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Abstract: Apolipoprotein E (apoE) is a ligand for members of the low-density lipoprotein receptor (LDLR) family and functions in plasma cholesterol homeostasis. A fluorescence-based assay has been employed in molecular studies of receptor–ligand interactions. Competition experiments revealed isoform-specific differences in binding of lipid-associated apoE N terminal (NT) domain to a recombinant soluble LDLR (sLDLR). In a similar manner, lipid-associated—but not lipid-free—full-length apoE3 showed binding activity to sLDLR. The molecular chaperone, receptor-associated protein, inhibited apoE3–NT–phospholipid complex binding to sLDLR. Kinetic studies of apoE3–NT–phospholipid complex interaction with sLDLR revealed time-dependent effects of apoE–NT isoform binding to sLDLR. The results reveal a discerning method for study of the molecular basis of ligand interactions that likely influence receptor function in maintenance of whole body cholesterol homeostasis. [Copyright &y& Elsevier]

Abstract: Apolipoprotein E (apoE) is a ligand for members of the low-density lipoprotein receptor (LDLR) family. Lipid-free apoE is not recognized by LDLR, yet interaction with lipid confers receptor recognition properties. Although lipid interaction is known to induce a conformational change in apoE, it is not known if the lipid composition of the resulting complex influences binding. Using reconstituted lipoprotein particles of apoE3 N-terminal (NT) domain and dimyristoylphosphatidylcholine (DMPC), maximal LDLR binding was observed at DMPC:apoE3-NT ratios >2.5:1 (w/w). ApoE3-NT lipid particles prepared with egg sphingomyelin were functional as LDLR ligands while complexes formed with the anionic phospholipids dimyristoylphosphatidylglycerol or dimyristoylphosphatidylserine (DMPS) were not. In the case of apoE3-NT, lipid particles comprised of a mixture of DMPC and DMPS, a DMPS concentration dependent inhibition of LDLR binding activity was observed. Thus, in addition to affecting apoE conformational status, the lipid composition of ligand particles can modulate LDLR binding activity. [Copyright &y& Elsevier]

Abstract: The homeodomain protein Hex can function either as a transcriptional repressor or activator in animals. Recent reports have indicated that Hex is involved in liver development. However, its target genes and interacting proteins are largely unknown. We found that Hex functionally interacted with hepatocyte nuclear factor (HNF) 1α to further stimulate its activity using reporter gene containing multiple copies of HNF1α-binding site of the L-type pyruvate kinase (L-PK) gene promoter or natural L-PK promoter. This stimulation required the homeodomain and the acidic carboxyl-terminal region of Hex. Over-expression of Hex in primary cultured hepatocytes resulted in stimulation of the L-PK gene expression. Glutathione S-transferase pull-down assay and co-immunoprecipitation revealed that Hex physically interacted with HNF1α in mammalian cells through the homeodomain of Hex and POU-homeodomain of HNF1α. Since HNF1α is an important liver-enriched transcription factor involved in liver differentiation, Hex may contribute to liver differentiation through interaction with HNF1α. [Copyright &y& Elsevier]

We report here the existence of interactions between a ubiquitin-conjugating enzyme, Rad6, from rice, Oryza sativa L. cv. Nipponbare (OsRad6), and Sgt1 (OsSgt1), a novel subunit of the SCF ubiquitin ligase complex. Rad6 is not only related to post-replicational repair but also to the proteasome system, while Sgt1 has a function in kinetochore assembly. The relationship between the two is unexpected, but of great interest. The open reading frames of OsRad6 and OsSgt1 encode predicted products of 152 and 367 amino acid residues, respectively, with molecular weights of 17.3 and 40.9 kDa. Two-hybrid and pull-down analyses indicated that OsRad6 binds to OsSgt1, and transcripts of both OsRad6 and OsSgt1 were found to be strongly expressed only in the proliferating tissues such as the shoot apical meristem, suggesting that their expression is cell cycle-dependent. The amount of the Rad6 mRNA in cultured cells increased rapidly after division was halted, and mRNA levels of Rad6 and Sgt1 were induced by UV- and DNA-damaging agents such as MMS or H2O2. The Rad6 pathway for repair or the proteasome system may thus require Sgt1 as ubiquitin-conjugating enzyme. [Copyright &y& Elsevier]

The deletion of the Hhex (Hematopoietically expressed homeobox) gene causes agenesis of the liver and polycystic liver disease depending on its timing. The present study was undertaken to determine the role of the Hhex gene in not only signaling cascades to cyst and abnormal bile duct formation but also the liver progenitor contribution to cystic development. Liver-specific Hhex knockout mice (Alb - Cre / Hhex loxP / loxP ) in adult stages were used. Wild-type and conditional knockout (cKO) livers were immunohistologically compared for cell growth, and gene expression of liver functions, biliary markers and cystic markers. In Hhex cKO livers, cyst formation and dilated intrahepatic bile ducts were noted, which resembled the histology of the von Meyenburg complex. Ki67 immunohistochemistry showed that the growth activity in bile ducts and cysts of cKO livers was elevated compared with that of wild-type livers. There were far fewer liver progenitor cells or bile ductule cells around portal veins of cKO livers than in wild-type livers. Several liver-enriched transcription factors, including Foxa1 and Foxa2, were heterogeneously expressed in bile ducts and cysts of cKO livers whereas their expression in wild-type bile ducts was comparatively homogeneous. PC1 and PC2 immunohistochemistry revealed their up-regulation in cysts of cKO livers. These data indicate that Hhex is not only required for proper bile duct morphogenesis, but is also involved in cyst formation through promoted cell growth. Liver progenitor cells may form cysts. Unbalanced expression of liver-enriched transcription factors might be involved in cyst formation. Hhex cKO mice may be a good animal model for hepatic cystic diseases. • Hhex is involved in bile duct development and cyst formation through growth control. • Liver progenitor cells may participate in cyst formation. • Cyst cells have unbalanced expression of liver-enriched transcription factors. • Hhex knockout mice may be a good animal model for hepatic cystic disease. [ABSTRACT FROM AUTHOR]

Abstract: Small compounds that activate the insulin-dependent signaling pathway have potential therapeutic applications in controlling type 2 diabetes mellitus. The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor that decreases expression of the phosphoenolpyruvate carboxykinase gene, a gluconeogenic enzyme gene. In this study, we screened for soybean isoflavones that can induce the rat SHARP-2 gene expression and analyzed their mechanism(s). Genistein and (S)-Equol, a metabolite of daidzein, induced rat SHARP-2 gene expression in H4IIE rat hepatoma cells. The (S)-Equol induction was mediated by both the phosphoinositide 3-kinase- and protein kinase C (PKC)-pathways. When a dominant negative form of atypical PKC lambda (aPKCλ) was expressed, the induction of SHARP-2 mRNA level by (S)-Equol was inhibited. In addition, Western blot analyses showed that (S)-Equol rapidly activated both aPKCλ and classical PKC alpha. Furthermore, the (S)-Equol induction was inhibited by treatment with a RNA polymerase inhibitor or a protein synthesis inhibitor. Finally, a reporter gene assay revealed that the transcriptional stimulation by (S)-Equol was mediated by nucleotide sequences located between −4687 and −4133 of the rat SHARP-2 gene. Thus, we conclude that (S)-Equol is an useful dietary supplement to control type 2 diabetes mellitus. [Copyright &y& Elsevier]

Abstract: Carbohydrate response element binding protein (ChREBP) is a transcription factor that activates liver glycolytic and lipogenetic enzyme genes in response to high carbohydrate diet. Here we report the transcriptional regulatory mechanisms for the rat ChREBP gene. Firstly, we determined the transcription initiation site and the nucleotide sequences of the rat ChREBP promoter region encompassing approximately 900bp from the ATG initiation codon. Reporter gene assays demonstrated that the major positive regulatory region exists in the nucleotide sequence between −163 and −32 of the ChREBP gene. This region contains a cluster of putative transcription factor binding elements that consist of two specificity protein 1 (Sp1) binding sites (−66 to −50 and −93 to −78), a sterol regulatory element (−101 to −110), and two nuclear factor-Y (NF-Y) binding sites (−23 to −19 and −131 to −127). Mutations introduced into these sites caused marked reduction of ChREBP promoter activities. Functional synergisms were observed between Sp1/NF-Y and Sp1/sterol regulatory element-binding protein. Additionally, electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that these factors bound to these elements. Thus, we conclude that functional synergisms between these transcription factors are critical for ChREBP gene transcription. [Copyright &y& Elsevier]

Abstract: Previously, we described a novel DNA polymerase, designated as OsPolI-like, from rice. The OsPolI-like showed a high degree of sequence homology with the DNA polymerase I of cyanobacteria and was localized in the plastid. Here, we describe two PolI-like polymerases, designated as AtPolI-like A and AtPolI-like B, from Arabidopsis thaliana. In situ hybridization analysis demonstrated expression of both mRNAs in proliferating tissues such as the shoot apical meristem. Analysis of the localizations of GFP fusion proteins showed that AtPolI-like A and AtPolI-like B were localized to plastids. AtPolI-like B expression could be induced by exposure to the mutagen H2O2. These results suggested that AtPolI-like B has a role in the repair of oxidation-induced DNA damage. Our data indicate that higher plants possess two plastid DNA polymerases that are not found in animals and yeasts. [Copyright &y& Elsevier]