Resource limitation and responses to rivals in males of the fruit fly Drosophila melanogaster.

Diet has a profound direct and indirect effect on reproductive success in both sexes. Variation in diet quality and quantity can significantly alter the capacity of females to lay eggs and of males to deliver courtship. Here, we tested the effect of dietary resource limitation on the ability of male Drosophila melanogaster to respond adaptively to rivals by extending their mating duration. Previous work carried out under ad libitum diet conditions showed that males exposed to rivals prior to mating significantly extend mating duration, transfer more ejaculate proteins and achieve higher reproductive success. Such adaptive responses are predicted to occur because male ejaculate production may be limited. Hence, ejaculate resources require allocation across different reproductive bouts, to balance current vs. future reproductive success. However, when males suffer dietary limitation, and potentially have fewer reproductive resources to apportion, we expect adaptive allocation of responses to rivals to be minimized. We tested this prediction and found that males held on agar-only diets for 5-7 days lost the ability to extend mating following exposure to rivals. Interestingly, extended mating was retained in males held on low yeast/sugar: no sugar/yeast diet treatments, but was mostly lost when males were maintained on 'imbalanced' diets in which there was high yeast: no sugar and vice versa. Overall, the results show that males exhibit adaptive responses to rivals according to the degree of dietary resource limitation and to the ratio of individual diet components.

Design of reflection retarders by use of nonnegative film-substrate systems.

A reflection-type film-substrate retarder is an optical device that changes the relative phase but not the relative amplitude of light upon reflection from a film-substrate system. While there are several such device designs based on the common negative film-substrate system, very little has been done with the other two categories of systems, zero and positive. The system category is determine by the relationship between the refractive indices of the ambient N0, film N1 and substrate N2. If N1 < square root of N0N2, the system is negative; if N1 = square root of N0N2, the system is zero; and if N1 > square root of N0N2, the system is positive. The design procedure and characteristics of zero-system reflection retarders are discussed. The polarization and ellipsometric properties of the positive system preclude the existence of a reflection retarder. First, a brief characterization of the zero and positive systems by means of constant-angle-of-incidence contours and constant-thickness contours of the ellipsometric function is presented and discussed. Then an algorithm outlining the design procedures is presented, and the characteristics of the obtained designs are optimized, analyzed, and discussed. The exact retarder is valid for a single wavelength at a set angle of incidence. The design tolerance to changes in the design parameters is analyzed and discussed. In general, N1 < or = square root of N1N2 is the condition to be satisfied to realize reflection-type retarders with film-substrate systems.

3-D pharmacophores in drug discovery.

In this chapter we review the use of 3-D pharmacophores in drug discovery. Recent advances are highlighted, including the application of pharmacophore descriptors generated both from ligands and protein binding sites. The application of 3-D pharmacophore fingerprints as molecular descriptors for similarity and diversity applications such as virtual screening, library design and QSAR is discussed. In addition, we highlight the quantification of structure-based diversity using site-derived fingerprints, and review virtual screening methods using both single refined hypotheses and the fingerprints of multiple potential hypotheses. Further, we discuss methods that take protein flexibility and molecular shape-into account. Each of the above techniques are reviewed with particular reference to the recent advances, advantages and challenges of each methodology.

The design of combinatorial libraries using properties and 3D pharmacophore fingerprints.

Molecular diversity and similarity methods relevant to drug-receptor interactions are key for the design of combinatorial libraries for lead discovery and optimization. BCUT chemistry-space values for ligands have been used for many diversity-related applications and incorporate receptor-relevant properties such as hydrogen bonding and polarizability. Three-dimensional (3D) multiple-point pharmacophore descriptors (fingerprints) quantify diversity (or similarity) in terms of combinations of three or four functional groups associated with non-covalent ligand-receptor binding. BCUTs and pharmacophore fingerprints have been effectively utilized to design diversity libraries, and also show promise for focused library design.

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.

Library design using BCUT chemistry-space descriptors and multiple four-point pharmacophore fingerprints: simultaneous optimization and structure-based diversity.

New applications of fingerprints of multiple potential 4-point three-dimensional (3D) pharmacophores in combinatorial library design and virtual screening are presented. Preliminary results demonstrating the feasibility of a simulated annealing process for combinatorial reagent selection that concurrently optimizes product diversity in BCUT chemistry space and in terms of unique 4-point pharmacophores are discussed, and the advantage of using a customized chemistry-space derived for the library design is demonstrated. In addition, an extension to the multiple pharmacophore method for structure-based design that uses the shape of the target site as an additional constraint is presented. This development enables the docking process to be quantified in terms of the number and identities of the pharmacophoric hypotheses that can be matched by a compound or a library of compounds. The design of an example combinatorial library based on the Ugi condensation reaction and a serine protease active site is described.

High-throughput and virtual screening: core lead discovery technologies move towards integration.

In addition to high-throughput screening (HTS), the main lead discovery technology employed by most pharmaceutical companies today is virtual screening (VS). Although the two techniques have somewhat different philosophical origins, they contain many synergies that can potentially enhance the lead discovery process. Here, we describe many of the latest developments in VS technology with particular emphasis on their potential impact on HTS in, for example, focussed screening and data mining. In addition, we highlight key issues that need to be addressed before the potential of such efforts can be fully realized.

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.

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.

Diversity assessment.

Several themes have been highlighted recently in both conferences and publications: the availability of product-focused and pharmacophore-based methods for the analysis and design of combinatorial libraries; the power of cell-based methods for molecular similarity, diversity and library design applications; methods for 'rational' diverse subset selection (with applicability to library design); the need for specialized optimization programs for the design of combinatorial libraries that maximize the use of common reagents; and the concept of 'drug-likeness' and its importance in the design of combinatorial libraries.

Similarity measures for rational set selection and analysis of combinatorial libraries: the Diverse Property-Derived (DPD) approach.

The generation of new chemical leads for biological targets is a very challenging task for researchers in the pharmaceutical industry. The design of representative screening sets and combinatorial libraries is central to achieving this objective. In this paper, we describe a novel molecular descriptor, the Diverse Property-Derived (DPD) code, that contains information about key molecular and physicochemical properties of a molecule. The utility of this descriptor is explored through its application for the selection of a maximally diverse representative screening set, through the selection of secondary screening sets to obtain more information concerning the structure-activity relationships (SAR) of a particular target receptor, and through the profiling of combinatorial libraries. The usefulness of physicochemical/molecular property descriptors, such as the DPD code, is discussed critically.

Diagnosis of X-linked adrenal hypoplasia congenita by mutation analysis of the DAX1 gene.

OBJECTIVE: To develop a rapid diagnostic approach to individuals with the X-linked cytomegalic form of adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH) due to mutations in DAX1, a new member of the nuclear hormone receptor gene superfamily. DESIGN: Molecular genetic diagnostic investigations of individuals with AHC and their relatives included polymerase chain reaction amplification of DAX1 for identification of intragenic mutations and fluorescence in situ hybridization with a cosmid containing the DAX1 gene for evaluation of larger deletions. PARTICIPANTS: Families that had males affected with AHC were evaluated for mutations involving the DAX1 gene. RESULTS: DAX1 mutations were identified in four families that had males affected with AHC. Two apparently independent pedigrees had an identical frame-shift mutation due to a single base pair deletion, and a third had a larger deletion involving the entire DAX1 locus. The fourth family was evaluated by fluorescence in situ hybridization for prenatal diagnosis, and both the DAX1 locus and the contiguous glycerol kinase region were deleted. CONCLUSIONS: Molecular genetic and molecular cytogenetic techniques represent rapid and complementary approaches to the diagnosis of mutations in the DAX1 gene responsible for AHC and the associated HH. Specific diagnosis of the cause of adrenal insufficiency in these boys permits anticipatory management of the HH and prenatal counseling for parents of the affected child and other members of their families.

Influence of curricular changes in health promotion and disease prevention on residents' behaviors over a five-year period.

BACKGROUND: Residents' compliance with guidelines for health promotion and disease prevention (HPDP) often fall short of the ideal. METHOD: After a year in which faculty developed clinical practice HPDP guidelines, an intervention consisting of curricular changes and chart modifications was planned and pilot-tested in 1985-86 at an ambulatory care clinic of the Family Practice Residency Program, University of Louisville School of Medicine. Approximately 24 residents per year (eight at each level of training) worked in the clinic throughout the study years. The HPDP curriculum required residents to attend 48 one-hour conferences conducted over a three-year period. Four retrospective chart audits were used to measure residents' compliance with the HPDP guidelines during (1) the year before the pilot test (104 charts), (2) the pilot year (113 charts), (3) the year after the pilot test--the first year of full implementation (100 charts), and (4) five years after the pilot test (100 charts). Data from the chart audits were analyzed by using a permutation test for decreasing trend. RESULTS: Although there was an increase in the residents' compliance with the HPDP guidelines during the pilot year, the residents' HPDP activities tended to return to baseline levels in the following years, despite the ongoing curriculum. CONCLUSION: The authors conclude that because the frequency of the residents' HPDP activities tended to return to baseline levels, clinical education alone is not enough to sustain residents' compliance with HPDP guidelines.

The results of 20 years of family practice residency training at the University of Louisville.

In response to shortages of generalist physicians, especially in rural areas, family practice residency programs were established at both of Kentucky's medical schools. In 1972 a family practice residency program became operational at the University of Louisville. A study was conducted to determine the effectiveness of this program. We were especially interested in those factors that were associated with a physician's choice to practice in rural or urban medically underserved areas. The records of the 100 program graduates were reviewed to determine the physician's gender, ethnic background, hometown, and current practice location. A questionnaire was mailed to program graduates to obtain additional information about factors that influence physician practice location. For the purpose of this study, "rural" was defined as a community of less than 25,000. Sixty-eight of the program's 100 graduates currently practice in Kentucky. Forty-nine graduates serve patients in rural or medically underserved urban areas, and of those, 31 are located in Kentucky. Whites and those from rural hometowns were more likely to practice in rural communities than minorities or those from urban hometowns. However, minorities were more likely than whites to practice in medically underserved urban areas. No association was found between gender and practice location. Initial interest in rural practice and participation in financial aid programs requiring service in a rural area were associated with rural practice location, but participation in rural rotations at the predoctoral or residency training level were not. We conclude that the family practice training program at the University of Louisville has been successful in training generalist physicians to serve rural and medically underserved urban populations.(ABSTRACT TRUNCATED AT 250 WORDS)

Syntheses and biological activities of potent potassium channel openers derived from (+/-)-2-oxo-1-pyridin-3-yl-cyclohexanecarbothioic acid methylamide: new potassium channel openers.

The syntheses and biological activities of (+/-)-2-(cyanomethylene)-1-pyridin-3-ylcyclohexanecarbothioic++ + acid methylamide (6) and trans-(+/-)-2-(cyanomethyl)-1-pyridin-3-ylcyclohexanecarbothioic acid methylamide (14) derived from (+/-)-2-oxo-1-pyridin-3-ylcyclohexanecarbothioic acid methylamide (4) are reported. Compounds were tested for antagonism of potassium-induced contraction of de-endothelialized rat aorta. The effects of modification of 6 and 14 on in vitro K(+)-channel opening activity are presented. These new series of potassium channel openers so derived are best exemplified by (+/-)-2-[2-(phenylsulfanyl)ethylidene]-1-pyridin-3-ylcyclohexan ecarbothioic acid methylamide (13d, RP 66266) and trans-(+/-)-2-[2-[(phenylsulfonyl)amino]ethyl]-1-pyridin-3- ylcyclohexanecarbothioic acid methylamide (25a, RP 66784), which have IC90 values of 3 and 0.3 nM, respectively. The potency of the most active compounds indicates a possible interaction at an extra binding site. The compounds described herein are potential antihypertensive and antianginal agents.

Leydig cell neoplasia in a patient with Reifenstein syndrome.

We present the rare coincidence of a Leydig cell tumor in both testicles of a patient with partial androgen insensitivity syndrome (PAIS). The clinical picture with perineoscrotal hypospadia, micropenis, gynecomastia and delayed puberty and the serum hormone levels with elevated concentrations of testosterone, luteinising hormone (LH) and follicle-stimulating hormone were entirely consistent with PAIS. Ultimately, the diagnosis was confirmed by determination of genital skin fibroblast androgen receptor binding capacity for 5 beta-dihydrotestosterone, which demonstrated a qualitatively abnormal androgen receptor. At 44 years of age, a nodule in the left testis led to orchidectomy. At that time, the right testis was inconspicuous sonographically. But 3 years later the right testis developed nodules and was removed. Review of testicular histology revealed the presence of Leydig cell hyperplasia (LCH), multifocal nodular hyperplasia and Leydig cell neoplasia (LCN) in both testes. Many micronodules of Leydig cells in transition from hyperplasia to neoplasia were also identified. The simultaneous development of histologically identical nodes of LCN independently from each other and a different sites of both tests indicates the presence of a tumorigenic factor acting on the Leydig cells. Furthermore, the observation of multiple foci of cells in all stages of transition from hyperplasia to neoplasia demonstrates the persistent process of transformation. We speculate, that in this patient the grossly elevated LH levels present over 30 years have enhanced, if not provoked, the formation of LCN. In addition, the defective androgen receptor might have prevented suppressive effects of androgens on the Leydig cells.

Synthesis and biological activity of trans(+-)-N-methyl-2-(3-pyridyl)-2-tetrahydrothiopyrancarbothioamide 1-oxide (RP 49356) and analogues: a new class of potassium channel opener.

The synthesis and biological activity of trans-(+-)-N-methyl-2-(3-pyridyl)-2-tetrahydrothiopyrancarbothioamid+ ++ e 1-oxide (8a, RP 49356) and analogues is reported. These compounds constitute a new structural class of K(+)-channel opener. The effects of changes in pyridyl group, thioamide, and thiane ring on in vitro K(+)-channel opening reactivity are discussed. A 3-pyridyl or 3-quinolyl group, a small N-alkyl thioamide function, and a thiane oxide ring, in which the sulfoxide is in a trans relationship to the thioamide, are preferred for activity. Selected compounds were tested intravenously in the normotensive anaesthetized rat for hypotensive effects, and the activities reflect their in vitro K(+)-channel opening activity. This led to further evaluation of compound 8a and the selection of the (-)-enantiomer 8b (RP 52891) for development as an antihypertensive and antianginal agent.