Uroporphyria and hepatic carcinogenesis induced by polychlorinated biphenyls-iron interaction: absence in the Cyp1a2(-/-) knockout mouse

Aryl hydrocarbon receptor ligands, such as polychlorinated biphenyls (PCBs), cause inhibition of the heme biosynthesis enzyme, uroporphyrinogen decarboxylase; this leads to uroporphyria and hepatic tumors, which are markedly enhanced by iron overload in C57BL/10 and C57BL/6 strains of mice. Cyp1a2(-/-) knockout mice were used to compare the effects of CYP1A2 expression on uroporphyria and liver carcinogenesis. PCBs in the diet (100ppm) of Cyp1a2(+/+) wild-type mice caused hepatic uroporphyria, which was strongly increased by iron-dextran (800mg Fe/kg). In contrast, uroporphyria was not detected in Cyp1a2(-/-) knockout mice, although expression of CYP1A1 and CYP2B10 was greatly induced. After 57 weeks on this diet, hepatic preneoplastic foci and tumors were seen in the Cyp1a2(+/+) mice; numbers and severity were enhanced by iron. No foci or tumors were detected in Cyp1a2(-/-) mice, although evidence for other forms of liver injury was observed. Our findings suggest a link not only between CYP1A2, iron metabolism, and the induction of uroporphyria by PCBs, but also with subsequent hepatocarcinogenesis.     

Leukocyte-derived hepatic lipase increases HDL and decreases en face aortic atherosclerosis in LDLr-/- mice expressing CETP

In addition to hepatic expression, cholesteryl ester transfer protein (CETP) and hepatic lipase (HL) are expressed by human macrophages. The combined actions of these proteins have profound effects on HDL structure and function. It is not known how these HDL changes influence atherosclerosis. To elucidate the role of leukocyte-derived HL on atherosclerosis in a background of CETP expression, we studied low density lipoprotein receptor-deficient mice expressing human CETP (CETPtgLDLr(-/-)) with a leukocyte-derived HL deficiency (HL(-/-) BM). HL(-/-) bone marrow (BM), CETPtgLDLr(-/-) mice were generated via bone marrow transplantation. Wild-type bone marrow was transplanted into CETPtgLDLr(-/-) mice to generate HL(+/+) BM, CETPtgLDLr(-/-) controls. The chimeras were fed a high-fat, high-cholesterol diet for 14 weeks to promote atherosclerosis. In female HL(-/-) BM, CETPtgLDLr(-/-) mice plasma HDL-cholesterol concentration during high-fat feeding was decreased 27% when compared with HL(+/+) BM, CETPtgLDLr(-/-) mice (P < 0.05), and this was associated with a 96% increase in en face aortic atherosclerosis (P < 0.05). In male CETPtgLDLr(-/-) mice, leukocyte-derived HL deficiency was associated with a 16% decrease in plasma HDL-cholesterol concentration and a 25% increase in aortic atherosclerosis. Thus, leukocyte-derived HL in CETPtgLDLr(-/-) mice has an atheroprotective role that may involve increased HDL levels.     

Osteopontin is associated with nuclear factor kappaB gene expression during tail-suspension-induced bone loss

Osteoporosis due to unloading-induced bone loss is a critical issue in the modern aging society. Although the mechanisms underlying this phenomenon are largely unknown, osteopontin (OPN) is one of the critical mediators required for unloading-induced bone loss [M. Ishijima, S.R. Rittling, T. Yamashita, K. Tsuji, H. Kurosawa, A. Nifuji, D.T. Denhardt, and M. Noda, Enhancement of osteoclastic bone resorption and suppression of osteoblastic bone formation in response to reduced mechanical stress do not occur in the absence of osteopontin, J Exp Med, 193 (2001) 399-404]. To clarify the molecular bases for OPN actions, we carried out microarray analyses on the genes expressed in the femoral bone marrow cells in wild type and OPN-/- mice. The removal of the mechanical load induced bone loss in wild type, but not in OPN-/- mice, as previously reported. Expression analysis of 9586 cDNAs on a microarray system revealed that OPN deficiency blocked tail-suspension-induced expression of ten genes (group A). This observation was confirmed based on semi-quantitative RT-PCR analyses. On the other hand, expression of four genes (group B) was not altered by tail suspension in wild type but was enhanced in OPN-deficient mice. NF-kappaB p105 subunit gene (Nfkb1) was found in group A and Bax in group B. p53 gene expression was upregulated by tail suspension in wild type mice, but it was no longer observed in OPN-/- mice. These data indicate that OPN acts to mediate mechanical stress signaling upstream to the genes encoding apoptosis-related molecules, and its action is associated with alteration of the genes.     

Bile acids override steatosis in farnesoid X receptor deficient mice in a model of non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, and the pathogenesis is still not well known. The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily and plays an essential role in maintaining bile acid and lipid homeostasis. In this study, we study the role of FXR in the pathogenesis of NFALD. We found that FXR deficient (FXR^−/−) mice fed methionine- and choline-deficient (MCD) diet had higher serum ALT and AST activities and lower hepatic triglyceride levels than wild-type (WT) mice fed MCD diet. Expression of genes involved in inflammation (VCAM-1) and fibrosis (α-SMA) was increased in FXR^−/− mice fed MCD diet (FXR^−/−/MCD) compared to WT mice fed MCD diet (WT/MCD). Although MCD diet significantly induced hepatic fibrosis in terms of liver histology, FXR^−/−/MCD mice showed less degree of hepatic steatosis than WT/MCD mice. Moreover, FXR deficiency synergistically potentiated the elevation effects of MCD diet on serum and hepatic bile acids levels. The super-physiological concentrations of hepatic bile acids in FXR^−/−/MCD mice inhibited the expression of genes involved in fatty acid uptake and triglyceride accumulation, which may be an explanation for less steatosis in FXR^−/−/MCD mice in contrast to WT/MCD mice. These results suggest that hepatic bile acids accumulation could override simple steatosis in hepatic injury during the progression of NAFLD and further emphasize the role of FXR in maintaining hepatic bile acid homeostasis in liver disorders and in hepatic protection.     

Single Nucleotide Polymorphisms of Cytokine Genes are Associated with Fibrosis of the Intrahepatic Bile Duct Wall in Human Clonorchiasis

This study examined the association of cytokine gene polymorphisms with intrahepatic bile duct wall fibrosis in human clonorchiasis. A total of 240 residents in Heilongjiang, China underwent ultrasonography, blood sampling, and stool examination. Single nucleotide polymorphism (SNP) sites for IFN-γ (+874 T/A), IL-10 (-1,082 G/A, -819 C/T, -592 C/A), TNF-α (-308 G/A), and TGF-β1 (codon 10 T/C, codon 25 G/C) genes were observed with the TaqMan allelic discrimination assay. No significant correlation was observed between individual cytokine gene polymorphisms and intrahepatic duct dilatation (IHDD). Among individuals with clonorchiasis of moderate intensity, the incidence of IHDD was high in those with IFN-γ intermediate-producing genotype, +874AT (80.0%, P = 0.177), and in those with TNF-α low-producing genotype, -308GG (63.0%, P = 0.148). According to the combination of IFN-γ and TNF-α genotypes, the risks for IHDD could be stratified into high (intermediate-producing IFN-γ and low producing TNF-α), moderate, and low (low-producing IFN-γ and high producing TNF-α) risk groups. The incidence of IHDD was significantly different among these groups (P = 0.022): 88.9% (odds ratio, OR = 24.0) in high, 56.5% (OR = 3.9) in moderate, and 25.0% (OR = 1) in low risk groups. SNP of IFN-γ and TNF-α genes may contribute to the modulation of fibrosis in the intrahepatic bile duct wall in clonorchiasis patients.     

Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.     

Protein kinase Cβ deficiency attenuates obesity syndrome of ob/ob mice by promoting white adipose tissue remodeling

To explore the role of leptin in PKCβ action and to determine the protective potential of PKCβ deficiency on profound obesity, double knockout (DBKO) mice lacking PKCβ and ob genes were created, and key parameters of metabolism and body composition were studied. DBKO mice had similar caloric intake as ob/ob mice but showed significantly reduced body fat content, improved glucose metabolism, and elevated body temperature. DBKO mice were resistant to high-fat diet-induced obesity. Moreover, PKCβ deficiency increased β-adrenergic signaling by inducing expression of β1- and β3-adrenergic receptors (β-ARs) in white adipose tissue (WAT) of ob/ob mice. Accordingly, p38(MAPK) activation and expression of PGC-1α and UCP-1 were increased in WAT of DBKO mice. Consistent with results of in vivo studies, inhibition of PKCβ in WAT explants from ob/ob mice also increased expression of above β-ARs. In contrast, induction of PGC-1α and UCP-1 expression in brown adipose tissue of DBKO mice was not accompanied by changes in the expression of these β-ARs. Collectively, these findings suggest that PKCβ deficiency may prevent genetic obesity, in part, by remodeling the catabolic function of adipose tissues through β-ARs dependent and independent mechanisms.     

The organoselenium compound 1,4-phenylenebis(methylene)selenocyanate inhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced tumorgenesis and enhances glutathione-related antioxidant levels in A/J mouse lung

Selenium, in the form of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) but not Se-enriched yeast (Se-yeast), was highly effective at inhibiting lung tumors induced by the tobacco specific nitrosamine (TSNA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice and at reducing NNK-induced DNA methylation and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in the lung. Our goal was to determine if p-XSC but not Se-yeast is effective at inducing levels of glutathione (GSH)-related antioxidants and reducing markers of GSH oxidation in the NNK-induced lung tumor model. In the first bioassay, 6-week-old mice were fed either control or experimental diets (containing 10 ppm as selenium from p-XSC or Se-yeast) and, beginning at 8 weeks of age, received NNK (3 micromol) by gavage once weekly for 8 weeks. After 18 weeks, p-XSC significantly reduced NNK-induced tumor burden by 74% (10.4 +/- 6.0 versus 2.7 +/- 1.5 tumors/mouse, P < 0.001) and tumor incidence from 96% to 68% (P < 0.01), whereas, Se-yeast had no effect. Lung GSH levels were unchanged by either NNK or Se-yeast, but were increased 70% in mice treated with both NNK and p-XSC (P < 0.01) and 41% in mice treated with p-XSC alone. In the second bioassay, the time course of effects of p-XSC was examined. As early as one week after initiation of p-XSC feeding lung and blood selenium levels were increased nearly six- and two-fold, respectively. Increases of 120% for GSH and 65% for Cys were observed in p-XSC groups compared to controls within one week after initiation of p-XSC feeding (P < 0.01). The levels of protein-bound:free GSH ratios and Cys ratios were significantly decreased in p-XSC-treated mice, regardless of NNK status, suggesting a decrease in the levels of oxidative stress. Altogether, these results indicate that p-XSC is a potent inducer of GSH and related thiol antioxidants in the lung leading to decreased levels of oxidative stress and suggest that p-XSC inhibits tumor formation, in part, by protecting against oxidative damage.     

The complete mitogenome of Apocheima cinerarius (Lepidoptera: Geometridae: Ennominae) and comparison with that of other lepidopteran insects

The complete mitochondrial genome (mitogenome) of a female flightless geometrid moth Apocheima cinerarius was found to be 15,722 bp in length, containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a control region. The A + T content of the complete mitogenome is 80.83%. The AT skew value ([A − T] / [A + T]) is 0.027. The 13 PCGs of the mitogenome start with typical ATN codons, except for cox1 with the start codon CGA. All the tRNA genes have typical cloverleaf secondary structures, except for trnSer(AGN). The secondary structures of rrnL and rrnS were predicted. Six structural domains including conserved regions (IV, V) and variable regions (I, II, III, VI) were identified in the secondary structure of rrnL. The secondary structure of rrnS consists of 3 structural domains. The control region of A. cinerarius begins with conserved motifs of “ATAGA” + 19-bp poly T. It also contains a microsatellite-like (TA)₂₆, a stem-and-loop structure, and a poly-A stretch. Phylogenetic analysis showed that Geometroidea is more closely related to Bombycoidea than to Noctuoidea. A. cinerarius is more closely related to Biston panterinaria than to Phthonandria atrilineata, which is in accordance with the conventional morphology-based classification.     

Gene expression profile of oxidant stress and neurodegeneration in transgenic mice deficient in alpha-tocopherol transfer protein

Alpha-tocopherol transfer protein (TTP) regulates the retention and secretion of alpha-tocopherol (alpha-T) by the liver. Deletion of the TTP gene (Ttpa) in mice results in systemic deficiency of alpha-T and neurological dysfunctions described in patients with mutated Ttpa. We have explored genome-wide changes in mRNAs from brain cortex and liver of Ttpa-deficient (Ttpa(-/-)) mice and wild-type (Ttpa(+/+)) mice. Selective inductions of genes regulated by antioxidant response elements were detected in Ttpa(-/-) livers compared to Ttpa(+/+) livers, suggesting increased oxidant stress in Ttpa(-/-) livers. The activation of cell proliferation pathways in Ttpa(-/-) livers was indicated by the induction of genes that encode growth factor-binding proteins, mitogen-activated protein kinase kinase 3, and apoptosis inhibitor 6. The induction of synuclein-alpha and repression of synuclein-beta genes was detected in Ttpa(-/-) cortex. This may predispose Ttpa(-/-) cortex to increased formation of synuclein-alpha aggregates and Lewy body, often associated with oxidant stress. Cortex of Ttpa(-/-) mice revealed repression of genes encoding synaptic proteins, protein kinase C family members, and myelin proteins. A 13-fold decrease in the expression of retinoic acid receptor-related orphan receptor-alpha mRNA predicts staggerer-like phenotype (ataxia and deficits of motor coordination) of Ttpa(-/-) mice. The repression of specific genes that determine synaptic plasticity and neuronal development may account for suppressed electrophysiological activities of cortex and impaired behavior in Ttpa(-/-) mice.     

Intermittent exposure to traces of green leaf volatiles triggers the production of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol in exposed plants

Intermittent exposure during a period of 3 weeks of undamaged Arabidopsis plants to trace amounts of volatiles emitted by freshly damaged Arabidopsis plants resulted in an increase of subsequent artificial-damage-induced production of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol in the exposed Arabidopsis plants when compared with Arabidopsis plants exposed to undamaged Arabidopsis plant volatiles (control plants). We previously showed that (Z)-3-hexen-1-yl acetate attracts a parasitic wasp, Cotesia glomerata. Thus, the induced production of this volatile explained our previously reported finding that, when artificially damaged, the exposed plants were more attractive to C. glomerata than control plants.     

Toxoplasma gondii: in vitro and in vivo activities of the hydroxynaphthoquinone 2-hydroxy-3-(1'-propen-3-phenyl)-1,4-naphthoquinone alone or combined with sulfadiazine

The compound 2-hydroxy-3-(1'-propen-3-phenyl)-1,4-naphthoquinone (PHNQ6) was evaluated for activity against Toxoplasma gondii, alone or combined with sulfadiazine. Treatment with PHNQ6 combined with sulfadiazine protected at least 70 and 90% of mice infected with RH and EGS strains, respectively. Mice were treated with PHNQ6 (50 mg/kg/day) alone or combined with sulfadiazine (40 mg/L) 30 days after infection with P strain. The number of brain cysts was lower in mice treated with PHNQ6 alone or combined with sulfadiazine compared to that in control mice. Degenerated bradyzoites were observed in animals treated with PHNQ6. Infectivity of bradyzoites treated with PHNQ6 alone or combined with sulfadiazine was inhibited after in vitro incubation.     

Chlamydia trachomatis pulmonary infection induces greater inflammatory pathology in immunoglobulin A deficient mice

Chlamydia trachomatis is an intracellular bacterial pathogen that primarily infects via mucosal surfaces. Using mice with a targeted disruption in IgA gene expression (IgA(-/-) mice), we have studied the contribution of IgA, the principal mucosal antibody isotype, in primary immune defenses against pulmonary C. trachomatis infection. Bacterial burden was comparable between IgA(-/-) and IgA(+/+) animals following C. trachomatis challenge. Serum and pulmonary anti-Chlamydia antibody levels were higher in IgA(-/-) animals, with the exception of IgA. Lung sections of challenged IgA(-/-) mice showed more extensive immunopathology than corresponding IgA(+/+) animals. Real-time PCR analysis demonstrated significantly greater IFN-gamma and TGF-beta mRNA expression in IgA(-/-) as compared to IgA(+/+) animals. Together, these results suggest that IgA may not be necessary for clearance of primary C. trachomatis infection. However, IgA(-/-) mice displayed exaggerated lung histopathology and altered cytokine production, indicating an important role for IgA in regulating C. trachomatis induced pulmonary inflammation and maintenance of mucosal homeostasis.     

Aureobasidium-Derived Soluble Branched (1,3-1,6) ��-Glucan (Sophy ��-glucan) Enhances Natural Killer Activity in Leishmania amazonensis-Infected Mice

The β-glucans derived from yeast cell walls have been reported for having many immunomodulatory activities in vivo and in vitro. In this study, Aureobasidium-derived soluble branched (1,3-1,6) β-glucan (Sophy β-glucan) was checked for natural killer (NK) activity and for the production of IFN-γ and IL-4 in Leishmania amazonensis infection. The main experiment was performed with a group of female C57BL/6 and BALB/c mice, orally supplemented with 5% of Sophy β-glucan and infected with promastogotes of L. amazonensis (1 × 10⁷) into the footpad. Increase in the footpad thickness with time was observed in BALB/c mice in spite of the oral Sophy β-glucan supplement, but it was less in C57BL/6 mice. The difference in overall mean footpad thickness between ''infection only'' versus ''infection + glucan'' groups was statistically significant (P < 0.001). High NK activity in C57BL/6 than BALB/c mice was observed in ''glucan only'' group compared to the control group and also in ''infection + glucan'' group compared to ''infection only'' group. The difference in the NK activity among these groups was significant (P < 0.05). The IFN-γ level increased at weeks 7 and 8 post-infection in C57BL/6 mice and was significantly high in ''infection + glucan'' group compared to the ''infection only'' group (P < 0.05). IL-4 levels did not increase up to detectable levels throughout the study. The results led a conclusion that Sophy β-glucan enhances NK activity and cellular immunity in L. amazonensis-infected mice.