Presentation of epitopes on genetically engineered peptides and selection of lymphoma-targeting moieties based on epitope biorecognition

A His-tagged coiled coil stem loop peptide with stable secondary structure was designed and biosynthesized. A series of oligopeptides related to the EBV envelope glycoprotein 350/220 N-terminal nonapeptide as potential CD21 receptor-binding epitopes were engineered into the loop region of the peptide scaffold. It was shown that these peptides had a stable alpha-helical coiled coil structure and assumed a monomeric form in PBS. Biorecognition of the epitopes was studied by immobilizing the epitope-containing peptides on complexed Ni2+-containing surfaces through His-Ni2+ chelation and incubating with purified soluble CD21 receptor or CD21+ cells. The results showed that the potential epitopes bound to CD21 and CD21+ cells at different affinities depending on oligopeptide structures. This approach allows for the evaluation of epitope biorecognizabilities and the selection of optimal oligopeptides among sequences for use as targeting moieties in the design of new lymphoma-targeting polymeric drug carriers.     

Pyranose Oxidase, a Major Source of H(2)O(2) during Wood Degradation by Phanerochaete chrysosporium, Trametes versicolor, and Oudemansiella mucida

The production of the H(2)O(2)-generating enzyme pyranose oxidase (POD) (EC 1.1.3.10) (synonym, glucose 2-oxidase), two ligninolytic peroxidases, and laccase in wood decayed by three white rot fungi was investigated by correlated biochemical, immunological, and transmission electron microscopic techniques. Enzyme activities were assayed in extracts from decayed birch wood blocks obtained by a novel extraction procedure. With the coupled peroxidase-chromogen (3-dimethylaminobenzoic acid plus 3-methyl-2-benzothiazolinone hydrazone hydrochloride) spectrophotometric assay, the highest POD activities were detected in wood blocks degraded for 4 months and were for Phanerochaete chrysosporium (149 mU g [dry weight] of decayed wood), Trametes versicolor (45 mU g), and Oudemansiella mucida (1.2 mU g), corresponding to wood dry weight losses of 74, 58, and 13%, respectively. Mn-dependent peroxidase activities in the same extracts were comparable to those of POD, while lignin peroxidase activity was below the detection limit for all fungi with the veratryl alcohol assay. Laccase activity was high with T. versicolor (422 mU g after 4 months), in trace levels with O. mucida, and undetectable in P. chrysosporium extracts. Evidence for C-2 specificity of POD was shown by thin-layer chromatography detection of 2-keto-d-glucose as the reaction product. By transmission electron microscopy-immunocytochemistry, POD was found to be preferentially localized in the hyphal periplasmic space of P. chrysosporium and O. mucida and associated with membranous materials in hyphae growing within the cell lumina or cell walls of partially and highly degraded birch fibers. An extracellular distribution of POD associated with slime coating wood cell walls was also noted. The periplasmic distribution in hyphae and extracellular location of POD are consistent with the reported ultrastructural distribution of H(2)O(2)-dependent Mn-dependent peroxidases. This fact and the dominant presence of POD and Mn-dependent peroxidase in extracts from degraded wood suggest a cooperative role of the two enzymes during white rot decay by the test fungi.     

Glucose-2-oxidase activity and accumulation of D-arabino-2-hexosulose in cultures of the basidiomyceteOudemansiella mucida

Submerged cultures of the basidiomyceteOudemansiella mucidd, strain III, accumulate D-arabino-2-hexosulose. The maximum yields during cultivations in shaker flasks or in a laboratory fermentor are 6–12 and 15 mg/ml, respectively (20–50 % conversion of substrate glucose). The accumulation is transient, the aldoketose being again utilized after glucose exhaustion. Its production is stimulated by fluoride ions. The enzyme responsible for the C₍₂₎-specific oxidation ofd-glucose acts as an intracellular oxidase with a maximum activity in the exponential phase of growth.d-arabino-2-Hexosulose was also detected in the cultivation medium of the wood-rotting fungiPleurotus ostreatus, Laetiporus sulphureus, andPhellinus abietis. Part III of the series Enzymatic activity of Basidiomycetes; part II:Folia Microbiol. 13, 334 (1968).     

Age structure and growth of<Emphasis Type="Italic"> Dracaena cinnabari</Emphasis> populations on Socotra

Unique Dracaena cinnabari woodlands on Socotra Island—relics of the Mio-Pliocene xerophile-sclerophyllous southern Tethys Flora—were examined in detail, especially with regard to their age structure. Detailed statistical analyses of sets of 50 trees at four localities were performed in order to define a model reflecting relationships between specific growth habit and actual age. The problematic nature of determining the age of an individual tree or specific populations of D. cinnabari is illustrated by three models relating to orders of branching, frequency of fruiting, etc. which allow the actual tree age to be calculated. Based on statistical analyses as well as direct field observations, D. cinnabari populations on Socotra do not regenerate to a great extent and their age structure generally indicates overmaturity. The unique Firmihin D. cinnabari woodland will reach the stage of intensive disintegration within 30–77 years with 95% probability. According to our analysis of dead trees, it is evident that, on average, D. cinnabari in populations at Firmihin dies after reaching 17 orders of peripheral branches.     

Degradation of microvascular brain endothelial cell beta-catenin after co-culture with activated neutrophils from patients undergoing cardiac surgery with prolonged cardiopulmonary bypass

The adhesion of highly activated neutrophils to cerebral microvascular endothelial cells (MVECs) may contribute to disruption and hyperpermeability of the blood-brain barrier (BBB) after cardiac surgery with prolonged cardiopulmonary bypass (CPB). A correlation between CPB duration and neutrophil-mediated BBB damage has not been investigated on the cellular level yet. Therefore, we studied the effects of neutrophils from cardiac surgery patients with CPB time <80 min (group I; n=8) and >80 min (group II; n=8) on the integrity of cultured porcine MVEC. Ex vivo, neutrophils of group II but not of group I significantly degraded the zonula adherens molecule beta-catenin whereas VE-cadherin and occludin were not modified. The transendothelial electric resistance as a measure for the integrity of the endothelial monolayers was reduced over time in both groups. In conclusion, prolonged CPB time entails neutrophil-mediated decrease in MVEC beta-catenin expression, and thus may be an important trigger for BBB disruption.     

Preparation of N-nosyl-3,4-epimines derived from levoglucosan by sodium borohydride reduction

Starting from 1,6-anhydro-beta-d-glucopyranose 1 (levoglucosan), N-o-nitrobenzenesulfonyl (nosyl) 3,4-epimino derivatives with d-allo, d-galacto, and d-talo configurations have been prepared via NaBH(4) reduction of suitably substituted azido tosylates. The benefits and limitations of this method over the classical LiAlH(4) reduction method are discussed.     

Loss of genetic variation in geographically marginal populations of Atriplex tatarica (Chenopodiaceae)

BACKGROUND AND AIMS: Genetic variability was estimated for Atriplex tatarica from 25 populations in the Czech Republic. Since its north-western range margin is in central Europe, a relationship between marginality and low within-population genetic diversity was tested in accordance with the Central-Marginal Model. METHODS: Population genetic diversity was expressed by assessing patterns of variation at 13 putatively neutral allozyme loci (comprising 30 putative alleles) within and between 25 natural populations of A. tatarica along a north-west-south-east transect in the Czech Republic. KEY RESULTS: Atriplex tatarica is a species of human-made habitats with a mixed mating system and wide geographic distribution. Overall, A. tatarica displayed moderate levels of genetic diversity in comparison with other herbaceous plants. The percentage of loci that were polymorphic was 47.1%, with average values of 1.55, 0.151 and 0.155 for the average number of alleles per polymorphic locus (A), observed heterozygosity (Ho) and expected heterozygosity (He), respectively. There was only weak evidence of inbreeding within populations (FIS=0.031) and significant population differentiation (FST=0.214). Analysis of the data provides no evidence for isolation-by-distance for the whole study area. However, Mantel tests were highly significant for the marginal Bohemian region and non-significant for the central Moravian region. While northern populations of A. tatarica showed significantly lower allelic richness (A=1.462) than populations from the southern part of the study area (A=1.615), they did not differ in observed heterozygosity (Ho), gene diversity (HS), inbreeding within populations (FIS) or population differentiation (FST), despite generally lower values of particular genetic measurements in the marginal region. CONCLUSIONS: Genetic diversity, with the exception of allelic richness, was not significantly lower at the margins of the species' range. This, therefore, provides only weak support for the predictions of the Central-Marginal Model.     

Heart failure and first dose hypotension after angiotensin converting enzyme inhibitors

The first-dose induced decrease in blood pressure in some patients following the administration of ACE inhibitors is a fact causing some concern among clinicians prescribing these drugs. However, an overview of clinical trials and the authors' own experience clearly point to the possibility of reducing the incidence and/or severity of first-dose hypotension. Apart from appropriate clinical measures to be taken, the choice of the ACE inhibitor seems to be of crucial importance as some (fosinopril, perindopril) produce less hypotension than others. Thus, with due circumspection, ACE inhibitors can safely retain their position as the cornerstone of the treatment of chronic heart failure.     

Cryo-FE-SEM & TEM immuno-techniques reveal new details for understanding white-rot decay of lignocellulose

High-resolution Cryo-Field Emission Scanning Electron Microscopy (HR-Cryo-FE-SEM) and immuno-cytochemistry were used to reveal novel details on the morphological events and spatial distribution of oxidoreductive enzymes during the degradation of birch wood by the white-rot fungi Phlebia radiata and mutant strain P radiata Cel 26. Cryo-observations of fractured fibres showed degradation across the cell wall by P. radiata (wild) to progress by delamination and removal of concentric orientated aggregates from the secondary S2 cell wall. Decay by P radiata Cel 26 progressed by removal of materials (lignin and hemicelluloses) between the aggregates (primarily cellulose) that remained even after advanced decay. With both decay patterns, extracellular slime materials were present uniting lumina hyphae with the attacked fibre wall. The extracellular slime material had two morphological forms: viz a fibrillar (often tripartite) and a 'gel-form', the former found in discrete bands progressing across the lumen onto the fibre wall. Using TEM immunocytochemistry, laccase, manganese peroxidase (MnP) and diarylpropane enzymes were localized in the periplasmic space of luminal hyphae, in association with the cell membrane, periplasmic vesicles and fungal cell wall. Extracellularly, the three enzymes were found associated with the slime and tripartite membranes and with the birch cell walls at all stages of attack through to middle lamella corner decay. Enzyme distribution was correlated with morphological changes in cell wall structure. The association of extracellular slime with these enzymes and sites of decay strongly suggests a major role for this matrix in fibre cell wall decomposition.     

Tablet Keyboard Configuration Affects Performance,Discomfort and Task Difficulty for Thumb Typing in a Two-Handed Grip

When holding a tablet computer with two hands, the touch keyboard configuration imposes postural constraints on the user because of the need to simultaneously hold the device and type with the thumbs. Designers have provided users with several possible keyboard configurations (device orientation, keyboard layout and location). However, potential differences in performance, usability and postures among these configurations have not been explored. We hypothesize that (1) the narrower standard keyboard layout in the portrait orientation leads to lower self-reported discomfort and less reach than the landscape orientation; (2) a split keyboard layout results in better overall outcomes compared to the standard layout; and (3) the conventional bottom keyboard location leads to the best outcomes overall compared to other locations. A repeated measures laboratory experiment of 12 tablet owners measured typing speed, discomfort, task difficulty, and thumb/wrist joint postures using an active marker system during typing tasks for different combinations of device orientation (portrait and landscape), keyboard layout (standard and split), and keyboard location (bottom, middle, top). The narrower standard keyboard with the device in the portrait orientation was associated with less discomfort (least squares mean (and S.E.) 2.9±0.6) than the landscape orientation (4.5±0.7). Additionally, the split keyboard decreased the amount of reaching required by the thumb in the landscape orientation as defined by a reduced range of motion and less MCP extension, which may have led to reduced discomfort (2.7±0.6) compared to the standard layout (4.5±0.7). However, typing speed was greater for the standard layout (127±5 char./min.) compared to the split layout (113±4 char./min.) regardless of device orientation and keyboard location. Usage guidelines and designers can incorporate these findings to optimize keyboard design parameters and form factors that promote user performance and usability for thumb interaction.