Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins

Phage display is an in vitro method for selecting polypeptides with desired properties from a large collection of variants. The insecticidal Cry toxins produced by Bacillus thuringiensis are highly specific to different insects. Various proteins such as cadherin, aminopeptidase-N (APN) and alkaline phosphatase (ALP) have been characterized as potential Cry-receptors. We used phage display to characterize the Cry toxin-receptor interaction(s). By employing phage-libraries that display single-chain antibodies (scFv) from humans or from immunized rabbits with Cry1Ab toxin or random 12-residues peptides, we have identified the epitopes that mediate binding of lepidopteran Cry1Ab toxin with cadherin and APN receptors from Manduca sexta and the interaction of dipteran Cry11Aa toxin with the ALP receptor from Aedes aegypti. Finally we displayed in phages the Cry1Ac toxin and discuss the potential for selecting Cry variants with improved toxicity or different specificity.     

Secretion pattern, ultrastructural localization and function of extracellular matrix molecules involved in eggshell formation

The chicken eggshell is a composite bioceramic containing organic and inorganic phases. The organic phase contains, among other constituents, type X collagen and proteoglycans (mammillan, a keratan sulfate proteoglycan, and ovoglycan, a dermatan sulfate proteoglycan), whose localization depends on a topographically defined and temporally regulated deposition. Although the distribution of these macromolecules in the eggshell has been well established, little is known about their precise localization within eggshell substructures and oviduct cells or their pattern of production and function during eggshell formation. By using immunofluorescent and immuno-ultrastructural analyses, we examined the distribution of these macromolecules in oviduct cells at different post-oviposition times. To understand the role of proteoglycan sulfation on eggshell formation, we studied the effects of inhibition of proteoglycan sulfation by treatment with sodium chlorate. We showed that these macromolecules are produced by particular oviduct cell populations and at precise post-oviposition times. Based on the precise ultrastructural localization of these macromolecules in eggshell substructures, the timing of the secretion of these macromolecules by oviduct cells and the effects on eggshell formation caused by the inhibition of proteoglycan sulfation, the putative role of mammillan is in the nucleation of the first calcite crystals, while that of ovoglycan is to regulate the growth and orientation of the later forming crystals of the chicken eggshell.     

APETALA3 and PISTILLATA homologs exhibit novel expression patterns in the unique perianth of Aristolochia (Aristolochiaceae)

Several lines of evidence suggest that sterile floral organs, collectively known as the perianth, have evolved multiple times during the evolution of the angiosperms. In the family Aristolochiaceae, the perianth is formed by two whorls of organs in the genus Saruma but by only one whorl in the remaining genera, including Aristolochia. Although the morphology of Saruma is similar in appearance to the core eudicot perianth, with leaf-like sepals and showy colored petals, the unipartite perianth of Aristolochia combines morphological aspects of both calyx and corolla. To investigate the organ identity program functioning in the novel perianth of Aristolochia, we identified homologs of the B-class genes APETALA3 (AP3) and PISTILLATA (PI) in both Saruma and Aristolochia. The expression patterns of these genes in Saruma indicate they are functioning in the development of the second whorl petaloid organs and third whorl stamens. In Aristolochia, however, the expression of AP3 and PI homologs in the perianth does not suggest a role in organ identity but, rather, in promoting late aspects of cell differentiation. The implications of these findings for the evolution of both petaloidy and B gene function are discussed.     

Surfactant strengthens the inhibitory effect of C-reactive protein on human lung macrophage cytokine release

In this study we investigated the effect of acute-phase levels of C-reactive protein (CRP) on cytokine production by pulmonary macrophages in the presence or absence of pulmonary surfactant. Both human alveolar and interstitial macrophages as well as human surfactant were obtained from multiple organ donor lungs. Precultured macrophages were stimulated with LPS alone or together with IFN-gamma in the presence or absence of CRP, surfactant, and combinations. Releases of TNF-alpha and of IL-1beta to the medium were determined. We found that CRP could modulate lung inflammation in humans by decreasing the production of proinflammatory cytokines by both alveolar and interstitial macrophages stimulated with LPS alone or together with IFN-gamma. The potential interaction between CRP and surfactant phospholipids did not overcome the effect of either CRP or surfactant on TNF-alpha and IL-1beta release by lung macrophages. On the contrary, CRP and pulmonary surfactant together had a greater inhibitory effect than either alone on the release of proinflammatory cytokines by lung macrophages.     

Characterization and regulation of NADP+-isocitrate dehydrogenase from Saccharopolyspora erythraea

NADP⁺-Isocitrate dehydrogenase (ICDH) activity was detected in cell-free extracts of Saccharopolyspora erythraea CA340, an erythromycin producer. Apparent K _m values for dl-isocitrate and NADP⁺ were 0.14 M and 0.026 M, respectively. ATP, ADP, GTP, citric acid, oxaloacetate, -ketoglutarate, glyoxalate and glyoxalate plus oxaloacetate, each at 1 mM concentration, caused 50, 20 10, 50, 25, 60, 20 and 50% inhibition of ICDH activity, respectively. Phosphoenolpyruvate, fructose 1,6-diphosphate and pyruvate had no effect. ICDH specific activity profile was growth-associated and activity with dextrose or fructose as sole carbon source, was twice of that obtained with lactose.     

Aluminum affects membrane physical properties in human neuroblastoma (IMR-32) cells both before and after differentiation

The capacity of Al(3+) to induce changes in the physical properties of plasma membrane from human neuroblastoma cells (IMR-32) was investigated, and the magnitude of the changes was compared with that obtained after cell differentiation to a neuronal phenotype. Similarly to our previous results in liposomes, Al(3+) (10 to 100 microM) caused a significant loss of membrane fluidity, being the differentiated cells more affected than the nondifferentiated cells. Al(3+) also increased the relative content of lipids in gel phase and promoted lipid rearrangement through lateral phase separation, with the magnitude of this effect being similar in nondifferentiated and differentiated cells. Since membrane physical properties depend on bilayer composition, we characterized the content of proteins, phospholipids, cholesterol, and fatty acids in the IMR-32 cells before and after differentiation. Differentiated cells had a significantly higher content of unsaturated fatty acids, creating an environment that favors Al(3+)-mediated effects on the bilayer fluidity. The neurotoxic effects of Al(3+) may be, at least in part, due to alterations of neuronal membrane physical properties, with potential consequences on the normal functioning of membrane-related cellular processes.     

Interaction of apolipoprotein A-I in three different conformations with palmitoyl oleoyl phosphatidylcholine vesicles

Interactions of apolipoprotein A-I (apoA-I) with cell membranes appear to be important in the initial steps of reverse cholesterol transport. The objective of this work was to examine the effect of three distinct conformations of apoA-I (lipid-free and in 78 A or 96 A reconstituted high density lipoproteins, rHDL) on its ability to bind to, and abstract lipids from, palmitoyl oleoyl phosphatidylcholine membrane vesicles (small unilamellar vesicles, SUV, and giant unilamellar vesicles, GUV). The molecular interactions were observed by two-photon fluorescence microscopy, and the binding parameters were quantified by gel-permeation chromatography or isothermal titration microcalorimetry. Rearrangement of apoA-I-containing particles after exposure to SUVs was examined by native gel electrophoresis. The results indicate that lipid-free apoA-I binds reversibly, with high affinity, to the vesicles but does not abstract a significant amount of lipid nor perturb the vesicle structure. The 96 A rHDL, where all the amphipathic helices of apoA-I are saturated with lipid within the particles, do not bind to vesicles or perturb their structure. In contrast, the 78 A rHDL have a region of apoA-I, corresponding to a few amphipathic helical segments, which is available for external or internal phospholipid binding. These particles bind to vesicles with measurable affinity (lower than lipid-free apoA-I), abstract lipids from the membranes, and form particles of larger diameters, including 96 A rHDL. We conclude that the conformation of apoA-I regulates its binding affinity for phospholipid membranes and its ability to abstract lipids from the membranes.