Identification of crucial hydrogen-bonding residues for the interaction of herpes simplex virus DNA polymerase subunits via peptide display, mutational, and calorimetric approaches

The catalytic subunit, Pol, of herpes simplex virus DNA polymerase interacts via its extreme C terminus with the processivity subunit, UL42. This interaction is critical for viral replication and thus a potential target for antiviral drug action. To investigate the Pol-binding region on UL42, we engineered UL42 mutations but also used random peptide display to identify artificial ligands of the Pol C terminus. The latter approach selected ligands with homology to residues 171 to 176 of UL42. Substitution of glutamine 171 with alanine greatly impaired binding to Pol and stimulation of long-chain DNA synthesis by Pol, identifying this residue as crucial for subunit interactions. To study these interactions quantitatively, we used isothermal titration calorimetry and wild-type and mutant forms of Pol-derived peptides and UL42. Each of three peptides corresponding to either the last 36, 27, or 18 residues of Pol bound specifically to UL42 in a 1:1 complex with a dissociation constant of 1 to 2 microM. Thus, the last 18 residues suffice for most of the binding energy, which was due mainly to a change in enthalpy. Substitutions at positions corresponding to Pol residue 1228 or 1229 or at UL42 residue 171 abolished or greatly reduced binding. These residues participate in hydrogen bonds observed in the crystal structure of the C terminus of Pol bound to UL42. Thus, interruption of these few bonds is sufficient to disrupt the interaction, suggesting that small molecules targeting the relevant side chains could interfere with Pol-UL42 binding.     

Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO-3 by a Cl-offdependent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO-3 secretion and caused the secretion of Cl- by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO-3 secretion to Cl- secretion because the uptake of HCO-3 across the basolateral membrane is mediated by a 4,4 '-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive Na+:HCO-3 cotransporter. Since the stoichiometry reported for Na+:HCO-3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO-3 entry and favor the secretion of Cl-. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO-3 and Cl- secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO-3 and a Cl- channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO-3 secretion in the human airways warrants greater attention.     

Potential of real-time measurement of GFP-fusion proteins

Building on the basic design concepts of Randers-Eichhorn [Biotechnol. Bioeng. 55 (1997) 921], an on-line, real-time robust, steam sterilisable optical sensor for monitoring green fluorescent protein (GFP) has been developed. A general cloning vector for fusion expression proteins was constructed, allowing expression of both GFP and the target protein as a fusion. Cultivations were carried out at the 20l scale with the signal from the sensor being relayed directly to the control system of the bioreactors. The production of GFP was then measured on-line, the signal was interfaced directly with other controlling parameters, thereby allowing the microbial process to be controlled directly based on recombinant protein expression. A positive expression correlation between on-line and off-line data was obtained. Protein accretion measured off-line was quantified using both LC-MS and plate reader assays. The potential of such a sensor for many aspects of process development is considerable and we have developed a working system which allows the optimisation of production conditions, for example, linking pH control directly to the fusion protein. Results are also presented that illustrate GFP does not alter the cultivation characteristics of the target protein when compared to the native construct. Whether GFP expressed as a fusion influences the solubility of the target protein is also discussed.     

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.     

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.