A multi-approach study of the interaction of the Cu(II) and Ni(II) ions with alanylglycylhistamine, a mimicking pseudopeptide of the serum albumine N-terminal residue

The protonation equilibria of alanylglycylhistamine (Ala-Gly-Ha) and the complexation of this ligand with Cu(II) and Ni(II) have been studied by pH-potentiometry, 1H and 14N NMR spectroscopy, electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), UV-Vis spectrophotometry and electron paramagnetic resonance (EPR). From pH approximately 2-12, the following complexes: MLH, MLH(-1), MLH(-2) and MLH(-3) are successively formed in aqueous solutions, the ligand under its neutral form being noted L. At physiological pH, the MLH(-2) complex is predominant. The coordination in this complex is assumed by one amino, two deprotonated peptide and one imidazole nitrogen atoms. The ESI-MS study confirmed the formation of the MLH(-1), MLH(-2) and MLH(-3) complexes. The structure of MLH(-2) was determined by single crystal X-ray analysis. CD and UV-Vis techniques allowed us to propose that the imidazole-N3 nitrogen acts as the anchor group for the coordination to the metal(II) ions rather than the amino group. At high pH values, the further deprotonation of the N-H imidazole group, leading to the formation of MLH(-3), occurs, as revealed by 1H NMR spectroscopy.     

Ovipositional site selection by Anopheles gambiae: influences of substrate moisture and texture

The influence of substrate moisture (hydration) and grain size (texture) on oviposition was quantified in choice tests using Anopheles gambiae sensu stricto Giles (Diptera: Culicidae) laboratory strains and gravid An. gambiae sensu lato from a natural population in Western Kenya. A strong, positive correlation was found between moisture content and the degree of egg-laying, which peaked at saturation with standing water. Soil moisture quantified as surface conductivity, was measured with an electronic leaf-wetness sensor slightly modified from a unit available commercially. Although An. gambiae females were sensitive to measurable differences in substrate moisture, they distributed eggs on both fully hydrated and less hydrated substrates. In contrast, An. gambiae females showed little response to substrate texture: they oviposited with equal frequency on all silica substrates of eight particle size classes, ranging from small pebbles (850 microm diameter) to very fine grains (< 38 microm diameter), when all were moist. Female An. gambiae laid more eggs on dark than white substrates against a light background, but did not discriminate between moist, pulverized black soapstone and moist black Kenyan soil taken from typical An. gambiae larval habitats. We conclude that hydration and visual contrast are critical ovipositional site qualities for An. gambiae, but substrate texture is not.     

Lipophilic versus hydrogen-bonding effect in P3 on potency and selectivity of valine aspartyl ketones as caspase 3 inhibitors

Caspase 3 is a cysteinyl protease that mediates apoptotic cell death. Its inhibition may have an important impact in the treatment of several degenerative diseases. The P1 aspartic acid residue is a required element of recognition for this enzyme that was maintained constant along with the adjacent natural valine as the P2 group. The thiobenzylmethylketone warhead on the aspartate was conveniently handled through solid-phase synthesis allowing modification in the P3 region that eventually led to simpler derivatives with increased potency against caspase 3. The key to such an effect is the introduction of hydroxyl group alpha to the P3 carbonyl.     

Expression of a modified ADP-glucose pyrophosphorylase large subunit in wheat seeds stimulates photosynthesis and carbon metabolism

ADP-glucose pyrophosphorylase (AGP) is the rate-limiting step in seed starch biosynthesis. Expression of an altered maize AGP large subunit (Sh2r6hs) in wheat (Triticum aestivum L.) results in increased AGP activity in developing seed endosperm and seed yield. The yield phenotype involves increases in both seed number and total plant biomass. Here we describe stimulation of photosynthesis by the seed-specific Sh2r6hs transgene. Photosynthetic rates were increased in Sh2r6hs-expressing plants under high light but not low light growth conditions, peaking at roughly 7 days after flowering (DAF). In addition, there were significant increases in levels of fructose, glucose, and sucrose in flag leaves at both 7 and 14 DAF. In seeds, levels of carbon metabolites at 7 and 14 DAF were relatively unchanged but increases in glucose, ADP-glucose, and UDP-glucose were observed in seeds from Sh2r6hs positive plants at maturity. Increased photosynthetic rates relatively early in seed development appear to be key to the Sh2r6hs enhanced yield phenotype as no yield increase or photosynthetic rate changes were found when plants were grown in a suboptimal light environment. These findings demonstrate that stimulation of biochemical events in both source and sink tissues is associated with Sh2r6hs expression.     

Inhibition of Notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett's-like metaplasia via KLF4

Barrett's esophagus (BE) is defined as an incomplete intestinal metaplasia characterized generally by the presence of columnar and goblet cells in the formerly stratified squamous epithelium of the esophagus. BE is known as a precursor for esophageal adenocarcinoma. Currently, the cell of origin for human BE has yet to be clearly identified. Therefore, we investigated the role of Notch signaling in the initiation of BE metaplasia. Affymetrix gene expression microarray revealed that BE samples express decreased levels of Notch receptors (NOTCH2 and NOTCH3) and one of the the ligands (JAG1). Furthermore, BE tissue microarray showed decreased expression of NOTCH1 and its downstream target HES1. Therefore, Notch signaling was inhibited in human esophageal epithelial cells by expression of dominant-negative-Mastermind-like (dnMAML), in concert with MYC and CDX1 overexpression. Cell transdifferentiation was then assessed by 3D organotypic culture and evaluation of BE-lineage specific gene expression. Notch inhibition promoted transdifferentiation of esophageal epithelial cells toward columnar-like cells as demonstrated by increased expression of columnar keratins (K8, K18, K19, K20) and glandular mucins (MUC2, MUC3B, MUC5B, MUC17) and decreased expression of squamous keratins (K5, K13, K14). In 3D culture, elongated cells were observed in the basal layer of the epithelium with Notch inhibition. Furthermore, we observed increased expression of KLF4, a potential driver of the changes observed by Notch inhibition. Interestingly, knockdown of KLF4 reversed the effects of Notch inhibition on BE-like metaplasia. Overall, Notch signaling inhibition promotes transdifferentiation of esophageal cells toward BE-like metaplasia in part via upregulation of KLF4. These results support a novel mechanism through which esophageal epithelial transdifferentiation promotes the evolution of BE.     

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin–Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.     

Increased Resistance to Penicillium Seed Rot in Transgenic Wheat Over‐expressing Puroindolines

Puroindolines (PINs) are the main components of the wheat grain hardness locus (Ha) and have in vitro antimicrobial activity against bacteria and fungi. Here, we examined the effect of variation in PINA and/or PINB content upon Penicillium sp. seed fungal growth inhibition. The Penicillium sp. assays were germination assays performed after incubating seeds in Penicillium sp. contaminated soil. The first set of wheat genotypes consisted of two sets of transgenic isolines created in the varieties ‘Bobwhite’ and ‘Hi‐Line’ having over‐expression of PINA and/or PINB. The second set of genotypes consisted of near‐isogenic lines (NILs) varying for mutations in PINA or PINB created in the varieties ‘Explorer’ and ‘Hank’. After incubation in Penicillium sp.‐infected soil, transgenic wheat seeds over‐expressing PINA in both ‘Hi‐Line’ and ‘Bobwhite’ and both PINs in ‘Hi‐Line’ exhibited significantly reduced fungal infection and increased germination. No significant differences in Penicillium sp. infection or germination rates were observed in seeds of the NILs. The results indicate that puroindolines native role in seeds is to increase seed viability and that when over‐expressed as transgenes, the puroindolines are effective antifungal proteins.