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The development of new antibiotics to treat infections caused by drug-resistant Gram-negative pathogens is of paramount importance as antibiotic resistance continues to increase worldwide.sup.1. Here we describe a strategy for the rational design of diazabicyclooctane inhibitors of penicillin-binding proteins from Gram-negative bacteria to overcome multiple mechanisms of resistance, including [beta]-lactamase enzymes, stringent response and outer membrane permeation. Diazabicyclooctane inhibitors retain activity in the presence of [beta]-lactamases, the primary resistance mechanism associated with [beta]-lactam therapy in Gram-negative bacteria.sup.2,3. Although the target spectrum of an initial lead was successfully re-engineered to gain in vivo efficacy, its ability to permeate across bacterial outer membranes was insufficient for further development. Notably, the features that enhanced target potency were found to preclude compound uptake. An improved optimization strategy leveraged porin permeation properties concomitant with biochemical potency in the lead-optimization stage. This resulted in ETX0462, which has potent in vitro and in vivo activity against Pseudomonas aeruginosa plus all other Gram-negative ESKAPE pathogens, Stenotrophomonas maltophilia and biothreat pathogens. These attributes, along with a favourable preclinical safety profile, hold promise for the successful clinical development of the first novel Gram-negative chemotype to treat life-threatening antibiotic-resistant infections in more than 25 years. A lead-optimization strategy combining porin permeation properties and biochemical potency leads to development of a new class of antibiotic based on broad inhibition of penicillin-binding proteins from Gram-negative bacteria., Author(s): Thomas F. Durand-Reville [sup.1] , Alita A. Miller [sup.1] , John P. O'Donnell [sup.1] , Xiaoyun Wu [sup.2] , Mark A. Sylvester [sup.1] , Satenig Guler [sup.1] [sup.3] , [...]

Unlike the soma, which ages during the lifespan of multicellular organisms, the germ line traces an essentially immortal lineage. Genomic instability in somatic cells increases with age, and this decline in somatic maintenance might be regulated to facilitate resource reallocation towards reproduction at the expense of cellular senescence. Here we show that Caenorhabditis elegans mutants with increased longevity exhibit a soma-to-germline transformation of gene expression programs normally limited to the germ line. Decreased insulin-like signalling causes the somatic misexpression of the germline-limited pie-1 and pgl family of genes in intestinal and ectodermal tissues. The forkhead boxO1A (FOXO) transcription factor DAF-16, the major transcriptional effector of insulin-like signalling, regulates pie-1 expression by directly binding to the pie-1 promoter. The somatic tissues of insulin-like mutants are more germline-like and protected from genotoxic stress. Gene inactivation of components of the cytosolic chaperonin complex that induce increased longevity also causes somatic misexpression of PGL-1. These results indicate that the acquisition of germline characteristics by the somatic cells of C. elegans mutants with increased longevity contributes to their increased health and survival., In genetic and functional genomic screens for gene inactivations that increase the lifespan of C. elegans, decreases in the insulin-like signalling pathway confer the largest increase in longevity. Inactivation of [...]

Author(s): Vasanthi S. Viswanathan [1]; Matthew J. Ryan [1]; Harshil D. Dhruv [2]; Shubhroz Gill [1]; Ossia M. Eichhoff [3]; Brinton Seashore-Ludlow [1]; Samuel D. Kaffenberger [4]; John K. Eaton [...]

We have developed human cervicovaginal organ culture systems to examine the initiating events in HIV transmission after exposure to various sources of HIV infectivity, including semen. Newly infected cells were detected in the cervical submucosa 3-4 days after exposure to a primary HIV isolate. At earlier times, extensive and stable binding occurred when cervical surfaces were exposed to virions or seminal cells. Cervical mucus provided some protection for the endocervical surface, by physically trapping virions and seminal cells. Confocal microscopy combined with 3D surface reconstruction revealed that virions could both bind to the external surface of the cervical epithelium and actually penetrate beneath the epithelial surface. In quantitative assays, pretreatment with a blocking antibody directed against [beta]1 integrin reduced HIV virion binding. Collectively, these results highlight a continuum of complex interactions that occurs when natural sources of HIV infectivity are deposited onto mucosal surfaces in the female reproductive tract. confocal microscopy | female reproductive tissue | HIV transmission | organ culture | tissue surface reconstruction

We reported recently that 1-bromopropane (1-BP; n-propylbromide, CAS Registry no. 106-94-5), an alternative to ozone-depleting solvents, is neurotoxic and exhibits reproductive toxicity in rats. The four most recent case reports [...]

Transcription by RNA polymerase II (pol II) requires the ordered binding of distinct protein complexes to catalyze initiation, elongation, termination, and coupled mRNA processing events. One or more proteins from each complex are known to bind pol II via the carboxy-terminal domain (CTD) of the largest subunit, Rpb1. How binding is coordinated is not known, but it might involve conformational changes in the CTD induced by the Ess1 peptidyl-prolyl cis/trans isomerase. Here, we examined the role of ESS1 in transcription by studying one of its multicopy suppressors, BYE1. We found that Bye1 is a negative regulator of transcription elongation. This led to the finding that Ess1 also inhibits elongation; Ess1 opposes elongation factors Dst1 and Spt4/5, and overexpression of ESS1 makes cells more sensitive to the elongation inhibitor 6-AU. In reporter gene assays, ess1 mutations reduce the ability, of elongation-arrest sites to stall polymerase. We also show that Ess1 acts positively in transcription termination, independent of its role in elongation. We propose that Ess1-induced conformational changes attenuate pol II elongation and help coordinate the ordered assembly of protein complexes on the CTD. In this way, Ess1 might regulate the transition between multiple steps of transcription.

Although high-energy phosphate metabolism is abnormal in failing hearts [congestive heart failure (CHF)], it is unclear whether oxidative capacity is impaired. This study used the mitochondrial uncoupling agent 2,4-dinitrophenol (DNP) to determine whether reserve oxidative capacity exists during the high workload produced by catecholamine infusion in hypertrophied and failing hearts. Left ventricular hypertrophy (LVH) was produced by ascending aortic banding in 21 swine; 9 animals developed CHF. Basal myocardial phosphocreatine (PCr)/ATP measured with [sup.31]P NMR spectroscopy was decreased in both LVH and CHF hearts (corresponding to an increase in free [ADP]), whereas ATP was decreased in hearts with CHF. Infusion of dobutamine and dopamine (each 20 [micro]g*[kg.sup.-1]*[min.sup.-1] iv) caused an approximate doubling of myocardial oxygen consumption (MV[O.sub.2]) in all groups and decreased PCr/ATP in the normal and LVH groups. During continuing catecholamine infusion, DNP (2-8 mg/kg iv) caused further increases of MV[O.sub.2] in normal and LVH hearts with no change in PCr/ATP. In contrast, DNP caused no increase in MV[O.sub.2] in the failing hearts; the associated decrease of PCr/ATP suggests that DNP decreased the mitochondrial proton gradient, thereby causing ADP to increase to maintain adequate ATP synthesis. heart failure; left ventricular hypertrophy; mitochondria; high-energy phosphates; nuclear magnetic resonance

This paper presents an object-oriented (OO) software framework for computer-aided structural analysis and design research, where different structural analysis methods and design procedures need to be implemented and investigated. The framework is designed with four basic modules: structure, load, analysis, and design. Each module includes a set of cooperating interfaces and classes. Through the predefined interfaces, the framework provides architecture for many structural design applications. A variety of similar entities, such as different design applications, design procedures, and analysis methods, can be built on this architecture by implementing the necessary interfaces. The clearly defined interactions between the modules accommodate the future extensions within the modules. The final OO design of the framework can be communicated by many well-known design patterns, and it is described by unified modeling language. The framework is then customized to the application for optimizing the configuration of energy dissipation devices in a given structure. By implementing a few interfaces, this paper illustrates how this OO framework accommodates changes, and how reusability and extensibility can be achieved. DOI: 10.1061/(ASCE)0887-3801(2003)17:2(113) CE Database subject headings: Computer software; Optimization; Damping; Structural analysis; Energy dissipation

Author(s): Xiping Wei [1]; Julie M. Decker [1]; Shuyi Wang [1]; Huxiong Hui [2]; John C. Kappes [2, 3]; Xiaoyun Wu [2]; Jesus F. Salazar-Gonzalez [2]; Maria G. Salazar [2]; [...]

The upper gastrointestinal tract is a principal route of HIV-1 entry in vertical transmission and after oral-genital contact. The phenotype of the newly acquired virus is predominantly R5 (CCR5-tropic) and not X4 (CXCR4-tropic), although both R5 and X4 viruses are frequently inoculated onto the mucosa. Here we show that primary intestinal (jejunal) epithelial cells express galactosylceramide, an alternative primary receptor for HIV-1, and CCR5 but not CXCR4. Moreover, we show that intestinal epithelial cells transfer R5, but not X4, viruses to CCR5[sup.+] indicator cells, which can efficiently replicate and amplify virus expression. Transfer was remarkably efficient and was not inhibited by the fusion blocker T-20, but was substantially reduced by colchicine and low (4 [degrees]C) temperature, suggesting endocytotic uptake and microtubule-dependent transcytosis of HIV-1. Our finding that CCR5[sup.+] intestinal epithelial cells select and transfer exclusively R5 viruses indicates a mechanism for the selective transmission of R5 HIV-1 in primary infection acquired through the upper gastrointestinal tract., Author(s): Gang Meng [1]; Xiping Wei [5]; Xiaoyun Wu [1]; Marty T. Sellers [2]; Julie M. Decker [5]; Zina Moldoveanu [3]; Jan M. Orenstein [6]; Martin F. Graham [7]; John [...]

A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases. The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci. This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps. The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome. The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized. The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome., A physical map affording ready access to all chromosomal regions is an essential prerequisite for the international effort to sequence the entire human genome. In the shorter term, it is [...]

A liver-directed gene therapy is used with concentrated retroviral preparations to obtain transduction of up to 15% of hepatocytes as established by a quantitative PCR assay. In vivo comparisons of gene regulatory elements are possible with these quantitative assays. Transduced gene expression will continue to prevent difficulties.

This study was performed to determine whether the fall in myocardial high-energy phosphates (HEP) that occurs during high workstates can be ascribed to either inadequate glycolytic pyruvate generation and conversion to acyl-CoA or limitation of long-chain fatty acid transport into the mitochondria. This was tested by using infusions of either pyruvate or butyrate in anesthetized dogs. Pyruvate was used because it bypasses the glycolytic sequence of reactions, activates pyruvate dehydrogenase, and increases mitochondrial NADH concentration ([[NADH.sub.m]]) in isolated myocardium, whereas butyrate enters the mitochondria without need for transport by the rate-limiting, palmitoyl-carnitine transporter. Increasing blood pyruvate from 0.16 [+ or -] 0.016 mM to [is greater than] 3 mM did not alter baseline HEP levels determined with [sup.31]P nuclear magnetic resonance, but caused an increase in the rate-pressure product and a modest increase in myocardial oxygen consumption ([MVO.sub.2]). Infusion of dobutamine + dopamine (each 20 [micro]g [multiplied by] [kg.sup.-1][multiplied by] [min.sup.-1] iv) increased [MVO.sub.2] and caused decreases of myocardial phosphocreatine (PCr)/ATP. Pyruvate partially reversed the decrease of HEP levels produced by catecholamine stimulation, whereas butyrate had no effect. Neither pyruvate nor butyrate caused an increase of [MVO.sup.2] during catecholamine infusion. Deoxymyoglobin was not detected by [sup.1.H] magnetic resonance spectroscopyy in any group. The data demonstrate that carbon substrate availability to the mitochondria is not the only cause of the reduction of PCr/ATP that occurs at high workstates. Supplemental pyruvate (but not butyrate) attenuated the reduction of PCr/ATP during the high workstates; this may have resulted from direct effects on intermediary metabolism or from other effects such as the free radical scavenging activity of pyruvate. catecholamines; oxygen consumption; magnetic resonance spectroscopy

Author(s): Xiping Wei; Julie M. Decker; Shuyi Wang; Huxiong Hui; John C. Kappes; Xiaoyun Wu; Jesus F. Salazar-Gonzalez; Maria G. Salazar; J. Michael Kilby; Michael S. Saag; Natalia L. Komarova; [...]