Studies on the Flavor of Green Tea

In order to investigate how the flavor of fresh tea-leaves turn into that of green tea, six essential oils were obtained from chopped fresh tea-leaves, whole tea-leaves, and their residues after steaming, green tea and black tea by steam-distillation. They are then compared with each other in regard to their flavor, amount of I₂-absorption, βγ-hexenol content and ultraviolet spectrum. It is concluded that the change mentioned above is produced partly by fleeing of βγ-hexenol and partly by some chemical change concerning βγ-hexenol.     

Kinetochore stretching inactivates the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) monitors the attachment of microtubules to the kinetochore and inhibits anaphase when microtubule binding is incomplete. The SAC might also respond to tension; however, how cells can sense tension and whether its detection is important to satisfy the SAC remain controversial. We generated a HeLa cell line in which two components of the kinetochore, centromere protein A and Mis12, are labeled with green and red fluorophores, respectively. Live cell imaging of these cells reveals repetitive cycles of kinetochore extension and recoiling after biorientation. Under conditions in which kinetochore stretching is suppressed, cells fail to silence the SAC and enter anaphase after a delay, regardless of centromere stretching. Monitoring cyclin B levels as a readout for anaphase-promoting complex/cyclosome activity, we find that suppression of kinetochore stretching delays and decelerates cyclin B degradation. These observations suggest that the SAC monitors stretching of kinetochores rather than centromeres and that kinetochore stretching promotes silencing of the SAC signal.     

Properties of the uptake and release of neurotransmitter glutamate in cerebral cortical tissue of guinea pigs

In order to investigate the dynamics of glutamate as a neurotransmitter and to avoid a complication by its metabolism, we studied the uptake and release of labeled non-metabolizabled-isomers of aspartate and glutamate in cerebral cortical slices and synaptosome preparation from guinea-pigs. The rate of uptake ofd-aspartate and glutamate was mutually inhibited in a non-competitive fashion, indicating that their uptake mechanisms are not exactly the same. By ouabain (0.05 mM), the uptake ofd-aspartate and glutamate into synaptosome preparation was less inhibited than that into cerebral slices. In synaptosome preparation most of the preloadedd-aspartate and glutamate was released by high-potassium (50 mM) stimulation, whereas in cerebral slices only a slight release was observed. However, when the slices were superfused with a medium free of sodium ions, which are absolutely necessary for the uptake, after preloaded with the labeled amino acids in the standard medium, a distinct release of radioactivity was induced by high-potassium stimulation. This potassium-induced release corresponded to only about 20% of the radioactivity accumulated in the slices. The accumulation ofd-aspartate and glutamate into cerebral slices was much larger on the basis of their protein content than that into synaptosome preparation, when a high concentration (1 mM) of the amino acids was added to the medium. These observations suggest that the uptake system ofd-aspartate and glutamate in cerebral slices is quite different from that in synaptosome preparation, and that the accumulation into cerebral slices is mainly localized in glial cells. In vivo the glial cell uptake is probably more important in removing the released neurotransmitter glutamate.Dedicated to Professor Yasuzo Tsukada.     

A new endo-beta-galactosidase acting on the Gal beta 1-3Gal linkage of the proteoglycan linkage region.

A new type of endo-beta-galactosidase acting on the linkage region of peptidochondroitin sulfate was isolated from the mid-gut gland of the mollusk Patinopecten. The purification procedure included ammonium sulfate precipitation, Sephacryl S-200HR gel filtration, DEAE-Sephacel chromatography, and TSKgel Phenyl-5PW RP high performance liquid chromatography. The purified enzyme was free from exoglycosidases, sulfatases, and phosphatases. The specificity of the enzyme was as follows. 1) It acted on the internal galactoside linkage of sugar chains; 2) it specifically hydrolyzed the galactosylgalactose (Gal beta 1-3Gal) linkage, but not the galactosylxylose (Gal beta 1-4Xyl) linkage in the linkage region of peptidoglycans; 3) the enzyme activity was unaffected by the type of glycosaminoglycan, chondroitin sulfate, dermatan sulfate or heparan sulfate used as a substrate; 4) keratan sulfate and some oligosaccharides from glycolipid were not degraded by the enzyme. These properties of the endo-beta-galactosidase characterize it as a new endo-beta-galactosidase with unique specificity.     

Chemical synthesis of diastereomeric diadenosine boranophosphates (ApbA) from 2'-O-(2-cyanoethoxymethyl)adenosine by the boranophosphotriester method

We have synthesized diastereomerically pure diadenosine 3',5'-boranophosphates (Ap(b)A) by using the boranophosphotriester method from ribonucleosides protected with the 2'-hydroxy protecting group 2-cyanoethoxymethyl (CEM). Melting curves of the triple-helical complex of the dimer Ap(b)A and 2poly(U) at high ionic strength revealed that presumptive (Sp)-Ap(b)A had a much higher affinity and presumptive (Rp)-Ap(b)A a much lower affinity for poly(U) than the natural dimer ApA did. In contrast, the affinities of these dimers for poly(dT) were similar. Both the (Rp)- and the (Sp)-boranophosphate diastereomers showed much higher resistance to digestion by snake venom phosphodiesterase and nuclease P1 than ApA did. They have potential for use as synthons to be incorporated into boranophosphate oligonucleotides. In particular, because oligonucleotides containing Sp boranophosphate nucleotides are expected to bind more strongly and specifically to RNA than natural oligoribonucleotides do, they may find application in the isolation and detection of functional RNA in basic research and diagnostics.     

Piezoelectric property of bundled peptide nanotubes stapled by bis‐cyclic‐β‐peptide

Cyclic tetra‐β‐peptides (CP4s) and a bis‐CP4 were synthesized to prepare peptide nanotubes (PNTs) by molecular stacking of cyclic peptides. The addition of bis‐CP4 to the PNT preparation afforded PNT bundles increasing the direct and converse piezoelectiric coefficients, which is ascribable to bis‐CP4 stapling PNTs into the parallel alignment of PNT dipoles.     

Interaction between collagens and glycosaminoglycans investigated using a surface plasmon resonance biosensor.

The interactions of glycosaminoglycans with collagens and other glycoproteins in extracellular matrix play important roles in cell adhesion and extracellular matrix assembly. In order to clarify the chemical bases for these interactions, glycosaminoglycan solutions were injected onto sensor surfaces on which collagens, fibronectin, laminin, and vitronectin were immobilized. Heparin bound to type V collagen, type IX collagen, fibronectin, laminin, and vitronectin; and chondroitin sulfate E bound to type II, type V, and type VII collagen. Heparin showed a higher affinity for type IX collagen than for type V collagen. On the other hand, chondroitin sulfate E showed the highest affinity for type V collagen. The binding of chondroitin sulfate E to type V collagen showed higher affinity than that of heparin to type V collagen. These data suggest that a novel characteristic sequence included in chondroitin sulfate E is involved in binding to type V collagen.     

A novel 4-methylumbelliferyl-beta-D-xyloside derivative, sulfate-O-3- xylosylbeta1-(4-methylumbelliferone), isolated from culture medium of human skin fibroblasts, and its role in methylumbelliferone-initiated glycosaminoglycan biosynthesis

Human skin fibroblasts were incubated in the presence of 4- methylumbelliferyl-beta-D-xyloside (Xyl-MU). The culture medium was recovered and Xyl-MU derivatives which were initiated by the Xyl-MU acting as a primer were purified. As a result, a novel Xyl-MU derivative was isolated, in addition to previously reported Xyl-MU derivatives such as glycosaminoglycan-MU, Gal-Gal-Xyl-MU, Gal-Xyl-MU, SA-Gal-Xyl-MU, Xyl-Xyl-MU, GlcA-Xyl-MU, and sulfate-GlcA-Xyl-MU. This Xyl-MU derivative was subjected to carbohydrate composition analysis, enzyme digestion, ion-spray mass spectrometric analysis, and Smith degradation. The results indicated that it was sulfate- O -3-Xyl-MU. When Xyl-MU was incubated with [35S]PAPS using a homogenate prepared from the same cultured skin fibroblasts, [35S]sulfate- O -3-Xyl-MU was produced. Moreover, when Xyl-MU was incubated with UDP-[3H]Gal, [3H]galactose was transferred to Xyl-MU, but when sulfate- O -3-Xyl-MU was incubated with UDP-[3H]Gal, [3H]galactose was not transferred. These results indicate that chain elongation from Xyl-MU is inhibited by sulfation of Xyl-MU, and that Xyl-MU sulfation is involved in the control of Xyl-MU-initiated glycosaminoglycan biosynthesis.