Tyrosol and tryptophol produced by <Emphasis Type="Italic">Ceratocystis adiposa</Emphasis>

Extracellular tyrosol and tryptophol were produced and isolated from the phytopathogen fungus Ceratocystis adiposa cultivated in a rich broth. These compounds have not been previously reported in this Ceratocystis species, as its identification was established through spectroscopic methods (¹H NMR, ¹³C NMR and MS). Productivity of tyrosol and tryptophol was 65.3 and 44 μg l⁻¹ day⁻¹, respectively, which were values higher than those reported to date. Tyrosol is a compound of pharmaceutical interest showing antioxidating activity, a property used in the treatment of atherosclerosis. Tryptophol is reported to have antibiotic and phytotoxic activity.     

Functions of cell surface galectin-glycoprotein lattices

Programmed remodeling of cell surface glycans by the sequential action of specific glycosyltransferases can control biological processes by generating or masking ligands for endogenous lectins. Galectins, a family of animal lectins with affinity for beta-galactosides, can form multivalent complexes with cell surface glycoconjugates and deliver a variety of intracellular signals to modulate cell activation, differentiation, and survival. Recent efforts involving genetic or biochemical manipulation of O-glycosylation and N-glycosylation pathways, as well as blockade of the synthesis of endogenous galectins, have illuminated essential roles for galectin-glycoprotein lattices in the control of biological processes including receptor turnover and endocytosis, host-pathogen interactions, and immune cell activation and homeostasis.     

Tools for glycomics: relative quantitation of glycans by isotopic permethylation using 13CH3I

Analysis of oligosaccharides by mass spectrometry (MS) has enabled the investigation of the glycan repertoire of organisms with high resolution and sensitivity. It is difficult, however, to correlate the expression of glycosyltransferases with the glycan structures present in a particular cell type or tissue because the use of MS for quantitative purposes has significant limitations. For this reason, in order to develop a technique that would allow relative glycan quantification by MS analysis between two samples, a procedure was developed for the isotopic labeling of oligosaccharides with (13)C-labeled methyl iodide using standard permethylation conditions. Separate aliquots of oligosaccharides from human milk were labeled with (12)C or (13)C methyl iodide; the labeled and non-labeled glycans were mixed in known proportions, and the mixtures analyzed by MS. Results indicated that the isotopic labeling described here was capable of providing relative quantitative data with a dynamic range of at least two orders of magnitude, adequate linearity, and reproducibility with a coefficient of variation that was 13% on average. This procedure was used to analyze N-linked glycans released from various mixtures of glycoproteins, such as alpha-1 acid glycoprotein, human transferrin, and bovine fetuin, using MS techniques that included matrix assisted laser desorption ionization-time of flight MS and electrospray ionization with ion cyclotron resonance-Fourier transformation MS. The measured (12)C:(13)C ratios from mixtures of glycans permethylated with either (12)CH(3)I or (13)CH(3)I were consistent with the theoretical proportions. This technique is an effective procedure for relative quantitative glycan analysis by MS.     

Four disulfide-bridged scorpion beta neurotoxin CssII: heterologous expression and proper folding in vitro

The gene of the four disulfide-bridged Centruroides suffusus suffusus toxin II was cloned into the expression vector pQE30 containing a 6His-tag and a FXa proteolytic cleavage region. This recombinant vector was transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The level of expression was 24.6 mg/l of culture medium, and the His tagged recombinant toxin (HisrCssII) was found exclusively in inclusion bodies. After solubilization the HisrCssII peptide was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisrCssII product obtained from the affinity chromatography step showed several peptide fractions having the same molecular mass of 9392.6 Da, indicating that HisrCssII was oxidized forming several distinct disulfide bridge arrangements. The multiple forms of HisrCssII after reduction eluted from the column as a single protein component of 9400.6 Da. Similarly, an in vitro folding of the reduced HisrCssII generated a single oxidized component of HisrCssII, which was cleaved by the proteolytic enzyme FXa to the recombinant CssII (rCssII). The molecular mass of rCssII was 7538.6 Da as expected. Since native CssII (nCssII) is amidated at the C-terminal residue whereas the rCssII is heterologously expressed in the format of free carboxyl end, there is a difference of 1 Da, when comparing both peptides (native versus heterologously expressed). Nevertheless, they show similar toxicity when injected intracranially into mice, and both nCssII and rCssII show the typical electrophysiological properties of beta-toxins in Na(v)1.6 channels, which is for the first time demonstrated here. Binding and displacement experiments conducted with radiolabelled CssII confirms the electrophysiological results. Several problems associated with the heterologously expressed toxins containing four disulfide bridges are discussed.     

Characterization of the interaction of two peptides from the N terminus of the NHR domain of HIV-1 gp41 with phospholipid membranes

The HIV-1 gp41 envelope glycoprotein is responsible for the membrane fusion between the virus and the target cell. According to recent models, the N-terminal coiled-coil (NHR) region of gp41 is involved in forming the interfaces between neighboring helices in the six-helix bundle, as well as in membrane binding and perturbation. In order to get new insights into the viral membrane fusion mechanism, two peptides, pFP15 and pFP23, pertaining to the first part of the gp41 NHR domain were studied regarding their structure and their ability to induce membrane leakage, aggregation, and fusion, as well as their affinity toward specific phospholipids by a variety of spectroscopic methods. Our results demonstrate that the first part of the NHR domain interacts with negatively charged phospholipid-containing model membranes, modifies the phase behavior of membrane phospholipids, and induces leakage and aggregation of liposomes, suggesting that it could be involved directly in the merging of the viral and target cell membranes working synergistically with other membrane-active regions of the gp41 glycoprotein to boost the fusion process. On the other hand, we suggest that this region of the NHR domain could be involved in the first steps of the destabilization of the HIV-1 gp41 six-helix bundle after its interaction with negatively charged phospholipid headgroups.     

The Sigma-1 receptor is an ER-localized type II membrane protein

The Sigma-1 receptor (S1R) is a transmembrane protein with important roles in cellular homeostasis in normal physiology and in disease. Especially in neurodegenerative diseases, S1R activation has been shown to provide neuroprotection by modulating calcium signaling, mitochondrial function and reducing endoplasmic reticulum (ER) stress. S1R missense mutations are one of the causes of the neurodegenerative Amyotrophic Lateral Sclerosis and distal hereditary motor neuronopathies. Although the S1R has been studied intensively, basic aspects remain controversial, such as S1R topology and whether it reaches the plasma membrane. To address these questions, we have undertaken several approaches. C-terminal tagging with a small biotin-acceptor peptide and BirA biotinylation in cells suggested a type II membrane orientation (cytosolic N-terminus). However, N-terminal tagging gave an equal probability for both possible orientations. This might explain conflicting reports in the literature, as tags may affect the protein topology. Therefore, we studied untagged S1R using a protease protection assay and a glycosylation mapping approach, introducing N-glycosylation sites. Both methods provided unambiguous results showing that the S1R is a type II membrane protein with a short cytosolic N-terminal tail. Assessments of glycan processing, surface fluorescence-activated cell sorting, and cell surface biotinylation indicated ER retention, with insignificant exit to the plasma membrane, in the absence or presence of S1R agonists or of ER stress. These findings may have important implications for S1R-based therapeutic approaches.     

Antigenotoxic Effect of Ascorbic Acid and Resveratrol in Erythrocytes of Ambystoma mexicanum,Oreochromis niloticus and Human Lymphocytes Exposed to Glyphosate

Glyphosate is a controversial herbicide. Its genotoxicity and presence in various ecosystems have been reported. The use of ascorbic acid and resveratrol could protect different organisms from glyphosate-induced genetic damage. In the present study, specific genetic damage induced by glyphosate was evaluated in erythrocytes of Oreochromis niloticus, Ambystoma mexicanum and human lymphocytes. Simultaneously, the antigenotoxic capacity of various concentrations of ascorbic acid and resveratrol was evaluated by means of pretreatment and simultaneous treatment protocols. The 0.03, 0.05 and 0.07 mM concentrations of glyphosate induced significant genotoxic activity (p < 0.05) in human lymphocytes and in erythrocytes of the species studied, and could cause genomic instability in these populations. The reduction in genetic damage observed in human lymphocytes exposed to high concentrations of glyphosate is only apparent: excessive genetic damage was associated with undetectable excessive tail migration length. A significant (p < 0.05) antigenotoxic effect of ascorbic acid and resveratrol was observed in all concentrations, organisms and protocols used. Both ascorbic acid and resveratrol play an important role in maintaining the integrity of DNA. Ascorbic acid in Oreochromis niloticus, Ambystoma mexicanum reduced glyphosate-induced genetic damage to a basal level. Therefore, our data indicate that these antioxidants could help preserve the integrity of the DNA of organisms exposed to glyphosate. The consumption of antioxidants is a useful tool against the genotoxicity of glyphosate.     

Swimming Marine Synechococcus Strains with Widely Different Photosynthetic Pigment Ratios Form a Monophyletic Group

Unicellular marine cyanobacteria are ubiquitous in both coastal and oligotrophic regimes. The contribution of these organisms to primary production and nutrient cycling is substantial on a global scale. Natural populations of marine Synechococcus strains include multiple genetic lineages, but the link, if any, between unique phenotypic traits and specific genetic groups is still not understood. We studied the genetic diversity (as determined by the DNA-dependent RNA polymerase rpoC1 gene sequence) of a set of marine Synechococcus isolates that are able to swim. Our results show that these isolates form a monophyletic group. This finding represents the first example of correspondence between a physiological trait and a phylogenetic group in marine Synechococcus. In contrast, the phycourobilin (PUB)/phycoerythrobilin (PEB) pigment ratios of members of the motile clade varied considerably. An isolate obtained from the California Current (strain CC9703) displayed a pigment signature identical to that of nonmotile strain WH7803, which is considered a model for low-PUB/PEB-ratio strains, whereas several motile strains had higher PUB/PEB ratios than strain WH8103, which is considered a model for high-PUB/PEB-ratio strains. These findings indicate that the PUB/PEB pigment ratio is not a useful characteristic for defining phylogenetic groups of marine Synechococcus strains.     

Characterization of Bilophila wadsworthia Isolates Using PCR Fingerprinting

Bilophila wadsworthia, an under-appreciated anaerobic organism, was originally described in 1989. Ninety-nine Bilophila wadsworthia isolates, recovered form environmental and clinical specimens in Germany and in Southern California, were examined in this study. Many isolates were recovered in mixed culture with facultative aerobic and other anaerobic bacteria. All isolates were identified by standard laboratory procedures, including gas–liquid chromatography (GLC). A PCR fingerprint assay was established to compare the profiles of clinical and environmental isolates to the type strain (ATCC 49260) and to an environmental (sewage) reference strain (DSM 11045, RZATAU) for intra-species differences. Two primers, one universal primer, M13 core, and one tDNA primer, T3B, were used individually to analyse the strains. Homogeneous PCR fingerprint profiles were found for the majority of strains using the M13 core primer; two PCR groups were determined with T3B, one matching the type strain and one matching the environmental reference strain (DSM 11045, RZATAU). Two urease negative strains, WAL 11470 (blood isolate from California) and TÜB 754 (intra-abdominal isolate from Germany) formed unique PCR fingerprint profiles with each of these primers. These results were confirmed by PCR fingerprinting using the T3A primer. These latter results suggest a possible genetic diversity in B. wadsworthia.