Fluorescence intensity decays of 2-aminopurine solutions: lifetime distribution approach

The fluorescent adenine analog 2-aminopurine (2AP) has been used extensively to monitor conformational changes and macromolecular binding events involving nucleic acids because its fluorescence properties are highly sensitive to changes in chemical environment. Furthermore, site-specific incorporation of 2AP permits local DNA and RNA conformational events to be discriminated from the global structural changes monitored by UV-Vis spectroscopy and circular dichroism. However, although the steady-state fluorescence properties of 2AP have been well defined in diverse settings, interpretation of 2AP fluorescence lifetime parameters has been hampered by the heterogeneous nature of multiexponential 2AP intensity decays observed across populations of microenvironments. To resolve this problem, we tested the utility of multiexponential versus continuous Lorentzian lifetime distribution models to describe fluorescence intensity decays from 2AP in diverse chemical backgrounds and within the context of RNA. Heterogeneity was introduced into 2AP intensity decays by mixing solvents of differing polarities or by adding quenchers under high viscosity to evaluate the transient effect. Heterogeneity of 2AP fluorescence within the context of a synthetic RNA hairpin was introduced by structural remodeling using a magnesium salt. In each case except folded RNA (which required a bimodal distribution), 2AP lifetime properties were well described by single Lorentzian distribution functions, abrogating the need to introduce additional discrete lifetime subpopulations. Rather, heterogeneity in fluorescence decay processes was accommodated by the breadth of each distribution. This approach also permitted solvent relaxation effects on 2AP emission to be assessed by comparing lifetime distributions at multiple wavelengths. Together, these studies provide a new perspective for the interpretation of 2AP fluorescence lifetime properties that will further the utility of this fluorophore in analyses of the complex and heterogeneous structural microenvironments associated with nucleic acids.     

Structure of the O-polysaccharide and classification of Proteus mirabilis strain G1 in Proteus serogroup O3.

The O-chain polysaccharide of the lipopolysaccharide (LPS) of a previously nonclassified strain of Proteus mirabilis termed G1 was studied by sugar analysis and 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, rotating-frame NOE (ROESY), H-detected 1H,13C HMQC, and heteronuclear multiple-bond correlation (HMBC) experiments. The following structure of the polysaccharide was established: [carbohydrate structure: see text] where D-GalA6(L-Lys) stands for N(alpha)-(D-galacturonoyl)-L-lysine. The structure of the O-polysaccharide of P. mirabilis G1 is similar, but not identical, to that of P. mirabilis S1959 and OXK belonging to serogroup O3. Immunochemical studies with P. mirabilis G1 and S1959 anti-(O-polysaccharide) sera revealed close LPS-based serological relatedness of P. mirabilis G1 and S1959, and therefore it was suggested to classify P. mirabilis G1 in serogroup O3 as a subgroup. P. mirabilis G1 and S1959 anti-(O-polysaccharide) sera also cross-reacted with LPS of P. mirabilis strains from two other serogroups containing D-GalA6(L-Lys) in the O-polysaccharide or in the core region.     

Gene structure and regulation of the murine epithelial calcium channels ECaC1 and 2.

The recently discovered epithelial calcium channels ECaC1 and ECaC2 are thought to play an important role in active calcium absorption in the intestine and kidney. Vitamin D-responsive elements (VDRE) were detected in the promoter sequence of human ECaC1 and regulation of ECaC by the steroid hormone 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) has been postulated. In this study we describe the structure of two murine ECaCs genes, each consisting of 15 exons localized on chromosome 6. Murine ECaC2 expression was found in many target tissues of 1,25-(OH)(2)D(3), including skin and osteoblastic cells, while ECaC1 expression is confined to the kidney. By screening the murine promoter sequences, we detected a putative VDRE in ECaC1 and an estrogen response element in ECaC2. However, experiments in mice with a mutant, nonfunctioning vitamin D receptor showed that expression of ECaC1 in the kidney and of ECaC2 in duodenum is regulated by calcium levels, but not by 1,25-(OH)(2)D(3). Also, estrogen-deficient ovariectomized (OVX) mice and OVX mice supplemented with estradiol showed unchanged duodenal ECaC2 expression compared with control mice. We conclude that ECaC expression in the kidney and the intestine is regulated by extracellular calcium but not by vitamin D or estrogen in vivo in mice.     

Steady-state and time-resolved fluorescence studies of conformational changes induced by cyclic AMP and DNA binding to cyclic AMP receptor protein from Escherichia coli.

cAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes in Escherichia coli. As binding of cAMP leads to undefined conformational changes in CRP, we performed a steady-state and time-resolved fluorescence study to show how the binding of the ligand influences the structure and dynamics of the protein. We used CRP mutants containing a single tryptophan residue at position 85 or 13, and fluorescently labeled with 1,5-I-AEDANS attached to Cys178. Binding of cAMP in the CRP-(cAMP)2 complex leads to changes in the Trp13 microenvironment, whereas its binding in the CRP-(cAMP)4 complex alters the surroundings of Trp85. Time-resolved anisotropy measurements indicated that cAMP binding in the CRP-(cAMP)2 complex led to a substantial increase in the rotational mobility of the Trp13 residue. Measurement of fluorescence energy transfer (FRET) between labeled Cys178 and Trp85 showed that the binding of cAMP in the CRP-(cAMP)2 complex caused a substantial increase in FRET efficiency. This indicates a decrease in the distance between the two domains of the protein from 26.6 A in apo-CRP to 18.7 A in the CRP-(cAMP)2 complex. The binding of cAMP in the CRP-(cAMP)4 complex resulted in only a very small increase in FRET efficiency. The average distance between the two domains in CRP-DNA complexes, possessing lac, gal or ICAP sequences, shows an increase, as evidenced by the increase in the average distance between Cys178 and Trp85 to approximately 20 A. The spectral changes observed provide new structural information about the cAMP-induced allosteric activation of the protein.     

Methodological Aspects of the Potential Use of Dendrochronological Techniques When Analyzing the Long-Term Impact of Tourism on Protected Areas

Intensification of pedestrian tourism causes damage to trees near tourist tracks, and likewise changes the soil structure. As a result, one may expect that annual amount of trees growing near tracks is significantly lower than deeper in the forest. However, during the study of the long-term impact of tourism on the environment (determined from tree increment dynamics), some methodological problems may occur. It is particularly important in protected areas where law and administrative regulations related to nature conservation force research to be conducted using small samples. In this paper we have analyzed the data collected in the Polish part of the Tatra National Park in the two study plots divided into two zones each: the area directly under the influence of the tourist''s trampling and the control group. The aim of such analyses was to present the potential effects of the factors which may affect the results of dendrochronological analysis: (i) small size of samples that affects their representativeness, (ii) spatial differences in the rates of the process, as a result of spatial variability of environmental factors and (iii) temporal differences in the rates of the process. This study confirms that the factors mentioned above could significantly influence the results and should be taken into consideration during the analysis.     

Modeling the formation of root apices

The formation of new root apices from small groups of cells with different cellular patterns has been simulated using an existing model based on growth tensors. To generate an apex, a steady growth field was used. The pattern of cells evolved to approach the steady state. Two extreme types of progressions have been obtained : one leading to an apex with a single or a few apical cells, and the other to an apex with a quiescent centre. The change of structure while applying a steady growth tensor indicates that development may involve a succession of discrete growth tensors.     

Cross-amplification of nonnative Acacia species in the Hawaiian Islands using microsatellite markers from Acacia koa

Thirty-one microsatellite markers, developed from Acacia koa, were evaluated for cross-amplification of seven nonnative species of Acacia in the Hawaiian Islands. The number of alleles per locus ranged from one to seven, with 16 of the 31 markers showing polymorphism in at least two of the nonnative Acacia species. Of the Acacia species examined, A. melanoxylon showed the highest percentage of amplified loci.     

Analysis of immune response: comparison of immunoblots after isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cytoplasmic protein extract from Brucella

Analysis of the immune response towards the facultative intracellular bacterium, Brucella melitensis, was studied by immunoblotting after either isoelectric focusing (IEF) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A cytoplasmic extract (CPE) of Brucella melitensis was used as antigen to analyse the response in 17 sera from naturally infected goats. CPE analysed by IEF exhibited 25 proteins within the pH range of 4.35 to 6. Immunoblotting revealed most of the stained bands around pH 4.5-5.4. CPE analysed by SDS-PAGE showed more than 20 silver stained proteins in the molecular range of 16-18 kDa to 70 kDa but immunoblotting revealed only 1 to 6 bands according to the sera tested. Because proteins are preserved in their native state with IEF, in contrast to SDS-PAGE treatment, this technique may be best suited for analysis of the overall response to natural infection.     

Reversible covalent binding of peptide nitriles to papain

The dissociation constants for reversible covalent binding of twelve peptide nitrile inhibitors to the active site of papain have been measured by means of fluorescence titration. The binding constants generally parallel the kinetic specificity constants (kcat/Km) for related papain substrates, supporting earlier suggestions that peptide nitriles behave as transition state analog inhibitors of papain. In ten cases the temperature dependence of binding was analyzed to determine the enthalpic and entropic contributions to the binding energy. A compensation plot of delta H vs. T delta S resulted in two parallel lines, one for 'specific' nitriles (i.e., N-Ac-L-aa-NHCH2CN; aa = Phe, Leu, Met) and the other for 'non-specific' nitriles (e.g., N-Ac-D-Phe-NHCH2CN, PhCH2CH2CONHCH2CN hippurylnitrile, etc.). For both specific and nonspecific nitriles representing an 1800-fold range of Kd values (0.27 microM-490 microM), the solvent deuterium isotope effect on binding (Kd(H2O)/Kd(D2O) = DKd) was very close to 2.0. This isotope effect could be accounted for entirely by the simple protonic change which occurs upon the reversible addition of the active site sulfhydryl of papain to the nitrile group of the peptide derivative to form a covalent thioimidate linkage. In contrast, six closely related non-nitrile ligands containing identical peptide side chains but having C-terminal groups incapable of binding covalently to papain had unmeasureably high dissociation constants. Collectively, these results indicate that strong binding of peptide nitrile substrate analogs to papain requires a combination of (1) hydrophobic interaction (especially at the P2 position), (2) specific intermolecular hydrogen bonding and (3) covalent interaction of the nitrile with the active site sulfhydryl group.