Critical role for CXC chemokine ligand 16 (SR-PSOX) in Th1 response mediated by NKT cells

The transmembrane chemokine CXCL 16 (CXCL16), which is the same molecule as the scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX), has been shown to mediate chemotaxis and adhesion of CXC chemokine receptor 6-expressing cells such as NKT and activated Th1 cells. We generated SR-PSOX/CXCL16-deficient mice and examined the role of this chemokine in vivo. The mutant mice showed a reduced number of liver NKT cells, and decreased production of IFN-gamma and IL-4 by administration of alpha-galactosylceramide (alphaGalCer). Of note, the alphaGalCer-induced production of IFN-gamma was more severely impaired than the production of IL-4 in SR-PSOX-deficient mice. In this context, SR-PSOX-deficient mice showed impaired sensitivity to alphaGalCer-induced anti-tumor effect mediated by IFN-gamma from NKT cells. NKT cells from wild-type mice showed impaired production of IFN-gamma, but not IL-4, after their culture with alphaGalCer and APCs from mutant mice. Moreover, Propionibacterium acnes-induced in vivo Th1 responses were severely impaired in SR-PSOX-deficient as well as NKT KO mice. Taken together, SR-PSOX/CXCL16 plays an important role in not only the production of IFN-gamma by NKT cells, but also promotion of Th1-inclined immune responses mediated by NKT cells.     

Structural insights into thermostabilization of leucine dehydrogenase from its atomic structure by cryo-electron microscopy

Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD⁺-dependent oxidoreductase that catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH of Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has been applied for the quantification or production of BCAAs. Here the cryo-electron microscopy (cryo-EM) structures of apo and NAD⁺-bound LDH are reported at 3.0 and 3.2?Å resolution, respectively. On comparing the structures, the two overall structures are almost identical, but it was observed that the partial conformational change was triggered by the interaction between Ser147 and the nicotinamide moiety of NAD⁺. NAD⁺ binding also enhanced the strength of oligomerization interfaces formed by the core domains. Such additional interdomain interaction is in good agreement with our experimental results showing that the residual activity of NAD⁺-bound form was approximately three times higher than that of the apo form after incubation at 80?°C. In addition, sequence comparison of three structurally known LDHs indicated a set of candidates for site-directed mutagenesis to improve thermostability. Subsequent mutation analysis actually revealed that non-conserved residues, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric thermostability.     

Replication of IMR‐32‐adapted JC virus clones in human embryonic kidney cells

It has been difficult to study JCV replication because of its restricted host range. In this study, JCV replication was examined using different clones in 293 cells. RT‐PCR assay revealed that large T antigen expression in cells transfected with IMR‐32‐adapted JCVs was significantly greater than in those transfected with Mad‐1 or CY. DNA replication assay and viral load verified that the IMR‐32‐adapted JCVs were replication‐competent in 293 cells, but not Mad‐1 or CY JCVs. These results suggest that a 293 culture system with IMR‐32‐adapted JCVs may be a useful tool for assessing replication of JCV in vitro.     

Direct Optical Microscopic Observation of the Microtubule Polymerization Intermediate Sheet Structure in the Presence of Gas7

The process of microtubule elongation is thought to consist of two stages—formation of a tubulin sheet structure and its closure into a tube. However, real-time observation of this process has been difficult. Here, by utilizing phospho-tau binding protein Gas7 (growth-arrest-specific protein 7), we visualized the polymer transformation process by dark-field microscopy. Upon elongation, thin and flexible structures, often similar to a curved hook, appeared at the end of microtubules. Electron microscopic observations supported the idea that these flexible structures are tubulin sheets. They maintained their length until they gradually became thick and rigid beginning in the central portion, resulting in straight microtubules. In the absence of Gas7, the sheet-like structure was rarely observed; moreover, when observed, it was fragile and engaged in typical dynamic instability. With Gas7, no catastrophe was observed. These results suggest that Gas7 enhances microtubule polymerization by stabilizing sheet intermediates and is a useful tool for analyzing microtubule transformation.     

Archetype JC virus efficiently propagates in kidney-derived cells stably expressing HIV-1 Tat

Pathogenic JCV with rearranged regulatory regions (PML-type) causes PML, a demyelinating disease, in the brains of immunocompromised patients. On the other hand, archetype JCV persistently infecting the kidney is thought to be converted to PML-type virus during JCV replication in the infected host under immunosuppressed conditions. In addition, Tat protein, encoded by HIV-1, markedly enhances the expression of a reporter gene under control of the JCV late promoter.
In order to examine the influence of Tat on JCV propagation, we used kidney-derived COS-7 cells, which only permit archetype JCV, and established COS-tat cells, which express HIV-1 Tat stably. We found that the extent of archetype JCV propagation in COS-tat cells is significantly greater than in COS-7 cells. On the other hand, COS-7 cells express SV40 T antigen, which is a strong stimulator of archetype JCV replication. The expression of SV40 T antigen was enhanced by HIV-1 Tat slightly according to real-time RT-PCR, this was not closely related to JCV replication in COS-tat cells. The efficiency of JCV propagation depended on the extent of expression of functional Tat. To our knowledge, this is the first report of increased production of archetype JCV in a culture system using cell lines stably expressing HIV-1 Tat. We propose here that COS-tat cells are a useful tool for studying the role of Tat in archetype JCV replication in the development of PML.     

Evaluation of an oral function promotion programme for the independent elderly in Japan

Objectives: The purpose of this study was to provide an oral function promotion programme for the independent elderly and evaluate the changes in oral health status and oral function. Background: Few studies have scientifically analysed and evaluated the effectiveness of oral function promotion programmes provided for the independent elderly. Materials and methods: The subjects were independent elderly females (mean age: 74.6 ± 6.3) recruited from senior citizens’ centres in Tokyo. The intervention group (n = 79) received a 3‐month oral function promotion programme, which included facial muscle and tongue exercises and salivary gland massages. The control group (n = 62) did not receive this programme. Results: In the intervention group, the tongue coating scores decreased and the organoleptic score of oral malodour fell. The amount of food debris in the oral cavity decreased and the tongue dryness improved. Furthermore, the salivary flow rate increased. The length of time for maintaining the tongue in the forward position increased from 11.2 s to 18.7 s, and the number of times for moving the tip of the tongue in a clockwise circular motion, counter‐clockwise circular motion and side‐to‐side motion within 30 s, increased from 14.5 to 20.6, 14.5 to 20.2, and 17.2 to 23.3 respectively. The number of times for movement of the lips significantly improved from 23.0 to 28.8 and the pronunciation of words was observed to be clearer. Conclusion: An oral function promotion programme was effective in improving the oral health status and oral function of an independent elderly population.     

Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes

Introduction

Osteoclastogenesis plays an important role in the bone erosion of rheumatoid arthritis (RA). Recently, Notch receptors have been implicated in the development of osteoclasts. However, the responsible Notch ligands have not been identified yet. This study was undertaken to determine the role of individual Notch receptors and ligands in osteoclastogenesis.

Methods

Mouse bone marrow-derived macrophages or human peripheral blood monocytes were used as osteoclast precursors and cultured with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) to induce osteoclasts. Osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining. K/BxN serum-induced arthritic mice and ovariectomized mice were treated with anti-mouse Delta-like 1 (Dll1) blocking monoclonal antibody (mAb).

Results

Blockade of a Notch ligand Dll1 with mAb inhibited osteoclastogenesis and, conversely, immobilized Dll1-Fc fusion protein enhanced it in both mice and humans. In contrast, blockade of a Notch ligand Jagged1 enhanced osteoclastogenesis and immobilized Jagged1-Fc suppressed it. Enhancement of osteoclastogenesis by agonistic anti-Notch2 mAb suggested that Dll1 promoted osteoclastogenesis via Notch2, while suppression by agonistic anti-Notch1 mAb suggested that Jagged1 suppressed osteoclastogenesis via Notch1. Inhibition of Notch signaling by a gamma-secretase inhibitor suppressed osteoclastogenesis, implying that Notch2/Dll1-mediated enhancement was dominant. Actually, blockade of Dll1 ameliorated arthritis induced by K/BxN serum transfer, reduced the number of osteoclasts in the affected joints and suppressed ovariectomy-induced bone loss.

Conclusions

The differential regulation of osteoclastogenesis by Notch2/Dll1 and Notch1/Jagged1 axes may be a novel target for amelioration of bone erosion in RA patients.