of three distinct mouse brain regions. The results of the whole cell recording demonstrated that α1 isoform predominated in layer-5 pyramidal cells at rostral motor cortex, while α3 isoform governed the pyramidal neurons at hippocampal CA1 region and to a lesser extent the layer-5 pyramidal neurons of parietal cortex. Furthermore, selective α isoform inhibition induced differential effects on distinct physiological properties even within the same brain region. In addition, our results supported the existence of synergism between two NaKA α isoforms. To conclude, this systematic study of NaKA α isoforms demonstrated their broader roles in neuronal functioning in a region-specific manner.

MAMP (microbe-associated molecular pattern)-induced changes in plasma membrane-associated proteins

Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins.

Cytotoxicity Assays as Predictors of the Safety and Efficacy of Antimicrobial Agents.

The development of safe antimicrobial agents is important for the effective treatment of pathogens. From a multitude of discovered inhibitory compounds only few antimicrobial agents are able to enter the market. Many antimicrobials are, on the one hand, quite effective in killing pathogens but, on the other hand, cytotoxic to eukaryotic cells. Cell health can be monitored by various methods. Plasma membrane integrity, DNA synthesis, enzyme activity, and reducing conditions within the cell are known indicators of cell viability and cell death. For a comprehensive overview, methods to analyze cytotoxic and hemolytic effects, e.g., lactate dehydrogenase release, cell proliferation analysis, cell viability analysis, and hemolysis assay of antimicrobial compounds on human cells, are described in this chapter.

Cold Shock Proteins: A Minireview with Special Emphasis on Csp-family of Enteropathogenic Yersinia.

Bacteria have evolved a number of mechanisms for coping with stress and adapting to changing environmental conditions. Many bacteria produce small cold shock proteins (Csp) as a response to rapid temperature downshift (cold shock). During cold shock, the cell membrane fluidity and enzyme activity decrease, and the efficiency of transcription and translation is reduced due to stabilization of nucleic acid secondary structures. Moreover, protein folding is inefficient and ribosome function is hampered. Csps are thought to counteract these harmful effects by serving as nucleic acid chaperons that may prevent the formation of secondary structures in mRNA at low temperature and thus facilitate the initiation of translation. However, some Csps are non-cold inducible and they are reported to be involved in various cellular processes to promote normal growth and stress adaptation responses. Csps have been shown to contribute to osmotic, oxidative, starvation, pH and ethanol stress tolerance as well as to host cell invasion. Therefore, Csps seem to have a wider role in stress tolerance of bacteria than previously assumed. Yersinia enterocolitica and Yersinia pseudotuberculosis are enteropathogens that can spread through foodstuffs and cause an enteric infection called yersiniosis. Enteropathogenic Yersinia are psychrotrophs that are able to grow at temperatures close to 0°C and thus they set great challenges for the modern food industry. To be able to efficiently control psychrotrophic Yersinia during food production and storage, it is essential to understand the functions and roles of Csps in stress response of enteropathogenic Yersinia.

Enhanced cell adhesion on bioinert ceramics mediated by the osteogenic cell membrane enzyme alkaline phosphatase

Functional bone and dental implant materials are required to guide cell response, offering cues that provide specific instructions to cells at the implant/tissue interface while maintaining full biocompatibility as well as the desired structural requirements and functions. In this work we investigate the influence of covalently immobilized alkaline phosphatase (ALP), an enzyme involved in bone mineralization, on the first contact and initial cell adhesion. To this end, ALP is covalently immobilized by carbodiimide-mediated chemoligation on two highly bioinert ceramics, alpha-alumina (Al2O3) and yttria-stabilized zirconia (Y-TZP) that are well-established for load-bearing applications. The physicochemical surface properties are evaluated by profilometry, zeta potential and water contact angle measurements. The initial cell adhesion of human osteoblasts (HOBs), human osteoblast-like cells (MG-63) and mesenchymal stromal cells (hMSCs) was investigated. Cell adhesion was assessed at serum free condition via quantification of percentage of adherent cells, adhesion area and staining of the focal adhesion protein vinculin. Our findings show that after ALP immobilization, the Al2O3 and Y-TZP surfaces gained a negative charge and their hydrophilicity was increased. In the presence of surface-immobilized ALP, a higher cell adhesion, more pronounced cell spreading and a higher number of focal contact points were found. Thereby, this work gives evidence that surface functionalization with ALP can be utilized to modify inert materials for biological conversion and faster bone regeneration on inert and potentially load-bearing implant materials.

Legume NADPH Oxidases Have Crucial Roles at Different Stages of Nodulation.

Plant NADPH oxidases, formerly known as respiratory burst oxidase homologues (RBOHs), are plasma membrane enzymes dedicated to reactive oxygen species (ROS) production. These oxidases are implicated in a wide variety of processes, ranging from tissue and organ growth and development to signaling pathways in response to abiotic and biotic stimuli. Research on the roles of RBOHs in the plant’s response to biotic stresses has mainly focused on plant-pathogen interactions; nonetheless, recent findings have shown that these oxidases are also involved in the legume-rhizobia symbiosis. The legume-rhizobia symbiosis leads to the formation of the root nodule, where rhizobia reduce atmospheric nitrogen to ammonia. A complex signaling and developmental pathway in the legume root hair and root facilitate rhizobial entrance and nodule organogenesis, respectively. Interestingly, several reports demonstrate that RBOH-mediated ROS production displays versatile roles at different stages of nodulation. The evidence collected to date indicates that ROS act as signaling molecules that regulate rhizobial invasion and also function in nodule senescence. This review summarizes discoveries that support the key and versatile roles of various RBOH members in the legume-rhizobia symbiosis.

Evolution of the α-Subunit of Na/K-ATPase from Paramecium to Homo sapiens: Invariance of Transmembrane Helix Topology.

Na/K-ATPase is a key plasma membrane enzyme involved in cell signaling, volume regulation, and maintenance of electrochemical gradients. The α-subunit, central to these functions, belongs to a large family of P-type ATPases. Differences in transmembrane (TM) helix topology, sequence homology, helix–helix contacts, cell signaling, and protein domains of Na/K-ATPase α-subunit were compared in fungi (Beauveria), unicellular organisms (Paramecia), primitive multicellular organisms (Hydra), and vertebrates (Xenopus, Homo sapiens), and correlated with evolution of physiological functions in the α-subunit. All α-subunits are of similar length, with groupings of four and six helices in the N- and C-terminal regions, respectively. Minimal homology was seen for protein domain patterns in Paramecium and Hydra, with high correlation between Hydra and vertebrates. Paramecium α-subunits display extensive disorder, with minimal helix contacts. Increases in helix contacts in Hydra approached vertebrates. Protein motifs known to be associated with membrane lipid rafts and cell signaling reveal significant positional shifts between Paramecium and Hydra vulgaris, indicating that regional membrane fluidity changes occur during evolution. Putative steroid binding sites overlapping TM-3 occurred in all species. Sites associated with G-protein-receptor stimulation occur both in vertebrates and amphibia but not in Hydra or Paramecia. The C-terminus moiety “KETYY,” necessary for the Na+ activation of pump phosphorylation, is not present in unicellular species indicating the absence of classical Na+/K+-pumps. The basic protein topology evolved earliest, followed by increases in protein domains and ordered helical arrays, correlated with appearance of α-subunit regions known to involve cell signaling, membrane recycling, and ion channel formation.

Colorimetric Assays of Na,K-ATPase.

The Na,K-ATPase is a plasma membrane enzyme that catalyzes active ion transport by the hydrolysis of ATP. Its activity in vivo is determined by many factors, particularly the concentration of intracellular sodium ions. It is the target of the cardiac glycoside class of drugs and of endogenous regulators. Its assay is often an endpoint in the investigation of physiological processes, and it is a promising drug target. As described in this unit, its enzymatic activity can be determined in extracts from tissues by test tube assay using a spectrophotometer or (32)P-ATP. The protocols in this chapter measure inorganic phosphate as the end product of hydrolysis of ATP.

ACTIVITIES OF PROXIMAL TUBULE ENZYMES AND ALBUMIN CONCENTRATION IN URINE OF CHILDREN TREATED WITH METHOTREXATE

In order to study methotrexate nephrotoxicity, the activities of proximal tubule epithelial cell membrane enzymes: alanine aminopeptidase (AAP) and gamma-glutamyltransferase (GGT), as well as of lysosomal N-acetyl-beta-D-glucosaminidase (NAG) and urinary albumin concentrations were determined in 12-h-urine samples of 30 patients with lymphoblastomous leukemia. The patients were i.v. receiving 4 individual methotrexate doses of 2000 mg/m 2 every 15 days followed by leucovorin as a protector. Control and methotrexate-treated group, each consisting of 30 examinees, included 4–10 years old children of both sexes. Statistically significant increase of AAP and GGT activities, expressed as U/mmol creatinine was observed after the first two ( p < 0.05), as well as after the remaining two therapies ( p < 0.01) in relation to the control. Enzymatic activities of these two enzymes decreased to control value before the second and the third methotrexate application, but they increased again after the third application and remained elevated up to the end of experiments. Significant increase of NAG activity expressed as U/mmol creatinine ( p < 0.01), as well as urinary albumin levels (mg/mmol creatinine; p < 0.01) were registered after the third methotrexate therapy and this elevation of the same statistical significance of the differences remained stable till the end of the therapy. Based on these results it can be concluded that during the time period of two first applications nephrotoxic methotrexate action is reversible and at the level of proximal tubule epithelial cell membranes. During the last two applications impairment is irreversible and at the level of cell organelles and glomerular filtration.