Genetic variation of Trigonobalanus verticillata,a primitive species of Fagaceae,in Malaysia revealed by chloroplast sequences and AFLP markers

The genetic variation of Trigonobalanus verticillata, the most recently described genus of Fagaceae, was studied using chloroplast DNA sequences and AFLP fingerprinting. This species has a restricted distribution that is known to include seven localities in tropical lower montane forests in Malaysia and Indonesia. A total of 75 individuals were collected from Bario, Kinabalu, and Fraser's Hill in Malaysia. The sequences of rbcL, matK, and three non-coding regions (atpB-rbcL spacer, trnL intron, and trnL-trnF spacer) were determined for 19 individuals from these populations. We found a total of 30 nucleotide substitutions and four length variations, which allowed identification of three haplotypes characterizing each population. No substitutions were detected within populations, while the tandem repeats in the trnL -trnF spacer had a variable repeat number of a 20-bp motif only in Kinabalu. The differentiation of the populations inferred from the cpDNA molecular clock calibrated with paleontological data was estimated to be 8.3 MYA between Bario and Kinabalu, and 16.7 MYA between Fraser's Hill and the other populations. In AFLP analysis, four selective primer pairs yielded a total of 431 loci, of which 340 (78.9%) were polymorphic. The results showed relatively high gene diversity (H(S) = 0.153 and H(T) = 0.198) and nucleotide diversity (pi(S) = 0.0132 and pi(T) = 0.0168) both within and among the populations. Although the cpDNA data suggest that little or no gene flow occurred between the populations via seeds, the fixation index estimated from AFLP data (F(ST) = 0.153 and N(ST) = 0.214) implies that some gene flow occurs between populations, possibly through pollen transfer.     

Asymmetrical,water-soluble phthalocyanine dyes for covalent labeling of oligonucleotides

Two new water-soluble, porphyrazine (Pz) dyes containing an isothiocyanate function for covalent linking have each been prepared by cross condensation of two different aromatic dinitriles, one containing carboxylates for solubilizing purposes and the other containing a nitro group for conversion into the labeling function. The initial mononitrotricarboxylato Pzs have been purified to homogeneity from the mixture of Pz congeners formed in the condensation reaction by anion exchange chromatography. The phthalocyanine dye 1 has an absorption maxima at 683 nm while the trinaphthoporphyrazine dye 2 has an absorption maxima at 755 nm, due to the increased size of the aromatic system. Both dyes were successfully conjugated to oligonucleotide primers, showing their potential for use in near-infrared-based DNA diagnostic applications.     

Studies of Pseudomonas aeruginosa azurin mutants: cavities in beta-barrel do not affect refolding speed

Pseudomonas aeruginosa azurin is a blue-copper protein with a Greek-key fold. Removal of copper produces an apoprotein with the same structure as holoazurin. To address the effects on thermodynamic stability and folding dynamics caused by small cavities in a beta-barrel, we have studied the behavior of the apo-forms of wild-type and two mutant (His-46-Gly and His-117-Gly) azurins. The equilibrium- and kinetic-folding and unfolding reactions appear as two-state processes for all three proteins. The thermodynamic stability of the two mutants is significantly decreased as compared with the stability of wild-type azurin, in accord with cavities in or near the hydrophobic interior having an overall destabilizing effect. Large differences are also found in the unfolding rates: the mutants unfold much faster than wild-type azurin. In contrast, the folding-rate constants are almost identical for the three proteins and closely match the rate-constant predicted from the native-state topology of azurin. We conclude that the topology is more important than equilibrium stability in determining the folding speed of azurin.     

Effect of high oxygen on placental function in short-term explant cultures

Ex situ culture of human gestational tissues has been routinely used as a model to investigate tissue function. The objective of this study was to determine the effect of varying oxygen concentrations on human term placental explants over a 24-h time period. Specifically, the effect of incubating placental explants in oxygen concentrations of 8%, 21% or 95% on tissue viability, metabolism and cell death was measured by assessing glucose consumption, lactate production, release of lactate dehydrogenase, parathyroid hormone-related protein (PTHrP), tumour necrosis factor-alpha (TNF-α) and 8-isoprostane, immunoreactivity for cleaved-caspase-9 and immunohistochemistry for the caspase-3-cleaved cytokeratin-18 neoepitope, M30. Exposure to higher oxygen concentrations significantly increased the rates of glucose consumption and lactate production. Apoptosis was significantly increased under conditions of higher oxygen as evidenced by increased M30 in placental explant sections. Similarly, hyperoxia significantly increased the releases of PTHrP, TNF-α and 8-isoprostane. Thus, incubation of placental explants with oxygen concentrations of 95% and, to a lesser extent, 21% oxygen was associated with the modulation of multiple cellular response pathways including those associated with tissue viability and cell death. These data are consistent with the hypothesis that hyperoxia activates pathways and mechanisms involved in cellular metabolism, necrosis and apoptosis, thereby shifting the balance from a steady state towards cell death.     

DNA sequencing: bench to bedside and beyond

Fifteen years elapsed between the discovery of the double helix (1953) and the first DNA sequencing (1968). Modern DNA sequencing began in 1977, with development of the chemical method of Maxam and Gilbert and the dideoxy method of Sanger, Nicklen and Coulson, and with the first complete DNA sequence (phage ϕX174), which demonstrated that sequence could give profound insights into genetic organization. Incremental improvements allowed sequencing of molecules >200 kb (human cytomegalovirus) leading to an avalanche of data that demanded computational analysis and spawned the field of bioinformatics. The US Human Genome Project spurred sequencing activity. By 1992 the first ‘sequencing factory’ was established, and others soon followed. The first complete cellular genome sequences, from bacteria, appeared in 1995 and other eubacterial, archaebacterial and eukaryotic genomes were soon sequenced. Competition between the public Human Genome Project and Celera Genomics produced working drafts of the human genome sequence, published in 2001, but refinement and analysis of the human genome sequence will continue for the foreseeable future. New ‘massively parallel’ sequencing methods are greatly increasing sequencing capacity, but further innovations are needed to achieve the ‘thousand dollar genome’ that many feel is prerequisite to personalized genomic medicine. These advances will also allow new approaches to a variety of problems in biology, evolution and the environment.