Enhancement of bacterial mutagenicity of bifunctional alkylating agents by expression of mammalian glutathione s-transferase

Recently, we inserted the plasmid vector pKK233-2 containing rat GSH S-transferase (GST) 5-5 cDNA into Salmonella typhimurium TA1535 and found that these bacteria [GST 5-5(+)] expressed the protein and produced mutations when ethylene or methylene dihalides were added [Thier, R., Taylor, J. B., Pemble, S. E., Ketterer, B., Persmark, M., Humphreys, W. G., and Guengerich, F. P. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8576-8580]. After exposure to the known GST 5-5 substrate 1,2-epoxy-3-(4′-nitrophenoxy)propane, the GST 5-5(+) strain showed fewer mutants than the bacteria transfected with the cDNA clone in a reverse orientation [GST 5-5(-)], suggesting a protective role of GST 5-5. However, mutations were considerably enhanced in the GST 5-5(+) strain [as compared to GST 5-5(-)] when 1,2,3,4-diepoxybutane (butadiene diepoxide) or 1,2-epoxy-4-bromobutane was added. The GST 5-5(+) and GST 5-5(-) bacterial stains showed similar responses to 1,2-epoxypropane, 3,4-epoxy-1-butene, and 1,4-dibromobutane. The results suggest that some bifunctional activated butanes are transformed to mutagenic products through GSH conjugation. We also found that the GST 5-5(+) strain showed enhanced mutagenicity with 1,4-dibromo-2,3-epoxybutane, 1,2-epoxy-3-bromopropane (epibromohydrin), and (±)-1,4-dibromo-2,3-dihydroxybutane. The possibility was considered that a 5-membered thialonium ion may be involved in the mutagenicity. Model thialonium compounds were rather stable to hydrolysis in aqueous solution at pH 7.4 and slowly alkylated 4-(4-nitrobenzyl)pyridine. The presence of a hydroxyl group β to the sulfur did not enhance reactivity. Mechanisms involving episulfonium ions are considered more likely. Potential oxidation products of the toxic pesticide 1,2-dibromo-3-chloropropane (DBCP) were also considered in this system. DBCP itself gave rather similar results in the two strains. Others have reported that oxidation of DBCP is required for mutagenicity, along with GST-catalyzed GSH conjugation [Simula, T. P., Glancey, M. J., Söderlund, E. J., Dybing, E., and Wolf, C. R. (1993) Carcinogenesis 14, 2303-2307]. The putative oxidation product 1,2-dibromopropional did not show a difference between the two strains. However, 1,3-dichloroacetone, a model for the putative oxidation product 1-bromo-3-chloroacetone, was considerably more mutagenic in the GST 5-5(+) strain.

Comparison of GST theta activity in liver and kidney of four species

Glutathione transferases (GSTs) catalyzing the conjugation of glutathione with electrophilic substrates are important enzymes in the metabolism of xenobiotics. Several isozymes exhibit polymorphisms in humans. The two deletion polymorphisms of hGSTM1 and hGSTT1 result in total loss of enzyme activity in homozygous null genotype (GSTM1*0 and GSTT1*0 respectively) individuals (Seidegård et al. 1988; Pemble et al. 1994). Individuals that are heterozygous for hGSTT1 show distinctly lower enzyme activities than individuals carrying two functional alleles of hGSTT1 (Wiebel et al. 1996). A similar effect is conceivable for the hGSTM1 polymorphism but has not been verified so far.

Detoxification enzyme activities (CYP1A1 and GST) in the skin of humpback whales as a function of organochlorine burdens and migration status

The activities of glutathione-s-transferase (GST) and cytochrome P-450 1A1 (CYP1A1) enzymes were measured in freshly extracted epidermis of live-biopsied, migrating, southern hemisphere humpback whales (Megaptera novaeangliae). The two quantified enzyme activities did not correlate strongly with each other. Similarly, neither correlated strongly with any of the organochlorine compound groups previously measured in the superficial blubber of the sample biopsy core, likely reflecting the anticipated low levels of typical aryl-hydrocarbon receptor ligands. GST activity did not differ significantly between genders or between northward (early migration) or southward (late migration) migrating cohorts. Indeed, the inter-individual variability in GST measurements was relatively low. This observation raises the possibility that measured activities were basal activities and that GST function was inherently impacted by the fasting state of the sampled animals, as seen in other species. These results do not support the implementation of CYP1A1 or GST as effective biomarkers of organochlorine contaminant burdens in southern hemisphere populations of humpback whales as advocated for other cetacean species. Further investigation of GST activity in feeding versus fasting cohorts may, however, provide some insight into the fasting metabolism of these behaviourally adapted populations.

Locality-sensitive hashing for protein classification

Determination of sequence similarity is a central issue in computational biology, a problem addressed primarily through BLAST, an alignment based heuristic which has underpinned much of the analysis and annotation of the genomic era. Despite their success, alignment-based approaches scale poorly with increasing data set size, and are not robust under structural sequence rearrangements. Successive waves of innovation in sequencing technologies – so-called Next Generation Sequencing (NGS) approaches – have led to an explosion in data availability, challenging existing methods and motivating novel approaches to sequence representation and similarity scoring, including adaptation of existing methods from other domains such as information retrieval. In this work, we investigate locality-sensitive hashing of sequences through binary document signatures, applying the method to a bacterial protein classification task. Here, the goal is to predict the gene family to which a given query protein belongs. Experiments carried out on a pair of small but biologically realistic datasets (the full protein repertoires of families of Chlamydia and Staphylococcus aureus genomes respectively) show that a measure of similarity obtained by locality sensitive hashing gives highly accurate results while offering a number of avenues which will lead to substantial performance improvements over BLAST..

Potential role of EPB41L3 (Protein 4.1B/Dal-1) as a target for treatment of advanced prostate cancer

Background: Loss of erythrocyte membrane protein band 4.1-like 3 (EPB41L3; aliases: protein 4.1B, differentially expressed in adenocarcinoma of the lung-1 (Dal-1)) expression has been implicated in tumor progression. Objective: To evaluate literature describing the role of EPB41L3 in tumorigenesis and metastasis, and to consider whether targeting this gene would be useful in the treatment of prostate cancer. Methods: A literature review of studies describing EPB41L3 and its aliases was conducted. Online databases (NCBI, SwissProt) were also interrogated to collect further data. Results/conclusion: A growing body of evidence supports a role for loss of EPB41L3 in tumor progression, including in prostate cancer. Therapeutic strategies that could be harnessed to upregulate EPB41L3 gene expression in prostate cancer cells are currently being developed.

Protein-energy malnutrition exists and is associated with negative outcomes in morbidly obese hospital patients

Prevalence of protein-energy malnutrition (PEM), food intake inadequacy and associated health-related outcomes in morbidly obese (Body Mass Index ≥ 40 kg/m2) acute care patients are unknown. This study reports findings in morbidly obese participants from the Australasian Nutrition Care Day Survey (ANCDS) conducted in 2010. The ANCDS was a cross-sectional survey involving acute care patients from 56 Australian and New Zealand hospitals. Hospital-based dietitians evaluated participants’ nutritional status (defined by Subjective Global Assessment, SGA) and 24-hour food intake (as 0%, 25%, 50%, 75%, and 100% of the offered food). Three months later, outcome data, including length of stay (LOS) and 90-day in-hospital mortality, were collected. Of the 3122 participants, 4% (n = 136) were morbidly obese (67% females, 55 ± 14 years, BMI: 48 ± 8 kg/m2). Eleven percent (n = 15) of the morbidly obese patients were malnourished, and most (n = 11/15, 73%)received standard hospital diets without additional nutritional support. Malnourished morbidly obese patients had significantly longer LOS and greater 90-day in-hospital mortality than well-nourished counterparts (23 days vs. 9 days, p = 0.036; 14% vs. 0% mortality, p = 0.011 respectively). Thirteen morbidly obese patients (10%) consumed only 25% of the offered meals with a significantly greater proportion of malnourished (n = 4, 27%) versus well-nourished (n = 9, 7%) (p = 0.018). These results provide new knowledge on the prevalence of PEM and poor food intake in morbidly obese patients in Australian and New Zealand hospitals. For the first time internationally, the study establishes that PEM is significantly associated with negative outcomes in morbidly obese patients and warrants timely nutritional support during hospitalisation.

Three-dimensional flow-through protein platform

We have developed a new protein microarray (Immuno-Flow Protein Platform, IFPP) that utilizes a porous nitrocellulose (NC) membrane with printed spots of capture probes. The sample is pumped actively through the NC membrane, to enhance binding efficiency and introduce stringency. Compared to protein microarrays assayed with the conventional incubation-shaking method the rate of binding is enhanced on the IFPP by at least a factor of 10, so that the total assay time can be reduced drastically without compromising sensitivity. Similarly, the sensitivity can be improved. We demonstrate the detection of 1 pM of C-reactive protein (CRP) in 70 mu L of plasma within a total assay time of 7 min. The small sample and reagent volumes, combined with the speed of the assay, make our IFPP also well-suited for a point-of-care/near-patient setting. The potential clinical application of the IFPP is demonstrated by validating CRP detection both in human plasma and serum samples against standard clinical laboratory methods.

One-step homogeneous C-reactive protein assay for saliva

Background: Cardiovascular disease is the leading cause of death in the world. Human C-reactive protein (CRP) has been used in the risk assessment of coronary events. Human saliva mirrors the body's health and well-being and is non-invasive, easy to collect and ideal for third world countries as well as for large patient screening. The aim was to establish a saliva CRP reference range and to demonstrate the clinical utility of salivary CRP levels in assessing the coronary events in a primary health care setting. Methods: We have used a homogeneous bead based assay to detect CRP levels in human saliva. We have developed a rapid 15 min (vs 90 min), sequential, one-step assay to detect CRP in saliva. Saliva was collected from healthy volunteers (n = 55, ages 20-70 years) as well as from cardiac patients (n = 28, ages 43-86 years). Results: The assay incubation time was optimised from 90 min to 15 mm and generated a positive correlation (n = 29, range 10-2189 pg/mL, r2 = 0.94; Passing Bablok slope 0.885. Intercept 0, p>0.10), meaning we could decrease the incubation time and produce equivalent results with confidence. The mean CRP level in the saliva of healthy human volunteers was 285 pg/mL and in cardiac patients was 1680 pg/mL (p<0.01). Analysis of CRP concentrations in paired serum and saliva samples from cardiac patients gave a positive correlation (r2 = 0.84, p<0.001) and the salivary CRP concentration capable of distinguishing healthy from diseased patients. Conclusions: The results suggest that this minimally invasive, rapid and sensitive assay will be useful in large patient screening studies for risk assessment of coronary events. (C) 2011 Elsevier B.V. All rights reserved.

Score-level multibiometric fusion based on Dempster-Shafer theory incorporating uncertainty factors

While existing multi-biometic Dempster-Shafer the- ory fusion approaches have demonstrated promising perfor- mance, they do not model the uncertainty appropriately, sug- gesting that further improvement can be achieved. This research seeks to develop a unified framework for multimodal biometric fusion to take advantage of the uncertainty concept of Dempster- Shafer theory, improving the performance of multi-biometric authentication systems. Modeling uncertainty as a function of uncertainty factors affecting the recognition performance of the biometric systems helps to address the uncertainty of the data and the confidence of the fusion outcome. A weighted combination of quality measures and classifiers performance (Equal Error Rate) are proposed to encode the uncertainty concept to improve the fusion. We also found that quality measures contribute unequally to the recognition performance, thus selecting only significant factors and fusing them with a Dempster-Shafer approach to generate an overall quality score play an important role in the success of uncertainty modeling. The proposed approach achieved a competitive performance (approximate 1% EER) in comparison with other Dempster-Shafer based approaches and other conventional fusion approaches.

Complementary surface-enhanced resonance raman spectroscopic biodetection of mixed protein solutions by chitosan- and silica-coated plasmon-tuned silver nanoparticles

Silver nanoparticles with identical plasmonic properties but different surface functionalities are synthesized and tested as chemically selective surface-enhanced resonance Raman (SERR) amplifiers in a two-component protein solution. The surface plasmon resonances of the particles are tuned to 413 nm to match the molecular resonance of protein heme cofactors. Biocompatible functionalization of the nanoparticles with a thin film of chitosan yields selective SERR enhancement of the anionic protein cytochrome b5, whereas functionalization with SiO2 amplifies only the spectra of the cationic protein cytochrome c. As a result, subsequent addition of the two differently functionalized particles yields complementary information on the same mixed protein sample solution. Finally, the applicability of chitosan-coated Ag nanoparticles for protein separation was tested by in situ resonance Raman spectroscopy.

Functionalized Ag nanoparticles with tunable optical properties for selective protein analysis

We present a preparation procedure for small sized biocompatibly coated Ag nanoparticles with tunable surface plasmon resonances. The conditions were optimised with respect to the resonance Raman signal enhancement of heme proteins and to the preservation of the native protein structure....