Temporal and spatial change of the investment in carnivory of the tropical Utricularia foliosa

Investment by bladderwort (Utricularia foliosa L.) in carnivory, in terms of biochemical composition (carbohydrates per bladder), elemental composition (carbon and nitrogen per bladder), and morphology of the bladders (length, depth, size of the trap door, and size of antennae), was estimated in seven plants located in Yahuarcaca creek (Colombian Amazon) five times from March to May 2005. The aims were to determine whether investment in carnivory varies temporally (over the growing season of the plant) and/or spatially, and if this potential change in carnivory investment varies according to nutrient conditions. The main differences in the investment in carnivory (changes in bladder number and bladder size, and changes in the size of the antennae) were among locations and there were not important differences over the growing season of the plant. Nitrogen and not phosphorus, was the element that stimulated the investment in carnivory. In addition to changes in bladder number and bladder size, we observed a new strategy to enhance prey capture under nitrogen limitation: changes in the size of the antennae. The size of the antennae was approximately 1.3 higher in those plants located in sites with low NO₃⁻. However, we did not observed changes in the carbon/nitrogen ratio of the bladders or in the relationship between bladder length with bladder depth or size of the trap door. The amount of carbohydrates per bladder was also 1.8 higher in those plants located in sites with low NO₃⁻ (0.13 μM) than those with higher NO₃⁻ concentration (0.39 μM). However, the amount of carbohydrates in the bladder was related with the abundance of periphyton and, hence, it is not possible to conclude that carbohydrate production was a strategy of the plant to enhance the capture of prey. Therefore, our findings do not support the carbohydrate mucilage lure speculations.     

BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies

The tricarboxylate reagent benzene-1,3,5-triacetic acid (BTA) was used to attach 5′-aminated DNA primers and templates on an aminosilanized glass surface for subsequent generation of DNA colonies by in situ solid-phase amplification. We have characterized the derivatized surfaces for the chemical attachment of oligonucleotides and evaluate the properties relevant for the amplification process: surface density, thermal stability towards thermocycling, functionalization reproducibility and storage stability. The derivatization process, first developed for glass slides, was then adapted to microfabricated glass channels containing integrated fluidic connections. This implementation resulted in an important reduction of reaction times, consumption of reagents and process automation. Innovative analytical methods for the characterization of attached DNA were developed for assessing the surface immobilized DNA content after amplification. The results obtained showed that the BTA chemistry is compatible and suitable for forming highly dense arrays of DNA colonies with optimal surface coverage of about 10 million colonies/cm² from the amplification of initial single-template DNA molecules immobilized. We also demonstrate that the dsDNA colonies generated can be quantitatively processed in situ by restriction enzymes digestion. DNA colonies generated using the BTA reagent can be used for further sequence analysis in an unprecedented parallel fashion for low-cost genomic studies.     

Molecular evaluation of foetuses with holoprosencephaly shows high incidence of microdeletions in the HPE genes

Holoprosencephaly (HPE), the most common structural malformation of the forebrain in humans, can be detected early during pregnancy using prenatal ultrasonography . Among foetuses with a normal karyotype, 14% have mutations in the four main HPE genes (SHH, ZIC2, SIX3 and TGIF). Genomic rearrangements have now been implicated in many genetic diseases, so we hypothesized that microdeletions in the major HPE genes may also be common in HPE foetuses with severe phenotype or other associated malformations. We screened the DNA obtained from 94 HPE foetuses with a normal karyotype for the presence of microdeletions involving the four major HPE genes (SHH, ZIC2, SIX3 and TGIF). Thirteen of the foetuses had a point mutation in one of the 4 genes and 81 had no known mutations. Quantitative multiplex PCR of short fluorescent fragments (QMPSF) analysis was used for rapid determination of HPE genes copy numbers and the identified microdeletions were confirmed by real time quantitative PCR, or fluorescent in situ hybridization (FISH) (if a cell line was available). Microdeletions were detected in 8 of 94 foetuses (8.5%) (2 in SHH, 2 in SIX3, 3 in ZIC2 and 1 in TGIF genes), and only among the 81 foetuses with a normal karyotype and no point mutations. These data suggest that microdeletions in the four main HPE genes are a common cause of prenatal HPE, as well as point mutations, and increase the total diagnosis rate close to ≈22.3% of foetuses with normal karyotype. Detection can be achieved by the QMPSF testing method that proved to be efficient for testing several genes in a single assay. Databases: SHH - OMIM: 600725; GenBank: NM_000193.2, ZIC2 - OMIM: 603073; GenBank: AF104902.1, SIX3 - OMIM: 603714; GenBank: NM_005413.1, TGIF - OMIM: 602630; GenBank: NM_003244.2, On-line Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/omim/, UCSC (http://www.genome.ucsc.edu/), Ensembl (http://www.ensembl.org/), Database of genomic variants (http://projects.tcag.ca/variation/genomeView.html)     

Recurrent hypoglycemia reduces the glucose sensitivity of glucose-inhibited neurons in the ventromedial hypothalamus nucleus

Recurrent hypoglycemia blunts the brain's ability to sense and respond to subsequent hypoglycemic episodes. Glucose-sensing neurons in the ventromedial hypothalamus nucleus (VMN) are well situated to play a role in hypoglycemia detection. VMN glucose-inhibited (GI) neurons, which decrease their firing rate as extracellular glucose increases, are extremely sensitive to decreased extracellular glucose. We hypothesize that recurrent hypoglycemia decreases the glucose sensitivity of VMN GI neurons. To test our hypothesis, 14- to 21-day-old Sprague-Dawley rats were subcutaneously injected with regular human insulin (4 U/kg) or saline (control) for three consecutive days. Blood glucose levels 1 h after insulin injection on day 3 were significantly lower than on day 1, reflecting an impaired ability to counteract hypoglycemia. On day 4, the glucose sensitivity of VMN GI neurons was measured using conventional whole cell current-clamp recording. After recurrent insulin-induced hypoglycemia, VMN GI neurons only responded to a glucose decrease from 2.5 to 0.1, but not 0.5, mM. Additionally, lactate supplementation also decreased glucose sensitivity of VMN GI neurons. Thus our findings suggest that decreases in glucose sensitivity of VMN GI neurons may contribute to the impairments in central glucose-sensing mechanisms after recurrent hypoglycemia.     

Arbuscular mycorrhizal fungi associated with Populus-Salix stands in a semiarid riparian ecosystem

This study examined the activity, species richness, and species composition of the arbuscular mycorrhizal fungal (AMF) community of Populus-Salix stands on the Verde River (Arizona, USA), quantified patterns of AMF richness and colonization along complex floodplain gradients, and identified environmental variables responsible for structuring the AMF community. Samples from 61 Populus-Salix stands were analyzed for AMF and herbaceous composition, AMF colonization, gravimetric soil moisture, soil texture, per cent organic matter, pH, and concentrations of nitrate, bicarbonate phosphorus and exchangeable potassium. AMF species richness declined with stand age and distance from and elevation above the channel and was positively related to perennial species cover and richness and gravimetric soil moisture. Distance from and elevation above the active channel, forest age, annual species cover, perennial species richness, and exchangeable potassium concentration all played a role in structuring the AMF community in this riparian area. Most AMF species were found across a wide range of soil conditions, but a subset of species tended to occur more often in hydric areas. This group of riparian affiliate AMF species includes several not previously encountered in the surrounding Sonoran desert.     

A new scenario for the quaternary history of European beech populations: palaeobotanical evidence and genetic consequences

Here, palaeobotanical and genetic data for common beech (Fagus sylvatica) in Europe are used to evaluate the genetic consequences of long-term survival in refuge areas and postglacial spread. Four large datasets are presented, including over 400 fossil-pollen sites, 80 plant-macrofossil sites, and 450 and 600 modern beech populations for chloroplast and nuclear markers, respectively. The largely complementary palaeobotanical and genetic data indicate that: (i) beech survived the last glacial period in multiple refuge areas; (ii) the central European refugia were separated from the Mediterranean refugia; (iii) the Mediterranean refuges did not contribute to the colonization of central and northern Europe; (iv) some populations expanded considerably during the postglacial period, while others experienced only a limited expansion; (v) the mountain chains were not geographical barriers for beech but rather facilitated its diffusion; and (vi) the modern genetic diversity was shaped over multiple glacial-interglacial cycles. This scenario differs from many recent treatments of tree phylogeography in Europe that largely focus on the last ice age and the postglacial period to interpret genetic structure and argue that the southern peninsulas (Iberian, Italian and Balkan) were the main source areas for trees in central and northern Europe.     

Triterpenoid saponins from the stem bark of Caryocar villosum

Five triterpenoid saponins, caryocarosides II-22 (3), III-22 (4), II-23 (5), III-23 (6), and II-24 (7), have been isolated from the methanol extract of the stem bark of Caryocar villosum, along with two known saponins (1-2). The seven saponins are glucuronides of hederagenin (II) or bayogenin (III). Caryocaroside II-24 (7) is an unusual galloyl ester saponin acylated on the sugar chain attached to C-28, the 3-O-alpha-L-rhamnopyranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->3)-beta-D-glucuronopyranosyl hederagenin-28-O-[2-O-galloyl-beta-D-glucopyranosyl] ester. The structures of the saponins were established on the basis of extensive NMR ((13)C, (1)H, COSY, TOCSY, HSQC, HMBC and ROESY) and ESI-MS studies. The cytotoxic activity of saponins 2 and 3 was evaluated in vitro against human keratinocytes. The DOPA-oxidase inhibition and the lipolytic activities were evaluated ex vivo using an explant of human adipose tissue.     

Cell-wall structure and anisotropy in procuste, a cellulose synthase mutant of Arabidopsis thaliana

In dark-grown hypocotyls of the Arabidopsis procuste mutant, a mutation in the CesA6 gene encoding a cellulose synthase reduces cellulose synthesis and severely inhibits elongation growth. Previous studies had left it uncertain why growth was inhibited, because cellulose synthesis was affected before, not during, the main phase of elongation. We characterised the quantity, structure and orientation of the cellulose remaining in the walls of affected cells. Solid-state NMR spectroscopy and infrared microscopy showed that the residual cellulose did not differ in structure from that of the wild type, but the cellulose content of the prc-1 cell walls was reduced by 28%. The total mass of cell-wall polymers per hypocotyl was reduced in prc-1 by about 20%. Therefore, the fourfold inhibition of elongation growth in prc-1 does not result from aberrant cellulose structure, nor from uniform reduction in the dimensions of the cell-wall network due to reduced cellulose or cell-wall mass. Cellulose orientation was quantified by two quantitative methods. First, the orientation of newly synthesised microfibrils was measured in field-emission scanning electron micrographs of the cytoplasmic face of the inner epidermal cell wall. The ordered transverse orientation of microfibrils at the inner face of the cell wall was severely disrupted in prc-1 hypocotyls, particularly in the early growth phase. Second, cellulose orientation distributions across the whole cell-wall thickness, measured by polarised infrared microscopy, were much broader. Analysis of the microfibril orientations according to the theory of composite materials showed that during the initial growth phase, their anisotropy at the plasma membrane was sufficient to explain the anisotropy of subsequent growth.     

Anchoring of a large set of markers onto a BAC library for the development of a draft physical map of the grapevine genome

Five hundred and six EST-derived markers, 313 SSR markers and 26 BAC end-derived or SCAR markers were anchored by PCR on a subset of a Cabernet Sauvignon BAC library representing six genome equivalents pooled in three dimensions. In parallel, the 12,351 EST clusters of the grapevine UniGene set (build #11) from NCBI were used to design 12,125 primers pairs and perform electronic PCR on 67,543 nonredundant BAC-end sequences. This in silico experiment yielded 1,140 positive results concerning 638 different markers, among which 602 had not been already anchored by PCR. The data obtained will provide an easier access to the regulatory sequences surrounding important genes (represented by ESTs). In total, 1,731 islands of BAC clones (set of overlapping BAC clones containing at least one common marker) were obtained and 226 of them contained at least one genetically mapped anchor. These assigned islands are very useful because they will link the genetic map and the future fingerprint-based physical map and because they allowed us to indirectly place 93 ESTs on the genetic map. The islands containing two or more mapped SSR markers were also used to assess the quality of the integrated genetic map of the grapevine genome.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .Didier Lamoureux and Anne Bernole contributed equally to this work.