Phylogeography of Ramalina menziesii,a widely distributed lichen‐forming fungus in western North America

The complex topography and climate history of western North America offer a setting where lineage formation, accumulation and migration have led to elevated inter‐ and intraspecific biodiversity in many taxa. Here, we study Ramalina menziesii, an epiphytic lichenized fungus with a range encompassing major ecosystems from Baja California to Alaska to explore the predictions of two hypotheses: (i) that the widespread distribution of R. menziesii is due to a single migration episode from a single lineage and (ii) that the widespread distribution is due to the formation and persistence of multiple lineages structured throughout the species' range. To obtain evidence for these predictions, we first construct a phylogenetic tree and identify multiple lineages structured throughout the species' range – some ancient ones that are localized and other more recent lineages that are widely distributed. Second, we use an isolation with migration model to show that sets of ecoregion populations diverged from each other at different times, demonstrating the importance of historical and current barriers to gene flow. Third, we estimated migration rates among ecoregions and find that Baja California populations are relatively isolated, that inland California ecoregion populations do not send out emigrants and that migration out of California coastal and Pacific Northwest populations into inland California ecoregions is high. Such intraspecific geographical patterns of population persistence and dispersal both contribute to the wide range of this genetically diverse lichen fungus and provide insight into the evolutionary processes that enhance species diversity of the California Floristic Province.     

Trade and legislation: consequences for the conservation of lichens in the Nepal Himalaya

Lichen harvest and trade are closely associated with the livelihood of most of the rural people in Western Nepal. The present study investigates the commercial collection of lichens, quantifies the traded volume and relates it to a market scenario, and discusses conservation measures in relation to established legal practices in Nepal. Data on lichen trade and revenue generated for the 12 years (2000–2011) were collected and analyzed from 74 districts of Nepal. Voucher specimens were deposited at TUCH (Tribhuvan University Central Herbarium) in Nepal. The lichens collected in West Nepal are mainly used in international trade, while those in East Nepal are used locally for food. A total of 20 commercially important species of lichens were identified from five trade centers and one local market. During 2000–2011, Nepal legally exported 2020 tons of lichens and collected NRs 25,293,305 (USD 240,000). The average annual quantity of turnover was 168 tons, though it is estimated that much was exported illegally. The hill districts in Nepal, which traded 1774 tons, were more important for the collection of commercial lichen species than the Mountainous and inner-Tarai districts, which traded 167 and 108 tons, respectively. Through the Forest Act, Forest Regulations and its amendment in 2011, the collection of lichens for harvest, trade and export in any crude or processed form was banned. However, the legislation lacks an effective implementation strategy, and sustainable harvest of lichen resources based on scientific data would better serve local livelihood and lichen conservation in Nepal.     

Expression of the GM2-activator protein in the methylotrophic yeast Pichia pastoris, purification, isotopic labeling, and biophysical characterization

The GM2-activator protein (GM2AP) belongs to a group of five small, nonenzymatic proteins that are essential cofactors for the degradation of glycosphingolipids in the lysosome. It mediates the interaction between the water-soluble enzyme beta-hexosaminidase A and its membrane-embedded substrate, ganglioside GM2, at the lipid-water interphase. Inherited defects in the gene encoding this glycoprotein cause a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. With the aim to establish a convenient eukaryotic system that allows the efficient production of functionally folded, glycosylated GM2AP and offers the potential of cost-efficient isotopic labeling for structural studies by NMR spectroscopy, we established the expression of recombinant GM2AP in the methylotrophic yeast Pichia pastoris. For the construction of expression plasmids, either the full cDNA encoding human GM2AP preproprotein was cloned in the expression vector pPIC3.5K, or the cDNA encoding only the mature form of GM2AP was inserted in the vector pPIC9K under control of the alcohol oxidase 1 promoter. Both plasmids led to the successful secretory expression of active, glycosylated GM2AP, which could easily be purified by Ni-NTA chromatography due to the hexahistidine tag introduced at the C-terminus. Remarkably, the expression of this membrane-active protein in P. pastoris was accompanied by two peculiarities which were not encountered in other expression systems for GM2AP: First, a significant fraction of the secreted protein existed in the form of aggregates, and second, considerable amounts of noncovalently bound lipids were associated with the recombinant protein. A three-step purification scheme was therefore devised consisting of Ni-NTA, reversed phase, and gel filtration chromatography, which finally yielded 10-12 mg of purified, monomeric GM2AP per liter of expression supernatant. MALDI- and ESI-TOF mass spectrometry were employed to assess the processing, homogeneity, and glycosylation pattern of the recombinant protein. Surface plasmon resonance spectroscopy allowed the interaction of GM2AP with immobilized liposomes to be studied. A modified version of FM22 minimal medium was then used in the cost-effective (15)N-labeling of GM2AP to assess its amenability for the structural investigation by NMR spectroscopy. Initial (15)N,(1)H-HSQC experiments show a well-folded protein and provide evidence for extensive conformational exchange processes within the molecule.     

Mice expressing only monosialoganglioside GM3 exhibit lethal audiogenic seizures

Gangliosides are a family of glycosphingolipids that contain sialic acid. Although they are abundant on neuronal cell membranes, their precise functions and importance in the central nervous system (CNS) remain largely undefined. We have disrupted the gene encoding GD3 synthase (GD3S), a sialyltransferase expressed in the CNS that is responsible for the synthesis of b-series gangliosides. GD3S-/- mice, even with an absence of b-series gangliosides, appear to undergo normal development and have a normal life span. To further restrict the expression of gangliosides, the GD3S mutant mice were crossbred with mice carrying a disrupted GalNAcT gene encoding beta1,4-N-acetylgalactosaminyltransferase. These double mutant mice expressed GM3 as their major ganglioside. In contrast to the single mutant mice, the double mutants displayed a sudden death phenotype and were extremely susceptible to induction of lethal seizures by sound stimulus. These results demonstrate unequivocally that gangliosides play an essential role in the proper functioning of the CNS.     

A STUDY OF SPATIAL FEATURES OF CLONES IN A POPULATION OF BRACKEN FERN,PTERIDIUM AQUILINUM (DENNSTAEDTIACEAE)

Allozymes were used to study the spatial attributes of clones (genets) comprising a population of Pteridium aquilinum (L.) Kuhn var. latiusculum (Desv.) Underw. ex Heller (bracken fern) in the Appalachian Mountains of Virginia. Ramets (individual leaves) were sampled at intervals of 165 m (or less in some cases) and genotyped for six polymorphic isozyme loci to produce a map depicting the spatial patterning of genets. Forty-five distinct genotypes were detected, 14 of which were sampled more than once, five of these more than four times. Genotype proportions at all loci except Pgm-1 conformed to Hardy-Weinberg expectations. Estimation of allele frequencies in the population used a “round-robin” approach that removed any upward bias for rare alleles that distinguish genets. Based on these allele frequencies, the probability that each genotype could arise independently and be sampled was calculated. Some genotypes represented by widely separated ramets had very low probabilities of re-encounter, documenting fragmentation of widespread genets. Coarse-scale mapping indicated a population consisting of many small genets and a few very large ones (up to 1,015 m across). The larger genets tended to be irregular in shape, fragmented, and overlapping. Fine scale mapping of individual fronds in spatially discrete patches of ramets revealed extensive intergrowth of genets, indicating that P. aquilinum exhibits a “guerrilla-type” clonal morphology.     

Vinculin phosphorylation by the src kinase. Interaction of vinculin with phospholipid vesicles

Vinculin phosphorylation by pp60src is stimulated by anionic phospholipids (Ito, S., Richert, N., and Pastan, I. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4628-4631). We have examined whether vinculin interacts with phospholipids, the specificity of the interactions, and a possible mechanism for the enhancement of vinculin phosphorylation by these phospholipids. 3H-labeled vinculin binds to phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. No binding to phosphatidylcholine or phosphatidylethanolamine was observed. The phospholipid binding specificity correlated with the ability of these phospholipids to enhance vinculin phosphorylation by the src kinase. Chlorpromazine (0.1 and 0.3 mM) inhibited both vinculin binding to phosphatidylinositol and the enhanced phosphorylation of vinculin by pp60src in the presence of phosphatidylinositol. Tryptic peptide maps of vinculin phosphorylated in the absence of phospholipid revealed three phosphorylated peptides. The same three peptides were phosphorylated in the presence of phospholipid. However, phosphorylation at one site was markedly increased. In the presence of phospholipid proteolysis of vinculin with both chymotrypsin and V8 protease was markedly enhanced and different peptide maps of vinculin were generated. Microheterogeneity of vinculin was observed with isoelectric focusing. All the isoforms (pI 5.45-5.8) were found to bind phospholipids and undergo phosphorylation by the src kinase. These results suggest that one way anionic phospholipids can enhance vinculin phosphorylation is by binding to vinculin and inducing a conformational change in the vinculin molecule.     

Vinculin, a cytoskeletal substrate of protein kinase C

Vinculin, a cytoskeletal protein localized at adhesion plaques, is a phosphoprotein containing phosphoserine, phosphothreonine, and phosphotyrosine. Vinculin has been previously shown to be a substrate for pp60src, a phosphotyrosine protein kinase, but the kinase(s) responsible for phosphorylation of the other amino acid residues is unknown. The present report examines the phosphorylation of vinculin by various serine- and threonine-specific protein kinases. Only protein kinase C, the calcium-activated phospholipid-dependent protein kinase, phosphorylates vinculin at a significant rate (24 nmol/min/mg) and displays marked specificity for vinculin. Both calcium and phosphatidylserine were required for vinculin phosphorylation by protein kinase C. In addition, both phorbol 12,13-dibutyrate (10 nM) and phorbol 12-myristate 13-acetate (10 nM) stimulated vinculin phosphorylation by protein kinase C at a limiting calcium concentration (10(-6) M). Tryptic peptide analysis revealed two major sites of phosphorylation. One site contained phosphoserine and the other contained phosphothreonine. When compared with tryptic maps of vinculin phosphorylated by src kinase, no overlapping phosphorylated peptides were found. The present findings coupled with the plasma membrane location of both these proteins suggest that vinculin may be a physiologic substrate for protein kinase C.     

Reduction of N-acetyl methionine sulfoxide: A simple assay for peptide methionine sulfoxide reductase

A simple and rapid assay has been developed to measure the enzymatic activity of peptide methionine sulfoxide reductase. The assay is based on the reduction of labeled N-acetylmethionine sulfoxide to N-acetylmethionine. The N-acetylmethionine can be separated from the substrate by extraction into ethyl acetate.     

Clonal population structure and genetic variation in sand-shinnery oak,Quercus havardii (Fagaceae)

We investigated clonal population structure and genetic variation in Quercus havardii (sand-shinnery oak), a deciduous rhizomatous shrub that dominates vegetation by forming uninterrupted expanses of ground cover over sandy deposits on the plains of western Texas, western Oklahoma, and eastern New Mexico. Isozyme electrophoresis (15 loci coding 11 enzymes) was used to recognize and map clones arrayed in a 2000-m transect (50-m sample intervals) and a 200 × 190 m grid (10-m sample intervals). Ninety-four clones were discovered, 38 in the transect and 56 in the grid, resulting in an estimated density of ～15 clones per hectare. Clones varied greatly in size (～100-7000 m), shape, and degree of fragmentation. The larger clones possessed massive interiors free of intergrowth by other clones, while the smaller clones varied in degree of intergrowth. The population maintained substantial levels of genetic variation (P = 60%, A = 2.5, H(exp) = 0.289) comparable to values obtained for other Quercus spp. and for other long-lived perennials. The population was outcrossing as evidenced by conformance of most loci to Hardy-Weinberg expected genotype proportions, although exceptions indicated a limited degree of population substructuring. These data indicate that despite apparent reproduction primarily through vegetative means, Q. havardii possesses conventional attributes of a sexual population.