An in vitro investigation of the cardiovascular effects of the 5-HT(4) receptor selective agonists, velusetrag and TD-8954.

The 5-HT(4) receptor agonists, and gastrointestinal (GI) prokinetic agents, cisapride and tegaserod, lack selectivity for the 5-HT(4) receptor. Cisapride is a potent human ether-à-go-go-related gene (hERG) potassium channel inhibitor while cisapride and tegaserod have significant affinity for 5-HT(1) and 5-HT(2) receptor subtypes. Marketing of both compounds was discontinued due to cardiovascular concerns (cardiac arrhythmias with cisapride and ischemic events with tegaserod). The reported association of tegaserod with ischemia has been postulated to involve coronary artery constriction or augmentation of platelet aggregation. This in vitro study investigated the effects of two of the new generation of highly selective 5-HT(4) receptor agonists, velusetrag and TD-8954, on canine, porcine and human coronary artery tone, human platelet aggregation and hERG potassium channel conductance. No significant off-target actions of velusetrag or TD-8954 were identified in these, and prior, studies. While cisapride inhibited potently the hERG channel currents, tegaserod failed to affect platelet aggregation, and had only a small contractile effect on the canine coronary artery at high concentrations. Tegaserod inhibited the 5-HT-induced contractile response in the porcine coronary artery. New generation 5-HT(4) receptor agonists hold promise for the treatment of patients suffering from GI motility disorders with a reduced cardiovascular risk.

Effects of telavancin on coagulation test results.

BACKGROUND: The lipoglycopeptide antibiotic, telavancin, may interfere with some laboratory coagulation tests including prothrombin time (PT) and activated partial thromboplastin time (aPTT). OBJECTIVE: To evaluate the effects of telavancin on PT and aPTT assays in common use. METHODS: Pooled normal human plasma was spiked with telavancin 10, 20, 100 or 200 µg/ml (equivalent to trough, 2 × trough, peak and 2 × peak clinical plasma concentrations, respectively) or diluent control (0.9% sodium chloride). Samples were analysed using 16 PT reagents and seven aPTT reagents. RESULTS: Telavancin 200 µg/ml (corresponding to 2 × peak clinical plasma concentration), produced significant PT prolongation (> 9% difference vs. diluent control) with all the 16 PT reagents (range 12% to > 600%). At lower telavancin concentrations, PT prolongation was dose-dependent and varied among reagents, but appeared greatest with preparations containing recombinant tissue factor. With telavancin 10 µg/ml (equivalent to trough), PT prolongation was 10% with HemosIL(®) PT-Fibrinogen Recombinant, while ranging from 5% to -1% with all other reagents. Significant (> 34% difference vs. baseline) and dose-dependent aPTT prolongation was observed with all the seven reagents in samples spiked with telavancin 100 or 200 µg/ml (range 65-142% at 200 µg/ml). aPTT reagents containing a silica activator appeared to be more sensitive to telavancin interference. Telavancin 10 µg/ml was not associated with increased aPTT with any of the reagents tested. CONCLUSIONS: Telavancin has the potential to prolong both PT and aPTT in vitro. It is recommended that samples for PT or aPTT be obtained just prior to a telavancin dose (trough).

Pharmacokinetics, serum inhibitory and bactericidal activity, and safety of telavancin in healthy subjects.

The pharmacokinetics, tolerability, and serum inhibitory and bactericidal titers of telavancin, a new rapidly bactericidal lipoglycopeptide with multiple mechanisms of action against gram-positive pathogens, were assessed in a two-part, randomized, double-blind, placebo-controlled, ascending-dose study with 54 healthy men. In part 1, single ascending intravenous doses of 0.25 to 15 mg/kg of body weight were studied. In part 2, multiple ascending doses (30-min infusions of 7.5 to 15 mg/kg/day) were studied over 7 days. Following the administration of multiple doses, steady state was achieved by days 3 to 4. At day 7 after the administration of telavancin at 7.5, 12.5, and 15 mg/kg/day, peak concentrations in plasma were 96.7, 151.3, and 202.5 microg/ml, respectively, and steady-state area-under-the-curve values were 700, 1,033, and 1,165 microg x h/ml, respectively. The elimination half-life ranged from 6.9 to 9.1 h following the administration of doses > or =5 mg/kg. Most adverse events were mild in severity. At 24 h postinfusion, serum from subjects given telavancin demonstrated potent bactericidal activity against methicillin-resistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae strains. The results suggest that telavancin may be an effective once-daily therapy for serious bacterial infections caused by these pathogens.

Protein affinity map of chemical space.

Affinity fingerprinting is a quantitative method for mapping chemical space based on binding preferences of compounds for a reference panel of proteins. An effective reference panel of <20 proteins can be empirically selected which shows differential interaction with nearly all compounds. By using this map to iteratively sample the chemical space, identification of active ligands from a library of 30,000 candidate compounds has been accomplished for a wide spectrum of specific protein targets. In each case, <200 compounds were directly assayed against the target. Further, analysis of the fingerprint database suggests a strategy for effective selection of affinity chromatography ligands and scaffolds for combinatorial chemistry. With such a system, the large numbers of potential therapeutic targets emerging from genome research can be categorized according to ligand binding properties, complementing sequence based classification.

Ligand-based protein alignment and isozyme specificity of glutathione S-transferase inhibitors.

Glutathione S-transferases (GST, E.C.2.5.1.18) comprise a family of detoxification enzymes. Elevated levels of specific GST isozymes in tumor cells are thought responsible for resistance to chemotherapeutics, which renders selective GST inhibitors potentially useful pharmaceutical agents. We discuss the development of a structure activity model that rationalizes the isozyme selectivity observed in a series of 12 glutathione (GSH) analogues. Enzymatic activity data was determined for human P1-1, A1-1, and M2-2 isozymes, and these data were then considered in light of structural features of these three GST proteins. A survey of all GST structures in the PDB revealed that GSH binds to these proteins in a single "bioactive" conformation. To focus on differences between binding sites, we exploited our finding of a common GSH conformation and aligned the GST x-ray structures using bound ligands rather than the backbones of the different proteins. Once aligned, binding site lipophilicity and electrostatic potentials were computed, visualized, and compared. Docking and energy minimization exercises provided additional refinements to a model of selectivity developed initially by visual analysis. Our results suggest that binding site shape and lipophilic character are key determinants of GST isozyme selectivity for close GSH analogues.

The effects of HIV counseling and testing on risk-related practices and help-seeking behavior.

In an earlier review of the behavioral effects of HIV counseling and testing (HIV CT), Higgins and colleagues (1991) found that the evidence regarding the ability of HIV CT to influence HIV-risk related practices was largely inconclusive. This article reviews 35 domestic and international studies published since that time to reassess the scientific data regarding the ability of HIV CT to motivate changes in risk-related practices and to promote help-seeking behavior. The studies identified for this review were grouped into four categories according to subject population: (1) men who have sex with men, (2) injection and other drug users, (3) women and heterosexual couples, and (4) mixed samples recruited from sexually transmitted disease (STD) clinics and other settings. Findings from the studies reviewed were generally mixed--many provided at least some evidence supporting the ability of HIV CT to motivate risk-reducing and help-seeking behavior, but others did not. The pattern of results varied substantially across, and within, study populations and were often limited by considerable methodological weaknesses.

Glubodies: randomized libraries of glutathione transferase enzymes.

BACKGROUND: The immunoglobulin framework has been mutagenized to engineer recombinant libraries of proteins as potential diagnostics and novel catalysts, although the often shallow binding cleft may limit the utility of this framework for binding diverse small organic molecules. By contrast, the glutathione S-transferase (GST) family of enzymes contains a deep binding cleft, which has evolved to accommodate a broad range of hydrophobic xenobiotics. We set out to determine whether GST molecules with novel ligand-binding characteristics could be produced by random mutagenesis of segments of the binding cleft. RESULTS: We have identified two ligand-recognition segments (LRSs) in human GST P1, which are near the active site in the folded protein, but have characteristics indicating that the integrity of their sequence is not essential for the overall structure or activity of the protein. Libraries of GST P1-derived proteins were produced by substituting randomized sequences for an LRS or inserting random sequences into an LRS. The recombinant proteins in the libraries, collectively designated as 'glubodies,' generally retain enzymatic activity but differ markedly both from each other and from the parent enzyme in sensitivity to inhibition by diverse small organic compounds. In some instances, a glubody is inhibited by completely novel structures. CONCLUSIONS: We have shown that a non-antibody framework can be used to create large libraries of proteins with a wide range of binding specificities for small organic molecules. The glubodies provide a rich source of data for correlating the structural and functional features of proteins relevant to ligand binding. The criteria applied for identifying an LRS in GST P1 are generally applicable to other protein frameworks.

Increasing the use of bleach and condoms among injecting drug users in Denver: outcomes of a targeted, community-level HIV prevention program.

OBJECTIVE: To evaluate the impact of an HIV risk-reduction program among injecting drug users (IDU) in Denver, Colorado. DESIGN: A targeted, community-level intervention study with multiple, time-phased, cross-sectional measurements assessing HIV high-risk behaviors among IDU in intervention and comparison sites. SETTING: Neighborhoods with high IDU prevalence in Denver, Colorado (intervention site) and Long Beach, California (non-intervention comparison site). PARTICIPANTS: Street-recruited IDU who had injected drugs in the previous 30 days and shared injection equipment in the previous 60 days to evaluate the use of bleach to clean injection equipment; or had sexual intercourse in the previous 30 days, to evaluate condom use. INTERVENTION: A prevention program in which peer volunteers were recruited and trained to distribute and discuss intervention kits that included condoms, bleach bottles and role model stories. MAIN OUTCOME MEASURES: Multiple cross-sectional surveys were conducted in the intervention and comparison sites to assess the impact of the intervention on the consistent use of bleach before sharing injection equipment and the consistent use of condoms for vaginal intercourse with steady and occasional partners. RESULTS: Between February 1991 and December 1993, 1997 IDU were interviewed, 890 at the intervention site and 1107 at the comparison site. In contrast to the comparison site, subjects from the intervention site reported significant increases in consistent use of bleach [odds ratio (OR), 2.6; 95% confidence interval (CI), 1.3-5.1; P < 0.001], and consistent use of condoms with occasional partners (OR, 13.6; 95% CI, 3.2-58.0; P < 0.001). CONCLUSION: This targeted, peer-based intervention was associated with significant HIV risk reduction among IDU in Denver and may be useful in other communities at risk for HIV infection.

Using formative research to lay the foundation for community level HIV prevention efforts: an example from the AIDS Community Demonstration Projects.

The AIDS Community Demonstration Projects provided community-level HIV prevention interventions to historically hard-to-reach groups at high risk for HIV infection. The projects operated under a common research protocol which encompassed formative research, intervention delivery, process evaluation, and outcome evaluation. A formative research process specifically focusing on intervention development was devised to assist project staff in identifying, prioritizing, accessing, and understanding the intervention target groups. This process was central to the creation of interventions that were acceptable and unique to the target populations. Intended to be rapid, the process took 6 months to complete. Drawn from the disciplines of anthropology, community psychology, sociology, and public health, the formative research process followed distinct steps which included (a) defining the populations at high-risk for HIV; (b) gathering information about these populations through interviews with persons who were outside of, but who had contact with, the target groups (such as staff from the health department and alcohol and drug treatment facilities, as well as persons who interacted in an informal manner with the target groups, such as clerks in neighborhood grocery stores and bartenders); (c) interviewing people with access to the target populations (gatekeepers), and conducting observations in areas where these high-risk groups were reported to gather (from previous interviews); (d) interviewing members of these groups at high risk for HIV infection or transmission; and (e) systematically integrating information throughout the process. Semistructured interview schedules were used for all data collection in this process. This standardized systematic method yielded valuable information about the focal groups in each demonstration project site. The method, if adopted by others, would assist community intervention specialists in developing interventions that are culturally appropriate and meaningful to their respective target populations.

Building a peer network for a community level HIV prevention program among injecting drug users in Denver.

As part of a multi-site Centers for Disease Control and Prevention-funded initiative, a community-level HIV prevention project targeting injection drug users was implemented in the FivePoints community in Denver, Colorado. The protocol for the initiative included the use of peer networks to conduct outreach and disseminate intervention materials to injecting drug users. Since April 1993, project staff established a peer network of 119 participants who distribute approximately 3,000 materials per month.

Predicting ligand binding to proteins by affinity fingerprinting.

BACKGROUND: There are many ways to represent a molecule's properties, including atomic-connectivity drawings, NMR spectra, and molecular orbital models. Prior methods for predicting the biological activity of compounds have largely depended on these physical representations. Measuring a compound's binding potency against a small reference panel of diverse proteins defines a very different representation of the molecule, which we call an affinity fingerprint. Statistical analysis of such fingerprints provides new insights into aspects of binding interactions that are shared among a wide variety of proteins. These analyses facilitate prediction of the binding properties of these compounds assayed against new proteins. RESULTS: Affinity fingerprints are reported for 122 structurally-diverse compounds using a reference panel of eight proteins that collectively are able to generate unique fingerprints for about 75% of the small organic compounds tested. Application of multivariate regression techniques to this database enables the creation of computational surrogates to represent new proteins that are surprisingly effective at predicting binding potencies. We illustrate this for two enzymes with no previously recognizable similarity to each other or to any of the reference proteins. Fitting of analogous computational surrogates to four other proteins confirms the generality of the method; when applied to a fingerprinted library of 5000 compounds, several sub-micromolar hits were correctly predicted. CONCLUSIONS: An affinity fingerprint database, which provides a rich source of data defining operational similarities among proteins, can be used to test theories of cryptic homology unexpected from current understanding of protein structure. Practical applications to drug design include efficient pre-screening of large numbers of compounds against target proteins using fingerprint similarities, supplemented by a small number of empirical measurements, to select promising compounds for further study.

Comparison of MICRO-ID Listeria method with conventional biochemical methods for identification of Listeria isolated from food and environmental samples: collaborative study.

Fourteen laboratories participated in a collaborative study to evaluate the ability of the MICRO-ID Listeria identification method to correctly identify Listeria isolated from food and environmental sources. Each collaborator received 60 isolates consisting of 51 Listeria and 9 non-Listeria cultures. All isolates were identified by conventional biochemical analyses in the principal laboratory. Cultures were checked for purity by Gram staining and examined for oxidase and catalase activities. Only Gram positive, oxidase negative, catalase positive cultures were tested with the method. Colonies from trypticase soy agar with 0.6% yeast extract were suspended in 4.6 mL physiological saline to a MacFarland No. 1 turbidity standard and used to inoculate the test strip. In addition, the hemolytic reaction of each isolate was determined by using the Christie-Atkins-Munch-Peterson (CAMP) test and by stabbing sheep blood agar. Identification of Listeria is based on the octal code obtained from the strip and the hemolytic reaction of the isolate. The MICRO-ID Listeria method agreed with conventinal biochemical identification for 98.0% of L. monocytogenes, 77.1% of L. seeligeri, 90.0% of L. ivanovii, 96.4% of L. grayi/L. murrayi, 73.9% of L. welshimeri, and 100% of L. innocua isolates. A large percentage of errors in identification of the L. seeligeri and L. ivanovii cultures was caused by inaccurate reading of the CAMP and hemolysis tests rather than errors in the test strip. The method was adopted first action by AOAC International.

Men's disclosure of HIV test results to male primary sex partners.

We evaluated disclosure of human immunodeficiency virus (HIV) antibody status to a main sex partner and the impact on the relationship in men who have sex with men and who are enrolled in the Acquired Immunodeficiency Syndrome (AIDS) Community Demonstration Projects cohorts. Eighty-nine percent of both seronegative and seropositive men disclosed the results to their main sex partner. Seventy percent of the seronegative men and 82% of the seropositive men who did so reported that the relationship remained "as strong as ever" after 6 months. Most men who did not disclose their test results to their main partner reported being "single" after 6 months.

Molecular characterization of a second abortive phage resistance gene present in Lactococcus lactis subsp. lactis ME2.

The fifth phage resistance factor from the prototype phage-insensitive strain Lactococcus lactis subsp. lactis ME2 has been characterized and sequenced. The genetic determinant for Prf (phage resistance five) was subcloned from the conjugative plasmid pTN20, which also encodes a restriction and modification system. Typical of other abortive resistance mechanisms, Prf reduces the efficiency of plaquing to 10(-2) to 10(-3) and decreases the plaque size and burst size of the small isometric-headed phage p2 in L. lactis subsp. lactis LM0230. However, normal-size plaques occurred at a frequency of 10(-4) and contained mutant phages that were resistant to Prf, even after repeated propagation through a sensitive host. Prf does not prevent phage adsorption or promote restriction and modification activities, but 90% of Prf+ cells infected with phage p2 die. Thus, phage infections in Prf+ cells are aborted. Prf is effective in both L. lactis subsp. lactis and L. lactis subsp. cremoris strains against several small isometric-headed phages but not against prolate-headed phages. The Prf determinant was localized by Tn5 mutagenesis and subcloning. DNA sequencing identified a 1,056-nucleotide structural gene designated abiC. Prf+ expression was obtained when abiC was subcloned into the lactococcal expression vector pMG36e. abiC is distinct from two other lactococcal abortive phage resistance genes, abiA (Hsp+, from L. lactis subsp. lactis ME2) and abi416 (Abi+, from L. lactis subsp. lactis IL416). Unlike abiA, the action of abiC does not appear to affect DNA replication. Thus, abiC represents a second abortive system found in ME2 that acts at a different point of the phage lytic cycle.

Conformational similarities between one-chain and two-chain tissue plasminogen activator (t-PA): implications to the activation mechanism on one-chain t-PA.

Tissue plasminogen activator (t-PA) is an exceptional serine protease, because unlike most other serine protease zymogens single-chain tissue plasminogen activator (sct-PA) possesses a substantial amount of proteolytic activity. The unusual reaction of sct-PA afforded the opportunity to directly compare the active site environment of sct-PA and two-chain tissue plasminogen activator (tct-PA) in solution through the application of a series of nitroxide spin labels and fluorophores. These labels, which have been previously shown to covalently label the catalytic serine of other serine proteases, inactivated both sct-PA and tct-PA. The labels can be divided into two classes: those which form tetrahedral complexes (sulfonates) and those which form trigonal complexes (anthranilates). Those which formed tetrahedral complexes were found to be insensitive to structural differences between sct-PA and tct-PA at the active site. In contrast, those which formed trigonal complexes could differentiate and monitor the sct-PA to tct-PA conversion by fluorescence spectroscopy. Models of the structure of sct-PA and tct-PA were constructed on the basis of the known X-ray structures of other serine protease zymogen and active enzyme forms. One of the nitroxide spin labels was modeled into the sct-PA and tct-PA structures in two possible orientations, both of which could be sensitive to structural differences between sct-PA and tct-PA. These models formed the structural rationale used to explain the results obtained with the "tetrahedral" and "trigonal" probes, as well as to offer a possible explanation for the unique reactivity of sct-PA.

A region of tissue plasminogen activator that affects plasminogen activation differentially with various fibrin(ogen)-related stimulators.

The dissolution of blood clots by plasmin is normally initiated in vivo by the activation of plasminogen to plasmin through the activity of tissue plasminogen activator (t-PA). The rate of plasminogen activation can be stimulated several orders of magnitude by the presence of fibrin-related proteins. Here we describe the kinetic analysis of both recombinant human t-PA (wild-type) and a t-PA variant produced by site-directed mutagenesis in which the original sequence from amino acids 296 to 299, KHRR, has been altered to AAAA. This tetra-alanine variant form of t-PA, K296A/H297A/R298A/R299A t-PA, we refer to as "KHRR" t-PA here. The plasminogen activating kinetics of wild-type t-PA (Activase alteplase) showed a catalytic efficiency which changed over 100-fold dependent on the stimulator in the assay. The lowest rate was in the absence of a stimulator. The following stimulators showed increasing ability to accelerate the catalytic efficiency of the reaction: fibrinogen, fragments of fibrinogen obtained by digestion with plasmin, fibrin, and slightly degraded fibrin. This increase in efficiency was driven primarily by decreases in the Michaelis constant (KM) of the reaction, whereas the catalytic rate constant (kcat) of the reaction did not change significantly. The "KHRR" variant of t-PA displayed novel kinetics with all stimulators tested. In the absence of a stimulator or with the poorer stimulators (fibrinogen and fibrinogen fragments), the KM values of the reaction with Activase alteplase and "KHRR" t-PA were similar. The kcat however, was lower with "KHRR" t-PA than with wild-type t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the effects of HIV antibody counseling and testing on risk behaviors.

OBJECTIVE: To review published abstracts, journal articles, and presentations for evidence of the effects of human immunodeficiency virus (HIV) antibody counseling and testing on risk behaviors. Studies reviewed focused on homosexual men, intravenous drug users in treatment programs, pregnant women, and other heterosexuals. DATA SOURCES: Peer-reviewed journals (January 1986 through July 1990) and published abstracts and oral presentations from the second (1986) through the sixth (1990) International Conferences on AIDS. STUDY SELECTION: We identified 66 studies that included data on the behavioral effects of HIV antibody counseling and testing. By consensus of the authors, 16 of these were excluded because of small sample size or inadequate study design. DATA EXTRACTION: Studies were assessed by the authors according to methodological strength (sample selection, inclusion of appropriate comparison groups, and inclusion of statistical tests of significance). DATA SYNTHESIS: All longitudinal studies of homosexual men reported reductions in risky behavior among both tested and untested men, and a few reported greater decreases among seropositive men than among seronegative men and those untested or unaware of their serostatus. For intravenous drug users in treatment, we found reductions in intravenous drug use and sexual risk behaviors regardless of counseling and testing experience. We found little evidence for the impact of counseling and testing on pregnancy and/or pregnancy termination rates for either seropositive or seronegative high-risk women. We noted substantial risk reduction among heterosexual couples with one infected partner. Findings among other heterosexuals at increased risk were scanty and mixed. CONCLUSIONS: Further studies should specifically address the behavioral consequences of counseling and testing in various settings.

AIDS Community Demonstration Projects for HIV prevention among hard-to-reach groups.

The AIDS Community Demonstration Projects are multicenter prevention projects directing community-based interventions to members of hard-to-reach groups at risk of infection from human immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome (AIDS). The projects are supported by the Centers for Disease Control (CDC). Interventions are derived from theories of behavior change and have as their goal reducing HIV and other sexually transmitted diseases in the communities. The current objectives, intentionally narrow to improve the project's specificity and clarity, are to increase the use of condoms in sexual activity and the use of bleach to clean injecting drug equipment. Additional objectives may be added. The impact of the interventions is seen in increases in the use of HIV counseling and testing services, decreases in all or specific sexual and drug-use risk behaviors, and requests for related social and public health services. A quasi-experimental research design is being used to evaluate the projects. Multiple evaluation measures are used, including a street-based interview with randomly identified respondents in both intervention and control communities. Success in facilitating HIV and AIDS risk reduction is being measured using a model of behavior change describing stages of change. Upon successful completion of these projects in 1994, CDC may be able to offer models of effective, feasible, and easy-to-monitor State and local health departments and community-based organizations.