Apparent Affinity Constant Research Articles

Measuring Equilibrium Bicarbonate Concentrations Directly in Cellular Mitochondria and in Human Serum Using Europium/Terbium Emission Intensity Ratios

A series of Eu and Tb complexes of four different chiral ligands incorporating an azaxanthone sensitiser has been evaluated as probes for the bicarbonate anion. Their binding affinities were assessed at ambient pH with bicarbonate, lactate, citrate, phosphate and serum albumin. Binding was signalled by modulation of circularly polarised luminescence and apparent affinity constants were measured by examining changes in emission intensity ratios. Competition experiments show that with these species and ATP present at normal physiological values, bicarbonate can be determined selectively over the concentration range 10 to 35 mM. Bicarbonate levels are also reported by using a mixture of Eu and Tb complexes of a common ligand, examining the ratio of red/green emitted light. These methods have been adapted for the determination of bicarbonate in human serum and used for the assessment of mitochondrial levels of bicarbonate in several different cell types with confocal microscopy.

A long-wavelength fluorescent squarylium cyanine dye possessing boronic acid for sensing monosaccharides and glycoproteins with high enhancement in aqueous solution.

Fluorescence sensing of saccharides and glycoproteins using a boronic acid functionalized squarylium cyanine dye (“SQ-BA”) is characterized in terms of synthetic, fluorometric, thermodynamic and kinetic parameters. In our previous work, this newly synthesized dye was successfully applied to the separation and quantification of Gram-positive bacteria by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF); however, the fundamental properties of the dye and its saccharide complexes still required elucidation, as presented in this paper. The dye itself forms nonemissive, soluble aggregates in aqueous solution. With the addition of a monosaccharide, the dye aggregate dissociates to form an emissive monomer accompanied by the formation of a cyclic cis-diol ester with long-wavelength emission (λex = 630 nm, λem = 660 nm). A very large fluorescence enhancement factor of 18× was observed for the sensing dye as a fructose complex at pH 10, yielding a limit of detection of 10 μM fructose. The relative order of fluorescence enhancement of SQ-BA with other monosaccharides was found to be: fructose > ribose > arabinose ≈ galactose > xylose > mannose > rhamnose > fucose ≈ glucose; and apparent affinity constants of 102.80, 102.08 and 100.86 M−1 were determined for fructose, ribose and glucose, respectively. Formation of the emissive complexes occurred within minutes, proving the kinetics of the sugar-dye interactions to be suitable for on-column labeling methods in CE-LIF. Furthermore, the sensing dye was successfully applied to glycoproteins, mucin type I–S and type III, which were detected with high sensitivity in batch aqueous solution as a result of the sugar-selective boronic acid-diol esterification as well as hydrophobic interactions.

Na+‐dependent transport of taurine is found only on the abluminal membrane of the blood‐brain barrier

Luminal and abluminal plasma membranes, isolated from bovine brain microvessels, were used to characterize Na+‐dependent and facilitative taurine transport. Initial transmembrane potential was − 59 mV 0, external Na+ was 126 mM, and T= 25°C. Apparent affinity constants of the taurine transporters were determined over a range of taurine concentrations from 0.24 μM to 11.4 μM. Abluminal membranes had Na+‐dependent taurine transport as well as facilitative transport; luminal membranes only had facilitative transport. Apparent Km's for facilitative and Na+‐dependent taurine transports were 0.06 ± 0.02 μM and 0.7 ± 0.1 μM, respectively. Na+‐dependent transport of taurine was voltage dependent between −25 to −101 mV. Transport was over 5 times greater at −101 mV compared with Vm was −25 mV. Sensitivity to external osmolality of Na+‐dependent transport was studied over a range of osmolalities (229 to 398 mOsm/kg H2O) using mannitol as the osmotic agent to adjust osmolality. For these experiments the concentration of Na+wasmaintained constant at 50 mM; the calculated transmembrane potential was −59 mV. Na+‐dependent transport was sensitive to osmolality with the greatest rate observed at 229 mOsm/kg H2O.

Characterization of cerebral glucose dynamics in vivo with a four‐state conformational model of transport at the blood–brain barrier

Determination of brain glucose transport kinetics in vivo at steady-state typically does not allow distinguishing apparent maximum transport rate (T(max)) from cerebral consumption rate. Using a four-state conformational model of glucose transport, we show that simultaneous dynamic measurement of brain and plasma glucose concentrations provide enough information for independent and reliable determination of the two rates. In addition, although dynamic glucose homeostasis can be described with a reversible Michaelis-Menten model, which is implicit to the large iso-inhibition constant (K(ii)) relative to physiological brain glucose content, we found that the apparent affinity constant (K(t)) was better determined with the four-state conformational model of glucose transport than with any of the other models tested. Furthermore, we confirmed the utility of the present method to determine glucose transport and consumption by analysing the modulation of both glucose transport and consumption by anaesthesia conditions that modify cerebral activity. In particular, deep thiopental anaesthesia caused a significant reduction of both T(max) and cerebral metabolic rate for glucose consumption. In conclusion, dynamic measurement of brain glucose in vivo in function of plasma glucose allows robust determination of both glucose uptake and consumption kinetics.

Comparison of three distinct ELLA protocols for determination of apparent affinity constants between Con A and glycoproteins

A procedure for determination of apparent affinity constants KDapp between Concanavalin A (Con A) and naturally d-mannose containing glycoproteins using enzyme-linked lectin assay (ELLA) is reported. Three distinct ELLA protocols are compared to each other with 3 different fitting models used (Liliom, Hill with and without a cooperativity factor). The glycoproteins were physisorbed on a highly charged polystyrene solid surface of immunoassay plates and the amount of lectin bound to the glycoproteins was determined by photometry. The interactions of Con A with five mannose-containing glycoproteins, invertase (INV), glucoamylase (GA), glucose oxidase (GOx), ovalbumin (OVA), and transferrin (TRF) were quantified with apparent affinity constant being in the range 2×10−7 to 9×10−6M. The strength of interaction between Con A and glycoproteins is discussed on the basis of glycan structure/exposure on the protein backbone for each glycoprotein.

Mass Spectrometry-assisted Study Reveals That Lysine Residues 1967 and 1968 Have Opposite Contribution to Stability of Activated Factor VIII

The A2 domain rapidly dissociates from activated factor VIII (FVIIIa) resulting in a dampening of the activity of the activated factor X-generating complex. The amino acid residues that affect A2 domain dissociation are therefore critical for FVIII cofactor function. We have now employed chemical footprinting in conjunction with mass spectrometry to identify lysine residues that contribute to the stability of activated FVIII. We hypothesized that lysine residues, which are buried in FVIII and surface-exposed in dissociated activated FVIII (dis-FVIIIa), may contribute to interdomain interactions. Mass spectrometry analysis revealed that residues Lys(1967) and Lys(1968) of region Thr(1964)-Tyr(1971) are buried in FVIII and exposed to the surface in dis-FVIIIa. This result, combined with the observation that the FVIII variant K1967I is associated with hemophilia A, suggests that these residues contribute to the stability of activated FVIII. Kinetic analysis revealed that the FVIII variants K1967A and K1967I exhibit an almost normal cofactor activity. However, these variants also showed an increased loss in cofactor activity over time compared with that of FVIII WT. Remarkably, the cofactor activity of a K1968A variant was enhanced and sustained for a prolonged time relative to that of FVIII WT. Surface plasmon resonance analysis demonstrated that A2 domain dissociation from activated FVIII was reduced for K1968A and enhanced for K1967A. In conclusion, mass spectrometry analysis combined with site-directed mutagenesis studies revealed that the lysine couple Lys(1967)-Lys(1968) within region Thr(1964)-Tyr(1971) has an opposite contribution to the stability of FVIIIa.

A Connection between the Binding Properties of Imprinted and Nonimprinted Polymers: A Change of Perspective in Molecular Imprinting

In the current paradigm for molecular imprinting, the imprinted binding sites exist as a consequence of the polymerization process around templates, and the properties of nonimprinted polymers (NIPs) have largely been overlooked. Thus, nothing can be affirmed a priori concerning the binding properties of NIPs. We propose an alternative view where the imprinting effect is due to the presence of a template molecule that enhances the pre-existing binding properties of a polymer. If a NIP shows no binding properties toward a target molecule, the corresponding imprinted polymer (MIP) will show a weak imprinting effect. On the other hand, if a NIP shows binding properties toward a target molecule, the corresponding MIP will show a significant imprinting effect. To verify this hypothesis, we prepared a 96-member combinatorial polymeric library in the absence of any template molecule. This library was screened for several potential ligands, and with no exceptions, the composition of the best-binding NIP produced a MIP with excellent binding properties, whereas a low-binding NIP formulation produced a MIP with comparable low binding. To validate these results, the binding properties toward naproxen and ibuprofen were measured for two combinatorial libraries of polymers prepared in the presence (MIP library) and the absence (NIP library) of the template molecule. The experiment's results showed a correlation between the apparent affinity constants measured for the NIP and MIP libraries, confirming the proposed hypothesis. Moreover, for closely related molecules, it was shown that binding selectivity is an emergent property derived from the imprinting process and not a property of NIPs.

Na+-dependent transport of taurine is found only on the abluminal membrane of the blood–brain barrier

Luminal and abluminal plasma membranes were isolated from bovine brain microvessels and used to identify and characterize Na+-dependent and facilitative taurine transport. The calculated transmembrane potential was −59mV at time 0; external Na+ (or choline under putative zero-trans conditions) was 126mM (T=25°C). The apparent affinity constants of the taurine transporters were determined over a range of taurine concentrations from 0.24μM to 11.4μM. Abluminal membranes had both Na+-dependent taurine transport as well as facilitative transport while luminal membranes only had facilitative transport. The apparent Km for facilitative and Na+-dependent taurine transport were 0.06±0.02μM and 0.7±0.1μM, respectively. The Na+-dependent transport of taurine was voltage dependent over the range of voltages studied (−25 to −101mV). The transport was over 5 times greater at −101mV compared to when Vm was −25mV. The sensitivity to external osmolality of Na+-dependent transport was studied over a range of osmolalities (229 to 398mOsm/kg H2O) using mannitol as the osmotic agent to adjust the osmolality. For these experiments the concentration of Na+ was maintained constant at 50mM, and the calculated transmembrane potential was −59mV. The Na+-dependent transport system was sensitive to osmolality with the greatest rate observed at 229mOsm/kg H2O.

Cation-dependent nutrient transport in shrimp digestive tract

Purified epithelial brush border membrane vesicles (BBMV) were produced from the hepatopancreas of the Atlantic White shrimp, Litopeneaus setiferus, using standard methods originally developed for mammalian tissues and previously applied to other crustacean and echinoderm epithelia. These vesicles were used to study the cation dependency of sugar and amino acid transport across luminal membranes of hepatopancreatic epithelial cells. (3)H-D: -glucose uptake by BBMV against transient sugar concentration gradients occurred when either transmembrane sodium or potassium gradients were the only driving forces for sugar accumulation, suggesting the presence of a possible coupled transport system capable of using either cation. (3)H-L: -histidine transport was only stimulated by a transmembrane potassium gradient, while (3)H-L: -leucine uptake was enhanced by either a sodium or potassium gradient. These responses suggest the possible presence of a potassium-dependent transporter that accommodates either amino acid and a sodium-dependent system restricted only to L: -leucine. Uptake of (3)H-L: -leucine was significantly stimulated (P < 0.05) by several metallic cations (e.g., Zn(2+), Cu(2+), Mn(2+), Cd(2+), or Co(2+)) at external pH values of 7.0 or 5.0 (internal pH 7.0), suggesting a potential synergistic role of the cations in the transmembrane transfer of amino acids. (3)H-L: -histidine influxes (15 suptakes) were hyperbolic functions of external [zinc] or [manganese], following Michaelis-Menten kinetics. The apparent affinity constant (e.g., K (m)) for manganese was an order of magnitude smaller (K (m) = 0.22 μM Mn) than that for zinc (K (m) = 1.80 μM Zn), while no significant difference (P > 0.05) occurred between their maximal transport velocities (e.g., J (max)). These results suggest that a number of cation-dependent nutrient transport systems occur on the shrimp brush border membrane and aid in the absorption of these important dietary elements.

Jacalin interaction with human immunoglobulin A1 and bovine immunoglobulin G1: Affinity constant determined by piezoelectric biosensoring

The affinity of the D-galactose-binding lectin from Artocarpus heterophyllus lectin, known as jacalin, with immonuglobulins (Igs) was determined by biofunctionalization of a piezoelectric transducer. This piezoelectric biofunctionalized transducer was used as a mass-sensitive analytical tool, allowing the real-time binding analysis of jacalin-human immunoglobulin A1 (IgA(1)) and jacalin-bovine IgG(1) interactions from which the apparent affinity constant was calculated. The strategy was centered in immobilizing jacalin on the gold electrode's surface of the piezoelectric crystal resonator using appropriate procedures based on self-assembling of 11-mercaptoundecanoic acid and 2-mercaptoethanol thiol's mixture, a particular immobilization strategy by which it was possible to avoid cross-interaction between the proteins over electrode's surface. The apparent affinity constants obtained between jacalin-human IgA(1) and jacalin-bovine IgG(1) differed by 1 order of magnitude [(8.0 ± 0.9) 10(5) vs (8.3 ± 0.1) 10(6) L mol(-1)]. On the other hand, the difference found between human IgA(1) and human IgA(2) interaction with jacalin, eight times higher for IgA(1), was attributed to the presence of O-linked glycans in the IgA(1) hinge region, which is absent in IgA(2). Specific interaction of jacalin with O-glycans, proved to be present in the human IgA(1) and hypothetically present in bovine IgG(1) structures, is discussed as responsible for the obtained affinity values.

Differential Calcium Signaling by Cone Specific Guanylate Cyclase-Activating Proteins from the Zebrafish Retina

Zebrafish express in their retina a higher number of guanylate cyclase-activating proteins (zGCAPs) than mammalians pointing to more complex guanylate cyclase signaling systems. All six zGCAP isoforms show distinct and partial overlapping expression profiles in rods and cones. We determined critical Ca2+-dependent parameters of their functional properties using purified zGCAPs after heterologous expression in E.coli. Isoforms 1–4 were strong, 5 and 7 were weak activators of membrane bound guanylate cyclase. They further displayed different Ca2+-sensitivities of guanylate cyclase activation, which is half maximal either at a free Ca2+ around 30 nM (zGCAP1, 2 and 3) or around 400 nM (zGCAP4, 5 and 7). Zebrafish GCAP isoforms showed also differences in their Ca2+/Mg2+-dependent conformational changes and in the Ca2+-dependent monomer-dimer equilibrium. Direct Ca2+-binding revealed that all zGCAPs bound at least three Ca2+. The corresponding apparent affinity constants reflect binding of Ca2+ with high (≤100 nM), medium (0.1–5 µM) and/or low (≥5 µM) affinity, but were unique for each zGCAP isoform. Our data indicate a Ca2+-sensor system in zebrafish rod and cone cells supporting a Ca2+-relay model of differential zGCAP operation in these cells.

Cation permeation through connexin 43 hemichannels is cooperative, competitive and saturable with parameters depending on the permeant species

Kinetics of permeation through connexin 43-EGFP hemichannels (Cx43-EGFP HCs) were evaluated in divalent cation-free solutions, which enhance HC open probability and thus, allow measurements during initial velocity. Three cations that become fluorescent upon binding to intracellular nucleic acids [ethidium (Etd), propidium (Prd) and 4′,6-diamidino-2-phenylindole (DAPI)] and Cx43-EGFP or Cx43 wild type HeLa cell transfectants (Cx43-EGFP- and Cx43-WT-HeLa cells, respectively) were used. Levels of Cx43-EGFP at the cell periphery and rate of dye uptake were directly related. The rate of uptake of each dye reached saturation consistent with a facilitated transport mechanism. Before saturation, the relation between rate of uptake and concentration of each dye was sigmoidal with Hill coefficients >1, indicating positive cooperativity of transport at low concentrations. The maximal rate of Etd uptake was not affected by the presence of DAPI and vice versa, but under each condition the apparent affinity constant of the main permeant molecule increased significantly consistent with competitive inhibition or competition for binding sites within the channel. Moreover, Cx43-EGFP and Cx43-WT HCs had similar permeability properties, indicating that EGFP bound to the C-terminal of Cx43 does not significantly alter the permeability of Cx43 HCs to positively charged molecules. Thus, competitive inhibition of permeation through hemichannels might contribute to cellular retention of essential molecules and/or uptake inhibition of toxic compounds.

Determination of apparent kinetic and stoichiometric parameters in a nitrifying fixed-bed reactor by in situ pulse respirometry

The determination of stoichiometric and kinetic parameters of biological processes occurring in fixed-bed reactors is usually achieved through mass balance methods or by the characterization of samples taken from the core of the reactor. Mass balance is time demanding and to obtain representative samples from the core of filter media is often difficult. A fast and non-invasive alternative method might be beneficial. In this paper we explored the potential for using in situ pulse respirometry in a submerged fixed-bed reactor for assessing biological processes. A nitrifying fixed-bed reactor was operated over 85 days while analyzing the dissolved oxygen response to pulses of ammonium. In situ pulse respirometry allowed us to determine the apparent maximum oxygen uptake rate and substrate affinity constant. Additionally, the apparent biomass growth yield was determined during the first 60 days. Unfortunately, after day 60, erratic biomass growth yield results were obtained, which we were unable to explain through hydrodynamic characterization of the reactor. However, it is concluded that in situ pulse respirometry is a potentially useful tool to characterize fixed-bed reactors as an alternative to other methods, such as mass balance.

Actin-Binding Proteins Mediate Regulation by Calcium of Polycystin-2 (tRPP2)

Polycystin-2 (PC2, TRPP2) is a member of the TRP superfamily that acts as a non-selective cation channel, with permeability to calcium. PC2 is implicated in calcium transport, and therefore, its regulation by calcium is relevant to its role(s) in cell function. Recent studies (Cantero & Cantiello, BJ 98(3):340a, 2010) demonstrated that PC2 channel activity from human syncytiotrophoblast is regulated by cytoplasmic calcium, such that low calcium (< 0.3 nM) renders the channel inactive. Titration of cytoplasmic calcium recovers PC2 channel activity, with a Hill coefficient of 4 and an apparent affinity constant of 1-5 nM. Interestingly, in vitro translated PC2, devoid of regulatory proteins was completely insensitive to intracellular calcium (ibid). In this study we further explored the nature of this regulation by assessing potential calcium-sensitive target proteins in the channel complex. Several calcium sensitive and insensitive actin-binding proteins (ABP) were studied in the in vitro translated protein. The actin-bundling protein α-actinin (250 nM) increased PC2 channel activity in the presence of high (10 μM) cytoplasmic calcium, but instead was inhibitory in its absence. The calcium insensitive G-actin binding protein profiling (4 nM), increased PC2 channel activity both in presence and absence of calcium, while the calcium-sensitive, actin-severing protein gelsolina (4 nM), regulated PC2 channel function in the presence, but not absence of calcium (10 μM). These data suggest that the formation of ABP-PC2 complexes confer distinct calcium regulatory functions to the channel, thus providing a novel cytoskeletal pathway for channel regulation. This response to cytoplasmic calcium by PC2 may provide functional diversity to the channel by choosing, and possibly exchanging ABP-structural partners.

A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo1[S

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.

Turnover and accessibility of a reentrant loop of the Na+-glutamate transporter GltS are modulated by the central cytoplasmic loop

GltS of Escherichia coli is a secondary transporter that catalyzes Na+-glutamate symport. The structural model of GltS shows two homologous domains with inverted membrane topology that are connected by a central loop that resides in the cytoplasm. Each domain contains a reentrant loop structure. Accessibility of the Cys residues in two GltS mutants in which Pro351 and Asn356 in the reentrant loop in the C-terminal domain were replaced by Cys is demonstrated to be sensitive to the catalytic state supporting a role for the reentrant loops in catalysis. Saturating concentrations of the substrate L-glutamate protected both mutants against inactivation by thiol reagents, while the presence of the co-ion Na+ stimulated the inactivation of both mutants. Insertion of the 10 kDa biotin acceptor domain (BAD) of oxaloacetate decarboxylase of Klebsiella pneumoniae in the central cytoplasmic loop blocked the access pathway from the periplasmic side of the membrane to the cysteine residues in mutants P351C and N356C in the reentrant loop. Kinetically, insertion of BAD increased the maximal rate of uptake 2.7-fold while leaving the apparent affinity constants for L-glutamate and Na+ unaltered. The data suggests that insertion of BAD in the central loop results in conformational changes at the translocation site that lower the activation energy of the translocation step without affecting the access pathway from the periplasmic side for substrate and co-ions. It is concluded that changes in the central loop that connects the two domains may have a regulatory function on the activity of the transporter.

The kinetics of ferrous ion oxidation by Leptospirillum ferriphilum in continuous culture: The effect of pH

The kinetics of ferrous ion oxidation by Leptospirillum ferriphilum were studied in continuous culture with a focus on the effect of solution pH (pH 0.8–2.0), assuming that the effect of pH on cell metabolism can be independently studied of reactor context and other reactions common in bioleach heaps. A simplified competitive ferric ion inhibition model and the Pirt Equation were used to analyze the experimental data. The results showed that the maximum specific activity of L. ferriphilum has a symmetrical bell-shaped curve relationship with pH. The maximum specific ferrous-iron oxidation rate, q Fe 2 + maxgave a highest value of 14.54 mmol Fe 2+(mmol C h) − 1 at pH 1.3, and was described by a quadratic function. The steady state carbon biomass in the reactor and the apparent affinity constant, K′ Fe 2 + , also increased with increase in pH; however, a slight increase in the carbon biomass was observed beyond pH 1.6. The results also showed that ferric ion precipitation is significant beyond pH 1.3 and about 13% total iron from the feed was lost at pH 2.0. The maximum biomass yield increased linearly with pH, while the culture maintenance coefficient was significantly small in all experiments and was minimum at pH 1.3. The values are indicative of actively growing chemostat cultures. This study shows that microbial ferrous ion oxidation by L. ferriphilum may be sustained at pH lower than pH 0.8 as the microbial activity is much higher than reported values for common mesophilic acidophiles. This may have implications on how bioleach heap operations can be started-up to improve metal recovery.

Stimulation of glucose transport in osteoblastic cells by parathyroid hormone and insulin-like growth factor I

Insulin and parathyroid hormone (PTH) regulate glucose metabolism in bone cells. In order to differentiate between the effects of these hormones and to compare the potency of insulin with that of insulin-like growth factor (IGF) I, we treated rat bone-derived osteoblastic (PyMS) cells for different time periods and at different concentrations with insulin, IGF I, or PTH, and measured [1-(14)C]-2-deoxy-D-glucose (2DG) uptake and incorporation of D-[U-(14)C] glucose into glycogen. 2DG uptake was Na-independent with an apparent affinity constant (K (M)) of ~2 mmol/l. Expression of the high affinity glucose transporters (GLUT), GLUT1 and GLUT3 but not of GLUT4, was found by Northern and Western analysis. Similar to the findings with primary rat osteoblasts, but distinct from those in rat fibroblasts, 2DG uptake and glycogen synthesis were increased in this cell line after exposure to low concentrations (0.1 nmol/l and above) of PTH. IGF I at low doses (0.3 nmol/l and above) or insulin at higher doses (1 nmol/l and above) stimulated 2DG uptake and [(3)H] thymidine incorporation into DNA. 2DG transport was enhanced already after 30 min of IGF I treatment whereas the effect of PTH became significant after 6 h. It is concluded that IGF I rather than insulin may be a physiological regulator of 2DG transport and glycogen synthesis in osteoblasts.

Colorimetric and fluorescent sensor constructing from the nanofibrous membrane of porphyrinated polyimide for the detection of hydrogen chloride gas

A novel sensor fabricated from the nanofibrous membrane of porphyrinated polyimide (PPI) for the rapid detection of trace amount of hydrogen chloride (HCl) gas is described. Covalently bonding of the porphyrin fluorophores into polyimide main chains overcomes the disadvantage of porphyrin aggregation and improves the physicochemical stability of polyimide simultaneously. The dual chromo- and fluorogenic responses of the nanofibrous membrane upon exposure to HCl gas are interpreted in terms of the out-of-plane distortion of porphyrin macrocycle, which ultimately affects its optical properties. UV–vis and fluorescence spectroscopies were used to further study the protonation of porphyrin moieties in polyimide. With large amount of available surface area and hence good gas accessibility, the nanofibrous membrane of porphyrinated polyimide shows unusually high sensitivity and fast response time in sensing application. An apparent binding affinity constant of (1.05±0.23)×104Lmol−1 was calculated from surface plasmon resonance (SPR) analysis, confirming that the porphyrinated nanofibrous membrane is an applicable material for constructing HCl-sensitive gas sensor.

UDP-N-acetylglucosamine enolpyruvyl transferase from Pseudomonas aeruginosa

Multi-drug resistant Pseudomonas aeruginosa (MDRPA) are emerging as a major threat in the hospitals as they have become resistant to current antibiotics. There is an immediate requirement of drugs with novel mechanisms as the pipeline of investigational drugs against these organisms is lean. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme that catalyzes the first committed step of bacterial cell wall biosynthesis is an ideal target for the discovery of novel antibiotics against Gram negative pathogens as they have only one copy of murA gene in its genome. We have performed biochemical characterization and comparative kinetic analysis of MurA from E. coli and P. aeruginosa. Both enzymes were active at broad range of pH with temperature optima of 37°C. Metal ions did not enhance the activity of both enzymes. These enzymes had an apparent affinity constant (K m ) for its substrate UDP-N-acetylglucosamine 36 ± 5.2 and 17.8 ± 2.5 μM and for phosphoenolpyruvate 0.84 ± 0.13 μM and 0.45 ± 0.07 μM for E. coli and P. aeruginosa enzymes respectively. Both the enzymes showed 5–7 fold shift in IC50 for the known inhibitor fosfomycin upon pre-incubation with the substrate UDP-N-acetylglucosamine. This observation was used to develop a novel rapid sensitive high throughput assay for the screening of MurA inhibitors.