Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

Evaluation of the metabochip genotyping array in African Americans and implications for fine mapping of GWAS-identified loci: the PAGE study

The Metabochip is a custom genotyping array designed for replication and fine mapping of metabolic, cardiovascular, and anthropometric trait loci and includes low frequency variation content identified from the 1000 Genomes Project. It has 196,725 SNPs concentrated in 257 genomic regions. We evaluated the Metabochip in 5,863 African Americans; 89% of all SNPs passed rigorous quality control with a call rate of 99.9%. Two examples illustrate the value of fine mapping with the Metabochip in African-ancestry populations. At CELSR2/PSRC1/SORT1, we found the strongest associated SNP for LDL-C to be rs12740374 (p = 3.5 × 10(-11)), a SNP indistinguishable from multiple SNPs in European ancestry samples due to high correlation. Its distinct signal supports functional studies elsewhere suggesting a causal role in LDL-C. At CETP we found rs17231520, with risk allele frequency 0.07 in African Americans, to be associated with HDL-C (p = 7.2 × 10(-36)). This variant is very rare in Europeans and not tagged in common GWAS arrays, but was identified as associated with HDL-C in African Americans in a single-gene study. Our results, one narrowing the risk interval and the other revealing an associated variant not found in Europeans, demonstrate the advantages of high-density genotyping of common and rare variation for fine mapping of trait loci in African American samples.     

Severe restriction of xenotropic murine leukemia virus-related virus replication and spread in cultured human peripheral blood mononuclear cells

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus recently isolated from human prostate cancer and peripheral blood mononuclear cells (PBMCs) of patients with chronic fatigue syndrome (CFS). We and others have shown that host restriction factors APOBEC3G (A3G) and APOBEC3F (A3F), which are expressed in human PBMCs, inhibit XMRV in transient-transfection assays involving a single cycle of viral replication. However, the recovery of infectious XMRV from human PBMCs suggested that XMRV can replicate in these cells despite the expression of APOBEC3 proteins. To determine whether XMRV can replicate and spread in cultured PBMCs even though it can be inhibited by A3G/A3F, we infected phytohemagglutinin-activated human PBMCs and A3G/A3F-positive and -negative cell lines (CEM and CEM-SS, respectively) with different amounts of XMRV and monitored virus production by using quantitative real-time PCR. We found that XMRV efficiently replicated in CEM-SS cells and viral production increased by >4,000-fold, but there was only a modest increase in viral production from CEM cells (<14-fold) and a decrease in activated PBMCs, indicating little or no replication and spread of XMRV. However, infectious XMRV could be recovered from the infected PBMCs by cocultivation with a canine indicator cell line, and we observed hypermutation of XMRV genomes in PBMCs. Thus, PBMCs can potentially act as a source of infectious XMRV for spread to cells that express low levels of host restriction factors. Overall, these results suggest that hypermutation of XMRV in human PBMCs constitutes one of the blocks to replication and spread of XMRV. Furthermore, hypermutation of XMRV proviruses at GG dinucleotides may be a useful and reliable indicator of human PBMC infection.     

Effect of amine fluoride–stannous fluoride preparations on oral yeasts in the elderly: a randomised placebo‐controlled trial

Objectives: Oral yeast infections are an emerging problem among medically compromised and frail elderly. Antifungal drug resistance is also increasing because of an increase in non‐albicans Candida strains in these populations. We therefore set out to study, in the randomised‐controlled trial setting if the use of a topical amine fluoride–stannous fluoride combination (AmF–SnF₂) could control oral Candida growth in the elderly. The hypothesis was based on earlier findings showing that in vitro this combination had antifungal efficacy. Methods: A total of 194 nursing home residents were randomised to receive either the test mouth rinse and toothpaste or a placebo twice daily for 8 months. Of these, 136 completed the trial. Saliva samples were taken using the oral rinse method, cultivated and the strain level identified with routine microbial methods. Compliance and use of preparations was assessed by a nurse. Results: Significantly at the end of the trial, less mucosal lesions were observed in the test group in comparison to controls. Total bacterial count decreased in both the groups during the trial. Candida albicans was the most prevalent strain detected both at baseline and 8 months later. Only a few subjects carried non‐albicans strains. The AmF–SnF₂ did not significantly affect mean oral Candida counts, but median Candida counts were reduced in the AmF–SnF₂ group while an increase was seen in the placebo group. However, the differences observed were not statistically significant. Compliance among the regular elderly users slightly increased during the trial for both the groups. Conclusion: The number of subjects with high Candida counts decreased in the AmF–SnF₂ group. Hence, the fluoride combination might be useful as a support therapy for oral candidiasis. Prevalence of non‐albicans Candida strains was low in this population.     

Direct Quantitation and Detection of Salmonellae in Biological Samples without Enrichment, Using Two-Step Filtration and Real-Time PCR

A new two-step filtration protocol followed by a real-time PCR assay based on SYBR green I detection was developed to directly quantitate salmonellae in two types of biological samples: i.e., chicken rinse and spent irrigation water. Four prefiltration filters, one type of final filter, and six protocols for recovery of salmonellae from the final filter were evaluated to identify an effective filtration protocol. This method was then combined with a real-time PCR assay based on detection of the invA gene. The best results were obtained by subsequent filtration of 100 ml of chicken rinse or 100 ml of spent irrigation water through filters with pore diameters of >40 μm to remove large particles and of 0.22 μm to recover the Salmonella cells. After this, the Salmonella cells were removed from the filter by vortexing in 1 ml of physiological saline, and this sample was then subjected to real-time quantitative PCR. The whole procedure could be completed within 3 h from sampling to quantitation, and cell numbers as low as 7.5 × 10² CFU per 100-ml sample could be quantified. Below this limit, qualitative detection of concentrations as low as 2.2 CFU/100 ml sample was possible on occasion. This study has contributed to the development of a simple, rapid, and reliable method for quantitation of salmonellae in food without the need for sample enrichment or DNA extraction.     

<Emphasis Type="Italic">In silico</Emphasis>discovery of gene-coding variants in murine quantitative trait loci using strain-specific genome sequence databases

Background  

The identification of genes underlying complex traits has been aided by quantitative trait locus (QTL) mapping approaches, which in turn have benefited from advances in mammalian genome research. Most recently, whole-genome draft sequences and assemblies have been generated for mouse strains that have been used for a large fraction of QTL mapping studies. Here we show how such strain-specific mouse genome sequence databases can be used as part of a high-throughput pipeline for the in silico discovery of gene-coding variations within murine QTLs. As a test of this approach we focused on two QTLs on mouse chromosomes 1 and 13 that are involved in physical dependence on alcohol.     

Rational design of an active avidin monomer

Homotetrameric chicken avidin that binds four molecules of biotin was converted to a monomeric form (monoavidin) by mutations of two interface residues: tryptophan 110 in the 1 --> 2 interface was mutated to lysine and asparagine 54 in the 1 --> 4 interface was converted to alanine. The affinity for biotin binding of the mutant decreased from K(d) approximately 10(-15) m of the wild-type tetramer to K(d) approximately 10(-7) m, which was studied by an optical biosensor IAsys and by a fluorescence spectroscopical method in solution. The binding was completely reversible. Conversion of the tetramer to a monomer results in increased sensitivity to proteinase K digestion. The antigenic properties of the mutated protein were changed, such that monoavidin was only partially recognized by a polyclonal antibody whereas two different monoclonal antibodies entirely failed to recognize the avidin monomer. This new monomeric avidin, which binds biotin reversibly, may be useful for applications both in vitro and in vivo. It may also shed light on the effect of intersubunit interactions on the binding of ligands.     

Changed energy state and increased mitochondrial beta-oxidation rate in liver of rats associated with lowered proton electrochemical potential and stimulated uncoupling protein 2 (UCP-2) expression: evidence for peroxisome proliferator-activated receptor-alpha independent induction of UCP-2 expression

Lowering of plasma triglyceride levels by hypolipidemic agents is caused by a shift in the liver cellular metabolism, which become poised toward peroxisome proliferator-activated receptor (PPAR) alpha-regulated fatty acid catabolism in mitochondria. After dietary treatment of rats with the hypolipidemic, modified fatty acid, tetradecylthioacetic acid (TTA), the energy state parameters of the liver were altered at the tissue, cell, and mitochondrial levels. Thus, the hepatic phosphate potential, energy charge, and respiratory control coefficients were lowered, whereas rates of oxygen uptake, oxidation of pyridine nucleotide redox pairs, beta-oxidation, and ketogenesis were elevated. Moderate uncoupling of mitochondria from TTA-treated rats was confirmed, as the proton electrochemical potential (Delta(p)) was 15% lower than controls. The change affected the Delta(Psi) component only, leaving the (Delta)pH component unaltered, suggesting that TTA causes induction of electrogenic ion transport rather than electrophoretic fatty acid activity. TTA treatment induced expression of hepatic uncoupling protein 2 (UCP-2) in rats as well as in wild type and PPARalpha-deficient mice, accompanied by a decreased double bond index of the mitochondrial membrane lipids. However, changes of mitochondrial fatty acid composition did not seem to be related to the effects on mitochondrial energy conductance. As TTA activates PPARdelta, we discuss how this subtype might compensate for deficiency of PPARalpha. The overall changes recorded were moderate, making it likely that liver metabolism can maintain its function within the confines of its physiological regulatory framework where challenged by a hypolipemic agent such as TTA, as well as others.