Direct determination of the π-helix structure and helix transition behavior of poly(β-phenethyl l-aspartate) via synchrotron X-ray diffraction

The helix-sense inversions of poly(β-phenethyl l -aspartate) (2P) and diblock copolymers (2P-3P), with 2P and poly(β-phenylpropyl l -aspartate) (3P) blocks, were studied in their solid states using synchrotron wide-angle X-ray diffraction and small-angle X-ray scattering. The characteristic parameters of the π-helix structure of 2P were directly determined in situ after the helix transition at a high temperature. In the 2P-3P block copolymers, the main chains of the 3P blocks initially convert from right- to left-handed α-helices, and then the 2P blocks convert irreversibly from right-handed α-helices to left-handed π-helices. The chemical structures of the side chains of poly(l -aspartic acid ester)s significantly affect their helix transition behaviors.     

Phosphoinositides and synaptic function in NG108-15 neuroblastoma x glioma hybrid cells.

1. The second-messengers system of bradykinin (BK) receptors was examined in NG108-15 neuroblastoma x glioma hybrid cells. 2. An application of BK induced an immediate outward (K+) current and acetylcholine (ACh) release, which are generated through inositol 1,4,5-trisphosphate (InsP3)-dependent Ca2+ ions. 3. Application of phorbol dibutyrate (a protein kinase C activator) produced a voltage-dependent inward current and inhibited another K+ (M)-current. 4. A similar current response has been produced by ACh in NG108-15 cells transfected with rodent muscarinic ACh receptor I and III subtype genes. 5. These results suggest a dual and time-dependent role for these two intracellular messengers in the control of neuronal signalling by BK and ACh.     

Characteristics on staining of leukocytes in experimental animals]

The staining characteristics of the peripheral blood cells from mouse, rat, guinea pig, rabbit, dog, marmoset and monkey were studied. In marmoset, it is easy to distinguish neutrophils from eosinophils by using the phosphate-buffered solution of pH 5 or 6. It was found in the special staining methods that neutrophil granules showed intense peroxidase and Sudan black B reactions in marmoset in comparison with those in the other species of experimental animals. Neutrophil granules rabbit was, however, intensely stained with esterase and acid phosphatase.     

Perithecial formation inGelasinospora reticulispora V. Microscopically versus photobiologically recognizable differentiation of hyphae

Morphological changes in the early development of photobiologically induced perithecium weve microscopically observed with hyphae in apically growing mycellum ofGelasinospora reticulispora. No morphological differentiation was recognized in any hyphae during the inductive darkness and the following lag period. Semispherical protrusion was first noticed on slender hyphae 7 to 9 hr after the photoinductive treatment, and it developed into a coil-shaped mass within 3 to 6 hr thereafter. This hyphal mass grew to a young perithecium having 50 to 100 μm in diameter ca. 24 hr after the photoinduction. The density of induced pertithecium was dependent upon spot size of irradiation; the smaller the exposing area of mycelium, the higher the density. The result indicated that most slender hyphae can be responsive to photobiological induction of perithecium.     

Immobilization and culture of coffee (Coffea arabica L.) cells on novel culture systems using a basket-shaped unit, “EGSTAR”

Summary A simple and new basket-shaped unit for agitation made of stainless steel (EGSTAR), in which immobilized coffee cells in Ca-alginate gel beads were packed, was placed in a jar fermentor (System-1). This system allowed the plant cells to grow submersed in the unit even at high agitation speed (650 rpm). Only a small amount of cells existed out of the EGSTAR. Most of the purine alkaloids produced were released into the medium. Suspended coffee cells in the jar fermentor were also possibly immobilized onto a polyurethane foam sheet fixed inside the net of the EGSTAR (System-2). The total cells in System-2 biotransformed theobromine to caffeine (77.9%). Other plant cell suspensions were also immobilized as efficiently as were the coffee cells in this system. Thus, System-2 is a simple and convenient system for immobilization of plant cells to produce secondary metabolites. This paper is Part 79 in the series of “Studies on Plant Tissue Cultures”. For Part 78, see Orihara, Y., Furuya, T., Hashimoto, N., Deguchi, Y., Tokoro, K., and Kanisawa, T., (1992)Phytochemistry 31: 827–831.     

Synchronous Activation of Cell Division by Light or Temperature Stimuli in the Dimorphic Yeast Schizosaccharomyces japonicus

Many fungi respond to light and regulate fungal development and behavior. A blue light-activated complex has been identified in Neurospora crassa as the product of the wc-1 and wc-2 genes. Orthologs of WC-1 and WC-2 have hitherto been found only in filamentous fungi and not in yeast, with the exception of the basidiomycete pathogenic yeast Cryptococcus. Here, we report that the fission yeast Schizosaccharomyces japonicus responds to blue light depending on Wcs1 and Wcs2, orthologs of components of the WC complex. Surprisingly, those of ascomycete S. japonicus are more closely related to those of the basidiomycete. S. japonicus reversibly changes from yeast to hyphae in response to environmental stresses. After incubation at 30°C, a colony of yeast was formed, and then hyphal cells extended from the periphery of the colony. When light cycles were applied, distinct dark- and bright-colored hyphal cell stripes were formed because the growing hyphal cells had synchronously activated cytokinesis. In addition, temperature cycles of 30°C for 12 h and 35°C for 12 h or of 25°C for 12 h and 30°C for 12 h during incubation in the dark induced a response in the hyphal cells similar to that of light. The stripe formation of the temperature cycles was independent of the wcs genes. Both light and temperature, which are daily external cues, have the same effect on growing hyphal cells. A dual sensing mechanism of external cues allows organisms to adapt to daily changes of environmental alteration.     

Organic anion transporter 1 (OAT1/SLC22A6) enhances bioluminescence based on d-luciferin–luciferase reaction in living cells by facilitating the intracellular accumulation of d-luciferin

Bioluminescence (BL) imaging based on d-luciferin (d-luc)–luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d-luc–luciferase reaction, an approach to increase intracellular levels of d-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293?cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293?cells, intracellular accumulation of d-luc was higher than that in control cells, and the specific d-luc uptake mediated by OAT1 was saturable with a Michaelis constant (K_m) of 0.23?μM. The interaction between OAT1 and d-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis–Menten kinetics with an apparent K_m of 0.36?μM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d-luc could be a rate-limiting step in the d-luc–luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d-luc. Our findings suggest that because OAT1 is capable of transporting d-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells.