Efficacy of Dietary Lipid Control in Healing High-Fat and High-Cholesterol Diet-Induced Fibrotic Steatohepatitis in Rats

Nonalcoholic steatohepatitis is related to lifestyle, particularly to dietary habits. We developed diet-induced fibrotic steatohepatitis model stroke-prone spontaneously hypertensive 5/Dmcr (SHRSP5/Dmcr) rats showing steatosis, hepatic inflammation, and severe fibrosis induced by high-fat and -cholesterol (HFC) diet feeding. We aimed to clarify the efficacy of dietary intervention on the disease before and after the appearance of fibrosis. Male SHRSP5/Dmcr rats were divided into 9 groups; of these, 6 groups were fed control or HFC diet for several weeks and the remaining 3 groups represented the dietary intervention groups, which were fed the control diet after HFC diet feeding for 2 (before the appearance of fibrosis) or 8 (after the appearance of fibrosis) weeks. Dietary intervention before the appearance of fibrosis significantly improved the steatosis and reset the increased serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and serum total cholesterol (TC) levels. However, dietary intervention after the appearance of fibrosis was unable to reset the levels of hepatic TC, serum ALT, and fibrogenesis-related markers and had only a minor influence on hepatic fibrosis, although it reset the increased expression of transforming growth factor (TGF)-β1 and α-smooth muscle actin (SMA). It was noted that dietary intervention improved the increased AST levels; however, aggregated CD68-positive cells were still observed around the fibrosis area, which may be related to the findings of inflammatory cytokine mRNAs. Taken together, dietary intervention for fibrotic steatohepatitis improved steatosis, although it could not completely improve fibrosis.     

Characterization of conserved arginine residues on Cdt1 that affect licensing activity and interaction with Geminin or Mcm complex

All organisms ensure once and only once replication during S phase through a process called replication licensing. Cdt1 is a key component and crucial loading factor of Mcm complex, which is a central component for the eukaryotic replicative helicase. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent rereplication. Here, we address the mechanism of DNA licensing using purified Cdt1, Mcm and Geminin proteins in combination with replication in Xenopus egg extracts. We mutagenized the 223th arginine of mouse Cdt1 (mCdt1) to cysteine or serine (R-S or R-C, respectively) and 342nd and 346th arginines constituting an arginine finger-like structure to alanine (RR-AA). The RR-AA mutant of Cdt1 could not only rescue the DNA replication activity in Cdt1-depleted extracts but also its specific activity for DNA replication and licensing was significantly increased compared to the wild-type protein. In contrast, the R223 mutants were partially defective in rescue of DNA replication and licensing. Biochemical analyses of these mutant Cdt1 proteins indicated that the RR-AA mutation disabled its functional interaction with Geminin, while R223 mutations resulted in ablation in interaction with the Mcm2～7 complex. Intriguingly, the R223 mutants are more susceptible to the phosphorylation-induced inactivation or chromatin dissociation. Our results show that conserved arginine residues play critical roles in interaction with Geminin and Mcm that are crucial for proper conformation of the complexes and its licensing activity.     

Cloning and characterization of a novel human gene encoding a zinc finger protein with 25 fingers

This study reports cloning and characterization of a human cDNA encoding a novel human zinc finger protein, ZFD25. ZFD25 cDNA is 6118 bp long and has an open reading frame of 2352 bp that encodes a 783 amino acid protein with 25 C2H2-type zinc fingers. The ZFD25 cDNA also contains a region with high sequence similarity to the Krüppel-associated box A and B domain in the 5'-untranslated region, suggesting that ZFD25 belongs to the Krüppel-associated box zinc finger protein family. The ZFD25 gene was localized to chromosome 7q11.2. Northern blot analysis showed that ZFD25 was expressed in a wide range of human organs. In cultured endothelial cells, the mRNA level was decreased upon serum starvation.     

Quercetin inhibited DNA synthesis and induced apoptosis associated with increase in c-fos mRNA level and the upregulation of p21WAF1CIP1 mRNA and protein expression during liver regeneration after partial hepatectomy

Quercetin, a widely distributed bioflavonoid, inhibited DNA synthesis in regenerating liver after partial hepatectomy. This inhibition was accompanied by apoptosis, evidenced by in situ end-labeling and gel electrophoresis of DNA fragmentation. Characteristic DNA fragmentation was detected as early as 2 h after injection. Northern blot analysis revealed that quercetin induced the increases in c-fos and p21WAF1CIP1 mRNA levels within 2 h. The expression of p21 protein was also enhanced, while p53 mRNA and protein levels were not affected by quercetin. These results suggest that quercetin-induced apoptosis is associated with the increase in c-fos mRNA level and the upregulation of p21 mRNA and protein expression, probably in a p53-independent pathway.     

Cytoplasmic domain is not essential for the cell adhesion activities of gicerin, an Ig-superfamily molecule

Gicerin is a cell adhesion molecule in the immunoglobulin (Ig) superfamily and is expressed abundantly during development in the nervous system. It has homophilic cell adhesion activity and also has heterophilic binding activity with NOF (neurite outgrowth factor) and mediates neurite extension. There are two isoforms of gicerin, one with a short (s-gicerin) and the other with a longer cytoplasmic domain (l-gicerin). We have reported that s-gicerin possesses stronger activities than l-gicerin during cell aggregation, in NOF-binding, and in neurite extension. In this study, we established cell lines which expressed a mutant-gicerin whose cytoplasmic domain was deleted and we compared the above three biological activities of the mutant-gicerin with those of s- and l-gicerin. We found that the mutant-gicerin retained all these activities, but the activities were weaker than those of s-gicerin and almost the same as those of l-gicerin. We concluded that the cytoplasmic domain of gicerin is not essential for optimal adhesive activities of gicerin, but might be involved in the regulation of its activities.     

Involvement of gicerin, a cell adhesion molecule, in development and regeneration of oviduct and metastasis of oviductal adenocarcinomas of the chicken

Gicerin is a novel cell adhesion molecule in the immunoglobulin superfamily and has both homophilic adhesion and heterophilic adhesive activity to neurite outgrowth factor (NOF), an extracellular matrix protein in the laminin family. We investigated the possible involvement of gicerin in oviductal development, regeneration, and metastasis of oviductal adenocarcinomas of the chicken. In the oviductal epithelium, gicerin was expressed strongly during development, disappeared after maturation, and reappeared during regeneration. NOF was constitutively expressed in the basement membrane of the epithelium. These molecules were expressed strongly in oviductal adenocarcinomas in both primary and metastatic lesions in the mesentery. An anti-gicerin antibody inhibited the attachment of adenocarcinoma cells to the mesentery in vitro. Many cells migrated from adenocarcinoma tissues on NOF, which were inhibited by an anti-gicerin antibody. These results suggest that gicerin might play a role in oviductal development and regeneration and also in the metastasis of adenocarcinomas.     

Bovine milk lactoferrin modulates the proliferative lymphocyte response to phytohemagglutinin

Bovine milk lactoferrin (2 to 20 g ml^–1) changed enhancement of [³H]thymidine incorporation by phytohemagglutinin-stimulated rat spleen lymphocytes into suppression as their lactoferrin-withdrawal incorporation increased to greater than 10000 cpm culture^–1 under the present isotope-labeling conditions. The enhancement disappeared by 15-min delayed addition of lactoferrin after addition of lectin. There was no lactoferrin effect when the cells were stimulated with 12-O-tetradecanoylphorbol-13-acetate plus ionomycin. Thus, lactoferrin has a certain extracellular effect on lymphocyte proliferation in response to the lectin.