Mechanism of action of choleragen.

Choleragen exerts its effect on cells through activation of adenylate cyclase. Choleragen initially interacts with cells through binding of the B subunit of the toxin to the ganglioside GM1 on the cell surface. Subsequent events are less clear. Patching or capping of toxin on the cell surface may be an obligatory step in choleragen action. Studies in cell-free systems have demonstrated that activation of adenylate cyclase by choleragen requires NAD. In addition to NAD, requirements have been observed for ATP, GTP, and calcium-dependent regulatory protein. GTP also is required for the expression of choleragen-activated adenylate cyclase. In preparations from turkey erythrocytes, choleragen appears to inhibit an isoproterenol-stimulated GTPase. It has been postulated that by decreasing the activity of a specific GTPase, choleragen would stabilize a GTP-adenylate cyclase complex and maintain the cyclase in an activated state. Although the holotoxin is most effective in intact cells, with the A subunit having 1/20th of its activity and the B subunit (choleragenoid) being inactive, in cell-free systems the A subunit, specifically the A1 fragment, is required for adenylate cyclase activation. The B protomer is inactive. Choleragen, the A subunit, or A1 fragment under suitable conditions hydrolyzes NAD to ADP-ribose and nicotinamide (NAD glycohydrolase activity) and catalyzes the transfer of the ADP-ribose moiety of NAD to the guandino group of arginine (ADP-ribosyltransferase activity). The NAD glycohydrolase activity is similar to that exhibited by other NAD-dependent bacterial toxins (diphtheria toxin, Pseudomonas exotoxin A), which act by catalyzing the ADP-ribosylation of a specific acceptor protein. If the ADP-ribosylation of arginine is a model for the reaction catalyzed by choleragen in vivo, then arginine is presumably an analog of the amino acid which is ADP-ribosylated in the acceptor protein. It is postulated that choleragen exerts its effects on cells through the NAD-dependent ADP-ribosylation of an arginine or similar amino acid in either the cyclase itself or a regulatory protein of the cyclase system.     

Some kinetic and chromatographic properties of detergent-dispersed adenylate cyclase.

Studies on the reaction kinetics and chromatographic properties of detergent-dispersed adenylate cyclase are described. Detergent-dispersed enzyme was prepared from whole rat cerebellum and from partially purified plasma membranes from rat liver. Data were simulated to fit kinetic models for which an inhibitor is added in constant proportion to the variable substrate. Models were chosen to distinguish whether the adenylate cyclase reaction may be controlled by an inhibitory action of free ATP--4 (or HATP--3) or by a stimulatory action of free divalent cations. The various kinetic models were then tested with the dispersed brain adenylate cyclase with both Mg++ and Mn++ and in two different buffer systems. The experimental data indicate that this enzyme has a distinct cation binding site, but exhibits no significant inhibition by HATP--3 or ATP--4. The detergent-dispersed adenylate cyclase both from liver plasma membranes and from brain have been chromatographed on anion exchange material and have been subjected to gel filtration. The presence of detergent was required for elution of cyclase activity from DEAE-Sephadex but was not required when DEAE-agarose was used. Dispersed brain cyclase was also chromatographed on agarose-NH(CH2)3NH(CH2)3-NH2 which exhibits both ionic and hydrophobic properties. Fifty percent of the applied activity was recovered with a fivefold increase in specific activity. The data suggest that the relative effectiveness of a given chromatographic procedure for detergent-dispersed adenylate cyclase may reflect the influence of both hydrophobic and ionic factors.     

Catecholamine binding to CNS adrenergic receptors.

The properties of 3H-catecholamine binding to alpha- and beta-adrenergic receptors in CNS are reviewed. 3H-epinephrine and 3H-norepinephrine label one class of alpha-receptors throughout the brain, with high affinities for agonists and some antagonists. Agonist affinities at this site are increased in low temperature conditions but are reduced by guanine nucleotides and monovalent cations. Divalent cations reverse both effects. This alpha-receptor may be coupled to adenylate cyclase by GTP and/or sodium, and uncoupled by divalent cations. 3H-epinephrine labels beta2, but not beta1, receptors in CNS, especially in bovine cerebellum. The same beta-receptor does not show agonist-specific GTP-sensitivity, but does exhibit Na+-sensitivity. This receptor appears to be linked to adenylate cyclase, and sodium rather than GTP may be the coupling agent.     

Vasopressin-sensitive kidney adenylate cyclase: modulation of the hormonal response.

Vasopressin-sensitive pig kidney adenylate cyclase is sensitive to several effectors, such as Mg2+, other divalent cations, and guanyl nucleotides. The purpose of the present study was to compare the main characteristics of adenylate cyclase activation by vasopressin, Mg2+, and GMPPNP, respectively. Mg2+ ions were shown to exert at least three different effects on adenylate cyclase. The substrate of the adenylate cyclase reaction is the Mg-ATP complex. Mg2+ interacts with an enzyme regulatory site. Finally, Mg2+ can modulate the hormonal response, with Mg2+ ions affecting the coupling function--that is, the quantitative relationship between receptor occupancy and adenylate cyclase activation. At all the magnesium concentrations tested, from 0.25 mM to 16 mM, adenylate cyclase activation was not a direct function of receptor occupancy. At low Mg2+ concentrations, adenylate cyclase activation dose-response curve to the hormone tended to be superimposable to the hormone dose-binding curve. These results suggest a role of magnesium at the coupling step between the hormone-receptor complex and adenylate cyclase response. Cobalt, but not calcium, ions could exert the same effects as Mg2+ ions on this coupling step. GMPPNP induced considerable adenylate cyclase activation (15 to 35 times the basal value). Activation by GMPPNP was highly time and temperature dependent. At 30 degrees C, a 20 to 60 min preincubation period in the presence of GMPPNP was needed to obtain maximal activation. The higher the dose of GMPPNP in the medium, the longer it took to reach equilibrium. At 15 degrees C, activation was still increasing with time after 3 hr preincubation in the presence of the nucleotide. GMPPNP was active in a 10(-8)M to 10(-5)M concentration range. Unlike the results obtained with lysine vasopressin, the kinetic characteristics of dose-dependent adenylate cyclase activation curves by GMPPNP were unaffected by varying Mg2+ concentrations except for the increase in velocity when raising Mg2+ concentration. It was not clear whether or not the activation processes by the hormone and by GMPPNP had common mechanisms.     

The cytochemical localization of adenylate cyclase activity in mucous and serous cells of the salivary gland.

The present study was undertaken to localize adenylate cyclase activity in salivary glands by cytochemical means. For the study, serous parotid glands and mixed sublingual glands of the rat were used. Pieces of the fixed glands were incubated with adenosine triphosphate (ATP) or adenylyl-imidodiphosphate (AMP-PNP) as substrate: inorganic pyrophosphate or PNP liberated upon the action of adenylate cyclase on the substrates is precipitated by lead ions at their sites of production. In both glands, the reaction product was detected along the myoepithelial cell membranes in contact with secretory cells, indicating that a high level of adenylate cyclase activity occurs in association with these cell membranes. The association with a high level of the enzyme activity might be related to the contractile nature of myoepithelial cells which are supposed to aid secretory cells in discharging secretion products. A high level of adenylate cyclase activity was also detected associated with serous secretory cells (acinar cells of the parotid gland and demilune cells of the sublingual gland), but not with mucous secretory cells. In serous cells, deposits of reaction product were localized along the extracellular space of the apical cell membrane bordering the lumen. This is the portion of the cell membrane which fuses with the granule membranes during secretion. Since the granule membranes are not associated with a detectable level of adenylate cyclase activity, it appears that the enzyme activity becomes activated or associated with the granule membranes as they become part of the cell membrane by fusion. The association with a high level of adenylate cyclase activity appears to be related to the ability of the membrane to fuse with other membranes. It is likely, since the luminal membrane of mucous cells which does not fuse with mucous granule membranes during secretion is not associated with a detectable enzyme activity.     

The size of adenylate cyclase and guanylate cyclase from the rat renal medulla.

The size distribution of adenylate cyclase from the rat renal medulla solubilized with the nonionic detergents Triton X-100 and Lubrol PX was determined by gel filtration and by centrifugation in sucrose density gradients made up in H2O or D2O. The physical parameters of the predominant form in Triton X-100 are s20,w, 5.9S; Strokes radius, 62 A; partial specific volume (v), 0.74 ml/g; mass, 159,000 daltons; f/f0, 1.6; axial ratio (prolate ellipsoid), 11. For the minor form the values are: s20w, 3.0; Stokes radius, 28 A; mass, 38,000 daltons; f/f0, 1.2. The corresponding values determined in Lubrol PX are similar. The value for V for the enzyme indicates that it binds less than 0.2 mg detergent/mg protein. Since interactions with detergents probably substitute for interactions with lipids and hydrophobic amino acid side chains, these findings suggest that no more than 5% of the surface of adenylate cyclase is involved in hydrophobic interactions with other membrane components. Thus, most of the mass of the enzyme is not deeply embedded in the lipid bilayer of the plasma membrane. Similar studies have been performed on the soluble guanylate cyclase of the rat renal medulla. In the absence of detergent, the molecular properties of this enzyme are: s20w, 6.3S; Stokes radius, 54 A, V, 0.75 ml/g; mass, 154,000 daltons f/f0, 1.4; Axial ratio, 7. The addition of 0.1% Lubrol PX to this soluble enzyme increases it activity two- to fourfold and changes the physical properties to: s20,w, 5.5S; Stokes radius, 62 A; V, 0.74 ml/g; mass, 148,000 daltons, f/f0, 1.6; axial ratio, 11. These results show that Lubrol PX activates the enzyme by causing a conformational change with unfolding on the polypeptide chain. Guanylate cyclase from the particulate cell fraction can be solubilized with Lubrol PX but has properties quite different from those of the enzyme in the soluble cell fraction. It is a heterogeneous aggregate with s20,w, 10S; Stokes radius, 65 A; mass about 300,000 daltons. The conditions which solubilize guanylate cyclase also solubilize adenylate cyclase and the two activities can be separated on the same sucrose gradient.     

The Escherichia coli adenylate cyclase complex: activation by phosphoenolpyruvate.

A model for the regulation of the activity of Escherichia coli adenylate cyclase is presented. It is proposed that Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) interacts in a regulatory sense with the catalytic unit of adenylate cyclase. The phosphoenolpyruvate (PEP)-dependent phosphorylation of Enzyme I is assumed to be associated with a high activity state of adenylate cyclase. The pyruvate or sugar-dependent dephosphorylation of Enzyme I is correlated with a low activity state of adenylate cyclase. Evidence in support of the proposed model involves the observation that Enzyme I mutants have low cAMP levels and that PEP increases cellular cAMP levels and, under certain conditions, activates adenylate cyclase, Kinetic studies indicate that various ligands have opposing effects on adenylate cyclase. While PEP activates the enzyme, either glucose or pyruvate inhibit it. The unique relationships of PEP and Enzyme I to adenylate cyclase activity are discussed.     

Effect of essential fatty acid deficiency on activity of liver plasma membrane enzymes in the rat.

Liver plasma membranes (LPM) were isolated from rats fed an essential fatty acid-supplemented diet (+EFA) or from rats fed an essential fatty acid-deficient diet (-EFA). The proportions of linoleate and arachidonate in membrane total fatty acids in the -EFA preparations were one-half or less than the values for the +EFA preparations. Basal, F-, or glucagon-stimulated adenylate cyclase activities were significantly lower in EFA-deficient livers than in nondeficient ones. Addition of GTP significantly enhanced glucagon-stimulated adrenylate cyclase in both groups, but extent of stimulation above basal was greater in EFA-deficient livers. Portal vein injection of glucagon in vivo resulted in significantly higher cAMP formation in +EFA livers than in -EFA livers. When glucagon was used in vitro at 1-1,000 nM, stimulation of adenylate cyclase remained lower in EFA-deficient membranes, but extent of stimulation above basal activity was larger in -EFA membranes than in +EFA. Total Na+, K+ (Mg2+)-ATPase from EFA-depleted LPM exhibited significantly higher values of apparent Km and Vmax-5'-Nucleotidase activity, in contrast, was considerably decreased in EFA-deficient rats. These findings show that, in animals, changes in unsaturated fatty acid composition can affect the properties of membrane-bound enzymes. These alterations could be due to changes in membrane physical properties and/or prostaglandin formation.     

Predictive value of the analogy between hormone-sensitive adenylate cyclase and light-sensitive photoreceptor cyclic GMP phosphodiesterase: a specific role for a light-sensitive GTPase as a component in the activation sequence.

We report experiments which involve a light sensitive GTPase in the light dependent activation of retinal rod 3'5'-cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE). The data suggest that the light activated GTPase is intermediate between rhodopsin and PDE in the light-dependent activation sequence. We list the many striking similarities between hormone sensitive adenylate cyclase and light activated PDE in order to emphasize that the findings presented herein may have predictive value for ongoing studies of the hormone sensitive adenylate cyclase specifically regarding the role of the hormone activated GTPase in the activation sequence.     

PARTIAL CHARACTERIZATION and LIGHT-INDUCED REGULATION OF GTP-BINDING PROTEINS IN Lemna paucicostata

Binding of GTP-binding proteins with [³⁵S]GTP7S in the extract containing membrane components of Lemna paucicostata 441 was inhibited by red or far red light by 20 to 25%, but blue light showed no or little effect. The plant used for the preparation of the extract was subjected to single darkness for 8 h, as both red and far red light inhibit flowering. The extract treated with 1% Lubrol was fractionated by gel filtration. Four species of GTP-binding proteins, GL1, GL2, GL3 and GL4 were detected with Km values 3, 7, 80 and 4 n M , respectively. GL1, GL2 and GL3 were ADP-ribosylated by pertussis toxin. The extract activated by [³⁵S]GTP-γS in darkness, under red light or under far red light was treated with 1% Lubrol and subsequent gel filtration of the extracts made it possible to detect GTP-binding protein with a small molecular weight only in an extract labeled in darkness. The reduction in the molecular weight of GTP-binding protein from the larger molecule associated with the binding of [³⁵S]GTPγS was confirmed by rechromatography of the larger molecule activated by [³⁵S]GTPγS in darkness. The binding of GL2 and/or GL3 with [³⁵S]GTPγS was suggested to be inhibited by red or far red light.     

Development of an efficient protein phosphatase-based colorimetric test for okadaic acid detection

Okadaic acid (OA), responsible for gastrointestinal problems, inhibits protein phosphatase 2A (PP2A). Therefore, the inhibition exerted by the toxin on PP2A could be used to detect the presence of OA in aqueous solution and in shellfish sample.In this work, two commercial PP2As (from ZEU Immunotec and Millipore) and one produced by molecular engineering (from GTP Technology) were tested. Enzymes were used in solution and also immobilized within a polymeric gel. In solution, best performances were obtained using PP2A purchased from ZEU Immunotec (Spain). OA was detected in aqueous solution in concentration as low as 0.0124 μg L⁻¹ using the enzyme from ZEU Immunotec whereas the detection limits were 0.47 μg L⁻¹ and 0.123 μg L⁻¹ with PP2As from Millipore and GTP Technology, respectively. Considering that the immobilization step contributes to stabilize the PP2A activity, enzymes were entrapped within a photopolymer and an agarose gel. These different polymeric matrices were optimized, tested and compared. Agarose gel seems to be a good alternative to the photopolymer largely used in our group. For instance, the IC₅₀ value obtained with the test based on PP2A from ZEU Immunotec immobilized within an agarose gel was 1.98 μg L⁻¹. This value was 1.8-fold lower than those obtained with the photopolymer test using the same enzyme. The proposed test is sensitive, fast and does not require expensive equipment. To evaluate the efficiency of the assay, PP inhibition tests based on PP2A from ZEU Immunotec in solution or immobilized within a gel were used for OA detection in contaminated shellfish.     

High-voltage paper electrophoretic assay for guanyl cyclase.

A simple, sensitive and rapid technique is described, permitting separation of cGMP from GMP, GDP and GTP by the use of unidirectional high-voltage paper electrophoresis. The recovery of labeled cGMP in the assay of guanyl cyclase, by this procedure is 85-90%; the blank values (no enzyme) are negligible.     

Regulation of dome formation in differentiated epithelial cell cultures.

Rat mammary (Rama 25) and dog kidney (MDCK) epithelial cell cultures formed 'domes' of cells due to fluid accumulation in focal regions between the culture dish and the cell monolayer. Addition of ouabain caused collapse of domes, suggesting that transport functions were required for maintenance of domes. Dome formation in both epithelial cell lines was stimulated by a broad spectrum of known inducers of erythroid differentiation in Fried erythroleukemia cells. Among these inducers were: 1) polar solvents such as dimethyl-sulfoxide, dimethylformamide, and hexamethylene bisacetamide; 2) purines such as hypoxanthine, inosine, and adenosine; 3) low-molecular-weight fatty acids such as n-butyrate; and 4) conditions expected to elevate levels of cyclic AMP. In the latter group were activators of adenylate cyclase such as cholera toxin and prostaglandin E 1; cyclic AMP phosphodiesterase inhibitors such as theophylline and 1-methyl-3-isobutylxanthine; and analogs of cyclic AMP. Induction of domes occurred 15--30 h after addition of inducer to the culture medium. Induction by chemicals was serum-dependent and required protein synthesis but not DNA synthesis. Induced dome formation was reversible after removal of inducer, requiring the continuous presence of inducer. Reversal was also observed after either either removal of serum or addition of inhibitors of protein synthesis. These results suggest that hypothesis that domes arise in these epithelial cultures by a process that is similar to cell differentiation and is influenced by cyclic AMP.     

Microfluidic tectonics platform: A colorimetric, disposable botulinum toxin enzyme-linked immunosorbent assay system

A fabrication platform for realizing integrated microfluidic devices is discussed. The platform allows for creating specific microsystems for multistep assays in an ad hoc manner as the components that perform the assay steps can be created at any location inside the device via in situ fabrication. The platform was utilized to create a prototype microsystem for detecting botulinum neurotoxin directly from whole blood. Process steps such as sample preparation by filtration, mixing and incubation with reagents was carried out on the device. Various microfluidic components such as channel network, valves and porous filter were fabricated from prepolymer mixture consisting of monomer, cross-linker and a photoinitiator. For detection of the toxoid, biotinylated antibodies were immobilized on streptavidin-functionalized agarose gel beads. The gel beads were introduced into the device and were used as readouts. Enzymatic reaction between alkaline phosphatase (on secondary antibody) and substrate produced an insoluble, colored precipitate that coated the beads thus making the readout visible to the naked eye. Clinically relevant amounts of the toxin can be detected from whole blood using the portable enzyme-linked immunosorbent assay (ELISA) system. Multiple layers can be realized for effective space utilization and creating a three-dimensional (3-D) chaotic mixer. In addition, external materials such as membranes can be incorporated into the device as components. Individual components that were necessary to perform these steps were characterized, and their mutual compatibility is also discussed.     

Modeling of concentration polarization and depolarization with high-frequency backpulsing

Rapid backpulsing to reduce membrane fouling during crossflow microfiltration and ultrafiltration is studied by solving the convection-diffusion equation for concentration polarization and depolarization during cyclic operation with transmembrane pressure reversal. For a fixed duration of reverse filtration, there is a critical duration of forward filtration which must not be exceeded if the formation of a cake or gel layer on the membrane surface is to be avoided. The theory also predicts an optimum duration of forward filtration which maximizes the net flux, since backpulsing at too high of frequency does not allow for adequate permeate collection during forward filtration relative to that lost during reverse filtration, whereas backpulsing at too low of frequency results in significant flux decline due to cake or gel buildup during each period of forward filtration. In general, short backpulse durations, low feed concentrations, high shear rates, and high forward transmembrane pressures give the highest net fluxes, whereas the magnitude of the reverse transmembrane pressure has a relatively small effect.Rapid backpulsing experiments with yeast suspended in deionized water performed with a flat-sheet crossflow microfiltration module and cellulose acetate membranes with 0.07 μm average pore diameter. The optimum forward filtration times were found to be 1.5, 3, and 5 s, respectively, for backpulse durations of 0.1, 0.2, and 0.3 s. Both theory and experiment gave net fluxes with backpulsing of about 85% of the clean membrane flux (0.022 cm/s = 790 l/m² h), whereas the long-term flux in the absence of backpulsing is an order-of-magnitude lower (0.0026 cm/s = 94 l/m² h).     

Radioimmunoassay kit formulation and its validation for serum progesterone using progesterone radiotracer purified by gel filtration

Purification of the radioiodinated progesterone tyrosine methyl ester conjugate by gel filtration and the development, optimization and clinical validation of a direct radioimmunoassay (RIA) of progesterone using this radiotracer are described. High purity radiotracer is essential for the error free performance of any RIA. Progesterone 11 hemisuccinate was conjugated to tyrosine methyl ester (TME) by the mixed anhydride method and this conjugate was then radioiodinated by the chloramine-T method. Purification of the radioiodinated product was carried out by gel filtration. About 12 batches of the radiotracer were prepared and purified. The purification by gel filtration gave reproducible elution pattern and purity. The radiotracer thus purified was found to have consistent quality as compared to that of any other purification methods. Non-specific binding of the radiotracer was found to be <5% in second antibody-polyethylene glycol based separation. 'Zero standard' binding was found to be consistently 50-60% in all the batches of radiotracer. The radiochemical purity of all the batches of radiotracer was >95% as checked by paper electrophoresis. The stability (retention of the immunoreactivity) of the radiotracer was two to three months. No appreciable changes in the assay characteristics were observed during this period. The assay involved 3 hours incubation of progesterone antibody with individual standards or sample and radiotracer at room temperature. The optimized assay was then validated for internal and external quality control parameters. A RIA kit was then formulated with this radiotracer for estimation of progesterone in serum. The assay kit consisted of lyophilized individual standards ranging from 0.25 to 50 ng/ml. The clinical performance of the developed kit was compared with that of a commercial ELISA kit and a correlation of 0.94 was observed.     

ATP-GTP-BINDING PROTEINS AND ENDOGENOUS ADP-RIBOSYL TRANSFERASE IN Lemna paucicostata 441

Abstract— A crude extract containing membrane components of Lemna paucicostata was treated with 1% Lubrol PX and fractionated by gel nitration. Binding activities to non-hydrolyzable analogues of ATP, [³⁵S]ATPγS (adenosine 5'[;γ-thio]triphosphate) and that of GTP, [³⁵S]GTPγS (guanosine 5'[γ-thiojtriphosphate) were detected in some fractions, and these activities were prevented in the presence of 0.1 mM ATP or GTP. ATP and GTP were 2 to 3 orders of magnitude more effective than CTP or UTP in preventing this binding activity. These fractions showed ATPase and GTPase activities with 1 nM [γ-³²P]ATP or [γ–³²P]GTP substrate. Analyses by sodium dodecyl sulfate polyacrylamide gel electrophoresis of these fractions after binding with [³⁵S]ATPγS or [³⁵S]GTP-γ S revealed that these fractions contained [³⁵S]ATPγS and [³⁵S]GTPγS binding proteins with molecular weights of 53 000 and 60 000, respectively. Both of these proteins were [³²P]ADP-ribosylated by endogenous ADP-ribosyl transferase. Three proteins with molecular weights of 11 000, 12 000 and 13 000 which could bind [³⁵S]ATP7S or [-³⁵S]GTP-γ S were ADP-ribosylated by endogenous ADP-ribosyl transferase. Pertussis toxin stimulated ADP-ribosylation of these proteins. Four proteins with molecular weight of 37 000, 50 000, 80 000 and 115 000 with P^SS]ATP7S and [^,3S]GTP7S binding activities were also detected. The signal transduction of light to underlying clock mechanism in Lemna may be controlled by ATP-GTP-binding proteins and by the ADP-ribosylation of these proteins.     

A homogeneous fluorescence resonance energy transfer system for monitoring the activation of a protein switch in real time

A homogeneous fluorescence resonance energy transfer (FRET) system for the real-time monitoring of exchange factor-catalyzed activation of a ras-like small GTPase is described. The underlying design is based on supramolecular template effects exerted by protein-protein interactions between the GTPase adenosine diphosphate ribosylation factor (ARF) and its effector protein GGA3. The GTPase is activated when bound to guanosine triphosphate (GTP) and switched off in its guanosine diphosphate (GDP)-bound state. Both states are accompanied by severe conformational changes that are recognized by GGA3, which only binds the GTPase "on" state. GDP-to-GTP exchange, i.e., GTPase activation, is catalyzed by the guanine nucleotide exchange factor cytohesin-2. When GGA3 and the GTPase ARF1 are labeled with thoroughly selected FRET probes, with simultaneous recording of the fluorescence of an internal tryptophan residue in ARF1, the conformational changes during the activation of the GTPase can be monitored in real time. We applied the FRET system to a multiplex format that allows the simultaneous identification and distinction of small-molecule inhibitors that interfere with the cytohesin-catalyzed ARF1 activation and/or with the interaction between activated ARF1-GTP and GGA3. By screening a library of potential cytohesin inhibitors, predicted by in silico modeling, we identified new inhibitors for the cytohesin-catalyzed GDP/GTP exchange on ARF1 and verified their increased potency in a cell proliferation assay.     

Molecular field analysis and 3D-quantitative structure-activity relationship study (MFA 3D-QSAR) unveil novel features of bile acid recognition at TGR5

Bile acids regulate nongenomic actions through the activation of TGR5, a membrane receptor that is G protein-coupled to the induction of adenylate cyclase. In this work, a training set of 43 bile acid derivatives is used to develop a molecular interaction field analysis (MFA) and a 3D-quantitative structure-activity relationship study (3D-QSAR) of TGR5 agonists. The predictive ability of the resulting model is evaluated using an external set of compounds with known TGR5 activity, and six bile acid derivatives whose unknown TGR5 activity is herein assessed with in vitro luciferase assay of cAMP formation. The results show a good predictive model and indicate a statistically relevant degree of correlation between the TGR5 activity and the molecular interaction fields produced by discrete positions of the bile acid scaffold. This information is instrumental to extend on a quantitative basis the current structure-activity relationships of bile acids as TGR5 modulators and will be fruitful to design new potent and selective agonists of the receptor.