Total evidence phylogeny of Gasterosteidae: combining molecular, morphological and behavioral data

Genealogical relationships among Gasterosteidae (Teleostei: Gasterosteiformes) were tested with 84 morphological, 48 behavioral, and 2879 molecular characters. Phylogenetic analysis of the combined data set identified a single (CI = 0.735) best‐supported hypothesis (Spinachia (Apeltes ((Pungitius + Culaea)(Gasterosteus aculeatus + G. wheatlandi)))). This hypothesis is identical to previous phylogenetic propositions proposed on the basis of behavioral, and behavioral plus morphological data. Our hypothesis, however, differed from a molecular‐based phylogeny in the placement of Apeltes. This analysis highlights the importance of combining all available evidence in order to produce the best‐supported proposition of genealogical relationships.     

Phosphorus and intraspecific density alter plant responses to arbuscular mycorrhizas

We investigated how phosphorus availability, intraspecific density, and their interaction affect plant responses to arbuscular mycorrhizas. Four facultatively mycotrophic species: chile, cilantro, tomato, and corn were examined separately in pot experiments that employed a tropical phosphorus-immobilizing soil. Each experiment comprised nine soluble phosphorus additions, two levels of intraspecific plant density, and inoculation with arbuscular mycorrhizal fungi or not. High phosphorus signi- ficantly diminished mycorrhizal colonization of corn, cilantro, and tomato, but not chile, which was highly variably colonized. Corn roots were colonized by other root-inhabiting fungi, and mycorrhizas significantly reduced colonization by these potential root parasites. High phosphorus significantly increased relative growth rates (RGR) of all species, and high density significantly reduced RGR of cilantro, tomato, and corn. Chile showed little growth at any but the highest phosphorus additions, and consequently had no RGR response to density or mycorrhizas. Mycorrhiza inoculation caused transient depression of corn growth during the first month, but mycorrhizas increased corn RGR during the second month of growth. Both RGR and dry weights at harvest, cilantro, tomato, and corn benefited from mycorrhizas at low phosphorus availability, but this benefit diminished or changed to disadvantage as phosphorus availability increased. At low phosphorus availability, high density increased the dry weight of mycorrhizal cilantro and thereby amplified the benefit of mycorrhizas. At high phosphorus availability, increased density diminished the effects of mycorrhizas on dry weight, reducing mycorrhiza benefit to tomato and chile and reducing mycorrhiza detriment to cilantro. This study demonstrates that for three of the four plant species examined, phosphorus availability, intraspecific density, and their interaction significantly modify plant responses to arbuscular mycorrhizas.     

Drug recognition of a DNA single strand break: nogalamycin intercalation between coaxially stacked hairpins.

Two DNA hairpin motifs (5'-GCGAAGC-3' and 5'-ACGA AGT-3'), both stabilized by a 5'-GAA loop, have been used to design novel intramolecular double hairpin structures (5'-GCGAAGCACGAAGT-3' and 5'-ACGAAGTGCG AAGC-3') in which coaxial stacking of the two hairpin components generates a double-stranded stem region effectively with a single-strand break in the middle of the sequence at either the TG or CA step between unconnected 3' and 5' terminal bases. We have investigated by NMR the conformation and dynamics of the DNA at the strand break site. We show that mutual stacking significantly enhances the stability of each hairpin. Further, the anthracycline antibiotic nogalamycin binds cleanly to the 5'-TG (5'-CA) site formed by the mutually stacked hairpins despite the break in the sugar-phosphate backbone on one strand. The complex resembles the structure of nogalamycin-DNA complexes with the drug bound at 5'-TG sites in intact duplex sequences, with pi-stacking interactions probably the single dominant stabilizing interaction.     

Localization of endothelial nitric-oxide synthase phosphorylated on serine 1179 and nitric oxide in Golgi and plasma membrane defines the existence of two pools of active enzyme.

The subcellular localization of endothelial nitric-oxide synthase (eNOS) is critical for optimal coupling of extracellular stimulation to nitric oxide production. Because eNOS is activated by Akt-dependent phosphorylation to produce nitric oxide (NO), we determined the subcellular distribution of eNOS phosphorylated on serine 1179 using a variety of methodologies. Based on sucrose gradient fractionation, phosphorylated-eNOS (P-eNOS) was found in both caveolin-1-enriched membranes and intracellular domains. Co-transfection of eNOS with Akt and stimulation of endothelial cells with vascular endothelial growth factor (VEGF) increased the ratio of P-eNOS to total eNOS but did not change the relative intracellular distribution between these domains. The proper localization of eNOS to intracellular membranes was required for agonist-dependent phosphorylation on serine 1179, since VEGF did not increase eNOS phosphorylation in cells transfected with a non-acylated, mistargeted form of eNOS. Confocal imaging of P-eNOS and total eNOS pools demonstrated co-localization in the Golgi region and plasmalemma of transfected cells and native endothelial cells. Finally, VEGF stimulated a large increase in NO localized in both the perinuclear region and the plasma membrane of endothelial cells. Thus, activated, phosphorylated eNOS resides in two cellular compartments and both pools are VEGF-regulated to produce NO.     

Local and global cerebral blood flow and glucose utilization in the α-galactosidase A knockout mouse model of Fabry disease

Fabry disease is an X-linked lysosomal disorder characterized by deficient alpha-galactosidase A activity and intracellular accumulations of glycosphingolipids, mainly globotriaosylceramide (Gb3). Clinically, patients occasionally present CNS dysfunction. To examine the pathophysiology underlying brain dysfunction, we examined glucose utilization (CMR(glc)) and cerebral blood flow (CBF) globally and locally in 18 brain structures in the alpha-galactosidase A gene knockout mouse. Global CMR(glc) was statistically significantly reduced by 22% in Fabry mice (p < 0.01). All 18 structures showed decreases in local CMR(glc) ranging from 14% to 33%. The decreases in all structures of the diencephalon, caudate-putamen, brain stem, and cerebellar cortex were statistically significant (p < 0.05). Global cerebral blood flow (CBF) and local CBF measured in the same 18 structures were lower in Fabry mice than in control mice, but none statistically significantly. Histological examination of brain revealed no cerebral infarcts but abundant Gb3 deposits in the walls of the cerebral vessels with neuronal deposits localized to the medulla oblongata. These results indicate an impairment in cerebral energy metabolism in the Fabry mice, but one not necessarily due to circulatory insufficiency.     

Sucrose Phosphate Synthase Activity Rises in Correlation with High-Rate Cellulose Synthesis in Three Heterotrophic Systems

Based on work with cotton fibers, a particulate form of sucrose (Suc) synthase was proposed to support secondary wall cellulose synthesis by degrading Suc to fructose and UDP-glucose. The model proposed that UDP-glucose was then channeled to cellulose synthase in the plasma membrane, and it implies that Suc availability in cellulose sink cells would affect the rate of cellulose synthesis. Therefore, if cellulose sink cells could synthesize Suc and/or had the capacity to recycle the fructose released by Suc synthase back to Suc, cellulose synthesis might be supported. The capacity of cellulose sink cells to synthesize Suc was tested by analyzing the Suc phosphate synthase (SPS) activity of three heterotrophic systems with cellulose-rich secondary walls. SPS is a primary regulator of the Suc synthesis rate in leaves and some Suc-storing, heterotrophic organs, but its activity has not been previously correlated with cellulose synthesis. Two systems analyzed, cultured mesophyll cells of Zinnia elegans L. var. Envy and etiolated hypocotyls of kidney beans (Phaseolus vulgaris), contained differentiating tracheary elements. Cotton (Gossypium hirsutum L. cv Acala SJ-1) fibers were also analyzed during primary and secondary wall synthesis. SPS activity rose in all three systems during periods of maximum cellulose deposition within secondary walls. The Z. elegans culture system was manipulated to establish a tight linkage between the timing of tracheary element differentiation and rising SPS activity and to show that SPS activity did not depend on the availability of starch for degradation. The significance of these findings in regard to directing metabolic flux toward cellulose will be discussed.