A pairwise chemical genetic screen identifies new inhibitors of glucose transport

Oxidative phosphorylation (OXPHOS) and glycolysis are the two main pathways that control energy metabolism of a cell. The Warburg effect, in which glycolysis remains active even under aerobic conditions, is considered a key driver for cancer cell proliferation, malignancy, metastasis, and therapeutic resistance. To target aerobic glycolysis, we exploited the complementary roles of OXPHOS and glycolysis in ATP synthesis as the basis for a chemical genetic screen, enabling rapid identification of novel small-molecule inhibitors of facilitative glucose transport. Blocking mitochondrial electron transport with antimycin A or leucascandrolide A had little effect on highly glycolytic A549 lung carcinoma cells, but adding known glycolytic inhibitors 2-deoxy-D-glucose, iodoacetate or cytochalasin B, rapidly depleted intracellular ATP, displaying chemical synthetic lethality. Based on this principle, we exposed antimycin A-treated A549 cells to a newly synthesized 955 member diverse scaffold small-molecule library, screening for compounds that rapidly depleted ATP levels. Two compounds potently suppressed ATP synthesis, induced G1 cell-cycle arrest and inhibited lactate production. Pathway analysis revealed that these novel probes inhibited GLUT family of facilitative transmembrane transporters but, unlike cytochalasin B, had no effect on the actin cytoskeleton. Our work illustrated the utility of a pairwise chemical genetic screen for discovery of novel chemical probes, which would be useful not only to study the system-level organization of energy metabolism but could also facilitate development of drugs targeting upregulation of aerobic glycolysis in cancer.     

Mathematical model of substance active transport via cytoplasmic membrane of microbial cell along concentration gradient

Applied Biochemistry and Biotechnology - Considering the process of methane generation by methanogenic bacteria as an example, the model describes the distribution of methane concentration inside...     

A review of chemical gradient systems for cell analysis

Microfluidic spatial and temporal gradient generators have played an important role in many biological assays such as in the analysis of wound healing, inflammation, and cancer metastasis. Chemical gradient systems can also be applied to other fields such as drug design, chemical synthesis, chemotaxis, etc. Microfluidic systems are particularly amenable to gradient formation, as the length scales used in chips enable fluid processes that cannot be conducted in bulk scale. In this review we discuss new microfluidic devices for gradient generation and applications of those systems in cell analysis.     

Improved biosensor for glucose based on glucose oxidase-immobilized silk fibroin membrane

Based on glucose oxidase-immobilized silk fibroin membrane and oxygen electrode, the authors have developed an amperometric glucose sensor in flow-injection analysis. After the sensor was improved by the configuration of oxygen electrode and a temperature control system was added to the electrode body, its sensitivity, analytical precision, and stability were enhanced greatly. The authors first introduced a tailing inhibitor-ion pair reagent into a buffer system in the biosensor so as to eliminate all interference from hemacyte, macromolecules, and small mol wt charged species besides electroactive specie ascorbate in complex matrices. A considerably serious tailing of the biosamples, such as whole blood, plasma, serum, or urine on the sensor, based on enzyme electrode, entirely disappeared, their response times were shortened, and base lines became more smooth and stable. The glucose sensor has a broad range of linear response for glucose (up to 25.0 mmol/L) and a good correlation (γ = 0.999) under conditions of control temperature 32.0°C and 1.6 mL/min 0.02 mol/L phosphate buffer containing 0.5% tailing inhibitor (v/v). Recoveries of glucose in these biosamples are within the range of 93.71–105.88%, and its repeatabilities for determining glucose, repeated 100 times, human blood dilution 125 times, and serum 128 times, are 1.81,2.48, and 2.91% (RSD), respectively. The correlation analysis for 200 serum samples showed that the correlation (γ) is 0.9934 between the glucose sensor and Worthington method for determining serum glucose used conventionally in a hospital laboratory. Moreover, the enzyme membrane used in the biosensor can be stored for a long time (over 2 yr) and measured repeatedly over 1000 times for biosamples. The glucose sensor is capable of detecting over 60 biosamples/hr.     

Preparation of a polymeric membrane with lipoamide structure and its application to electron transport across membrane

A lipoamide (LAm) structure was introduced into a polymeric membrane by chemical modification of poly(γ-methyl-D -glutamate) (PMG). A. redox reaction proceeded across the membrane mediated by pendant LAm groups as solid carriers. It is suggested that the electron transport process in the membrane is derived from the exchange reaction between reduced LAm (thiol) and LAm (disulfide).     

Biosensors for determination of glucose with glucose oxidase immobilized on an eggshell membrane

A glucose biosensor using an enzyme-immobilized eggshell membrane and oxygen electrode for glucose determination has been fabricated. Glucose oxidase was covalently immobilized on an eggshell membrane with glutaraldehyde as a cross-linking agent. The glucose biosensor was fabricated by positioning the enzyme-immobilized eggshell membrane on the surface of a dissolved oxygen sensor. The detection scheme was based on the depletion of dissolved oxygen content upon exposure to glucose solution and the decrease in the oxygen level was monitored and related to the glucose concentration. The effect of glutaraldehyde concentration, pH, phosphate buffer concentration and temperature on the response of the glucose biosensor has been studied in detail. Common matrix interferents such as ethanol, d-fructose, citric acid, sodium benzoate, sucrose and l-ascorbic acid did not give significant interference. The resulting sensor exhibited a fast response (100 s), high sensitivity (8.3409 mg L⁻¹ oxygen depletion/mmol L⁻¹ glucose) and good storage stability (85.2% of its initial sensitivity after 4 months). The linear response is 1.0×10⁻⁵ to 1.3×10⁻³ mol L⁻¹ glucose. The glucose content in real samples such as commercial glucose injection preparations and wines was determined, and the results were comparable to the values obtained from a commercial glucose assay kit based on a spectrophotometric method.     

Lipid membrane technology for chemical and biosensor development

New developments and applications of selective biosensors specifically designed for clinical assay and invasive biological analysis are revolutionizing chemical sensing. One area which has remained largely unaddressed consists of the chemical sensing systems found in nature, which offer splendid examples of efficient and sensitive devices from which much can be learned. Efforts to devise lipid-based biosensors, which are very limited models of the highly refined natural system, have recently attracted much attention and financing. The superior sensing characteristics demonstrated by the prototype systems tested to date indicate that this technology may provide the basis for the ultimate artificial biosensor of the future.     

A study of the transport of ions against their concentration gradient across ion-exchange membranes using the network method

The network method has been used to analyze the conditions that favour the uphill transport across ion-exchange membranes. A model for the Nernst-Planck-Poisson equations describing the ionic transport in such system is proposed, including the Donnan equilibrium relations at the membrane/solution interfaces. With this model and the electric circuit simulation program PSPICE, the transient response of the system under open circuit conditions (I=0) and the response of the system subject to an applied potential difference are simulated. The ionic concentrations and electric potential profiles, as well as the electric current density, the ionic fluxes and the charge density, have been obtained as a function of time.     

Effect of chemical modification of ion-exchange membrane MA-40 on its electrochemical characteristics

Transport characteristics of commercial heterogeneous anion-exchange membranes MA-40 and MA-41 are studied, together with those of membrane MA-40M fabricated by treating the MA-40 surfaces with a strong polyelectrolyte complex. It is demonstrated that, after modification, the electrical conductivity of MA-40M in an NaOH solution increases. At overlimiting currents, the water dissociation rate on this membrane decreases as compared with the initial membrane. At the same time, no noticeable change in the rate of transport of counter-ions (ions Cl^-) through the membrane at a fixed potential drop is discovered at under-and overlimiting currents. The MA-40M membrane behavior is explained by the conversion of secondary and tertiary functional ammonium groups in the near-surface membrane layers approximately 80 μm thick into quaternary groups during the treatment by the polyelectrolyte.     

Preparation of PVA membrane for immobilization of GOD for glucose biosensor

A membrane was prepared using polyvinyl alcohol (PVA) with low and high degree of polymerization (DOP), acetone, benzoic acid (BA) and was cross-linked by UV treatment. Membrane composition was optimized on the basis of swelling index. Membrane prepared with 12% low DOP and 8% high DOP of PVA, 2% BA, dissolved in buffer containing 20% acetone and cross-linked with UV treatment exhibited lower swelling index. Fourier transform infrared (FTIR) study of the membranes showed appearance of a strong band at approximately 2337 cm(-1) when UV was used for cross-linking in the presence of benzoic acid. Scanning electron microscope (SEM) study revealed that membrane cross-linked with UV treatment was smoother. Glucose oxidase (GOD)-PVA membrane was associated with the dissolved oxygen (DO) probe for biosensor reading. Glucose was detected on the basis of depletion of oxygen, when immobilized GOD oxidizes glucose to gluconolactone. A wide detection range, 0.9-225 mg/dl was estimated from the linear range of calibration plot of biosensor reading. Membranes were reused for 32 reactions without significant loss of activity and stored for 30 days (approximately 90% activity) at 4 degrees C. Membranes were also used with real blood samples.     

Radiation grafting of acrylic acid onto polytetrafluoroethylene films for glucose oxidase immobilization and its application in membrane biosensor

Radiation induced grafting of acrylic acid (AA) onto 40 μm polytetrafluoroethylene (PTFE) films was carried out by the direct method of multiple (discrete) and single irradiation form ⁶⁰Co source at different doses up to 100 kGy and room temperature. Depending on the method, the grafting takes place either on the surface layer or within the polymer matrix. The graft copolymers synthesized (PTFE-g-PAA) were transformed into ionomers by treatment with KOH. Both forms were used as carriers for immobilization of enzymes. The copolymers in H- and K-forms were activated by the acylazide method and glucose oxidase (GOD) was immobilized on them. The most suitable proved to be the ionomers PTEE-g-COOK obtained by single irradiation, possessing activity of ca. 120 mU/cm². Enzyme biosensor was designed based on Clark-type electrode and the active membranes prepared, where the membrane plays both the roles of enzyme and oxygen membrane. It can be used for determination of glucose in solutions.     

Pressurized membrane indicator system for fluorogenic-based fiber-optic chemical sensors

A new kind of fiber-optic-based chemical sensor for the detection of chemical species in solution is described. The sensor uses a fluorogenic indicator that is forced through an ultrafiltration membrane into the analyte solution. Complex formation between the indicator molecule and target ion occurs at the membrane/solution interface, where light from a bifurcated fiber-optic cable stimulates fluorescence. Fluorescence emission from the sample is transmitted back up the cable to a photodiode detector. Characterization of the sensor was performed using the response of the indicator, 8-hydroxyquinoline-5-sulfonic acid, to magnesium ion. The sensor responds linearly to increasing and decreasing concentration changes on time scales of 1 s. Chemical modification of the indicator solution to enhance sensitivity is demonstrated. The use of an internal standard to correct for variations in indicator flow-rate and pressure is discussed.     

Enzyme electrode for glucose based on an iodide membrane sensor

The construction of a new type of enzyme electrode for the potentiometric determination of glucose is reported. The electrode response is based on the enzymecatalyzed reactions: The highly selective iodide sensor monitors the local decrease in the iodide activity at the electrode surface. The properties of the above reactions were examined kinetically, with flow-stream techniques and potentiometric detection. The glucose electrode constructed and the use of flow-stream experiments with two iodide sensors provided accurate and convenient glucose determinations in the absence of some oxidizing and reducing agents.     

Activated platinum electrodes as transducer for a glucose sensor using glucose oxidase in a photopolymer membrane

A planar platinum electrode was covered by a photopolymer membrane containing glucose oxidase (GOD) to construct an amperometric glucose sensor. The application of a photopolymer system in membrane formation gives the opportunity to manufacture cheap biosensors with good reproducibility by means of automated techniques and to miniaturise sensors using photolithography. The electrodes were pretreated mechanically and chemically resulting in a half-wave potential (E_1/2) of the H₂O₂ oxidation shifted towards more negative potentials. This shift allows the determination of glucose at a low working potential (300 mV vs. SCE) without addition of mediators. The important advantage of such applied potential decreasing lies in minimising the interference of oxidisable substances such as uric acid, bilirubin and paracetamol. The selectivity to ascorbic acid could also be proved without the application of additional protection layers. The glucose sensor developed has a high life-time, selectivity and sensitivity and a linear working concentration range from 0.05 up to 10 mmol/l of glucose. The sensor was used for the glucose determination in human serum samples with a very good correlation to a common photometric reference method. Received: 13 July 1996 / Revised: 11 September 1996 / Accepted: 14 September 1996     

Electrical oscillation in lipid-impregnated porous membrane filter and its modulation by a light-driven proton pump,bacteriorhodopsin

A lipid-impregnated porous membrane filter (placed between two chambers) was able to display reproducibly an electrical oscillation under constant current stimulation when alamethicin (a channel-forming polypeptide) and protamine (a basic protein) were added to the chamber whose salt concentration was higher than that of the other and the membrane was left for 12 h. In addition, bacteriorhodopsin, which is a light-driven proton pump, was found to control the oscillation. The oscillatory phenomenon is considered to be driven by the alternating change in the ion selectivity of the membrane between cation and anion.     

Coupled pervaporation/mass spectrometry for investigating membrane mass transport phenomena

The integration of organophilic pervaporation into processes of varying feed concentration, such as bioconversions, chemical reactions, or analytical sample preparation, requires not only the understanding of mass transport phenomena across the membrane under steady-state conditions, but also the insight into the transient response of the pervaporation membrane to changes as they occur in practice. For this purpose, a laboratory-scale pervaporation unit was coupled to a mass spectrometer for on-line permeate analysis, maintaining the overall pervaporation operating conditions controllable independently, and without introducing any inert gases for sample transfer.The experimental set-up was employed for investigating the transport of aroma compounds across a POMS–PEI composite membrane, focusing in particular on the so-called “membrane conditioning”; the possible synergetic effect of ethanol on the flux of one model aroma compound, ethyl hexanoate; the application of the system proposed to the rapid screening of the effect of the hydrodynamic upstream conditions on the degree of concentration polarisation.The method proposed proved to be robust and flexible, not only allowing insights into transient mass transport phenomena otherwise not attainable, but also reducing experimental workload significantly when characterising the effect of varying operating conditions on the pervaporation performance.     

Fluctuation theorem for channel-facilitated membrane transport of interacting and noninteracting solutes

In this paper, we discuss the fluctuation theorem for channel-facilitated transport of solutes through a membrane separating two reservoirs. The transport is characterized by the probability, P(n)(t), that n solute particles have been transported from one reservoir to the other in time t. The fluctuation theorem establishes a relation between P(n)(t) and P-(n)(t): The ratio P(n)(t)/P-(n)(t) is independent of time and equal to exp(nbetaA), where betaA is the affinity measured in the thermal energy units. We show that the same fluctuation theorem is true for both single- and multichannel transport of noninteracting particles and particles which strongly repel each other.