Isohumulones modulate blood lipid status through the activation of PPARα

Isohumulones derived from hops are the major bitter compounds in beer. It was recently reported that isohumulones activated peroxisome proliferator-activated receptors (PPARs) α and γ in vitro and modulated glucose and lipid metabolism in vivo. In this study, we examined the effects of isomerized hop extract (IHE) primarily containing isohumulones in C57BL/6N male mice and found that such treatment increased their liver weight and reduced their plasma triglyceride and free fatty acid levels. Microarray analysis and quantitative real time PCR (QPCR) showed that IHE dose-dependently upregulated the expression of a battery of hepatic genes that are involved in microsomal ω-oxidation and peroxisomal and mitochondrial β-oxidation. These effects were common in both genders and very similar to those found with the PPARα agonist, fenofibrate (FF). Moreover, these effects were not found in PPARα-deficient mice. Thus, our results strongly suggest that IHE intake upregulates the expression of key genes that are involved in hepatic fatty acid oxidation, and that it ameliorates the blood lipid profile by activating PPARα.     

Helicobacter pylori activates NF-kappaB via the alternative pathway in B lymphocytes

Helicobacter pylori causes various gastroduodenal diseases including gastric MALT lymphoma, but the mechanism underlying H. pylori-induced carcinogenesis is not known. The alternative pathway for NF-kappaB activation, which involves the processing of NF-kappaB2/p100 to p52, has been implicated in lymphocyte survival, attenuated apoptosis, and secondary lymphoid tissue development. In this study, we investigated H. pylori-induced activation of NF-kappaB through the alternative pathway in B lymphocytes. In immunoblot and EMSA, H. pylori induced NF-kappaB2/p100 processing to p52 and subsequent nuclear accumulation in IM-9 (human B cell line) cells and human peripheral blood B cells, but not in AGS (human gastric cancer cell line) cells. The activation of the alternative pathway was LPS-dependent but not cag pathogenicity island-dependent. Alternative pathway activation by H. pylori was associated with attenuated apoptosis. The expression levels of B lymphocyte chemoattractant, EBI-1 ligand chemokine, and stromal cell-derived factor-1alpha mRNAs were up-regulated in cocultured human B cells and in infected human gastric mucosa. In the infected mucosa, NF-kappaB2/p100 and p52 were detected immunohistochemically in the cytoplasm and nuclear compartments of lymphocytes, but not in epithelial cells. In summary, H. pylori activates the alternative NF-kappaB pathway in B lymphocytes. The effects on chemokine production and antiapoptosis mediated by H. pylori-induced processing of NF-kappaB2/p100 to p52 may drive lymphocytes to acquire malignant potential.     

Safe And Efficient Collection of Cytokine-Mobilized Peripheral Blood Cells From Cynomolgus Monkeys (Macaca fascicularis) with Human Newborn-Equivalent Body Weights.

Hematopoietic stem cells in bone marrow can be mobilized into peripheral blood by cytokine administration. Cytokine-mobilized peripheral blood stem cells are of great use in clinical applications. We previously established a modified procedure for the collection of cytokine-mobilized peripheral blood cells from rhesus monkeys (Macaca mulata) using a commercially available apparatus originally developed for human subjects. In this study, we examined the efficacy and safety of this method with even smaller macaques, cynomolgus monkeys (Macaca fascicularis), which are equivalent to human newborns in body weight (mean = 3.3 kg). Using the manufacturer's unmodified protocol (n=6), one monkey died of cardiac failure and three developed severe anemia. In contrast, using our modified procedure (n=6), no such complication was observed in any animal. In addition, the harvested nuclear cell, mononuclear cell and CD34(+) cell counts were significantly higher with the modified method. The modified method should allow safe and efficient collection of cytokine-mobilized peripheral blood cells from non-human primates as small as human newborns in a non-invasive manner.     

Mass outbreak of food poisoning disease caused by small amounts of staphylococcal enterotoxins A and H

It was believed that food poisoning in Osaka in 2000 was due to small amounts of staphylococcal enterotoxin A (SEA) in reconstituted milk. Results of this study clearly indicate that SEH was also present in the raw material of reconstituted milk, indicating that the food poisoning was caused by multiple staphylococcal enterotoxins.     

Improvement in the proliferative activity of human-human hybridomas at low cell density by transfection with bFGF gene

Highly purified recombiaant basic fibroblast growth factor (rbFGF) and acidic FGF (aFGF) stimulated the proliferation of human-human (h-h) hybridomas to the extent of over four-fold from a low cell density such as 1×10³ cells per ml in a serum-free medium in 24-well plates. The stimulatory effect of rbFGF was also observed in various lymphoid cell lines. Expecting that FGF could be an autocrine growth factor, we introduced bFGF gene into a h-h hybridoma using an expression plasmid induced by dexamethasone. The transformed cells thus obtained, HPO-75.11bbFGF-7, were able to grow well from a low inoculum density in a serum-free medium and antibody production was also increased when bFGF gene expression was induced. The transformed cells could grow at clonal density in a serum-free medium in 96-well plates, though the original cells could not. We also obtained a more practical transfectant, HPO-75.29-H74, using a high-shear stress adapted clone as the recipient and an expression plasmid having bFGF gene under the control of metallothioneine-I promoter. The HOP-75.29-H74 cells were capable of growing and producing human monoclonal antibody against hepatitis B virus surface antigen from an inoculum density of 1×10³ cells per ml in an agitation vessel without addition of an inducer.     

Discovery of benzimidazole derivatives as orally active renin inhibitors: Optimization of 3,5-disubstituted piperidine to improve pharmacokinetic profile

We previously identified 2-tert-butyl-4-[(3-methoxypropyl)amino]-N-(2-methylpropyl)-N-[(3S,5R)-5-(morpholin-4-ylcarbonyl)piperidin-3-yl]pyrimidine-5-carboxamide 3 as a potent renin inhibitor. Since 3 showed unacceptably low bioavailability (BA) in rats, structural modification, using SBDD and focused on physicochemical properties was conducted to improve its PK profile while maintaining renin inhibitory activity. Conversion of the amino group attached at the 4-position of pyrimidine to methylene group improved PK profile and decreased renin inhibitory activity. New central cores with carbon side chains were explored to improve potency. We had designed a series of 5-membered azoles and fused heterocycles that interacted with the lipophilic S3 pocket. In the course of modification, renin inhibitory activity was enhanced by the formation of an additional hydrogen bonding with the hydroxyl group of Thr77. Consequently, a series of novel benzimidazole derivatives were discovered as potent and orally bioavailable renin inhibitors. Among those, compound 13 exhibited more than five-fold of plasma renin inhibition than aliskiren in cynomolgus monkeys at dose ratio.     

Rpn9 Is Required for Efficient Assembly of the Yeast 26S Proteasome

We have isolated the RPN9 gene by two-hybrid screening with, as bait, RPN10 (formerly SUN1), which encodes a multiubiquitin chain receptor residing in the regulatory particle of the 26S proteasome. Rpn9 is a nonessential subunit of the regulatory particle of the 26S proteasome, but the deletion of this gene results in temperature-sensitive growth. At the restrictive temperature, the Deltarpn9 strain accumulated multiubiquitinated proteins, indicating that the RPN9 function is needed for the 26S proteasome activity at a higher temperature. We analyzed the proteasome fractions separated by glycerol density gradient centrifugation by native polyacrylamide gel electrophoresis and found that a smaller amount of the 26S proteasome was produced in the Deltarpn9 cells and that the 26S proteasome was shifted to lighter fractions than expected. The incomplete proteasome complexes were found to accumulate in the Deltarpn9 cells. Furthermore, Rpn10 was not detected in the fractions containing proteasomes of the Deltarpn9 cells. These results indicate that Rpn9 is needed for incorporating Rpn10 into the 26S proteasome and that Rpn9 participates in the assembly and/or stability of the 26S proteasome.     

Adhesion-mediated self-renewal abilities of Ph+ blastoma cells

The Philadelphia chromosome-positive blastoma, maintained by serial subcutaneous transplantation in nude mice, is a highly proliferating biological mass consisting of homogenous CD34⁺CD38⁻ myeloblastoid cells. These cells newly evolved from pluripotent leukemia stem cells of chronic myeloid leukemia in the chronic phase. Therefore, this mass may provide a unique tool for better understanding cellular and molecular mechanisms of self-renewal of leukemia stem cells. In this paper, we demonstrated that intravenously injected blastoma cells can cause Ph+ blastic leukemia with multiple invasive foci in NOD/SCID mice but not in nude mice. In addition, using an in vitro culture system, we clearly showed that blastoma cell adhesion to OP9 stromal cells accelerates blastoma cell proliferation that is associated with up-regulation of BMI1 gene expression; increased levels of β-catenin and the Notch1 intra-cellular domain; and changed the expression pattern of variant CD44 forms, which are constitutively expressed in these blastoma cells. These findings strongly suggest that adhesion of leukemic stem cells to stromal cells via CD44 might be indispensable for their cellular defense against attack by immune cells and for maintenance of their self-renewal ability.