Evaluation of Superoxide Scavenging Activities of Hamamelis Extract and Hamamelitannin

Hamamelitannin, which is a component of bark extract of hamamelis (Hamamelis virginior L.), was found to be a potent scavenger of superoxide anion radicals. Superoxide anion scavenging activity of the compound was evaluated by ESR-spin trap method using DMPO (5,5'-dimethyl-1-pyrroline-N-oxide) as a spin trapping agent. The IC₅₀ value (the concentration producing 50% inhibition of superoxide anion radicals) of hamamelitannin was found to be 1.38 ± 0.06 μM much lower than that of ascorbic acid (23.31 ± 2.23 μM). Supporting the superoxide scavenging activity of hamamelitannin, the compound showed both suppresive ability against depolymelization of hyaluronic acid and protective ability against cytotoxicity induced by superoxide anion radicals. Hamamelitannin increased the survival rate of fibroblast to 85.5 ± 3.3%, compared with that of control (27.2 ± 4.3%).     

Development of simulation models for protein folding in a thermal annealing process -I: a simulation of BPTI folding by the pearl necklace model

A model system is proposed to simulate the folding processesof proteins during thermal annealing. This system consists offour subsystems: (i) the pearl necklace model with isotropicinter-residue interactions; (ii) the extended pearl necklacemodel with anisotropic interaction potentials; (iii) moltenglobule phase dynamics; and (iv) final generation of the three-dimensionalstructure of a given protein. In this paper results obtainedwith the pearl necklace model are reported. This model consistsof spherical elements and virtual bonds of 3.8 Å in lengthand is intended to sinudate dynamical processes at relativelyhigh temperature where entropic terms play a dominant role.Inter-residue interactions are composed of spherical soft repulsivepotentials and hydrophobic interactions inherent to respectiveresidues. A simulation of folding processes of BPTI startingfrom the fully extended conformation indicated that intermediates,even at early stages of folding, are not randomly coiled butassume organized structures that resemble, to some extent, thenative conformation.     

Identification of photo-inactive phytochrome A in etiolated seedlings and photo-active phytochrome B in green leaves of the aurea mutant of tomato

The contents of spectrophotometrically measurable phytochrome A (PhyA) and phytochrome B (PhyB) and the corresponding immunochemically detectable apoproteins (PHYA and PHYB) were examined in dark- and light-grown tissues of the aurea mutant of tomato and its wild-type (WT). The amount of PHYA in etiolated aurea seedlings was found to be about 20% of that in the WT; this PHYA showed no photoreversible changes in absorbance, no downregulation of the level of PHYA in light-grown seedlings, and no differential proteolysis of Pr and Pfr species in vitro which was seen in the case of the WT. By contrast, the amount of PHYB in aurea seedlings was not significantly different from that in WT seedlings. Phytochrome isolated from green leaves of the aurea mutant and purified by ion-exchange chromatography showed a red/far-red reversible spectral change, and its elution profile during chromatography was essentially similar to that of PHYB. The results indicate that aurea is a mutant that is deficient in photoactive PhyA at the etiolated stage, when it contains a spectrally inactive PHYA. However, the mutant contains spectrally active PhyB in its green tissue as does the WT.     

In vivo cross-linking of nucleosomal histones catalyzed by nuclear transglutaminase in starfish sperm and its induction by egg jelly triggering the acrosome reaction.

A histone heterodimer, designated as p28, which contains an Nepsilon(gamma-glutamyl)lysine cross-link between Gln9 of histone H2B and Lys5 or Lys12 of histone H4, is present in starfish (Asterina pectinifera) sperm. Treatment of sperm nuclei with micrococcal nuclease produced soluble chromatin, which was size-fractionated by sucrose-gradient centrifugation to give p28-containing oligonucleosome and p28-free mononucleosome fractions, indicating that the cross-link is internucleosomal. When sperm nuclei were incubated with monodansylcadaverine, a fluorescent amine, in the presence or absence of Ca(2+), histone H2B was modified only in the presence of Ca(2+). Gln9, in the N-terminal region, was modified, but the other Gln residues located in the internal region were not, suggesting that the modification takes place on the surface of the nucleosome core by the in situ action of a Ca(2+)-dependent nuclear transglutaminase. Treatment of sperm with the egg jelly, which activates Ca(2+) influx to induce the acrosome reaction, resulted in a significant elevation of the p28 content in the nucleus. This is the first demonstration of an in vivo activation of transglutaminase leading to the formation of a cross-link in intracellular proteins.     

A new method of inhalation anesthesia with nasopharyngeal insufflation in rat experiment.

We have established a new method of anesthesia with nasopharyngeal insufflation for intraoral procedure in rodents. Twelve male Wistar rats weighing 330-390 g were used in this study. Insertion of a feeding tube 1.0 mm in diameter coated with 2% xylocaine jelly was inserted into the nasal cavity approximately 25 mm from the naris, and anesthetization with mixed gas of 100% oxygen with 3-4% enflurane at 0.25-0.5 l/min flow rate was achieved. Using this anesthetic method, a chronic experiment comprising 1-h/day experimental procedure was carried out for 14 days. This method enabled, 1) simple and safe operation of the induction, emergence and anesthetic depth, 2) experimental procedures on the dental/oral region, 3) avoidance of the dyspnea and tachypnea, and 4) avoidance of cumulative effects in daily anesthesia.     

Characterization of a Dopamine-Releasing Action of 6R-l-erythro-Tetrahydrobiopterin: Comparison with a 6S-Form

Abstract: 6R-l -erythro-Tetrahydrobiopterin (6R-BH₄) is a cofactor for aromatic l -amino acid hydroxylases and nitric oxide synthase. Recently, we have reported that independently of its cofactor activities, 6R-BH₄ acts from the outside of neurons in the brain to enhance the release of monoamine neurotransmitters such as dopamine. To characterize the pharmacological properties of the action, we examined the effects of 6S-BH₄, a diastereoisomer of 6R-BH₄, on dopamine release in the rat striatum by using brain microdialysis and compared its effects with those of 6R-BH₄. Perfusion of 6S-BH₄ or 6R-BH₄ through the dialysis probe increased extracellular dopamine levels (an index of in vivo dopamine release) concentration dependently; the maximal increase by 6S-BH₄, was one-sixth of that by 6R-BH₄. 6S-BH₄ increased extracellular DOPA levels in the presence of NSD 1015, an inhibitor of aromatic l -amino acid decarboxylase (an index of in vivo tyrosine hydroxylase activity), to an extent similar to the increase induced by 6R-BH₄. The increase in the DOPA levels induced by either of the pteridines was abolished after pretreatment of rats with α-methyl-p-tyrosine (an inhibitor of tyrosine hydroxylase). Under the same conditions, the 6S-BH₄-induced dopamine release was abolished, but most of the 6R-BH₄-induced increase persisted. Coadministration of 6S-BH₄ with 6R-BH₄ inhibited the increase in dopamine release induced by 6R-BH₄ alone. These results show that 6R-BH₄ stimulates dopamine release by acting at the specific recognition site on the neuronal membrane, and that 6S-BH₄ acts as an antagonist of 6R-BH₄ at this site, although it has cofactor activities.     

A comparison of screening methods for antioxidant activity in seaweeds

The inhibition of lipid peroxidation and radical scavenging effects were studied to evaluate the antioxidant activity for extracts of 17 species of seaweed. The antioxidant effect was evaluated by determination of lipoxygenase activity and by α, α-diphenyl-β-picrylhydrazyl (DPPH) decolorization. Lipoxygenase activity was depressed in the presence of aqueous and ethanol extracts of 4 algal species; Sargassum species had the highest antioxidant activity of all the species examined. The ethanol extracts of one Sargassum species showed competitive inhibition with the substrate. The same species also showed radical scavenging activity in the DPPH decolorization test. Comparison of these results shows no relationship between enzyme inhibition and radical scavenging activity. This revised version was published online in August 2006 with corrections to the Cover Date.