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PubMed2005

Coincidence planar imaging for dynamic [18F]FDG uptake in nude mice with tumors and inflammation: correlated with histopathology and micro-autoradiography.

The Institute of Nuclear Energy Research of Taiwan has developed a dynamic coincidence detection device for positron emitted radiotracer pharmacodynamic study in small mice models. In this study, we set up an experimental paradigm by determining [fluorine-18]-2-deoxy-2-fluoro-D-glucose ([18F]FDG) dynamic uptake in tumors and inflammations in nude mice as the foundation for future applications in therapy development. Histopathology and micro-autoradiography of these tumors and inflammations were obtained for confirmation. Dynamic coincidence planar images of six tumors and two inflammations in nude mice were acquired over 4 hours immediately after injection of 25.9 MBq of [18F]FDG into the right thigh of each animal. After image reconstruction, the lesion-to-background ratios were calculated in regions of interest over the lesion and contralateral thigh to determine the equilibrium status of the radiotracer. All mice were sacrificed for histopathologic examination and six of the mice were examined with micro-autoradiography. [18F]FDG uptake in tumors and inflammations both reached equilibrium about 3 hours after injection. At equilibrium, [18F]FDG uptake into tumors was two to four times higher than the background. Uptake into the 4-day and 8-day inflammations was 2.3 and 5.5 times higher than the background, respectively. Histopathology showed macrophage and neutrophil infiltration around the tumors and in the inflammations. Micro-autoradiography showed dense silver grains in the granulation tissue surrounding the tumors and inflammations. The preliminary results suggested that dynamic [18F]FDG coincidence planar imaging can help in determining the suitable time for static [18F]FDG imaging in nude mice models. The optimal time for static [18F]FDG positron emission tomography imaging was around 3 hours after injection. The paradigm for determining a dynamic [18F]FDG uptake pattern was demonstrated for future new therapeutic drug experimental use.

Animals↗
PubMed2003

Identity proton-transfer reactions from C-H, N-H, and O-H acids. An ab initio, DFT, and CPCM-B3LYP aqueous solvent model study.

Identity proton-transfer reactions between 21 acids, Y-X-H, and their conjugate bases, (-)X-Y, were studied according to the reaction scheme, Y-X-H + (-)X-Y --> (Y-X-H...(-)X-Y)(cx) --> [Y-X...H...X-Y](ts) --> (Y-X..H-X-Y)(cx) --> Y-X(-) + H-X-Y, where cx indicates an ion-molecule complex and ts indicates the proton-transfer transition state. All species were optimized at the MP2/6-311+G level, and these geometries were used for single-point calculations by other methods: coupled-cluster, DFT (gas phase), and a polarizable continuum aqueous solvent model (COSMO). All methods gave enthalpies of deprotonation which correlate well with experimental measurements of deltaH(ACID) (gas) or pK(a) (aq). Calculated gas-phase enthalpies of deprotonation (deltaH(ACID)) and enthalpies of activation (deltaH(#)) are poorly correlated except for small, carefully selected sets. This result stands in contrast to the many aqueous phase Brönsted correlations of kinetic and equilibrium acid strength. On the other hand, gas-phase enthalpies of complexation and deltaH(#) are well correlated, indicating that factors which stabilize the transition state are at work in the bimolecular ion-molecule complex although to a smaller degree. We infer that intermoiety electrostatic and other interactions, similar within the complex and the transition state, but absent in the separated reactants (products), cause the lack of correlation between deltaH(ACID) and the other two quantities. Such differences are strongly attenuated in water because reactants and products do interact with polar/polarizable matter (the solvent) if not with each other. Charge distributions (NPA) were computed, allowing calculation of Bernasconi's "transition state imbalance parameter". Such measures provide intuitively satisfactory trends, but only if the reaction termini, X, are kept the same. As X is made more electronegative, the magnitude of the apparent imbalance increases, a result of greater negative charge on X in the transition state. This result gives additional support for the importance of the ion-triplet structure, [YX(-)...H(+)...(-)XY], to the stability of the transition state. Additional qualitative support for this conclusion is provided by the inverse relationship between the activation barrier and the charge on the in-flight hydrogen in the transition state, and by the dominance of polar over resonance substituent effects on the stability of the transition state. Calculations also show that the "nitroalkane anomaly", well established in solution, does not exist in the gas phase. The COSMO model partly reproduces this anomaly and performs adequately except when strong, specific intermolecular forces such as hydrogen bonding between solvent and anions are important.

Journal Article↗
PubMed2005

An ab initio and density functional theory study of keto-enol equilibria of hydroxycyclopropenone in gas and aqueous solution phase.

Keto-enol tautomerism in hydroxycyclopropenone (2-hydroxy-2-cyclopropen-1-one) has been studied using ab initio methods, the B3LYP functional of density functional theory, as well as complete basis set (CBS-QB3 and CBS-APNO) and G3 methods. Absolute and relative energies were calculated with each of the methods, whereas computations of geometries and harmonic frequencies for hydroxycyclopropenone and 1,2-cyclopropanedione were computed in the gas phase but were limited to HF, MP2 and CCSD levels of theory, and the B3LYP functional, in combination with the 6-31++G** basis set. Using the MP2/6-31++G** gas phase optimized structure, each species was then optimized fully in aqueous solution by employing the polarizable continuum model (PCM) self-consistent reaction field approach, in which HF, MP2 and B3LYP levels of theory were utilized, with the same 6-31++G** basis set. In both gas and aqueous solution phases, the keto form is higher in energy for all of the model chemistries considered. The presence of the solvent, however, is found to have very little effect on the bond lengths, angles and harmonic frequencies. From the B3LYP/6-31++G** Gibbs free energy, the keto-enol tautomeric equilibrium constant for 2-hydroxy-2-cyclopropen-1-one <==> 1,2-cyclopropanedione is computed to be K(T)(gas) = 2.35 x 10(-6), K(T)(aq) = 5.61 x 10(-14). It is concluded that the enol form is overwhelmingly predominant in both environments, with the effect of the solvent shifting the direction of equilibrium even more strongly in the favor of hydroxycyclopropenone. The almost exclusive nature of this species is attributed to stabilization resulting from aromaticity. Confirmation is provided by comparison of the simulated vibrational spectra of hydroxycyclopropenone with the measured infrared spectrum in an argon matrix.

Journal Article↗
PubMed2001

From 'captive' agonism to insurmountable antagonism: demonstrating the power of analytical pharmacology.

1. Mathematical modelling is useful in pharmacology, allowing the investigator to obtain insights into the biological processes under study that may not always be intuitively obvious. Examples are presented in this review using the pharmacology of the muscarinic acetylcholine receptor (mAChR) agonist xanomeline. 2. Xanomeline possesses a novel mode of action that involves persistent binding to the M1 mAChR, yielding a fraction of agonist in the receptor compartment that continually activates the receptor, despite extensive washout, as assessed in functional assays measuring the cumulative production of M1 mAChR-mediated L-[3H]-citrulline. This persistent effect was reversed by the antagonist atropine, but re-established upon the removal of atropine. Thus, xanomeline may represent the first 'captive' agonist of the mAChR. 3. Atropine was equally potent at reversing the effect of persistently bound xanomeline and preventing the effect of added xanomeline. Application of standard quantitative equilibrium models of agonist-antagonist interaction to these data suggested that the interaction between xanomeline and atropine satisfied the criteria of competitivity in each case. 4. Subsequent real-time assays of M1 mAChR-mediated intracellular calcium mobilization found that atropine inhibited the effects of xanomeline in an insurmountable manner. 5. The discrepancy between the modes of antagonism in the various functional assays could be reconciled in a dynamic receptor model of antagonism within a transient response system and subsequent Monte Carlo simulations allowed for the development of an optimized analytical procedure to quantify antagonist potency under such conditions of response fade. 6. These types of studies exemplify the diagnostic and integrative features of analytical pharmacology.

Animals↗
PubMed1990

Partial Purification and Characterization of a 3'- Phosphoadenosine 5' -Phosphosulfate: Desulfoglucosinolate Sulfotransferase from Cress (Lepidium sativum).

A 3' -phosphoadenosine 5' -phosphosulfate (PAPS):desulfoglucosinolate sulfotransferase (EC 2.8.2-) was extensively purified from light-grown cress (Lepidium sativum L.) seedlings by gel filtration and concanavalin A-Sepharose 4B, Matrex Gel Green A, and Mono Q fast protein liquid chromatography. The purified enzyme, which required bovine serum albumin for stabilization, had a native molecular weight of 31,000 +/- 5,000 and an apparent isoelectric point of 5.2. Using PAPS (K(m) 60 micromolar) as sulfur donor, it catalyzed the sulfation of desulfobenzylglucosinolate (K(m) 82 micromolar), desulfo-p-hydroxybenzylglucosinolate (K(m) 670 micromolar), and desulfoallylglucosinolate (K(m) 6.5 millimolar) at an optimal pH of 9.0. All other potential substrates tested, including flavonoids, flavonoid glycosides, cinnamic acids, and phenylacetaldoxime, were not sulfated. Sulfotransferase activity was stimulated by MgCl(2), MnCl(2) and reducing agents and inhibited by ZnCl(2), PbNO(3) NiCl(2) and the reaction product PAP. The thiol reagents N-ethylmaleimide, p-chloromercuriphenylsulfonic acid, and 5,5' -dithio-bis-(2-nitrobenzoic acid) were also potent inhibitors, but the enzyme was protected from covalent modification by beta-mercaptoethanol. The kinetics of desulfobenzylglucosinolate sulfation were consistent with a rapid equilibrium ordered mechanism with desulfobenzylglucosinolate binding first and PAPS second.

Journal Article↗
PubMed1998

Characterization of a novel group of basic small heat shock proteins in Xenopus laevis A6 kidney epithelial cells.

In this study, we report the detection of a new group of five stress-inducible basic small heat shock proteins (BShsps) in Xenopus laevis kidney epithelial A6 cells by means of two-dimensional non-equilibrium pH gradient gel electrophoresis. These basic 30-kDa small hsps are distinct from the previously described X. laevis acidic hsp30 family on the basis of their charge and lack of cross-reactivity with an hsp30 antibody. Furthermore, at least two of the five BShsps were present constitutively, an observation that has not been made with the acidic hsp30 family. The heat inducibility of the BShsps was regulated at the level of transcription as indicated by their inhibited synthesis in the presence of the transcriptional inhibitor actinomycin D. Furthermore, the optimal temperature of BShsp induction, temporal pattern of synthesis, and induction of BShsps by other stressors such as herbimycin A and sodium arsenite were similar to those reported for the acidic hsp30 family. This study suggests that X. laevis contains at least two unique groups of small heat shock proteins that are coordinately expressed.

Animals↗
PubMed1992

Role of carnitine and carnitine palmitoyltransferase as integral components of the pathway for membrane phospholipid fatty acid turnover in intact human erythrocytes.

The deacylation and reacylation process of phospholipids is the major pathway of turnover and repair in erythrocyte membranes. In this paper, we have investigated the role of carnitine palmitoyltransferase in erythrocyte membrane phospholipid fatty acid turnover. The role of acyl-L-carnitine as a reservoir of activated acyl groups, the buffer function of carnitine, and the importance of the acyl-CoA/free CoA ratio in the reacylation process of erythrocyte membrane phospholipids have also been addressed. In intact erythrocytes, the incorporation of [1-14C]palmitic acid into acyl-L-carnitine, phosphatidylcholine, and phosphatidylethanolamine was linear with time for at least 3 h. The greatest proportion of the radioactivity was found in acyl-L-carnitine. Competition experiments using [1-14C]palmitic and [9,10-3H]oleic acid demonstrated that [9,10-3H]oleic acid was incorporated preferentially into the phospholipids and less into acyl-L-carnitine. When an erythrocyte suspension was incubated with [1-14C]palmitoyl-L-carnitine, radiolabeled palmitate was recovered in the phospholipid fraction, and the carnitine palmitoyltransferase inhibitor, 2-tetradecylglycidic acid, completely abolished the incorporation. ATP depletion decreased incorporation of [1-14C]palmitic and/or [9,10-3H]oleic acid into acyl-L-carnitine, but the incorporation into phosphatidylcholine and phosphatidylethanolamine was unaffected. In contrast, ATP depletion enhanced the incorporation into phosphatidylcholine and phosphatidylethanolamine of the radiolabeled fatty acid from [1-14C]palmitoyl-L-carnitine. These data are suggestive of the existence of an acyl-L-carnitine pool, in equilibrium with the acyl-CoA pool, which serves as a reservoir of activated acyl groups. The carnitine palmitoyltransferase inhibition by 2-tetradecylglycidic acid or palmitoyl-D-carnitine caused a significant reduction of radiolabeled fatty acid incorporation into membrane phospholipids, only when intact erythrocytes were incubated with [9,10-3H]oleic acid. These latter data may be explained by the differences in rates and substrates specificities between acyl-CoA synthetase and the reacylating enzymes for palmitate and oleate, which support the importance of carnitine palmitoyltransferase in modulating the optimal acyl-CoA/free CoA ratio for the physiological expression of the membrane phospholipids fatty acid turnover.

Acylation↗
PubMedDate not supplied

[Further study of spontaneous virus production using transplantable HEp-2 cells as a model].

Two maxima of optic density were observed at zones of gravity 1.27 g/ml and 1.15-1.16 g/ml by sedimentation equilibrium in sucrose gradient of cultural fluid, obtained from the transplantable cells of the HEP-2 strain and concentrated by ultracentrifugation. These fractions thus isolated were tested for presence of RNA- and DNA-dependent DNA-polymerase. The structures with the density of 1.15-1.16 g/ml were identified with the oncornaviruses on the basis of characteristics flotating density, presence of RNA-dependent DNA-polymerase. Analyses of products of RNA- and DNA-dependent polymerases reaction, flotating density of oncornaviral nucleotides in sucrose and CsCl gradients are presented. The optimal conditions for reverse-transcriptase reaction of virions of D type viruses are characterized.

Cell Line↗
PubMed1986

Protons, osmolytes, and fitness of internal milieu for protein function.

The composition of the intracellular milieu shows striking similarities among widely different species. Only certain values of intracellular pH, values that generally reflect alphastat regulation, and only narrow ranges of inorganic ion concentrations are found in the cytoplasm of the cells of most animals, plants, and microorganisms. In water-stressed organisms only a few types of low-molecular-weight organic molecules (osmolytes) are accumulated. These highly conserved characteristics of the intracellular fluids reflect the need to maintain critical features of macromolecules within narrow ranges optimal for life. For proteins these features include maintaining adequate rates of catalysis, a high level of regulatory responsiveness, and a precise balance between stability and lability of structure (tertiary conformation, subunit assembly, and multiprotein complexes). The optimal values for these functional and structural features of proteins often lie near the midrange of possible values for these properties, and only under specific conditions of intracellular pH, ionic strength, and osmolyte composition are these optimal midrange values conserved. In dormant cells the departure of solution conditions from values that are optimal for protein function and structure may be instrumental in reducing or shutting down metabolic functions. Seen from a broad evolutionary perspective, the evolution of the intracellular milieu is an important complement to macromolecular evolution. In certain instances appropriate modifications of the internal milieu may reduce the need for adaptive amino acid replacements in proteins.

Acid-Base Equilibrium↗
PubMed2005

Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP.

The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use.

Biotechnology↗
PubMed2006

Methylene blue adsorption from aqueous solution by dehydrated peanut hull.

Dyes are colour organic compounds which can colorize the other substances. These substances usually presents in the effluent water of many industries, such as textiles, leather, paper, printing and cosmetics. To observe the potential feasibility of removing colour, peanut hull as an agricultural by-product was dehydrated with sulphuric acid (DPH) and used for adsorption of methylene blue (MB) from aqueous solution. The effects of various parameters such as initial methylene blue concentrations, temperatures and particle sizes were examined and optimal experimental conditions were determined. Adsorption data were well described by the Langmuir model, although they could be modelled by the Freundlich model as well. The adsorption process followed the pseudo-second order kinetic model. The mass transfer model as intraparticle diffusion was applied to the experimental data to examine the mechanisms of rate controlling step. It was found that at the higher initial MB concentration, intraparticle diffusion is becoming significant controlling step. The thermodynamic constants of the adsorption process were also evaluated by using the Langmuir constants related to the equilibrium of adsorption at different temperatures. The results in this study indicated that dehydrated peanut hull was a good adsorbent for removing methylene blue.

Adsorption↗
PubMed2000

The preparation and characterization of an immobilized l-glutamic decarboxylase and its application for determination of l-glutamic acid.

This paper is to study the preparation and characterization of an immobilized L-glutamic decarboxylase (GDC) and develop a sensitive method for the determination of L-glutamate using a new biosensor, which consists of an enzyme column reactor of GDC immobilized on a novel ion exchange resin (carboxymethyl-copolymer of allyl dextran and N.N'-methylene-bisacrylamide CM-CADB) and ion analyzer coupled with a CO(2) electrode. The conditions for the enzyme immobilization were optimized by the parameters: buffer composition and concentration, adsorption equilibration time, amount of enzyme, temperature, ionic strength and pH. The dynamic response of Na(2)HPO(4)-citric acid buffer system selected is much better than that of the others, 0.10 M HAc-0.10 M NaAc and 0.10 M sodium citrate-0.10 M citric acid. The initial rate of the enzyme reaction v(0) in this buffer system is 1.76 mol. l(-1) min(-1), moreover, the rate of the enzyme reaction appears linear in the first 4 min. The optimum adsorption equilibrium time is around 6 h. The amount of enzyme adsorbed on CM-CADB resin affects the response to substrate L-glutamic acid, the widest range of linearity is obtained with over 30 mg (GDC)/g(resin). The GDC activity immobilized on CM-CADB reaches a maximum when the immobilization temperature was kept around 40 degrees C. pH was kept at 4.4 when measuring the activity of the immobilized GDC. No variation of the activity of immobilized GDC is observed when the capacity is over 2.5 meq/g.(CM-CADB resin). The properties of the immobilized enzyme on CM-CADB were characterized. No significant improvement can be achieved when the substrate concentration exceeds 12.00 mmol/l, where the activity of immobilized GDC is equal to 1.58 mmol/l.min.g. The optimum pH is found to be 5.2, which changes 0.2 unit, comparing with that of the free GDC (5.0). The optimum temperature is found to be around 48 degrees C, which is lower than that of free GDC (55 degrees C). The critical temperature of the free GDC and the immobilized GDC is approximately 50 degrees C and 45 degrees C, respectively. The half-life of the activity is 127 days when the immobilized enzyme was stored in the cold (4 degrees C). An immobilized GDC enzyme column reactor matched with a flow injection system-ion analyzer coupled with CO(2) electrode-data collection system made up the original form of the apparatus of biosensor for determining of L-glutamic acid. The determination conditions are that the buffer solution is 0.10 M Na(2)HPO(4)-0.05 M citric acid at pH 4.4 and t = 37 degrees C. The limit of detection is 1.0 x 10(-)(5) M. The linearity response is in the range of 5 x 10 (-2) - 5 x 10 (-5) M. The equation of linear regression of the calibration curve is y = 43.3x + 181.6 (y is the milli-volt of electrical potential response, x is the logarithm of the concentration of the substrate of L-glutamic acid). The correlation coefficient equals 0.99. The coefficient of variation equals 2.7%.

Journal Article↗
PubMed1994

[Microbiologic criteria of the choice of antibiotic for antibiotic prophylaxis in surgery].

The choice of an antibiotic for antimicrobial prophylaxis is based on microbiological, pharmacokinetic, chemical and pharmacodynamic parameters. The knowledge of the bacterial flora allows the identification of bacteria which could be responsible for postsurgical infections. These floras are complex and their equilibrium can be modified by various factors, such as hospitalization, co-existing disease, medico-surgical procedure, administration of antibiotics, which cause the selection of the so-called hospital-bacteria feared by therapists. The infection will develop according to the quantity of bacteria that have been introduced and to their virulence, which is often altered by local factors, especially the biomaterials. The knowledge of pharmacodynamic parameters such as bactericidal activity, postantibiotic effect, activity on virulence factors (bacterial adhesion), allows the refinement of the choice of the antibiotic and the optimization of its posology.

Anti-Bacterial Agents↗
PubMed2004

Chemokine-binding specificity of soluble chemokine-receptor analogues: identification of interacting elements by chimera complementation.

The specificity of chemokine-receptor interactions plays a central role in the regulation of leukocyte migration in inflammatory responses. Herein, we describe a soluble mimic of CC chemokine receptor 2 (CCR2), dubbed CROSS-N(2)E3(2), which incorporates the N-terminal region (N) and third extracellular loop (E3) elements of CCR2 displayed on the surface of a soluble protein scaffold. CROSS-N(2)E3(2) binds to the CCR2 ligand monocyte chemoattractant protein-1 (MCP-1) with a dissociation equilibrium constant of 1.1 +/- 0.1 microM but does not bind to the cognate chemokines of the receptor CCR3 (eotaxin-1, -2, and -3). Similarly, a soluble analogue of CCR3 (CROSS(5)-N(3)E3(3)) binds to eotaxin-1, -2, and -3 but not to MCP-1. Thus, these receptor analogues have the same specificity as the natural receptors. Using soluble proteins containing N and E3 elements from different receptors (CROSS-N(2)E3(3) and CROSS-N(3)E3(2)), we demonstrate that both receptor elements are required for optimal binding to the cognate chemokines. In addition, we report the binding affinities of all four CROSS proteins to a panel of two wild-type and six chimeric chemokines. These complementation studies indicate the regions of the chemokines that interact with each element of the receptors, allowing us to deduce the orientations of the receptor extracellular elements relative to the bound chemokines.

Amino Acid Sequence↗
PubMed1997

Pharmacological characterization of type 1alpha metabotropic glutamate receptor-stimulated [35S]-GTPgammaS binding.

1. The activation of G proteins by type 1alpha metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1alpha receptor has been studied by use of a [35S]-guanosine 5'-[gamma-thio]triphosphate ([35S]-GTPgammaS) binding assay. 2. L-Glutamate increased the rate of [35S]-GTPgammaS binding in a concentration-dependent manner (-logEC50 (M) 5.25 +/- 0.07), with an optimal (62.4 +/- 1.6%) increase over basal binding being observed following 60 min incubation at 30 degrees C with 70 pM [35S]-GTPgammaS, 1 microM GDP, 10 mM MgCl2, 100 mM NaCl and 100 microg membrane protein ml(-1). The L-glutamate (100 microM)-stimulated increase in [35S]-GTPgammaS binding was totally prevented in the presence of the group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine (300 microM). 3. Quantitative analysis of the affinity and number of G proteins activated by a maximally effective concentration of L-glutamate revealed an equilibrium dissociation constant (K(D)) for [35S]-GTPgammaS binding of 0.76 +/- 0.20 nM and a maximal number of GTPgammaS-liganded G proteins (Bmax) of 361 +/- 30 fmol mg(-1) protein. 4. Metabotropic glutamate receptor agonists, quisqualate (-logEC50 (M) 6.74 +/- 0.06), 1S,3R-ACPD (4.64 +/- 0.08) and (S)-3,5-dihydroxyphenylglycine (5.16 +/- 0.23) also increased [35S]-GTPgammaS binding in a concentration-dependent manner, with the latter two agents behaving as partial agonists. 5. (+)-alpha-Methylcarboxyphenylglycine (300 microM) caused a parallel rightward shift of the L-glutamate concentration-effect curve for [35S]-GTPgammaS binding, allowing an antagonist equilibrium dissociation constant (K(D)) of 34.0 +/- 7.8 microM to be calculated for this mGluR antagonist. 6. Pretreatment of BHK-mGluR1alpha cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100 ng ml(-1), 24 h) before membrane preparation resulted in a marked decrease in agonist-stimulated [35S]-GTPgammaS binding (by 66.0 +/- 0.9%), and an altered concentration-effect relationship for agonist-stimulated [35S]-GTPgammaS binding by the residual PTX-insensitive G-protein population. 7. The modulation of [35S]-GTPgammaS binding by agonists and antagonists in membranes from recombinant cells provides an excellent system in which to study mGluR interactions with PTX-sensitive and -insensitive G proteins.

Animals↗
PubMed2006

Exact tests of Hardy-Weinberg equilibrium and homogeneity of disequilibrium across strata.

Detecting departures from Hardy-Weinberg equilibrium (HWE) of marker-genotype frequencies is a crucial first step in almost all human genetic analyses. When a sample is stratified by multiple ethnic groups, it is important to allow the marker-allele frequencies to differ over the strata. In this situation, it is common to test for HWE by using an exact test within each stratum and then using the minimum P value as a global test. This approach does not account for multiple testing, and, because it does not combine information over strata, it does not have optimal power. Several approximate methods to combine information over strata have been proposed, but most of them sum over strata a measure of departure from HWE; if the departures are in different directions, then summing can diminish the overall evidence of departure from HWE. An exact stratified test is more appealing because it uses the probability of genotype configurations across the strata as evidence for global departures from HWE. We developed an exact stratified test for HWE for diallelic markers, such as single-nucleotide polymorphisms (SNPs), and an exact test for homogeneity of Hardy-Weinberg disequilibrium. By applying our methods to data from Perlegen and HapMap--a combined total of more than five million SNP genotypes, with three to four strata and strata sizes ranging from 23 to 60 subjects--we illustrate that the exact stratified test provides more-robust and more-powerful results than those obtained by either the minimum of exact test P values over strata or approximate stratified tests that sum measures of departure from HWE. Hence, our new methods should be useful for samples composed of multiple ethnic groups.

Computer Simulation↗
PubMed2004

Reconstructing biological networks using conditional correlation analysis.

MOTIVATION: One of the present challenges in biological research is the organization of the data originating from high-throughput technologies. One way in which this information can be organized is in the form of networks of influences, physical or statistical, between cellular components. We propose an experimental method for probing biological networks, analyzing the resulting data and reconstructing the network architecture. METHODS: We use networks of known topology consisting of nodes (genes), directed edges (gene-gene interactions) and a dynamics for the genes' mRNA concentrations in terms of the gene-gene interactions. We proposed a network reconstruction algorithm based on the conditional correlation of the mRNA equilibrium concentration between two genes given that one of them was knocked down. Using simulated gene expression data on networks of known connectivity, we investigated how the reconstruction error is affected by noise, network topology, size, sparseness and dynamic parameters. RESULTS: Errors arise from correlation between nodes connected through intermediate nodes (false positives) and when the correlation between two directly connected nodes is obscured by noise, non-linearity or multiple inputs to the target node (false negatives). Two critical components of the method are as follows: (1) the choice of an optimal correlation threshold for predicting connections and (2) the reduction of errors arising from indirect connections (for which a novel algorithm is proposed). With these improvements, we can reconstruct networks with the topology of the transcriptional regulatory network in Escherichia coli with a reasonably low error rate.

Algorithms↗
PubMed2001

Properties and modulation of alpha human atrial natriuretic peptide (alpha-hANP)-formed ion channels.

Using the lipid bilayer technique we have optimized recording conditions and confirmed that alpha human atrial natriuretic peptide [alpha-hANP(1-28)] forms single ion channels. The single channel currents recorded in 250/50 mM KCl cis/trans chambers show that the ANP-formed channels were heterogeneous, and differed in their conductance, kinetic, and pharmacological properties. The ANP-formed single channels were grouped as: (i) H202- and Ba2+-sensitive channel with fast kinetics; the nonlinear current-voltage (I-V) relationship of this channel had a reversal potential (Erev) of -28.2 mV, which is close to the equilibrium potential for K+ (EK = -35 mV) and a maximal slope conductance (gmax) of 68 pS at positive potentials. Sequential ionic substitution (KCl, K gluconate and choline Cl) of the cis solution suggests that the current was carried by cations. The fast channel had three modes (spike mode, burst mode, and open mode) that differed in their kinetics but not in their conductance properties. (ii) A large conductance channel possessing several subconductance levels that showed time-dependent inactivation at positive and negative membrane potentials (Vm). The inactivation ratio of the current at the end of the voltage step (Iss) to the initial current (Ii) activated immediately after the voltage step, (Iss/Ii), was voltage dependent and described by a bell-shaped curve. The maximal current-voltage (I-V) relationship of this channel, which had an Erev of +17.2 mV, was nonlinear and the value of gmax was 273 pS at negative voltages. (iii) A transiently-activated channel: the nonlinear I-V relationship of this channel had an Erev of -29.8 mV and the value of gmax was 160 pS at positive voltages. We propose that the voltage-dependence of the ionic currents and the kinetic parameters of these channel types indicate that if they were formed in vivo and activated by cytosolic factors they could change the membrane potential and the electrolyte homeostasis of the cell.

Amino Acid Sequence↗