Characterization of virulence genes of enteroinvasive Escherichia coli by TnphoA mutagenesis: identification of invX, a gene required for entry into HEp-2 cells.

While enteroinvasive Escherichia coli (EIEC) and shigellae are genotypically nearly identical, a difference has been reported in the infective dose to humans: EIEC is 10,000-fold less infectious than shigellae. A possible basis for this difference lies in the inherent invasiveness of these bacteria toward epithelial cells. Thus, despite the high degree of homology between the invasion plasmids of EIEC and shigellae, substantial differences in genetic organization and/or sequence may exist. We have undertaken a systematic genetic analysis of the EIEC plasmid pSF204, using transposon mutagenesis. Congo red-negative TnphoA insertion mutants (Pcr- PhoA-) and TnphoA fusion mutants (PhoA+) were isolated and screened for the ability to invade cultured HEp-2 cells. Most invasion-negative (Inv-) mutations mapped to a 30-kb segment of the invasion plasmid, including homologs of the Shigella flexneri ipa, mxi, and spa genes. Inv- PhoA+ fusions in the EIEC ipaC, mxiG, mxiJ, mxiM, and mxiD homologs and in a proposed new gene, named invX, located downstream of the spa region were identified and characterized. This analysis indicates the presence of the ipaC, mxiG, mxiJ, mxiM, mxiD, and invX gene products in the EIEC cell envelope and demonstrates a strict requirement for these genetic loci in invasion. Overall, our results suggest a high degree of genetic, structural, and functional homology between the EIEC and S. flexneri large invasion plasmids.     

Development of a liquid chromatographic method for bioanalytical applications with sildenafil

An improved HPLC method was developed for the determination of sildenafil concentrations in plasma. Analysis of sildenafil in plasma samples was simplified by utilizing a one-step liquid-liquid extraction after alkaline treatment of only 1 ml of plasma. The lower limit of quantitation was 10 ng/ml with a coefficient of variation of less than 20%. A linear range was found to exist from 10 to 1000 ng/ml. This HPLC method was validated with precisions (coefficient of variation, C.V.) for inter- and intra-day runs of 0.41-11.15% and 0.36-8.05%, respectively, and accuracies (the relative error of the mean, REM) for inter- and intra-day runs of -8.72-6.81% and 0.41-11.15%, respectively. A bioavailability study of sildenafil was performed on one normal healthy male volunteer by analyzing sildenafil plasma concentrations with this validated HPLC method. Results demonstrated that this HPLC method is appropriate for applications to bioavailability studies of sildenafil. In addition, an example of the influence of the co-administration of grapefruit juice on sildenafil pharmacokinetics in a healthy volunteer is presented.     

Picky: oligo microarray design for large genomes

MOTIVATION: Many large genomes are getting sequenced nowadays. Biologists are eager to start microarray analysis taking advantage of all known genes of a species, but existing microarray design tools were very inefficient for large genomes. Also, many existing tools operate in a batch mode that does not assure best designs. RESULTS: Picky is an efficient oligo microarray design tool for large genomes. Picky integrates novel computer science techniques and the best known nearest-neighbor parameters to quickly identify sequence similarities and estimate their hybridization properties. Oligos designed by Picky are computationally optimized to guarantee the best specificity, sensitivity and uniformity under the given design constrains. Picky can be used to design arrays for whole genomes, or for only a subset of genes. The latter can still be screened against a whole genome to attain the same quality as a whole genome array, thereby permitting low budget, pathway-specific experiments to be conducted with large genomes. Picky is the fastest oligo array design tool currently available to the public, requiring only a few hours to process large gene sets from rice, maize or human.     

Deconvoluting lung evolution: from phenotypes to gene regulatory networks

Speakers in this symposium presented examples of respiratoryregulation that broadly illustrate principles of evolution fromwhole organ to genes. The swim bladder and lungs of aquaticand terrestrial organisms arose independently from a commonprimordial "respiratory pharynx" but not from each other. Pathwaysof lung evolution are similar between crocodiles and birds buta low compliance of mammalian lung may have driven the developmentof the diaphragm to permit lung inflation during inspiration.To meet the high oxygen demands of flight, bird lungs have evolvedseparate gas exchange and pump components to achieve unidirectionalventilation and minimize dead space. The process of "screening"(removal of oxygen from inspired air prior to entering the terminalunits) reduces effective alveolar oxygen tension and potentiallyexplains why nonathletic large mammals possess greater pulmonarydiffusing capacities than required by their oxygen consumption.The "primitive" central admixture of oxygenated and deoxygenatedblood in the incompletely divided reptilian heart is actuallyco-regulated with other autonomic cardiopulmonary responsesto provide flexible control of arterial oxygen tension independentof ventilation as well as a unique mechanism for adjusting metabolicrate. Some of the most ancient oxygen-sensing molecules, i.e.,hypoxia-inducible factor-1alpha and erythropoietin, are up-regulatedduring mammalian lung development and growth under apparentlynormoxic conditions, suggesting functional evolution. Normalalveolarization requires pleiotropic growth factors acting viahighly conserved cell–cell signal transduction, e.g.,parathyroid hormone-related protein transducing at least partlythrough the Wingless/int pathway. The latter regulates morphogenesisfrom nematode to mammal. If there is commonality among thesediverse respiratory processes, it is that all levels of organization,from molecular signaling to structure to function, co-evolveprogressively, and optimize an existing gas-exchange framework.     

Alveolar diffusion-perfusion interactions during high-altitude residence in guinea pigs.

We previously reported in weanling guinea pigs raised at high altitude (HA; 3,800 m) an elevated lung diffusing capacity estimated by morphometry from alveolar-capillary surface area, harmonic mean blood-gas barrier thickness, and pulmonary capillary blood volume (Vc) compared with litter-matched control animals raised at an intermediate altitude (IA; 1,200 m) (Hsia CCW, Polo Carbayo JJ, Yan X, Bellotto DJ. Respir Physiol Neurobiol 147: 105-115, 2005). To determine if HA-induced alveolar ultrastructural changes are associated with improved alveolar function, we measured lung diffusing capacity for carbon monoxide (DLCO), membrane diffusing capacity for carbon monoxide (DMCO), Vc, pulmonary blood flow, and lung volume by a rebreathing technique in litter-matched male weanling Hartley guinea pigs raised at HA or IA for 4 or 12 mo. Separate control animals were also raised and studied at sea level (SL). Resting measurements were obtained in the conscious nonsedated state. In HA animals compared with corresponding IA or SL controls, lung volume and hematocrit were significantly higher while pulmonary blood flow was lower. At a given pulmonary blood flow, DLCO and DMCO were higher in HA-raised animals than in control animals without a significant change in Vc. We conclude that 1) HA residence enhanced physiological diffusing capacity corresponding to that previously estimated on the basis of structural adaptation, 2) adaptation in diffusing capacity and its components should be interpreted with respect to pulmonary blood flow, and 3) this noninvasive rebreathing technique could be used to follow adaptive responses in small animals.