Phylogeography and genetic ancestry of tigers (Panthera tigris)

Eight traditional subspecies of tiger (Panthera tigris),of which three recently became extinct, are commonly recognized on the basis of geographic isolation and morphological characteristics. To investigate the species' evolutionary history and to establish objective methods for subspecies recognition, voucher specimens of blood, skin, hair, and/or skin biopsies from 134 tigers with verified geographic origins or heritage across the whole distribution range were examined for three molecular markers: (1) 4.0 kb of mitochondrial DNA (mtDNA) sequence; (2) allele variation in the nuclear major histocompatibility complex class II DRB gene; and (3) composite nuclear microsatellite genotypes based on 30 loci. Relatively low genetic variation with mtDNA,DRB,and microsatellite loci was found, but significant population subdivision was nonetheless apparent among five living subspecies. In addition, a distinct partition of the Indochinese subspecies P. t. corbetti in to northern Indochinese and Malayan Peninsula populations was discovered. Population genetic structure would suggest recognition of six taxonomic units or subspecies: (1) Amur tiger P. t. altaica; (2) northern Indochinese tiger P. t. corbetti; (3) South China tiger P. t. amoyensis; (4) Malayan tiger P. t. jacksoni, named for the tiger conservationist Peter Jackson; (5) Sumatran tiger P. t. sumatrae; and (6) Bengal tiger P. t. tigris. The proposed South China tiger lineage is tentative due to limited sampling. The age of the most recent common ancestor for tiger mtDNA was estimated to be 72,000-108,000 y, relatively younger than some other Panthera species. A combination of population expansions, reduced gene flow, and genetic drift following the last genetic diminution, and the recent anthropogenic range contraction, have led to the distinct genetic partitions. These results provide an explicit basis for subspecies recognition and will lead to the improved management and conservation of these recently isolated but distinct geographic populations of tigers.     

Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor

UTP activates P2Y₂ receptors in both 1321N1 cell transfectants expressing the P2Y₂ receptor and human HT-29 epithelial cells expressing endogenous P2Y₂ receptors with an EC₅₀ of 0.2- 1.0 M. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC₅₀ for UTP-induced receptor desensitization was 0.3 - 1.0 M for both systems). Desensitization and down-regulation of the P2Y₂ nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y₂ receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C-terminal truncation mutants of the P2Y,₂ receptor. In these cells, potent P2Y₂ receptor desensitization was observed after a 5 min exposure to UTP. Full receptor responsiveness returned 5-10 min after removal of UTP. Thapsigargin, an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca²⁺]_i, after addition of desensitizing concentrations of UTP, indicating that P2Y₂ receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca²⁺]_i induced by UTP in 1321N1 cell transfectants expressing the P2Y₂ receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y₂ receptor phosphorylation/dephosphorylation regulate receptor desensitization/resensitization. A 5 min preincubation of 1321N1 cell transfectants with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), reduced the subsequent response to UTP by about 50% whereas co-incubation of PMA with UTP caused a greater inhibition in the response. The protein phosphatases - 1 and -2A inhibitor, okadaic acid, partially blocked resensitization of the receptor. Furthermore, C-terminal truncation mutants of the P2Y₂ receptor that eliminated several potential phosphorylation sites including two for PKC were resistant to UTP-, but not phorbol ester-induced desensitization. Down regulation of protein kinase C isoforms prevented phorbol ester-induced desensitization but had no effect on agonist-induced desensitization of wild type or truncation mutant receptors. These results suggest that phosphorylation of the C-terminus of the P2Y₂ receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y₂ nucleotide receptor desensitization may help optimize a promising cystic fibrosis pharmacotherapy based on the activation of anion secretion in airway epithelial cells by P2Y₂ receptor agonists.     

ANATOMICAL DISTINCTIONS BETWEEN THE PISTILLATE SPIKELETS OF THE SPECIES OF WILD-RICE (ZIZANIA,POACEAE)

The most reliable macromorphological characters that can be used to discriminate between the annual species of the genus Zizania are found in the pistillate spikelet. One aspect of this morphology is a textural dimorphism. The pistillate lemmas and paleas of Z. aquatica are chartaceous (papery) whereas those of Z. palustris are coriaceous (leathery). Pistillate lemmas and paleas of the two perennial species, Z. texana and Z. latifolia, are also chartaceous. To determine the anatomical basis for the nature of this character, pistillate lemmas and paleas were either fixed, treated with hydrofluoric acid, and sectioned; or fresh material was sectioned on a freezing microtome. Those with a chartaceous texture were found to have a single layer of thin-walled, subepidermal fibers whereas those with a coriaceous texture had at least two layers of thick-walled, subepidermal fibers.     

The relationship between Chironomus plumosus burrows and the spatial distribution of pore-water phosphate, iron and ammonium in lake sediments

1. To study the influence of chironomids on the distribution of pore‐water concentrations of phosphate, iron and ammonium, we conducted a laboratory experiment using mesocosms equipped with two‐dimensional pore‐water samplers, filled with lake sediment and populated with different densities of Chironomus plumosus. 2. Specially designed mesocosms were used in the study. A 6‐mm deep space between the front plate and the pore‐water sampler at the back plate was just thick enough to allow the chironomids to live undisturbed, yet thin enough to force all the burrows into a two‐dimensional plane. 3. The courses of the burrows were observed during the experiment as oxidised zones surrounding them, as well as being identified with an X‐ray image taken at the end of the experiment. 4. We investigated the relationship between C. plumosus burrows and spatial patterns of pore‐water composition. Concentrations of the three ions were significantly less around ventilated burrows (54% to 24%), as bioirrigation caused a convective exchange of pore‐water enriched with dissolved species compared with the overlying water, and also because oxygen imported into the sediment resulting in nitrification of ammonium, oxidation of iron(II) and a co‐precipitation of phosphate with Fe(III) oxyhydroxides. 5. In mesocosms with chironomids, new (redox) interfaces occurred with diffusive pore‐water gradients perpendicular to the course of burrows and the site of major phosphate, ammonium and iron(II) release shifted from the sediment surface to the burrow walls.     

Fused dimerization increases expression,solubility, and activity of bacterial dehydratase enzymes

FabA and FabZ are the two dehydratase enzymes in Escherichia coli that catalyze the dehydration of acyl intermediates in the biosynthesis of fatty acids. Both enzymes form obligate dimers in which the active site contains key amino acids from both subunits. While FabA is a soluble protein that has been relatively straightforward to express and to purify from cultured E. coli, FabZ has shown to be mostly insoluble and only partially active. In an effort to increase the solubility and activity of both dehydratases, we made constructs consisting of two identical subunits of FabA or FabZ fused with a naturally occurring peptide linker, so as to force their dimerization. The fused dimer of FabZ (FabZ‐FabZ) was expressed as a soluble enzyme with an ninefold higher activity in vitro than the unfused FabZ. This construct exemplifies a strategy for the improvement of enzymes from the fatty acid biosynthesis pathways, many of which function as dimers, catalyzing critical steps for the production of fatty acids.     

Phytoplankton patchiness in Winam Gulf,Lake Victoria: a study using principal component analysis of in situ fluorescent excitation spectra

1. In order to characterise phytoplankton patchiness at fine scales, a profiling multiwavelength fluorometer was cast at numerous locations throughout Winam Gulf in Lake Victoria to measure fluorescent excitation spectra, which are indicators of both phytoplankton diversity and coloured dissolved organic matter (CDOM). 2. Processing the spectral data with principal component analysis (PCA) revealed that linear combinations of four fundamental ‘base’ spectra could explain almost all of the variation in spectral measurements. Three of the base spectra were associated with spatially distinct patches of phytoplankton containing different species assemblages, while the fourth base spectrum was due to CDOM fluorescence. 3. The locations of the phytoplankton patches were traced to the south‐east of Winam Gulf, the western end of the Rusinga Channel and the open waters of Lake Victoria adjacent to Winam Gulf, respectively. The high CDOM fluorescence was traced mainly to relatively deep water in the Rusinga Channel. 4. The phytoplankton and CDOM patchiness were interpreted in the context of physical and chemical gradients that were measured at the site at the same scale as the spectral data. Strong relationships were found between the gradients in spectral data and other environmental variables, which suggested several underlying explanations for the phytoplankton and CDOM patchiness.     

Why only some plants emit isoprene

Isoprene (2‐methyl‐1,3‐butadiene) is emitted from many plants and it appears to have an adaptive role in protecting leaves from abiotic stress. However, only some species emit isoprene. Isoprene emission has appeared and been lost many times independently during the evolution of plants. As an example, our phylogenetic analysis shows that isoprene emission is likely ancestral within the family Fabaceae (= Leguminosae), but that it has been lost at least 16 times and secondarily gained at least 10 times through independent evolutionary events. Within the division Pteridophyta (ferns), we conservatively estimate that isoprene emissions have been gained five times and lost two times through independent evolutionary events. Within the genus Quercus (oaks), isoprene emissions have been lost from one clade, but replaced by a novel type of light‐dependent monoterpene emissions that uses the same metabolic pathways and substrates as isoprene emissions. This novel type of monoterpene emissions has appeared at least twice independently within Quercus, and has been lost from 9% of the individuals within a single population of Quercus suber. Gain and loss of gene function for isoprene synthase is possible through relatively few mutations. Thus, this trait appears frequently in lineages; but, once it appears, the time available for evolutionary radiation into environments that select for the trait is short relative to the time required for mutations capable of producing a non‐functional isoprene synthase gene. The high frequency of gains and losses of the trait and its heterogeneous taxonomic distribution in plants may be explained by the relatively few mutations necessary to produce or lose the isoprene synthase gene combined with the assumption that isoprene emission is advantageous in a narrow range of environments and phenotypes.     

Glucuronidation of Lipophilic Substrates: Preparation of 3-Benzo[a]pyrenyl-β-D-glucopyranosiduronic Acid in Multimilligram Quantities by Microsomal UDP-Glucuronyl Transferase

convenient method for the enzyraic conversion of multimilli-gram quantities of 3-hydroxybenzo[a]pyrene to 3-benzo[a]pyrenyl-β-D-glucopyranosiduronic acid in 90% yield is described. Commer-cially available freeze-dried rabbit liver microsomes were incubated in the presence of UDPGA, 3-hydroxybenzo[a]pyrene, and Triton X-100 detergent (Figure 1). The course of the biosynthetic reaction was followed by fluorimetry. The glucuronide product was extracted from the acidified incubation supernate with ethyl acetate and the acid function of the glucuronide was utilized in an acid-base extraction procedure to purify the glucuronide from biological and unreacted starting material. The glucuronide pre-cipitated from ethyl acetate and was collected by centrifugation.

High pressure liquid chromatography and spectroscopic techniques were used to verify the structure and purity of 3-benzo[a]-pyrenyl-β-D-glucopyranosiduronic acid.