Physiological adaptations of the gut in the Lake Magadi tilapia, Alcolapia grahami, an alkaline- and saline-adapted teleost fish

We describe the gut physiology of the Lake Magadi tilapia (Alcolapia grahami), specifically those aspects associated with feeding and drinking while living in water of unusually high carbonate alkalinity (titratable base=245 mequiv l(-1)) and pH (9.85). Drinking of this highly alkaline lake water occurs at rates comparable to or higher than those seen in marine teleosts. Eating and drinking take place throughout the day, although drinking predominates during hours of darkness. The intestine directly intersects the esophagus at the anterior end of the stomach forming a 'T', and the pyloric sphincter, which comprises both smooth and striated muscle, is open when the stomach is empty and closed when the stomach is full. This unique configuration (a functional trifurcation) allows imbibed alkaline water to bypass the empty stomach, thereby avoiding a reactive mixing with acidic gastric fluids, and minimizes interference with a full stomach. No titratable base was present in the stomach, where the mean pH was 3.55, but the intestine was progressively more alkaline (foregut 6.96, midgut 7.74, hindgut 8.12, rectum 8.42); base levels in the intestinal fluid were comparable to those in lake water. The gut was highly efficient at absorbing water (76.6%), which accompanied the absorption of Na(+) (78.5%), titratable base (80.8%), and Cl(-) (71.8%). The majority of Na(+), base and water absorption occurred in the foregut by an apparent Na(+) plus base co-transport system. Overall, more than 70% of the intestinal flux occurred via Na(+) plus base co-transport, and less than 30% by Na(+) plus Cl(-) co-transport, a very different situation from the processes in the intestine of a typical marine teleost.     

Canopy transpiration of Jeffrey pine in mesic and xeric microsites: O<Subscript>3</Subscript> uptake and injury response

Canopy transpiration of mature Jeffrey pine was compared in "mesic" and "xeric" microsites differing in topographical position, bole growth, and the level of drought stress experienced. Diurnal and seasonal course of canopy transpiration was monitored with thermal dissipation probes in 1999 and 2000. Mid-canopy measures of diurnal foliar stomatal conductance (gs) were taken in June and August in 1999. In early summer, there was little difference between trees in either microsite with regard to gs (55 mmol H2O m⁻²s⁻¹), canopy transpiration (4.0 l h⁻¹), and total duration of active transpiration (12 h >0.03 l h⁻¹). In late summer, xeric trees had a lower daily maximum gs (by 30%), a greater reduction in whole canopy transpiration relative to the seasonal maximum (66 vs 79%), and stomata were open 2 h less per day than in mesic trees. Based on leaf-level gas exchange measurements, trees in mesic sites had an estimated 46% decrease in O3 uptake from June to August. Xeric trees had an estimated 72% decrease over the same time period. A multivariate analysis of morphological and tissue chemistry attributes in mid-canopy elucidated differences in mesic and xeric tree response. Mesic trees exhibited more O3 injury than xeric trees based on reduced foliar nitrogen content and needle retention in mid-canopy.     

Measurement of dissociation constants of inhibitors binding to Src SH2 domain protein by non-covalent electrospray ionization mass spectrometry

A mass spectrometric protocol for identifying ligands with a wide range of affinities (3-101 microM) and quantitative spectral analysis for non-covalent interactions have been developed using Src SH2 as a target. Dissociation constants of five compounds, three with a phospho moiety, one with a sulphonic acid group and one with carboxylic acid groups only, were determined using one-ligand one-binding-site, two-ligands one-binding site and one-ligand two-binding-sites models. The Kd values determined by ESI-MS of the three compounds containing the phospho moiety (3.2-7.9 microM) were comparable to those obtained from a solution equilibrium fluorescence polarization assay. The compound with a sulphonate group is a much weaker binding ligand (Kd=101 microM by ESI, >300 microM by FP) towards the Src SH2 protein. Two complexes with different stoichiometric ratios 1:1 and 2:1 (ligand-protein) were observed by ESI-MS for the ligand GIXXX630X. Analysis of binding isotherms indicated the presence of two binding sites for the ligand with Kd values of 9.3 and 193 microM. These data confirmed that, for these polar compounds, non-covalent ESI-MS can measure affinity which very closely reflects the affinity measured under true solution equilibrium conditions. ESI-MS has several key advantages over many solution methods: it can identify the existence of and measure the affinity of complexes other than simple 1:1 ligand-enzyme complexes. Moreover, ESI-MS competition experiments can be readily performed to yield data on whether two ligands bind simultaneously or competitively at the same time as measuring the affinity of the ligand.     

Synthesis and NMR solution structure of an alpha-helical hairpin stapled with two disulfide bridges

Helical coiled-coils and bundles are some of the most common structural motifs found in proteins. Design and synthesis of alpha-helical motifs may provide interesting scaffolds that can be useful as host structures to display functional sites, thus allowing the engineering of novel functional miniproteins. We have synthesized a 38-amino acid peptide, alpha2p8, encompassing the alpha-helical hairpin present in the structure of p8MTCP1, as an alpha-helical scaffold particularly promising for its stability and permissiveness of sequence mutations. The three-dimensional structure of this peptide has been solved using homonuclear two-dimensional NMR techniques at 600 MHz. After sequence specific assignment, a total of 285 distance and 29 dihedral restraints were collected. The solution structure of alpha2p8 is presented as a set of 30 DIANA structures, further refined by restrained molecular dynamics, using simulated annealing protocol with the AMBER force field. The RMSD values for the backbone and all heavy atoms are 0.65+/-0.25 and 1.51+/-0.21 A, respectively. Excised from its protein context, the alpha-hairpin keeps its native structure: an alpha-helical coiled-coil, similar to that found in superhelical structures, with two helices spanning residues 4-16 and 25-36, and linked by a short loop. This motif is stabilized by two interhelical disulfide bridges and several hydrophobic interactions at the helix interface, leaving most of its solvent-exposed surface available for mutation. This alpha-helical hairpin, easily amenable to synthetic chemistry and biological expression system, may represent a stable and versatile scaffold to display new functional sites and peptide libraries.     

Comparison of artificial light photobioreactors and other production systems using Porphyridium cruentum

A new type of preparative photobioreactor for high quality production of microalgae is developed for hatchery-nursery of marine animals, as well as for fine chemicals extraction. Of modular conception, two artificial light photobioreactors in plastic and stainless steel are designed so as to provide strictly controlled conditions in an attempt to increase quality and diminish cost prices. They are assessed for production of Porphyridum cruentum and compared to conventional transparent tanks and solar photobioreactors. The concentration and productivity obtained are ten-fold higher than with hatchery tanks, which leads to a significant drop in cost price of biomass. Comparison is also made with a 10 m² solar photobioreactor operated in the south of France, for which biomass cost price is half that of 1.5 m² artificial light photobioreactor. Extrapolations erasing size discrepancy show that the cost price of the two technologies are not very different. This revised version was published online in June 2006 with corrections to the Cover Date.     

Angiovision: Aortic stent-graft placement by augmented angionavigation

The endovascular treatment of abdominal aortic aneurysm (EVAR - EndoVascular Aneurism Repair) has become an alternative therapy to conventional open surgery. The objective of Angiovision project was to contribute to the improvement of EVAR procedures in terms of accuracy and customization of the interventional strategy. Our approach was mainly based on the use of patient-specific data, computer-aided endovascular navigation and numerical simulation. We proposed a new FEM-based patient-specific simulation solution to estimate the deformations caused by the introduction of stiff endovascular devices. We developed and implemented an original endovascular navigation system to augment the intraoperative fluoroscopy with the visualization of deformed preoperative aorto-iliac structure, and related information concerning vessel wall (calcifications), as well as stent-graft virtual deployment. The proposed solution, that requires only lightweight computer equipment, can be used in a standard operating room in order to optimize device placement (from a single angle of incidence). Evaluation is currently under way.     

W::Neo: a novel dual-selection marker for high efficiency gene targeting in Drosophila

We have recently developed a so-called genomic engineering approach that allows for directed, efficient and versatile modifications of Drosophila genome by combining the homologous recombination (HR)-based gene targeting with site-specific DNA integration. In genomic engineering and several similar approaches, a "founder" knock-out line must be generated first through HR-based gene targeting, which can still be a potentially time and resource intensive process. To significantly improve the efficiency and success rate of HR-based gene targeting in Drosophila, we have generated a new dual-selection marker termed W::Neo, which is a direct fusion between proteins of eye color marker White (W) and neomycin resistance (Neo). In HR-based gene targeting experiments, mutants carrying W::Neo as the selection marker can be enriched as much as fifty times by taking advantage of the antibiotic selection in Drosophila larvae. We have successfully carried out three independent gene targeting experiments using the W::Neo to generate genomic engineering founder knock-out lines in Drosophila.