Vitamin E status and metabolism in adult and aged aryl hydrocarbon receptor null mice

The aryl hydrocarbon receptor (AhR) is involved in regulation of mechanisms for detoxification of xenobiotics, as well as vitamin A metabolism. Vitamin E is a fat-soluble nutrient whose metabolism is initialized via the cytochrome P450 system. Thus, AhR absence could alter hepatic regulation of α-tocopherol metabolism. To test this hypothesis, we assessed vitamin E status in adult (2-5 m) and old (21-22 m), wild-type and AhR-null mice. Plasma α-tocopherol concentrations in AhR-null mice (2.3±1.2 μmol/L, n=19) were lower than those of wild-type mice (3.2±1.2, n=17, P=.0131); those in old mice (3.2±1.2, n=20) were higher than those of adults (2.2±1.0, n=16, P=.0075). Hepatic α-tocopherol concentrations were not different between genotypes, but were nearly double in old (32±8 nmol/g, n=20) as compared with adult mice (17±2, n=16, P<.0001). Hepatic Cyp3a concentrations in AhR-null mice were greater than those in wild-type mice (P=.0011). Genotype (P=.0047), sex (P<.0001) and age (P<.0001) were significant modifiers of liver α-tocopherol metabolite (α-CEHC) concentrations. In general, Cyp3a concentrations correlated with hepatic α-tocopherol (r=0.3957, P<.05) and α-CEHC (r=0.4260, P<.05) concentrations. Since there were no significant genotype differences in the hepatic α- or γ-tocopherol concentrations, AhR-null mice did not have dramatically altered vitamin E metabolism. Since they did have higher hepatic α-CEHC concentrations, these data suggest metabolism was up-regulated in the AhR-null mice in order to maintain the hepatic tocopherol concentrations similar to those of wild-type mice.     

Rapid PCR-based test for identifying Candida albicans by using primers derived from the pH-regulated KER1 gene

A PCR-based method in combination with a simple, reliable and inexpensive DNA extraction procedure for rapid detection of Candida albicans clinical isolates is described here. The extraction protocol is based on a combination of chemical (NaOH and detergents) and physical (boiling) treatments, thus avoiding many of the problems inherent in the currently available DNA extraction protocols (basically the use of expensive and/or toxic chemical reagents), and may be useful for daily clinical routine. The PCR-based system described here uses a single pair of primers (SC1F and SC1R) deduced from the C. albicans-specific KER1 gene sequence. These primers amplify a 670-bp fragment of the KER1 gene. All the clinical C. albicans isolates generated the expected 670-bp amplicon. Other non-albicans Candida species, including the azole-resistant C. krusei and C. glabrata, and the very closely related C. dubliniensis, failed to amplify any DNA fragment. The PCR results reported here suggest that amplification with SC1F and SC1R primers is species specific and, consequently, may be useful for specifically identifying C. albicans strains.     

Biofilm formation by Candida albicans mutants for genes coding fungal proteins exhibiting the eight-cysteine-containing CFEM domain

Several features and functions of a Candida albicans gene, PGA10 (also designated as RBT51), coding for a putative polypeptide species belonging to a subset of fungal proteins containing an eight-cysteine domain referred as CFEM (Common in several Fungal Extracellular Membrane proteins), are described. The ORF of the gene (ORF19.5674) encoded a protein of 250 amino acids, with a predicted molecular mass of 25.17 kDa. The product of the PGA10 gene also exhibited some features reminiscent of a class II-type hydrophobin. Deletion of PGA10 resulted in a cascade of pleiotropic effects, mostly affecting cell-surface-related properties. Thus, the null pga10Delta mutant displayed an increased sensitivity to cell-wall-perturbing agents and formed fragile biofilms that appeared partially split and weakly attached to the substratum. The biofilm-forming ability of several C. albicans mutants with single, double and triple deletions of genes encoding other protein species also containing the CFEM domain (RBT5 and WAP1/CSA1) was determined. These mutants also exhibited an abnormal ability to form biofilms. Overall, the evidence presented here suggests that fungal proteins containing the CFEM domain (Pga10p/Rbt51p, Rbt5p and Wap1p/Csa1p) may play a key role in the formation, development and/or maintenance of the biofilm structure in C. albicans.     

Unravelling the complexity of salt marsh ‘Fucus cottonii’ forms (Phaeophyceae,Fucales)

Genetic affiliation, nuclear DNA content, and gamete functioning were examined in small salt marsh Fucus from three localities in western Ireland. Individuals with small and dioecious receptacles were found at all localities, but production of germlings was only evident at Locality 1. Here, the Fucus vegetation formed a morphological cline from F. vesiculosus with bladders in the mid-intertidal to small Fucus individuals lacking bladders in the salt marsh of the upper intertidal. Measurements of nuclear DNA content ranged from 1–1.8 pg at this locality, with F. vesiculosus individuals in the lower range. At the two other localities, the small salt marsh Fucus consisted of distinct morphological entities. Microsatellite analyses revealed that individuals at Locality 2 were derived mainly from F. vesiculosus, whereas those from Locality 3 were hybrids between F. vesiculosus and F. spiralis with greatest affiliation to F. spiralis. While the small salt marsh Fucus forms from Locality 2 had high nuclear DNA content (c. 4 pg) and were probably octoploids, the small salt marsh Fucus from Locality 3 formed two groups: one with high (3.9–4.6 pg) and one with low (1.5–1.9 pg) nuclear DNA content. Nuclear DNA content measured in individuals from Locality 3 varied between 1.1–2.8 pg in F. vesiculosus and 2–3.5 pg in F. spiralis, and showed a more or less stepwise increase in both species, consistent with polyploidy. We hypothesize that the small salt marsh Fucus forms originate from genome size changes in the parental taxa.     

An Invasive Mammal (the Gray Squirrel,Sciurus carolinensis) Commonly Hosts Diverse and Atypical Genotypes of the Zoonotic Pathogen Borrelia burgdorferi Sensu Lato

Invasive vertebrate species can act as hosts for endemic pathogens and may alter pathogen community composition and dynamics. For the zoonotic pathogen Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, recent work shows invasive rodent species can be of high epidemiological importance and may support host-specific strains. This study examined the role of gray squirrels (Sciurus carolinensis) (n = 679), an invasive species in the United Kingdom, as B. burgdorferi sensu lato hosts. We found that gray squirrels were frequently infested with Ixodes ricinus, the main vector of B. burgdorferi sensu lato in the United Kingdom, and 11.9% were infected with B. burgdorferi sensu lato. All four genospecies that occur in the United Kingdom were detected in gray squirrels, and unexpectedly, the bird-associated genospecies Borrelia garinii was most common. The second most frequent infection was with Borrelia afzelii. Genotyping of B. garinii and B. afzelii produced no evidence for strains associated with gray squirrels. Generalized linear mixed models (GLMM) identified tick infestation and date of capture as significant factors associated with B. burgdorferi sensu lato infection in gray squirrels, with infection elevated in early summer in squirrels infested with ticks. Invasive gray squirrels appear to become infected with locally circulating strains of B. burgdorferi sensu lato, and further studies are required to determine their role in community disease dynamics. Our findings highlight the fact that the role of introduced host species in B. burgdorferi sensu lato epidemiology can be highly variable and thus difficult to predict.     

Eicosapentaenoic acid inhibits tumour necrosis factor-α-induced lipolysis in murine cultured adipocytes

Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid with beneficial effects in obesity and insulin resistance. High levels of proinflammatory cytokine tumour necrosis factor-α (TNF-α) in obesity promote lipolysis in adipocytes, leading to the development of insulin resistance. Thus, the aims of the present study were to analyze the potential antilipolytic properties of EPA on cytokine-induced lipolysis and to investigate the possible mechanisms involved. The EPA effects on basal and TNF-α-induced lipolysis were determined in both primary rat and 3T3-L1 adipocytes. Treatment of primary rat adipocytes with EPA (100 and 200 μM) significantly decreased basal glycerol release (P<.01) and prevented cytokine-induced lipolysis in a dose-dependent manner (P<.001). Moreover, EPA decreased TNF-α-induced activation of nuclear factor-κB and extracellular-related kinase 1/2 phosphorylation. In addition, the antilipolytic action of EPA was stimulated by the AMP-kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-b-d-ribofuranoside and blocked by the AMPK-inhibitor compound C. Moreover, we found that EPA stimulated hormone-sensitive lipase (HSL) phosphorylation on serine-565, which further supports the involvement of AMPK in EPA's antilipolytic actions. Eicosapentaenoic acid treatment (24 h), alone and in the presence of TNF-α,? also decreased adipose triglyceride lipase (ATGL) protein content in cultured adipocytes. However, oral supplementation with EPA for 35 days was able to partially reverse the down-regulation of HSL and ATGL messenger RNA observed in retroperitoneal adipose tissue of high-fat-diet-fed rats. These findings suggest that EPA inhibits proinflammatory cytokine-induced lipolysis in adipocytes. This effect might contribute to explain the insulin-sensitizing properties of EPA.     

Essential role of insulin and insulin-like growth factor 1 receptor signaling in cardiac development and function

Cardiovascular disease is the leading cause of death in people with type 2 diabetes and is linked to insulin resistance even in the absence of diabetes. Here we show that mice with combined deficiency of the insulin receptor and insulin-like growth factor 1 (IGF-1) receptor in cardiac and skeletal muscle develop early-onset dilated cardiomyopathy and die from heart failure within the first month of life despite having a normal glucose homeostasis. Mice lacking the insulin receptor show impaired cardiac performance at 6 months, and mice lacking the insulin receptor plus one Igf1r allele have slightly increased mortality. By contrast, mice lacking the IGF-1 receptor or the IGF-1 receptor plus one Ir allele appear normal. Morphological characterization and oligonucleotide array analysis of gene expression demonstrate that prior to development of these physiological defects, mice with combined deficiency of both insulin and IGF-1 receptors have a coordinated down-regulation of genes encoding components of the electron transport chain and mitochondrial fatty acid beta-oxidation pathways and altered expression of contractile proteins. Thus, while neither the insulin receptor nor IGF-1 receptor in muscle is critical for glucose homeostasis during the first month of life, signaling from these receptors, particularly the insulin receptor, is required for normal cardiac metabolism and function.