Differences In Basal Expression Research Articles

Interspecies Differences in 6PPD-Quinone Toxicity Across Seven Fish Species: Metabolite Identification and Semiquantification.

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q) is a recently identified contaminant that originates from the oxidation of the tire antidegradant 6PPD. 6PPD-Q is acutely toxic to select salmonids at environmentally relevant concentrations, while other fish species display tolerance to concentrations that surpass those measured in the environment. The reasons for these marked differences in sensitivity are presently unknown. The objective of this research was to explore potential toxicokinetic drivers of species sensitivity by characterizing biliary metabolites of 6PPD-Q in sensitive and tolerant fishes. For the first time, we identified an O-glucuronide metabolite of 6PPD-Q using high-resolution mass spectrometry. The semiquantified levels of this metabolite in tolerant species or life stages, including white sturgeon (Acipenser transmontanus), chinook salmon (Oncorhynchus tshawytscha), westslope cutthroat trout (Oncorhynchus clarkii lewisi), and nonfry life stages of Atlantic salmon (Salmo salar), were greater than those in sensitive species, including coho salmon (Oncorhynchus kisutch), brook trout (Salvelinus fontinalis), and rainbow trout (Oncorhynchus mykiss), suggesting that tolerant species might detoxify 6PPD-Q more effectively. Thus, we hypothesize that differences in species sensitivity are a result of differences in basal expression of biotransformation enzyme across various fish species. Moreover, the semiquantification of 6PPD-Q metabolites in bile extracted from wild-caught fish might be a useful biomarker of exposure to 6PPD-Q, thereby being valuable to environmental monitoring and risk assessment.

Orotic acid induces apoptotic death in ovarian adult granulosa tumour cells and increases mitochondrial activity in normal ovarian granulosa cells

Orotic acid (OA) is a natural product that acts as a precursor in the pyrimidine nucleotide biosynthesis pathway. Most studies concerning administration of OA focus on its therapeutic effects; however, its effect on tumours is unclear. We aimed to determine whether treatment with OA influences the viability and apoptosis of normal (HGrC1) and tumour-derived (KGN) human ovarian granulosa cells. The effects of OA (10–250 μM) on viability and apoptosis of both cell lines were determined by using alamarBlue and assessing caspase-3/7 activity, respectively. Annexin V binding and loss of membrane integrity were evaluated in KGN cells. The cell cycle and proliferation of HGrC1 cells were assessed by performing flow cytometric and DNA content analyses, respectively. The influence of OA (10 and 100 μM) on cell cycle- and apoptosis-related gene expression was assessed by RT-qPCR in both cell lines. Mitochondrial activity was analysed by JC-1 staining in HGrC1 cells. In KGN cells, OA reduced viability and increased caspase-3/7 activity, but did not affect mRNA expression of Caspase 3, BAX, and BCL2. OA enhanced proliferation and mitochondrial activity in HGrC1 cells without activating apoptosis. This study demonstrates that the anti-cancer properties of OA in ovarian granulosa tumour cells are not related to changes in apoptosis-associated gene expression, but to increased caspase-3/7 activity. Thus, OA is a promising therapeutic agent for ovarian granulosa tumours. Further, our results suggest that differences in basal expression of cell cycle- and apoptosis-related genes between the two cell lines are responsible for their different responses to OA.

Gene expression of Lysozyme C and Lysozyme G in diploid and triploid Astyanax lacustris experimentally infected with Aeromonas jandaei

Triploid fish are becoming increasingly popular in commercial aquaculture due to their sterility, which results in improved meat quality and better growth rates as a result of the lack of sexual maturation. However, triploid fish are generally perceived as being more susceptible to diseases and less able to cope with suboptimal environmental conditions. The present study aimed to evaluate the effects of triploidy on the expression of C-type and G-type lysozymes (LysC and LysG) in lambaris (Astyanax lacustris) that were challenged with a pathogenic strain of Aeromonas jandaei. The animals were induced to triploidy as described in the literature. We infected 10 diploid and 10 triploid fish with 104 CFU of A. jandaei in 0.1 mL of PBS by intraperitoneal injection, a dose capable of triggering an immune response in the animals. Fish from the control groups were inoculated with 0.1 mL of sterile PBS. Molecular cloning of Lysozyme C and Lysozyme G was performed to characterize these genes, and the analysis of gene expression for LysC and LysG of A. lacustris diploids and triploids was conducted using qPCR. The collected data revealed that there were no significant differences in basal expression between diploid and triploid uninfected fish. However, the challenged fish of both ploidies exhibited an increase in expression compared to the control group. The results of study indicate that infected diploid fish exhibit significantly higher lysozyme expression in the spleen compared to infected triploid fish. While basal lysozyme expression levels between diploid and triploid fish remain unaffected, our findings suggest that triploidy may lower lysozyme expression in response to infection, with diploid fish exhibiting higher levels of expression.

Age-associated reduction in angiogenic capacity associates with impaired functions of neuropeptide Y

Impaired angiogenesis is associated with aging and several age-related pathologies, including ischemic vascular disease and delayed wound healing. However, such a reduction in angiogenic capacity could also inform therapeutic applications aiming at inhibiting tumor vascularization. Neuropeptide Y (NPY) and its Y receptors (Rs) - Y2R and Y5R - have been implicated in angiogenic responses associated with cardiovascular disease and cancer. Yet, changes in the NPY system associated with aging are not clear. Therefore, we sought to determine whether alterations in the NPY system are involved in age-associated impairment of angiogenesis. We hypothesized that NPY-mediated angiogenesis is diminished with age due to downregulation of the peptide, its receptors, and converting enzyme - cleaving full-length NPY to a Y2R agonist, as well as downstream signaling pathways. To test our hypothesis, the angiogenic profiles of human microvascular endothelial cells (HMVECs) from 24- and 90-year-old subjects were assessed via proliferation and mRNA expression. This analysis included dose-response and cross-inhibition assays with NPY, fibroblast growth factor 2 (FGF2), and vascular endothelial growth factor (VEGF) systems. Also, basal plasma levels of NPY were compared in old (24 months) and young (3 months) Balb/c mice. Further, NPY-mediated aortic sprouting was assessed in Y2R knockout (KO) mice. Angiogenic stimulation via NPY, FGF, and VEGF was observed in HMVECs from both age groups, with lower basal proliferation levels and less responsiveness to all three factors in aged HMVECs (p<0.05). While qRT-PCR analysis revealed no differences in basal expression of the NPY system, Y2R and DPPIV were induced by NPY in young HMVECs only. These results suggest that the decreased responsiveness of aged HMVECs to NPY stimulation is caused by impairment of NPY receptor function and/or signaling pathways. NPY stimulation increased mRNA expression of FGF2, FGFR1 and 2, and VEGFR2 in young HMVECs only. Further, anti-FGF2 neutralizing antibody and VEGFR2/Fc chimera, which is a potent VEGF antagonist, blocked the mitogenic effect of NPY in HMVECs from both age groups; while NPY receptor antagonists had no effect on HMVEC proliferation stimulated by FGF2 and VEGF (p<0.05). Moreover, despite completely impaired NPY-induced sprouting of Y2KO aortic rings, the response to FGF2 and VEGF remained intact. These data indicate that the FGF2 and VEGF pathways mediate angiogenic activities of NPY. In summary, our results show age-associated impairment of NPY-induced angiogenesis is due to a diminished mitogenic response of endothelium to the peptide via loss of induction of its angiogenic Y2R and DPPIV. This is also associated with reduced stimulation of downstream FGF2 and VEGF signaling. Future studies will determine if impaired angiogenesis in aging can be rescued with stimulation of the NPY angiogenic system and how these manipulations can improve revascularization of ischemic tissue. NIH R03 AG-20795 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MO420: Effects of Indoxyl Sulfate and Apixaban in Liver Gene Expression

Abstract BACKGROUND AND AIMS Uraemic toxins accumulate in the blood and tissues of patients with chronic kidney disease (CKD). Previous animal studies have shown that chronic kidney disease (CKD) not only alters the elimination of drugs excreted by the kidneys, but it also impacts the metabolism of drugs subject to non-renal clearance, which involves mainly the liver and the gut. The use of apixaban, an oral anticoagulant, has been approved in the USA for dialysis patients. However, the coexistence of thrombotic and haemorrhagic risk in patients with CKD makes dose adjustment difficult. The liver has a major role in drug metabolization. Hepatocytes express high levels of AhR, a ligand-inducible transcription factor that mediates the induction of various liver cytochrome P450 enzymes by xeno and endobiotic. Tryptophan-derived uraemic toxins (TDUT) are AhR agonists. Indoxyl sulfate (IS) is the main TDUT involved in uraemic syndrome. IS increases the expression and activity of P-glycoprotein (P-gp) in the liver. Apixaban metabolism is mediated by P-gp and Cyp3a4 (Cyp3a11 in mice). We aim to study the effect of apixaban and IS in the expression of drug metabolism genes. Our hypothesis is that AhR activation by IS could severely modify drug metabolism during CKD. METHOD C57BL/6J Wild-type mice purchased from The Jackson Laboratory were fed ad libitum with a standard diet. At 10 weeks of age, mice drinking water was substituted with a 5% sucrose water solution with either 0.1% indoxyl sulphate or KCl (control) added at equivalent concentrations. 48-h before sacrifice some mice were gavaged with an apixaban solution (0.6 mg/mL) twice a day with the last dose given 4 h prior to sacrifice. All mice were sacrificed at 11 weeks of age. Liver samples were stored in an RNA-later solution at −20°C. Samples were thawed and lysed in Trizol using the Tissue-Ruptor system (Qiagen). RNA was extracted and purified with chloroform and precipitated with isopropanol. RNA concentration was estimated by spectrophotometry. Gene expression was analysed by q-RT-PCR using Gusb as a housekeeping gene. Apixaban levels were quantified by LC-MS. Kruskal–Wallis followed by a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli (q < 0.05) was performed using GraphPad Prism 9.2.1. RESULTS Four groups of mice per sex were thus created: WT-KCl, WT-IS, WT-KCl Apix, WT-IS Apix. In the IS/KCl model we observe higher mRNA basal expression of Abcb1a (q = 0.008) and Sult1a1 (q = 0.006) and lower basal expression of Abcg2 (q = 0.0005) in females compared to males when treated with KCl. IS treatment increases Cyp1a2 expression in females (q = 0.0139) and Cyp1a1 in both males (q = 0.0145) and females (q = 0.026). In the IS/KCl-Apix model the same sex-related differences are maintained for Abcb1a (q = 0.0031), Sult1a1 (q = 0.0316) and Abcg2 (q < 0.0001). In males, CYP2e1 expression is increased by apixaban in KCl (q = 0.067) and IS (q = 0.0278) treated mice. Moreover, apixaban counteracts the increased expression of Cyp1a1 induced by IS (q = 0.0177). The expression of Cyp3a11 is augmented in males treated with KCl-apixaban (q = 0.0143), an effect that seems to be reversed by IS (q = 0.0066). In females, apixaban has no effect in gene expression. Apixaban serum concentration is higher in KCl (q = 0.0350) and IS (q = 0.0019) treated females when compared to males. CONCLUSION The effect of IS as an agonist of AhR in the liver is confirmed by the increased expression of Cyp1a1. Apixaban provokes a remarkable increase of Cyp3a11 in male mice, which could lead to higher degradation rates decreasing its activity. IS seems to reverse this effect. This could lead to increased activity of apixaban during CKD which could derive in an increased risk of bleeding. Higher BCRP rates could explain lower rates of apixaban in male serum. Apixaban did not alter gene expression in females. Clinical trials in CKD are generally biased regarding sex (fewer women represented) possibly masking important sex-dependent drug adverse effects. Our results show great differences in basal expression of various genes, confirming the importance to study men and women separately with respect to drug metabolism.

Regulation of Brown Adipose Tissue Function by HuR

Stimulating the metabolic function of brown adipose tissue (BAT) and/or inducing the beiging of white adipocytes (WAT) to BAT‐like cells represent potential therapeutic strategies for increasing energy expenditure and reducing obesity. HuR (ELAVL1) is an RNA binding protein that mediates gene expression through stabilization of mRNA targets and is highly expressed in both WAT and BAT. We identified an inverse correlation between HuR expression and obesity in humans via the METabolic Syndrome In Men study of Finnish men. Thus, we hypothesized that HuR expression in adipocytes plays a critical role in maintaining metabolic homeostasis.Our results show that, compared to control littermates, adipocyte‐specific HuR‐null (adipo‐HuR−/−) mice display a lean phenotype with decreased fat mass and increased energy expenditure, independent of diet. Interestingly, adipo‐HuR−/− mice also show a disruption of BAT structure and function, with increased lipid droplet size, decreased BAT density, and an inability to thermoregulate when subjected to cold. Examination of skeletal muscle suggests that the lean phenotype in adipo‐HuR−/− mice is due to compensatory shivering to thermoregulate in the absence of functional BAT, a result consistent with UCP1−/− mice. Surprisingly, we found no difference in basal expression of UCP1 in BAT from control and adipo‐HuR−/− mice. However, HuR is activated downstream of β3 adrenergic stimulation and appears to play a role in cold/β3 induced UCP1 expression. Additionally, adipo−HuR−/− mice show a decrease in mitochondrial density in both BAT and subcutaneous (sc) WAT, as well as a decrease in brown/beige adipocyte marker genes (PRDM16, Elovl3) in scWAT.In conclusion, our results demonstrate that HuR plays a central role in the structure and function of BAT. Work is ongoing to identify the transcriptome wide changes in HuR‐dependent gene expression as well as the potential role of HuR in beiging of WAT.Support or Funding InformationThis work was partially funded by a University of Cincinnati Heart, Lung, and Vascular Institute Pilot Grant (MT, APO).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Resistant and susceptible cacao genotypes exhibit defense gene polymorphism and unique early responses to Phytophthora megakarya inoculation

Key genes potentially involved in cacao disease resistance were identified by transcriptomic analysis of important cacao cultivars. Defense gene polymorphisms were identified which could contribute to pathogen recognition capacity. Cacao suffers significant annual losses to the water mold Phytophthora spp. (Oomycetes). In West Africa, P. megakarya poses a major threat to farmer livelihood and the stability of cocoa production. As part of a long-term goal to define key disease resistance genes in cacao, here we use a transcriptomic analysis of the disease-resistant cacao clone SCA6 and the susceptible clone NA32 to characterize basal differences in gene expression, early responses to infection, and polymorphisms in defense genes. Gene expression measurements by RNA-seq along a time course revealed the strongest transcriptomic response 24h after inoculation in the resistant genotype. We observed strong regulation of several pathogenesis-related genes, pattern recognition receptors, and resistance genes, which could be critical for the ability of SCA6 to combat infection. These classes of genes also showed differences in basal expression between the two genotypes prior to infection, suggesting that prophylactic expression of defense-associated genes could contribute to SCA6's broad-spectrum disease resistance. Finally, we analyzed polymorphism in a set of defense-associated receptors, identifying coding variants between SCA6 and NA32 which could contribute to unique capacities for pathogen recognition. This work is an important step toward characterizing genetic differences underlying a successful defense response in cacao.

Influence of sex and developmental stage on acute hepatotoxic and inflammatory responses to liver procarcinogens in the mouse.

The incidence of liver cancer is higher in men than in women. This sex difference is also observed in murine tumor induction models that result in the appearance of liver tumors in adult mice following their exposure on postnatal days 8 and/or 15 to carcinogens such as 4-aminobiphenyl (ABP) or diethylnitrosamine (DEN). Previous studies performed in adult mice showed that acute hepatotoxic and inflammatory responses to high-dose DEN exposure were greater in males than in females, leading to the suggestion that these responses could account for the sex difference in tumor development. We also recently observed that female but not male mice exposed postnatally to ABP had slightly increased expression of the antioxidant defense genes Nqo1 and Ggt1, which are regulated by the oxidative stress response protein nuclear factor erythroid 2-related factor 2 (NRF2), while expression of Hmox1 was increased in both sexes. The goal of the present study was therefore to compare selected acute hepatotoxic, inflammatory and oxidative stress defense responses to ABP, DEN, or the prototype hepatotoxicant carbon tetrachloride (CCl4), in male and female mice exposed to these chemicals either postnatally or as adults. Exposure of adult mice to ABP, DEN or CCl4 produced a 2-fold greater acute elevation in serum levels of the hepatotoxicity biomarker alanine aminotransferase (ALT) in males than in females, while levels of the inflammatory biomarker interleukin-6 (IL-6) showed no sex difference. However, treatment of immature mice with either ABP or DEN using standard tumor-inducing postnatal exposure protocols produced no increase in serum ALT or IL-6 levels in either males or females, while CCl4 produced a 40-fold ALT elevation but with no sex difference. Basal expression of the NRF2-responsive gene Nqo1 was higher in adult females than in males, but there was no sex difference in basal expression of Ggt1 or Hmox1. Sexually immature animals showed no sex difference in basal expression of any of the three genes. Postnatal DEN exposure modestly increased the expression of Ggt1 only in male mice and Nqo1 in both sexes, while CCl4 slightly increased expression of Ggt1 in both males and females and Nqo1 only in females. Taken together, our results make it unlikely that acute hepatotoxic, inflammatory or NRF2-activated gene responses account for the male predominance in liver tumor growth following postnatal carcinogen exposure in mice. Our findings also suggest that acute toxicity studies performed in adult mice should be interpreted with caution when extrapolating potential mechanisms to liver carcinogenesis models that commonly use postnatally exposed mice.

Type I interferon responses are impaired in latently HIV infected cells.

Background The latent HIV-1 reservoir represents the primary barrier to the eradication of HIV-1 infection. The design of novel reservoir-clearance strategies, however, is impeded in part by the inability to distinguish latently HIV-infected cells from uninfected cells. Significant impairment of the type I interferon (IFN-I) response is observed during productive HIV-1 infection. Although this remains poorly described in the context of latent HIV-1 infection, presence of potential defects may serve as a novel therapeutic target. Therefore, IFN-I pathways were characterized using two latently HIV-1-infected cell lines, U1 and OM10.1, in comparison to their respective uninfected parental U937 and HL60 cell lines.FindingsConstitutive expression and induction of important mediators of IFN-I signaling including IFNα/β cytokines, IFNAR1, MHC-I, ISG15, and PKR were evaluated following exogenous IFNα or poly(I:C) treatment. Differences in basal expression of IFNAR1, MHC-I, and PKR were observed between the latently HIV-1 infected and uninfected cell lines. In parallel, significant impairments in the induction of MHC-I, ISG15 and PKR, as well as secretion of IFNα/β cytokines were observed in response to appropriate exogenous stimulation within the two latently HIV-infected U1 and OM10.1 cells, relative to their HIV-uninfected parental cells.ConclusionsIn comparison to the HIV-uninfected U937 and HL60 cell lines, widespread defects in IFN-I responsiveness were observed within the latently HIV-infected U1 and OM10.1 cells. These impairments represent novel therapeutic targets, which may be amenable to strategies currently employed in cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0302-9) contains supplementary material, which is available to authorized users.

The effect of taurine and β-alanine supplementation on taurine transporter protein and fatigue resistance in skeletal muscle from mdx mice.

This study investigated the effect of taurine and β-alanine supplementation on muscle function and muscle taurine transporter (TauT) protein expression in mdx mice. Wild-type (WT) and mdx mice (5months) were supplemented with taurine or β-alanine for 4weeks, after which in vitro contractile properties, fatigue resistance and force recovery, and the expression of the TauT protein and proteins involved in excitation-contraction (E-C) coupling were examined in fast-twitch muscle. There was no difference in basal TauT protein expression or basal taurine content between mdx than WT muscle. Supplementation with taurine and β-alanine increased and reduced taurine content, respectively, in muscle from WT and mdx mice but had no effect of TauT protein. Taurine supplementation reduced body and muscle mass, and enhanced fatigue resistance and force recovery in mdx muscle. β-Alanine supplementation enhanced fatigue resistance in WT and mdx muscle. There was no difference in the basal expression of key E-C coupling proteins [ryanodine receptor 1 (RyR1), dihydropyridine receptor (DHPR), sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA1) or calsequestrin 1 (CSQ1)] between WT and mdx mice, and the expression of these proteins was not altered by taurine or β-alanine supplementation. These findings suggest that TauT protein expression is relatively insensitive to changes in muscle taurine content in WT and mdx mice, and that taurine and β-alanine supplementation may be viable therapeutic strategies to improve fatigue resistance of dystrophic skeletal muscle.

Epigenetic regulation of the 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) in colon cancer cells

Calcitriol is the hormonally active form of vitamin D and has anti-proliferative and pro-apoptotic effects. Calcitriol and its precursor calcidiol (25(OH)D3) are degraded by the 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1). This enzyme is overexpressed in colorectal tumors, however, the mechanisms of this overexpression remain to be elucidated. CYP24A1 mRNA level differs among colorectal cancer cell lines and range from almost undetectable to high. Since DNA methylation and histone acetylation regulate CYP24A1 gene expression in prostate cancer cell lines, we investigated whether epigenetic mechanisms could explain the differences in basal expression of CYP24A1 in colon cancer cells. Methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) treatment resulted in an over 50-fold induction of CYP24A1 mRNA expression in Coga1A and HT-29 cells but in no response in Caco2/AQ and Coga13 cells. This finding is supported by a strong increase in CYP24A1 activity after DAC treatment in Coga1A (35%). In addition, calcitriol and DAC had synergistic effects on CYP24A1 gene transcription. Interestingly, the CYP24A1 promoter was not methylated in Coga1A and HT-29 (<5%), while in Caco2/AQ it was 62% methylated. This suggests that DNA demethylation must activate genes upstream of CYP24A1 rather than act on the gene itself. However, transcriptional regulators of CYP24A1 such as vitamin D receptor (VDR), retinoid X receptor (RXR), specificity protein 1 (SP1), or mediator complex subunit 1 (MED1) were not upregulated. We conclude that in colon cancer cells, CYP24A1 gene expression is inducible by methyltransferase and some histone deacetylase inhibitors in a cell line-dependent manner. This effect does not correlate with the methylation state of the promoter and therefore must affect genes upstream of CYP24A1.This article is part of a Special Issue ‘Vitamin D Workshop’.

RNA-Binding Protein HuD Controls Insulin Translation

Although expression of the mammalian RNA-binding protein HuD was considered to be restricted to neurons, we report that HuD is present in pancreatic β cells, where its levels are controlled by the insulin receptor pathway. We found that HuD associated with a 22-nucleotide segment of the 5' untranslated region (UTR) of preproinsulin (Ins2) mRNA. Modulating HuD abundance did not alter Ins2 mRNA levels, but HuD overexpression decreased Ins2 mRNA translation and insulin production, and conversely, HuD silencing enhanced Ins2 mRNA translation and insulin production. Following treatment with glucose, HuD rapidly dissociated from Ins2 mRNA and enabled insulin biosynthesis. Importantly, HuD-knockout mice displayed higher insulin levels in pancreatic islets, while HuD-overexpressing mice exhibited lower insulin levels in islets and in plasma. In sum, our results identify HuD as a pivotal regulator of insulin translation in pancreatic β cells.

T2084 Comparison of Small Bowel and Colonic Mucosal Permeability in Diarrhea-Predominant Irritable Bowel Syndrome and Health by Urinary Saccharide Excretion

Background: A significant proportion of patients with irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) suffer from psychological co-morbidities, i.e. depression. Recently, we identified 16 interferon (IFN)-responsive genes (ISGs) that were associated with IFN-α induced depression during therapy of chronic HCV infection as well as with severe endogenous depressive episodes. Based upon data that a large proportion of patients with chronic bowel disorders suffer from depressive disorders, we aimed to investigate, whether these “depression ISGs” may play a role in patients with IBS and IBD. Methods: 11 consecutive IBS (all constipation predominant) and 12 IBD patients (7 ulcerative colitis, 5 Crohn's disease) as well as 14 healthy controls were included. Basal expression of ISGs and the internal expression of IFN-α, -β, and -γ was analyzed in whole blood (PAXgene blood RNA tubes) using quantitative real-time PCR. Additionally, PBMC were isolated from each patient by density gradient centrifugation and were stimulated In Vitro with IFN-α (0, 100, and 1000 IU/ mL for 16h). The upregulation of ISGs was analyzed by real-time PCR. HRQOL (SF-36, physical [PCS] and mental component score [MCS]) and affective disorders (Hospital Anxiety and Depression Scale) were also assessed. Results: We found highly significant differences in the basal expression of 11 out of 14 tested ISGs (p<0.001 for 10 genes) in IBS and IBD patients vs. healthy controls. Basal expression of ISGs associated with depression and of “classical” ISGs (IFIT1, ISG15, and MX1) was significantly lower in 13/ 14 genes in IBS and IBD patients compared to healthy controls. Basal expression of IFN-β was significantly upregulated in IBS and IBD patients (p<0.001), while there was no difference in basal expression of IFN-α and IFN-γ. Comparing IBS and IBD patients, no significant differences in basal gene expression were detected. PBMC of IBS and IBD patients were hyper-responsive to exogenous stimulation with IFN-α compared to healthy controls leading to significantly higher induction of selected ISGs. Summary: In patients with refractory IBS and chronic IBD a marked activation of the type I IFN system and a hyper-responsiveness to stimulation with type I IFNs can be observed. This is associated with a response pattern that is also found in patients with depressive disorders and may thus explain at least some of the psychopathological abnormalities in these patients.

Time-course comparison of xenobiotic activators of CAR and PPARα in mouse liver

Constitutive androstane receptor (CAR) and peroxisome proliferator activated receptor (PPAR)α are transcription factors known to be primary mediators of liver effects, including carcinogenesis, by phenobarbital-like compounds and peroxisome proliferators, respectively, in rodents. Many similarities exist in the phenotypes elicited by these two classes of agents in rodent liver, and we hypothesized that the initial transcriptional responses to the xenobiotic activators of CAR and PPARα will exhibit distinct patterns, but at later time-points these biological pathways will converge. In order to capture the global transcriptional changes that result from activation of these nuclear receptors over a time-course in the mouse liver, microarray technology was used. First, differences in basal expression of liver genes between C57Bl/6J wild-type and Car-null mice were examined and 14 significantly differentially expressed genes were identified. Next, mice were treated with phenobarbital (100 mg/kg by gavage for 24 h, or 0.085% w/w diet for 7 or 28 days), and liver gene expression changes with regards to both time and treatment were identified. While several pathways related to cellular proliferation and metabolism were affected by phenobarbital in wild-type mice, no significant changes in gene expression were found over time in the Car-nulls. Next, we determined commonalities and differences in the temporal response to phenobarbital and WY-14,643, a prototypical activator of PPAR α. Gene expression signatures from livers of wild-type mice C57Bl6/J mice treated with PB or WY-14,643 were compared. Similar pathways were affected by both compounds; however, considerable time-related differences were present. This study establishes common gene expression fingerprints of exposure to activators of CAR and PPARα in rodent liver and demonstrates that despite similar phenotypic changes, molecular pathways differ between classes of chemical carcinogens.

Regulation of Sulfotransferase Enzymes by Prototypical Microsomal Enzyme Inducers in Mice

In the present study, the regulation of the mRNA of 11 sulfotransferases (Sults) and two 3'-phosphoadenosine 5'-phosphosulfate synthase (PAPSs) isozymes by 15 microsomal enzyme inducers (MEI) in livers of male mice and five MEIs in livers of female mice was examined. These MEIs represent the transcriptionally mediated pathways: aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), peroxisomal proliferator-activated receptor alpha (PPARalpha), and NF-E2-related factor 2 (Nrf2). AhR ligands suppress the expression of Sults, especially the Sult1 isoenzymes in female mice. CAR activators up-regulate several Sults and PAPSs2 in female but not in male mice. PXR ligands cause marked induction of Sult1e1 in male, Sult2a1/2a2 in female, and PAPSs2 in both male and female mice. PPARalpha ligands do not have a marked effect on Sult expression in males, but they tend to suppress the expression of several Sult isoforms in female mice. Nrf2 activators appear to induce the mRNA expression of Sults in male and have mixed effects in female mice. In silico analysis indicated the presence of putative binding sites for all five transcription factors in the promoter region of many Sult and PAPSs isoforms. In conclusion, induction of Sults by typical MEIs is not as marked as the induction of P450 enzymes in mice. In addition to gender differences in basal expression of Sults, there is also a marked gender difference in the inducibility of various Sult isoenzymes in mice by MEIs.

Characterization of the aryl hydrocarbon receptor repressor and a comparison of its expression in Atlantic tomcod from resistant and sensitive populations

Atlantic tomcod from the Hudson River, USA, are resistant to cytochrome P4501A1 (CYP1A1) mRNA induction and early life stage toxicities induced by coplanar polychlorinated biphenyls (PCBs) or tetrachlorodibenzo-p-dioxins but not polycyclic aromatic hydrocarbons. We sought to determine if basal expression or inducibility of aryl hydrocarbon receptor repressor (AHRR) mRNA is higher in tomcod from the resistant Hudson River population than in those from sensitive populations. Tomcod AHRR cDNA was characterized and its expression quantified in different tissues and life stages of tomcod from the Hudson River, Miramichi River, Canada (sensitive), and among environmentally exposed tomcod from these two sources and the St. Lawrence River, Canada. Phylogenetic analysis revealed that tomcod AHRR falls within the clade of other vertebrate aryl hydrocarbon receptors (AHRs) but is most closely related to the four previously identified AHRR genes. Induction of AHRR mRNA was observed in all tissues of PCB77-treated juvenile tomcod of Miramichi River descent, and expression differed among tissues and was significantly related to levels of CYPIAI mRNA expression. Aryl hydrocarbon receptor repressor mRNA was similarly inducible in F2 embryos of Miramichi and Hudson River descent by benzo[a]pyrene but less by PCB77 in Hudson River offspring. A significant, positive correlation was observed between CYP1A1 mRNA and AHRR mRNA concentrations in environmentally exposed tomcod from the three rivers. We conclude that differences in basal expression or inducibility of AHRR mRNA are not the mechanistic basis of resistance but that levels of AHRR often mirror those of CYP1A1, suggesting that a common AHR pathway-related mechanism may modulate expression of both genes.

Metalloproteinase and inhibitor expression profiling of resorbing cartilage reveals pro-collagenase activation as a critical step for collagenolysis

Excess proteolysis of the extracellular matrix (ECM) of articular cartilage is a key characteristic of arthritis. The main enzymes involved belong to the metalloproteinase family, specifically the matrix metalloproteinases (MMPs) and a group of proteinases with a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS). Chondrocytes are the only cell type embedded in the cartilage ECM, and cell-matrix interactions can influence gene expression and cell behaviour. Thus, although the use of monolayer cultures can be informative, it is essential to study chondrocytes encapsulated within their native environment, cartilage, to fully assess cellular responses. The aim of this study was to profile the temporal gene expression of metalloproteinases and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), reversion-inducing cysteine-rich protein with Kazal motifs (RECK), and α2-macroglobulin (α2M), in actively resorbing cartilage. The addition of the pro-inflammatory cytokine combination of interleukin-1 (IL-1) + oncostatin M (OSM) to bovine nasal cartilage induces the synthesis and subsequent activation of pro-metalloproteinases, leading to cartilage resorption. We show that IL-1+OSM upregulated the expression of MMP-1, -2, -3, -9, 12, -13, -14, TIMP-1, and ADAMTS-4, -5, and -9. Differences in basal expression and the magnitude of induction were observed, whilst there was no significant modulation of TIMP-2, -3, RECK, or ADAMTS-15 gene expression. IL-1+OSM downregulated MMP-16,TIMP-4, and α2M expression. All IL-1+OSM-induced metalloproteinases showed marked upregulation early in the culture period, whilst inhibitor expression was reduced throughout the stimulation period such that metalloproteinase production would be in excess of inhibitors. Moreover, although pro-collagenases were upregulated and synthesized early (by day 5), collagenolysis became apparent later with the presence of active collagenases (day 10) when inhibitor levels were low. These findings indicate that the activation cascades for pro-collagenases are delayed relative to collagenase expression, further confirm the coordinated regulation of metalloproteinases in actively resorbing cartilage, and support the use of bovine nasal cartilage as a model system to study the mechanisms that promote cartilage degradation.

Apoptosis and cell cycle arrest in oral lichen planus: Hypothesis on their possible influence on its malignant transformation

The quantitative importance of cell cycle arrest and apoptosis mechanisms in oral lichen planus (OLP) was analysed in order to assess the cell response to T lymphocyte aggression and establish a hypothesis on the influence of these phenomena in the malignant transformation process. The TUNEL assay and immunohistochemical methods were used to detect caspase-3, bax, and p21 in 32 tissue samples of oral mucosa with OLP and in 20 samples of normal oral mucosa. Positivity for TUNEL, caspase-3 and p21 was significantly more frequent in cases than in controls (p<0.001). Both TUNEL and caspase-3 positivity was significantly greater in the basal versus suprabasal layer (p=0.004 and 0.052, respectively). The basal and suprabasal expression of p21 was significantly higher in cases with a more intense liquefaction degeneration (p<0.01). There was no significant difference in basal expression of bax between cases and controls. The quantitative importance of apoptosis was small in OLP. Epithelial cells attacked in OLP have a very low response to apoptosis and cell cycle arrest mechanisms, which may produce an epithelial substrate that favours malignant transformation.

Basal and Reovirus-Induced Beta Interferon (IFN-β) and IFN-β-Stimulated Gene Expression Are Cell Type Specific in the Cardiac Protective Response

Viral myocarditis is an important human disease, with a wide variety of viruses implicated. Cardiac myocytes are not replenished yet are critical for host survival and thus may have a unique response to infection. Previously, we determined that the extent of reovirus induction of beta interferon (IFN-beta) and IFN-beta-mediated protection in primary cardiac myocyte cultures was inversely correlated with the extent of reovirus-induced cardiac damage in a mouse model. Surprisingly, and in contrast, the IFN-beta response did not determine reovirus replication in skeletal muscle cells. Here we compared the IFN-beta response in cardiac myocytes to that in primary cardiac fibroblast cultures, a readily replenished cardiac cell type. We compared basal and reovirus-induced expression of IFN-beta, IRF-7 (an interferon-stimulated gene [ISG] that further induces IFN-beta), and another ISG (561) in the two cell types by using real-time reverse transcription-PCR. Basal IFN-beta, IRF-7, and 561 expression was higher in cardiac myocytes than in cardiac fibroblasts. Reovirus T3D induced greater expression of IFN-beta in cardiac myocytes than in cardiac fibroblasts but equivalent expression of IRF-7 and 561 in the two cell types (though fold induction for IRF-7 and 561 was higher in fibroblasts than in myocytes because of the differences in basal expression). Interestingly, while reovirus replicated to equivalent titers in cardiac myocytes and cardiac fibroblasts, removal of IFN-beta resulted in 10-fold-greater reovirus replication in the fibroblasts than in the myocytes. Together the data suggest that the IFN-beta response controls reovirus replication equivalently in the two cell types. In the absence of reovirus-induced IFN-beta, however, reovirus replicates to higher titers in cardiac fibroblasts than in cardiac myocytes, suggesting that the higher basal IFN-beta and ISG expression in myocytes may play an important protective role.

Differential expression of TGF-beta1 and TGF-beta3 in serosal tissues of human intraperitoneal organs and peritoneal adhesions

Elevated local expression of transforming growth factor (TGF-beta) has been associated with increased incidence of peritoneal adhesion formation. In this study we determine whether differences in basal expression of TGF-beta in serosal tissue of peritoneal organs correlate with incidence of adhesion formation. Serosal tissue of parietal peritoneum, uterus, oviduct, ovary, omentum, large and small bowels as well as adhesions, skin, fascia, subcutaneous tissue, peritoneal fluid and serum were collected from 57 subjects with/without adhesions who were undergoing abdominal/pelvic surgery. To determine TGF-beta1 and TGF-beta3 mRNA and protein expression, total RNA and protein were isolated from these tissues and along with the fluids, subjected to quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA) respectively. Tissue sections were immunostained for TGF-beta1 and TGF-beta3 protein. We found that TGF-beta1 and TGF-beta3 mRNA and protein are expressed in these tissues and present in peritoneal fluids and serum, with considerable variations in level of their expression. Comparatively, there was more variation in TGF-beta1 than TGF-beta3 expression without age or gender relation. Adhesions express a significantly higher TGF-beta1 mRNA and have the highest TGF-beta1:TGF-beta3 ratio, with lowest concentrations and ratio detected in omentum, small and large bowels; in contrast uterus expresses higher TGF-beta3, with lowest concentrations detected in subcutaneous tissue and large bowels (P < 0.05). A similar trend was also observed for total (active + latent) TGF-beta1 protein expression, with low active TGF-beta1 that was not significantly different among the tissue extracts and fluids. However, the lowest active:total TGF-beta1 ratio was found in adhesions and ovary. In subjects with adhesions, the adhesions express significantly more TGF-beta1 compared to parietal peritoneum (P < 0.05). Immunoreactive TGF-beta1 and TGF-beta3 protein were present in various cell types in these tissues with intensity reflecting their mRNA and protein expression. In conclusion, we provided evidence that serosal tissue of various peritoneal organs and adhesions express TGF-beta1 and TGF-beta3. Since TGF-beta is expressed differently in these tissues and tissue injury often alters the expression of TGF-beta, we propose that tissues with a higher basal expression of TGF-beta may become predisposed to develop more adhesions compared to others.