Prostaglandin F2 alpha increases responsiveness of pulmonary airways in dogs

We studied the effect of prostaglandin F2 alpha (PGF2 alpha) on the responsiveness of pulmonary airways in dogs. Airway responsiveness was assessed by determining the bronchoconstrictor response to increasing concentrations of acetylcholine aerosol delivered to the airways. In each of five dogs, we determined responsiveness during treatment with physiologic saline, histamine, or PGF2 alpha aerosols. The doses of histamine and PGF2 alpha were determined by establishing the largest dose of each which could be given to the dog without causing bronchoconstriction (subthreshold doses). We found that airway responsiveness was not significantly different during histamine treatment than after saline, however, responsiveness increased during treatment with PGF2 alpha. In addition, the hyperresponsiveness induced by PGF2 alpha was prevented by pretreatment with the ganglion blocking drug hexamethonium (5 mg/kg given intravenously). The results show that PGF2 alpha specifically increases the responsiveness of pulmonary airways in doses that do not cause bronchoconstriction, and suggest that the hyperresponsiveness involves a neural mechanism such as increased responsiveness of airway sensory nerves.     

Design and synthesis of MMP inhibitors with appended fluorescent tags for imaging and visualization of matrix metalloproteinase enzymes

We describe the synthesis, MMP-2 and 9 potency, and in vitro evaluation of a series of α-sulfone hydroxmate MMP inhibitors conjugated to a series of dyes with different absorption/emission lamina maxima’s that can be used to visualize tumors.     

Exploring the effectiveness of tazobactam against ceftazidime resistant Escherichia coli: insights from the comparison between susceptibility testing and beta-lactamase inhibition

Thirteen clinical isolates of Escherichia coli resistant to ceftazidime that possessed an AmpC and other (beta-lactamases were identified. The effectiveness of different formulations of piperacillin/tazobactam to other beta-lactams was compared. Antibiotic susceptibility testing, polymerase chain reaction, amplification of blaTEM, blaSHV and blaAmpC, and enzyme-linked immunosorbent assays to identify AmpC beta-lactamases were performed. Hydrolysis rates were obtained and residual enzymatic activity was determined. Cefepime and ertapenem were more active than piperacillin/tazobactam. In contrast, increasing the relative proportion of tazobactam improved susceptibility testing. Twenty micromolar tazobactam inhibited total beta-lactamase activity (as measured by nitrocefin hydrolysis rates) by greater than 75% against all isolates tested: in 11 of 13 E. coli isolates, total beta-lactamase activity was inhibited by 90%. The observed differences between MIC determinations and susceptibility to enzymatic inactivation by tazobactam against E. coli containing AmpC and other -lactamases may be due to the final tazobactam concentration achieved in the periplasmic space. Factors determining this are critical considerations in assessing beta-lactamase inhibitor potency.     

Phylo-VISTA: interactive visualization of multiple DNA sequence alignments

MOTIVATION: The power of multi-sequence comparison for biological discovery is well established. The need for new capabilities to visualize and compare cross-species alignment data is intensified by the growing number of genomic sequence datasets being generated for an ever-increasing number of organisms. To be efficient these visualization algorithms must support the ability to accommodate consistently a wide range of evolutionary distances in a comparison framework based upon phylogenetic relationships. RESULTS: We have developed Phylo-VISTA, an interactive tool for analyzing multiple alignments by visualizing a similarity measure for multiple DNA sequences. The complexity of visual presentation is effectively organized using a framework based upon interspecies phylogenetic relationships. The phylogenetic organization supports rapid, user-guided interspecies comparison. To aid in navigation through large sequence datasets, Phylo-VISTA leverages concepts from VISTA that provide a user with the ability to select and view data at varying resolutions. The combination of multiresolution data visualization and analysis, combined with the phylogenetic framework for interspecies comparison, produces a highly flexible and powerful tool for visual data analysis of multiple sequence alignments. AVAILABILITY: Phylo-VISTA is available at http://www-gsd.lbl.gov/phylovista. It requires an Internet browser with Java Plug-in 1.4.2 and it is integrated into the global alignment program LAGAN at http://lagan.stanford.edu     

Understanding resistance to beta-lactams and beta-lactamase inhibitors in the SHV beta-lactamase: lessons from the mutagenesis of SER-130

Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 beta-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G beta-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a reduction in kcat/Km for ampicillin. Marked increases in the dissociation constant for the preacylation complex, KI, of clavulanic acid (SHV-1, 0.14 microm; S130G, 46.5 microm) and tazobactam (SHV-1, 0.07 microm; S130G, 4.2 microm) were observed. In contrast, the k(inact)s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for beta-lactams and beta-lactamase inhibitors. These results underscore the mechanistic versatility of class A beta-lactamases and have implications for the design of novel beta-lactamase inhibitors.     

hORFeome v3.1: a resource of human open reading frames representing over 10,000 human genes

Complete sets of cloned protein-encoding open reading frames (ORFs), or ORFeomes, are essential tools for large-scale proteomics and systems biology studies. Here we describe human ORFeome version 3.1 (hORFeome v3.1), currently the largest publicly available resource of full-length human ORFs (available at ). Generated by Gateway recombinational cloning, this collection contains 12,212 ORFs, representing 10,214 human genes, and corresponds to a 51% expansion of the original hORFeome v1.1. An online human ORFeome database, hORFDB, was built and serves as the central repository for all cloned human ORFs (http://horfdb.dfci.harvard.edu). This expansion of the original ORFeome resource greatly increases the potential experimental search space for large-scale proteomics studies, which will lead to the generation of more comprehensive datasets.     

Structures of SHV-1 β-Lactamase with Penem and Penam Sulfone Inhibitors That Form Cyclic Intermediates Stabilized by Carbonyl Conjugation

Bacterial β-lactamase enzymes are in large part responsible for the decreased ability of β-lactam antibiotics to combat infections. The inability to overcome β-lactamase mediated resistance spurred the development of inhibitors with penems and penam sulfones being amongst the most potent and broad spectrum mechanism-based inactivators. These inhibitors form covalent, “suicide-type” inhibitory intermediates that are attached to the catalytic S70 residue. To further probe the details of the mechanism of β-lactamase inhibition by these novel compounds, we determined the crystal structures of SHV-1 bound with penem 1, and penam sulfones SA1-204 and SA3-53. Comparison with each other and with previously determined crystal structures of members of these classes of inhibitors suggests that the final conformation of the covalent adduct can vary greatly amongst the complex structures. In contrast, a common theme of carbonyl conjugation as a mechanism to avoid deacylation emerges despite that the penem and penam sulfone inhibitors form different types of intermediates. The detailed insights gained from this study could be used to further improve new mechanism-based inhibitors of these common class A serine β-lactamases.     

Exogenous fluorescent tracer agents based on pegylated pyrazine dyes for real-time point-of-care measurement of glomerular filtration rate

Novel pyrazine carboxamides bearing hydrophilic poly(ethylene glycol) (PEG) moieties were designed, synthesized, and evaluated for use as fluorescent glomerular filtration rate (GFR) tracer agents. Among these, compounds 4d and 5c that contain about 48 ethylene oxide units in the PEG chain exhibited the most favorable physicochemical and renal clearance properties. In vitro studies show that these two compounds have low plasma protein binding, a necessary condition for renal excretion. In vivo animal model results show that 4d and 5c have a higher urine recovery of the injected dose than iothalamate (a commonly considered gold standard GFR agent). Pharmacokinetic studies show that these two compounds exhibit a plasma clearance equivalent to iothalamate, but with a faster (i.e. lower) terminal half-life than iothalamate (possibly from restricted distribution into the extracellular space due to large molecular size and hydrodynamic volume). Furthermore, the plasma clearance of 4d and 5c remained unchanged upon blockage of the tubular secretion pathway with probenecid, a necessary condition for establishment of clearance via glomerular filtration exclusively. Finally, noninvasive real-time monitoring of this class of compounds was demonstrated by pharmacokinetic clearance of 5c by optical measurements in rat model, which correlates strongly with plasma concentration of the tracer. Hence, 4d and 5c are promising candidates for translation to the clinic as exogenous fluorescent tracer agents in real-time point-of-care monitoring of GFR.