[Selection of reliable reference genes for qRT-PCR analysis in human non-cancerous gastric tissue].

UNLABELLED: Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in complex diseases like obesity and gastritis. However, variations in amount of starting material, enzymatic efficiency and presence of amplification inhibitors can lead to quantification errors. Hence, the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Human gastric tissue has been the least investigated for stability of reference gene expression. In this study, three popular algorithms, GeNorm, NormFinder and BestKeeper were used to evaluate the reference gene stability. CONCLUSION: HPRT1 and GAPDH are the best performing pair of reference genes for qRT-PCR profiling experiments involving non-malignant gastric tissue samples.

The effect of dopamine receptor blockade on motor behavior in Aplysia californica.

The mammalian D1- and D2-like receptor blockers SCH-23390 and raclopride were used to block receptors in Aplysia californica, and the effect on reflexes and escape behavior was examined. Four groups of 20 young adults were each injected with SCH-23390, raclopride, SCH-23390+raclopride, or seawater. The drug (0.0125 mg/g of body weight) was injected 2 mm anterior to the parapodia. After the injection of either SCH-23390 or SCH-23390+raclopride, there was a significant increase in parapodia opening (P<.001), siphon withdrawal (P<.05), and galloping following tail pinch (P<.01) compared to raclopride-injected or control animals. The data showed that blockade of receptors by SCH-23390, but not raclopride, produced significant changes in motor behavior in A. californica.

Dopamine receptor subtype density as a function of age in Aplysia californica.

The age-associated changes in dopamine subtype receptors were examined in Aplysia californica. The density of the subtype receptors D1, D2, D3 and D4 was examined in the ganglia from 4.5-, 6-, 8-, 9- and 12-month animals. Receptor analysis was performed by examining the binding of radiolabeled ligands to the individual subtypes. [3H]SCH23390 and [3H]Clozapine were used to analyze D1 and D4 specific binding. [3H]Quinpirole was used for determining D2 and D3 specific binding. Specific binding was found to be present for all four receptor subtypes. All receptor subtypes showed an increase in density from 4.5 to 6 months. From 6 to 8 months D2 and D3 decreased, while D1 and D4 increased. D4 showed the strongest increase. All four subtypes examined showed decreases from 8 to 12 months. ANOVA results indicated age was a significant factor in the subtype receptor density for all receptor types.

Serotonin levels as a function of age in Aplysia californica.

The neurotransmitter serotonin (5-HT) plays an important role in a number of behaviors in Aplysia californica some of which have been shown to vary with age. We were thus interested in examining the age-dependence of 5-HT in A. californica. Because animals of the same age can have very different weights, and weight alone is reliably known for wild-caught animals, we also examined the variation of 5-HT with weight. Serotonin was measured in the ring and abdominal ganglia combined, in lab-reared animals from 3 to 12 months post-hatch across a wide weight range. Serotonin increased rapidly from 4 to 6 months, and more slowly from 6 to 13 months. Serotonin scaled by soluble ganglion protein increased from 3 to 6-7 months, reached a maximum, and then decreased again. Serotonin, but not scaled 5-HT, increased significantly with weight across the whole weight range. Animals of the same weight, but different ages, had different 5-HT levels, as did young animals of the same age but different weight. Serotonin varied significantly with both age and weight, with the age-dependence being the more significant.

Age dependent changes in serotonin and dopamine receptors in Aplysia californica.

Age related changes in dopaminergic and serotonergic receptors were examined in Aplysia californica. In this study dopamine (DA) and serotonin (5-HT) receptor levels were examined for animals belonging to 4-, 5-, 6-, 8-, 9- and 12-month age groups. Receptors analysis was performed using radio-labeled d-[3H] lysergic acid diethylamide (LSD) as the specific ligand. Specific binding for 5-HT was found to be significantly greater than that for DA in the young (4-month post-hatch) animals. The total DA and 5-HT receptor levels changed significantly with age. Dopamine levels increased from 5.34 fmol/mg of protein at 4 months to 19.11 fmol/mg at 12 months. Serotonin receptor levels increased from 7.35 fmol/mg at 4 months to 20.45 at 12 months.

Melanin and melanogenesis in adipose tissue: possible mechanisms for abating oxidative stress and inflammation?

Obesity has become a worldwide epidemic and can lead to multiple chronic diseases. Adipose tissue is increasingly thought to play an active role in obesity-related pathologies such as insulin resistance and non-alcoholic fatty liver disease. Obesity has been strongly associated with systemic inflammation and, to a lesser degree, with oxidative stress, although the causal relationships among these factors are unclear. A recent study demonstrating an expression of the components of the melanogenic pathway and the presence of melanin in visceral adipose has raised questions regarding the possible role of melanogenesis in adipose tissue. As this study also found larger amounts of melanin in the adipose tissue of obese patients relative to lean ones, we hypothesize that melanin, a pigment known for its antioxidant and anti-inflammatory properties, may scavenge reactive oxygen species and abate oxidative stress and inflammation in adipose tissue. This review considers the evidence to support such a hypothesis, and speculates on the role of melanin within adipocytes. Furthermore, we consider whether the α-melanocyte-stimulating hormone or its synthetic analogues could be used to stimulate melanin production in adipocytes, should the hypothesis be supported in future experiments.

Differential expression of miRNAs in the visceral adipose tissue of patients with non-alcoholic fatty liver disease.

BACKGROUND: Progression of non-alcoholic fatty liver disease (NAFLD) can be facilitated by soluble molecules secreted by visceral adipose tissue (VAT). MicroRNAs (miRNAs) are likely to regulate some of these molecular pathways involved in pathogenesis of NAFLD. AIM: To profile miRNA expression in the visceral adipose tissue of patients with NAFLD. METHODS: Visceral adipose tissue samples were collected from NAFLD patients and frozen. Patients with biopsy-proven NAFLD were divided into non-alcoholic steatohepatitis (NASH) (n = 12) and non-NASH (n = 12) cohorts controlled for clinical and demographic characteristics. Extracted total RNA was profiled using TaqMan Human MicroRNA arrays. Univariate Mann-Whitney comparisons and multivariate regression analysis were performed to compare miRNA profiles. RESULTS: A total of 113 miRNA differentially expressed between NASH patients and non-NASH patients (P < 0.05). Of these, seven remained significant after multiple test correction (hsa-miR-132, hsa-miR-150, hsa-miR-433, hsa-miR-28-3p, hsa-miR-511, hsa-miR-517a, hsa-miR-671). Predicted target genes for these miRNAs include insulin receptor pathway components (IGF1, IGFR13), cytokines (CCL3, IL6), ghrelin/obestatin gene, and inflammation-related genes (NFKB1, RELB, FAS). In addition, two miRNA species, hsa-miR-197 and hsa-miR-99, were significantly associated with pericellular fibrosis in NASH patients (P < 0.05). Levels of IL-6 in the serum negatively correlated with the expression levels of all seven miRNAs capable of down regulating IL-6 encoding gene. CONCLUSIONS: miRNA expression from VAT may contribute to the pathogenesis of NAFLD - a finding which may distinguish relatively simple steatosis from NASH. This could help identify potential targets for pharmacological treatment regimens and candidate biomarkers for NASH.

Adipokines and cytokines in non-alcoholic fatty liver disease.

BACKGROUND: Several adipocytokines have been implicated in the pathogenesis non-alcoholic fatty liver disease (NAFLD). AIM: To assess adipocytokines in NAFLD patients and controls. METHODS: A total of 95 patients (26 non-alcoholic steatohepatitis (NASH), 19 simple steatosis (SS), 38 obese controls and 12 non-obese controls) were included. Fasting serum insulin, glucose, visfatin, resistin, adiponectin, tumour necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8) and IL-6 were determined. Univariate and multivariate analyses were used to compare groups and determine associations. RESULTS: Serum TNF-alpha and IL-8 were higher in NAFLD patients when compared with both obese and non-obese controls. Analysis involving all patients revealed a significant correlation between serum TNF-alpha and IL-8 (P < 6.319e-08), and between IL-6 and IL-8 (P < 5.271e-15). Homeostatic model assessment scores negatively correlated with adiponectin in NAFLD (P < 0.0032). Serum visfatin was higher in all three obese groups than in non-obese controls (P < 0.02, P < 0.002 and P < 0.008). Visfatin in NASH patients was lower than SS and obese controls. Although TNF-alpha was associated with NAFLD (P < 0.02), it was interdependent on visfatin. In comparison to SS, four factors were independently associated with NASH: age, alanine aminotransferase, IL-8 and adiponectin (P < 0.05). Multivariate analysis indicated that TNF-alpha was the only independent predictor of fibrosis in NASH (P < 0.0004). CONCLUSION: These findings support a complex interaction between adipocytokines and the pathogenesis of NAFLD.

Expression profile of ovarian tumors: distinct signature of Sertoli-Leydig cell tumor.

Epithelial ovarian tumors are the most common subtype of ovarian cancer. In this study, we reveal distinct expression signatures of previously uncharacterized ovarian carcinoma subtypes, including endometrioid component of mixed ovarian tumor and Sertoli-Leydig tumor. Both subtypes were compared to the most common and well-characterized ovarian epithelial carcinoma of the serous type. These comparisons were performed by complementaryDNA (cDNA) microarrays allowing high-fidelity measurements of the expression levels of 39,360 human individual cDNA species representing both known and unknown human genes. Functional analysis of differentially expressed genes in Sertoli-Leydig tumor revealed an upregulation in sonic hedgehog pathway, deregulation of several metabolic pathways especially in amino acid metabolism and overexpression of genes associated with protein synthesis, including ribosomal genes.

Destruction of lichen chemical defenses by a fungal.

Lichen secondary metabolites are known to inhibit various animal consumers and pathogenic microorganisms. Nevertheless, many obligate fungal pathogens have evolved a tolerance to these inhibitory lichen compounds. We recently discovered a new lichen pathogen in the fungal genus Fusarium that is not only tolerant of lichen compounds but also able to degrade many of these compounds. This organism was discovered in field investigations of a different lichenicolous fungus, Marchandiomyces corallinus, which was found growing on lichens (Lasallia papulosa and L. pensylvanica) that normally inhibit its growth. Subsequent experiments established that M. corallinus is found on Lasallia species only when Fusarium is also present. We hypothesized that Fusarium altered the inhibitory chemistry of Lasallia spp. and permitted colonization by M. corallinus. A laboratory experiment to test this hypothesis demonstrated that sterilized tissues of Lasallia papulosa exposed to Fusarium for 30 d are readily degraded by M. corallinus; control tissues left in sterile water for 30 d continue to inhibit growth of M. corallinus. High performance liquid chromatography (HPLC) established that the lichen compound lecanoric acid, one of several lichen compounds that inhibit growth of M. corallinus, is degraded by extracellular enzymes produced by this newly discovered Fusarium. Taken together, our results demonstrate that enzymatic degradation of lichen compounds permits colonization of lichens by fungi that would otherwise be chemically excluded.

Iron-binding compounds produced by wood-decaying basidiomycetes.

The chrome azurol-S universal siderophore assay and the rapid paper electrophoresis siderophore assay were used to screen 10 wood-decaying basidiomycete isolates for the formation of iron-chelating compounds. All 10 isolates were positive for chrome azurol-S reactivity on solid plating medium and in liquid cultures, and 9 of the 10 isolates produced fluorescent iron-binding compounds in the paper electrophoresis assay.

4-hydroxy-2-nonylquinoline: a novel iron chelator isolated from a bacterial cell membrane.

The membrane associated iron chelator of Pseudomonas aeruginosa has been extracted from membranes of iron-rich cells with ethanol and purified by reverse phase HPLC. Using 13C NMR and FAB mass spectroscopy, the structure of the chelator has been determined to be 4-hydroxy-2-nonylquinoline. This compound has been previously isolated and named pseudan IX, a name which we use here. We synthesized pseudan IX and show that the spectral properties of the synthesized compound and the purified compound are nearly identical. Also purified from the ethanol extract of membranes is 4-hydroxy-2-heptylquinoline, i.e., pseudan VII. Bacterially purified pseudan IX binds iron as indicated by the incorporation of radiolabeled iron into the chelator and by the formation of pink micelles in a concentrated ethanol extract. The formation of pink micelles upon addition of iron to the synthesized compound indicates that it binds iron.