Sphingosylphosphorylcholine induces stress fiber formation via activation of Fyn-RhoA-ROCK signaling pathway in fibroblasts

Sphingosylphosphorylcholine (SPC), a bioactive sphingolipid, has recently been reported to modulate actin cytoskeleton rearrangement. We have previously demonstrated Fyn tyrosine kinase is involved in SPC-induced actin stress fiber formation in fibroblasts. However, Fyn-dependent signaling pathway remains to be elucidated. The present study demonstrates that RhoA-ROCK signaling downstream of Fyn controls stress fiber formation in SPC-treated fibroblasts. Here, we found that SPC-induced stress fiber formation was inhibited by C3 transferase, dominant negative RhoA or ROCK. SPC activated RhoA, which was blocked by pharmacological inhibition of Fyn activity or dominant negative Fyn. Constitutively active Fyn (ca-Fyn) stimulated stress fiber formation and localized with F-actin at the both ends of stress fibers, both of which were prevented by Fyn translocation inhibitor eicosapentaenoic acid (EPA). In contrast, inhibition of ROCK abolished only the formation of stress fibers, without affecting the localization of ca-Fyn. These results allow the identification of the molecular events downstream SPC in stress fiber formation for a better understanding of stress fiber formation involving Fyn.     

Th17 cell-derived IL-17 is dispensable for B cell antibody production

IL-17, which is preferentially produced by Th17 cells, is important for host defense against pathogens and is also involved in the development of autoimmune and allergic disorders. Antibody (Ab) production was shown to be impaired in IL-17-deficient mice, suggesting that IL-17 may promote B cell activation and direct secretion of Ab. However, the precise role of IL-17 in Ab production by B cells remains unclear. In the present study, we found constitutive expression of IL-17R in murine splenic B cells. Nevertheless, IL-17, IL-17F or IL-25 alone could not induce Ab production by B cells even in the presence of agonistic anti-CD40 Ab. IL-17 also could not affect IFN-γ-, IL-4- or TGF-β1-mediated Ig class-switching. Furthermore, in co-cultures of B cells and IL-17(-/-) CD4(+) T cells or IL-17(-/-) Th17 cells, IL-17 deficiency did not influence Ab production by B cells in vitro, suggesting that Th17 cell-derived IL-17 was not required for B cell Ab production through T cell-B cell interaction in vitro. Thus, in vivo, IL-17 may be indirectly involved in Ab production by enhancing production of B cell activator(s) by other immune cells.     

Activation of beta1,3-N-acetylglucosaminyltransferase-2 (beta3Gn-T2) by beta3Gn-T8. Possible involvement of beta3Gn-T8 in increasing poly-N-acetyllactosamine chains in differentiated HL-60 cells

Enzymatic activities of some glycosyltransferases are markedly increased via complex formation with other transferases or cofactor proteins. We previously showed that beta1,3-N-acetylglucosaminyltransferase-2 (beta3Gn-T2) and beta3Gn-T8 can form a heterodimer in vitro and that the complex exhibits much higher enzymatic activity than either enzyme alone (Seko, A., and Yamashita, K. (2005) Glycobiology 15, 943-951). Here we examined this activation and the biological significance of complex formation in differentiated HL-60 cells. beta3Gn-T2 and -T8 were co-immunoprecipitated from the lysates of both-transfected COS-7 cells, indicating their association in vivo. We prepared inactive mutants of both enzymes by destroying the DXD motifs. The mixture of mutated beta3Gn-T2 and intact beta3Gn-T8 did not exhibit any activation, whereas the mixture of intact beta3Gn-T2 and mutated beta3Gn-T8 had increased activity, indicating the activation of beta3Gn-T2 via complex formation. Next, we compared expression levels of beta3Gn-T1-T8 in HL-60 cells and DMSO-treated differentiated HL-60 cells, which produce larger poly-N-acetyllactosamine chains. The expression level of beta3Gn-T8 in the differentiated cells was 2.6-fold higher than in the untreated cells. Overexpression of beta3Gn-T8, but not beta3Gn-T2, induced an increase in poly-N-acetyllactosamine chains in HL-60 cells. These results raise a possibility that up-regulation of beta3Gn-T8 in differentiated HL-60 cells increases poly-N-acetyllactosamine chains by activating intrinsic beta3Gn-T2.     

Bap31 is an itinerant protein that moves between the peripheral endoplasmic reticulum (ER) and a juxtanuclear compartment related to ER-associated Degradation

Certain endoplasmic reticulum (ER)-associated degradation (ERAD) substrates with transmembrane domains are segregated from other ER proteins and sorted into a juxtanuclear subcompartment, known as the ER quality control compartment. Bap31 is an ER protein with three transmembrane domains, and it is assumed to be a cargo receptor for ER export of some transmembrane proteins, especially those prone to ERAD. Here, we show that Bap31 is a component of the ER quality control compartment and that it moves between the peripheral ER and a juxtanuclear ER or ER-related compartment distinct from the conventional ER–Golgi intermediate compartment. The third and second transmembrane domains of Bap31 are principally responsible for the movement to and recycling from the juxtanuclear region, respectively. This cycling was blocked by depolymerization of microtubules and disruption of dynein–dynactin function. Overexpression of Sar1p and Arf1 mutants affected Bap31 cycling, suggesting that this cycling pathway is related to the conventional vesicular transport pathways.     

Scaffold-free cartilage by rotational culture for tissue engineering

Our objective was to investigate the hypothesis that tissue-engineered cartilage with promising biochemical, mechanical properties can be formed by loading mechanical stress under existing cell-cell interactions analogous to those that occur in condensation during embryonic development. By loading dedifferentiated chondrocytes with mechanical stress under existing cell-cell interactions, we could first form a scaffold-free cartilage tissue with arbitrary shapes and a large size with promising biological, mechanical properties. The cartilage tissue which constituted of chondrocytes and ECM produced by inoculated dedifferentiated chondrocytes to a high porous simple mold has arbitrary shapes, and did not need any biodegradable scaffold to control the shape. In contrast, scaffold-free cartilage tissue cultured under static conditions could not keep their shapes; it was fragile tissue. The possibility of scaffold-free organ design was suggested because the cartilage tissue increases steadily in size with culture time; indeed, the growth of cartilage tissue starting from an arbitrary shape might be predictable by mathematical expression. For tissue-engineered cartilage formation with arbitrary shapes, biochemical and mechanical properties, loading dedifferentiated chondrocytes with mechanical stress under existing cell-cell interactions has prominent effects. Therefore, our scaffold-free cartilage model loaded mechanical stress based on a simple mold system may be applicable for tissue-engineered cartilage.     

Quality evaluation of Astragali Radix using a multivariate statistical approach

The quality of 43 Astragali Radix samples collected in China and Mongolia was evaluated using multivariate statistical analysis of data obtained from liquid chromatography-ion trap-time of flight (LC-IT-TOF) mass spectrometry. The samples were classified into four characteristic groups and most of the marker compounds were identified by elemental composition data and the results of MS/MS analysis. The approach provides useful information and gives an overview of the difference between crude drugs originating from different production environments and the genetic nature of the medicinal plants. In addition, the ease with which particular marker compounds could be identified and the effectiveness of the comparison by means of multivariate statistics, such as principal component analysis (PCA), indicates that this method could be utilized for the establishment of standardization and quality control procedures for crude drugs.     

Kid-mediated chromosome compaction ensures proper nuclear envelope formation

Toward the end of mitosis, neighboring chromosomes gather closely to form a compact cluster. This is important for reassembling the nuclear envelope around the entire chromosome mass but not individual chromosomes. By analyzing mice and cultured cells lacking the expression of chromokinesin Kid/kinesin-10, we show that Kid localizes to the boundaries of anaphase and telophase chromosomes and contributes to the shortening of the anaphase chromosome mass along the spindle axis. Loss of Kid-mediated anaphase chromosome compaction often causes the formation of multinucleated cells, specifically at oocyte meiosis II and the first couple of mitoses leading to embryonic death. In contrast, neither male meiosis nor somatic mitosis after the morula-stage is affected by Kid deficiency. These data suggest that Kid-mediated anaphase/telophase chromosome compaction prevents formation of multinucleated cells. This protection is especially important during the very early stages of development, when the embryonic cells are rich in ooplasm.     

The binding of VIP36 and alpha-amylase in the secretory vesicles via high-mannose type glycans

Vesicular integral protein of 36 kDa (VIP36) is an intracellular lectin recognizing high-mannose type glycans and is highly expressed in salivary glands, especially the parotid gland, which secretes alpha-amylase in large quantities. Here immunoelectron microscopy demonstrated that VIP36 was primarily localized to secretory vesicles in the glandula parotis of the rat, where alpha-amylase also resided. A secretory vesicle fraction, prepared by Percoll density gradient centrifugation, contained both VIP36 and alpha-amylase. Moreover, alpha-amylase that was localized to these secretory vesicles contained high-mannose type glycans. In addition, VIP36 coprecipitated with alpha-amylase in an endo H treatment-sensitive manner. These results suggest that VIP36 is involved in the secretion of alpha-amylase in the rat parotid gland.