Regulation and expression of human Fabs under the control of the Escherichia coli arabinose promoter, PBAD

BACKGROUND: The L-arabinose operon from E. coli contains an inducible promoter PBAD which has been extensively studied for the control of gene expression. PBAD has a number of potential advantages over Plac, and has been used successfully to promote high level expression of recombinant proteins. OBJECTIVES: The aim of this study was to investigate PBAD as an alternative system to Plac for the bacterial expression of recombinant Fabs. STUDY DESIGN: The promoter PBAD from the E. coli arabinose operon araBAD and the gene encoding the regulator of this promoter, were cloned into the phagemid expression vector MCO1. Expression of human recombinant tetanus toxoid (TT) and c-erbB2 Fabs under the control of PBAD was compared at two induction temperatures with the same Fabs produced under the control of Plac. RESULTS: Expression of TT and c-erbB2 Fabs under the control of PBAD was comparable to Fab expression from Plac. However, highly expressed TT Fab under the control of PBAD was localised to the soluble periplasmic fraction whereas under the control of Plac, there was greater leakage of Fab into the culture supernatant. In addition, Fab expression from PBAD could be more tightly repressed than from Plac. CONCLUSION: PBAD is a useful and cheaply inducible alternative to the more commonly used Plac for the rapid expression of soluble recombinant human antibody fragments.     

Contact Sex Signals in Two Sympatric Spider Species, Tegenaria domestica and Tegenaria pagana

Bioassays in two sympatric spider species, Tegenaria domestica and T. pagana, demonstrate that the total female chemical extract, and in particular the polar fraction, are involved in the sexual behavior of males and reproductive isolation mechanisms. In T. domestica female attractiveness was correlated with changes in 18 chemical compounds. In T. pagana little difference was observed between 12 compounds in extracts of attractive and unattractive females. 12- + 10-Methyloctacosane was not detected in attractive females of either species. Reproductive isolation was linked not only to quantitative changes in 18 chemical compounds detected in both species, but also to nine compounds present in one or the other species.     

A note on the confidence properties of reference priors for the calibration model

Reference priors are often argued to enjoy better frequentist optimality and confidence properties than other noninformative alternatives. In this paper, we study the coverage performances of two reference priors for the linear calibration model, and show that the mode of the posterior distribution is asymptotically close to the quantity of interest in some region of the parameter space. In these cases, the nominal confidence level of HPD regions is met in the frequentist sense and the reference priors can be argued to be frequency valid. Limitations of the reference priors are also exposed since they cannot guarantee a good uniform frequentist coverage over the parameter space.     

The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169

Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanasecleaving primarily cellobiose units from the non-reducing endof cellulose chains. The ß-l,4 glycosidic bond iscleaved by acid catalysis with an aspartic acid, D221, as thelikely proton donor, and another aspartate, D175, probably ensuringits protonation and stabilizing charged reaction intermediates.The catalytic base has not yet been identified experimentally.The refined crystal structure of CBHII also shows a tyrosineresidue, Y169, located close enough to the scissile bond tobe involved in catalysis. The role of this residue has beenstudied by introducing a mutation Y169F, and analysing the kineticand binding behaviour of the mutated CBHII. The crystal structureof the mutated enzyme was determined to 2.0 Å resolutionshowing no changes when compared with the structure of nativeCBHII. However, the association constants of the mutant enzymefor cellobiose and cellotriose are increased threefold and for4-methylumbelliferyl cellobioside over 50-fold. The catalyticconstants towards cellotriose and cellotetraose are four timeslower for the mutant. These data suggest that Y169, on interactingwith a glucose ring entering the second subsite in a narrowtunnel, helps to distort the glucose ring into a more reactiveconformation. In addition, a change in the pH activity profilewas observed. This indicates that Y169 may have asecond rolein the catalysis, namely to affect the protonation state ofthe active site carboxylates, D175 and D221.     

High resolution solid state C n.m.r. of Canadian peats

Peat samples from three locations in Quebec, Canada, were characterized by ¹³C CP/MAS n.m.r. spectroscopy. The n.m.r. analysis indicates that the peats contain a significant amount of unaltered plant components including cellulose, hemicellulose, lignin, waxes and resins. Integration of specific regions of the spectra can give semi-quantitative estimates of some of these plant components and, in limited cases, information about the degree of decomposition can be obtained from the ¹³C CP/MAS n.m.r. spectrum. Dipolar dephasing experiments and difference spectroscopy were found to be valuable techniques for spectra assignments and enhancement of compositional differences.     

Isolation of Aroma-Bearing Material from Lactobacillus helveticus Culture and Cheese

In whey permeate supplemented with amino acids and inoculated with Lactobacillus helveticus, methionine was necessary for good aroma development. The aroma of Lactobacillus helveticus cultures could be removed by passing the culture fluid through either acid or basic ion-exchange resins. When the ion-exchange resins were eluted with appropriate volatile acid or base and the eluates were evaporated, the residues slowly generated volatile aroma compounds. Several acetamides were identified in culture extracts. The culture eluates from acid ion-exchange resins were fractionated into a caramelized-burned and a cereal-like component. The burned component was a complex of carbonyls with amino acids, and the burned flavor was attributed to 2,5-dimethyl-4-hydroxy-3[2H] furanone. The cereal component contained a number of volatile amines among which pyr-rolidine had the greatest aroma impact. Various pyrazines, indole, and skatole also were identified. Aqueous cheese extracts yielded aroma-bearing fractions that behaved like the culture aromas on ion-exchange chromatography. The cheese extracts contained aromas with the same gas chromatographic retention times as those in the cultures.     

Immediate usability: a case study of public access design for a community photo library

This paper describes a novel instantiation of a digital photo library in a public access system. It demonstrates how designers can utilize characteristics of a target user community (social constraints, trust, and a lack of anonymity) to provide capabilities, such as unrestricted annotation and uploading of photos, which would be impractical in other types of public access systems. It also presents a compact set of design principles and guidelines for ensuring the immediate usability of public access information systems. These principles and guidelines were derived from our experience developing PhotoFinder Kiosk, a community photo library. Attendees of a major HCI conference (CHI 2001 Conference on Human Factors in Computing Systems) successfully used the tool to browse and annotate collections of photographs spanning 20 years of HCI-related conferences, producing a richly annotated photo history of the field of human–computer interaction. Observations and usage log data were used to evaluate the tool and develop the guidelines. They provide specific guidance for practitioners, as well as a useful framework for additional research in public access interfaces.     

Infants' detection of shearing motion in random-dot displays.

Random-dot kinematograms were used to estimate infants' thresholds for shearing motion in the absence of flicker and position cues. The principal advantage of these stimuli is that changes in dot position are camouflaged by the presence of numerous matching dots, thus necessitating the detection of motion before extraction of local pattern features. 13- and 20-wk-old infants were tested with a forced-choice preferential looking technique. The target stimulus resembled a vertically oriented corrugated pattern that oscillated at 1 Hz, if, and only if, shearing motion was detected. Infants were tested at different velocities, ranging from 0.7°/sec to 5.6°/sec, and the results revealed minimum velocity thresholds of 3.5°/sec and 1.2°/sec for 13- and 20-wk-old infants, respectively. Possible interpretations for these results based on position- or flicker-sensitive mechanisms are considered and are found inconsistent with the overall pattern of results. It is concluded that infants detect shearing motion in random-dot displays with a motion-sensitive mechanism. (PsycINFO Database Record (c) 2010 APA, all rights reserved)