Streamlined yeast colorimetric reporter activity assays using scanners and plate readers

Two-hybrid systems have become favored tools for detection and analysis of protein interactions because of their low cost and ease of use compared to biochemical or biophysical interaction technologies. It is possible to augment the utility of two-hybrid systems and derivative systems such as dual-bait two-hybrid systems by adapting strategies that speed the analysis of the relative strength of a series of protein-protein associations. This report describes two simple techniques that employ either a flatbed scanner or a plate reader to quantitate the activity of colorimetric reporters such as LacZ or GusA commonly used in two-hybrid approaches.     

Increased tau phosphorylation on mitogen-activated protein kinase consensus sites and cognitive decline in transgenic models for Alzheimer's disease and FTDP-17: evidence for distinct molecular processes underlying tau abnormalities

Abnormal tau phosphorylation occurs in several neurodegenerative disorders, including Alzheimer's disease (AD) and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Here, we compare mechanisms of tau phosphorylation in mouse models of FTDP-17 and AD. Mice expressing a mutated form of human tau associated with FTDP-17 (tau(V337M)) showed age-related increases in exogenous tau phosphorylation in the absence of increased activation status of a number of kinases known to phosphorylate tau in vitro. In a "combined" model, expressing both tau(V337M) and the familial amyloid precursor protein AD mutation APP(V717I) in a CT100 fragment, age-dependent tau phosphorylation occurred at the same sites and was significantly augmented compared to "single" tau(V337M) mice. These effects were concomitant with increased activation status of mitogen-activated protein kinase (MAPK) family members (extracellular regulated kinases 1 and 2, p38, and c-Jun NH(2)-terminal kinase) but not glycogen synthase kinase-3alphabeta or cyclin-dependent kinase 5. The increase in MAPK activation was a discrete effect of APP(V717I)-CT100 transgene expression as near identical changes were observed in single APP(V717I)-CT100 mice. Age-dependent deficits in memory were also associated with tau(V337M) and APP(V717I)-CT100 expression. The data reveal distinct routes to abnormal tau phosphorylation in models of AD and FTDP-17 and suggest that in AD, tau irregularities may be linked to processing of APP C-terminal fragments via specific effects on MAPK activation status.     

Paraoxonase 1 (PON1) status and risk of insecticide exposure

© 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:182–183, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20079     

Insect host location: a volatile situation

Locating a host plant is crucial for a phytophagous (herbivorous) insect to fulfill its nutritional requirements and to find suitable oviposition sites. Insects can locate their hosts even though the host plants are often hidden among an array of other plants. Plant volatiles play an important role in this host-location process. The recognition of a host plant by these olfactory signals could occur by using either species-specific compounds or specific ratios of ubiquitous compounds. Currently, most studies favor the second scenario, with strong evidence that plant discrimination is due to central processing of olfactory signals by the insect, rather than their initial detection. Furthermore, paired or clustered olfactory receptor neurons might enable fine-scale spatio-temporal resolution of the complex signals encountered when ubiquitous compounds are used.     

Theoretical models of selection and mutation on quantitative traits

Empirical studies of quantitative genetic variation have revealed robust patterns that are observed both across traits and across species. However, these patterns have no compelling explanation, and some of the observations even appear to be mutually incompatible. We review and extend a major class of theoretical models, 'mutation-selection models', that have been proposed to explain quantitative genetic variation. We also briefly review an alternative class of 'balancing selection models'. We consider to what extent the models are compatible with the general observations, and argue that a key issue is understanding and modelling pleiotropy. We discuss some of the thorny issues that arise when formulating models that describe many traits simultaneously.     

Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric

There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesistrade mark technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassemblytrade mark technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degrees C higher than the wild type enzyme while retaining the same specific activity at 50 degrees C. Optimal temperature of the evolved enzyme was 70 degrees C, which is 20 degrees C higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process.