Characterization of Borrelia Species Isolated from Ixodid Ticks,Ixodes ovatus

Thirty-two Borrelia isolates were obtained from the adult stage of ixodid ticks, Ixodes ovatus, collected in various localities in Japan. Borrelial isolates were cultivated and analyzed by polyacrylamide gel electrophoresis, with monoclonal antibodies, by pulsed field gel electrophoresis, and by genomic Southern hybridization. All borrelial isolates showed similar protein profiles and monoclonal antibody reactivities, while plasmid profiles were rather diverse. Genomic hybridization using rRNA gene probes demonstrated the genetic similarities of those isolates. We found no significant differences among the borrelial isolates tested, and the restriction fragment length polymorphism patterns of I. ovatus isolates were quite distinct from those of borrelial strains associated with Lyme disease. Therefore, the isolates of Borrelia obtained from I. ovatus were thought to fall into different genospecies.     

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.     

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.     

Phosphorylation of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) by Proline-Directed Protein Kinases and Its Dephosphorylation

Abstract: Excitatory amino acid (EAA) neurotransmitters may play a role in the pathophysiology of traumatic injury to the CNS. Although NMDA receptor antagonists have been reported to have therapeutic efficacy in animal models of brain injury, these compounds may have unacceptable toxicity for clinical use. One alternative approach is to inhibit the release of EAAs following traumatic injury. The present study examined the effects of administration of a novel sodium channel blocker and EAA release inhibitor, BW1003C87, or the NMDA receptor-associated ion channel blocker magnesium chloride on cerebral edema formation following experimental brain injury in the rat. Animals (n = 33) were subjected to fluid percussion brain injury of moderate severity (2.3 atm) over the left parietal cortex. Fifteen minutes after injury, the animals received a constant infusion of BW1003C87 (10 mg/kg, i.v.), magnesium chloride (300 µmol/kg, i.v.), or saline over 15 min (2.75 ml/kg/15 min). In all animals, regional tissue water content in brain was assessed at 48 h after injury, using the wet weight/dry weight technique. In saline-treated control animals, fluid percussion brain injury produced significant regional brain edema in injured left parietal cortex ( p < 0.001), the cortical area adjacent to the site of maximal injury ( p < 0.001), left hippocampus ( p < 0.001), and left thalamus ( p = 0.02) at 48 h after brain injury. Administration of BW1003C87 15 min postinjury significantly reduced focal brain edema in the cortical area adjacent to the site of maximal injury ( p < 0.02) and left hippocampus ( p < 0.01), whereas magnesium chloride attenuated edema in left hippocampus ( p = 0.02). These results suggest that excitatory neurotransmission may play an important role in the pathogenesis of posttraumatic brain edema and that pre- or post-synaptic blockade of glutamate receptor systems may attenuate part of the deleterious sequelae of traumatic brain injury.     

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.     

Platelet talin is phosphorylated by calyculin A

Calyculin A and okadaic acid, potent and cell permeable inhibitors of type 1 and type 2A protein phosphatases, inhibit platelet aggregation and secretion. However, the relationship between phosphatase inhibition and inhibition of platelet function is not well understood. We found that in unstimulated platelets, talin (P235) was phosphorylated at threonine residues by calyculin A. Furthermore, the extent of talin phosphorylation by calyculin A was closely correlated with its inhibition of thrombin-induced platelet aggregation. Since the binding of talin to platelet glycoprotein IIb/IIIa complex has been shown to be affected by its phosphorylation, these results suggest that type 1 and/or type 2A protein phosphatases may play a role in the regulation of membrane-cytoskeleton interaction through dephosphorylation of talin.