Cell Wall β-(1,6)-Glucan of Saccharomyces cerevisiae: STRUCTURAL CHARACTERIZATION AND IN SITU SYNTHESIS*S?

Despite its essential role in the yeast cell wall, the exact composition of the β-(1,6)-glucan component is not well characterized. While solubilizing the cell wall alkali-insoluble fraction from a wild type strain of Saccharomyces cerevisiae using a recombinant β-(1,3)-glucanase followed by chromatographic characterization of the digest on an anion exchange column, we observed a soluble polymer that eluted at the end of the solvent gradient run. Further characterization indicated this soluble polymer to have a molecular mass of ∼38 kDa and could be hydrolyzed only by β-(1,6)-glucanase. Gas chromatographymass spectrometry and NMR (¹H and ¹³C) analyses confirmed it to be a β-(1,6)-glucan polymer with, on average, branching at every fifth residue with one or two β-(1,3)-linked glucose units in the side chain. This polymer peak was significantly reduced in the corresponding digests from mutants of the kre genes (kre9 and kre5) that are known to play a crucial role in the β-(1,6)-glucan biosynthesis. In the current study, we have developed a biochemical assay wherein incubation of UDP-[¹⁴C]glucose with permeabilized S. cerevisiae yeasts resulted in the synthesis of a polymer chemically identical to the branched β-(1,6)-glucan isolated from the cell wall. Using this assay, parameters essential for β-(1,6)-glucan synthetic activity were defined.The cell wall of Saccharomyces cerevisiae and other yeasts contains two types of β-glucans. In the former yeast, branched β-(1,3)-glucan accounts for ∼50–55%, whereas β-(1,6)-glucan represents 10–15% of the total yeast cell wall polysaccharides, each chain of the latter extending up to 140–350 glucose residues in length. The amount of 3,6-branched glucose residues varies with the yeast species: 7, 15, and 75% in S. cerevisiae, Candida albicans, and Schizosaccharomyces pombe, respectively (1). β-(1,6)-Glucan stabilizes the cell wall, since it plays a central role as a linker for specific cell wall components, including β-(1,3)-glucan, chitin, and mannoproteins (2, 3). However, the exact structure of the β-(1,6)-glucan and the mode of biosynthesis of this polymer are largely unknown. In S. pombe, immunodetection studies suggested that synthesis of this polymer backbone begins in the endoplasmic reticulum, with extension occurring in the Golgi (4) and final processing at the plasma membrane. In S. cerevisiae, Montijn and co-workers (5), by immunogold labeling, detected β-(1,6)-glucan at the plasma membrane, suggesting that the synthesis takes place largely at the cell surface.More than 20 genes, including the KRE gene family (14 members) and their homologues, SKN1 and KNH1, have been reported to be involved in β-(1,6)-glucan synthesis in S. cerevisiae, C. albicans, and Candida glabrata (6–10). Among all of these genes, the ones that seem to play the major synthetic role are KRE5 and KRE9, since their disruption caused significant reduction (100 and 80%, respectively, relative to wild type) in the cell wall β-(1,6)-glucan content (11–13).To date, the biochemical reaction responsible for the synthesis of β-(1,6)-glucan and the product synthesized remained unknown. Indeed, in most cases, when membrane preparations are incubated with UDP-glucose, only linear β-(1,3)-glucan polymers are produced, although some studies have reported the production of low amounts of β-(1,6)-glucans by membrane preparations (14–17). These data suggest that disruption of the fungal cell prevents or at least has a strong negative effect on β-(1,6)-glucan synthesis. The use of permeabilized cells, which allows substrates, such as nucleotide sugar precursors, to be readily transported across the plasma membrane, is an alternative method to study in situ cell wall enzyme activities (18–22). A number of methods have been developed to permeabilize the yeast cell wall (23), of which osmotic shock was successfully used to demonstrate β-(1,3)-glucan and chitin synthase activities (20, 24). Herein, we describe the biochemical activity responsible for β-(1,6)-glucan synthesis using permeabilized S. cerevisiae cells and UDP-[¹⁴C]glucose as a substrate. We also have analyzed the physicochemical parameters of this activity and chemically characterized the end product and its structural organization within the mature yeast cell wall.     

Characterization of glucuronic acid containing glycolipid in Aspergillus fumigatus mycelium

A glucuronic acid containing glycerolipid was isolated from the filamentous fungi Aspergillus fumigatus. This acidic glycolipid was extracted from the membrane of mycelium and purified by two successive chromatographic steps on DEAE-Sephadex and Silica columns. Chemical structural analysis was performed using methylation, gas-chromatography, gas-chromatography-mass spectrometry, nano-electrospray mass spectrometry and ¹H/¹³C NMR spectra. The corresponding structure is a 3-(O-α-glucuronyl)-1,2-diacyl-sn-glycerol, where acyl chains are mainly C_16:0, C_18:0, C_18:1, and C_18:2. This α-GlcA-diacylglycerol is not present in fungal conidia. This acidic glycerolipid is described here for the first time in a fungal species. Two homologs of UDP-glucose dehydrogenase that convert UDP-glucose into UDP-glucuronic acid, are present in A. fumigatus genome, UGD1 and UGD2. Gene deletion showed that only UGD1 is essential for the biosynthesis of GlcA-DG. However, no particular phenotype has been observed in the Ugd1Δ mutant. Biological function of this acidic glycolipid remains unknown in A. fumigatus.     

The challenge for genetic epidemiologists: how to analyze large numbers of SNPs in relation to complex diseases

Background

Molecular genetic approaches have much to offer population biology. Despite recent advances, convenient techniques to develop and screen highly-resolving markers can be limiting for some applications and taxa. We describe an improved PCR-based, cloning-free, nuclear marker development procedure, in which single-stranded conformation polymorphism (SSCP) plays a central role. Sequence-variable alleles at putative nuclear loci are simultaneously identified and isolated from diploid tissues. Based on a multiple allele alignment, locus-specific primers are designed in conserved regions, minimizing 'null' alleles. Using two undescribed endemic Australian Collembola as exemplars, we outline a comprehensive approach to generating and validating suites of codominant, sequence-yielding nuclear loci for previously unstudied invertebrates.

Results

Six markers per species were developed without any baseline genetic information. After evaluating the characteristics of each new locus via SSCP pre-screening, population samples were genotyped on the basis of either DNA sequence, restriction site, or insertion/deletion variation, depending on which assay was deemed most appropriate. Polymorphism was generally high (mean of nine alleles per locus), and the markers were capable of resolving population structuring over very fine spatial scales (<100 km). SSCP coupled with targeted DNA sequencing was used to obtain genotypic, genic and genealogical information from six loci (three per species). Phylogeographic analysis identified introns as being most informative.

Conclusion

The comprehensive approach presented here feasibly overcomes technical hurdles of (i) developing suitably polymorphic nuclear loci for non-model organisms, (ii) physically isolating nuclear allele haplotypes from diploid tissues without cloning, and (iii) genotyping population samples on the basis of nuclear DNA sequence variation.     

Biologically relevant effects of mRNA amplification on gene expression profiles

Background  

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results.     

HeatMapper: powerful combined visualization of gene expression profile correlations, genotypes, phenotypes and sample characteristics

Background  

Accurate interpretation of data obtained by unsupervised analysis of large scale expression profiling studies is currently frequently performed by visually combining sample-gene heatmaps and sample characteristics. This method is not optimal for comparing individual samples or groups of samples. Here, we describe an approach to visually integrate the results of unsupervised and supervised cluster analysis using a correlation plot and additional sample metadata.     

Do distribution volumes and clearances relate to tissue volumes and blood flows? A computer simulation

Background

Thoracotomy is associated with severe pain that may persist for years. Acupuncture is a complementary therapy with a proven role in pain control. A randomized trial showed that acupuncture was effective in controlling pain after abdominal surgery, but the efficacy of this technique for the treatment of thoracotomy pain has not been established. We developed a novel technique for convenient application of acupuncture to patients undergoing thoracotomy, and in a Phase II trial evaluated the safety of this intervention and the feasibility of doing a randomized trial.

Methods

Adult patients scheduled for unilateral thoracotomy with preoperative epidural catheter placement received acupuncture immediately prior to surgery. Eighteen semi-permanent intradermal needles were inserted on either side of the spine, and four were inserted in the legs and auricles. Needles were removed after four weeks. Using a numerical rating scale, pain was measured on the first five postoperative days. After discharge, pain was assessed using the Brief Pain Inventory at 7, 30, 60 and 90 days.

Results

Thirty-six patients were treated with acupuncture. Of these, 25, 23, and 22 patients provided data at 30, 60, and 90 days, respectively. The intervention was well tolerated by patients with only one minor and transient adverse event of skin ulceration.

Conclusion

The rate of data completion met our predefined criterion for determining a randomized trial to be feasible (at least 75% of patients tolerated the intervention and provided evaluable data). This novel intervention is acceptable to patients undergoing thoracotomy and does not interfere with standard preoperative care. There was no evidence of important adverse events. We are now testing the hypothesis that acupuncture significantly adds to standard perioperative pain management in a randomized trial.     

Reversal of profound vecuronium-induced neuromuscular block under sevoflurane anesthesia: sugammadex versus neostigmine

Background

The perioperative period is characterized by an intense inflammatory response. Perioperative inflammation promotes postoperative morbidity and increases mortality. Blunting the inflammatory response to surgical trauma might thus improve perioperative outcomes. We are studying three interventions that potentially modulate perioperative inflammation: corticosteroids, tight glucose control, and light anesthesia.

Methods/Design

The DeLiT Trial is a factorial randomized single-center trial of dexamethasone vs placebo, intraoperative tight vs. conventional glucose control, and light vs deep anesthesia in patients undergoing major non-cardiac surgery. Anesthetic depth will be estimated with Bispectral Index (BIS) monitoring (Aspect medical, Newton, MA). The primary outcome is a composite of major postoperative morbidity including myocardial infarction, stroke, sepsis, and 30-day mortality. C-reactive protein, a measure of the inflammatory response, will be evaluated as a secondary outcome. One-year all-cause mortality as well as post-operative delirium will be additional secondary outcomes. We will enroll up to 970 patients which will provide 90% power to detect a 40% reduction in the primary outcome, including interim analyses for efficacy and futility at 25%, 50% and 75% enrollment.

Discussion

The DeLiT trial started in February 2007. We expect to reach our second interim analysis point in 2010. This large randomized controlled trial will provide a reliable assessment of the effects of corticosteroids, glucose control, and depth-of-anesthesia on perioperative inflammation and morbidity from major non-cardiac surgery. The factorial design will enable us to simultaneously study the effects of the three interventions in the same population, both individually and in different combinations. Such a design is an economically efficient way to study the three interventions in one clinical trial vs three.

Trial registration

This trial is registered at Clinicaltrials.gov #: NTC00433251     

Arbuscular mycorrhiza partially protect chicory roots against oxidative stress induced by two fungicides, fenpropimorph and fenhexamid

The present work examined the oxidative stress induced by different concentrations (0.02 and 0.2 mg l-1) of two sterol biosynthesis inhibitor fungicides (fenpropimorph and fenhexamid) in non-target chicory root colonised or not by Glomus intraradices in a monoxenic system. The fungicides were found to cause oxidative damage by increasing lipid peroxidation measured by malondialdehyde production in non-colonised roots. Detoxification of the H₂O₂ product was measured at 0.2 mg l-1 of fenpropimorph by an increase in peroxidase activities suggesting an antioxidant capacity in these roots. Moreover, this study pointed out the ability of arbuscular mycorrhiza to alleviate partially the oxidative stress in chicory roots, probably by lowering reactive oxygen species concentrations, resulting from increases in antioxidant defences. Our results suggest that the enhanced fungicide tolerance in the AM symbiosis could be related to less cell membrane damage.