Legionella hijacks the host Golgi-to-ER retrograde pathway for the association of Legionella-containing vacuole with the ER

Legionella pneumophila (L. pneumophila) is a gram-negative bacterium that replicates in a compartment that resembles the host endoplasmic reticulum (ER). To create its replicative niche, L. pneumophila manipulates host membrane traffic and fusion machineries. Bacterial proteins called Legionella effectors are translocated into the host cytosol and play a crucial role in these processes. In an early stage of infection, Legionella subverts ER-derived vesicles (ERDVs) by manipulating GTPase Rab1 to facilitate remodeling of the Legionella-containing vacuole (LCV). Subsequently, the LCV associates with the ER in a mechanism that remains elusive. In this study, we show that L. pneumophila recruits GTPases Rab33B and Rab6A, which regulate vesicle trafficking from the Golgi to the ER, to the LCV to promote the association of LCV with the ER. We found that recruitment of Rab6A to the LCV depends on Rab33B. Legionella effector SidE family proteins, which phosphoribosyl-ubiquitinate Rab33B, were found to be necessary for the recruitment of Rab33B to the LCV. Immunoprecipitation experiments revealed that L. pneumophila facilitates the interaction of Rab6 with ER-resident SNAREs comprising syntaxin 18, p31, and BNIP1, but not tethering factors including NAG, RINT-1, and ZW10, which are normally required for syntaxin 18-mediated fusion of Golgi-derived vesicles with the ER. Our results identified a Rab33B-Rab6A cascade on the LCV and the interaction of Rab6 with ER-resident SNARE proteins for the association of LCV with the ER and disclosed the unidentified physiological role of SidE family proteins.     

Prospectively Isolated Cancer-Associated CD10+ Fibroblasts Have Stronger Interactions with CD133+ Colon Cancer Cells than with CD133? Cancer Cells

Although CD133 has been reported to be a promising colon cancer stem cell marker, the biological functions of CD133⁺ colon cancer cells remain controversial. In the present study, we investigated the biological differences between CD133⁺ and CD133⁻ colon cancer cells, with a particular focus on their interactions with cancer-associated fibroblasts, especially CD10⁺ fibroblasts. We used 19 primary colon cancer tissues, 30 primary cultures of fibroblasts derived from colon cancer tissues and 6 colon cancer cell lines. We isolated CD133⁺ and CD133⁻ subpopulations from the colon cancer tissues and cultured cells. In vitro analyses revealed that the two populations showed similar biological behaviors in their proliferation and chemosensitivity. In vivo analyses revealed that CD133⁺ cells showed significantly greater tumor growth than CD133⁻ cells (P = 0.007). Moreover, in cocultures with primary fibroblasts derived from colon cancer tissues, CD133⁺ cells exhibited significantly more invasive behaviors than CD133⁻ cells (P<0.001), especially in cocultures with CD10⁺ fibroblasts (P<0.0001). Further in vivo analyses revealed that CD10⁺ fibroblasts enhanced the tumor growth of CD133⁺ cells significantly more than CD10⁻ fibroblasts (P<0.05). These data demonstrate that the in vitro invasive properties and in vivo tumor growth of CD133⁺ colon cancer cells are enhanced in the presence of specific cancer-associated fibroblasts, CD10⁺ fibroblasts, suggesting that the interactions between these specific cell populations have important roles in cancer progression. Therefore, these specific interactions may be promising targets for new colon cancer therapies.     

Induction of erythroid differentiation by cytoplast fusion in mouse erythroleukemia (Friend) cells

An intracellular activity, which is induced by dimethyl sulfoxide (DMSO) or hexamethylenebisacetamide (HMBA) and leads to erythroid differentiation in mouse Friend cells, was characterized by cell fusion between genetically marked intact cells and cytoplasts. For this, a procedure for rapid selection of cybrids was devised by sensitizing non-fused cells with oligomycin. We were able to demonstrate that cytoplasts derived from DMSO- (or HMBA)-treated cells trigger erythroid differentiation upon fusion with UV-irradiated cells. The activity in the cytoplasts remained only transiently and its induction was inhibited by biologically active phorbol esters or cycloheximide. The activity, however, was not induced in cytoplasts by directly treating them with DMSO (or HMBA). These results indicate that (1) the intracellular erythroid-inducing activity is located in cytoplasts, (2) it acts in trans and induces erythroid differentiation as a dominant factor and (3) its production requires de novo nuclear protein synthesis. The mechanisms of the induction of the intracellular activity and of how it triggers erythroid differentiation are discussed.     

Involvement of protein kinase C in Ca(2+)-independent contraction of rat uterine smooth muscle

The contribution of protein kinase C to the contraction by oxytocin of rat uterine longitudinal smooth muscle in Ca(2+)-free solution was investigated. Immunological analysis revealed that type II (beta) and III (alpha) protein kinase C subspecies were present in rat uterine smooth muscle. The pretreatment of a diacylglycerol kinase inhibitor R59022 to accumulate diacylglycerol potentiated the Ca(2+)-independent contraction. The contractile activity was diminished with the depletion of protein kinase C, when the contraction was evoked repeatedly by oxytocin during the prolonged exposure to a tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate. These results suggested the involvement of protein kinase C in oxytocin-induced contraction in Ca(2+)-free solution.     

Developmental partitioning of SYK and ZAP70 prevents autoimmunity and cancer

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Development of a microbioreactor for glycoconjugate synthesis

A microbioreactor immobilized with a synthase-type mutant enzyme, Endo-M-N175Q (glycosynthase) of endo-β-N-acetylglucosaminidase derived from Mucor hiemalis (Endo-M), was constructed and used for glycoconjugate synthesis. The transglycosylation was performed with a reaction mixture containing an oxazoline derivative of sialo complex-type glycoside (SG), which was prepared from a sialo complex-type glycopeptide SGP derived from hen egg yolk, as a glycosyl donor and N-Fmoc-N-acetylglucosaminyl-l-asparagine [Fmoc-Asn(GlcNAc)-OH] as an acceptor. The reaction mixture was injected into a glycosynthase microbioreactor at a constant flow rate. Highly efficient and nearly stoichiometric transglycosylation occurred in the microbioreactor, and the transglycosylation product was eluted from the other end of the reactor. The glycosynthase microbioreactor was stable and could be used repeatedly for a long time.     

Association Between Moringness‐Eveningness Preference and Mental/Physical Premenstrual Symptoms in Japanese Females 12 to 31 Years of Age

This study investigates the relationship between circadian typology, i.e., morningness‐eveningness (M‐E) preference, and the occurrence and severity of premenstrual mental and physical symptoms among 154 young Japanese female university students (range, 18 to 31 yrs; mean±S.D., 20.69±3.69 yrs) and 417 junior high school students (range, 12 to 15 yrs; mean±S.D., 14.29±0.67 yrs) living in an urban or suburban area of Kochi prefecture. Female university students experienced melancholy mood more frequently than did males, and the female university students who frequently became melancholy were more evening‐typed than those who did not experience melancholy. Female university students who experienced frequent fluctuations in mood and/or menstrual pain were more evening‐typed than those who were not so affected. M‐E preference of junior high school students was not correlated with stability of mood or frequency of menstrual pain. In urban areas, however junior high school students who had very stable menstrual cycles were significantly more morning‐typed than those whose menstrual cycles were not stable. In suburban areas, the bedtimes of female junior high students who had stable menstrual cycles were significantly earlier than those whose menstrual cycle duration was not stable. A physiological relationship between the circadian system, M‐E, and attributes of the menstrual cycle seems to be present in adolescent female Japanese junior high school students.     

Regulation of RhoA Signaling by the cAMP-dependent Phosphorylation of RhoGDIα

RhoA plays a pivotal role in regulating cell shape and movement. Protein kinase A (PKA) inhibits RhoA signaling and thereby induces a characteristic morphological change, cell rounding. This has been considered to result from cAMP-induced phosphorylation of RhoA at Ser-188, which induces a stable RhoA-GTP-RhoGDIα complex and sequesters RhoA to the cytosol. However, few groups have shown RhoA phosphorylation in intact cells. Here we show that phosphorylation of RhoGDIα but not RhoA plays an essential role in the PKA-induced inhibition of RhoA signaling and in the morphological changes using cardiac fibroblasts. The knockdown of RhoGDIα by siRNA blocks cAMP-induced cell rounding, which is recovered by RhoGDIα-WT expression but not when a RhoGDIα-S174A mutant is expressed. PKA phosphorylates RhoGDIα at Ser-174 and the phosphorylation of RhoGDIα is likely to induce the formation of a active RhoA-RhoGDIα complex. Our present results thus reveal a principal molecular mechanism underlying G_s/cAMP-induced cross-talk with G_q/G₁₃/RhoA signaling.     

Presentation of Various Tactile Sensations Using Micro-Needle Electrotactile Display

Tactile displays provoke tactile sensations by artificially stimulating tactile receptors. While many types of tactile displays have been developed, electrotactile displays that exploit electric stimulation can be designed to be thin, light, flexible and thus, wearable. However, the high voltages required to stimulate tactile receptors and limited varieties of possible sensations pose problems. In our previous work, we developed an electrotactile display using a micro-needle electrode array that can drastically reduce the required voltage by penetrating through the high-impedance stratum corneum painlessly, but displaying various tactile sensations was still a challenge. In this work, we demonstrate presentation of tactile sensation of different roughness to the subjects, which is enabled by the arrangement of the electrodes; the needle electrodes are on the fingertip and the ground electrode is on the fingernail. With this arrangement, the display can stimulate the tactile receptors that are located not only in the shallow regions of the finger but also those in the deep regions. It was experimentally revealed that the required voltage was further reduced compared to previous devices and that the roughness presented by the display was controlled by the pulse frequency and the switching time, or the stimulation flow rate. The proposed electrotactile display is readily applicable as a new wearable haptic device for advanced information communication technology.