Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

A (1-->6)-linked beta-mannopyrananan, pseudonigeran, and a (1-->4)-linked beta-xylan, isolated from the lichenised basidiomycete Dictyonema glabratum

Extraction of Dictyonema glabratum with hot 2% (w/v) aqueous KOH at 100 degrees C, followed by neutralisation and freeze-thawing, gave an insoluble glucan. The residue was further extracted by a similar process, but with hot 10% (w/v) aqueous KOH, furnishing a mixture of glucan, mannan and xylan. The mannan and xylan were obtained via precipitation of its copper complex with Fehling's solution, leaving the glucan in the supernatant. The insoluble complex was finally purified through gel permeation chromatography. Methylation analysis, one- and two-dimensional nuclear magnetic resonance examination showed the polysaccharides to be a (1-->3)-linked alpha-glucan (pseudonigeran) and a (1-->4)-linked beta-xylan, both not previously encountered in lichens, and a newly discovered (1-->6)-linked beta-mannan.     

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.      

O-Glycosylation in Cell Wall Proteins in Scedosporium prolificans Is Critical for Phagocytosis and Inflammatory Cytokines Production by Macrophages

In this study, we analyze the importance of O-linked oligosaccharides present in peptidorhamnomannan (PRM) from the cell wall of the fungus Scedosporium prolificans for recognition and phagocytosis of conidia by macrophages. Adding PRM led to a dose-dependent inhibition of conidia phagocytosis, whereas de-O-glycosylated PRM did not show any effect. PRM induced the release of macrophage-derived antimicrobial compounds. However, O-linked oligosaccharides do not appear to be required for such induction. The effect of PRM on conidia-induced macrophage killing was examined using latex beads coated with PRM or de-O-glycosylated PRM. A decrease in macrophage viability similar to that caused by conidia was detected. However, macrophage killing was unaffected when beads coated with de-O-glycosylated PRM were used, indicating the toxic effect of O-linked oligosaccharides on macrophages. In addition, PRM triggered TNF-α release by macrophages. Chemical removal of O-linked oligosaccharides from PRM abolished cytokine induction, suggesting that the O-linked oligosaccharidic chains are important moieties involved in inflammatory responses through the induction of TNF-α secretion. In summary, we show that O-glycosylation plays a role in the recognition and uptake of S. prolificans by macrophages, killing of macrophages and production of pro- inflammatory cytokines.     

First report on polysaccharides of Asterochloris and their potential role in the lichen symbiosis

A structural study of the carbohydrates from the aposymbiotically cultured Asterochloris sp., the algal symbiont of the lichen Cladina confusa was carried out for the first time. A xylorhamnogalactofuranan was purified and was predominated by (1-->3)-linked galactofuranosyl units with sidechains in position 6 on approximately 6.4% of the units. The sidechains have galactofuranosyl units 5-O and 6-O-substituted, as well rhamnopyranosyl units 2-O, 3-O and 2,3-di-O-substituted. Xylose was detected only as nonreducing end units, together with galactofuranosyl units. Amylose and a beta-(1-->4)-xylan were also present. These polysaccharides have not been found in the symbiotic thallus of C. confusa, which contained only glucans, galactomannoglucan and galactoglucomannan. A potential role of these carbohydrates in lichen recognition proccess is also discussed.     

Effects of aerobic exercise therapy and cognitive behavioural therapy on functioning and quality of life in amyotrophic lateral sclerosis: protocol of the FACTS-2-ALS trial

Background

Neuropathic pain must be correctly diagnosed for optimal treatment. The questionnaire named Neuropathic Pain Symptom Inventory (NPSI) was developed in its original French version to evaluate the different symptoms of neuropathic pain. We hypothesized that the NPSI might also be used to differentiate neuropathic from non-neuropathic pain.

Methods

We translated the NPSI into German using a standard forward-backward translation and administered it in a case-control design to patients with neuropathic (n = 68) and non-neuropathic pain (headache and osteoarthritis, n = 169) to validate it and to analyze its discriminant properties, its sensitivity to change, and to detect neuropathic pain subgroups with distinct profiles.

Results

Using a sum score (the NPSI-G score), we found sensitivity to change (r between 0.37 and 0.5 for pain items of the graded chronic pain scale) and could distinguish between neuropathic and other pain on a group basis, but not for individual patients. Post hoc development of a discriminant score with optimized diagnostic properties to distinguish neuropathic pain from non-neuropathic pain resulted in an instrument with high sensitivity (91%) and acceptable specificity (70%). We detected six different pain profiles in the patient group with neuropathic pain; three profiles were found to be distinct.

Conclusions

The NPSI-G potentially combines the properties of a diagnostic tool and an instrument to identify subtypes of neuropathic pain.     

Fatty acid composition of the tropical lichen Teloschistes flavicans and its cultivated symbionts

Fatty acid components, in both the free and combined form of the intact tropical lichen Teloschistes flavicans, and its isolated photobiont and mycobiont, were analyzed by GC-MS of derived methyl esters. Its rDNA analysis confirmed that the isolated cultured symbionts belong to the genera Trebouxia and Teloschistes, respectively. The fatty acid composition of the lichen did not correspond to those found in the isolated symbionts, suggesting that the fatty acid metabolism is markedly influenced by the symbiosis. Differences in the fatty acid composition in the lichen were observed during the summer (27 degrees C), when the main fatty acids were saturated and in the winter (22 degrees C) when an increase of unsaturated fatty acids occurred. Similar differences of composition were also observed for the cultured mycobiont at different temperatures. The increase in the unsaturation level at low temperatures would maintain the membrane fluidity. Our results are the first on the fatty acids of a tropical lichen and suggest that it is sensitive to small temperature variations, which influences its saturated and unsaturated fatty acid composition.     

Glucans of lichenized fungi: significance for taxonomy of the genera Parmotrema and Rimelia

The glucans of lichenized fungi are an important class of polysaccharides with structural and chemotaxonomic roles. The water-insoluble glucans of the genus Parmotrema (P. austrosinense, P. delicatulum, P. mantiqueirense, P. schindleri, and P. tinctorum) and those of Rimelia (R. cetrata and R. reticulata), were investigated in order to evaluate the significance in chemotyping, with nigeran [(1-->3),(1-->4)-alpha-glucan] and lichenan [(1-->3),(1-->4)-beta-glucan] characterized using (1)H and (13)C NMR, methylation analysis, and controlled Smith degradations. Results from all species were similar, suggesting that glucan chemistry does not support separation of Rimelia from Parmotrema.     

TLR4 recognizes Pseudallescheria boydii conidia and purified rhamnomannans

Pseudallescheria boydii (Scedosporium apiospermum) is a saprophytic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections, particularly mycetoma, in immunocompromised and immunocompetent hosts. We have previously shown that highly purified α-glucan from P. boydii activates macrophages through Toll-like receptor TLR2, however, the mechanism of P. boydii recognition by macrophage is largely unknown. In this work, we investigated the role of innate immune receptors in the recognition of P. boydii. Macrophages responded to P. boydii conidia and hyphae with secretion of proinflammatory cytokines. The activation of macrophages by P. boydii conidia required functional MyD88, TLR4, and CD14, whereas stimulation by hyphae was independent of TLR4 and TLR2 signaling. Removal of peptidorhamnomannans from P. boydii conidia abolished induction of cytokines by macrophages. A fraction highly enriched in rhamnomannans was obtained and characterized by NMR, high performance TLC, and GC-MS. Preparation of rhamnomannans derived from P. boydii triggered cytokine release by macrophages, as well as MAPKs phosphorylation and IκBα degradation. Cytokine release induced by P. boydii-derived rhamnomannans was dependent on TLR4 recognition and required the presence of non-reducing end units of rhamnose of the rhamnomannan, but not O-linked oligosaccharides from the peptidorhamnomannan. These results imply that TLR4 recognizes P. boydii conidia and this recognition is at least in part due to rhamnomannans expressed on the surface of P. boydii.