Molecular Weight and Intrinsic Viscosity of Konjac Gluco-mannan

For the purpose of making clear the macromolecular chemical properties of konjac gluco-mannan, the light scattering and viscosity measurements were carried out for the aqueous solutions of the partially methylated derivatives, and following results were obtained: 1) The weight-average molecular weight, Mw, and the root mean square of radius of gyration, <S²>^1/2, were 100×l0⁴~120×l0⁴ and 1100~1300 Å, respectively. 2) The 2> versus Mw relationship was represented by the equation of <S²>=4.20×10^?1 Mw^1.08, and this fact suggested a random coil as a molecular form in the solution. 3) The intrinsic viscosity, [η], versus Mw relationship was represented by the equation of [η]=6.37×10^?4 Mw^0.74, which was considered to be useful for the purpose of estimating the molecular weight of konjac gluco-mannan by viscometric method.     

Studies on Components of Mammalian Urine

Hexahydrohippuric acid was detected from the urine of cattles together with hippuric and phenaceturic acids as one of the conjugated compounds with glycine. Furthermore, cyclohexanecarboxylic acid was also detected. These experimental results suggest the interesting synthetic processes in vivo or in rumen of the cattle, though both acids have not determined whether they are metabolites of cattle or synthesized by miccroorganisms in rumen.     

Composition of Acid-sensitive Fraction in Soybean Proteins

An acid-sensitive fraction (ASF) was prepared from defatted soybean meals by two procedures. ASF1 was prepared by precipitation at pH 4.5 followed by removal of 1 m NaCl-soluble materials from the precipitate. ASF2 was prepared by precipitation in solution containing 1 m NaCl at pH 4.5. The protein components of the two fractions were analyzed by gel electrophoresis in a dissociating-buffer system and found to contain β-conglycinin, glycinin and whey proteins. In addition to these, several other bands appeared.

Appreciable amounts of lipid (8.2% in ASF1 and 8.8% in ASF2) were also found in the fractions. They were separated by column chromatography and thin-layer chromatography. Glycolipids were the major components of the lipids. Both glycolipid and phospholipid fractions contained slower-moving materials on thin-layer chromatography.     

Liver-targeted siRNA delivery by polyethylenimine (PEI)-pullulan carrier

Recently, small interfering RNA (siRNA)-based therapeutics have been used to treat diseases. Efficient and stable siRNA delivery into disease cells is important in the use of this agent for treatment. In the present study, pullulan was introduced into polyethylenimine (PEI) for liver targeting. PEI/siRNA or pullulan-containing PEI/siRNA complexes were delivered into mice through the tail vein either by a hydrodynamics- or non-hydrodynamics-based injection. The incidence of mortality was found to increase with an increase in the nitrogen/phosphorus (N/P) ratio of PEI/siRNA complexes. Moreover, the hydrodynamics-based injection increased mice mortality. Introduction of pullulan into PEI dramatically reduced mouse death after systemic injection. After systemic injection, the PEI/fluorescein-labeled siRNA complex increased the level of fluorescence in the lung and the PEI-pullulan/siRNA complex led to an increased fluorescence level in the liver. These results suggest that the PEI-pullulan polymer may be a useful, low toxic means for efficient delivery of siRNA into the liver.     

Cell-based chemical genetic screen identifies damnacanthal as an inhibitor of HIV-1 Vpr induced cell death

Viral protein R (Vpr), one of the human immunodeficiency virus type 1 (HIV-1) accessory proteins, contributes to multiple cytopathic effects, G2 cell cycle arrest and apoptosis. The mechanisms of Vpr have been intensely studied because it is believed that they underlie HIV-1 pathogenesis. We here report a cell-based small molecule screen on Vpr induced cell death in the context of HIV-1 infection. From the screen of 504 bioactive compounds, we identified damnacanthal (Dam), a component of noni [corrected] as an inhibitor of Vpr induced cell death. Our studies illustrate a novel efficient platform for drug discovery and development in anti-HIV therapy which should also be applicable to other viruses.     

Thioredoxin-1 contributes to protection against DON-induced oxidative damage in HepG2 cells

Leucocytes are susceptible to the toxic effects of deoxynivalenol (DON), which is a trichothecene mycotoxin produced by a number of fungi including Fusarium species. One mechanism of action is mediated by reactive oxygen species (ROS). The liver is an important target for toxicity caused by foreign compounds including mycotoxins. On the other hand, little is known about the influence of the redox state on hepatocytes treated with DON. The present study investigated the effect of DON on the cytosolic redox state and antioxidative system in the human hepatoma cell line HepG2. The cell viability of human monocyte cell line THP-1 or leukemia cell line KU812 treated with 2.5 and 5???mol/l DON were significantly reduced. However, HepG2 cells showed no toxic effects under the same conditions and did not exhibit an increased oxidative state. Further experiments showed that thioredoxin-1 (Trx-1) protein levels but not glutathione increased in the cells treated with 10???mol/l DON. In addition, the enhancement of Trx-1 content was repressed by antioxidants. These results suggest that DON-induced accumulation of Trx-1 in HepG2 cells plays one of the key roles in protection against cytotoxicity caused by DON and that the mechanism may be mediated by the antioxidant properties of Trx-1.     

Valsartan, independently of AT1 receptor or PPARγ, suppresses LPS-induced macrophage activation and improves insulin resistance in cocultured adipocytes

Macrophages are integrated into adipose tissues and interact with adipocytes in obese subjects, thereby exacerbating adipose insulin resistance. This study aimed to elucidate the molecular mechanism underlying the insulin-sensitizing effect of the angiotensin II receptor blocker (ARB) valsartan, as demonstrated in clinical studies. Insulin signaling, i.e., insulin receptor substrate-1 and Akt phosphorylations, in 3T3-L1 adipocytes was impaired markedly by treatment with tumor necrosis factor-α (TNFα) or in the culture medium of lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages, and valsartan had no effects on these impairments. However, in contrast, when cocultured with RAW 264.7 cells using a transwell system, the LPS-induced insulin signaling impairment in 3T3-L1 adipocytes showed almost complete normalization with coaddition of valsartan. Furthermore, valsartan strongly suppressed LPS-induced productions of cytokines such as interleukin (IL)-1β, IL-6, and TNFα with nuclear factor-κB activation and c-Jun NH(2)-terminal kinase phosphorylation in RAW 264.7 and primary murine macrophages. Very interestingly, this effect of valsartan was also observed in THP-1 cells treated with angiotensin II type 1 (AT1) siRNA or a peroxisome proliferator-activated receptor-γ (PPARγ) antagonist as well as macrophages from AT1a receptor-knockout mice. We conclude that valsartan suppresses the inflammatory response of macrophages, albeit not via PPARγ or the AT1a receptor. This suppression appears to secondarily improve adipose insulin resistance.     

Aminoguanidine normalizes ET-1-induced aortic contraction in type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats by suppressing Jab1-mediated increase in ET(A)-receptor expression

Circulating levels of endothelin (ET)-1 are increased in the diabetic state, as is endogenous ET(A)-receptor-mediated vasoconstriction. However, the responsible mechanisms remain unknown. We hypothesized that ET-1-induced vasoconstriction is augmented in type 2 diabetes with hyperglycemia through an increment in advanced glycation end-products (AGEs). So, we investigated whether treatment with aminoguanidine (AG), an inhibitor of AGEs, would normalize the ET-1-induced contraction induced by ET-1 in strips of thoracic aortas isolated from OLETF rats at the chronic stage of diabetes. In such aortas (vs. those from age-matched genetic control LETO rats): (1) the ET-1-induced contraction was enhanced, (2) the levels of HIF1α/ECE1/plasma ET-1 and plasma CML-AGEs were increased, (3) the ET-1-stimulated ERK phosphorylation mediated by ET(A)-R was increased, (4) the expression level of Jab1-modified ET(A)-R protein was reduced, and (5) the expression level of O-GlcNAcylated ET(A)-R protein was increased. Aortas isolated from such OLETF rats that had been treated with AG (50mg/kg/day for 10 weeks) exhibited reduced ET-1-induced contraction, suppressed ET-1-stimulated ERK phosphorylation accompanied by down-regulation of ET(A)-R, and increased modification of ET(A)-R by Jab1. Such AG-treated rats exhibited normalized plasma ET-1 and CML-AGE levels, and their aortas exhibited decreased HIF1α/ECE1 expression. However, such AG treatment did not alter the elevated levels of plasma glucose or insulin, or systolic blood pressure seen in OLETF rats. These data from the OLETF model suggest that within the timescale studied here, AG normalizes ET-1-induced aortic contraction by suppressing ET(A)-R/ERK activities and/or by normalizing the imbalance between Jab1 and O-GlcNAc in type 2 diabetes.     

The design and optimization of a series of 2-(pyridin-2-yl)-1H-benzimidazole compounds as allosteric glucokinase activators

The optimization of a series of benzimidazole glucokinase activators is described. We identified a novel and potent achiral benzimidazole derivative as an allosteric GK activator. This activator was designed and synthesized via removal of the chiral center of the lead compound, 6-(N-acylpyrrolidin-2-yl)benzimidazole. The activator exhibited good PK profiles in rats and dogs, and significant hypoglycemic efficacy at 1 mg/kg po dosing in a rat OGTT model. The binding site and binding mode of the benzimidazole class of GKA with GK protein was confirmed by X-ray crystallographic analysis.     

A Novel RNA-Binding Protein,Ossa/C9orf10, Regulates Activity of Src Kinases To Protect Cells from Oxidative Stress-Induced Apoptosis

During the process of tumor progression and clinical treatments, tumor cells are exposed to oxidative stress. Tumor cells are frequently resistant to such stress by producing antiapoptotic signaling, including activation of Src family kinases (SFKs), although the molecular mechanism is not clear. In an attempt to identify the SFK-binding proteins selectively phosphorylated in gastric scirrhous carcinoma, we identified an uncharacterized protein, C9orf10. Here we report that C9orf10 (designated Ossa for oxidative stress-associated Src activator) is a novel RNA-binding protein that guards cancer cells from oxidative stress-induced apoptosis by activation of SFKs. Exposure to oxidative stress such as UV irradiation induces the association of Ossa/C9orf10 with regulatory domains of SFKs, which activates these kinases and causes marked tyrosine phosphorylation of C9orf10 in turn. Tyrosine-phosphorylated Ossa recruits p85 subunits of phosphatidylinositol 3-kinase (PI3-kinase) and behaves as a scaffolding protein for PI3-kinase and SFKs, which activates the Akt-mediated antiapoptotic pathway. On the other hand, the carboxyl terminus of Ossa has a distinct function that directly binds RNAs such as insulin-like growth factor II (IGF-II) mRNA and promotes the extracellular secretion of IGF-II. Our findings indicate that Ossa is a dual-functional protein and might be a novel therapeutic target which modulates the sensitivity of tumors to oxidative stress.Tumor cells are exposed to oxidative stress in various situations in vivo. Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H₂O₂) are generated by exposure of cancer cells to hypoxia, followed by reperfusion; radiotherapy; photodynamic therapy; and some chemotherapeutic agents such as cisplatin (6, 25). This production of ROS generally induces apoptosis, whereas some tumor cells become resistant to this kind of apoptosis by some mechanism such as elevated expression of antioxidant thiols in the cells (17, 27).Src family kinases (SFKs) play important roles in various cell functions such as cell proliferation, cell adhesion, and cell migration (26), and the activities of SFKs often correlate with the malignant potential of cancer and a poor prognosis (37). Activation of c-Src is observed after the cells are exposed to oxidative stress (1, 9, 11, 34), and the activation of SFKs contributes to the resistance to apoptosis induced upon cellular stress. For instance, treatment of cells with oxidative stress such as UV irradiation or H₂O₂ causes apoptotic cell death, which is rescued by expression of v-Src (28). In our attempt to identify the key molecules that promote the expansion of gastric scirrhous carcinoma in vivo by mediating signals from activated SFKs, we identified an uncharacterized protein, C9orf10.C9orf10 (Homo sapiens chromosome 9 open reading frame 10) was originally found by the human genome sequence project as an annotated protein, and the gene was mapped to chromosome 9q22.31 (12). C9orf10 was recently detected within the Purα-containing mRNA-protein complex in the brain, although no functional information about this protein is available (14). We show that C9orf10 protects cells from apoptosis through activation of SFKs in response to oxidative stress. The kinase activity of SFKs is regulated by two intramolecular interactions. The inactive form is achieved by interaction of the SH2 domain with the phosphorylated C-terminal tail and association of the SH3 domain with a polyproline type II helix formed by the linker region between the SH2 domain and the catalytic domain (30). C9orf10 functions as a novel activator of SFKs that unfolds the inactive form of SFKs by association with both the SH2 and SH3 domains of SFKs. Tyrosine phosphorylation of C9orf10 is induced by the activated SFKs in turn, producing scaffolds to recruit phosphatidylinositol 3-kinase (PI3-kinase) and activate PI3-kinase-Akt signaling, which plays a key role in protecting cancer cells from oxidative stress-induced apoptosis. Therefore, we named C9orf10 Ossa (oxidative stress-associated Src activator).We also showed that the carboxyl terminus of Ossa directly binds to RNA, suggesting a distinct role for Ossa as an RNA-binding protein. As one of the target RNAs, Ossa directly binds to insulin-like growth factor II (IGF-II) mRNA, which subsequently enhances the extracellular secretion of IGF-II. Because an increase in IGF-II promotes cell proliferation, the RNA-binding function of Ossa may also contribute to the survival of cancer cells in vivo.Scirrhous gastric carcinoma diffusely infiltrates a broad region of the stomach and is frequently associated with metastasis to lymph nodes and peritoneal dissemination and therefore has the worst prognosis among the various types of gastric cancer (35). Blocking of the survival signaling mediated by Ossa, which sensitizes the cancer cells to stress-induced apoptosis, may be a novel therapeutic approach for gastric scirrhous carcinoma cells.