Analysis of early processes in wound-induced vascular regeneration using TED3 and ZeHB3 as molecular markers

Interruption of the vascular bundles of Zinnia internodes induced transdifferentiation of cells into tracheary elements (TEs) or sieve elements (SEs) within 4 d of wounding. The early stage of the regeneration processes was analyzed using two molecular marker genes, TED3 and ZeHB3, which are expressed specifically in TE precursor cells and immature phloem cells, respectively. An increase in the numbers of TED3 and ZeHB3 mRNA-expressing cells always preceded an increase in the numbers of TEs and SEs formed. The earliest sign of vascular differentiation was the appearance 24 h after wounding of a layer(s) of TED3 mRNA-expressing cells in the inter- and intrafascicular cambial-like regions along the severed vascular bundles. In contrast, the number of ZeHB3 mRNA-expressing cells decreased dramatically along the severed bundles 24 h after wounding, and increased again 36 h after wounding. These results clearly indicate that xylem and phloem differentiation are not synchronized during vascular regeneration. Treatment with 10(-3) M colchicine abolished the expression of ZeHB3 mRNA in pith parenchyma, but not TED3 mRNA; this suggests that cell division is a prerequisite for the transdifferentiation of pith parenchymal cells into immature phloem cells expressing ZeHB3. In contrast, transdifferentiation of pith parenchymal cells to TE precursor cells does not require preceding cell division. However, the inhibition of cell division prevented the formation of both radial files of TEs and the cambial-like layer(s) of TED3 mRNA-expressing cells, and, ultimately, vascular regeneration altogether. These results imply that wound-induced cambial-like activity in and between severed vascular bundles is essential for vascular regeneration.     

Neurotrophin-3 is required for appropriate establishment of thalamocortical connections

Ca2+-permeable AMPARs are inwardly rectifying due to block by intracellular polyamines. Neuronal activity regulates polyamine synthesis, yet whether this affects Ca2+-AMPAR-mediated synaptic transmission is unknown. We test whether 4 hr of increased visual stimulation regulates glutamatergic retino-tectal synapses in Xenopus tadpoles. Tectal neurons containing Ca2+-AMPARs form a gradient along the rostro-caudal developmental axis. These neurons had inwardly rectifying AMPAR-mediated EPSCs. Four hours of visual stimulation or addition of intracellular spermine increased rectification in immature neurons. Polyamine synthesis inhibitors blocked the effect of visual stimulation, suggesting that visual activity regulates AMPARs via the polyamine synthesis pathway. This modulation resulted in changes in the integrative properties of tectal neurons. Regulation of polyamine synthesis by physiological stimuli is a novel form of modulation of synaptic transmission important for understanding the short-term effects of enhanced sensory experience during development.     

Alpha-glucosidase inhibitor from the seeds of balsam pear (Momordica charantia) and the fruit bodies of Grifola frondosa

Alpha-glucosidase inhibitory activities were found in aqueous methanol extracts of the seeds of Momordica charantia and the fruit bodies of Grifola frondosa. An active principle against the enzyme prepared from rat small intestine acetone powders was isolated and characterized. The structure of the isolated compound was identified as D-(+)-trehalose by FDMS, 1H-, 13C-NMR, and [alpha]D measurements. The inhibitory activity of trehalose was compared with 1-deoxynojirimycin. Trehalose showed 45% inhibitory activity at the concentration of 2 x 10(-3) m, but 1-deoxynojirimycin had 52% inhibitory activity at 1 x 10(-7) M.     

Production of antibacterial violet pigment by psychrotropic bacterium RT102 strain

The antibacterial action of violet pigment, a mixture of violacein and deoxyviolacein, isolated from phychrotrophic bacterium RT102 strain was examined, and the operational conditions for the effective production of violet pigment were studied. The antibacterial activity of the violet pigment was confirmed for several bacteria such asBacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, andPseudomonas aeruginosa, and the high concentration of violet pigment, above about 15 mg/L, caused not only growth inhibition but also death of cells. The growth properties of RT102 strain were clarified under various incubation conditions such as pH, temperature, and dissolved oxygen concentration. The maximum violet pigment concentration,i.e. 3.7 g/L, and the maximum productivity of violet pigment,i.e. 0.12 g L⁻¹h⁻¹, were obtained in a batch culture of pH 6, 20°C, and 1 mg/L of dissolved oxygen concentration.     

Evaluation of the anti-oxidative effect (in vitro) of tea polyphenols

Forty-three polyphenols from tea leaves were evaluated for their anti-oxidative effect against lipid peroxidation by the ferric thiocyanate method in vitro. Among these, 1,4,6-tri-O-galloyl-beta-D-glucose (hydrolyzable tannin) showed the highest anti-oxidative activity against lipid peroxidation, even stronger than that of 3-tert.-butyl-4-hydroxyanisole (BHA). The assay demonstrates that tea polyphenols, except for desgalloylated dimeric proanthocyanidins that possess a catechin structure in the upper unit and desgalloylated flavan-3-ols, and excepting theaflavin 3,3'-di-O-gallate, had more anti-oxidative activity than that of alpha-tocopherol. The chemical structure-activity relationship shows that the anti-oxidative action advanced with the condensation of two molecules of flavan-3-ols as well as with 3-O-acylation in the flavan skeleton such as that by galloyl, (3'-O-methyl)-galloyl, and p-coumaroyl groups.     

DPPH radical scavengers from dried leaves of oregano (Origanum vulgare)

1,1-Dipehnyl-2-picrylhydrazyl (DPPH) radical scavenging activities were found in the extract of dried leaves of oregano (Origanum vulgare). The water-soluble active ingredients were isolated, and their structures were determined to be 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate and 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl 4-O-methylprotocatechuate by (1)H-, (13)C-NMR, DEPT, HMQC, and HMBC spectral analyses, and by NOE experiments. The DPPH radical scavenging activities of these compounds were compared with those of rutin, quercetin and rosmarinic acid at a concentration of 2 x 10(-5) M. The scavenging activity of 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate was almost the same as that of quercetin and rosmarinic acid, but that of 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybennzyl 4-O-methylprotocatechuate was less than that of quercetin, rosmarinic acid and 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate. The amount of 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate was estimated to be 3.8 mg/1 g of dried leaves by an HPLC analysis.     

Effects of deficiencies of STAMs and Hrs,mammalian class E Vps proteins,on receptor downregulation

The STAM family proteins, STAM1 and STAM2/EAST/Hbp, are phosphotyrosine proteins that contain SH3 domains and ubiquitin-interacting motifs. Their yeast homologue, Hse1, and its binding protein, Vps27, are involved in the vacuolar membrane transport machinery. Here we show that STAM1 and STAM2 are localized to the endosomal membrane. Some of these complexes contain Eps15, an endocytic protein, which accumulates in clumps upon expression of a dominant-negative form of Vps4-A, an AAA-type ATPase, that is required for normal endosome function. These results support the idea that the STAMs are mammalian vacuolar protein sorting (Vps) proteins. We also demonstrate that ligand-mediated epidermal growth factor receptor (EGFR) degradation is partially but not completely impaired in both Hrs(-/-) and STAM1(-/-)STAM2(-/-) mouse embryonic fibroblasts. Furthermore, endosome swelling is seen in both Hrs(-/-) and STAM1(-/-)STAM2(-/-) cells. These results suggest that the STAMs and Hrs play important roles in the mammalian endosomal/vacuolar protein sorting pathway.     

Collective movement of epithelial cells on a collagen gel substrate

Collective cell movement acts as an efficient strategy in many physiological events, including wound healing, embryonic development, and morphogenesis. We found that epithelial cells (Madin-Darby canine kidney cell) migrated collectively along one direction on a collagen gel substrate. Time-lapse images of Madin-Darby canine kidney cells cultured on type-I collagen gels and glass substrates were captured by phase contrast microscopy equipped with an incubation system. On the gel substrate, the directions of cell movement gradually converged on one direction as the number of cells increased, whereas the cells moved randomly on the glass substrate. We also observed "leader" cells, which extended large lamellae and were accompanied by many "follower" cells, migrating in the direction of oriented collagen fibers. The mean-squared displacement of each cell movement and the spatial correlation function calculated from the spatial distribution of cell velocity were obtained as functions of observation time. In the case of the gel substrate, the spatial correlation length increased gradually, representing the collectiveness of multicellular movement.