Absorption and plasma kinetics of collagen tripeptide after peroral or intraperitoneal administration in rats

Collagen tripeptide (CTP) is a collagen-derived compound containing a high concentration of tripeptides with a Gly-X-Y sequence. In this study, the concentrations and metabolites of CTP were monitored in rat plasma after its administration. We performed a quantitative analysis using high-performance liquid chromatography tandem mass spectrometry according to the isotopic dilution method with stable isotopes. We confirmed that the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp were transported into the plasma. Dipeptides, which are generated by degradation of the N- or C-terminus of the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp, were also present in plasma. The plasma kinetics for peroral and intraperitoneal administration was similar. In addition, tripeptides and dipeptides were detected in no-administration rat blood. The pharmacokinetics were monitored in rats perorally administered with Gly-[³H]Pro-Hyp. Furthermore, CTP was incorporated into tissues including skin, bone, and joint tissue. Thus, administering collagen as tripeptides enables efficient absorption of tripeptides and dipeptides.     

Properties of lead deposits in cell walls of radish (Raphanus sativus) roots

Various mechanisms are involved in detoxification of heavy metals such as lead (Pb) in plant cells. Most of the Pb taken up by plants accumulates in their roots. However, the detailed properties of Pb complexes in roots remain unclear. We have investigated the properties of Pb deposits in root cell walls of radish (Raphanus sativus L.) seedlings grown on glass beads bed containing Pb pellets, which are the source of Pb-contamination in shooting range soils. Pb deposits were tightly bound to cell walls. Cell wall fragments containing about 50,000 ppm Pb were prepared from the roots. After extracting Pb from the cell wall fragments using HCl, Pb ions were recombined with the Pb-extracted cell wall fragments in a solution containing Pb acetate. When the cell wall fragments were treated with pectinase (E.C. 3.2.1.15) and were chemically modified with 1-ethyl-3-dimethylamino-propylcarboimide, the Pb-rebinding ability of the treated cell wall fragments decreased. When acid-treated cell wall fragments were incubated in a solution containing Pb²⁺ and excess amounts of a chelating agent, Pb recombined with the cell wall fragments were measured to estimate the affinity between Pb²⁺ and the cell wall fragments. Our data show that Pb²⁺ binds to carboxyl groups of cell walls. The source of the carboxyl groups is suggested to be pectic compounds. A stability constant of the Pb-cell wall complex was estimated to be about 10⁸. The role of root cell walls in the mechanism underlying heavy metal tolerance was discussed.     

Met Is the Most Frequently Amplified Gene in Endometriosis-Associated Ovarian Clear Cell Adenocarcinoma and Correlates with Worsened Prognosis

Clear cell adenocarcinoma of the ovary (OCC) is a chemo-resistant tumor with a relatively poor prognosis and is frequently associated with endometriosis. Although it is assumed that oxidative stress plays some role in the malignant transformation of this tumor, the characteristic molecular events leading to carcinogenesis remain unknown. In this study, an array-based comparative genomic hybridization (CGH) analysis revealed Met gene amplification in 4/13 OCC primary tumors and 2/8 OCC cell lines. Amplification of the AKT2 gene, which is a downstream component of the Met/PI3K signaling pathway, was also observed in 5/21 samples by array-based CGH analysis. In one patient, both the Met and AKT2 genes were amplified. These findings were confirmed using fluorescence in situ hybridization, real-time quantitative PCR, immunoblotting, and immunohistochemistry. In total, 73 OCC cases were evaluated using real-time quantitative PCR; 37.0% demonstrated Met gene amplification (>4 copies), and 8.2% had AKT2 amplification. Furthermore, stage 1 and 2 patients with Met gene amplification had significantly worse survival than patients without Met gene amplification (p<0.05). Met knockdown by shRNA resulted in reduced viability of OCC cells with Met amplification due to increased apoptosis and cellular senescence, suggesting that the Met signaling pathway plays an important role in OCC carcinogenesis. Thus, we believe that targeted inhibition of the Met pathway may be a promising treatment for OCC.     

The crystal structure of the amidohydrolase VinJ shows a unique hydrophobic tunnel for its interaction with polyketide substrates

VinJ is an amidohydrolase belonging to the serine peptidase family that catalyzes the hydrolysis of the terminal aminoacyl moiety of a polyketide intermediate during the biosynthesis of vicenistatin. Herein, we report the crystal structure of VinJ. VinJ possesses a unique hydrophobic tunnel for the recognition of the polyketide chain moiety of its substrate in the cap domain. Taken together with the results of phylogenetic analysis, our results suggest that VinJ represents a new amidohydrolase family that is different from the known α/β hydrolase type serine peptidases.     

OX40 ligand regulates splenic CD8 dendritic cell-induced Th2 responses in vivo

In mice, splenic conventional dendritic cells (cDCs) can be separated, based on their expression of CD8α into CD8⁻ and CD8⁺ cDCs. Although previous experiments demonstrated that injection of antigen (Ag)-pulsed CD8⁻ cDCs into mice induced CD4 T cell differentiation toward Th2 cells, the mechanism involved is unclear. In the current study, we investigated whether OX40 ligand (OX40L) on CD8⁻ cDCs contributes to the induction of Th2 responses by Ag-pulsed CD8⁻ cDCs in vivo, because OX40–OX40L interactions may play a preferential role in Th2 cell development. When unseparated Ag-pulsed OX40L-deficient cDCs were injected into syngeneic BALB/c mice, Th2 cytokine (IL-4, IL-5, and IL-10) production in lymph node cells was significantly reduced. Splenic cDCs were separated to CD8⁻ and CD8⁺ cDCs. OX40L expression was not observed on freshly isolated CD8⁻ cDCs, but was induced by anti-CD40 mAb stimulation for 24 h. Administration of neutralizing anti-OX40L mAb significantly inhibited IL-4, IL-5, and IL-10 production induced by Ag-pulsed CD8⁻ cDC injection. Moreover, administration of anti-OX40L mAb with Ag-pulsed CD8⁻ cDCs during a secondary response also significantly inhibited Th2 cytokine production. Thus, OX40L on CD8⁻ cDCs physiologically contributes to the development of Th2 cells and secondary Th2 responses induced by Ag-pulsed CD8⁻ cDCs in vivo.     

Functional dissection of the N-terminal sequence of Clostridium sp. G0005 glucoamylase: identification of components critical for folding the catalytic domain and for constructing the active site structure

Applied Microbiology and Biotechnology - Clostridium sp. G0005 glucoamylase (CGA) is composed of a β-sandwich domain (BD), a linker, and a catalytic domain (CD). In the present study, CGA was...     

Interaction of monocytes with vascular endothelial cells synergistically induces interferon gamma-inducible protein 10 expression through activation of specific cell surface molecules and cytokines

To further understand the regulatory mechanisms involved in the process of angiogenesis, the present study was designed to determine the expression and regulation of interferon gamma-inducible protein 10 (IP-10) in peripheral blood monocytes and human umbilical vein endothelial cells (HUVECs). We found that the interaction of monocytes with HUVECs resulted in synergistic increases in IP-10 expression and secretion, which consequently inhibited endothelial tube formation in vitro. Induction of IP-10 was mediated via specific cell surface molecules, as indicated by the finding that IP-10 secretion was significantly inhibited by anti-CD40 ligand antibody, and to a lesser extent by anti-CD40 antibody. Furthermore, we examined the effects of soluble mediators, such as inflammatory and immune cytokines on IP-10 secretion. Addition of interleukin (IL)-1, as well as interferon gamma, induced a marked augmentation of IP-10 secretion by unstimulated monocytes, unstimulated HUVECs, and co-cultures of the two cell types. In contrast, IL-10, recognized as an anti-inflammatory cytokine, significantly inhibited IP-10 secretion by co-cultures. Our results suggest that the interaction of monocytes with endothelial cells results in synergistic increases in IP-10 expression and secretion, which contribute to the regulation of angiogenesis and initiation of inflammatory vascular diseases.     

Apical transport of influenza A virus ribonucleoprotein requires Rab11-positive recycling endosome

Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs). Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM). However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE) in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD) of Rab11 family interacting proteins (Rab11-FIPs). Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking.