Leptin accelerates pronuclear formation following intracytoplasmic sperm injection of porcine oocytes: Possible role for MAP kinase inactivation

Leptin, a multifunctional hormone, is present in mammalian oocytes and follicular fluids and cumulus cells. While leptin modulates oocyte maturation in vitro which seems to result in enhancement of embryo development, it is unclear whether leptin treatment of oocytes affects cytoplasmic maturation and fertilization processes. In order to gain a better understanding of the role of leptin during oocyte maturation, we examined microtubule and microfilament assembly following oocyte maturation and blastocyst formation, mitogen-activated protein kinase (MAPK) activity, and pronuclear formation following parthenogenetic stimuli or intracytoplasmic sperm injection (ICSI) in leptin-treated oocytes. Addition of 10 or 100 ng/ml leptin during oocyte maturation did not increase the proportion of metaphase II oocytes, but enhanced development to blastocyst stage by day 7 (P < 0.01) after parthenogenetic activation (PA), accompanied by increased cell number. However there was no effect on the number of apoptotic cells in blastocysts. Following maturation in the presence of leptin, there were more oocytes with normal spindle formation. MAPK activity decreased more rapidly, and pronuclear formation was accelerated after parthenogenetic activation or ICSI of leptin-treated oocytes. These results suggested that exogeneous leptin enhanced spindle assembly and accelerated pronuclear formation following fertilization, possibly via the MAPK pathway.     

Curcumin Prevents Palmitoylation of Integrin β4 in Breast Cancer Cells

Curcumin has been shown to mitigate cancer phenotypes such as invasive migration, proliferation, and survival by disrupting numerous signaling pathways. Our previous studies showed that curcumin inhibits integrin β4 (ITG β4)-dependent migration by blocking interaction of this integrin with growth factor receptors in lipid rafts. In the current study, we investigated the possibility that curcumin inhibits ITG β4 palmitoylation, a post-translational modification required for its lipid raft localization and signaling activity. We found that the levels of ITG β4 palmitoylation correlated with the invasive potential of breast cancer cells, and that curcumin effectively reduced the levels of ITG β4 palmitoylation in invasive breast cancer cells. Through studies of ITG β4 palmitoylation kinetics, we concluded curcumin suppressed palmitoylation independent of growth factor-induced phosphorylation of key ITG β4 Ser and Tyr residues. Rather, curcumin blocked autoacylation of the palmitoyl acyltransferase DHHC3 that is responsible for ITG β4 palmitoylation. Moreover, these data reveal that curcumin is able to prevent the palmitoylation of a subset of proteins, but not indiscriminately bind to and block all cysteines from modifications. Our studies reveal a novel paradigm for curcumin to account for much of its biological activity, and specifically, how it is able to suppress the signaling function of ITG β4 in breast cancer cells.     

Enzymatic reduction of shogaol: a novel biotransformation pathway for the alpha,beta-unsaturated ketone system.

A novel reductive metabolism of 1-(4-hydroxy-3-methoxyphenyl)-deca-4-ene-3-one (shogaol), a pungent principle of ginger, was investigated in rat liver in vitro. Ethyl acetate-extractable metabolites of shogaol formed by incubation of this alpha,beta-unsaturated ketone with rat liver cytosolic fraction fortified with NADPH or NADPH-generating system were isolated, and two major metabolites were identified as 1-(4-hydroxy-3-methoxyphenyl)-decan-3-one (paradol) and 1-(4-hydroxy-3-methoxy)-decan-3-ol (reduced paradol). 1-(4-hydroxy-3-methoxyphenyl)-deca-1-ene-3-one (dehydroparadol), a non-pungent analog of shogaol, formed the same metabolites as did shogaol under similar incubation conditions. Paradol appears to be an intermediate in the reductive metabolism of the alpha,beta-unsaturated ketone moiety of shogaol to the corresponding saturated alcohol.     

Phylogenomics of <Emphasis Type="Italic">Bartheletia paradoxa</Emphasis> reveals its basal position in Agaricomycotina and that the early evolutionary history of basidiomycetes was rapid and probably not strictly bifurcating

The higher level phylogeny of fungi has been addressed in previous studies, but for those analyses, either taxon sampling or gene sampling was low, or some basal lineages important for the inference of basidiomycete phylogeny were lacking. Here, a phylogenomic analysis based on highly conserved genes and including the enigmatic species Bartheletia paradoxa from Ginkgo biloba is presented. While phylogenetic analyses including also less conserved parts of core eukaryotic genes yielded a basal position for the extremophile genus Wallemia with low support, an exclusion of highly variable parts of these genes suggested Bartheletia paradoxa as the most basal member of the Agaricomycotina, but again with low support. Network analyses suggest a network-like evolution at the base of the Basidiomycota, supported by phylogenies based on single genes and gene clusters with shared topology. When further removing noise by removing poorly resolving genes, strong but not maximum support was obtained for Bartheletia paradoxa being the sister lineage to all other Agaricomycotina. We speculate that the lack of support for the early splits in Agaricomycotina and Basidiomycota can probably be explained by rapid radiation, linked to major evolutionary developments, such as, in the case of Basidiomycota, the advent of basidia in the last common ancestor.     

Evidence for the targeting by 2-oxo-dehydrogenase enzymes in the T cell response of primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease that includes the presence of lymphoid infiltrates in portal tracts, high titer autoantibodies against pyruvate dehydrogenase-E2 (PDH-E2) and branched chain ketoacid dehydrogenase-E2 (BCKD-E2), and biliary tract destruction. The mechanism by which the autoimmune response is induced, the specificity of damage to the biliary epithelium, and the role of T cells in PBC are still unknown. To address these issues, we have taken advantage of a mouse mAb, coined C355.1, and studied its reactivity against a panel of liver tissue from normal subjects as well as a panel of liver specimens from patients with PBC, progressive sclerosing cholangitis, and chronic active hepatitis (CAH). C355.1, much like human autoantibodies to PDH-E2, reacts exclusively by immunoblotting with PDH-E2, binds to the inner lipoyl domain of the protein, and inhibits PDH-E2 activity in vitro. In addition, we have also attempted to develop cloned T cell lines that react with PDH-E2 and/or BCKD-E2 using liver biopsies from patients with PBC, compared with CAH. Although monoclonal C355.1 produced typical mitochondrial fluorescence on sections of normal liver, pancreas, lung, heart, thyroid, and kidney, it produced a distinct and intense reactivity when used to stain the bile ducts of patients with PBC. Nine of 13 PBC liver biopsies studied herein contained bile ducts on light microscopy, all of which reacted intensely at a 1:100 culture supernatant dilution of monoclonal C355.1. In contrast, although bile ducts of liver specimens from normals, CAH, and progressive sclerosing cholangitis also reacted with C355.1, such reactivity was exclusively mitochondrial and readily detectable only at a dilution of 1:2. More importantly, we generated CD4+, CD8-, alpha beta TCR+ cloned T cell lines from patients with PBC, but not from CAH, that produced IL-2 specifically in response to PDH-E2 or BCKD-E2.     

Hepatic DNA and RNA adduct formation from the carcinogen 7-hydroxymethyl-12-methylbenz[a]anthracene and its electrophilic sulfuric acid ester metabolite in preweanling rats and mice

DNA and RNA adducts that were chromatographically identical to those formed in vitro on reaction of 7-sulfooxymethyl-12-methyl-benz[a]anthracene with guanine and adenine nucleosides were formed in the livers of rats and mice given i.p. injections of 7-hydroxymethyl- or 7-sulfooxymethyl-12-methyl-benz[a]anthracene. Considerably higher levels of these hepatic adducts were obtained from the latter short-lived electrophilic ester than from the hydroxymethyl compound. These observations are consistent with the finding of rat liver cytosolic sulfotransferase activity for 7-hydroxymethyl-12-methylbenz[a]anthracene (Watabe et al., Science 215, 403, 1982). Formation of these hepatic adducts from 7-hydroxymethyl-12-methylbenz[a]anthracene was inhibited by prior administration to rats of dehydroepiandrosterone, an inhibitor of the sulfotransferase activity for this hydroxymethyl hydrocarbon.