Critical function of death-associated protein 3 in T cell receptor-mediated apoptosis induction

Death-associated protein 3 (DAP3) is crucial for promoting apoptosis induced by various stimulations. This report demonstrates that DAP3 is also important for T cell receptor (TCR)-mediated apoptosis induction in immature thymocytes. Enforced expression of DAP3 accelerated the negative selection in developing thymocytes, using the reaggregate thymus organ culture system. In addition, expression of DAP3 accelerated TCR-mediated apoptosis induction in DO11.10 cells. We also demonstrated that DAP3 translocates into the nucleus during TCR-mediated apoptosis in a Nur77 dependent manner. It is concluded that DAP3 is critical for TCR-mediated induction of apoptosis at the downstream of Nur77.     

Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.     

Interaction of angiotensin II type 1 receptor blockers with P-gp substrates in Caco-2 cells and hMDR1-expressing membranes

AimsThe inhibitory effect of angiotensin II type 1 receptor blockers (ARBs) on P-glycoprotein (P-gp) was examined to evaluate their clinical drug–drug interaction (DDI) potential.Main methodsWe performed an inhibition study on the vectorial transport of digoxin, a typical substrate for P-gp, using a human colonic adenocarcinoma cell line, Caco-2 cells, and verapamil-stimulated ATPase activity using human multidrug resistance 1 (hMDR1)-expressing membrane.Key findingsThe vectorial transport of digoxin was inhibited by candesartan cilexetil, irbesartan and telmisartan with the IC₅₀ values of 14.7, 34.0 and 2.19 µM, respectively. Those values were 7.4–426-fold higher than their theoretical clinical gastrointestinal concentration [I] at doses in clinical DDI studies. Other ARBs failed to show interaction with P-gp.SignificanceIt was demonstrated that candesartan cilexetil, irbesartan and telmisartan had the potential to inhibit the transport of various drugs via P-gp. Telmisartan, which caused an increase in the serum digoxin concentration in humans, had a sufficiently high [I]/IC₅₀ value, suggesting that DDI between digoxin and telmisartan was caused by the inhibition of digoxin efflux via intestinal P-gp.     

Treatment with apolipoprotein A-1 mimetic peptide reduces lupus-like manifestations in a murine lupus model of accelerated atherosclerosis

Introduction

The purpose of this study was to evaluate the effects of L-4F, an apolipoprotein A-1 mimetic peptide, alone or with pravastatin, in apoE^-/-Fas^-/-C57BL/6 mice that spontaneously develop immunoglobulin G (IgG) autoantibodies, glomerulonephritis, osteopenia, and atherosclerotic lesions on a normal chow diet.

Methods

Female mice, starting at eight to nine weeks of age, were treated for 27 weeks with 1) pravastatin, 2) L-4F, 3) L-4F plus pravastatin, or 4) vehicle control, followed by disease phenotype assessment.

Results

In preliminary studies, dysfunctional, proinflammatory high-density lipoproteins (piHDL) were decreased six hours after a single L-4F, but not scrambled L-4F, injection in eight- to nine-week old mice. After 35 weeks, L-4F-treated mice, in the absence/presence of pravastatin, had significantly smaller lymph nodes and glomerular tufts (P_{L, LP} < 0.05), lower serum levels of IgG antibodies to double stranded DNA (dsDNA) (P_L < 0.05) and oxidized phospholipids (oxPLs) (P_{L, LP} < 0.005), and elevated total and vertebral bone mineral density (P_{L, LP} < 0.01) compared to vehicle controls. Although all treatment groups presented larger aortic root lesions compared to vehicle controls, enlarged atheromas in combination treatment mice had significantly less infiltrated CD68⁺ macrophages (P_LP < 0.01), significantly increased mean α-actin stained area (P_LP < 0.05), and significantly lower levels of circulating markers for atherosclerosis progression, CCL19 (P_{L, LP} < 0.0005) and VCAM-1 (P_L < 0.0002).

Conclusions

L-4F treatment, alone or with pravastatin, significantly reduced IgG anti-dsDNA and IgG anti-oxPLs, proteinuria, glomerulonephritis, and osteopenia in a murine lupus model of accelerated atherosclerosis. Despite enlarged aortic lesions, increased smooth muscle content, decreased macrophage infiltration, and decreased pro-atherogenic chemokines in L-4F plus pravastatin treated mice suggest protective mechanisms not only on lupus-like disease, but also on potential plaque remodeling in a murine model of systemic lupus erythematosus (SLE) and accelerated atherosclerosis.     

Piwi expression in archeocytes and choanocytes in demosponges: insights into the stem cell system in demosponges

SUMMARY Little is known about the stem cells of organisms early in metazoan evolution. To characterize the stem cell system in demosponges, we identified Piwi homologs of a freshwater sponge, Ephydatia fluviatilis, as candidate stem cell (archeocyte) markers. EfPiwiA mRNA was expressed in cells with archeocyte cell morphological features. We demonstrated that these EfPiwiA‐expressing cells were indeed stem cells by showing their ability to proliferate, as indicated by BrdU‐incorporation, and to differentiate, as indicated by the coexpression of EfPiwiA with cell‐lineage‐specific genes in presumptive committed archeocytes. EfPiwiA mRNA expression was maintained in mature choanocytes forming chambers, in contrast to the transition of gene expression from EfPiwiA to cell‐lineage‐specific markers during archeocyte differentiation into other cell types. Choanocytes are food‐entrapping cells with morphological features similar to those of choanoflagellates (microvillus collar and a flagellum). Their known abilities to transform into archeocytes under specific circumstances and to give rise to gametes (mostly sperm) indicate that even when they are fully differentiated, choanocytes maintain pluripotent stem cell‐like potential. Based on the specific expression of EfPiwiA in archeocytes and choanocytes, combined with previous studies, we propose that both archeocytes and choanocytes are components of the demosponge stem cell system. We discuss the possibility that choanocytes might represent the ancestral stem cells, whereas archeocytes might represent stem cells that further evolved in ancestral multicellular organisms.     

Effects of protein conformational changes on separation performance in electrostatic interaction chromatography: unfolded proteins and PEGylated proteins

As it is important to understand how protein conformational changes affect the separation performance in ion exchange chromatography (IEC), we investigated two model systems, unfolded proteins (lysozyme and bovine serum albumin) with urea and dithiothreitol, and PEGylated proteins (lysozyme attached with polyethyleneglycol molecular weight 5000). Linear gradient elution IEC experiments were carried out and the data were analysed by our model previously presented in order to obtain the binding site value B and the peak salt concentration I(R). Unfolded proteins (bovine serum albumin and lysozyme) with urea and dithiothreitol showed weaker retention and larger binding site values compared with the values for native proteins. Multiple PEGylated lysozyme peaks were separated, and eluted earlier than the native peak appeared. There is a good correlation between B and I(R) for PEGylated lysozymes.     

Rat organic solute carrier protein 1 (rOscp1) mediated the transport of organic solutes in Xenopus laevis oocytes: isolation and pharmacological characterization of rOscp1

Rat organic solute carrier protein 1 (rOscp1) was isolated from a rat testis cDNA library. Isolated rOscp1 cDNA consisted of 1089 base pairs that encoded a 363-amino acid protein, and the amino acid sequence was 88% and 93% identical to that of human OSCP1 (hOSCP1) and mouse Oscp1 (mOscp1), respectively. The message for rOscp1 is highly detected in rat testis. When expressed in X. oocytes, rOscp1 mediated the high affinity transport of p-aminohippurate (PAH) with a Km value of 15.7+/-1.9 microM, and rOscp1-mediated organic solutes were exhibited in time- and Na+-independent manners. rOscp1 also transported various structurally heterogenous compounds such as testosterone, dehydroepiandrosterone sulfate (DHEA-S), and taurocholate with some differences in substrate specificity compared with hOSCP1. Immunohistochemical analysis revealed that the rOscp1 protein is localized in the basal membrane side of Sertoli cells as observed in mouse testis [Kobayashi et al., 2007; Kobayashi, Y., Tsuchiya, A., Hayashi, T., Kohyama, N., Ohbayashi, M., Yamamoto, T., 2007. Isolation and characterization of polyspecific mouse organic solute carrier protein 1 (mOscp1). Drug Metabolism and Disposition 35 (7), 1239-1245]. Thus, the present results indicate that a newly isolated cDNA clone, rOscp1, is a polyspecific organic solute carrier protein with some differences in substrate specificity compared with human and mouse OSCP1.     

Structural insights into unique substrate selectivity of Thermoplasma acidophilum D-aldohexose dehydrogenase

The d-aldohexose dehydrogenase from the thermoacidophilic archaea Thermoplasma acidophilum (AldT) belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and catalyzes the oxidation of several monosaccharides with a preference for NAD⁺ rather than NADP⁺ as a cofactor. It has been found that AldT is a unique enzyme that exhibits the highest dehydrogenase activity against d-mannose. Here, we describe the crystal structures of AldT in ligand-free form, in complex with NADH, and in complex with the substrate d-mannose, at 2.1 Å, 1.65 Å, and 1.6 Å resolution, respectively. The AldT subunit forms a typical SDR fold with an unexpectedly long C-terminal tail and assembles into an intertwined tetramer. The d-mannose complex structure reveals that Glu84 interacts with the axial C2 hydroxyl group of the bound d-mannose. Structural comparison with Bacillus megaterium glucose dehydrogenase (BmGlcDH) suggests that the conformation of the glutamate side-chain is crucial for discrimination between d-mannose and its C2 epimer d-glucose, and the conformation of the glutamate side-chain depends on the spatial arrangement of nearby hydrophobic residues that do not directly interact with the substrate. Elucidation of the d-mannose recognition mechanism of AldT further provides structural insights into the unique substrate selectivity of AldT. Finally, we show that the extended C-terminal tail completely shuts the substrate-binding pocket of the neighboring subunit both in the presence and absence of substrate. The elaborate inter-subunit interactions between the C-terminal tail and the entrance of the substrate-binding pocket imply that the tail may play a pivotal role in the enzyme activity.     

Analysis of a nuclease activity of catalytic domain of Thermus thermophilus MutS2 by high-accuracy mass spectrometry

Electrospray ionization with Fourier-transform ion cyclotron resonance mass spectrometry (ESI–FT ICR MS) is a powerful tool for analyzing the precise structural features of biopolymers, including oligonucleotides. Here, we described the detailed characterization of a newly discovered nuclease activity of the C-terminal domain of Thermus thermophilus MutS2 (ttMutS2). Using this method, the length, nucleotide content and nature of the 5′- and 3′-termini of the product oligonucleotides were accurately identified. It is revealed that the C-terminal domain of ttMutS2 incised the phosphate backbone of oligodeoxynucleotides non-sequence-specifically at the 3′ side of the phosphates. The simultaneous identification of the innumerable fragments was achieved by the extremely high-accuracy of ESI–FT ICR MS.     

Modulation of the chemokines KC and MCP-1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice

Activation of the aryl hydrocarbon receptor (AhR) by TCDD may lead to the induction of proinflammatory cytokines in various cell types and organs such as liver leading to active chronic inflammation. Here we studied the expression of the chemokines keratinocyte chemoattractant (KC) and monocyte chemoattractant protein 1 (MCP-1) in different organs of mice after exposure to TCDD. TCDD exposure led to an early and clear induction of KC in liver and spleen on day 1 which was sustained over a period of 10 days. The level of MCP-1 mRNA was induced by TCDD on day 1 in spleen, lung, kidney, and liver, which was further increased at day 7. Increase of KC and MCP-1 at day 7 in liver, thymus, kidney, adipose, and heart was associated with elevated levels of the macrophage marker F4/80, indicating the infiltration of macrophages in these organs. Induction of KC requires a functional AhR since mice with a mutation in the AhR nuclear localization domain (AhR(nls)) were found to be resistant to TCDD-induced expression of KC. These results are the first showing the induction of the chemokines KC and MCP-1 in multiple organs of mice associated with an increase of the macrophage marker F4/80 indicating the involvement in TCDD's inflammatory response like infiltration of macrophages.