Ultrasonographic and CT accuracy in localising surgical- or necropsy- confirmed solitary hepatic masses in dogs.

OBJECTIVES: To estimate the sensitivity and specificity of ultrasound and CT for localising solitary hepatic masses to particular liver divisions. MATERIALS AND METHODS: Dogs diagnosed with a solitary liver mass by ultrasound and/or CT, with surgical or necropsy confirmation within 1 month of imaging. Ultrasound reports were reviewed for mass location. CT scans were reviewed by two radiologists and mass location was determined by consensus agreement. Sensitivity and specificity were calculated for ultrasound and CT for each liver division. RESULTS: Fourteen of 71 dogs had CT only, 27 of 71 had ultrasound only and 30 of 71 had both. Location was correctly predicted in 42 of 57 dogs (74%) by ultrasound and in 37 of 44 dogs (84%) by CT. Both CT and ultrasound had high specificity for localising masses in all divisions. Sensitivity varied among divisions and was highest for left division masses. CLINICAL SIGNIFICANCE: The results suggest that either imaging modality is appropriate for presurgical planning. Prospective studies are recommended to help identify additional factors that may aid in determination of hepatic mass location.

Just-in-Time Compound Pooling Increases Primary Screening Capacity without Compromising Screening Quality.

Compound pooling, or multiplexing more than one compound per well during primary high-throughput screening (HTS), is a controversial approach with a long history of limited success. Many issues with this approach likely arise from long-term storage of library plates containing complex mixtures of compounds at high concentrations. Due to the historical difficulties with using multiplexed library plates, primary HTS often uses a one-compound-one-well approach. However, as compound collections grow, innovative strategies are required to increase the capacity of primary screening campaigns. Toward this goal, we have developed a novel compound pooling method that increases screening capacity without compromising data quality. This method circumvents issues related to the long-term storage of complex compound mixtures by using acoustic dispensing to enable "just-in-time" compound pooling directly in the assay well immediately prior to assay. Using this method, we can pool two compounds per well, effectively doubling the capacity of a primary screen. Here, we present data from pilot studies using just-in-time pooling, as well as data from a large >2-million-compound screen using this approach. These data suggest that, for many targets, this method can be used to vastly increase screening capacity without significant reduction in the ability to detect screening hits.

Temperature effects on the depuration of Vibrio parahaemolyticus and Vibrio vulnificus from the American oyster (Crassostrea virginica).

This study investigated temperature effects on depuration for reducing Vibrio parahaemolyticus and Vibrio vulnificus in American oyster (Crassostrea virginica). Raw oysters were inoculated with 5-strain cocktail of V. parahaemolyticus or V. vulnificus to levels of 10(4) to 10(5) MPN (most probable number)/g and depurated in artificial seawater (ASW) at 22, 15, 10, and 5 degrees C. Depuration of oysters at 22 degrees C had limited effects on reducing V. parahaemolyticus or V. vulnificus in the oysters. Populations of V. parahaemolyticus and V. vulnificus were reduced by 1.2 and 2.0 log MPN/g, respectively, after 48 h of depuration at 22 degrees C. Decreasing water temperature to 15 degrees C increased the efficacy of depuration in reducing V. parahaemolyticus and V. vulnificus in oysters. Reductions of V. parahaemolyticus and V. vulnificus in oysters increased to 2.1 and 2.9 log MPN/g, respectively, after 48 h of depuration at 15 degrees C. However, depurations at 10 and 5 degrees C were less effective than at 15 degrees C in reducing the Vibrio spp. in oysters. Extended depuration at 15 degrees C for 96 h increased reductions of V. parahaemolyticus and V. vulnificus in oysters to 2.6 and 3.3 log MPN/g, respectively.

Bromoperoxidase activity in microplantlet suspension cultures of the macrophytic red alga Ochtodes secundiramea.

Bromoperoxidase is an enzyme found in marine macroalgae that catalyzes the bromination of organic substrates. Photosynthetic microplantlet suspension cultures derived from the macrophytic red alga Ochtodes secundiramea were shown to possess bromoperoxidase. The optimum pH for O. secundiramea bromoperoxidase activity in cell-free extracts was 6.0, and the half-saturation constant for bromination of the exogeneous substrate monochlorodimedone (MCD) was 18 microM. O. secundiramea microplantlets were cultivated in a bubble-column photobioreactor at an incident light intensity of 38 microE x m(-2) x s(-1) (71% of light-saturated photosynthesis, 10:14 light:dark photoperiod), and the kinetics of cell growth and bromoperoxidase production were followed. At these conditions, the specific growth rate was 0.052 x day(-1). The lowest specific bromoperoxidase activity of 0.3 micromol MCD x g(-1) cell x min(-1) occurred during the midexponential phase of growth, and then increased steeply to 1.9 micromol MCD x g(-1) cell x min(-1) during the late stationary phase, suggesting that bromoperoxidase production was part of secondary metabolism. The estimated bromoperoxidase content in the cell mass at late stationary phase was 67 microg x g(-1) dry cell mass, demonstrating that bioreactor production of marine bromoperoxidase is feasible.

Library design and virtual screening using multiple 4-point pharmacophore fingerprints.

The use of multiple potential 4-point three-dimensional (3-D) pharmacophores for the design of combinatorial libraries and for virtual screening is discussed. These 3-D pharmacophoric fingerprints can be calculated from both ligands and complementary to a protein site, with a common frame of reference, and can be very rapidly searched to identify common and different 4-point pharmacophoric shapes in compounds and protein sites. A new extension to the method for structure-based design is reported that uses the shape of the target site as an additional constraint. This enables the docking process, for example in library design and virtual screening, to be quantified in terms of how many, and which, pharmacophoric hypotheses can be matched by a compound or a library of compounds.

Proximate factors mediating "contact" calls in adult female baboons (Papio cynocephalus ursinus) and their infants.

"Contact" calls are widespread in social mammals and birds, but the proximate factors that motivate call production and mediate their contact function remain poorly specified. Field study of chacma baboons (Papio cynocephalus ursinus) revealed that contact barks in adult females were motivated by separation both from the group at large and from their dependent infants. A variety of social and ecological factors affect the probability of separation from either one or both. Results of simultaneous observations and a playback experiment indicate that the contact function of calling between mothers and infants was mediated by occasional maternal retrieval rather than coordinated call exchange. Mothers recognized the contact barks of their own infants and often were strongly motivated to locate them. However, mothers did not produce contact barks in reply unless they themselves were at risk of becoming separated from the group.

Development of infant baboons' responses to graded bark variants.

We studied the development of infant baboons' (Papio cynocephalus ursinus) responses to conspecific 'barks' in a free-ranging population in the Okavango Delta, Botswana. These barks grade from tonal, harmonically rich calls into calls with a more noisy, harsh structure. Typically, tonal variants are given when the signaller is at risk of losing contact with the group or a particular individual ('contact barks'), whereas harsh variants are given in response to predators ('alarm barks'). We conducted focal observations and playback experiments in which we presented variants of barks recorded from resident adult females. By six months of age, infants reliably discriminated between typical alarm and contact barks and they responded more strongly to intermediate alarm calls than to typical contact barks. Infants of six months and older also recognized their mothers by voice. The ability to discriminate between different call variants developed with increasing age. At two and a half months of age, infants failed to respond at all, whereas at four months they responded irrespective of the call type that was presented. At six months, infants showed adult-like responses by responding strongly to alarm barks but ignoring contact barks. We concluded that infants gradually learn to attach the appropriate meaning to alarm and contact barks.

Aminoisoquinolines: design and synthesis of an orally active benzamidine isostere for the inhibition of factor XA.

The design, synthesis and SAR of sulfonamidopyrrolidinone fXa inhibitors incorporating a new benzamidine isostere, namely aminoisoquinolines, is described. These inhibitors have higher Caco-2 cell permeability than comparable benzamidines and attain higher levels of exposure upon oral dosing. The most potent member 14b (fXa Ki=6 nM) is selective against other serine proteases of interest (>600 fold).

Design and structure-activity relationships of potent and selective inhibitors of blood coagulation factor Xa.

The discovery of a series of non-peptide factor Xa (FXa) inhibitors incorporating 3-(S)-amino-2-pyrrolidinone as a central template is described. After identifying compound 4, improvements in in vitro potency involved modifications of the liphophilic group and optimizing the angle of presentation of the amidine group to the S1 pocket of FXa. These studies ultimately led to compound RPR120844, a potent inhibitor of FXa (K(i) = 7 nM) which shows selectivity for FXa over trypsin, thrombin, and several fibrinolytic serine proteinases. RPR120844 is an effective anticoagulant in both the rat model of FeCl(2)-induced carotid artery thrombosis and the rabbit model of jugular vein thrombus formation.

Sulfonamidopyrrolidinone factor Xa inhibitors: potency and selectivity enhancements via P-1 and P-4 optimization.

Sulfonamidopyrrolidinones were previously disclosed as a selective class of factor Xa (fXa) inhibitors, culminating in the identification of RPR120844 as a potent member with efficacy in vivo. Recognizing the usefulness of the central pyrrolidinone template for the presentation of ligands to the S-1 and S-4 subsites of fXa, studies to optimize the P-1 and P-4 groups were initiated. Sulfonamidopyrrolidinones containing 4-hydroxy- and 4-aminobenzamidines were discovered to be effective inhibitors of fXa. X-ray crystallographic experiments in trypsin and molecular modeling studies suggest that our inhibitors bind by insertion of the 4-hydroxybenzamidine moiety into the S-1 subsite of the fXa active site. Of the P-4 groups examined, the pyridylthienyl sulfonamides were found to confer excellent potency and selectivity especially in combination with 4-hydroxybenzamidine. Compound 20b (RPR130737) was shown to be a potent fXa inhibitor (K(i) = 2 nM) with selectivity against structurally related serine proteinases (>1000 times). Preliminary biological evaluation demonstrates the effectiveness of this inhibitor in common assays of thrombosis in vitro (e.g. activated partial thromboplastin time) and in vivo (e.g. rat FeCl(2)-induced carotid artery thrombosis model).

New 4-point pharmacophore method for molecular similarity and diversity applications: overview of the method and applications, including a novel approach to the design of combinatorial libraries containing privileged substructures.

A new 4-point pharmacophore method for molecular similarity and diversity that rapidly calculates all potential pharmacophores/pharmacophoric shapes for a molecule or a protein site is described. The method, an extension to the ChemDiverse/Chem-X software (Oxford Molecular, Oxford, England), has also been customized to enable a new internally referenced measure of pharmacophore diversity. The "privileged" substructure concept for the design of high-affinity ligands is presented, and an example of this new method is described for the design of combinatorial libraries for 7-transmembrane G-protein-coupled receptor targets, where "privileged" substructures are used as special features to internally reference the pharmacophoric shapes. Up to 7 features and 15 distance ranges are considered, giving up to 350 million potential 4-point 3D pharmacophores/molecule. The resultant pharmacophore "key" ("fingerprint") serves as a powerful measure for diversity or similarity, calculable for both a ligand and a protein site, and provides a consistent frame of reference for comparing molecules, sets of molecules, and protein sites. Explicit "on-the-fly" conformational sampling is performed for a molecule to enable the calculation of all geometries accessible for all combinations of four features (i.e., 4-point pharmacophores) at any desired sampling resolution. For a protein site, complementary site points to groups displayed in the site are generated and all combinations of four site points are considered. In this paper we report (i) the details of our customized implementation of the method and its modification to systematically measure 4-point pharmacophores relative to a "special" substructure of interest present in the molecules under study; (ii) comparisons of 3- and 4-point pharmacophore methods, highlighting the much increased resolution of the 4-point method; (iii) applications of the 4-point potential pharmacophore descriptors as a new measure of molecular similarity and diversity and for the design of focused/biased combinatorial libraries.

Ligand-receptor 3-D similarity studies using multiple 4-point pharmacophores.

A new method for 3-D similarity is presented based on the multiple potential 4-point 3-D pharmacophores expressed by ligands and complementary to receptors. These are calculated for ligands taking conformational flexibility into account, and for receptors through the use of complementary site-points. Through this common frame of reference both ligand-ligand and ligand-receptor similarity studies are possible. The application of the method to selectivity between different serine proteases (thrombin, factor Xa and trypsin) is discussed, and the need to use 4-point pharmacophores rather than 3-point pharmacophores is illustrated. A novel refinement to the potential pharmacophore method that uses a "special" feature to give a relative measure of similarity/diversity is also discussed.

Identification and initial structure-activity relationships of a novel class of nonpeptide inhibitors of blood coagulation factor Xa.

The discovery and some of the basic structure-activity relationships of a series of novel nonpeptide inhibitors of blood coagulation Factor Xa is described. These inhibitors are functionalized beta-alanines, exemplified by 2a. Docking experiments placing 2a in the active site of Factor Xa implied that the most expeditious route to enhancing in vitro potency was to modify the group occupying the S3 site of the enzyme. Increasing the hydrophobic contacts between the inhibitor and the enzyme in this region led to 8, which has served as the prototype for this series. In addition, an enantioselective synthesis of these substituted beta-alanines was also developed.

The acoustic features of vowel-like grunt calls in chacma baboons (Papio cyncephalus ursinus): implications for production processes and functions.

The acoustic features of 216 baboon grunts were investigated through analysis of field-recorded calls produced by identified females in known contexts. Analyses addressed two distinct questions: whether the acoustic features of these tonal sounds could be characterized using a source-filter approach and whether the acoustic features of grunts varied by individual caller and social context. Converging evidence indicated that grunts were produced through a combination of periodic laryngeal vibration and a stable vocal tract filter. Their acoustic properties closely resembled those of prototypical human vowel sounds. In general, variation in the acoustic features of the grunts was more strongly related to caller identity than to the social contexts of calling. However, two acoustic parameters, second formant frequency and overall spectral tilt, did vary consistently depending on whether the caller was interacting with an infant or participating in a group move. Nonetheless, in accordance with the general view that identity cueing is a compelling function in animal communication, it can be concluded that much of the observed variability in grunt acoustics is likely to be related to this aspect of signaling. Further, cues related to vocal tract filtering appear particularly likely to play an important role in identifying individual calling animals.

Pregnenolone sulfate antagonizes dizocilpine amnesia: role for allopregnanolone.

Pregnenolone (PREG) is metabolized in brain to progesterone (PROG), 5 alpha-dihydroprogesterone (5 alpha-DHP) and allopregnanolone (ALLO). Infusion of adrenalectomized/castrated rats with PREG sulfate prevented the cognition deficit elicited by the ionotropic glutamate receptor antagonists, dizocilpine and CPPene. Using a new gas chromatographic/mass-spectrometric method, we demonstrated that PREG sulfate infusion markedly increased the PREG, PROG, 5 alpha-DHP and ALLO brain content. The increase in 5 alpha-DHP and ALLO, but not PREG or PROG content and the antagonism of dizocilpine amnesia observed by injecting rats with PREG sulfate was reversed by inhibiting the conversion of PROG to 5 alpha-DHP with the 5 alpha-reductase blocker SKF 105111. We and others have shown that ALLO potently modulate GABAA receptor function whereas 5 alpha-DHP fails to induce rapid changes in neurotransmitter receptor function. Thus it is possible to suggest that the increase in the brain content of ALLO, rather than 5 alpha-DHP, mediates the effect of PREG sulfate on dizocilpine- or CPPene-induced cognition deficit.

The responses of female baboons (Papio cynocephalus ursinus) to anomalous social interactions: evidence for causal reasoning?

Baboons' (Papio cynocephalus ursinus) understanding of cause-effect relations in the context of social interactions was examined through use of a playback experiment. Under natural conditions, dominant female baboons often grunt to more subordinate mothers when interacting with their infants. Mothers occasionally respond to these grunts by uttering submissive fear barks. Subjects were played causally inconsistent call sequences in which a lower ranking female apparently grunted to a higher ranking female, and the higher ranking female apparently responded with fear barks. As a control, subjects heard a sequence made causally consistent by the inclusion of grunts from a 3rd female that was dominant to both of the others. Subjects responded significantly more strongly to the causally inconsistent sequences, suggesting that they recognized the factors that cause 1 individual to give submissive vocalizations to another.

Gas chromatographic-mass fragmentographic quantitation of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and its precursors in blood and brain of adrenalectomized and castrated rats.

Coupling high performance liquid chromatography with gas chromatography-mass fragmentography has made it possible to simultaneously measure subpicomolar concentrations of allopregnanolone and its precursors, pregnenolone, progesterone and 5 alpha-dihydroprogesterone (5 alpha-DHP) in various brain areas. Allopregnanolone was measured in the brain of adrenalectomized/castrated (ADX/CX) rats in nanomolar concentrations long after peripheral sources of allopregnanolone were removed. A partial decrease (approximately 30%) in the content of allopregnanolone was found in the brains of ADX/CX rats compared to sham-operated rats. Moreover, the content of allopregnanolone in brains of sham-operated as well as ADX/CX rats was nonuniformly distributed (olfactory bulb > striatum > cortex > hippocampus) and was one to two orders of magnitude higher than in plasma or liver. Infusion of pregnenolone sulfate in ADX/CX rats elicited a fourfold increase in 5 alpha-DHP and progesterone content and a seven- to eightfold increase in the content of allopregnanolone in brain but not in liver or plasma. Furthermore, the content of allopregnanolone in brain increased to the same extent in both sham-operated and ADX/CX rats following pregnenolone sulfate infusion. The 5 alpha-reductase inhibitor, (17 beta)17[[bis(1-methylethyl)amino]carbonyl] androstane-3,5-diene-3-carboxylic acid (SKF 105111), reduced the brain content of allopregnanolone and blocked the increased formation of allopregnanolone in brain following pregnenolone sulfate infusion. The results clearly demonstrate that the synthesis of allopregnanolone from 5 alpha-DHP and progesterone occurs in the brain and that a significant amount of allopregnanolone is synthesized locally in brain from its precursors. These experiments suggest that the brain, like adrenals and gonads, is a steroidogenic organ which produces allopregnanolone as one of its own most important physiologically relevant steroids.

Mitochondrial diazepam-binding inhibitor receptor complex agonists antagonize dizocilpine amnesia: putative role for allopregnanolone.

In rats trained to retain a passive avoidance response or to retrieve a learned task in the radial and water maze tests, a pretreatment with 2-hexyl-3-indoleacetamide (FGIN-1-27) (IC50 57 mumol/kg p.o.) or 4' chlorodiazepam (4'CD) (15 mumol/kg i.p.), two steroidogenic ligands at the mitochondria diazepam-binding inhibitor receptor complex (MDRC), antagonized the performance deficit elicited by dizocilpine (0.3 mumol/kg i.p.), a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. The 1-(2-chlorophenyl)-N-methyl-N-(-1-methyl-propyl)-3-isoquinoline carboxamide (PK-11195), an antagonist at MDRC in vivo, failed to modify the disruptive effect of dizocilpine in the passive avoidance response but reversed the FGIN-1-27- or 4' CD-induced antagonism of dizocilpine behavioral actions. Pretreatment with pregnenolone sulfate (48 mumol/kg i.p.), 3 alpha, 21-dihydroxy-5 alpha-pregnan-20-one (THDOC) (15 mumol/kg i.v.) and 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) (15 mumol/kg i.v.) also reduced the passive avoidance retention deficit elicited by dizocilpine. The (17-beta)-17-[[bis(1-methylethyl)amino[carbonyl]androstane-3,5-diene-3- carboxylic acid (SKF-105111), a 5 alpha-reductase inhibitor, blocked the antagonism of dizocilpine behavioral actions by pregnenolone sulfate or by FGIN-1-27 but not those caused by THDOC or allopregnanolone either in normal or adrenalectomized-castrated rats. Thus, it is inferred that the amnesic effect of dizocilpine is counteracted by FGIN-1-27, 4'CD and pregnenolone sulfate because of their ability to increase brain accumulation of allopregnanolone.