ESTimating plant phylogeny: lessons from partitioning

Background  

While Expressed Sequence Tags (ESTs) have proven a viable and efficient way to sample genomes, particularly those for which whole-genome sequencing is impractical, phylogenetic analysis using ESTs remains difficult. Sequencing errors and orthology determination are the major problems when using ESTs as a source of characters for systematics. Here we develop methods to incorporate EST sequence information in a simultaneous analysis framework to address controversial phylogenetic questions regarding the relationships among the major groups of seed plants. We use an automated, phylogenetically derived approach to orthology determination called OrthologID generate a phylogeny based on 43 process partitions, many of which are derived from ESTs, and examine several measures of support to assess the utility of EST data for phylogenies.     

Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery

Multiphoton fluorescence photobleaching recovery (MP-FPR) is a technique for measuring the three-dimensional (3D) mobility of fluorescent molecules with 3D spatial resolution of a few microns. A brief, intense flash of mode-locked laser light pulses excites fluorescent molecules via multiphoton excitation in an ellipsoidal focal volume and photobleaches a fraction. Because multiphoton excitation of fluorophores is intrinsically confined to the high-intensity focal volume of the illuminating beam, the bleached region is restricted to a known, three-dimensionally defined volume. Fluorescence in this focal volume is measured with multiphoton excitation, using the attenuated laser beam to measure fluorescence recovery as fresh unbleached dye diffuses in. The time course of the fluorescence recovery signal after photobleaching can be analyzed to determine the diffusion coefficient of the fluorescent species. The mathematical formulas used to fit MP-FPR recovery curves and the techniques needed to properly utilize them to acquire the diffusion coefficients of fluorescently labeled molecules within cells are presented here. MP-FPR is demonstrated on calcein in RBL-2H3 cells, using an anomalous subdiffusion model, as well as in aqueous solutions of wild-type green fluorescent protein, yielding a diffusion coefficient of 8.7 x 10(-7) cm(2)s(-1) in excellent agreement with the results of other techniques.     

Interaction of lead(II) with beta-cyclodextrin in alkaline solutions

Polarographic and UV-spectrophotometric investigations of Pb(II) complex formation with beta-cyclodextrin have showed that the complexation of Pb(II) ions begins at pH >10. The formation of lead(II) 1:1 complex with the beta-cyclodextrin anion was observed at pH 10-11.5. The logarithm of the stability constant of this complex compound is 15.9+/-0.3 (20 degrees C, ionic strength 1.0), and the molar extinction coefficient value is ca. 5500 (lambda(max)=260 nm). With further increase in solution pH the Pb-beta-cyclodextrin complex decomposes and converts to Pb(OH)(2) or Pb(OH)(3)(-) hydroxy-complexes. This process occurs with a decrease in Pb(II) complexation degree. The latter result could be explained by a decrease in the beta-cyclodextrin anion activity. Neither Pb(OH)(2) nor Pb(OH)(3)(-) encapsulation into beta-CD cavity was observed.     

Evidence of two-step deprotonation of D-mannitol in aqueous solution

Deprotonation of D-mannitol was studied in aqueous basic solutions by means of potentiometry and (13)C NMR spectroscopy. Two-step dissociation in the pH range from 12 to 13.8 was shown, and successive dissociation constants K(a1) and K(a2) were determined. In a solution with ionic strength I = 1.0 M (NaOH + NaNO(3)) pK(a1) = 13.1 +/- 0.1 and pK(a2) = 13.8 +/- 0.2. With increasing ionic strength from 0.75 to 3.0 M, both pK(a1) and pK(a2) values decrease. Deprotonation-induced chemical shifts in pH-variable (13)C NMR spectra show that the OH-groups next to internal carbon atoms C-3 and C-4 dissociate to a greater extent compared to OH-groups next to external carbon atoms C-1 and C-6.     

Distribution of MT1 Melatonin Receptor Promoter-Driven RFP Expression in the Brains of BAC C3H/HeN Transgenic Mice

The pineal hormone melatonin activates two G-protein coupled receptors (MT₁ and MT₂) to regulate in part biological functions. The MT₁ and MT₂ melatonin receptors are heterogeneously distributed in the mammalian brain including humans. In the mouse, only a few reports have assessed the expression of the MT₁ melatonin receptor expression using 2-iodomelatonin binding, in situ hybridization and/or polymerase chain reaction (PCR). Here, we described a transgenic mouse in which red fluorescence protein (RFP) is expressed under the control of the endogenous MT₁ promoter, by inserting RFP cDNA at the start codon of MTNR1a gene within a bacterial artificial chromosome (BAC) and expressing this construct as a transgene. The expression of RFP in the brain of this mouse was examined either directly under a fluorescent microscope or immunohistochemically using an antibody against RFP (RFP-MT₁). RFP-MT₁ expression was observed in many brain regions including the subcommissural organ, parts of the ependyma lining the lateral and third ventricles, the aqueduct, the hippocampus, the cerebellum, the pars tuberalis, the habenula and the habenula commissure. This RFP-MT₁ transgenic model provides a unique tool for studying the distribution of the MT₁ receptor in the brain of mice, its cell-specific expression and its function in vivo.     

Vomeronasal/accessory olfactory system and pheromonal recognition

Pregnancy block in mice requires exposure of recently mated females to urinary pheromones of a strange male, and when working with inbred strains this invariably requires urine from an outbred line. The pheromones which induce oestrus and early puberty in mice have been identified as the brevicomins and dihydrothiazoles. Since the same vomeronasal, neural and neuroendocrine pathways are also activated in pregnancy block, these compounds are likely candidates for pregnancy blocking pheromones. However, these relatively simple chemicals lack the capacity to code for differing mouse strains. Since large quantities of the polymorphic major urinary proteins from the lipocalin family found in urine serve as transporters for the dihydrothiazoles and brevicomins, and differ across strains, then these proteins must participate in pheromone recognition in the context of pregnancy block.      

A cross-sectional study of polycyclic aromatic hydrocarbon-DNA adducts and polymorphism of glutathione S-transferases among heavy smokers by race/ethnicity.

Differences in lung cancer risk by race/ethnicity have been observed among smokers. To determine whether these observations might reflect differences in the formation of carcinogen-DNA adducts, we analysed blood specimens (n=151) collected from smokers who were recruited for possible participation in an antioxidant vitamin intervention study. Mononuclear cells were analysed for polycyclic aromatic hydrocarbon (PAH)-DNA adducts by competitive enzyme-linked immunosorbent assay. Genotypes of glutathione S-transferase M1 and P1 (GSTM1 and GSTP1), enzymes involved in the detoxification of PAH metabolites, were determined by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism, respectively. GSTM1 was present in 65 out of 88 (73.4%), 16 out of 32 (50.0%) and 16 out of 29 (54.8%) of African-Americans, Caucasians and Latinos, respectively (p=0.022). Homozygosity for the GSTP1 codon 105 variant was found in 25.6%, 6.3% and 10.0% of African-Americans, Caucasians and Latinos, respectively (p=0.023). Regression analysis of the log-transformed adduct levels confirmed that Caucasian and Latino subjects had lower PAH-DNA adduct levels than African-American subjects, after adjustment for gender, education, alpha-tocopherol and beta-carotene levels, and GSTM1 status. Further adjustment for age and current smoking habits had no impact on these findings. Although crude analysis suggested that the GSTM1-positive genotype may be associated with lower PAH-DNA levels in Caucasians (but not in African-Americans or Latinos), a formal test for interaction between GSTM1 and ethnicity was not significant. We found no association between adduct levels and GSTP1 genotype. Although the mechanism is unclear, ethnic differences in DNA damage levels may in part explain why African-Americans have higher lung cancer incidence rates than other ethnic groups.