Arabidopsis irregular xylem8 and irregular xylem9: implications for the complexity of glucuronoxylan biosynthesis

Mutations of Arabidopsis thaliana IRREGULAR XYLEM8 (IRX8) and IRX9 were previously shown to cause a collapsed xylem phenotype and decreases in xylose and cellulose in cell walls. In this study, we characterized IRX8 and IRX9 and performed chemical and structural analyses of glucuronoxylan (GX) from irx8 and irx9 plants. IRX8 and IRX9 are expressed specifically in cells undergoing secondary wall thickening, and their encoded proteins are targeted to the Golgi, where GX is synthesized. 1H-NMR spectroscopy showed that the reducing end of Arabidopsis GX contains the glycosyl sequence 4-beta-D-Xylp-(1-->4)-beta-D-Xylp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-GalpA-(1-->4)-D-Xylp, which was previously identified in birch (Betula verrucosa) and spruce (Picea abies) GX. This indicates that the reducing end structure of GXs is evolutionarily conserved in woody and herbaceous plants. This sequence is more abundant in irx9 GX than in the wild type, whereas irx8 and fragile fiber8 (fra8) plants are nearly devoid of it. The number of GX chains increased and the GX chain length decreased in irx9 plants. Conversely, the number of GX chains decreased and the chain length heterodispersity increased in irx8 and fra8 plants. Our results suggest that IRX9 is required for normal GX elongation and indicate roles for IRX8 and FRA8 in the synthesis of the glycosyl sequence at the GX reducing end.     

Isolation of high-lipid content strains of the marine microalga <Emphasis Type="Italic">Tetraselmis suecica</Emphasis> for biodiesel production by flow cytometry and single-cell sorting

Biodiesel from algae is considered an alternative for a third generation of biofuels. However, most microalgae are not lipogenic during fast growth periods, but high-lipid content occurs at resting stages. Microalgae biomass production for biodiesel needs continuous high volumetric and aerial yields and large amount of neutral lipid in the biomass. These requirements are similar to demanding a marathon runner to be obese. We show that by using cell sorting capabilities of flow cytometers, in combination with the lipid-soluble fluorescent dye Nile Red, we can isolate and select cells with a high and stable lipid content. In our study, we were able to select the equivalent of a stable “fat marathon runner” through three sorting events obtained from wild populations of Tetraselmis suecica.     

Pollen morphology and its taxonomic significance in the tribe Gochnatieae (Compositae, Gochnatioideae)

In the context of recent molecular phylogenies of the basal grades of Compositae, we investigated the utility of pollen morphology within the tribe Gochnatieae. The pollen of 64 species of Anastraphia, Cnicothamnus, Cyclolepis, Gochnatia, Pentaphorus, and Richterago was studied using light microscopy and scanning electron microscopy. In addition, three extra-Gochnatieae genera (Ianthopappus, Leucomeris, and Nouelia) were examined as they were traditionally morphologically related to members of the tribe Gochnatieae. Three of the species of Gochnatieae were examined using transmission electron microscopy. Two pollen types, and two new subtypes, have been recognized on the basis of the pollen shape, size, and exine sculpture. The pollen features of Gochnatia sect. Moquiniastrum and G. cordata are similar and distinctive within the genus and support the recently re-circumscribed section Hedraiophyllum. Within the species with echinate pollen surface, the distinctive spine length of Anastraphia supports its recent resurrection as a genus. The identity of Pentaphorus could not be supported by pollen features as was for other morphological characteristics. The pollen features shared across Cyclolepis, Ianthopappus, Leucomeris, Nouelia and Gochnatia sect. Moquiniastrum, as well as those shared by Richterago and Anastraphia could be a result of parallel evolution.     

Streamlining DNA Barcoding Protocols: Automated DNA Extraction and a New cox1 Primer in Arachnid Systematics

Background

DNA barcoding is a popular tool in taxonomic and phylogenetic studies, but for most animal lineages protocols for obtaining the barcoding sequences—mitochondrial cytochrome C oxidase subunit I (cox1 AKA CO1)—are not standardized. Our aim was to explore an optimal strategy for arachnids, focusing on the species-richest lineage, spiders by (1) improving an automated DNA extraction protocol, (2) testing the performance of commonly used primer combinations, and (3) developing a new cox1 primer suitable for more efficient alignment and phylogenetic analyses.

Methodology

We used exemplars of 15 species from all major spider clades, processed a range of spider tissues of varying size and quality, optimized genomic DNA extraction using the MagMAX Express magnetic particle processor—an automated high throughput DNA extraction system—and tested cox1 amplification protocols emphasizing the standard barcoding region using ten routinely employed primer pairs.

Results

The best results were obtained with the commonly used Folmer primers (LCO1490/HCO2198) that capture the standard barcode region, and with the C1-J-2183/C1-N-2776 primer pair that amplifies its extension. However, C1-J-2183 is designed too close to HCO2198 for well-interpreted, continuous sequence data, and in practice the resulting sequences from the two primer pairs rarely overlap. We therefore designed a new forward primer C1-J-2123 60 base pairs upstream of the C1-J-2183 binding site. The success rate of this new primer (93%) matched that of C1-J-2183.

Conclusions

The use of C1-J-2123 allows full, indel-free overlap of sequences obtained with the standard Folmer primers and with C1-J-2123 primer pair. Our preliminary tests suggest that in addition to spiders, C1-J-2123 will also perform in other arachnids and several other invertebrates. We provide optimal PCR protocols for these primer sets, and recommend using them for systematic efforts beyond DNA barcoding.     

Cryopyrin-induced interleukin 1beta secretion in monocytic cells: enhanced activity of disease-associated mutants and requirement for ASC

Several autoinflammatory disorders are associated with missense mutations within the nucleotide-binding oligomerization domain of cryopyrin. The mechanism by which cryopyrin mutations cause inflammatory disease remains elusive. To understand the molecular bases of these diseases, we generated constructs to express three common cryopyrin disease-associated mutations, R260W, D303N, and E637G, and compared their activity with that of the wild-type protein. All cryopyrin mutant proteins tested were found to induce potent NF-kappaB activity when compared with the wild-type protein. This activation was dependent on the expression of ASC, an adaptor protein previously suggested to mediate cryopyrin signaling. When the disease-associated mutants were expressed in monocytic THP-1 cells (which express endogenous ASC), each induced spontaneous IL-1beta secretion, whereas wild-type protein did not. In the absence of stimuli, wild-type cryopyrin was unable to bind to ASC, whereas the three mutants coimmunoprecipitated with ASC, suggesting a mechanism involved in the constitutive activation of mutant proteins. The induction of cryopyrin activity by enforced oligomerization in THP-1 cells resulted in ASC binding and the secretion of IL-1beta, an effect that was abolished by the inhibition of ASC expression with small interfering RNAs. Thus, cryopyrin-mediated IL-1beta secretion requires ASC in monocytic cells. Further, these results indicate that cryopyrin disease-associated mutants are constitutively active and able to induce NF-kappaB activation and IL-1beta secretion at least in part by an increased ability to interact with ASC.     

Both α1- and α2-adrenoceptors in the Insular Cortex Are Involved in the Cardiovascular Responses to Acute Restraint Stress in Rats

The insular cortex (IC) is a limbic structure involved in cardiovascular responses observed during aversive threats. However, the specific neurotransmitter mediating IC control of cardiovascular adjustments to stress is yet unknown. Therefore, in the present study we investigated the role of local IC adrenoceptors in the cardiovascular responses elicited by acute restraint stress in rats. Bilateral microinjection of different doses (0.3, 5, 10 and 15 nmol/100 nl) of the selective α₁-adrenoceptor antagonist WB4101 into the IC reduced both the arterial pressure and heart rate increases elicited by restraint stress. However, local IC treatment with different doses (0.3, 5, 10 and 15 nmol/100 nl) of the selective α₂-adrenoceptor antagonist RX821002 reduced restraint-evoked tachycardia without affecting the pressor response. The present findings are the first direct evidence showing the involvement of IC adrenoceptors in cardiovascular adjustments observed during aversive threats. Our findings indicate that IC noradrenergic neurotransmission acting through activation of both α₁- and α₂-adrenoceptors has a facilitatory influence on pressor response to acute restraint stress. Moreover, IC α₁-adrenoceptors also play a facilitatory role on restraint-evoked tachycardiac response.     

Overview of the Distribution,Habitat Association and Impact of Exotic Ants on Native Ant Communities in New Caledonia

Ants are among the most ubiquitous and harmful invaders worldwide, but there are few regional studies of their relationships with habitat and native ant communities. New Caledonia has a unique and diverse ant fauna that is threatened by exotic ants, but broad-scale patterns of exotic and native ant community composition in relation to habitat remain poorly documented. We conducted a systematic baiting survey of 56 sites representing the main New Caledonian habitat types: rainforest on ultramafic soils (15 sites), rainforest on volcano-sedimentary soils (13), maquis shrubland (15), Melaleuca-dominated savannas (11) and Acacia spirorbis thickets (2). We collected a total of 49 species, 13 of which were exotic. Only five sites were free of exotic species, and these were all rainforest. The five most abundant exotic species differed in their habitat association, with Pheidole megacephala associated with rainforests, Brachymyrmex cf. obscurior with savanna, and Wasmannia auropunctata and Nylanderia vaga present in most habitats. Anoplolepis gracilipes occurred primarily in maquis-shrubland, which contrasts with its rainforest affinity elsewhere. Multivariate analysis of overall ant species composition showed strong differentiation of sites according to the distribution of exotic species, and these patterns were maintained at the genus and functional group levels. Native ant composition differed at invaded versus uninvaded rainforest sites, in the absence of differences in habitat variables. Generalised Myrmicinae and Forest Opportunists were particularly affected by invasion. There was a strong negative relationship between the abundance of W. auropunctata and native ant abundance and richness. This emphasizes that, in addition to dominating many ant communities numerically, some exotic species, and in particular W. auropunctata, have a marked impact on native ant communities.     

Sandhoff disease in Argentina: high frequency of a splice site mutation in the HEXB gene and correlation between enzyme and DNA-based tests for heterozygote detection

The level of -hexosaminidase activity in plasma and leukocytes and the frequency of three known HEXB mutations were studied in an Argentinean deme with high incidence of infantile Sandhoff disease. Two mutations were previously identified in one of two Sandhoff patients from the region, a splice mutation, IVS-2+1 GA, and a 4-bp deletion, CTTT_782–785. These mutations, and a 16kb deletion from the 5' end of the HEXB gene common in non-Argentineans, were screened in 9 Sandhoff patients (all unrelated), 24 obligate heterozygotes, 33 additional individuals belonging to families with affected members, and 64 randomly ascertained individuals from the high risk region. Of 31 independent alleles examined, including those of the two patients previously reported, 30 had the IVS-2 splice mutation and only the originally reported patient had the CTTT deletion. The 16-kb deletion was not observed. Further, among the 57 unaffected members of families with a previous history of Sandhoff disease, and absolute correlation was found between carrier diagnosis by enzyme assay of leukocytes and the DNA-based tests for mutation. One of the 64 controls was classified as a carrier by enzyme assay but did not have one of the three mutations screened. We conclude that a single mutation predominates in this Argentinean population and that the DNA-based test can be an effective supplement or alternative to enzyme-based testing.