The Saccharomyces cerevisiae och1 mutant shows a deficiency in the mannose outer chain elongation at the non‐permissive temperature. We have cloned the OCH1 gene by complementation of temperature sensitive (ts) phenotype for growth. The integrant of OCH1 gene in the yeast chromosome can complement the ts phenotype and shows the same mapping position as that of the och1 mutation, indicating that the cloned gene is the true gene for mutation. The OCH1 gene disruptant is not lethal but ts for cell growth, and lacks mannose outer chains. The OCH1 gene sequence predicts a 55 kDa protein consisting of 480 amino acids. It contains four potential asparagine‐linked (N‐linked) glycosylation sites and a single transmembrane region near the N‐terminus. In vitro translation/translocation analysis revealed that the large C‐terminal region of the OCH1 protein is located at the lumenal side of microsomal membranes with some sugar modification, indicating a type II membrane topology. The OCH1 protein was detected in yeast membrane fractions as four forms of 58–66 kDa, which correspond to the size of a glycoprotein containing four N‐linked sugar chains the length of which is almost the same or slightly larger than the inner core (Man8GlcNAc2) formed in the endoplasmic reticulum (ER). Finally, the OCH1 gene was found to encode a novel mannosyltransferase which specifically transfers [14C]mannose to the unique acceptor, the core‐like oligosaccharide of cell wall mannan accumulated in the och1 disruptant.

Multireference perturbation theory with complete active space self-Ž . consistent field CASSCF reference functions is applied to the study of the valence ª * excited states of 1,3-butadiene, 1,3,5-hexatriene, 1,3,5,7-octatetraene, and 1,3,5,7,9-decapentaene. Our focus was put on determining the nature of the two lowestlying singlet excited states, 1 1 B q and 2 1 A y , and their ordering. The 1 1 B q state is a singly u g u excited state with an ionic nature originating from the HOMO ª LUMO one-electron transition while the covalent 2 1 A y state is the doubly excited state which comes mainly g Ž . 2 Ž . 2 from the HOMO ª LUMO transition. The active-space and basis-set effects are taken into account to estimate the excitation energies of larger polyenes. For butadiene, the 1 1 B q state is calculated to be slightly lower by 0.1 eV than the doubly excited 2 1 A y u g state at the ground-state equilibrium geometry. For hexatriene, our calculations predict the two states to be virtually degenerate. Octatetraene is the first polyene for which we predict that the 2 1 A y state is the lowest excited singlet state at the ground-state g geometry. The present theory also indicates that the 2 1 A y state lies clearly below the g 1 1 B q state in decapentaene with the energy gap of 0.4 eV. The 0᎐0 transition and the u emission energies are also calculated using the planar C relaxed excited-state 2 h geometries. The covalent 2 1 A y state is much more sensitive to the geometry variation g than is the ionic 1 1 B q state, which places the 2 1 A y state significantly below the 1 1 B q u g u state at the relaxed geometry.

Epidermal growth factor receptor (EGFR) at membrane microdomains plays an essential role in the growth control of epidermal cells, including cancer cells derived therefrom. Ligand-dependent activation of EGFR tyrosine kinase is known to be inhibited by ganglioside GM3, but to a much lesser degree by other glycosphingolipids. However, the mechanism of the inhibitory effect of GM3 on EGFR tyrosine kinase has been ambiguous. The mechanism is now defined by binding of N-linked glycan having multiple GlcNAc termini to GM3 through carbohydrate-to-carbohydrate interaction, based on the following data: (i) EGFR (molecular mass, Ϸ170 kDa) has N-linked glycan with GlcNAc termini, as probed by mAb (J1) or lectin (GS-II); (ii) GS-II-bound EGFR also bound to anti-EGFR Ab as well as to GM3-coated beads; (iii) GM3 inhibitory effect on EGFR tyrosine kinase was abrogated in vitro by coincubation with glycan having multiple GlcNAc termini and in cell culture in situ incubated with the same glycan; and (iv) cells treated with swainsonine, which increased expression of complex-type and hybridtype glycans with GlcNAc termini, displayed higher inhibition of EGFR kinase by GM3 than swainsonine-untreated control cells. A similar effect was observed with 1-deoxymannojirimycin, which increased hybrid-type structure in addition to major accumulation of high mannose-type glycan. These findings indicate that N-linked glycan with GlcNAc termini linked to EGFR is the target to interact with GM3, causing inhibition of EGF-induced EGFR tyrosine kinase.carbohydrate-to-carbohydrate interaction ͉ N-linked glycan ͉ glycosphingolipid ͉ ganglioside ͉ oligosaccharide Fr.B

Glycosylphosphatidylinositol (GPI) is a conserved posttranslational modification to anchor cell surface proteins to plasma membrane in all eukaryotes. In yeast, GPI mediates cross-linking of cell wall mannoproteins to ␤1,6-glucan. We reported previously that the GWT1 gene product is a target of the novel anti-fungal compound, 1-[4-butylbenzyl]isoquinoline, that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae (Tsukahara, K., Hata, K., Sagane, K., Watanabe, N., Kuromitsu, J., Kai, J., Tsuchiya, M., Ohba, F., Jigami, Y., Yoshimatsu, K., and Nagasu, T. (2003) Mol. Microbiol. 48, 1029 -1042). In the present study, to analyze the function of the Gwt1 protein, we isolated temperature-sensitive gwt1 mutants. The gwt1 cells were normal in transport of invertase and carboxypeptidase Y but were delayed in transport of GPI-anchored protein, Gas1p, and were defective in its maturation from the endoplasmic reticulum to the Golgi. The incorporation of inositol into GPI-anchored proteins was reduced in gwt1 mutant, indicating involvement of GWT1 in GPI biosynthesis. We analyzed the early steps of GPI biosynthesis in vitro by using membranes prepared from gwt1 and ⌬gwt1 cells. The synthetic activity of GlcN-(acyl)PI from GlcN-PI was defective in these cells, whereas ⌬gwt1 cells harboring GWT1 gene restored the activity, indicating that GWT1 is required for acylation of inositol during the GPI synthetic pathway. We further cloned GWT1 homologues in other yeasts, Cryptococcus neoformans and Schizosaccharomyces pombe, and confirmed that the specificity of acyl-CoA in inositol acylation, as reported in studies of endogenous membranes (Franzot, S. P., and Doering, T. L. (1999) Biochem. J. 340, 25-32), is due to the properties of Gwt1p itself and not to other membrane components.

A complete active space valence bond ͑CASVB͒ method is proposed which is particularly adapted to chemical interpretation. A CASVB wave function can be obtained simply by transforming a canonical CASSCF function and readily interpreted in terms of the well known classical VB resonance structures. The method is applied to the ground and excited states of benzene, butadiene, and the ground state of methane. The CASVB affords a clear view of the wave functions for the various states. The electronic excitation is represented in a VB picture as rearrangements of the spin couplings or as charge transfers which involve breaking covalent bonds and forming new ionic bonds. The former gives rise to covalent excited states and the latter to ionic excited states. The physical reasons why it is so difficult to describe the ionic excited states at the CASSCF level with a single active space and why the lowest 1 1 B 2 ϩ state in cis-butadiene is so stabilized compared to the corresponding 1 1 B u ϩ state in the trans isomer are easily identified in view of a VB picture. The CASVB forms a useful bridge from molecular orbital theory to the familiar concepts of chemists.

Carbon nanomaterials, such as carbon nanohorns and carbon nanotubes, have attracted considerable attention for their biomedical applications. We report here the first application of carbon nanohorns (CNHs) as potent laser therapeutic agents for highly selective elimination of microorganisms. This is the first report, supported by direct observations, of the highly selective elimination of yeast and bacteria (Saccharomyces cerevisiae and Escherichia coli) by employing molecular recognition element–CNH complexes and a near-infrared laser.

We identified an insect neuropeptide, namely, allatostatin 1 from Drosophila melanogaster, that transfects living NIH 3T3 and A431 human epidermoid carcinoma cells and transports quantum dots (QDs) inside the cytoplasm and even the nucleus of the cells. QD-conjugated biomolecules are valuable resources for visualizing the structures and functions of biological systems both in vivo and in vitro. Here, we selected allatostatin 1, Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2, conjugated to streptavidin-coated CdSe-ZnS QDs. This was followed by investigating the transfection of live mammalian cells with QD-allatostatin conjugates, the transport of QDs by allatostatin inside the nucleus, and the proliferation of cells in the presence of allatostatin. Also, on the basis of dose-dependent proliferation of cells in the presence of allatostatin we identified that allatostatin is not cytotoxic when applied at nanomolar levels. Considering the sequence similarity between the receptors of allatostatin in D. melanogaster and somatostatin/galanin in mammalian cells, we expected interactions and localization of allatostatin to somatostatin/galanin receptors on the membranes of 3T3 and A431 cells. However, with QD conjugation we identified that the peptide was delivered inside the cells and localized mainly to the cytoplasm, microtubules, and nucleus. These results indicate that allatostatin is a promising candidate for high-efficiency cell transfection and nucleus-specific cell labeling. Also, the transport property of allatostatin is promising with respect to label/drug/gene delivery and high contrast imaging of live cells and cell organelles. Another promising application of allatostatin is that the transport of QDs inside the nucleus would lift the limit of general photodynamic therapy to nucleus-specific photodynamic therapy, which is expected to be more efficient than photosensitization at the cell membrane or in the cytoplasm as a result of the short lifetime of singlet oxygen.

Epidermal growth factor receptor (EGFR), an N-glycosylated transmembrane protein with an intracellular kinase domain, undergoes dimerization by ligand binding resulting in activation of the kinase domain and phosphorylation. Ganglioside GM3 containing sialyllactose inhibits the tyrosine kinase activity of EGFR through carbohydrate to carbohydrate interactions (CCI) between N-glycans with GlcNAc termini on EGFR and oligosaccharides on GM3. In this study, we provide further evidence for CCI between EGFR and GM3. (i) In vitro and in situ, the inhibitory effect of GM3 on EGFR tyrosine kinase was much higher in A431 cells upon exposure of the GlcNAc termini of the N-glycans to glycosidase treatment (neuraminidase and ␤-galactosidase) than in untreated A431 cells. Furthermore, the GM3-mediated inhibition was abrogated by co-incubation with N-glycan containing terminal GlcNAc. (ii) In situ, inhibition of EGFR phosphorylation by GM3 was not observed in ␣-mannosidase IB (ManIB)-knocked down A431 cells that accumulate high mannose-type N-glycans. (iii) EGFR binding to GM3 was enhanced in glycosidase-treated cells that accumulated GlcNAc termini, whereas GM3 did not bind to EGFR from ManIBknocked down cells that accumulated high mannose-type N-glycans. These results indicate that GM3-mediated inhibition of EGFR phosphorylation is caused by interaction of GM3 with GlcNAc-terminated N-glycan on EGFR. Epidermal growth factor receptor (EGFR)4 is a 170-kDa transmembrane glycoprotein with an intracellular tyrosine kinase domain (1). EGFR is expressed in epithelial cells, especially in basement membrane of stratified epithelium and squamous epithelium, and is overexpressed in solid cancers such as carcinoma cells. EGFR belongs to the erbB transmembrane receptor family, and plays a role in cell proliferation and differentiation of the epithelial surface. Ligand (EGF or ␣-celluline) binding leads to dimerization of EGFR and tyrosine phosphorylation, which then activates intracellular signal transducers such as mitogen-activated protein kinase (MAPK), signal transducers and activators of transcription (STAT), Akt, etc. (2, 3).The involvement of oligosaccharides on EGFR function has been widely studied. Twelve N-linked oligosaccharides (N-glycans) are attached to EGFR (1), and loss of these N-glycans reduced EGFR activation (4). Moreover, Taniguchi's group (5-8) also reportedthatEGFRfunctionwaschangedinanN-glycanstructuredependent manner. They demonstrated the biological significance of the core fucosylation of N-glycans on EGFR in intracellular signaling. Mice lacking fucosyltransferase 8 (Fut8 Ϫ/Ϫ mice) have provided insight into the molecular mechanism of core fucosylationregulated cell growth and cell differentiation. Studies using these mice demonstrated that a decrease in core fucose on EGFR caused a reduction in EGFR phosphorylation, which in turn reduced the affinity of EGF for EGFR (8). A bisecting N-acetylglucosamine (␤-1,4-GlcNAc) modification of N-glycans also affects EGFR function (5). The addition of bisecting GlcNAc in...