Improved enantioselective analysis of polyunsaturated hydroxy fatty acids in psoriatic skin scales using high-performance liquid chromatography

Enantioselective analysis is used as a valuable tool for determining the biological origin of chiral derivatives of arachidonic, 11,14-eicosadienoic and linoleic acid in psoriatic skin scales and for clarifying their role in pathogenesis. This paper reports on a simple and rapid enantioselective determination (without any derivatization) of the fatty acid derivatives 13(R,S)-hydroxyoctadecadienoic acid [13(R,S)-HODE], 9(R,S)-hydroxyoctadecadienoic acid [9(R,S)-HODE] and 12(R,S)-hydroxyeicosatetraenoic acid [12(R,S)-HETE], using high-performance liquid chromatography (HPLC) with Chiralpak AD as the chiral selector and electrospray ionisation mass spectrometry (ESI-MS). The enantiomeric distribution of 12-HETE, 9-HODE and 13-HODE in psoriatic skin scales of untreated patients (untreated during the last 4 weeks before sampling) was evaluated in comparison to psoriatic skin scales of patients underlying systemic treatment. The enantiomeric distribution of 12-HETE and 9-HODE showed no remarkable differences, whilst samples of patients under systemic treatment exhibited a lower predominance of 13(S)-HODE than samples of untreated patients. Furthermore, the effect of UVB phototherapy on the enantiomeric distribution of 12-HETE, 9-HODE and 13-HODE was studied and a semiquantitation of these compounds in psoriatic skin scales performed. The detected amounts of 9-HODE in samples of untreated patients were remarkably lower than those in samples of patients underlying systemic treatment. In the case of UVB phototherapy, no influence on the enantiomeric distribution could be observed.     

ScaC, an adaptor protein carrying a novel cohesin that expands the dockerin-binding repertoire of the Ruminococcus flavefaciens 17 cellulosome

A new gene, designated scaC and encoding a protein carrying a single cohesin, was identified in the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 as part of a gene cluster that also codes for the cellulosome structural components ScaA and ScaB. Phylogenetic analysis showed that the sequence of the ScaC cohesin is distinct from the sequences of other cohesins, including the sequences of R. flavefaciens ScaA and ScaB. The scaC gene product also includes at its C terminus a dockerin module that closely resembles those found in R. flavefaciens enzymes that bind to the cohesins of the primary ScaA scaffoldin. The putative cohesin domain and the C-terminal dockerin module were cloned and overexpressed in Escherichia coli as His(6)-tagged products (ScaC-Coh and ScaC-Doc, respectively). Affinity probing of protein extracts of R. flavefaciens 17 separated in one-dimensional and two-dimensional gels with recombinant cohesins from ScaC and ScaA revealed that two distinct subsets of native proteins interact with ScaC-Coh and ScaA-Coh. Furthermore, ScaC-Coh failed to interact with the recombinant dockerin module from the enzyme EndB that is recognized by ScaA cohesins. On the other hand, ScaC-Doc was shown to interact specifically with the recombinant cohesin domain from ScaA, and the ScaA-Coh probe was shown to interact with a native 29-kDa protein spot identified as ScaC by matrix-assisted laser desorption ionization-time of flight mass spectrometry. These results suggest that ScaC plays the role of an adaptor scaffoldin that is bound to ScaA via the ScaC dockerin module, which, via the distinctive ScaC cohesin, expands the range of proteins that can bind to the ScaA-based enzyme complex.     

Ruminococcus albus 8 mutants defective in cellulose degradation are deficient in two processive endocellulases, Cel48A and Cel9B, both of which possess a novel modular architecture

The cellulolytic bacterium Ruminococcus albus 8 adheres tightly to cellulose, but the molecular biology underpinning this process is not well characterized. Subtractive enrichment procedures were used to isolate mutants of R. albus 8 that are defective in adhesion to cellulose. Adhesion of the mutant strains was reduced 50% compared to that observed with the wild-type strain, and cellulose solubilization was also shown to be slower in these mutant strains, suggesting that bacterial adhesion and cellulose solubilization are inextricably linked. Two-dimensional polyacrylamide gel electrophoresis showed that all three mutants studied were impaired in the production of two high-molecular-mass, cell-bound polypeptides when they were cultured with either cellobiose or cellulose. The identities of these proteins were determined by a combination of mass spectrometry methods and genome sequence data for R. albus 8. One of the polypeptides is a family 9 glycoside hydrolase (Cel9B), and the other is a family 48 glycoside hydrolase (Cel48A). Both Cel9B and Cel48A possess a modular architecture, Cel9B possesses features characteristic of the B(2) (or theme D) group of family 9 glycoside hydrolases, and Cel48A is structurally similar to the processive endocellulases CelF and CelS from Clostridium cellulolyticum and Clostridium thermocellum, respectively. Both Cel9B and Cel48A could be recovered by cellulose affinity procedures, but neither Cel9B nor Cel48A contains a dockerin, suggesting that these polypeptides are retained on the bacterial cell surface, and recovery by cellulose affinity procedures did not involve a clostridium-like cellulosome complex. Instead, both proteins possess a single copy of a novel X module with an unknown function at the C terminus. Such X modules are also present in several other R. albus glycoside hydrolases and are phylogentically distinct from the fibronectin III-like and X modules identified so far in other cellulolytic bacteria.     

Penicillium thiersii, Penicillium angulare and Penicillium decaturense, new species isolated from wood-decay fungi in North America and their phylogenetic placement from multilocus DNA sequence analysis

We describe three new fungicolous species on the basis of phenotypic and phylogenetic differences from known species. Penicillium thiersii, P. angulare and Penicillium decaturense are described. Penicillium thiersii phenotypically is identified on the basis of several characteristics including growth rates, vesicle size and condium shape and roughening. Penicillium angulare is related most closely to P. adametzioides but differs from it by restricted growth rates and conidiophores greater than 60 μm in length. Penicillium decaturense is related most closely to P. miczynskii but differs from that species by growth rate, minimum growth temperature and pigment production on MEA. Multilocus phylogenetic analysis confirmed the genetic distinctiveness of P. decaturense and the closely related species P. miczynskii, P. chrzaszczii and P. manginii. Penicillium rivolii is a synonym of P. waksmanii on the basis of this analysis. Analysis of the EF-1α gene shows rapid changes of position, number and length of introns between the species, suggesting a recent evolutionary origin for the introns.     

Thermobifida fusca family-6 cellulases as potential designer cellulosome components

During the course of our studies on the structure-function relationship of cellulosomes, we were interested in converting the free cellulase system of the aerobic bacterium, Thermobifida fusca, to a cellulosomal system. For this purpose, the cellulose-binding modules (CBM) of two T. fusca family-6 cellulases, endoglucanase Cel6A and exoglucanase Cel6B, were replaced by divergent dockerin modules. Thus far, family-6 cellulases have not been shown to be members of natural cellulosome systems. The resultant chimaeric proteins, 6A-c and t-6B, respectively, were purified and found to interact specifically and stoichiometrically with their corresponding cohesin modules, indicating their suitability for use as components in 'designer cellulosomes'. Both chimaeric enzymes showed somewhat decreased but measurable levels of activity on carboxymethyl cellulose, consistent with the known endo- and exo-glucanase character of the parent enzymes. The activity of 6A-c on phosphoric acid swollen cellulose was also consistent with that of the wild-type endoglucanase Cel6A. The startling finding of the present research was the extent of degradation of this substrate by the chimaeric enzyme t-6B. Wild-type exoglucanase Cel6B exhibited very low activity on this substrate, while the specific activity of t-6B was 14-fold higher than the parent enzyme.     

Plasmid‐free T7‐based Escherichia coli expression systems

In order to release host cells from plasmid‐mediated increases in metabolic load and high gene dosages, we developed a plasmid‐free, T7‐based E. coli expression system in which the target gene is site‐specifically integrated into the genome of the host. With this system, plasmid‐loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid‐free system was proven in chemostat cultivation for 40 generations in a non‐induced and for 10 generations in a fully induced state. For this reason plasmid‐free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid‐free systems in upstream‐processing. Biotechnol. Bioeng. 2010. 105: 786–794. © 2009 Wiley Periodicals, Inc.     

Identification of low molecular weight pyroglutamate A{beta} oligomers in Alzheimer disease: a novel tool for therapy and diagnosis

N-terminally truncated Aβ peptides starting with pyroglutamate (AβpE3) represent a major fraction of all Aβ peptides in the brain of Alzheimer disease (AD) patients. AβpE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length Aβ. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of AβpE3 and studied the potential involvement of oligomeric AβpE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall Aβ plaque load and AβpE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight AβpE3 oligomers.     

Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution

The innate binding specificity of different carbohydrate-binding modules (CBMs) offers a versatile approach for mapping the chemistry and structure of surfaces that contain complex carbohydrates. We have employed the distinct recognition properties of a double His-tagged recombinant CBM tagged with semiconductor quantum dots for direct imaging of crystalline cellulose at the molecular level of resolution, using transmission and scanning transmission electron microscopy. In addition, three different types of CBMs from families 3, 6, and 20 that exhibit different carbohydrate specificities were each fused with either green fluorescent protein (GFP) or red fluorescent protein (RFP) and employed for double-labeling fluorescence microscopy studies of primary cell walls and various mixtures of complex carbohydrate target molecules. CBM probes can be used for characterizing both native complex carbohydrates and engineered biomaterials.     

Cutting edge: allogeneic CD4+CD25+Foxp3+ T regulatory cells suppress autoimmunity while establishing transplantation tolerance

An important unresolved question with regard to T regulatory (Treg) cell specificity and suppressive activity is whether allogeneic Treg cells inhibit self-reactive T cells. In the present study, this issue was addressed using IL-2Rbeta-deficient mice that develop rapid lethal autoimmunity due to impaired production of Treg cells. We show that adoptive transfer of completely MHC-mismatched Treg cells into IL-2Rbeta(-/-) mice resulted in life-long engraftment of the donor cells, which exhibited skewed reactivity toward host alloantigens, and prevented autoimmunity. Thus, Treg cells that underwent thymic selection by peptide/MHC class II complexes distinct from those recognized by autoreactive T cells, still effectively suppress autoimmunity. Remarkably, when such animals were skin grafted, they exhibited dominant tolerance to those grafts bearing MHC molecules that were shared with donor Treg cells. Collectively, these data demonstrate that effective engraftment by allogeneic Treg cells controls autoimmunity and results in permissive conditions for long-term acceptance of allografts.