Pharmacogenomic profiling of an oxidative stress-mediated spongiform encephalopathy

The majority of cellular superoxide is generated in the mitochondria as a by-product of normal oxidative metabolism. In the mitochondria, superoxide is detoxified by manganese superoxide dismutase (SOD2). Mice lacking SOD2 demonstrate a multifaceted neonatal lethal phenotype, including a spongiform encephalopathy that is preventable through antioxidant treatment. The molecular events behind the observed pathology in the cortex of these mice are unknown. We hypothesized that the lack of SOD2 would result in significant changes in cortical gene expression and that therapeutically beneficial antioxidant treatment would normalize the expression of some genes, providing insight into the mechanism by which mitochondrial oxidative stress results in neurodegeneration. We report the identification of gene expression profiles associated with this paradigm, which characterize the degree of response to the pharmacologic intervention. We have identified specific pathways targeted by endogenous oxidative stress, including glutathione metabolism, iron metabolism, and cell-survival pathways centering on the kinase AKT. The normalization of expression of some of these pathways by antioxidant treatment suggests approaches to treating disease in which endogenous oxidative stress plays a role.     

Crystal structure of a phage Twort group I ribozyme-product complex

Group I introns are catalytic RNAs capable of orchestrating two sequential phosphotransesterification reactions that result in self-splicing. To understand how the group I intron active site facilitates catalysis, we have solved the structure of an active ribozyme derived from the orf142-I2 intron from phage Twort bound to a four-nucleotide product RNA at a resolution of 3.6 A. In addition to the three conserved domains characteristic of all group I introns, the Twort ribozyme has peripheral insertions characteristic of phage introns. These elements form a ring that completely envelops the active site, where a snug pocket for guanosine is formed by a series of stacked base triples. The structure of the active site reveals three potential binding sites for catalytic metals, and invokes a role for the 2' hydroxyl of the guanosine substrate in organization of the active site for catalysis.     

A zinc(II)/lead(II)/cadmium(II)-inducible operon from the Cyanobacterium anabaena is regulated by AztR, an alpha3N ArsR/SmtB metalloregulator

A novel Zn(II)/Pb(II)/Cd(II)-responsive operon that consists of genes encoding a Zn(II)/Pb(II) CPx-ATPase efflux pump (aztA) and a Zn(II)/Cd(II)/Pb(II)-specific SmtB/ArsR family repressor (aztR) has been identified and characterized from the cyanobacterium Anabaena PCC 7120. In vivo real time quantitative RT-PCR assays reveal that both aztR and aztA expression are induced by divalent metal ions Zn(II), Cd(II), and Pb(II) but not by other divalent [Co(II), Ni(II)] or monovalent metal ions [Cu(I) and Ag(I)]. The introduction of a plasmid containing the azt operon into a Zn(II)/Cd(II)-hypersensitive Escherichia coli strain GG48 functionally restores Zn(II) and Pb(II) resistance with a limited effect on Cd(II) resistance. Gel mobility shift assays and aztR O/P-lacZ induction experiments confirm that AztR is the metal-regulated repressor of this operon. In vitro biochemical and mutagenesis studies indicate that AztR contains a sole metal-binding site, designated the alpha3N site, that binds Zn(II), Cd(II), and Pb(II) with a high affinity. Optical absorption spectra of Co(II)- and Cd(II)-substituted AztR and (113)Cd NMR spectroscopy of (113)Cd(II)-substituted AztR reveal that the sole alpha3N site in AztR is a CadC-like distorted tetrahedral S(3)(N,O) metal site. The first metal-coordination shell in the AztR alpha3N site differs from other alpha3N family members that sense Cd(II)/Pb(II) and those alpha5 repressors that sense Zn(II)/Co(II). Our results reveal that the alpha3N site in AztR mediates derepression of the azt operon in the presence of Zn(II), as well as Cd(II) and Pb(II); this might have provided Anabaena with an evolutionary advantage to adapt to heavy-metal-rich environments, while maintaining homeostasis of an essential metal ion, Zn(II).     

Comparative susceptibility of sweetpotato weevil (Coleoptera: Brentidae) to selected insecticides

The response of sweetpotato weevil, Cylas formicarius (F.) (Coleoptera: Brentidae), to insecticides used for its control was tested in laboratory bioassays. A glass vial bioassay technique was used to determine the susceptibility of two cohorts of sweetpotato weevil to selected insecticides. Vials were treated with methyl parathion, bifenthrin, cyfluthrin, carbaryl, and phosmet. Sweetpotato weevils demonstrated a mortality response to increasing concentrations of all insecticides tested, and our results indicated decreases in susceptibility of the Louisiana cohort of sweetpotato weevil compared with the Texas cohort for all insecticides tested. Methyl parathion was the most toxic chemical tested for both cohorts, followed by the pyrethroids, cyfluthrin and bifenthrin. Phosmet exhibited moderate toxicity compared with other chemicals tested, whereas sweetpotato weevils were least susceptible to carbaryl. Significant differences in lethal concentration (LC)50 and LC90 values for cyfluthrin and bifenthrin, the LC50 values for methyl parathion and phosmet, and the LC90 values for carbaryl were observed between the two cohorts. This study documents baseline toxicological data for five insecticides in two populations of sweetpotato weevil and demonstrates that susceptibility to all insecticides tested is lower for the Louisiana population compared with the Texas population.     

Application of 13C-[2] - and 13C-[1,2] acetate in metabolic labelling studies of yeast and insect cells

The advantage of using ¹³C-labelled glucose in metabolic studies is that it is an important carbon and energy source for almost all biotechnologically and medically important organisms. On the other hand, the disadvantage is its relatively high cost in the labelling experiments. Looking for cheaper alternatives we found that ¹³C-[2] acetate or ¹³C-[1,2] acetate is a prospective compound for such experiments. Acetate is well incorporated by many organisms, including mammalian and insect cell cultures as preferred source of acetyl-CoA. Our experimental results using ¹³C NMR demonstrated that acetate was efficiently incorporated into glutamate and alanine secreted by the insect cell culture. Using D-stat culture of Saccharomyces uvarum on glucose/¹³C-acetate mineral media we demonstrated that the labelling patterns of proteinogenic amino acids can be well predicted on the basis of specific substrate consumption rates using the modified scheme of yeast metabolism and stoichiometric modelling. According to this scheme aspartate and alanine in S. uvarum under the experimental conditions used is synthesised in the mitochondria. Synthesis of alanine in the mitochondria was also demonstrated for Spodoptera frugiperda. For both organisms malic enzyme was also operative. For S. uvarum it was shown that the activity of malic enzyme is sufficient for supporting the mitochondrial biosynthetic reactions with NADPH.     

Comparative genome analysis of the high pathogenicity Salmonella Typhimurium strain UK-1

Salmonella enterica serovar Typhimurium, a gram-negative facultative rod-shaped bacterium causing salmonellosis and foodborne disease, is one of the most common isolated Salmonella serovars in both developed and developing nations. Several S. Typhimurium genomes have been completed and many more genome-sequencing projects are underway. Comparative genome analysis of the multiple strains leads to a better understanding of the evolution of S. Typhimurium and its pathogenesis. S. Typhimurium strain UK-1 (belongs to phage type 1) is highly virulent when orally administered to mice and chickens and efficiently colonizes lymphoid tissues of these species. These characteristics make this strain a good choice for use in vaccine development. In fact, UK-1 has been used as the parent strain for a number of nonrecombinant and recombinant vaccine strains, including several commercial vaccines for poultry. In this study, we conducted a thorough comparative genome analysis of the UK-1 strain with other S. Typhimurium strains and examined the phenotypic impact of several genomic differences. Whole genomic comparison highlights an extremely close relationship between the UK-1 strain and other S. Typhimurium strains; however, many interesting genetic and genomic variations specific to UK-1 were explored. In particular, the deletion of a UK-1-specific gene that is highly similar to the gene encoding the T3SS effector protein NleC exhibited a significant decrease in oral virulence in BALB/c mice. The complete genetic complements in UK-1, especially those elements that contribute to virulence or aid in determining the diversity within bacterial species, provide key information in evaluating the functional characterization of important genetic determinants and for development of vaccines.     

Role of sindbis virus capsid protein region II in nucleocapsid core assembly and encapsidation of genomic RNA

Sindbis virus is an enveloped positive-sense RNA virus in the alphavirus genus. The nucleocapsid core contains the genomic RNA surrounded by 240 copies of a single capsid protein. The capsid protein is multifunctional, and its roles include acting as a protease, controlling the specificity of RNA that is encapsidated into nucleocapsid cores, and interacting with viral glycoproteins to promote the budding of mature virus and the release of the genomic RNA into the newly infected cell. The region comprising amino acids 81 to 113 was previously implicated in two processes, the encapsidation of the viral genomic RNA and the stable accumulation of nucleocapsid cores in the cytoplasm of infected cells. In the present study, specific amino acids within this region responsible for the encapsidation of the genomic RNA have been identified. The region that is responsible for nucleocapsid core accumulation has considerable overlap with the region that controls encapsidation specificity.     

Therapeutic perspectives for the treatment of Huntington's disease: treating the whole body

Huntington's disease (HD) is a tremendously debilitating disorder that strikes relatively young individuals and progresses rapidly over the next ten to fifteen years inducing a loss of cognitive and motor skills and eventually death occurs. The primary locus of the disorder is a polyglutamine expansion of the protein product of the huntingtin (htt) gene. The htt protein appears to be a scaffolding protein that orchestrates the complex assembly of multiple intracellular proteins involved in multiple processes, including vesicular movement and cell metabolism. The htt protein is ubiquitously expressed in human tissues but the predominance of the interest in the pathology lies in its effects on the central nervous system (CNS). Most of the current therapeutics for HD thus have been targeted at preventing neuronal damage in the CNS, however, a considerable body of evidence has been accumulating to suggest that the maintenance of a healthy nervous system is tightly linked with peripheral physiological health. Therefore treatment of both the peripheral and central pathophysiologies of HD could form the basis of a more effective HD therapeutic strategy.