Light-evoked somatosensory perception of transgenic rats that express channelrhodopsin-2 in dorsal root ganglion cells

In vertebrate somatosensory systems, each mode of touch-pressure, temperature or pain is sensed by sensory endings of different dorsal root ganglion (DRG) neurons, which conducted to the specific cortical loci as nerve impulses. Therefore, direct electrical stimulation of the peripheral nerve endings causes an erroneous sensation to be conducted by the nerve. We have recently generated several transgenic lines of rat in which channelrhodopsin-2 (ChR2) transgene is driven by the Thy-1.2 promoter. In one of them, W-TChR2V4, some neurons were endowed with photosensitivity by the introduction of the ChR2 gene, coding an algal photoreceptor molecule. The DRG neurons expressing ChR2 were immunohistochemically identified using specific antibodies to the markers of mechanoreceptive or nociceptive neurons. Their peripheral nerve endings in the plantar skin as well as the central endings in the spinal cord were also examined. We identified that ChR2 is expressed in a certain population of large neurons in the DRG of W-TChR2V4. On the basis of their morphology and molecular markers, these neurons were classified as mechanoreceptive but not nociceptive. ChR2 was also distributed in their peripheral sensory nerve endings, some of which were closely associated with CK20-positive cells to form Merkel cell-neurite complexes or with S-100-positive cells to form structures like Meissner's corpuscles. These nerve endings are thus suggested to be involved in the sensing of touch. Each W-TChR2V4 rat showed a sensory-evoked behavior in response to blue LED flashes on the plantar skin. It is thus suggested that each rat acquired an unusual sensory modality of sensing blue light through the skin as touch-pressure. This light-evoked somatosensory perception should facilitate study of how the complex tactile sense emerges in the brain.     

Valosin-containing protein/p97 interacts with sperm-activating and sperm-attracting factor (SAAF) in the ascidian egg and modulates sperm-attracting activity

Sperm chemotaxis toward an egg is observed in many animals, and the control of sperm-attracting activity is thought to play an important role in ensuring fertilization. However, the mechanism underlying the release of a sperm attractant from an egg is still obscure. In this study, we examined the systems involved in the release of sperm-activating and sperm-attracting factor (SAAF), which is the sperm attractant of the ascidian Ciona intestinalis. Here, we show that the egg acquires sperm-attracting activity after germinal vesicle breakdown. Further, since the cytoplasmic extracts of immature oocytes exhibit no sperm-attracting activity, the SAAF in oocytes may be activated after germinal vesicle breakdown. We found 13 SAAF-binding proteins in an egg plasma membrane extract and identified five proteins by proteomic analysis: valosin-containing protein (VCP)/p97, proteasome alpha 2 subunit, MGC97756 protein, proteasome subunit Y, and beta-tubulin. In particular, the interaction between VCP/p97 and SAAF was confirmed by a pull-down assay. VCP/p97 is initially localized in the germinal vesicle, and during oocyte maturation, it shifts to the endoplasmic reticulum in the cortical regions. Thus, VCP/p97 is a potential modulator of SAAF release from the egg.     

Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae

Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning. We explored the possibility of employing more than one PCR fragment to introduce various mutations in a single step, and found that when up to five PCR fragments were co-transformed into yeast, the recombination frequency was maintained as the number of fragments was increased. All transformants expressed the model membrane protein, while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination.     

Resistance to collagen-induced arthritis in SHPS-1 mutant mice

SHPS-1 is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is abundantly expressed on dendritic cells and macrophages. Here we show that mice expressing a mutant form of SHPS-1 fail to develop type-II collagen (CII)-induced arthritis (CIA), a model for rheumatoid arthritis in humans. Histological examinations of the arthritic paws from immunized wild-type mice revealed that cartilage was destroyed in association with marked mononuclear cell infiltration, while only mild cell infiltration was observed in immunized SHPS-1 mutant mice. Consistently, the serum levels of both IgG and IgG2a specific to CII and of IL-1β in immunized SHPS-1 mutant mice were markedly reduced compared with those apparent for wild-type mice. The CII-induced proliferation of, and production of cytokines by, T cells from immunized SHPS-1 mutant mice were reduced compared to wild-type cells. These results suggest that SHPS-1 is essential for development of CIA.     

Roles of integral protein in membrane permeabilization by amphidinols

Amphidinols (AMs) are a group of dinoflagellate metabolites with potent antifungal activity. As is the case with polyene macrolide antibiotics, the mode of action of AMs is accounted for by direct interaction with lipid bilayers, which leads to formation of pores or lesions in biomembranes. However, it was revealed that AMs induce hemolysis with significantly lower concentrations than those necessary to permeabilize artificial liposomes, suggesting that a certain factor(s) in erythrocyte membrane potentiates AM activity. Glycophorin A (GpA), a major erythrocyte protein, was chosen as a model protein to investigate interaction between peptides and AMs such as AM2, AM3 and AM6 by using SDS-PAGE, surface plasmon resonance, and fluorescent-dye leakages from GpA-reconstituted liposomes. The results unambiguously demonstrated that AMs have an affinity to the transmembrane domain of GpA, and their membrane-permeabilizing activity is significantly potentiated by GpA. Surface plasmon resonance experiments revealed that their interaction has a dissociation constant of the order of 10 microM, which is significantly larger than efficacious concentrations of hemolysis by AMs. These results imply that the potentiation action by GpA or membrane integral peptides may be due to a higher affinity of AMs to protein-containing membranes than that to pure lipid bilayers.     

Wheat germ agglutinin stains dispersed post-golgi vesicles after treatment with the cytokinesis inhibitor psychosine

The galactosylsphingosine psychosine (Psy) is one of the sphingolipids and induce the formation of multinuclear cells in several cell lines by inhibiting cytokinesis. In the present report, we show that intracellular organelles, including wheat germ agglutinin (WGA)-positive vesicles and early endosomes, are selectively dispersed by Psy. WGA is a conventional Golgi marker and WGA-positive vesicles appeared to co-localize with the Golgi apparatus in untreated cells. Psy treatment induced the dispersal of WGA-positive vesicles without affecting the structure of the Golgi apparatus, resulting in discrimination of WGA-positive vesicles from the Golgi apparatus. In sharp contrast to this effect of Psy, WGA-positive vesicles were not affected by brefeldin A treatment, which induced the disappearance of the Golgi apparatus. Immunostaining with anti-TGN46 antibodies revealed that a large portion of the WGA-positive vesicles were derived from the trans-Golgi network. Notably, the dispersed WGA-positive vesicles did not stain with anti-syntaxin 6, another marker of the trans-Golgi network. During cytokinesis, WGA-positive vesicles in the cytoplasm decreased, and WGA staining accumulated at the cleavage furrow, which was apparently inhibited by the presence of Psy. These data suggest that the transport of WGA-positive vesicles to the cleavage furrow is associated with the progression of cytokinesis.     

Allele-selective effect of PA28 in MHC class I antigen processing

PA28 is an IFN-gamma-inducible proteasome activator and its genetic ablation causes complete loss of processing of certain Ags, but not all of them. The reason why this occurs and how PA28 influences the formation of peptide repertoires for MHC class I molecules remains unknown. In this study, we show the allele-specific role of PA28 in Ag processing. Retrovirus-transduced overexpression of PA28alpha decreased expression of K(d) (D(d)) while it increased K(b) and L(d) on the cell surface. By contrast, overexpression of PA28alphaDeltaC5, a mutant carrying a deletion of its five C-terminal residues and capable of attenuating the activity of endogenous PA28, produced the opposite effect on expression of those MHC class I molecules. Moreover, knockdown of both PA28alpha and beta by small-interfering RNA profoundly augmented expression of K(d) and D(d), but not of L(d), on the cell surface. Finally, we found that PA28-associated proteasome preferentially digested within epitopic sequences of K(d), although correct C-terminal flankings were removed, which in turn hampered production of K(d) ligands. Our results indicate that whereas PA28 negatively influences processing of K(d) (D(d)) ligands, thereby, down-regulating Ag presentation by those MHC class I molecules, it also efficiently produces K(b) (L(d)) epitopes, leading to up-regulation of the MHC molecules.     

Characterization of a tobacco TPK-type K+ channel as a novel tonoplast K+ channel using yeast tonoplasts

The tonoplast K(+) membrane transport system plays a crucial role in maintaining K(+) homeostasis in plant cells. Here, we isolated cDNAs encoding a two-pore K(+) channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco cells. Two of the four variants of NtTPK1 contained VHG and GHG instead of the GYG signature sequence in the second pore region. All four products were functional when expressed in the Escherichia coli cell membrane, and NtTPK1 was targeted to the tonoplast in tobacco cells. Two of the three promoter sequences isolated from N. tabacum cv. SR1 were active, and expression from these was increased approximately 2-fold by salt stress or high osmotic shock. To determine the properties of NtTPK1, we enlarged mutant yeast cells with inactivated endogenous tonoplast channels and prepared tonoplasts suitable for patch clamp recording allowing the NtTPK1-related channel conductance to be distinguished from the small endogenous currents. NtTPK1 exhibited strong selectivity for K(+) over Na(+). NtTPK1 activity was sensitive to spermidine and spermine, which were shown to be present in tobacco cells. NtTPK1 was active in the absence of Ca(2+), but a cytosolic concentration of 45 microM Ca(2+) resulted in a 2-fold increase in the amplitude of the K(+) current. Acidification of the cytosol to pH 5.5 also markedly increased NtTPK1-mediated K(+) currents. These results show that NtTPK1 is a novel tonoplast K(+) channel belonging to a different group from the previously characterized vacuolar channels SV, FV, and VK.     

Enzymatic synthesis of spacer-linked divalent glycosides carrying N-acetylglucosamine and N-acetyllactosamine: analysis of cross-linking activities with WGA

Divalent glycosides carrying N-acetyl-d-glucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) were designed and prepared as glycomimetics. First, hexan-1,6-diyl bis-(2-acetamido-2-deoxy-beta-d-glucopyranoside) (GlcNAc-Hx-GlcNAc) and 3,6-dioxaoct-1,8-diyl bis-(2-acetamido-2-deoxy-beta-d-glucopyranoside) (GlcNAc-Doo-GlcNAc) were enzymatically synthesized by transglycosylation of an N,N'N',N'-tetraacetylchitotetraose [(GlcNAc)(4)] donor with a primary diol acceptor, utilizing a chitinolytic enzyme from Amycolatopsis orientalis. The resulting divalent glycosides were further converted to the respective hexan-1,6-diyl bis-[beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside] (LacNAc-Hx-LacNAc) and 6-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-hexyl beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside (LacNAc-Hx-GlcNAc), and respective 3,6-dioxaoct-1,8-diyl bis-[beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside] (LacNAc-Doo-LacNAc) and 8-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-3,6-dioxaoctyl beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside (LacNAc-Doo-GlcNAc) by galactosyltransferase. The interaction of wheat germ agglutinin (WGA) with a series of divalent glycosides and related compounds were studied using a biosensor based on surface plasmon resonance (SPR) and by precipitation analysis. Our results demonstrated that divalent glycosides carrying GlcNAc on both sides and GlcNAc and LacNAc on each side are capable of precipitating WGA as divalent ligands, but that the corresponding monovalent controls and divalent glycosides carrying LacNAc on both sides are unable to precipitate the lectin and bind as univalent ligands.