Veratrol-<Emphasis Type="Italic">O</Emphasis>-demethylase of <Emphasis Type="Italic">Acetobacterium dehalogenans</Emphasis>: ATP-dependent reduction of the corrinoid protein

The anaerobic veratrol O-demethylase mediates the transfer of the methyl group of the phenyl methyl ether veratrol to tetrahydrofolate. The primary methyl group acceptor is the cobalt of a corrinoid protein, which has to be in the +1 oxidation state to bind the methyl group. Due to the negative redox potential of the cob(II)/cob(I)alamin couple, autoxidation of the cobalt may accidentally occur. In this study, the reduction of the corrinoid to the superreduced [Co^I] state was investigated. The ATP-dependent reduction of the corrinoid protein of the veratrol O-demethylase was shown to be dependent on titanium(III) citrate as electron donor and on an activating enzyme. In the presence of ATP, activating enzyme, and Ti(III), the redox potential versus the standard hydrogen electrode (E _SHE) of the cob(II)alamin/cob(I)alamin couple in the corrinoid protein was determined to be −290 mV (pH 7.5), whereas E _SHE at pH 7.5 was lower than −450 mV in the absence of either activating enzyme or ATP. ADP, AMP, or GTP could not replace ATP in the activation reaction. The ATP analogue adenosine-5′-(β,γ-imido)triphosphate (AMP-PNP, 2–4 mM) completely inhibited the corrinoid reduction in the presence of ATP (2 mM).     

Cotton boll age influences feeding preference by brown stink bug (Heteroptera: Pentatomidae)

Cotton plants were infested with brown stink bug, Euschistus servus (Say), to define cotton boll age classes (based on heat unit accumulation beyond anthesis) that are most frequently injured during each of the initial 5 wk of flowering. Bolls from each week were grouped into discrete age classes and evaluated for the presence of stink bug injury. Brown stink bug injured significantly more bolls of age class B (approximately 165-336 heat units), age class C (approximately 330-504 heat units), and age class D (approximately 495-672 heat units) during the initial 3 wk in both years and in week 5 in 2002 compared with other boll ages. Generally, the frequency of injured bolls was lowest in age class A (< or = 168 heat units) during these periods. The preference by brown stink bug for boll age classes B, C, and D within a week was similar when ages were combined across all 5 wk. Based on these data, bolls that have accumulated 165.2 through 672 heat units beyond anthesis (approximately 7-27-d-old) are more frequently injured by brown stink bug when a range of boll ages are available. The boll ages in our studies corresponded to a boll diameter of 1.161-3.586 cm with a mid-range of 2.375 cm. A general protocol for initiating treatments against stink bugs is to sample bolls for evidence of injury as an indicator of presence of infestations in cotton. Sampling bolls within a defined range, which is most likely to be injured, should improve the precision of this method in detecting economic stink bug infestations in cotton.     

Agonists and partial agonists of rhodopsin: retinals with ring modifications

Activation of the visual pigment rhodopsin is initiated by isomerization of its retinal chromophore to the all-trans geometry, which drives the conformation of the protein to the active state. We have examined by FTIR spectroscopy the impact of a series of modifications at the ring of retinal on the activation process and on molecular interactions within the binding pocket. Deletion of ring methyl groups at C1 and C5 or replacement of the ring in diethyl or ethyl-methyl acyclic analogues resulted in partial agonists, for which the conformational equilibrium between the Meta I and Meta II photoproduct is shifted from the active Meta II side to the inactive Meta I side. While the Meta II states of these artificial pigments had a conformation similar to those of native Meta II, the Meta I states were different. Modifications on the ring of retinal had a particular impact on the interaction of Glu 122 within the ring-binding pocket and are shown to interfere with the Glu 134-mediated proton uptake during formation of Meta II. We further found, upon partial deletion of ring constituents, a decrease of the entropy change of the transition from Meta I to Meta II by up to 50%, while the concomitant reduction of the enthalpy term was less pronounced. These findings underline the particular importance of the ring and the ring methyl groups and are discussed in a model of receptor activation.     

Functional Anatomy: A Taxonomic Proposal

It is argued that medical science requires a classificatory system that (a) puts functions in the taxonomic center and (b) does justice ontologically to the difference between the processes which are the realizations of functions and the objects which are their bearers. We propose formulae for constructing such a system and describe some of its benefits. The arguments are general enough to be of interest to all the life sciences.     

A rapid algorithm for processing digital physiologic signals: Application to skeletal muscle contractions

Quantifying mechanical output is fundamental to understanding metabolism that fuels muscle contraction and more recent attempts to understand signal transduction and gene regulation. The latter requires long-term application of exercise protocols that result in large amounts of data on muscle performance. The purpose of this study was to develop software for automated quantification of skeletal muscle contractions. An in situ mouse sciatic nerve stimulation model was used to produce contractions over a broad range of frequencies and recorded as both digital and analog signals using a PC analog to digital converter board and chart recorder, respectively. Spectral analysis of the noise components formed the basis for designing a smoothing Chebyshev filter. Algorithms implemented in custom software identified twitches and estimated baseline levels from the smoothed signal. The time to peak force, peak force, tension-time integral, and half-relaxation time were determined for each twitch after baseline correction. The automated results were compared to those obtained from manual measurements of the analog signal. Bland–Altman analysis of the parameters computed from digital signals compared with the corresponding measurements by manual planometry demonstrates the agreement of the digital processing algorithm with planometry over a wide range of twitch characteristics. This program may also be used to study the mechanics of other preparations from isolated muscles, human proximal limb performance, and other digital physiologic signals. Adaptation of the filter function is required to apply the analysis to another experimental apparatus with differing noise characteristics. A full version of the program and instructions for its use are available for download at www.rad.msu.edu.     

Saliva samples are a viable alternative to blood samples as a source of DNA for high throughput genotyping

ABSTRACT: BACKGROUND: The increasing trend for incorporation of biological sample collection within clinical trials requires sample collection procedures which are convenient and acceptable for both patients and clinicians. This study investigated the feasibility of using saliva-extracted DNA in comparison to blood-derived DNA, across two genotyping platforms: Applied Biosystems Taqman TM and Illumina Beadchip TM genome-wide arrays. METHOD: Patients were recruited from the Pharmacogenetics of Breast Cancer Chemotherapy (PGSNPS) study. Paired blood and saliva samples were collected from 79 study participants. The Oragene DNA Self-Collection kit (DNAgenotek(R)) was used to collect and extract DNA from saliva. DNA from EDTA blood samples (median volume 8 ml) was extracted by GenProbe, Livingstone, UK. DNA yields, standard measures of DNA quality, genotype call rates and genotype concordance between paired, duplicated samples were assessed. RESULTS: Total DNA yields were lower from saliva (mean 24 ug, range 0.2-52 ug) than from blood (mean 210 ug, range 58-577 ug) and a 2-fold difference remained after adjusting for the volume of biological material collected. Protein contamination and DNA fragmentation measures were greater in saliva DNA. 78/79 saliva samples yielded sufficient DNA for use on Illumina Beadchip arrays and using Taqman assays. Four samples were randomly selected for genotyping in duplicate on the Illumina Beadchip arrays. All samples were genotyped using Taqman assays. DNA quality, as assessed by genotype call rates and genotype concordance between matched pairs of DNA was high (>97%) for each measure in both blood and saliva-derived DNA. CONCLUSION: We conclude that DNA from saliva and blood samples is comparable when genotyping using either Taqman assays or genome-wide chip arrays. Saliva sampling has the potential to increase participant recruitment within clinical trials, as well as reducing the resources and organisation required for multicentre sample collection.     

Microbial and chemical characterization of underwater fresh water springs in the Dead Sea

Due to its extreme salinity and high Mg concentration the Dead Sea is characterized by a very low density of cells most of which are Archaea. We discovered several underwater fresh to brackish water springs in the Dead Sea harboring dense microbial communities. We provide the first characterization of these communities, discuss their possible origin, hydrochemical environment, energetic resources and the putative biogeochemical pathways they are mediating. Pyrosequencing of the 16S rRNA gene and community fingerprinting methods showed that the spring community originates from the Dead Sea sediments and not from the aquifer. Furthermore, it suggested that there is a dense Archaeal community in the shoreline pore water of the lake. Sequences of bacterial sulfate reducers, nitrifiers iron oxidizers and iron reducers were identified as well. Analysis of white and green biofilms suggested that sulfide oxidation through chemolitotrophy and phototrophy is highly significant. Hyperspectral analysis showed a tight association between abundant green sulfur bacteria and cyanobacteria in the green biofilms. Together, our findings show that the Dead Sea floor harbors diverse microbial communities, part of which is not known from other hypersaline environments. Analysis of the water's chemistry shows evidence of microbial activity along the path and suggests that the springs supply nitrogen, phosphorus and organic matter to the microbial communities in the Dead Sea. The underwater springs are a newly recognized water source for the Dead Sea. Their input of microorganisms and nutrients needs to be considered in the assessment of possible impact of dilution events of the lake surface waters, such as those that will occur in the future due to the intended establishment of the Red Sea-Dead Sea water conduit.     

Differentiation and visualization of diverse cellular phenotypic responses in primary high-content screening

High-throughput screening, based on subcellular imaging, has become a powerful tool in lead discovery. Through the generation of high-quality images, not only the specific target signal can be analyzed but also phenotypic changes of the whole cell are recorded. Yet analysis strategies for the exploration of high-content screening results, in a manner that is independent from predefined control phenotypes, are largely missing. The approach presented here is based on a well-established modeling technique, self-organizing maps (SOMs), which uses multiparametric results to group treatments that create similar morphological effects. This report describes a novel visualization of the SOM clustering by using an image of the cells from each node, with the most representative cell highlighted to deploy the phenotype described by each node. The approach has the potential to identify both expected hits and novel cellular phenotypes. Moreover, different chemotypes, which cause the same phenotypic effects, are identified, thus facilitating "scaffold hopping."