Development of a polyvalent assay system for lead identification

In an effort to identify new approaches to lead discovery a polyvalent assay was developed to allow identification of weak inhibitors. This approach involves the polyvalent display of a protein binder off a Tenta-gel scaffold and the generation of a polyvalent display of protein by biotinylation followed by complexation with fluorescently labeled streptavidin. Subsequent exposure of the streptavidin complexed protein to Tenta-gel beads with active protein binders results in fluorescent beads, which are easily viewed under a fluorescent microscope.     

Integrin alpha4beta1-dependent adhesion to ADAM 28 (MDC-L) requires an extended surface of the disintegrin domain

ADAMs (a disintegrin and metalloprotease) are a family of proteins that possess functional adhesive and proteolytic domains. ADAM 28 (MDC-L) is expressed by human lymphocytes and contains a disintegrin-like domain that serves as a ligand for the leukocyte integrin, alpha4beta1. To elucidate which residues comprise the alpha4beta1 binding site in the ADAM 28 disintegrin domain, a charge-to-alanine mutagenesis strategy was utilized. Each alanine substitution mutant was evaluated and compared to the native sequence for its ability to support cell adhesion of the T-lymphoma cell line, Jurkat. This approach identified ADAM 28 residues Lys(437), Lys(442), Lys(455), Lys(459), Lys(460), Lys(469), and Glu(476) as being essential for alpha4beta1-dependent cell adhesion. The epitope for a function-blocking monoclonal antibody, Dis 1-1, was localized to the N-terminal end of the ADAM 28 disintegrin domain using these same charge-to-alanine mutants. Three distinct molecular models based upon the known structures of snake venom disintegrins suggested that residues contributing to alpha4beta1 recognition are aligned on one face of the domain. This study demonstrates that residues located outside of the disintegrin loop participate in integrin recognition of mammalian disintegrins.     

Effects of indomethacin, luteinizing hormone (LH), prostaglandin E2 (PGE2), trilostane, mifepristone, ethamoxytriphetol (MER-25) on secretion of prostaglandin E (PGE), prostaglandin F2alpha (PGF2alpha) and progesterone by ovine corpora lutea of pregnancy or the estrous cycle

Two experiments were conducted to determine the luteotropin of pregnancy in sheep and to examine autocrine and paracrine roles of progesterone and estradiol-17 beta on progesterone secretion by the ovine corpus luteum (CL). Secretion of progesterone per unit mass by day-8 or day-11 CL of the estrous cycle was similar to day-90 CL of pregnancy (P > or = 0.05). In experiment 1, secretion of progesterone in vitro by slices of CL from ewes on day-8 of the estrous cycle was increased (P < or = 0.05) by LH or PGE2. Secretion of progesterone in vitro by CL slices from day-90 pregnant ewes was not affected by LH (P > or = 0.05) while PGE2 increased (P < or = 0.05) secretion of progesterone. Day 8 ovine CL of the estrous cycle did not secrete (P > or = 0.05) detectable quantities of PGF2alpha or PGE while day-90 ovine CL of pregnancy secreted PGE (P < or = 0.05) but not PGF2alpha. Secretion of progesterone and PGE in vitro by day-90 CL of pregnancy was decreased (P < or = 0.05) by indomethacin. The addition of PGE2, but not LH, in combination with indomethacin overcame the decreases in progesterone by indomethacin (P < or = 0.05). In experiment 2, secretion of progesterone in vitro by day-11 CL of the estrous cycle was increased at 4-h (P < or = 0.05) in the absence of treatments. Both day-11 CL of the estrous cycle and day-90 CL of pregnancy secreted detectable quantities of PGE and PGF2alpha (P < or = 0.05). In experiment 1, PGF2alpha secretion by day-8 CL of the estrous cycle and day-90 ovine CL of pregnancy was undetectable, but was detectable in experiment 2 by day-90 CL. Day 90 ovine CL of pregnancy also secreted more PGE than day-11 CL of the estrous cycle (P < or = 0.05), whereas day-8 CL of the estrous cycle did not secrete detectable quantities of PGE (P > or = 0.05). Trilostane, mifepristone, or MER-25 did not affect secretion of progesterone, PGE, or PGF2alpha by day- 11 CL of the estrous cycle or day-90 CL of pregnancy (P > or = 0.05). It is concluded that PGE2, not LH, is the luteotropin at day-90 of pregnancy in sheep and that progesterone does not modify the response to luteotropins. Thus, we found no evidence for an autocrine or paracrine role for progesterone or estradiol-17 36 on luteal secretion of progesterone, PGE or PGF2alpha.     

Short-term treatment with a controlled internal drug releasing (CIDR) device and FSH to induce fertile estrus and increase prolificacy in anestrous ewes

The objectives were to evaluate, in anestrous ewes, the effectiveness of a CIDR-G device (0.3 g progesterone) administered for 5 d to induce estrus; and FSH (Folltropin; 55 mg NIH-FSH-P1 equivalent) in saline:propylene glycol (1:4) 24 h before insert removal (Day 0), to increase ovulation rate and prolificacy. Ewes of mixed breeding were assigned at random to 3 treatments: control (C; n = 125), 5 d progesterone (P5; n = 257) and 5 d progesterone plus FSH (P5F; n = 271). Intact rams were joined at insert removal and ewes were observed every 24 h for 3 d. On Day 14, the ovulation rates of all ewes detected in estrus in the treated groups were determined using transrectal ultrasonography. Rams were removed on Day 26 to 31. Ewes were examined for pregnancy then, and again 20 to 25 d later to detect ewes that conceived to the second service period. Percentage of ewes marked by rams was higher in progesterone-treated (77%) than in C (20%; P < 0.01), but did not differ between P5 and P5F. The ovulation rate (1.95+/-0.04) did not differ due to FSH. Conception (68%) and pregnancy (52%) rates were higher in progesterone-treated (P < 0.01) than in C (0%) ewes. Estrous response varied quadratically with time after ram introduction, and the conception rate varied quadratically with the time of observation of onset of estrus. Over two service periods more progesterone-treated than C ewes lambed (65 vs 45%; P < 0.01). Lambs born per ewe exposed (0.7+/-0.1, 1.0+/-0.1, and 1.1+/-0.1 for C, P5 and P5F, respectively) was increased by progesterone (P < 0.05). Litter size to the first service period (1.59+/-0.04) and overall (1.54+/-0.03) did not differ among treatment groups. FSH-treated ewes tended to have more lambs (1.67+/-0.1) than did ewes receiving progesterone alone (1.5+/-0.1; P = 0.06) and than did ewes lambing to the second service period (1.5+/-0.1; P = 0.06). In summary, a 5-d progesterone pre-treatment of anestrous ewes induced estrous cycles and increased the pregnancy rates. A single injection of FSH only tended to increase litter size.     

Anti-HBV specific beta-L-2'-deoxynucleosides

A unique series of simple unnatural L-nucleosides that specifically inhibit hepatitis B virus (HBV) replication has been discovered. These molecules have in common a hydroxyl group in the 3'-position (3'-OH) of the beta-L-2'-deoxyribose sugar that confers antiviral activity specifically against hepadnaviruses. Replacement of the 3'-OH broadens activity to other viruses. Substitution in the base decreases antiviral potency and selectivity. Human DNA polymerases and mitochondrial function are not effected. Plasma viremia is reduced up to 8 logs in a woodchuck model of chronic HBV infection. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.     

Monoval-LdC: efficient prodrug of 2'-deoxy-beta-L-cytidine (L-dC), a potent and selective anti-HBV agent

In order to improve the oral bioavailability of LdC, valinyl esters were prepared as prodrugs. We report here the syntheses of the 3'-mono-, 5'-mono, and 3',5'-di-O-valinyl esters of LdC. The comparison of their ease of synthesis, their physicochemical properties, as well as their pharmacokinetic parameters in cynomologus monkeys has revealed 3'-mono-O-valinyl derivative as the most promising of the studied prodrugs. This compound is being developed as a new anti-HBV agent.     

Regulation of taurine transporter expression by NO in cultured human retinal pigment epithelial cells

Taurine is activelytransported at the retinal pigment epithelial (RPE) apical membrane inan Na⁺- and Cl-dependent manner. Diabetes mayalter the function of the taurine transporter. Because nitric oxide(NO) is a molecule implicated in the pathogenesis of diabetes, we askedwhether NO would alter the activity of the taurine transporter incultured ARPE-19 cells. The activity of the transporter was stimulatedin the presence of the NO donor 3-morpholinosydnonimine. Thestimulatory effects of 3-morpholinosydnonimine were not observed duringthe initial 16-h treatment; however, stimulation of taurine uptake waselevated dramatically above control values with 20- and 24-htreatments. Kinetic analysis revealed that the stimulation wasassociated with an increase in the maximal velocity of the transporterwith no significant change in the substrate affinity. The NO-induced increase in taurine uptake was inhibited by actinomycin D and cycloheximide. RT-PCR analysis and nuclear run-on assays provided evidence for upregulation of the transporter gene. This study providesthe first evidence of an increase in taurine transporter geneexpression in human RPE cells cultured under conditions of elevatedlevels of NO.

     

Outcomes of breeding soundness evaluation of 2898 yearling bulls subjected to different classification systems

This study is a retrospective analysis comparing data on yearling bull breeding soundness evaluation (BSE) subjected to 3 different classification systems: the Society for Theriogenology (SFT) 1983 and 1993 systems, and the Western Canadian Association of Bovine Practitioners (WC) 1993 system. Data were collected at 5 performance bull-test stations located in South Carolina and Tennessee from 1986 through 1996. Yearling bulls (n=2898) that were 10 to 20 mo of age were used in the analysis. To simplify analysis, bulls were determined to be either satisfactory or unsatisfactory potential breeders (including those classified as questionable, deferred or unsatisfactory). Data were analyzed 1) within location where a location was treated as an individual experiment, 2) with breeds and locations collapsed, and 3) within age-group where each age-group was treated as an individual experiment. An ANOVA was performed using GLM procedures of SAS, and this model was used to generate least square means for the proportion of bulls classified as satisfactory and the 5 possible unsatisfactory outcomes due to inadequacies in scrotal circumference, spermatozoal morphology, spermatozoal motility, a combination of inadequate spermatozoal morphology and motility, or physical abnormalities. Inadequate scrotal circumference or physical abnormality did not affect differences for BSE outcomes among systems. Using the SFT93 system, bulls failed at a higher rate due to inadequate spermatozoal morphology (P < 0.05) than when subjected to the SFT83 system. In the WC93 system, a higher percentage of bulls failed due to inadequate spermatozoal motility and to a combination of inadequate spermatozoal morphology and motility than in the other 2 systems (P < 0.05). The proportion of bulls in this data failing under the WC93 system appears to be the result of overestimation.