Determination of the orientational distribution and orientation factor for transfer between membrane-bound fluorophores using a confocal microscope

Orientational fluorophores have been a useful tool in physical chemistry, biochemistry, and more recently structural biology due to the polarized nature of the light they emit and that fact that energy can be transferred between them. We present a practical scheme in which measurements of the intensity of emitted fluorescence can be used to determine limits on the mean and distribution of orientation of the absorption transition moment of membrane-bound. uorophores. We demonstrate how information about the orientation of. uorophores can be used to calculate the orientation factor k(2) required for use in FRET spectroscopy. We illustrate the method using images of AlexaFluor probes bound to MscL mechanosensitive transmembrane channel proteins in spherical liposomes.

Comparative reproductive biology of two Florida pawpaws Asimina reticulata Chapman and Asimina tetramera Small

I investigated the phenology and breeding systems of two Florida endemic pawpaws, Asimina reticulata, widespread in peninsular Florida, and A. tetramera, a federally endangered species limited to two counties on the Atlantic Coastal Ridge. The purpose of this study was to determine if differences contribute to the rarity of Asimina tetramera compared with A. reticulata. The study was conducted in sand pine scrub sites with the largest populations of A. tetramera in the two counties. Flowering seasons differ for the two species. Both species are hermaphroditic and strongly protogynous. Pollination experiments show that neither species is autogamous and the primary breeding mechanism is outcrossing, although low levels of geitonogamous pollination occur in mature scrub habitats. High levels of inbreeding depression were noted in both species at both sites but inbreeding depression was relaxed the first year post-fire. Fruit set in mature habitats may be pollinator limited. ^ I studied insects associated with the flowers in sand pine scrub habitat in southeastern Florida from 1994–1996. The most commonly represented orders were Coleoptera (25 spp.), Lepidoptera. (3 spp.) and Hymenoptera. (3 spp.). All Coleoptera. were flower visitors; one species, Euphoria sepulchralis (Fabricius)(Scarabeaidae), visited flowers of the two Asimina species at both sites. Eurytides marcellus (Cramer) (Lepidoptera: Papilionidae) eggs and larvae were observed on both species of Asimina during each year of the study. ^ Resource management techniques were applied to a mature sand pine scrub community in Jonathan Dickinson State Park in southeastern Florida for the management of Asimina tetramera. Manipulations conducted in 1996 included combinations of fire and mechanical treatments. I measured effects of these treatments on flowering and fruit set on A. tetramera and found cutting and burning was most effective in increasing flowering, followed by burning. Mechanical cutting and mulching had no significant effect. ^

Stabilization of troponin I for applications in clinical chemistry and forensic medicine. Cardiac troponin I: A time of death marker

Cardiac troponin I (cTnI) is one of the most useful serum marker test for the determination of myocardial infarction (MI). The first commercial assay of cTnI was released for medical use in the United States and Europe in 1995. It is useful in determining if the source of chest pains, whose etiology may be unknown, is cardiac related. Cardiac TnI is released into the bloodstream following myocardial necrosis (cardiac cell death) as a result of an infarct (heart attack). In this research project the utility of cardiac troponin I as a potential marker for the determination of time of death is investigated. The approach of this research is not to investigate cTnI degradation in serum/plasma, but to investigate the proteolytic breakdown of this protein in heart tissue postmortem. If our hypothesis is correct, cTnI might show a distinctive temporal degradation profile after death. This temporal profile may have potential as a time of death marker in forensic medicine. The field of time of death markers has lagged behind the great advances in technology since the late 1850's. Today medical examiners are using rudimentary time of death markers that offer limited reliability in the medico-legal arena. Cardiac TnI must be stabilized in order to avoid further degradation by proteases in the extraction process. Chemically derivatized magnetic microparticles were covalently linked to anti-cTnI monoclonal antibodies. A charge capture approach was also used to eliminate the antibody from the magnetic microparticles given the negative charge on the microparticles. The magnetic microparticles were used to extract cTnI from heart tissue homogenate for further bio-analysis. Cardiac TnI was eluted from the beads with a buffer and analyzed. This technique exploits banding pattern on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a western blot transfer to polyvinylidene fluoride (PVDF) paper for probing with anti-cTnI monoclonal antibodies. Bovine hearts were used as a model to establish the relationship of time of death and concentration/band-pattern given its homology to human cardiac TnI. The final concept feasibility was tested with human heart samples from cadavers with known time of death. ^

Mechanistic study of arsenic uptake, transformation and tolerance in arsenic hyperaccumulator Pteris vittata

Pteris vittata, the first reported arsenic hyperaccumulating plant, is potentially used in phytoremediation of arsenic, as it can accumulate up to 2.3% of arsenic in its fronds. In this study, the mechanisms of arsenic tolerance, uptake and transformation were studied in the plant. Arsenic species were analyzed by HPLC-AFS. Results showed that arsenic was mainly accumulated in leaflets, and inorganic arsenate and arsenite were only species in P. vittata. Arsenite was the predominant species in leaflets, whereas arsenate was the predominant species in roots. Arsenic induced the synthesis of thiol containing compounds in P. vittata. As-induced thiol was purified by a novel method: covalent chromatography following preparative HPLC. The purified thiol was characterized as a phytochelatin with two units (PC2). ^ In P. vittata, enhanced tolerance likely results from unusual intracellular detoxification mechanisms. Although PC-dependent sequestration of arsenic into vacuoles is essential for nonhyperaccumulators, this sequestration is not the major arsenic tolerance mechanisms in this arsenic hyperaccumulator. PC-independent sequestration of arsenic is likely the major arsenic tolerance mechanism. PC-dependent arsenic detoxification is probably a supplement to this major mechanism. ^ Interactions between arsenic and phosphate were studied. Under hydroponic condition, arsenic supply decreased the concentrations of phosphate in roots. In soil, arsenic increased the concentrations of phosphate in roots. Arsenic concentrations in rachises and leaflets were not affected by arsenic supply in either hydroponic or soil system. Phosphate decreased arsenic accumulation in roots, rachises and leaflets in the hydroponic system. ^ The uptake kinetics of arsenate, arsenite, monomethyl arsinic acid (MMA), dimethyl arsonic acid, and phosphate were studied in P. vittata. Phosphate uptake systems in Pteris vittata cannot distinguish phosphate and As(V), resulting in As hyperaccumulation. Arsenic hyperaccumulation in this plant is an inevitable consequence during phosphate acquisition. Arsenate, arsenite and MMA are transported via the phosphate uptake systems. The co-transport of arsenite/phosphate and MMA/phosphate is reported for the first time in plants. These unique phenomena are useful for understanding arsenic hyperaccumulation and the evolution of this capacity in P. vittata. ^

Multimode Analysis of Nanoscale Biomolecular Interactions

Biomolecular interactions, including protein-protein, protein-DNA, and protein-ligand interactions, are of special importance in all biological systems. These interactions may occer during the loading of biomolecules to interfaces, the translocation of biomolecules through transmembrane protein pores, and the movement of biomolecules in a crowded intracellular environment. The molecular interaction of a protein with its binding partners is crucial in fundamental biological processes such as electron transfer, intracellular signal transmission and regulation, neuroprotective mechanisms, and regulation of DNA topology. In this dissertation, a customized surface plasmon resonance (SPR) has been optimized and new theoretical and label free experimental methods with related analytical calculations have been developed for the analysis of biomolecular interactions. Human neuroglobin (hNgb) and cytochrome c from equine heart (Cyt c) proteins have been used to optimize the customized SPR instrument. The obtained Kd value (~13 µM), from SPR results, for Cyt c-hNgb molecular interactions is in general agreement with a previously published result. The SPR results also confirmed no significant impact of the internal disulfide bridge between Cys 46 and Cys 55 on hNgb binding to Cyt c. Using SPR, E. coli topoisomerase I enzyme turnover during plasmid DNA relaxation was found to be enhanced in the presence of Mg2+. In addition, a new theoretical approach of analyzing biphasic SPR data has been introduced based on analytical solutions of the biphasic rate equations. In order to develop a new label free method to quantitatively study protein-protein interactions, quartz nanopipettes were chemically modified. The derived Kd (~20 µM) value for the Cyt c-hNgb complex formations matched very well with SPR measurements (Kd ~16 µM). The finite element numerical simulation results were similar to the nanopipette experimental results. These results demonstrate that nanopipettes can potentially be used as a new class of a label-free analytical method to quantitatively characterize protein-protein interactions in attoliter sensing volumes, based on a charge sensing mechanism. Moreover, the molecule-based selective nature of hydrophobic and nanometer sized carbon nanotube (CNT) pores was observed. This result might be helpful to understand the selective nature of cellular transport through transmembrane protein pores.

Lasting mantle scars lead to perennial plate tectonics

R.N.P. and P.J.H. are grateful for funding from an NSERC Discovery Grant. Computations were performed on the GPC supercomputer at the SciNet HPC Consortium. SciNet is funded by the Canada Foundation for Innovation under the auspices of Compute Canada, the Government of Ontario, Ontario Research Fund—Research Excellence and the University of Toronto. Numerical calculations were done using a modified version of the SOPALE (2000) software. The SOPALE modelling code was originally developed by Philippe Fullsack at Dalhousie University with Chris Beaumont and his Geodynamics group.

Oligodeoxynucleotide synthesis using protecting groups and a linker cleavable under non-nucleophilic conditions

Oligodeoxynucleotides (ODNs) containing latent electrophilic groups can be highly useful in antisense drug development and many other applications such as chemical biology and medicine, where covalent cross-linking of ODNs with mRNA, protein and ODN is required. However, such ODN analogues cannot be synthesized using traditional technologies due to the strongly nucleophilic conditions used in traditional deprotection/cleavage process. To solve this long lasting and highly challenging problem in nucleic acid chemistry, I used the 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) function to protect the exo-amino groups on the nucleobases dA, dC and dG, and to design the linker between the nascent ODN and solid support. These protecting groups and linker are completely stable under all ODN synthesis conditions, but can be readily cleaved under non-nucleophilic and nearly neutral conditions. As a result, the new ODN synthesis technology is universally useful for the synthesis of electrophilic ODNs. The dissertation is mainly comprised of two portions. In the first portion, the development of the Dmoc-based linker for ODN synthesis will be described. The construction of the dT-Dmoc-linker required a total of seven steps to synthesize. The linker was then anchored to the solid support―controlled pore glass (CPG). In the second portion, the syntheses of Dmoc-protected phosphoramidites ODN synthesis monomers including Dmoc-dC-amidite, Dmoc-dA-amidite, Dmoc-dG-amidite are described. The protection of dC and dA with 1,3-dithian-2-yl-methyl 4-nitrophenyl carbonate proceeded smoothly giving Dmoc-dC and Dmoc-dA in good yields. However, when the same acylation procedure was applied for the synthesis of Dmoc-dG, very low yield was obtained. This problem was later solved using a highly innovative and environmentally benign procedure, which is expected to be widely useful for the acylation of the exo-amino groups on nucleoside bases. The reactions to convert the Dmoc-protected nucleosides to phosphoramidite monomers proceeded smoothly with high yields. Using the Dmoc phosphoramidite monomers dA, dC, dG and the commercially available dT, and the Dmoc linker, four ODN sequences were synthesized. In all cases, excellent coupling yields were obtained. ODN deprotection/cleavage was achieved by using non-nucleophilic oxidative conditions. The new technology is predicted to be universally useful for the synthesis of ODNs containing one or more electrophilic functionalities.

The decrease of NAD(P)H:quinone oxidoreductase 1 activity and increase of ROS production by NADPH oxidases are early biomarkers in doxorubicin cardiotoxicity

info:eu-repo/semantics/publishedVersion

Cardioprotective Mechanism Of S-nitroso-n-acetylcysteine Via S-nitrosated Betadrenoceptor-2 In The Ldlr-/- Mice.

Previous studies from our group have demonstrated the protective effect of S-nitroso-N-acetylcysteine (SNAC) on the cardiovascular system in dyslipidemic LDLr-/- mice that develop atheroma and left ventricular hypertrophy after 15 days on a high fat diet. We have shown that SNAC treatment attenuates plaque development via the suppression of vascular oxidative stress and protects the heart from structural and functional myocardial alterations, such as heart arrhythmia, by reducing cardiomyocyte sensitivity to catecholamines. Here we investigate the ability of SNAC to modulate oxidative stress and cell survival in cardiomyocytes during remodeling and correlation with β₂-AR signaling in mediating this protection. Ventricular superoxide (O₂⁻) and hydrogen peroxide (H₂O₂) generation was measured by HPLC methods to allow quantification of dihydroethidium (DHE) products. Ventricular histological sections were stained using terminal dUTP nick-end labeling (TUNEL) to identify nuclei with DNA degradation (apoptosis) and this was confirmed by Western blot for cleaved caspase-3 and caspase-7 protein expression. The findings show that O₂⁻ and H₂O₂ production and also cell apoptosis were increased during left ventricular hypertrophy (LVH). SNAC treatment reduced oxidative stress during on cardiac remodeling, measured by decreased H₂O₂ and O₂⁻ production (65% and 52%, respectively), and a decrease in the ratio of p-Ser1177 eNOS/total eNOS. Left ventricle (LV) from SNAC-treated mice revealed a 4-fold increase in β₂-AR expression associated with coupling change to Gi; β₂-ARs-S-nitrosation (β₂-AR-SNO) increased 61%, while apoptosis decreased by 70%. These results suggest that the cardio-protective effect of SNAC treatment is primarily through its anti-oxidant role and is associated with β₂-ARs overexpression and β₂-AR-SNO via an anti-apoptotic pathway.

The inducing NO-vasodilation by chemical reduction of coordinated nitrite ion in cis-[Ru(NO(2))L(bpy)(2)](+) complex

The synthesis of [Ru(NO(2)) L(bpy)(2)](+) (bpy = 2,2'-bipyridine and L = pyridine (py) and pyrazine (pz)) can be accomplished by addition of [Ru(NO) L(bpy) 2](PF(6))(3) to aqueous solutions of physiological pH. The electrochemical processes of [Ru(NO2) L(bpy) 2]+ in aqueous solution were studied by cyclic voltammetry and differential pulse voltammetry. The anodic scan shows a peak around 1.00 V vs. Ag/AgCl attributed to the oxidation process centered on the metal ion. However, in the cathodic scan a second peak around-0.60 V vs. Ag/AgCl was observed and attributed to the reduction process centered on the nitrite ligand. The controlled reduction potential electrolysis at-0.80 V vs. Ag/AgCl shows NO release characteristics as judged by NO measurement with a NO-sensor. This assumption was confirmed by ESI/MS(+) and spectroelectrochemical experiment where cis-[Ru(bpy)(2)L(H(2)O)](2+) was obtained as a product of the reduction of cis-[Ru(II)(NO(2)) L(bpy)(2)](+). The vasorelaxation observed in denuded aortic rings pre-contracted with 0.1 mu mol L(-1) phenylephrine responded with relaxation in the presence of cis-[RuII(NO2) L(bpy) 2]+. The potential of rat aorta cells to metabolize cis-[RuII(NO(2)) L(bpy)(2)](+) was also followed by confocal analysis. The obtained results suggest that NO release happens by reduction of cis-[RuII(NO(2)) L(bpy)(2)](+) inside the cell. The maximum vasorelaxation was achieved with 1 x 10(-5) mol L(-1) of cis-[RuII(NO(2)) L(bpy)(2)](+) complex.

Vascular Endothelial Growth Factor Genetic Polymorphisms and Haplotypes in Women with Migraine

Vascular endothelial growth factor (VEGF) production is regulated by growth factors and inflammatory cytokines, and VEGF plays a role in migraine. We examined for the first time whether three functional polymorphisms in the promoter region of VEGF gene (C(-2578)A, G(-1154A), and G(-634C)) and VEGF haplotypes are associated with migraine. We studied 114 healthy women without migraine and 175 women with migraine (129 without aura, and 46 with aura). We found no differences in the distributions of VEGF genotypes and alleles (p > 0.05). However, the CAC haplotype was more frequent in controls than in migraine patients, and the AGC haplotype was more frequent in patients with migraine with aura than in controls (both p < 0.05). These findings suggest that VEGF haplotypes affect susceptibility to migraine.

Male sleeping aggregations of solitary oil-collecting bees in Brazil (Centridini, Tapinotaspidini, and Tetrapediini; Hymenoptera: Apidae)

Males of solitary bees usually spend the night in clusters on small branches of plants, cavities and flowers. The individuals usually return to the same location each evening during their life, exhibiting site fidelity to a particular plant. We report on the sleeping roosts of the males of some oil-collecting bees of the genera Centris, Paratetrapedia, Lanthanomelissa, Monoeca, and Tetrapedia, as well as the host plants. We discuss the role of the male clusters to the associated plants.

The novel mitochondrial gene arrangement of the cattle tick, Boophilus microplus: Fivefold tandem repetition of a coding region

We sequenced across all of the gene boundaries in the mitochondrial genome of the cattle tick, Boophilus microplus, to determine the arrangement of its genes. The mtDNA of B. microplus has a coding region, composed of tRNA(Glu) and 60 bp of the 3' end of ND1, that is repeated five times. Boophilus microplus is the first coelomate animal known to have more than two copies of a coding sequence. The mitochondrial genome of B, microplus has other unusual features, including (1) reduced T arms in tRNAs, (2) an AT bias in codon use, (3) two control regions that have evolved in concert, (4) three gene rearrangements, and (5) a stem-loop between tRNA(Gln) and tRNA(Phe). The short T arms and small control regions (CRs) of B. microplus and other ticks suggest strong selection for small genomes. Imprecise termination of replication beyond its origin, which can account for the evolution of tandem repeats of coding regions in other mitochondrial genomes, cannot explain the evolution of the fivefold repeated sequence in the mitochondrial genome of B. microplus. Instead, slipped-strand mispairing or recombination are the most plausible explanations for the evolution of these tandem repeats.

An "openable," high-strength gradient set for orthopedic MRI

A novel three-axis gradient set and RF resonator for orthopedic MRT has been designed and constructed. The set is openable and may be wrapped around injured joints. The design methodology used was the minimization of magnetic field spherical harmonics by simulated annealing. Splitting of the longitudinal coil presents the major design challenge to a fully openable gradient set and in order to efficiently design such coils, we have developed a new fast algorithm for determining the magnetic field spherical harmonics generated by an are of multiturn wire. The algorithm allows a realistic impression of the effect of split longitudinal designs. A prototype set was constructed based on the new designs and tested in a 2-T clinical research system. The set generated 12 mT/m/A with a linear region of 12 cm and a switching time of 100 mu s, conforming closely with theoretical predictions. Preliminary images from the set are presented. (C) 1999 Academic Press.

Patterns of variation within self-incompatibility loci

Diverse self-incompatibility (SI) mechanisms permit flowering plants to inhibit fertilization by pollen that express specificities in common with the pistil. Characteristic of at least two model systems is greatly reduced recombination across large genomic tracts surrounding the S-locus, which regulates SI. In three angiosperm families, including the Solanaceae, the gene that controls the expression of gametophytic SI in the pistil encodes a ribonuclease (S-RNase). The gene that controls pollen SI expression is currently unknown, although several candidates have recently been proposed. Although each candidate shows a high level of polymorphism and complete allelic disequilibrium with the S-RNase gene, such properties may merely reflect tight linkage to the S-locus, irrespective of any functional role in SI. We analyzed the magnitude and nature of nucleotide variation, with the objective of distinguishing likely candidates for regulators of SI from other genes embedded in the S-locus region. We studied the S-RNase gene of the Solanaceae and 48A, a candidate for the pollen gene in this system, and we also conducted a parallel analysis of the regulators of sporophytic SI in Brassica, a system in which both the pistil and pollen genes are known. Although the pattern of variation shown by the pollen gene of the Brassica system is consistent with its role as a determinant of pollen specificity, that of 48A departs from expectation. Our analysis further suggests that recombination between 48A and S-RNase may have occurred during the interval spanned by the gene genealogy, another indication that 48A may not regulate SI expression in pollen.