Effect of coix on plasma, liver, and fecal lipid components in the rat fed on lard- or soybean oil-cholesterol diet

To determine the influence of dietary coix on lipid metabolism, the effect of coix on plasma, liver, and fecal lipid components was studied using Sprague-Dawley male rats. All rats were divided into four groups, and the rats of each group were fed the coix-lard diet, coix-soybean oil diet, or the respective control diets (containing 1% cholesterol each) for 27 days. Plasma and liver cholesterol levels in the coix-lard diet group significantly decreased as compared with those in the control group, whereas there was no effect on the fecal excretion of cholesterol. The decreases in the concentrated liver triglyceride and the increases in the fecal excretion of triglyceride were found in coix-soybean oil diet group. Moreover, liver and fecal phospholipid levels in both coix diet groups significantly increased. But there were no significant changes in plasma and fecal bile acids in either coix diet group. These results suggest the possibilities that coix may have an inhibitory action on cholesterol synthesis in liver, a facilitating effect on biliary excretion of triglyceride, and an acceleratory action on phospholipid synthesis in liver.     

Demonstration of endothelin (ET) receptors on cultured rabbit chondrocytes and stimulation of DNA synthesis and calcium influx by ET-1 via its receptors

Endothelin (ET) receptors on chondrocytes were demonstrated using cultured rabbit costal chondrocytes. After crosslinking the receptors on the cells with ¹²⁵ I-ET-1, two major bands of 43 kDa and 46 kDa were separated by SDS-PAGE. Scatchard analysis demonstrated two classes of ET receptors with K_d values of 1 × 10^?10 M and 5 × 10^?9 M. The numbers of high- and low- affinity receptors were 1 × 10⁴ and 2 × 10⁵ per cell, respectively. The binding of ET-1 to chondrocytes was increased by treatment with PTH, DBcAMP, TGF-β1, IL-1β, RA and EGF. ET-1 stimulated DNA synthesis in cultured rabbit chondrocytes. ET-1 also stimulated calcium incorporation through the cell membrane of chondrocytes. These findings indicate that ET-1 has a physiological effect on chondrocytes via its receptors on the cells.     

Molecular monitoring of bleomycin-induced pulmonary fibrosis by cDNA microarray-based gene expression profiling.

Pulmonary fibrosis is a progressive disorder whose molecular pathology is poorly understood. Here we developed an in-house cDNA microarray ("lung chip") originating from a lung-normalized cDNA library. By using this lung chip, we analyzed global gene expression in a murine model of bleomycin-induced fibrosis and selected 82 genes that differed by more than twofold intensity in at least one pairwise comparison with controls. Cluster analysis of these selected genes showed that the expression of genes associated with inflammation reached maximum levels at 5 days after bleomycin administration, while genes involved in the development of fibrosis increased gradually up to 14 days after bleomycin treatment. These changes in gene expression signature were well correlated with observed histopathological changes. The results show that microarray analysis of animal disease models is a powerful approach to understanding the gene expression programs that underlie these disorders.     

Apoptosis induction by epigallocatechin gallate involves its binding to Fas

Epigallocatechin gallate (EGCG) is known to induce apoptosis in various types of tumor cells, but the precise mechanism by which EGCG induces apoptosis remains to be elucidated. The Fas-Fas ligand system is one of the major pathways operating in the apoptotic cascade. The aim of this study was to examine the possibility that EGCG-binding to Fas triggers the Fas-mediated apoptosis. The EGCG treatment of human monocytic leukemia U937 cells resulted in elevation of caspase 8 activity and fragmentation of caspase 8. The DNA ladder formation caused by the EGCG treatment was inhibited by the caspase 8 inhibitor. These findings suggested the involvement of the Fas-mediated cascade in the EGCG-induced apoptosis in U937 cells. Affinity chromatography revealed the binding between EGCG and Fas. Thus, the results suggest that EGCG-binding to Fas, presumably on the cell surface, triggers the Fas-mediated apoptosis in U937 cells.     

Mouse coq7/clk-1 orthologue rescued slowed rhythmic behavior and extended life span of clk-1 longevity mutant in Caenorhabditis elegans

The coq7/clk-1 gene was isolated from the long-lived mutant of Caenorhabditis elegans and was suggested to play a regulatory role in biological rhythm and longevity. The mouse COQ7 is homologous to Saccharomyces cerevisiae COQ7/CAT5 that is required for the biosynthesis of coenzyme Q (ubiquinone), an essential messenger in mitochondrial respiration. In the present study, we characterized the expression and processing of mouse COQ7. We found that COQ7 is highly expressed in tissues with high energy demand such as heart, muscle, liver, and kidney in mice. Biochemical analysis revealed that COQ7 is targeted to mitochondria where it is processed to mature form. Transgenic expression of mouse coq7 completely rescued the slowed rhythmic behaviors of clk-1 such as defecation. In life-span analysis, transgenic expression reverted the extended life span of clk-1 to the comparable level with wild-type control. These data strongly suggested that coq7 plays a pivotal role in the regulation of biological rhythms and the determination of life span in mammalian species.     

Selective isolation and distribution of the genus Planomonospora in soils

For the screening of bioactive compounds and study of global distribution, a selective isolation method for Planomonospora strains by centrifugation from soil is examined. Planomonospora strains produced characteristic sporangia on the humic acid-trace salts gellan gum medium (pH 9.0) so that this genus was readily recognized on the isolation plate. High yields of motile spores were obtained by using a flooding solution containing 0.1% skim milk in 5 mM N-cyclohexyl-2-amino-ethanesulfonic acid (pH 9.0) followed by incubating the preparation at 32 degreesC for 90 min, centrifuging it at 1000 x g for 10 min, and further incubation at 32 degreesC for 60 min after centrifugation. By combining the techniques described above, we isolated 246 Planomonospora strains from 137 of the 1200 soil samples examined. Ninety-four percent of these strains were recovered from neutral to slightly alkaline soils (pH 7.0 to 9.0). Strains of P. venezuelensis group were obtained from 13 soil samples (1.1%), which were collected in Bolivia, Cyprus, Egypt, Greece, India, Japan, New Caledonia, and Turkey. Strains of this group appear widely distributed in the soil of tropical to temperate regions. To our knowledge, this is the first record that strains of this group have been isolated from a location other than Venezuela.     

X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3 and -7 in distinct modes

The inhibitor of apoptosis proteins (IAP) regulates cell death by inhibiting caspases. The region of X-linked (X) IAP containing the second baculovirus IAP repeat domain (BIR2) is sufficient for inhibiting caspase-3 and -7. In this study, we found that the modes of inhibition of these two caspases were different: caspase-3 is inhibited in a competitive manner whereas caspase-7 inhibition occurs through a mixed competitive and noncompetitive mechanism. Binding assays revealed that the inhibition of caspase-3 by XIAP was totally dependent on the interaction between the active site of caspase-3 and the linker region between the BIR1 and BIR2 domains of XIAP. In contrast, the active site and the NH(2)-terminal region of caspase-7 bound to the linker region and the BIR2, respectively. Moreover the BIR2 with a mutated linker region, which inhibited caspase-3 very weakly, still bound to and inhibited caspase-7. Furthermore, a chimeric caspase-7/3 comprising the NH(2)-terminal portion of caspase-7 and COOH-terminal portion of caspase-3 was inhibited by XIAP by a mixed competitive and noncompetitive mechanism. Our results suggest that the linker region between BIR1 and BIR2 domains is responsible for active site-directed, competitive inhibition of both caspase-3 and -7, whereas the BIR2 itself is involved in noncompetitive inhibition of caspase-7.     

Human intelectin is a novel soluble lectin that recognizes galactofuranose in carbohydrate chains of bacterial cell wall

Galactofuranosyl residues are present in various microorganisms but not in mammals. In this study, we identified a human lectin binding to galactofuranosyl residues and named this protein human intelectin (hIntL). The mature hIntL was a secretory glycoprotein consisting of 295 amino acids and N-linked oligosaccharides, and its basic structural unit was a 120-kDa homotrimer in which 40-kDa polypeptides were bridged by disulfide bonds. The hIntL gene was split into 8 exons on chromosome 1q21.3, and hIntL mRNA was expressed in the heart, small intestine, colon, and thymus. hIntL showed high levels of homology with mouse intelectin, Xenopus laevis cortical granule lectin/oocyte lectin, lamprey serum lectin, and ascidian galactose-specific lectin. These homologues commonly contained no carbohydrate recognition domain, which is a characteristic of C-type lectins, although some of them have been reported as Ca(2+)-dependent lectins. Recombinant hIntL revealed affinities to d-pentoses and a d-galactofuranosyl residue in the presence of Ca(2+), and recognized the bacterial arabinogalactan of Nocardia containing d-galactofuranosyl residues. These results suggested that hIntL is a new type lectin recognizing galactofuranose, and that hIntL plays a role in the recognition of bacteria-specific components in the host.     

O-helix mutant T664P of Thermus aquaticus DNA polymerase I: altered catalytic properties for incorporation of incorrect nucleotides but not correct nucleotides

Previous studies indicate that the O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) plays an important role in the replication fidelity of the enzyme. This study examines the role of Thr-664, which lies in the middle of the O-helix of Taq pol I. A mutant of Taq Pol I with a proline substitution of Thr-664 (T664P) exhibits much lower replication fidelity than the wild type enzyme in a forward mutation assay. T664P produces base substitution, single-base deletion, and single-base insertion errors at 20-, 5, and 50-fold higher rates than wild type, respectively. In specific activity and steady-state kinetic experiments, T664P was catalytically robust for insertion of correct nucleotides. In contrast, it incorporated incorrect nucleotides 6.1- to 10-fold more efficiently than wild type at a template dC. Mismatched primer termini were extended by T664P 4.2- to 9.5-fold more efficiently than wild type. These data imply that the O-helix with a proline at position 664 functions like wild type Taq pol I for correct nucleotide incorporations, but bends and enlarges the catalytic pocket of the enzyme and increases the rate of nucleotide misincorporation.