Efficient construction of a highly useful phage-displayed human antibody repertoire

We have constructed a highly useful phage-displayed human antibody repertoire with limited cloning efforts. Our strategy was to maximize diversity during the first steps of library construction through the use of various lymphoid sources from several donors, inclusion of different immunoglobulin isotypes, and performance of multiple separate amplification reactions with all possible combinations within a complex primer set. The resulting variable region collections were cloned to form a moderate size library, composed by 4.25x10(8) single chain antibody fragments. This repertoire was successfully used to retrieve binders to seven model antigens: six proteins and one 12 aa peptide. Binding affinities reached nanomolar and even subnanomolar range. Sequence diversity and V-gene usage variability among binders were proven. Our approach was not focused on absolute library size, but on a high quality sampling of variable regions from the human antibody repertoire.     

The Arabidopsis‐related halophyte Thellungiella halophila: boron tolerance via boron complexation with metabolites?

Tolerance to boron (B) is still not completely understood. We tested here the hypothesis that Thellungiella halophila, an Arabidopsis thaliana-related 'extremophile' plant, with abundance of B in its natural environment, is tolerant to B, and examined the potential mechanisms of this tolerance. With 1-10 mm B applied ([B](ext)) to Thellungiella and Arabidopsis grown in hydroponics, the steady-state accumulated B concentration ([B](int)) in the root was below [B](ext), and was similar in both, suggesting both extrude B actively. Whether grown in soil or hydroponically, the shoot [B](int) was higher in Arabidopsis than in Thellungiella, suggesting more effective net B exclusion by Thellungiella root. Arabidopsis exhibited toxicity symptoms including reduced shoot fresh weight (FW), but Thellungiella was not affected, even at similar levels of shoot-accumulated [B](int) (about 10 to 40 mm B in 'shoot water'), suggesting additional B tolerance mechanism in Thellungiella shoot. At [B](ext) = 5 mm, the summed shoot concentration of the potentially B-binding polyhydroxyl metabolites (malic acid, fructose, glucose, sucrose and citric acid) in Arabidopsis was below [B](int) , but in Thellungiella it was over twofold higher than [B](int) , and therefore likely to allow appreciable 1:2 boron-metabolite complexation in the shoot. This, we suggest, is an important component of Thellungiella B tolerance mechanism.     

Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.     

Isolation of a novel neutralizing antibody fragment against human vascular endothelial growth factor from a phage-displayed human antibody repertoire using an epitope disturbing strategy

Following the clinical success of Bevacizumab, a humanized monoclonal antibody that blocks the interaction between vascular endothelial growth factor (VEGF) and its receptors, the search for new neutralizing antibodies targeting this molecule has continued until now. We used a human VEGF variant containing three mutations in the region recognized by Bevacizumab to direct antibody selection towards recognition of other epitopes. A total of seven phage-displayed antibody fragments with diverse binding properties in terms of inter-species cross-reactivity and sensitivity to chemical modifications of the antigen were obtained from a human phage display library. All of them were able to recognize not only the selector mutated antigen, but also native VEGF. One of these phage-displayed antibody fragments, denominated 2H1, was shown to compete with the VEGF receptor 2 for VEGF binding. Purified soluble 2H1 inhibited in a dose dependent manner the ligand-receptor interaction and abolished VEGF-dependent proliferation of human umbilical vein endothelial cells. Our epitope disturbing strategy based on a triple mutant target antigen was successful to focus selection on epitopes different from a known one. Similar approaches could be used to direct phage isolation towards the desired specificity in other antigenic systems.     

Bacterial species and evolution: Theoretical and practical perspectives

A discussion of the species problem in modern evolutionary biology serves as the point of departure for an exploration of how the basic science aspects of this problem relate to efforts to map bacterial diversity for practical pursuits—for prospecting among the bacteria for useful genes and gene-products. Out of a confusing array of species concepts, the Cohesion Species Concept seems the most appropriate and useful for analyzing bacterial diversity. Techniques of allozyme analysis and DNA fingerprinting can be used to put this concept into practice to map bacterial genetic diversity, though the concept requires minor modification to encompass cases of complete asexuality. Examples from studies of phenetically definedBacillus species provide very partial maps of genetic population structure. A major conclusion is that such maps frequently reveal deep genetic subdivision within the phenetically defined specles; divisions that in some cases are clearly distinct genetic species. Knowledge of such subdivisions is bound to make prospecting within bacterial diversity more effective. Under the general concept of genetic cohesion a hypothetical framework for thinking about the full range of species conditions that might exist among bacteria is developed and the consequences of each such model for species delineation, and species identification are discussed. Modes of bacterial evolution, and a theory of bacterial speciation with and without genetic recombination, are examined. The essay concludes with thoughts about prospects for very extensive mapping of bacterial diversity in the service of future efforts to find useful products. In this context, evolutionary biology becomes the handmaiden of important industrial activities. A few examples of past success in commercializing bacterial gene-products from species ofBacillus and a few other bacteria are reviewed.