Catalytic properties of yeast protein kinase C: difference between the yeast and mammalian enzymes.

With bovine myelin basic protein as a model common substrate, protein kinases C (PKC) purified from yeast (Saccharomyces cerevisiae) and mammalian tissue (rat brain) were shown to exhibit clearly different catalytic properties. The major sites of phosphorylation in bovine myelin basic protein by the yeast PKC were identified: Thr-19, Thr-34, and Thr-65. These sites are distinctly different from those for the mammalian PKC: Ser-8, Ser-46, Ser-55, Ser-110, Ser-132, Ser-151, and Ser-161, which were previously identified [Kishimoto, A., Nishiyama, K., Nakanishi, H., Uratsuji, Y., Nomura, H., Takeyama, Y., & Nishizuka, Y. (1985) J. Biol. Chem. 160, 12492-12499]. The results suggest that the yeast and mammalian enzymes may play distinct roles in cellular regulation. No evidence is available, however, that a yeast-type PKC exists in mammalian tissues. An oligopeptide containing the sequence around Thr-19 of bovine myelin basic protein, Lys-Tyr-Leu-Ala-Ser-Ala-Ser-Thr(19)-Met-Asp-His-Ala, can be used as a substrate for selective assaying of the yeast PKC.     

Uptake of adenine by purine permeases of Coffea canephora

Purine permeases (PUPs) mediate the proton-coupled uptake of nucleotide bases and their derivatives into cytosol. PUPs facilitate uptake of adenine, cytokinins and nicotine. Caffeine, a purine alkaloid derived from xanthosine, occurs in only a few eudicot species, including coffee, cacao, and tea. Although caffeine is not an endogenous metabolite in Arabidopsis and rice, AtPUP1 and OsPUP7 were suggested to transport caffeine. In this study, we identified 15 PUPs in the genome of Coffea canephora. Direct uptake measurements in yeast demonstrated that CcPUP1 and CcPUP5 facilitate adenine – but not caffeine – transport. Adenine uptake was pH-dependent, with increased activity at pH 3 and 4, and inhibited by nigericin, a potassium–proton ionophore, suggesting that CcPUP1 and CcPUP5 function as proton-symporters. Furthermore, adenine uptake was not competitively inhibited by an excess amount of caffeine, which implies that PUPs of C. canephora have evolved to become caffeine-insensitive to promote efficient uptake of adenine into cytosol.     

Double assembly composed of lectin association with columnar molecular assembly of cyclic tri-beta-peptide having sugar units

A novel double assembly was prepared by association between a columnar molecular assembly of cyclic tri-beta-peptides having sugar units and lectins. The NMR, FT-IR, and circular dichroism (CD) spectroscopy as well as computational calculations revealed that this compound took a flat and C3 symmetrical conformation and that the amide N-H and C=O groups protruded vertically to the ring plane. This disk-shaped molecule stacked one by one to form a columnar structure via intermolecular hydrogen bonds between the amide groups. WGA lectin moderately bound to this columnar assembly to form a double assembly. Another lectin (Con A) disturbed the columnar structure upon strong binding, and RCA lectin showed no binding. Fluorescence spectroscopy revealed that the association between WGA lectin and columnar assembly of cyclic glycopeptide could be achieved due to the high density of the hydroxyl groups on the assembly surface (cluster effects). Interestingly, after cross-linking the lectins bound to the columnar assembly (the double assembly) by glutaraldehyde, the core column of cyclic tri-beta-peptides could be washed away to leave the protein nanotube.     

Habitat use by endangered Japanese crayfish (<Emphasis Type="Italic">Cambaroides japonicus</Emphasis>) in low-gradient streams of southern Hokkaido,Japan: reach and microhabitat-scale analysis

The Japanese crayfish (Cambaroides japonicus), the only native crayfish in Japan, is endangered and has experienced rapid population declines. We surveyed the habitat requirements of Japanese crayfish at the reach and microhabitat scales in semi-natural low-gradient streams. Habitat use by Japanese crayfish differed between the spatial scales. Reach-scale analysis revealed that the bed slope was the only positive predictor of crayfish density. This finding indicates that relatively high-gradient reaches, such as headwater reaches, are an important habitat for the conservation of Japanese crayfish in low-gradient streams. Microhabitat-scale analysis showed that crayfish density was positively affected by substrate coarseness and the presence of instream vegetation cover (bank vegetation, woody debris, and leaf patches), whereas it was negatively affected by distance from the stream edge. Coarse substrates and vegetation cover may function as shelters from water flows and predators during low flow periods. The use of stream-edge areas may allow quick access to refugia and enable the crayfish to avoid unexpected flood disturbance and predation. These habitat characteristics should therefore be preserved for the conservation of Japanese crayfish, and scale-dependent habitat characteristics should be considered in future conservation plans.     

Artificial ladder-shaped polyethers that inhibit maitotoxin-induced Ca2+ influx in rat glioma C6 cells

Maitotoxin (MTX) is a ladder-shaped polyether produced by the epiphytic dinoflagellate Gambierdiscus toxicus. It is known to elicit potent toxicity against mammals and induce influx of Ca(2+) into cells. An artificial ladder-shaped polyether possessing a 6/7/6/6/7/6/6 heptacyclic ring system, which was designed for elucidating interactions with transmembrane proteins, was found to be the most potent inhibitor against MTX-induced Ca(2+) influx that has ever been reported.     

Discovery of CS-2100, a potent, orally active and S1P3-sparing S1P1 agonist

S1P(3)-sparing S1P(1) agonists have attracted attention as a suppressant of autoimmunity with reduced side effects. Our synthetic efforts and extensive SAR studies led to the discovery of 10b named CS-2100 with the EC(50) value of 4.0 nM for human S1P(1) and over 5000-fold selectivity against S1P(3). The in vivo immunosuppressive efficacy was evaluated in rats on host versus graft reaction and the ID(50) value was determined at 0.407mg/kg. The docking studies of CS-2100 with the homology model of S1P(1) and S1P(3) showed that the ethyl group on the thiophene ring of CS-2100 was sterically hindered by Phe263 in S1P(3), not in the case of Leu276 in S1P(1). This observation gives an explanation for the excellent S1P(3)-sparing characteristic of CS-2100.     

Transferrin receptor-1 suppresses neurite outgrowth in neuroblastoma Neuro2A cells

Transferrin receptor-1 (TfR1) is a cell membrane-associated glycoprotein responsible for incorporation of the iron bound to transferrin through an endocytotic process from the circulating blood. Iron is believed to play a dual role as an active center of the electron transfer system in mitochondria and as an endogenous cytotoxin through promoted generation of reactive oxygen species in different eukaryotic cells. In this study, we evaluated expression profiles of different genes related to iron mobilization across plasma membranes in neuronal cells. Marked mRNA expression was seen for various iron-related genes such as TfR1 in cultured mouse neocortical neurons, while TfR1 mRNA levels were more than doubled during culture from 3 to 6days. In mouse embryonal carcinoma P19 cells endowed to differentiate into neuronal and astroglial lineages, a transient increase was seen in both mRNA and corresponding protein for TfR1 in association with neuronal marker expression during culture with all-trans retinoic acid (ATRA). In neuronal Neuro2A cells cultured with ATRA, moreover, neurite was elongated together with increased expression of both mRNA and protein for TfR1. Overexpression of TfR1 significantly decreased the length of neurite elongated, however, while significant promotion was invariably seen in the neurite elongation in Neuro2A cells transfected with TfR1 siRNA as well as in Neuro2A cells cultured with an iron chelator. These results suggest that TfR1 would be highly expressed by neurons rather than astroglia to play a negative role in the neurite outgrowth after the incorporation of circulating transferrin in the brain.     

Interaction of integrin alpha(v)beta3 with nectin. Implication in cross-talk between cell-matrix and cell-cell junctions

Cell-matrix and cell-cell junctions cross-talk together, and these two junctions cooperatively regulate cell movement, proliferation, adhesion, and polarization. However, the mechanism of this cross-talk remains unknown. An immunoglobulin-like cell-cell adhesion molecule nectin first trans-interacts with each other to form cell-cell adhesion and induces activation of Rap1, Cdc42, and Rac small G proteins through c-Src. Trans-interacting nectin then recruits another cell-cell adhesion molecule cadherin to the nectin-based cell-cell adhesion sites and forms adherens junctions (AJs). Here, we show that integrin alpha(v)beta3 functionally and physically associates with nectin. Integrin alpha(v)beta3 colocalized with nectin at the nectin-based cell-cell adhesion sites. The association of integrin alpha(v)beta3 with nectin was direct and was mediated through their extracellular regions. This interaction was necessary for the nectin-induced signaling. Focal adhesion kinase, which relays the integrin-initiated outside-in signals to the intracellular signaling molecules, was also involved in the nectin-induced signaling. During the formation of AJs, the high affinity form of integrin alpha(v)beta3 co-localized with nectin at the primordial cell-cell contact sites, and then after the establishment of AJs, this high affinity form of integrin alpha(v)beta3 was converted to the low affinity form, which continued to co-localize with nectin. Thus, integrin alpha(v)beta3 and nectin play pivotal roles in the cross-talk between cell-matrix and cell-cell junctions and the formation of cadherin-based AJs.