A novel microtiter plate based method for identification of B‐cell epitopes

A new type of microtiter plate capable of binding biomolecules covalently in a one step procedure was used to map linear B‐cell epitopes in two different proteins using a peptide‐based solid phase immunoassay. The method was compared with a conventional immobilization method using passive adsorption to microtiter plates. An array of 15‐mer peptides, overlapping by five amino acids, representing the entire sequences of ubiquitin and murine tumor necrosis factor‐α, respectively, was synthesized. The peptides were immobilized covalently using the new, specialized microtiter plates or non‐covalently using conventional ELISA microtiter plates of the high binder type. Subsequently, specific antisera to ubiquitin or murine tumor necrosis factor‐α were added to identify potential linear B‐cell epitopes. All peptides, which were recognized on the conventional microtiter plates, were also recognized on the plates with the covalently bound peptides. In addition, the covalent immobilization method revealed epitopes that were not identified using the method for non‐covalent binding although the peptides were in fact present on the non‐covalent binding surface. The interaction with the hydrophobic surface of the conventional microtiter plate apparently interfered negatively with antibody recognition. The covalently binding microtiter plates described here could be useful for identification of new B‐cell epitopes in protein antigens. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Phylogenetic Identification and Substrate Uptake Patterns of Sulfate-Reducing Bacteria Inhabiting an Oxic-Anoxic Sewer Biofilm Determined by Combining Microautoradiography and Fluorescent In Situ Hybridization

We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes. The MAR-FISH analysis revealed that Desulfobulbus hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB. Approximately 9 and 27% of Desulfobulbus cells detected with probe 660 could take up [¹⁴C]propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments. Furthermore, more than 40% of Desulfobulbus cells incorporated acetate under anoxic conditions. SRB were also numerically important members of H₂-utilizing and ¹⁴CO₂-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H₂-utilizing bacteria hybridized with probe EUB338. A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the Desulfomicrobium hypogeium and the Desulfovibrio desulfuricans MB lineages, were found to be important H₂ utilizers in this biofilm. The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date. These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms.     

Phospholipase A(2) activity towards vesicles of DPPC and DMPC-DSPC containing small amounts of SMPC.

Phospholipase A(2) (PLA(2)) is an interfacially active enzyme whose hydrolytic activity is known to be enhanced in one-component phospholipid bilayer substrates exhibiting dynamic micro-heterogeneity. In this study the activity of PLA(2) towards large unilamellar vesicles composed of DPPC:SMPC and DMPC:DSPC:SMPC is investigated using fluorescence and HPLC techniques. Phase diagrams of the mixtures are established by differential scanning calorimetry and the PLA(2) activity, monitored by the lag time, is correlated with the phase behavior of the mixtures. In addition, the degree of lipid hydrolysis in the DMPC:DSPC:SMPC lipid mixtures is detected by HPLC. The PLA(2) activity is found to be significantly increased in the temperature range of the coexistence region where the lipid mixtures exhibit lateral gel-fluid phase separation. Furthermore, in the entire temperature range it is demonstrated that PLA(2) preferentially hydrolyzes the short chain DMPC lipid. This discriminative effect becomes less pronounced when the asymmetric lipid SMPC is present in the lipid substrate. Inclusion of SMPC into either DPPC or DMPC:DSPC vesicles prolongs the lag time. The results clearly show that the PLA(2) activity is significantly enhanced by lipid bilayer micro-heterogeneity in both one-component and multi-component lipid bilayer substrates. The PLA(2) activity measurements are discussed in terms of dynamic gel-fluid lipid domain formation due to density fluctuations and static lipid domain formation due to gel-fluid phase separation.     

aRNA-longSAGE: a new approach to generate SAGE libraries from microdissected cells

Large-scale gene expression analyses of microdissected primary tissue are still difficult because generally only a limited amount of mRNA can be obtained from microdissected cells. The introduction of the T7-based RNA amplification technique was an important step to reduce the amount of RNA needed for such analyses. This amplification technique produces amplified antisense RNA (aRNA), which so far has precluded its direct use for serial analysis of gene expression (SAGE) library production. We describe a method, termed ‘aRNA-longSAGE’, which is the first to allow the direct use of aRNA for standard longSAGE library production. The aRNA-longSAGE protocol was validated by comparing two aRNA-longSAGE libraries with two Micro-longSAGE libraries that were generated from the same RNA preparations of two different cell lines. Using a conservative validation approach, we were able to verify 68% of the differentially expressed genes identified by aRNA-longSAGE. Furthermore, the identification rate of differentially expressed genes was roughly twice as high in our aRNA-longSAGE libraries as in the standard Micro-longSAGE libraries. Using our validated aRNA-longSAGE protocol, we were able to successfully generate longSAGE libraries from as little as 40 ng of total RNA isolated from 2000–3000 microdissected pancreatic ductal epithelial cells or cells from pancreatic intraepithelial neoplasias.     

Quantifying training load and intensity in elite male ice hockey according to game-related contextual variables

We aimed to quantify training load (TL) and intensity during practice sessions according to game-related contextual variables (game outcome, opponent standard, game location) in an elite male ice hockey team. Practice data were collected using a wearable 200-Hz accelerometer, heart rate (HR) recording, and session-rating of perceived exertion (s-RPE) throughout 23 sessions (n = 306 files). The reference team performed a greater number of accelerations, decelerations, spent longer time > 85% maximum HR (t85%HR_max) and reported greater s-RPE after losing a game compared to a win (r = 0.13–0.19). Moreover, a lower number of accelerations, decelerations, t85%HR_max and s-RPE (r = 0.15–0.45) were found before playing against a top-ranked opponent. In contrast, more accelerations, decelerations, longer t85%HR_max and greater s-RPE were observed after playing against a top-ranked team opponent (r = 0.15–0.41). The players performed more accelerations/min, spent more t85%HR_max and reported greater s-RPE before playing an away game (r = 0.13–0.22). Weekly TL seems to slightly increase after losing a game, when preparing a game against a weaker opponent, after playing against a stronger opponent, and when preparing an away game. On the other hand, training intensity seems not to be affected by game-related contextual variables. Thus, ice hockey practitioners involved with TL monitoring should consider the interplay of the numerous variables that influence the volume of prescribed training and the actual training responses in each individual player.     

Ecological stoichiometry and nutrient partitioning in two insect herbivores responsible for large‐scale forest disturbance in the Fennoscandian subarctic

1. Outbreaks of herbivorous insects can have large impacts on regional soil carbon (C) storage and nutrient cycling. In northernmost Europe, population outbreaks of several geometrid moth species regularly cause large‐scale defoliation in subarctic birch forests. An improved understanding is required of how leaf C and nutrients are processed after ingestion by herbivores and what this means for the quantity and quality of different materials produced (frass, bodies). 2. In this study, larvae of two geometrid species responsible for major outbreaks (Epirrita autumnata and Operophtera brumata) were raised on exclusive diets of Betula pubescens var. czerepanovii (N. I. Orlova) Hämet Ahti and two other abundant understorey species (Betula nana, Vaccinium myrtillus). The quantities of C, nitrogen (N) and phosphorus (P) ingested and allocated to frass, bodies and (in the case of C) respired were recorded. 3. Overall, 23%, 70% and 48% of ingested C, N and P were allocated to bodies, respectively, rather than frass and (in the case of C) respiration. Operophtera brumata consistently maintained more constant body stoichiometric ratios of C, N and P than did E. autumnata, across the wide variation in physico‐chemical properties of plant diet supplied. 4. These observed differences and similarities on C and nutrient processing may improve researchers' ability to predict the amount and stoichiometry of frass and bodies generated after geometrid outbreaks.     

Problems and paradigms: Dynamic lipid-bilayer heterogeneity: A mesoscopic vehicle for membrane function?

The lipid-bilayer component of cell membranes is an aqueous bimolecular aggregate characterized by a heterogeneous lateral organization of its molecular constituents. The heterogeneity may be sustained statically as well as dynamically. On the basis of recent experimental and theoretical progress in the study of the physical properties of lipid-bilayer membranes, it is proposed that the dynamically heterogeneous membrane states are important for membrane functions such as transport of matter across the membrane and enzymatic activity. The heterogeneous membrane states undergo significant structural changes in response to changes in compositional, thermodynamic, and environmental conditions. The diverse effects of a variety of molecular compounds interacting with membranes, such as cholesterol and drugs like anaesthetics, may be understood in terms of the ability of these compounds to affect and modulate the dynamic membrane heterogeneity.     

Kinetics of Cellobiohydrolase (Cel7A) Variants with Lowered Substrate Affinity

Cellobiohydrolases are exo-active glycosyl hydrolases that processively convert cellulose to soluble sugars, typically cellobiose. They effectively break down crystalline cellulose and make up a major component in industrial enzyme mixtures used for deconstruction of lignocellulosic biomass. Identification of the rate-limiting step for cellobiohydrolases remains controversial, and recent reports have alternately suggested either association (on-rate) or dissociation (off-rate) as the overall bottleneck. Obviously, this uncertainty hampers both fundamental mechanistic understanding and rational design of enzymes with improved industrial applicability. To elucidate the role of on- and off-rates, respectively, on the overall kinetics, we have expressed a variant in which a tryptophan residue (Trp-38) in the middle of the active tunnel has been replaced with an alanine. This mutation weakens complex formation, and the population of substrate-bound W38A was only about half of the wild type. Nevertheless, the maximal, steady-state rate was twice as high for the variant enzyme. It is argued that these opposite effects on binding and activity can be reconciled if the rate-limiting step is after the catalysis (i.e. in the dissociation process).