Quantitation of mercapturic acid conjugates of 4-hydroxy-2-nonenal and 4-oxo-2-nonenal metabolites in a smoking cessation study

The breakdown of polyunsaturated fatty acids (PUFAs) under conditions of oxidative stress results in the formation of lipid peroxidation (LPO) products. These LPO products such as 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) can contribute to the development of cardiovascular and neurodegenerative diseases and cancer. Conjugation with glutathione, followed by further metabolism to mercapturic acid (MA) conjugates, can mitigate the effects of these LPO products in disease development by facilitating their excretion from the body. We have developed a quantitative method to simultaneously assess levels of 4-oxo-2-nonen-1-ol (ONO)-MA, HNE-MA, and 1,4-dihydroxy-2-nonene (DHN)-MA in human urine samples utilizing isotope-dilution mass spectrometry. We are also able to detect 4-hydroxy-2-nonenoic acid (HNA)-MA, 4-hydroxy-2-nonenoic acid lactone (HNAL)-MA, and 4-oxo-2-nonenoic acid (ONA)-MA with this method. The detection of ONO-MA and ONA-MA in humans is significant because it demonstrates that HNE/ONE branching occurs in the breakdown of PUFAs and suggests that ONO may contribute to the harmful effects currently associated with HNE. We were able to show significant decreases in HNE-MA, DHN-MA, and total LPO-MA in a group of seven smokers upon smoking cessation. These data demonstrate the value of HNE and ONE metabolites as in vivo markers of oxidative stress.     

Pollen performance of Raphanus sativus (Brassicaceae) declines in response to elevated [CO2]

Although increases in atmospheric [CO₂] are known to affect plant physiology, growth and reproduction, understanding of these effects is limited because most studies of reproductive consequences focus solely on female function. Therefore, we examined the effects of CO₂ enrichment on male function in the annual Raphanus sativus. Pollen donors grown under elevated [CO₂] initially sired a higher proportion of seeds per fruit than ambient [CO₂]-grown plants when each was tested against two different standard competitors; however, by the end of the 5-month experiment, these pollen donors sired fewer seeds than ambient [CO₂]-grown plants and produced a lower proportion of viable pollen grains. The results of this experiment confirm that elevated [CO₂] can alter reproductive success. Additionally, the change in response to elevated [CO₂] over time varied among pollen donor families; thus, changes in [CO₂] could act as a selective force on this species.     

Targeted quantitative analysis of superoxide dismutase 1 in cisplatin-sensitive and cisplatin-resistant human ovarian cancer cells

Protein quantification in a complex protein mixture presents a daunting task in biochemical analysis. Antibody-based immunoassays are traditional methods for protein quantification. However, there are issues associated with accuracy and specificity in these assays, especially when the changes are small (e.g., <2-fold). With recent developments in mass spectrometry, monitoring a selected peptide, thus protein, in a complex biological sample has become possible. In this study, we demonstrate a simple mass spectrometry-based method for selective measurement of a moderately low abundant protein, superoxide dismutase 1 (SOD1), in cisplatin-sensitive and cisplatin-resistant human ovarian cancer cells. Selected-reaction-monitoring (SRM) technology was employed to specifically analyze the target peptides in a pair of human ovarian cancer cell lines: 2008/2008-C13*5.25 (cisplatin-sensitive/cisplatin-resistant, respectively). The observed 1.47-fold higher expression in the resistant cell line is consistent with findings by other approaches. This robust liquid chromatography/mass spectrometry (LC/MS) method provides a powerful tool for targeted proteomic verification and/or validation studies.     

Environmental Management for Malaria Control: Knowledge and Practices in Mvomero,Tanzania

Environmental conditions play an important role in the transmission of malaria; therefore, regulating these conditions can help to reduce disease burden. Environmental management practices for disease control can be implemented at the community level to complement other malaria control methods. This study assesses current knowledge and practices related to mosquito ecology and environmental management for malaria control in a rural, agricultural region of Tanzania. Household surveys were conducted with 408 randomly selected respondents from 10 villages and qualitative data were collected through focus group discussions and in-depth interviews. Results show that respondents are well aware of the links between mosquitoes, the environment, and malaria. Most respondents stated that cleaning the environment around the home, clearing vegetation around the home, or draining stagnant water can reduce mosquito populations, and 63% of respondents reported performing at least one of these techniques to protect themselves from malaria. It is clear that many respondents believe that these environmental management practices are effective malaria control methods, but the actual efficacy of these techniques for controlling populations of vectors or reducing malaria prevalence in the varying ecological habitats in Mvomero is unknown. Further research should be conducted to determine the effects of different environmental management practices on both mosquito populations and malaria transmission in this region, and increased participation in effective techniques should be promoted.     

Mechanistic studies of agmatine deiminase from multiple bacterial species

One subfamily of guanidino group-modifying enzymes (GMEs) consists of the agmatine deiminases (AgDs). These enzymes catalyze the conversion of agmatine (decarboxylated arginine) to N-carbamoyl putrescine and ammonia. In plants, viruses, and bacteria, these enzymes are thought to be involved in energy production, biosynthesis of polyamines, and biofilm formation. In particular, we are interested in the role that this enzyme plays in pathogenic bacteria. Previously, we reported the initial kinetic characterization of the agmatine deiminase from Helicobacter pylori and described the synthesis and characterization the two most potent AgD inactivators. Herein, we have expanded our initial efforts to characterize the catalytic mechanisms of AgD from H. pylori as well as Streptococcus mutans and Porphyromonas gingivalis. Through the use of pH rate profiles, pK(a) measurements of the active site cysteine, solvent isotope effects, and solvent viscosity effects, we have determined that the AgDs, like PADs 1 and 4, utilize a reverse protonation mechanism.     

Isolation and characterization of microsatellite loci in Santalum lanceolatum and Santalum leptocladum (Santalaceae)

? Premise of the study: Microsatellite primers were developed for the first time in the native Australian sandalwood species Santalum lanceolatum. ? Methods and Results: Using an enrichment cloning protocol, five novel polymorphic codominant loci were developed and characterized in S. lanceolatum and S. leptocladum. In addition to these, three existing microsatellite loci from other sandalwood species were successfully amplified and characterized for S. lanceolatum and S. leptocladum. Among the eight loci, allelic diversity ranged from 4 to 29. ? Conclusions: Primers will be useful for studies of clonality, genetic diversity and spatial genetic structure in wild populations. When coupled with other molecular techniques will help investigate the relationship between S. lanceolatum and S. leptocladum, species of commercial and conservation interest.     

Mapping Spatial Relationships between Residues in the Ligand-Binding Domain of the 5-Ht3 Receptor Using a Molecular Ruler

The serotonin 5-HT₃ receptor (5-HT₃R) is a member of the Cys-loop ligand-gated ion channel family. We used a combination of site-directed mutagenesis, homology modeling, and ligand-docking simulations to analyze antagonist-receptor interactions. Mutation of E236, which is near loop C of the binding site, to aspartate prevents expression of the receptor on the cell surface, and no specific ligand binding can be detected. On the other hand, mutation to glutamine, asparagine, or alanine produces receptors that are expressed on the cell surface, but decreases receptor affinity for the competitive antagonist d-tubocurarine (dTC) 5-35-fold. The results of a double-mutant cycle analysis employing a panel of dTC analogs to identify specific points of interactions between the dTC analogs and E236 are consistent with E236 making a direct physical interaction with the 12 -OH of dTC. dTC is a rigid molecule of known three-dimensional structure. Together with previous studies linking other regions of dTC to specific residues in the binding site, these data allow us to define the relative spatial arrangement of three different residues in the ligand-binding site: R92 (loop D), N128 (loop A), and E236 (near loop C). Molecular modeling employing these distance constraints followed by molecular-dynamics simulations produced a dTC/receptor complex consistent with the experimental data. The use of the rigid ligands as molecular rulers in conjunction with double-mutant cycle analysis provides a means of mapping the relative positions of various residues in the ligand-binding site of any ligand-receptor complex, and thus is a useful tool for delineating the architecture of the binding site.     

The importance of aquatic vegetation in beaver diets and the seasonal and habitat specificity of aquatic‐terrestrial ecosystem linkages in a subarctic environment

The extent and ecological significance of trophic linkages across ecosystem boundaries have been the subject of considerable recent research attention. North American beavers Castor canadensis engineer terrestrial influences in aquatic ecosystems by constructing terrestrial food caches near their lodges and aquatic influences in terrestrial ecosystems by building dams and flooding low lying areas. However, it is poorly resolved to what extent beavers rely on aquatic food sources and whether this reliance is greater during winter when ice cover physically confines beavers to aquatic habitats or during summer when warm, ice free water promotes the growth and accessibility of aquatic vegetation. Working in a subarctic region, we surveyed the abundance of aquatic and terrestrial food sources in and around lotic and lentic environments and estimated their contributions to beaver diets during open water and ice covered periods using carbon and nitrogen stable isotope analysis of hair samples. Ponds had four times more aquatic vegetation than streams, but terrestrial habitats around ponds had less than half as much shrub cover as habitats adjacent to streams. Beaver diets in this subarctic environment are estimated to be comprised of 60 to 80% aquatic vegetation, with beavers occupying ponds consuming more aquatic vegetation in winter than beavers occupying streams, which rely more on terrestrial shrubs cached near their lodge. Collectively, these results show how the influence of physical barriers on ecosystem linkages can be modified by habitat‐ and season‐specific abundances of preferred resources and the potential for animals to consume food in ecosystems and seasons different from where and when the food was harvested.     

Inhibition of phospholipase C disrupts cytoskeletal organization and gravitropic growth in Arabidopsis roots

The phospholipase protein superfamily plays an important role in hormonal signalling and cellular responses to environmental stimuli. There is also growing evidence for interactions between phospholipases and the cytoskeleton. In this report we used a pharmacological approach to investigate whether inhibiting a member of the phospholipase superfamily, phospholipase C (PLC), affects microtubules and actin microfilaments as well as root growth and morphology of Arabidopsis thaliana seedlings. Inhibiting PLC activity using the aminosteroid U73122 significantly inhibited root elongation and disrupted root morphology in a concentration-dependent manner, with the response being saturated at 5 μM, whereas the inactive analogue U73343 was ineffective. The primary root appeared to lose growth directionality accompanied by root waving and formation of curls. Immunolabelling of roots exposed to increasingly higher U73122 concentrations revealed that the normal transverse arrays of cortical microtubules in the elongation zone became progressively more disorganized or depolymerized, with the disorganization appearing within 1 h of incubation. Likewise, actin microfilament arrays also were disrupted. Inhibiting PLC using an alternative inhibitor, neomycin, caused similar disruptions to both cytoskeletal organization and root morphology. In seedlings gravistimulated by rotating the culture plates by 90°, both U73122 and neomycin disrupted the normal gravitropic growth of roots and etiolated hypocotyls. The effects of PLC inhibitors are therefore consistent with the notion that, as with phospholipases A and D, PLC likewise interacts with the cytoskeleton, alters growth morphology, and is involved in gravitropism.     

Generation of a conditional null allele of NADPH oxidase activator 1 (NOXA1)

NADPH oxidase complexes are multiprotein assemblies that generate reactive oxygen species in a variety of mammalian tissues. The canonical phagocytic oxidase consists of a heterodimeric, enzymatic core comprised of the transmembrane proteins, CYBB andCYBA and is regulated, in part, by an “organizing” function of NCF1 and an “activating” activity of NCF2. In contexts outside of the phagocyte, these regulatory functions may be encoded not only by NCF1 and NCF2, but also alternatively by their respective paralogues, NOXO1 and NOXA1. To allow tissue‐specific dissection of Noxa1 function in mouse, we have generated an allele of Noxa1 suitable for conditional inactivation. Moreover, by crossing Noxa1 conditional allele carriers to B6.129S4‐Meox2^tm1(Cre)Sor/J mice, we have generated first, Noxa1‐null heterozygotes, and ultimately, Noxa1‐null homozygotes. Through the thoughtful use of tissue‐specific, Cre‐expressing mouse strains, the Noxa1 conditional allele will offer insight into the roles of NOXA1 in the variety of tissues in which it is expressed. genesis 48:568–575, 2010. © 2010 Wiley‐Liss, Inc.