Membrane insertion and dissociation processes of a model transmembrane helix

An important subject for elucidating membrane protein (MP) folding is how transmembrane helices (TMHs) insert into and dissociate from membranes. We investigated helix dissociation kinetics and insertion topology by means of intervesicular transfer of the fluorophore-labeled completely hydrophobic model transmembrane helix NBD-(LALAAAA)(3)-NH(2) (NBD = 7-nitro-2-1,3-benzoxadiazol-4-yl). The peptide forms a topologically stable transmembrane helix, which is in a monomer-antiparallel dimer equilibrium [Yano, Y., Takemoto, T., Kobayashi, S., Yasui, H., Sakurai, H., Ohashi, W., Niwa, M., Futaki, S., Sugiura, Y., and Matsuzaki, K. (2002) Biochemistry 41, 3073-3080]. The helix transfer kinetics, representing the helix dissociation process, was monitored by fluorescence recovery of the quenched peptide in donor vesicles containing a quencher upon its transfer to acceptor vesicles without the quencher. The transfer kinetics and vesicle concentration dependence demonstrated that the transfer was mediated by monomer in the aqueous phase. Furthermore, the activation enthalpy was estimated to be +17.7 +/- 1.3 kcal mol(-1). Helix insertion topology, detected by chemical quenching of the NBD group in the outer leaflet by dithionite ions, was found to be controlled by transmembrane electric potential-helix macro dipole interaction. On the basis of these observations, a model for the helix insertion/dissociation processes was discussed.     

Novel cyclohexene derivatives as anti-sepsis agents: synthetic studies and inhibition of NO and cytokine production

In order to develop an anti-sepsis agent, a series of cyclohexene derivatives were synthesized and evaluated for their biological activities. Through modification of the sulfonamide spacer moiety depicted by formula II, it was found that the benzylsulfone derivative 10a had potent inhibitory activity against the production of NO. Further modifications of the phenyl ring, ester moiety, and benzyl position of benzylsulfone derivatives III were carried out. Among these compounds, (R)-(+)-10a and (6R, 1S)-(+)-22a showed strong inhibitory activity not only against NO production but also against inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in vitro. Furthermore, (R)-(+)-10a and (6R, 1S)-(+)-22a protected mice from LPS-induced lethality in a dose-dependent manner.     

Inhibition of ornithine decarboxylase induction of interferon (alpha + beta) and its reversal by dibutyryl adenosine 3',5'-monophosphate

Interferon (alpha + beta) given to C3H/HeN mice intraperitoneally inhibited increases in the activities of adenylate cyclase and ornithine decarboxylase after partial hepatectomy. The inhibition of ornithine decarboxylase was prevented by administration of dibutyryl cAMP. Core (2'-5')oligo(adenylate), i.e. A2'p5'A2'p5'A or (A2'p)2A, as well as interferon inhibited the increases in these two enzymes caused by partial hepatectomy. The inhibition by (A2'p)2A of ornithine decarboxylase activity was reversed by dibutyryl cAMP. These results suggested that the activity of interferon was similar to that of (A2'p)2A and that the inhibition of ornithine decarboxylase induction caused by these agents resulted from the inhibition of adenylate cyclase activity.     

Angulin-1 seals tricellular contacts independently of tricellulin and claudins

Tricellular tight junctions (tTJs) are specialized tight junctions (TJs) that seal the intercellular space at tricellular contacts (TCs), where the vertices of three epithelial cells meet. Tricellulin and angulin family membrane proteins are known constituents of tTJs, but the molecular mechanism of tTJ formation remains elusive. Here, we investigated the roles of angulin-1 and tricellulin in tTJ formation in MDCK II cells by genome editing. Angulin-1–deficient cells lost the plasma membrane contact at TCs with impaired epithelial barrier function. The C terminus of angulin-1 bound to the TJ scaffold protein ZO-1, and disruption of their interaction influenced the localization of claudins at TCs, but not the tricellular sealing. Strikingly, the plasma membrane contact at TCs was formed in tricellulin- or claudin-deficient cells. These findings demonstrate that angulin-1 is responsible for the plasma membrane seal at TCs independently of tricellulin and claudins.     

Night-interrupting light inhibits diapause induction in the Kanzawa spider mite, Tetranychus kanzawai Kishida (Acari: Tetranychidae)

The inhibitory effects of the timing, intensity (I_I) and period (I_T) of night-interrupting light on diapause induction of the Kanzawa spider mite (Tetranychus kanzawai) were investigated in a series of laboratory experiments. During a light and dark period of 8 and 16 h d⁻¹, respectively, a single 1-h night-interrupting light was applied at early (E), middle (M), and late (L) parts of the dark period: i.e., at 3, 7.5, and 12 h after the start of the dark period, respectively. No interrupting light was applied in the control treatment. The incidence of diapause was significantly lower in the M treatment (63%) compared to the control treatment (100%). In the E and L treatments, more than 90% of females entered diapause, which was comparable to the control treatment. Since the longest consecutive dark period during the E and L treatments was longer than the critical dark period (CDP) of 10.5-11 h d⁻¹, during which 50% of females entered diapause, the night-interrupting light probably failed to prevent diapause induction. However, in the M treatment, the longest consecutive dark period was shorter than the CDP; therefore, the night-interrupting light inhibited diapause induction. Moreover, the inhibitory effects of night-interrupting light in the M treatment increased as I_I and I_T increased. The dose of night-interrupting light (I_I × I_T) was significantly negatively related to the incidence of diapause. The median effective dose for 50% disturbance of diapause induction was 2.5 kJ m⁻² at wavelengths between 350 and 1050 nm. Our results suggest that the longest consecutive dark period and the dose of night-interrupting light should both be considered when a lighting-based physical control is applied to inhibit diapause induction and consequent overwintering of T. kanzawai in commercial agricultural fields.     

Stem cells from human exfoliated deciduous teeth (SHED) enhance wound healing and the possibility of novel cell therapy

Background aimsIn recent years, stem cells from human exfoliated deciduous teeth (SHED) have received attention as a novel stem cell source with multipotent potential. We examined the effect on wound-healing promotion with unique stem cells from deciduous teeth as a medical waste.MethodsAn excisional wound-splinting mouse model was used and the effect of wound healing among SHED, human mesenchymal stromal cells (hMSCs), human fibroblasts (hFibro) and a control (phosphate-buffered saline; PBS) was evaluated by macroscopy, histology and enzyme-linked immunosorbent assay (ELISA), and the expression of hyaluronan (HA), which is related to wound healing, investigated.ResultsSHED and hMSCs accelerated wound healing compared with hFibro and the control. There was a statistically significant difference in wound healing area among hFibro, hMSCs and SHED compared with the control after day 5. At days 7 and 14 after cell transplantation, the histologic observation showed that transplanted PKH26-positive cells were surrounded by human HA binding protein, especially in hMSCs and SHED. HA expression volume values were 1558.41 ± 60.33 (control), 2092.75 ± 42.56 (hFibro), 2342.07 ± 188.10 (hMSCs) and 2314.85 ± 164.91 (SHED) ng/mg, respectively, and significantly higher in hMSCs and SHED compared with hFibro and control at days 7 and 14 (P < 0.05).ConclusionsOur results show that SHED hMSCs have similar effects of wound-healing promotion as hFibro and controls. This implies that SHED might offer a unique stem cell resource and the possibility of novel cell therapies for wound healing in the future.     

Phagocytosis by rat peritoneal mast cells: independence of IgG Fc-mediated and C3-mediated signals

Phagocytosis of sheep erythrocytes (E) bearing rabbit immunoglobulin and rat C3 by rat peritoneal mast cells was quantitated by using 51Cr-labeled E and was confirmed by electron microscopy. The relative importance of IgG Fc receptor interaction in C3-mediated phagocytosis was assessed. Removal of any traces of IgG antibody by absorption of IgM antibody with protein A-Sepharose and absorption of the other reagents with sheep E had no effect on the phagocytosis of C3b- and C3bi-coated cells. IgG antibody enhanced the phagocytosis of intermediates prepared with IgM, purified components and rat C3 (EaIgMClgp4hu oxy2hu3brat) with a graded dose response, but was only additive and not synergistic with C3. The independence of the C3- and Fc-mediated signals was confirmed by using chemically produced polymers of rabbit IgG to inhibit phagocytosis. These polymers, especially tetramers and higher aggregates, completely blocked ingestion of EAIgG, but not that of EAIgMC1423b or -3bi. When IgG was substituted for IgM in the C3b intermediate, the IgG polymers inhibited about 50% of the phagocytosis. Cumulatively, the data demonstrated that in the case of rat mast cells, the stimuli to phagocytosis induced by C3 and IgG are independent; either is sufficient by itself.     

Mutational analyses of the photosynthetic reaction center-bound triheme cytochrome subunit and cytochrome c2 in the purple bacterium Rhodovulum sulfidophilum

The purple photosynthetic bacterium Rhodovulum sulfidophilum has an unusual reaction center- (RC-) bound cytochrome subunit with only three hemes, although the subunits of other purple bacteria have four hemes. To understand the electron-transfer pathway through this subunit, three mutants of R. sulfidophilum were constructed and characterized: one lacking the RC-bound cytochrome subunit, another one lacking cytochrome c(2), and another one lacking both of these. The mutant lacking the RC-bound cytochrome subunit was grown photosynthetically with about half the growth rate of the wild type, indicating that the presence of the cytochrome subunit, while not indispensable, is still advantageous for the photosynthetic electron transfer to support its growth. The mutant lacking both the cytochrome subunit and cytochrome c(2) showed a slower rate of growth by photosynthesis (about a fourth of that of the wild type), indicating that cytochrome c(2) is the dominant electron donor to the RC mutationally devoid of the cytochrome subunit. On the other hand, the mutant lacking only the cytochrome c(2) gene grew photosynthetically as fast as the wild type, indicating that cytochrome c(2) is not the predominant donor to the RC-bound triheme cytochrome subunit. We further show that newly isolated soluble cytochrome c-549 with a redox midpoint potential of +238 mV reduced the photooxidized cytochrome subunit in vitro, suggesting that c-549 mediates the cytochrome c(2)-independent electron transfer from the bc(1) complex to the RC-bound cytochrome subunit. These results indicate that the soluble components donating electrons to the RC-bound triheme cytochrome subunit are somewhat different from those of other purple bacteria.     

Nuclear behavior during the formation of appressoria byAlternaria alternata

Nuclear behavior in the developmental process of appressoria inAlternaria alternata was investigated. In pregerminated conidia, approximately 94% of the conidial cells were uninucleate. The migration of a nucleus into an elongating germ tube from a germinating conidium was confirmed after 2h of incubation at 24±1°C in PDB. Peak frequencies of binucleate and trinucleate germ tubes were detected 1 and 2h after the peak frequency of uninucleate germ tubes, respectively. Four-and five-nucleate germ tubes did not show marked peak frrequencies. A marked peak frequency of the six-nucleate germ tubes occurred about 1 h after the peak frequency of the trinucleate germ tubes, suggesting that the nuclei in the trinucleate germ tubes each divided once within 1 h. The significance of early establishment of multinucleate appressorial cells in the colonization of host plants by pathogenicA. alternata was discussed.