Intracellular Enzyme Activity Research Articles

Dissecting the Function of IAP (Inhibitor of Apoptosis) Protein Domains in Inhibiting an Apoptotic Caspase

Apoptosis (programmed cell death) is an essential process in all animals for normal development and tissue regeneration. Apoptosis also eliminates cells at risk for becoming cancerous or spreading a viral infection. Apoptosis requires the activity of intracellular caspase enzymes that are normally kept inhibited in living cells by inhibitor of apoptosis proteins (IAPs). Certain apoptosis‐promoting proteins bind IAPs to relieve caspase inhibition. IAPs contain 2–3 BIR (baculovirus IAP repeat) domains with intervening linkers. In many IAPs, residues in a linker bind and block the caspase active site but the BIR domains also influence caspase inhibition. To determine how the BIRs contribute to caspase inhibition, we compared the ability of different fragments of the Drosophila IAP DIAP1 to inhibit a Drosophila caspase using purified recombinant proteins and an in vitro caspase assay. 2 μM of a DIAP1 fragment containing the BIR1 and BIR2 domains with intervening linker inhibited the caspase by 72 ± 1%. Removing either BIR domain reduced the inhibition to 39–45 ± 10%. Neither BIR domain alone inhibited the caspase even at 6 μM. However, 6 μM added BIR1 domain interfered with the 2 μM BIR1‐linker, reducing its caspase inhibition from 56 ± 1% to 30 ± 2%. An affinity chromatography binding assay detected direct BIR1 domain binding to the caspase. This suggests that the BIR1 interfered with the BIR1‐linker by binding to and blocking a critical BIR1‐binding site on the caspase. Thus, the BIR1 likely functions within DIAP1 by helping anchor the linker to the caspase active site. The BIR2 domain did not interfere with the linker‐BIR2 nor show direct binding to the caspase, indicating that its mechanism for enhancing caspase inhibition by DIAP1 must differ from that of the BIR1 domain. The role of the BIR2 as well as of other linker residues is being further investigated.Support or Funding InformationThis research was funded by a Kutztown University Research Grant to M. Junker and a Kutztown University BEARS grant and a Kutztown University Undergraduate Research Committee Grant to M. Storm.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparison of ATP sulfurylase 2 from selenium hyperaccumulator Stanleya pinnata and non-accumulator Stanleya elata reveals differential intracellular localization and enzyme activity levels

BackgroundThe plant Stanleya pinnata hyperaccumulates Se up to 0.5% of its dry weight in organic forms, whereas the closely related Stanleya elata does not hyperaccumulate Se. ATP sulfurylase (ATPS) can catalyze the formation of adenosine 5′-phosphoselenate (APSe) from ATP and selenate. We investigated the S. pinnata ATPS2 isoform (SpATPS2) to assess its possible role in Se hyperaccumulation. MethodsATPS expression and activity was compared in the two Stanleya species. The ATPS2 protein sequences were modeled. Sub-cellular locations were analyzed using GFP fusions. Enzyme activity of purified recombinant SpATPS2 was measured. ResultsATPS2 transcript levels were six-fold higher in roots of S. pinnata relative to S. elata. Overall root ATPS enzyme activity was two-fold elevated in S. pinnata. Cloning and sequencing of SpATPS2 and S. elata ATPS2 (SeATPS2) showed the predicted SeATPS2 to be canonical, while SpATPS2, although very similar in its core structure, has unique features, including an interrupted plastid targeting signal due to a stop codon in the 5′ region of the coding sequence. Indeed GFP fusions revealed that SpATPS2 had exclusive cytosolic localization, while SeATPS2 showed dual localization in plastids and cytosol. SpATPS2 activity was inhibited by both sulfate and selenate, indicating that the enzyme acts on both substrates. ConclusionsThe ATPS2 from S. pinnata differs from non-accumulator ATPS2 in its elevated expression and sub-cellular localization. It likely acts on both selente and sulfate substrates. General significanceThese observations shed new light on the role of ATPS2 in the evolution of Se hyperaccumulation in plants. This article is part of a Special Issue entitled Selenium research in biochemistry and biophysics - 200 year anniversary issue, edited by Dr. Elias Arnér and Dr. Regina Brigelius-Flohe.

Rapid Analysis and Guided Isolation of Astragalus Isoflavonoids by UHPLC-DAD-MSn and Their Cellular Antioxidant Defense on High-Glucose-Induced Mesangial Cell Dysfunction.

Isoflavonoids, including isoflavones, isoflavans, and pterocarpans, the principal components in Astragalus membranaceus, have a great deal of versatile health-promoting benefits. In this work, as a continuation of our search for bioactive constituents from A. membranaceus, a fast high-performance liquid chromatography-diode array detection-multiple-stage mass spectrometry method was first used to analyze the isoflavonoid profile of A. membranaceus roots extract. Twelve diverse isoflavonoids in subclasses of isoflavones, isoflavans, and pterocarpans present in glycoside/aglycone pair forms were tentatively characterized; of those 12, eight major isoflavonoids were finally isolated and simultaneously quantified by the established fast UHPLC method. Furthermore, the results confirmed for the first time that Astragalus isoflavonoid aglycones could attenuate mesangial cell proliferation and extracellular matrix (ECM) accumulation triggered by high glucose levels, and the primary mechanism might be via protecting intracellular antioxidant enzymes activities and enhancing endogenous antioxidant function to lower levels of cellular oxidative damage induced by high glucose levels. Collectively, diverse Astragalus isoflavonoid antioxidants have the potential to ameliorate high-glucose-induced mesangial cell dysfunction through the regulation of cellular antioxidant defense.

Condensed Tannins from Longan Bark as Inhibitor of Tyrosinase: Structure, Activity, and Mechanism.

In this study, the content, structure, antityrosinase activity, and mechanism of longan bark condensed tannins were evaluated. The findings obtained from mass spectrometry demonstrated that longan bark condensed tannins were mixtures of procyanidins, propelargonidins, prodelphinidins, and their acyl derivatives (galloyl and p-hydroxybenzoate). The enzyme analysis indicated that these mixtures were efficient, reversible, and mixed (competitive is dominant) inhibitor of tyrosinase. What's more, the mixtures showed good inhibitions on proliferation, intracellular enzyme activity and melanogenesis of mouse melanoma cells (B16). From molecular docking, the results showed the interactions between inhibitors and tyrosinase were driven by hydrogen bond, electrostatic, and hydrophobic interactions. In addition, high levels of total phenolic and extractable condensed tannins suggested that longan bark might be a good source of tyrosinase inhibitor. This study would offer theoretical basis for the development of longan bark condensed tannins as novel food preservatives and medicines of skin diseases.

Easy and Efficient Permeabilization of Cyanobacteria for in vivo Enzyme Assays Using B-PER.

Cyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO2 and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. A better understanding of activities of enzymes involved in the central carbon metabolism might lead to increased product yields. Currently, cell-free lysates are widely used for the determination of intracellular enzyme activities. However, due to thick cell walls in cyanobacteria, lysis of cyanobacterial cells is inefficient and often laborious. The present protocol describes an easy and efficient method to permeabilize cyanobacterial cells, without lysing them, and direct usage of the permeabilized cells for the determination of metabolic enzyme activities in vivo.

Co-metabolic degradation of iomeprol by a Pseudomonas sp. and its application in biological aerated filter systems

ABSTRACTThe non-ionic water-soluble X-ray contrast agent iomeprol (IOM) enters the water supply through sewage treatment plants, which can cause considerable environmental harm. In this study, Pseudomonas sp. I-24 (I-24) was tested for its ability to remove IOM from water via co-metabolic pathways. The optimum removal rate of IOM by I-24 was 38.43% ± 3.70% when starch served as the source of external carbon, and its co-metabolism of IOM conformed to the first-order kinetics. The highest activity of intracellular enzyme (degrading enzyme) extracted from I-24 was 0.143 ± 0.005 mU in starch condition. The Michaelis constant of the degrading enzyme was found to be 91.08 μmol L−1. However, glucose and maltose showed the best promotive effects on the growth and electron transport activity of I-24, indicating that overgrowth may result in competitive inhibition and a reduced degradation rate of IOM. Adding I-24 and degrading enzymes to biological aerated filters increased IOM removal rates without affecting CODMn removal.

A Water-Soluble Galactose-Decorated Cationic Photodynamic Therapy Agent Based on BODIPY to Selectively Eliminate Biofilm.

A multitude of serious chronic infections are involved in bacterial biofilms that are difficult to eradicate. Here, a water-soluble galactose-functionalized cationic 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-based photodynamic therapy agent was synthesized for selectively eliminating the bacterial biofilm. These conjugates can capture bacteria to form aggregations through electrostatic interaction and then generate a large number of reactive oxygen species (ROS) under visible light irradiation to kill the bacteria without the emergence of bacterial resistance. Simultaneously, this agent could effectively inhibit and eradicate both Gram-positive and Gram-negative bacterial biofilms. The in-depth analysis of the antimicrobial mechanism confirmed that the conjugates can quickly bind on the bacterial surface, irreversibly disrupt the bacterial membrane, and distinctly inhibit intracellular enzyme activity, ultimately leading to the bacterial death. Importantly, these conjugates are highly selective toward bacterial cells over mammalian cells as well as no cytotoxicity to A549 cells and no discernible hemolytic activity. Collectively, this water-soluble galactose-decorated cationic BODIPY-based photodynamic therapy agent design provides promising insights for the development of therapy for antibiotic-resistant bacteria.

Effect of preliminary stresses on the resistance of Escherichia coli and Staphylococcus aureus toward non-thermal plasma (NTP) challenge

As the development of hurdle technology, cross-protection of various stresses for pathogens posed the potential risk to food safety and public health. This study tried to explore various preliminary stresses including acidity, osmosis, oxidation, heat and cold on the resistance of microbial cells toward the non-thermal plasma (NTP) exposure. The results indicated that short-term (4h) exposure of Staphylococcus aureus and Escherichia coli to acidity, osmosis, oxidation, heat and cold stresses did not lead to the resistance to the subsequent NTP treatment. On the contrary, acidity, osmosis and heat preadaptation increased the vulnerability of E. coli cells to NTP treatment. After exposing S. aureus to osmosis, oxidation, heat and cold stress for longer period (24h), the reduction level showed significantly (P<0.05) higher. Interestingly, long-term (24h) preliminary exposure of acidic stress exhibited protective effect for S. aureus against the following NTP exposure with less damage in cell membrane integrity, membrane potential and intracellular enzyme activity. It might be due to the protein production for oxidative stress response during preliminary acidic adaptation. In general, the obtained result helped to grasp better understanding of the microbial stress response to NTP treatment and provided insight for the future research in order to accelerate the development of NTP technology in food industry.

Ursolic Acid, a Natural Nutraceutical Agent, Targets Caspase3 and Alleviates Inflammation-Associated Downstream Signal Transduction.

ScopeUrsolic acid (UA) is a pentacyclicterpenoid carboxylic acid that is present in a wide variety of plant foods. There are many beneficial health effects that are attributed to the properties of UA. However, the specific cellular targets of UA and the mechanism underlying downstream signal transduction processes linked to the anti‐inflammation pathway have not been thoroughly elucidated to date.Methods and resultsChemical biology strategies such as target fishing, click reaction synthesis of a UA probe and molecular imaging were used to identify potential target proteins of UA. Cysteinyl aspartate specific proteinase 3 (CASP3) and its downstream signaling pathway were verified as potential targets by molecular docking, intracellular enzyme activity evaluation and accurate pathway analysis. The results indicated that UA acted on CASP3, ERK1 and JNK2 targets, alleviated inflammation‐associated downstream multiple signal transduction factors, including ERK1, NF‐κB and STAT3, and exhibited anti‐inflammation activities.ConclusionAs a natural dietary supplement, UA demonstrated anti‐inflammation activity via inhibition of CASP3 and shows the potential to improve the therapy effect of several inflammation‐associated diseases.

THE PHENOTYPE AND METABOLISM RELATIONSHIP OF BLOOD NEUTROPHILS IN PATIENTS WITH WIDESPREAD PURULENT PERITONITIS IN THE POSTOPERATIVE PERIOD DYNAMICS

The aim of the study was to examine the relationship of the phenotype and metabolism of neutrophils in patients with widespread purulent peritonitis (WPP) in the dynamics of the postoperative period. The study involved 27 patients with acute surgical diseases and injuries of abdominal organs complicated by WPP. Blood sampling was performed prior to surgery (pre-operative period) and at 7, 14 and 24 day post-operative period. As controls 67 respect healthy people were examined. Research blood neutrophilic granulocytes phenotype was performed by f low cytometry using a direct immunof luorescence whole peripheral blood. The levels of surface receptor expression was assessed by the mean f luorescence intensity. The NADand NADP-dependent dehydrogenases activity in the blood neutrophils studied using bioluminescence method. It was found that in patients with WPP in the preoperative period in the peripheral blood increased content of CD62L+-, HLA-DR+and CD64+-neutrophils. High levels of CD62L+-cells stored within 24 postoperative days, whereas the amount of HLA-DR+and CD64+-neutrophils on 24 postoperative day is reduced to the level of controls. The dynamics of changes in the content of CD64+-cells in the peripheral blood of patients with WPP corresponds to the expression level of CD64-receptor on the membrane of neutrophilic granulocytes. The metabolism of blood neutrophils in patients with WPP in the preand postoperative period is characterized by high intensity of the substrate stream on the citric acid cycle, low activity of NADP-dependent glutamate dehydrogenase and aerobic reaction of lactate dehydrogenase. In the preoperative period and within 14 days of the postoperative period in neutrophil granulocytes of the patients revealed high activity of anaerobic lactate dehydrogenase reaction characterized by increased activity of anaerobic glycolysis. In the late postoperative period the intensity of anaerobic energy in the neutrophils of patients with WPP was reduced to the control level. The final stage of post-operative treatment the metabolism in the blood neutrophils of patients with WPP is also characterized by the activation of the pentose phosphate cycle, low activity of malate-aspartate shunt mitochondria and high intensity of the substrate interaction between citric acid cycle and reactions of amino acid metabolism. Using correlation analysis set dysregulation between phenotype and the system of intracellular metabolism of neutrophils which can be determined by the migration of activated cells in the inf lammatory focus as well as changes in the activity of intracellular enzymes under different regulatory factors and including postoperative therapy methods peritonitis.

PH-Sensitive degradable polymeric micelles for bio-triggered targeted anti-tumor drug delivery

Sanitation is a critical step to reduce microbial load in many process industries. To assess and monitor the efficacy of sanitation processes, there is a need to rapidly measure microbial resistance to sanitizers and determine the critical concentration of sanitizers above which significant reduction in microbial load can be achieved in a complex environment. In this study, a rapid and label-free magnetic resonance (MR) imaging and relaxometry method was developed to measure resistance of Escherichia coli and Listeria monocytogenes to hydrogen peroxide based on the activity of intracellular catalase enzymes. Intracellular catalase activity is a measure of resistance of bacteria to hydrogen peroxide as these enzymes can degrade hydrogen peroxide to oxygen and water. Using sub-lethal and lethal concentration of hydrogen peroxide, the resistance of both the exponential and stationary growth phase E. coli suspended in PBS and in high organic content solution was characterized. The results show that in stationary growth phase E. coli, molecular oxygen, generated by the interaction of endogenous catalases with 1% H2O2, results in an 8 fold increase in MR imaging signal intensity and a 22 fold decrease in spin-lattice relaxation time (T1) compared to the controls. Furthermore, the resistance of E. coli and L. monocytogenes to H2O2, measured using the MR method, was correlated with the bacterial reduction, quantified with traditional plate counting method. The comparison between the two methods shows the MR method developed in this study can determine the critical concentration of H2O2 above which a significant reduction in bacterial culturability (at least 4 logs) was achieved. Overall, this study demonstrates the potential of MR imaging and relaxometry method to predict the efficacy of H2O2 in high organic load environment.

Synergism between thioredoxin reductase inhibitor ethaselen and sodium selenite in inhibiting proliferation and inducing death of human non-small cell lung cancer cells

New effective treatment for human non-small cell lung cancer (NSCLC) is needed. The thioredoxin (Trx) system composes of thioredoxin reductase (TrxR), Trx and NADPH. In this study, we combined an organic selenium compound--TrxR inhibitor ethaselen (BBSKE) with low dosage sodium selenite to inhibit proliferation and induce death of NSCLC cells, and identified underlying mechanisms. Synergistic anti-proliferation effect of BBSKE and selenite was found in human NSCLC cell lines, A549, NCI-H1299 and NCI-1266. A significant increase of apoptosis, necrosis and autophagy were observed in the group of BBSKE plus selenite in A549 cells. The autophagy induced by BBSKE and selenite inhibited apoptosis and necrosis. In addition, BBSKE plus selenite induced G2/M arrest, which was verified by the alteration of gene and protein expression of cell cycle regulatory complexes. The intracellular enzyme activity of TrxR was remarkably decreased by cotreatment of BBSKE and selenite. Besides, the mRNA and protein level of TrxR1 and Trx1 were significantly inhibited by cotreatment of BBSKE and selenite. HEK 293 cells overexpressing TrxR1 were more sensitive to BBSKE plus selenite. The nuclear translocation of Trx1 and Ref-1, as well as expression of Ref-1 and AP-1 were inhibited by combination treatment. In short, BBSKE synergizes selenite in inhibiting proliferation and inducing death of NSCLC cells; BBSKE combined with selenite may be a treatment strategy for NSCLC.

Degradation of Cocaine and Benzoylecgonine in Sewage Sludge Batch Tests

The degradation studies of cocaine and its principal metabolites, benzoylecgonine, as emerging pollutants were carried out using various sludge types collected from the RAF Molesworth Sewage Treatment Work (STW) to determine their distribution and persistence in aquatic environment. The methodology, initially characterized the various sludge types before the laboratory batch tests were carried out at different temperatures and times. The results, after 3 hours equilibration of batch samples, showed the removal rates for cocaine and benzoylecgonine to be 91.0% and 90.6%, respectively. The partition coefficients (Kd) for the two substances ranged from 2.7 – 31.9 Kg L-1. Degradation of compounds was found to be both biotic and abiotic and the two processes produced many short chain compounds and several metabolites that include cocaethylene and ecgonine methylester. Also at 4 ± 0.5o C, the degradation of substances occurred slowly, at 5 and 10% but was relatively greater at 19 ± 0.5o C. Compounds degradation was also influenced by both extracellular and intracellular enzyme activities. The batch study is an innovative method in calculating degradation rates of compounds from sewage treatment works. The capabilities of generating removal rates of drugs have also been clearly demonstrated in this work and can be applied to any organic compounds in real life (STWs) situations

Anti-neuroinflammatory Activity of Elephantopus scaber L. via Activation of Nrf2/HO-1 Signaling and Inhibition of p38 MAPK Pathway in LPS-Induced Microglia BV-2 Cells.

Elephantopus scaber L. (family: Asteraceae) has been traditionally utilized as a folkloric medicine and scientifically shown to exhibit anti-inflammatory activities in various in vivo inflammatory models. Given the lack of study on the effect of E. scaber in neuroinflammation, this study aimed to investigate the anti-neuroinflammatory effect and the underlying mechanisms of ethyl acetate fraction from the leaves of E. scaber (ESEAF) on the release of pro-inflammatory mediators in lipopolysaccharide (LPS)-induced microglia cells (BV-2). Present findings showed that ESEAF markedly attenuated the translocation of NF-κB to nucleus concomitantly with the significant mitigation on the LPS-induced production of NO, iNOS, COX-2, PGE2, IL-1β, and TNF-α. These inflammatory responses were reduced via the inhibition of p38. Besides, ESEAF was shown to possess antioxidant activities evident by the DPPH and SOD scavenging activities. The intracellular catalase enzyme activity was enhanced by ESEAF in the LPS-stimulated BV-2 cells. Furthermore, the formation of ROS induced by LPS in BV-2 cells was reduced upon the exposure to ESEAF. Intriguingly, the reduction of ROS was found in concerted with the activation of Nrf2 and HO-1. It is conceivable that the activation promotes the scavenging power of antioxidant enzymes as well as to ameliorate the inflammatory response in LPS-stimulated BV-2 cells. Finally, the safety profile analysis through oral administration of ESEAF at 2000 mg/kg did not result in any mortalities, adverse effects nor histopathologic abnormalities of organs in mice. Taken altogether, the cumulative findings suggested that ESEAF holds the potential to develop as nutraceutical for the intervention of neuroinflammatory disorders.

P 016 - Cytoprotective effects of Chilean wild currants (Ribes spp.) against oxidative stress mediated by enhancing the activity of cellular antioxidant enzymes

The South American Ribes species are very appreciated for their sweet taste. Continuing our research on Ribes species, we decided to investigate the cytoprotective activity of this fruits against oxidative stress. Phenolic-enriched extracts (PEE), the anthocyanin fraction (AF) and copigments fraction (CF) were prepared in the lab. The cytoprotective effect of the Ribes was evaluated using human gastric epithelial cells (AGS). Cells were incubated overnight with the PEE, AF or CF at different concentrations. Then, AGS cells were exposed to H2O2 for 2 hour and viability was measured by means of the MTT reduction assay. In another experimental approach, cells were scrapped, collected by centrifugation and lysed to determine the activity of intracellular antioxidant enzymes. A significant cytoprotective effect was observed in a dose-dependent manner for all species studied. The highest activity was found for R. punctatum with 55.0 ± 1.8% of cell viability against 33.0 ± 0.4% of survival of untreated cells. No significant difference between PEE, AF or CF in the cytoprotective effect. A significant increase in the activity of superoxide dismutase by all Ribes species was found in a dose dependent manner. Our results on the protective potential of South American Ribes species provides evidence on the potential of native currants to be developed as functional foods.

Young leaves of reed (Phragmites communis) suppress melanogenesis and oxidative stress in B16F10 melanoma cells

This study investigated the effects young leaves of reed (Phragmites communis) water extract (YLR) on melanogenesis and oxidative stress using B16F10 cells. YLR decreased the intracellular melanin content, protein expression and enzyme activity of tyrosinase in a dose-dependent manner. YLR significantly decreased the gene and protein expression of melanogeneis-related proteins, such as microphthalmia-associated transcription factor (MITF), and tyrosinase-related protein-1 and -2. In addition, YLR up-regulated the melanogenesis inhibitory proteins, extracellular signal-regulated kinase (ERK) and protein kinase B (AKT), while it dose-dependently down-regulated p38 and cAMP response element-binding protein (CREB). Moreover, YLR significantly reduced H2O2-induced reactive oxygen species levels in B16F10 cells and showed antioxidant activity based on DPPH and ABTS free radical scavenging activity and SOD-like activity. These results suggest that YLR have anti-melanogensis properties that function through regulation of the CREB/MITF/tyrosinase pathway in B16F10 cells and antioxidant activity. Overall, these findings indicate that YLR has the potential for use in treatment of skin disorders and skin-whitening.

Antimicrobial effect of black pepper petroleum ether extract for the morphology of Listeria monocytogenes and Salmonella typhimurium.

This study aimed to evaluate the effects of black pepper petroleum extract (BPPE) on pathogenic bacteria. The extraction from black pepper showed intense antimicrobial activity against the Gram-positive Listeria monocytogenes ATCC 19115 and the Gram-negative bacteria Salmonella typhimurium ATCC 14028. The minimum inhibitory concentrations of BPPE against L. monocytogenes and S. typhimurium were 0.625 and 1.25mg/ml, respectively. Detection of Alkaline phosphatase outside the cell revealed that BPPE treatment destroyed the cell wall integrity. BPPE also altered the membrane integrity, thereby causing leaching of 260 and 280nm UV-absorbing materials into the medium, particularly, nucleic acids and proteins. Propidium iodide infiltration experiments also indicated that BPPE treatment altered the permeability of bacterial cell membrane. Moreover, Na+/K+-ATPase activity was inhibited by BPPE. And the results of scanning electron microscopy showed that BPPE treatment damaged the morphology of the tested bacteria. These results indicated that BPPE could destroy cell wall integrity, alter the permeability of cell membrane, and inhibit the activity of intracellular enzyme, which could kill bacteria.

Influence of co-existed tetrabromobisphenol A (TBBPA) and hexavalent chromium on the cellular characteristics of Pycnoporus sanguineus during their removal and reduction

Simultaneous TBBPA removal and Cr(VI) reduction by Pycnoporus sanguineus together with the effect of these co-existed pollutants on the fungal cellular characteristics were investigated in this study, aiming at illuminating the mechanism involved in the interactions between contaminants and microbial cells. The results revealed that Cr(VI) reduction and TBBPA removal declined from 92.5%, 75.4–30.6%, 44.8% when Cr(VI) concentration increased from 5 to 40mg/L, respectively. The removal efficiencies for Cr(VI) and TBBPA reached 61.4% and 94% separately under the optimum concentration of TBBPA at 10mg/L. Subsequent analyses indicated that the negative effect of Cr(VI) of high concentrations on Cr(VI) reduction and TBBPA removal was mainly attributed to the inhibition of fungal growth, intracellular proteins synthesis, cell viability and ATP enzyme activity. Compared with the moderate impact of TBBPA, the cell membrane of P. sanguineus was impaired severely and the surface morphology and intracellular structure changed dramatically in the presence of high concentration of Cr(VI) (above 10mg/L). This study also suggested that high level of TBBPA (15 and 20mg/L) promoted the synthesis of intracellular proteins and improved ATP enzyme activity within the first 48h of the reaction for enhancing the transportation and transformation of TBBPA.

Glycoengineering of Esterase Activity through Metabolic Flux-Based Modulation of Sialic Acid.

This report describes the metabolic glycoengineering (MGE) of intracellular esterase activity in human colon cancer (LS174T) and Chinese hamster ovary (CHO) cells. In silico analysis of carboxylesterases CES1 and CES2 suggested that these enzymes are modified with sialylated N-glycans, which are proposed to stabilize the active multimeric forms of these enzymes. This premise was supported by treating cells with butanolylated ManNAc to increase sialylation, which in turn increased esterase activity. By contrast, hexosamine analogues not targeted to sialic acid biosynthesis (e.g., butanoylated GlcNAc or GalNAc) had minimal impact. Measurement of mRNA and protein confirmed that esterase activity was controlled through glycosylation and not through transcription or translation. Azide-modified ManNAc analogues widely used in MGE also enhanced esterase activity and provided a way to enrich targeted glycoengineered proteins (such as CES2), thereby providing unambiguous evidence that the compounds were converted to sialosides and installed into the glycan structures of esterases as intended. Overall, this study provides a pioneering example of the modulation of intracellular enzyme activity through MGE, which expands the value of this technology from its current status as a labeling strategy and modulator of cell surface biological events.

Structure-Activity Relationship of Membrane-Targeting Cationic Ligands on a Silver Nanoparticle Surface in an Antibiotic-Resistant Antibacterial and Antibiofilm Activity Assay.

To explore the structure-activity relationship of membrane-targeting cationic ligands on a silver nanoparticle surface in an antibiotic-resistant antibacterial and antibiofilm activity assay, a series of functionalized silver nanocomposites were synthesized. Tuning the structural configuration, molecular weight, and side-chain length of the cationic ligands on the nanoparticle surface provided silver nanocomposites with effective antibacterial activity against both antibiotic-resistant Gram-negative and Gram-positive bacteria, including bacterial biofilms. These silver nanocomposites did not trigger hemolytic activity. Significantly, the bacteria did not develop resistance to the obtained nanocomposites even after 30 generations. A study of the antibacterial mechanism confirmed that these nanocomposites could irreversibly disrupt the membrane structure of bacteria and effectively inhibit intracellular enzyme activity, ultimately leading to bacterial death. The silver nanocomposites (64 μg/mL) could eradicate 80% of an established antibiotic-resistant bacterial biofilm. The strong structure-activity relationship toward antibacterial and antibiofilm activity suggests that variations in the conformational property of the functional ligand could be valuable in the discovery of new nano-antibacterial agents for treating pathogenic bacterial infections.