An unusual lanostane-type triterpenoid,spiroinonotsuoxodiol, and other triterpenoids from Inonotus obliquus

An unusual lanostane-type triterpenoid, spiroinonotsuoxodiol (1), and two lanostane-type triterpenoids, inonotsudiol A (2) and inonotsuoxodiol A (3), were isolated from the sclerotia of Inonotus obliquus. Their structures were determined to be (3S,7S,9R)-3,7-dihydroxy-7(8 → 9)abeo-lanost-24-en-8-one (1), lanosta-8,24-dien-3β,11β-diol (2), and (22R)-3β,22-dihydroxylanosta-8,24-dien-11-one (3) on the basis of NMR spectroscopy, including 1D and 2D (¹H–¹H COSY, NOESY, HMQC, HMBC) NMR, and FABMS. Compounds 1–3 showed moderate activity against cultured P388, L1210, HL-60 and KB cells.     

Alpha-tocopherol induces calnexin in renal tubular cells: another protective mechanism against free radical-induced cellular damage

Pre-administration of alpha-tocopherol is protective against oxidative renal tubular damage and subsequent carcinogenesis by ferric nitrilotriacetate (Fe-NTA) in rats. We searched for mechanisms other than the scavenging effect of alpha-tocopherol with microarray analyses, which implicated calnexin, a chaperone for glycoproteins. Renal mRNA levels of calnexin significantly increased 3h after an injection of Fe-NTA in rats fed a standard diet whereas those fed an alpha-tocopherol-supplemented diet showed an increase prior to injection, but after injection showed a decrease in renal calnexin mRNA levels, with unaltered protein levels. In experiments using LLC-PK1 cells, addition of alpha-tocopherol was protective against oxidative stress by H2O2, concomitant with calnexin induction. Knockdown of calnexin by siRNA significantly reduced this protection. Furthermore, COS-7 cells transfected with the calnexin gene were more resistant to H2O2. Together with the fact that alpha-tocopherol induced N-acetylglucosaminyltransferase 3, our data suggest that alpha-tocopherol modifies glycoprotein metabolism partially by conferring mild ER stress. This adds another molecular mechanism of alpha-tocopherol toward cancer prevention.     

Osteogenic differentiation of human dental papilla mesenchymal cells

We isolated dental papilla from impacted human molar and proliferated adherent fibroblastic cells after collagenase treatment of the papilla. The cells were negative for hematopoietic markers but positive for CD29, CD44, CD90, CD105, and CD166. When the cells were further cultured in the presence of beta-glycerophosphate, ascorbic acid, and dexamethasone for 14 days, mineralized areas together with osteogenic differentiation evidenced by high alkaline phosphatase activity and osteocalcin contents were observed. The differentiation was confirmed at both protein and gene expression levels. The cells can also be cryopreserved and, after thawing, could show in vivo bone-forming capability. These results indicate that mesenchymal type cells localize in dental papilla and that the cells can be culture expanded/utilized for bone tissue engineering.     

Differential analysis of D-beta-Asp-containing proteins found in normal and infrared irradiated rabbit lens

Although proteins are generally composed of l-alpha-amino acids, d-beta-aspartic acid (Asp)-containing proteins have been reported in various elderly tissues. Our previous study detected several d-beta-Asp-containing proteins in a rabbit lens derived from epithelial cell line by Western blot analysis of a 2D-gel using a polyclonal antibody that is highly specific for d-beta-Asp-containing proteins. The identity of each spot was subsequently determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Ms-Fit online database searching algorithm. In this study, we discovered novel d-beta-Asp-containing proteins from rabbit lens. The results indicate that beta-crystallin A3, beta-crystallin A4, beta-crystallin B1, beta-crystallin B2, beta-crystallin B3, gamma-crystallin C, gamma-crystallin D, and lambda-crystallin in rabbit lens contain d-beta-Asp residues. Furthermore, the occurrence of d-beta-Asp residues increases with infrared ray (IR) irradiation. Additionally, some d-beta-Asp-containing proteins only appear after IR irradiation. One such protein is the alpha-enolase, which shows homology to tau-crystallin.     

Effects of copper metabolism on neurological functions in Wistar and Wilson's disease model rats

Behavioral functions of Wistar and Long-Evans Cinnamon (LEC) rats, Wilson's disease animal model, were compared by measuring the open-field, acoustic startle reflex and prepulse inhibition (PPI), and shuttle-box avoidance learning tests with or without oral supplementation with copper or D-penicillamine, copper chelator. All of the LEC rats, irrespective of the treatment, exhibited higher locomotor activity, a decreased habituation to startle response or a lower PPI, compared with Wistar rats. The copper content of all brain regions examined, except for the medulla oblongata of LEC rats, was significantly lower than those in Wistar rats. Besides, in the region of the striatum and the nucleus accumbens of the LEC rats, lower content of norepinephrine, and higher content of dopamine and serotonin were observed compared with Wistar rats. Although copper supplementation did not affect the brain copper content, it reduced the PPI in both Wistar and LEC rats. In contrast, D-penicillamine supplementation decreased both the brain copper content and locomotor activity, and enhanced the startle amplitude only in Wistar rats. These findings suggest that an imbalance in copper homeostasis affects monoamine metabolism and behavioral functions.     

Expression and function of the B and T lymphocyte attenuator (BTLA/CD272) on human T cells

Co-signal receptors provide crucial activating or attenuating signals for T cells. The B and T lymphocyte attenuator (BTLA/CD272) is a third member of co-inhibitory receptors, which belongs to the CD28 immunoglobulin-superfamily. Using monoclonal antibodies (mAbs) against human BTLA, we show that BTLA is constitutively expressed on most CD4+ and CD8+ T cells and its expression progressively decreases upon T cell activation. Polarized Th1 and Th2 cells contained both BTLA-positive and BTLA-negative populations, but the extended culture diminished BTLA expression. Cross-linking BTLA with an agonistic mAb inhibited T cell proliferation and the production of the cytokines IFN-gamma and IL-10 in response to anti-CD3 stimulation. BTLA-mediated inhibition of T cell activation occurred during both primary CD4+ T cell responses and secondary CD4+ and CD8+ T cell responses, suggesting that BTLA ligation sends a constitutive "off" signal to T cells and thus might play an important role in the maintenance of T cell tolerance.     

Diagnostic surface expression of SWAP-70 on HIV-1 infected T cells

Following immunization with HIV-1 infected cells, a hybridoma cell line termed 9F11 was established from the P3U-1 myeloma line fused with lymphocytes from a trans-chromosome (TC) mouse, that harbors human chromosomes containing immunoglobulin genes. The 9F11 human IgM monoclonal antibody (9F11 Ab) reacts with HIV-1 infected MOLT4 cells but not with uninfected MOLT4 cells, and causes immune cytolysis with homologous human complement at a concentration as low as 0.4 microg/ml. This Ab was used to perform immunoscreening of a cDNA expression library derived from HIV-1 infected cells. All positive cDNA clones contained SWAP-70 cDNA. SWAP-70 RNA and protein expression are much stronger in HIV-1 infected cells. SWAP-70 was also detected on the surface of HIV-1 infected cells by flow cytometric analysis. The monocyte cell line U937 cells expresses SWAP-70 on its cell surface regardless of whether it was infected with HIV-1. Furthermore, among PBMCs surface expression of SWAP-70 was detected on CD21+, CD56+ and CD14+ cells. Although CD3+ cells scarcely express SWAP-70 on their surface, once activated, they become positive. SWAP-70 may therefore serve as a marker for T cell differentiation as well as for HIV-1 infection.     

Altered glucose homeostasis in mice lacking the receptor protein tyrosine phosphatase sigma

Several protein tyrosine phosphatases (PTPs) expressed in insulin sensitive-tissues are proposed to attenuate insulin action and could act as key regulators of the insulin receptor (IR) signaling pathway. Among these PTPs, RPTPsigma is expressed in relatively high levels in insulin-target tissues. We show that RPTPsigma-/- knockout mice have reduced plasma glucose and insulin concentrations in the fasted state compared with their wild-type siblings. The knockout animals were also more sensitive to exogenous insulin as assayed by insulin-tolerance tests. Despite increased whole-body insulin sensitivity, tyrosine phosphorylation of the IR was not increased in muscle of RPTPsigma-/- animals, as would be expected in insulin-sensitive animals. Instead, the levels of IR tyrosine phosphorylation and PI3-kinase activity were reduced in the muscle of knockout animals stimulated with insulin in vivo. However, insulin-stimulated Akt serine phosphorylation was essentially identical between both groups of mice. Accordingly, muscles isolated from RPTPsigma-/- mice did not have a significant increase in glucose uptake in response to insulin, suggesting that RPTPsigma did not play a direct role in this process. Taken together, our results suggest an indirect modulation of the IR signaling pathways by RPTPsigma. Since low dose injection of growth hormone (GH) normalized the response to exogenous insulin in RPTPsigma-/- mice, we propose that the insulin hypersensitivity observed in RPTPsigma-/- mice is secondary to their neuroendocrine dysplasia and GH/IGF-1 deficiency.     

Phosphorylation and up-regulation of diacylglycerol kinase gamma via its interaction with protein kinase C gamma

Diacylglycerol (DAG) acts as an allosteric activator of protein kinase C (PKC) and is converted to phosphatidic acid by DAG kinase (DGK). Therefore, DGK is thought to be a negative regulator of PKC activation. Here we show molecular mechanisms of functional coupling of the two kinases. gammaPKC directly associated with DGKgamma through its accessory domain (AD), depending on Ca2+ as well as phosphatidylserine/diolein in vitro. Mass spectrometric analysis and mutation studies revealed that gammaPKC phosphorylated Ser-776 and Ser-779 in the AD of DGKgamma. The phosphorylation by gammaPKC resulted in activation of DGKgamma because a DGKgamma mutant in which Ser-776 and Ser-779 were substituted with glutamic acid to mimic phosphorylation exhibited significantly higher activity compared with wild type DGKgamma and an unphosphorylatable DGKgamma mutant. Importantly, the interaction of the two kinases and the phosphorylation of DGKgamma by gammaPKC could be confirmed in vivo, and overexpression of the AD of DGKgamma inhibited re-translocation of gammaPKC. These results demonstrate that localization and activation of the functionally correlated kinases, gammaPKC and DGKgamma, are spatio-temporally orchestrated by their direct association and phosphorylation, contributing to subtype-specific regulation of DGKgamma and DAG signaling.