Development of multiplexed protein profiling and detection using near infrared detection of reverse-phase protein microarrays

Protein microarrays have been recently employed for signal pathway profiling and high-throughput protein expression analysis. Reversephase arrays, where the array consists of immobilized analytes and lysates has especially shown promise in low abundance analyte detection and signal pathway profiling using phospho-specific antibodies. A limitation to current reverse phase array methodology is the inability to multiplex proteomic-based endpoints as each array can only report one analyte endpoint. In this study, we report on the use of a dual dye based approach that can effectively double the number of endpoints observed per array allowing, for example, both phosphospecific and total protein levels to be measured and analyzed at once. The method utilizes antibody bound dyes that emit in the infrared spectral region as a means of sensitive and specific detection.     

Fluorescent Amplified Fragment Length Polymorphism Analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis Isolates

DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis. All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.     

Association Between Body Fat Response to Exercise Training and Multilocus ADR Genotypes

Objectives : To examine the contribution of adrenergic receptor (ADR) gene polymorphisms and their gene‐gene interactions to the variability of exercise training‐induced body fat response. Research Methods and Procedures : This was an intervention study that used a volunteer sample of 70 healthy, sedentary men (n = 29) and postmenopausal women (n = 41) 50 to 75 years of age, with a BMI ≤37 kg/m², from the Washington, DC, metropolitan area. Participants completed 6 weeks of dietary stabilization (American Heart Association diet) before 24 weeks of supervised aerobic exercise training. Diet was maintained throughout the intervention. Change in percent total body fat, percent trunk fat, and fat mass by DXA in ADR genotype groups (Glu¹²/Glu⁹ α2b‐ADR, Trp64Arg β3‐ADR, and Gln27Glu β2‐ADR) at baseline and after 24 weeks of aerobic exercise training was measured. Results : In multivariate analysis (covariates: age, gender, and baseline value of phenotype), best fit models for percent total body and trunk fat response to exercise training retained main effects of all three ADR gene loci and the effects of each gene‐gene interaction (p = 0.009 and 0.003, respectively). Similarly, there was a trend for the fat mass response model (p = 0.03). The combined genetic factors explained 17.5% of the overall model variability for percent total body fat, 22% for percent trunk fat, and 10% for fat mass. Discussion : The body fat response to exercise training in older adults is associated with the combined effects of the Glu¹²/Glu⁹ α2b‐, Trp64Arg β3‐, and Gln27Glu β2‐ADR gene variants and their gene‐gene interactions.     

Assessment of Potential Predation Costs of Male Decoration and Courtship Display in Wolf Spiders Using Video Digitization and Playback

We used video digitization and playback techniques to examine the potential predation risk of leg decorations and visual displays of male wolf spiders, which vary across the genus Schizocosa (Araneae: Lycosidae). Video images of courting males of four Schizocosa species were modified by adding or removing tufts and presented on LCD minitelevisions to the wolf spider Hogna helluo, a common predator of Schizocosa. Predatory responses of H. helluo varied significantly among stimuli and were highest for S. ocreata (which has decorative tufts and leg-waving displays) and lower for S. rovneri, S. duplex, and S. uetzi (which lack decorations and visual displays). Removal of tufts from S. ocreata significantly reduced predatory responses of H. helluo, but addition of tufts to other species had no effect. Results suggest that leg decorations may increase detection of active leg-waving displays and thereby increase predation risk.     

Foliar physiology of yellow-poplar ( Liriodendron tulipifera L.) exposed to O3 and elevated CO2 over five seasons.

The chronic effects of ozone (O₃) alone or combined with elevated carbon dioxide (CO₂) on the foliar physiology of unfertilized field-grown yellow-poplar ( Liriodendron tulipifera L.) seedlings were studied from 1992 to 1996. Within open-top chambers, juvenile trees were exposed to the following: charcoal-filtered air (CF); 1× ambient ozone (1XO₃); 1.5× ambient ozone (1.5XO₃); 1.5× ambient ozone plus 700 ppm carbon dioxide (1.5XO₃+CO₂); or chamberless open-air (OA). Seasonal 24-h mean ambient O₃ concentrations ranged from 32 to 46 ppm over the five seasons. Averaged over 5 years, midseason net photosynthesis at saturating light ( A _sat) was reduced by 14% ( P =0.029) and stomatal conductance ( g _s) was reduced, albeit non-significant, by 13% ( P =0.096) in upper canopy foliage exposed to 1.5XO₃-air relative to CF controls. There were no significant differences over the 5 years in A _sat and g _s between trees grown in 1XO₃- and 1.5XO₃-air. Our results support the hypothesis that the magnitude of O₃ effects on A _sat and g _s decreases as saplings age. When averaged over the five seasons of exposure, total chlorophyll concentration ( chl) was not significantly affected by exposure to elevated O₃; however, in 1.5XO₃+CO₂-air, foliar chl was reduced by 33% relative to all others ( P <0.001). In 1.5XO₃+CO₂-air, A _sat was 1.4–1.9 times higher ( P <0.001) and g _s was 0.7 times lower ( P =0.022) than all others. O₃ uptake in juvenile trees exposed to elevated O₃ plus elevated CO₂ (1.5XO₃+CO₂-air) was most comparable to trees exposed to ambient air (1XO₃) throughout the study. These findings suggest that elevated CO₂ may minimize the negative effects of O₃ by reducing O₃ uptake through decreased stomatal conductance.     

Near-infrared fluorescence detection permits accurate imaging of loading controls for Western blot analysis

Housekeeping proteins are typically chosen as internal loading controls for Western blot analysis because of their high, relatively constant expression. It was previously reported that antibodies against beta-actin did not reliably identify differences in sample loading, and extended antibody incubations caused a failure to discriminate differences in target protein levels. Here, beta-actin and GAPDH were evaluated as loading controls using near-infrared fluorescence. A load-dependent response in signal intensity was observed over a 250-fold range of sample concentrations, with R(2) values as high as 0.9939. Longer antibody incubations continued to detect differences in protein level and load-dependent responses became more linear.     

Identifying and characterizing the biological targets of metallotherapeutics: Two approaches using Au(I)-protein interactions as model systems

A significant challenge in the field of medicinal inorganic chemistry is the identification of biological targets of metal-based drugs and the characterization of the metal–biomolecule adducts. A classic example is Au(I), which has long been used to treat rheumatoid arthritis despite a poor understanding of its biological targets due to the lability, reactivity, and “spectroscopic silence” that are characteristic of Au(I). Here, we report two qualitative methods for characterizing Au(I)–protein adducts: a thiol-reactive probe that facilitates the identification of biological cysteine–Au(I) adducts and a photoreactive Au(I) complex that produces a covalent bond between the Au(I) complex and the biomolecule.     

Metal binding domains 3 and 4 of the Wilson disease protein: solution structure and interaction with the copper(I) chaperone HAH1

The Wilson disease protein or ATP7B is a P 1B-type ATPase involved in human copper homeostasis. The extended N-terminus of ATP7B protrudes into the cytosol and contains six Cu(I) binding domains. This report presents the NMR structure of the polypeptide consisting of soluble Cu(I) binding domains 3 and 4. The two domains exhibit ferredoxin-like folds, are linked by a flexible loop, and act independently of one another. Domains 3 and 4 tend to aggregate in a concentration-dependent manner involving nonspecific intermolecular interactions. Both domains can be loaded with Cu(I) when provided as an acetonitrile complex or by the chaperone HAH1. HAH1 forms a 70% complex with domain 4 that is in fast exchange with the free protein in solution. The ability of HAH1 to form a complex only with some domains of ATP7B is an interesting property of this class of proteins and may have a signaling role in the function of the ATPases.