An enhanced monomeric blue fluorescent protein with the high chemical stability of the chromophore

Commonly used monomeric blue fluorescent proteins suffer from moderate brightness. The brightest of them, mTagBFP, has a notably low chemical stability over time. Prolonged incubation of mTagBFP leads to its transition from a blue fluorescent state with absorbance at 401 nm to a non-fluorescent state with absorbance at 330 nm. Here, we have determined the chemical structure of the degraded product of the blue mTagBFP-like chromophore. On the basis of mTagBFP we have developed an improved variant, named mTagBFP2. mTagBFP2 exhibits 2-fold greater chemical stability and substantially higher brightness in live cells than mTagBFP. mTagBFP2 is also 1.2-fold and 1.7-fold more photostable than mTagBFP in widefield and confocal microscopy setups, respectively. mTagBFP2 maintains all other beneficial properties of the parental mTagBFP including the high pH stability and fast chromophore formation. The enhanced photostability and chromophore chemical stability of mTagBFP2 make it a superior protein tag. mTagBFP2 performs well in the numerous protein fusions and surpasses mTagBFP as a donor in Förster resonance energy transfer with several green fluorescent protein acceptors.     

APP substitutions V715F and L720P alter PS1 conformation and differentially affect Abeta and AICD generation

The 37-43 amino acid Abeta peptide is the principal component of beta-amyloid deposits in Alzheimer's disease (AD) brain, and is derived by serial proteolysis of the amyloid precursor protein (APP) by beta- and gamma-secretase. gamma-Secretase also cleaves APP at Val50 in the Abeta numbering (epsilon cleavage), resulting in the release of a fragment called APP intracellular domain (AICD). The aim of this study was to determine whether amino acid substitutions in the APP transmembrane domain differentially affect Abeta and AICD generation. We found that the APPV715F substitution, which has been previously shown to dramatically decrease Abeta40 and Abeta42 while increasing Abeta38 levels, does not affect in vitro generation of AICD. Furthermore, we found that the APPL720P substitution, which has been previously shown to prevent in vitro generation of AICD, completely prevents Abeta generation. Using a fluorescence resonance energy transfer (FRET) method, we next found that both the APPV715F and APPL720P substitutions significantly increase the distance between the N- and C-terminus of presenilin 1 (PS1), which has been proposed to contain the catalytic site of gamma-secretase. In conclusion, both APPV715F and APPL720P change PS1 conformation with differential effects on Abeta and AICD production.     

Increased sedoheptulose-1,7-bisphosphatase activity in transgenic tobacco plants stimulates photosynthesis and growth from an early stage in development

Activity of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase) was increased by overexpression of an Arabidopsis (Arabidopsis thaliana) cDNA in tobacco (Nicotiana tabacum) plants. In plants with increased SBPase activity, photosynthetic rates were increased, higher levels of Suc and starch accumulated during the photoperiod, and an increase in leaf area and biomass of up to 30% was also evident. Light saturated photosynthesis increased with increasing SBPase activity and analysis of CO2 response curves revealed that this increase in photosynthesis could be attributed to an increase in ribulose 1,5-bisphosphate regenerative capacity. Seedlings with increased SBPase activity had an increased leaf area at the 4 to 5 leaf stage when compared to wild-type plants, and chlorophyll fluorescence imaging of these young plants revealed a higher photosynthetic capacity at the whole plant level. Measurements of photosynthesis, made under growth conditions integrated over the day, showed that mature plants with increased SBPase activity fixed 6% to 12% more carbon than equivalent wild-type leaves, with the young leaves having the highest rates. In this paper, we have shown that photosynthetic capacity per unit area and plant yield can be increased by overexpressing a single native plant enzyme, SBPase, and that this gives an advantage to the growth of these plants from an early phase of vegetative growth. This work has also shown that it is not necessary to bypass the normal regulatory control of SBPase, exerted by conditions in the stroma, to achieve improvements in carbon fixation.     

Expression of silicatein in spicules from the Baikalian sponge Lubomirskia baicalensis

Lake Baikal harbors the largest diversity of sponge species [phylum Porifera] among all freshwater biotopes. The abundantly occurring species Lubomirskia baicalensis was used to study the seasonal silicatein metabolism; the spicules of this species have an unusually thick axial filament, consisting of silicatein, which remains constant in diameter during their growth. In the course of maturation, the size of the silicic acid shell grows, until the final diameter of the spicules of about 8 microm is reached. The seasonal content of silicatein was assessed by use of antibodies raised against silicatein; they stained specifically the axial filaments. In addition we determined, by application of the enzyme-linked immunosorbent assay system, that the proteinaceous content of the spicules, the silicatein, increases from spring to late summer by 8-fold. As molecular markers to quantify the seasonal changes in expression levels of genes coding for proteins/enzymes, the genes for the calumenin-like protein and the kinesin-related protein, were selected. The expression of calumenin-like gene, involved in the intracellular signaling, is highest during September, whereas the expression of the kinesin-related protein does not change during the annual course. These results suggest that the highest metabolic activity of L. baicalensis occurs in late summer (September), in parallel with the highest accumulation of silicatein, a structural protein/enzyme of the spicules.     

Heterometallic Ni/Zn amine complexes possessing extended 2D and 3D hydrogen-bonded networks prepared from zinc oxide

Five novel heterometallic Ni/Zn coordination compounds [Ni(en)₃][ZnCl₄] (1), [Ni(en)(Hea)₂][ZnCl₄] (2), [Ni(dien)₂][ZnCl₄] (3), [Ni(en)₃][ZnCl₄] · 2DMSO (4) and [Ni(en)₃][Zn(NCS)₄] · CH₃CN (5), where en = ethylenediamine (ethane-1,2-diamine), Hea = monoethanolamine (2-aminoethanol) and dien = diethylenetriamine (1,4,7-triazaheptane), have been synthesized by means of the open-air reaction of zinc oxide, nickel chloride (or nickel powder), NH₄X (X = Cl, NCS) and ligand (en, dien, Hea) in non-aqueous solvents, such as DMSO, DMF, CH₃OH and CH₃CN. The choice of a counter-anion in the initial ammonium salt as well as selection of the ligand and solvent provides an effortless approach to the controlled assembly of two- or three-dimensional extended networks assisted by hydrogen bonding. Crystallographic investigations reveal that 1, 2 and 5 possess 3D, while 3 and 4 exhibit 2D H-bonded crystal structures. The structures of the compounds exhibit six-coordinated Ni(II) centers and four-coordinated Zn(II) centers in distorted octahedral and tetrahedral geometries, respectively.     

Novel spectroscopic technique for in situ monitoring of collagen fibril alignment in gels

Development of collagen fibril alignment in contracting fibroblast-populated and externally tensioned acellular collagen gels was studied using elastic scattering spectroscopy. Spectra of the backscattered light (320-860 nm) were acquired with a 2.75-mm source-detector separation probe placed perpendicular to the gel surface and rotated to achieve different angles to the collagen fibril alignment. Backscatter was isotropic for noncontracted/unloaded gels (disorganized matrix). As gels were contracted/externally loaded (collagen alignment developed), anisotropy of backscatter increased: more backscatter was detected perpendicular than parallel to the direction of the fibril alignment. An "anisotropy factor" (AF) was calculated to characterize this effect as the ratio of backscatter intensities at orthogonal positions. Before contraction (or zero strain) the AF was close to unity at all wavelengths. In contrast, at 72 h, backscatter anisotropy varied from AF(400 nm) = 2.14 +/- 0.29 to AF(700 nm) = 3.04 +/- 0.48. It also increased over threefold up to a strain of 20%. The AF strongly correlated with the contraction time/strain. Different directions of the backscatter were detected in gel zones with known differences in the matrix alignment. Thus, backscatter anisotropy allows in situ nondestructive determination of collagen fibril alignment and quantitative monitoring of its development.     

Screening assays for cholinesterases resistant to inhibition by organophosphorus toxicants

Methods to measure resistance to inhibition by organophosphorus toxicants (OP) for mutants of butyrylcholinesterase (EC 3.1.1.8; BChE) and acetylcholinesterase (EC 3.1.1.7; AChE) enzymes were devised. Wild-type cholinesterases were completely inhibited by 0.1 mM echothiophate or 0.001 mM diisopropylfluorophosphate, but human BChE mutants G117H, G117D, L286H, and W231H and snake AChE mutant HFQT retained activity. Tissues containing a mixture of cholinesterases could be assayed for amount of G117H BChE. For example, the serum of transgenic mice expressing human G117H BChE contained 0.5 microg/ml human G117H BChE, 2 microg/ml wild-type mouse BChE, and 0.06 microg/ml wild-type mouse AChE. The oligomeric structure of G117H BChE in the serum of transgenic mice was determined by nondenaturing gel electrophoresis followed by staining for butyrylthiocholine hydrolysis activity in the presence of 0.1 mM echothiophate. Greater than 95% of the human G117H BChE in transgenic mouse serum was a tetramer. To visualize the distribution of G117H BChE in tissues of transgenic mice, sections of small intestine were treated with echothiophate and then stained for BChE activity. Both wild-type and G117H BChE were in the epithelial cells of the villi. These assays can be used to identify OP-resistant cholinesterases in culture medium and in animal tissues.