Slow Sodium Channel Inactivation and Use-dependent Block Modulated by the Same Domain IV S6 Residue

Voltage- and/or conformation-dependent association and dissociation of local anesthetic-class drugs from a putative receptor site in domain IV S6 of the sodium channel and slow conformation transitions of the drug-associated channel have been proposed as mechanisms of use- and frequency-dependent reduction in sodium current. To distinguish these possibilities, we have explored the reactivity to covalent modification by thiols and block of the mutations F1760C and F1760A at the putative receptor site of the cardiac sodium channel expressed as stable cell lines in HEK-293 cells. Both mutations decreased steady-state fast inactivation, shifting V1/2h from −86 ± 1.3 mV (WT) to −72.3 ± 1.4 mV (F1760C) and −67.7 ± 1 mV (F1760A). In the absence of drug, the F1760C mutant channel displayed use-dependent current reduction during pulse-train stimulation, and faster onset of slow inactivation. This mutant also retained some sensitivity to lidocaine. In contrast, the F1760A mutant showed no use-dependent current reduction or sensitivity to lidocaine. The covalent-modifying agent MTS-ET enhanced use-dependent current reduction of the F1760C mutant channel only. The use-dependent reduction in current of the covalently modified channel completely recovered with rest. Lidocaine produced no additional block during exposure to MTS-ET-treated cells (MTS-ET 43 ± 2.7%: MTS-ET lidocaine 47 ± 4.5%), implying interaction at a common binding site. The data suggest that use-dependent binding at the F1760 site results in enhanced slow inactivation rather than alteration of drug association and dissociation from that site and may be a general mechanism of action of sodium-channel blocking agents.     

Antifungal effects of the volatile oils from Allium plants against Trichophyton species and synergism of the oils with ketoconazole

In an attempt to develop stable and safe antifungal agents from natural products (daily foodstuffs in particular), the activities of essential oils from Allium sativum for. pekinense, A. cepa, and A. fistulosum against three Trichophyton species responsible for severe mycoses in humans were investigated and compared with activity of allicin in this study. The fungistatic activities of Allium oils were evaluated by the broth dilution method and disk diffusion assay. The combined effects of Allium oils with ketoconazole were tested by the checkerboard titer test. Among the tested oils, A. sativum for. pekinense oil exhibited the strongest inhibition of growth of T. rubrum, T. erinacei, and T. soudanense with MICs (minimum inhibiting concentrations) of 64microg/ml, while the activities of A. cepa and A. fistulosum were relatively mild. The inhibiting activities of the oils on Sabouraud agar plates were dose dependent against Trichophyton species. Additionally, these oils showed significant synergistic antifungal activity when combined with ketoconazole in the checkerboard titer test and disk diffusion test.     

Effects of p-benzoquinone and melatonin on amyloid fibrillogenesis of hen egg-white lysozyme

More than 20 different human proteins can fold abnormally resulting in the formation of pathological deposits and several lethal degenerative diseases. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained far from complete. In this work, utilizing hen egg-white lysozymes as a model system, two objectives were pursued: (1) to search for suitable conditions for producing amyloid fibrils and (2) to investigate inhibitory activities of two potential molecules against lysozyme fibril formation. Via numerous spectroscopic analyses and electron microscopy, our results showed that the formation of lysozyme amyloid fibrils at pH 2.0 was considerably increased by the addition of salt. Moreover, the inhibition of lysozyme amyloid formation by either p-benzoquinone or melatonin followed a concentration-dependent fashion. Furthermore, p-benzoquinone, in comparison with melatonin, served as a more effective inhibitor against amyloid fibril formation of lysozyme. We believe that a better understanding of how hen egg-white lysozymes aggregate will not only aid in deciphering the molecular mechanism of amyloid fibrillogenesis, but also shed light on a rational design of effective therapeutics for amyloidogenic diseases.     

Preliminary Estimation of the Factors Influencing the Distribution of the Midlittoral Crab Portumnus lysianassa (Herbst, 1796)

In order to acquire information on the factors influencing the distribution of the Mediterranean endemic species Portumnus lysianassa (Herbst, 1796), which lives in the soft substratum midlittoral zone, 83 stations were selected along almost the entire Greek coast. In each sampling station, dredging was carried out, just above the rise of the surf zone, and sediment samples were taken with a core sampler. Two faunal samples were also taken by means of a quadrat from the lower midlittoral zone. The preliminary results of this study show that: P. lysianassa prefers to burrow in the rise of the surf zone; wherever the sediment becomes very coarse or very fine the crab populations decline; there is a correlation between the population abundance of the midlittoral bivalve Donacilla cornea (Poli, 1795) and P. lysianassa, and, moreover, this crab species is related to the composition of the midlittoral assemblage; also, P. lysianassa abundance is negatively correlated with the organic matter content of the sediment; in very exposed areas, D. cornea can be replaced or co-exist with the infralittoral species Donax trunculus (Linnaeus, 1758), and, in these cases, P. lysianassa can be replaced by the closely related infralittoral species Portumnus latipes (Pennant, 1777); when low temperatures or intensive wave action, as well as disturbance caused by swimmers in summer prevail, the population of this crab descends in the upper infralittoral zone.     

Transformation with a gene for myo-inositol O-methyltransferase enhances the cold tolerance of Arabidopsis thaliana

In this study, we report a function of myo-inositol-O-methyltransferase (Imt1) in response to low temperature stress using transgenic Arabidopsis thaliana. Imt1 gene was constructed identical to the Imt1 gene from a halophyte Mesembryanthemum crystallinum. After cold stress, the Imt1 transgenic plants exhibited stronger growth than the wild type plants. The elevated cold tolerance of the Imt1 over-expressing plants was confirmed by the lower electrolyte leakage and accumulation of malondialdehyde, but higher proline and soluble sugar contents in transgenic than wild type plants.     

Unusual mitochondrial genome structure of the freshwater goby Odontobutis platycephala: rearrangement of tRNAs and an additional non-coding region

Mitochondrial DNA was isolated from the Korean freshwater gobioid fish Odontobutis platycephala by long-polymerase chain reaction with conserved primers and this mtDNA was sequenced by primer walking using flanking sequences as sequencing primers. The resultant O. platycephala mtDNA sequence was found to be 17 588 bp in size with a mostly conserved structural organization when compared with that of other teleost fish. Rearrangements of tRNAs (tRNA-Ser, tRNA-Leu, tRNA-His) and an additional non-coding region (533 bp) were present between the ND4 and ND5 genes. In the present paper, the basic characteristics of the O. platycephala mitochondrial genome is reported, including its structural organization, base composition of rRNAs, tRNAs and protein-encoding genes, characteristics of mitochondrial tRNAs and the peculiar rearrangement features of some parts of the mtDNA. Phylogenetic analysis performed using the cytochrome b gene sequences of 16 Korean freshwater fishes (15 gobioids) with the Bayesian algorithm showed that O. platycephala forms a clade (1·00 of posterior probability) with other species of Odontobutis . This suggests that the observed rearrangement between the ND4 and ND5 genes in the O. platycephala mitogenome reflects independent events.     

Antimicrobial and cytotoxic activity of 18 prenylated flavonoids isolated from medicinal plants: Morus alba L., Morus mongolica Schneider, Broussnetia papyrifera (L.) Vent, Sophora flavescens Ait and Echinosophora koreensis Nakai

Antimicrobial activity of the 18 prenylated flavonoids, which were purified from five different medicinal plants, was evaluated by determination of MIC using the broth microdilution methods against four bacterial and two fungal microorganisms (Candida albicans, Saccaromyces cerevisiae, Escherichia coli, Salmonella typhimurium, Staphylococcus epidermis and S. aureus). Papyriflavonol A, kuraridin, sophoraflavanone D and sophoraisoflavanone A exhibited a good antifungal activity with strong antibacterial activity. Kuwanon C, mulberrofuran G, albanol B, kenusanone A and sophoraflavanone G showed strong antibacterial activity with 5–30 μg/ml of MICs. Morusin, sanggenon B and D, kazinol B, kurarinone, kenusanone C and isosophoranone were effective to only gram positive bacteria, and broussochalcone A was effective to C. albicans. IC₅₀ values of papyriflavonol A, kuraridin, sophoraflavanone D, sophoraisoflavanone A and broussochalcone A in HepG2 cells were 20.9, 37.8, 39.1, 22.1, and 22.0 μg/ml, respectively. These results support the use of prenylated flavonoids in Asian traditional medicine to treat microbial infection and indicate a high potential for prenylated flavonoids as antimicrobial agents as well as anti-inflammatory agents.     

Viscoelastic properties of plasticized methylcellulose and chemically crosslinked methylcellulose

Films of methylcellulose (MC), poly(ethylene glycol)400 (PEG400) plasticized MC, and MC gels (MC crosslinked with glutaraldehyde (GA)) were prepared by casting from aqueous solutions. The swelling test has shown that the MC gels were insoluble in water and that their crosslinking density increased with increasing GA and HCl concentrations. The effect of the addition of PEG400 or GA to MC was investigated through dynamic mechanical analysis (DMA). The DMA analysis of PEG400/MC blends has shown that PEG400 was compatible with MC and was an effective plasticizer since the curves of tan δ against temperature exhibited single peaks (corresponding to a single glass transition temperature), which were displaced to lower values with increasing PEG400 content. The thermogravimetric analysis (TGA) indicated that the thermal stability of MC was not affected by the chemical crosslinking. The tensile strength was slightly increased through crosslinking while the elongation was slightly decreased. The presence of moisture in MC hydrogels decreased the tensile strength and enhanced the elongation while the addition of PEG400 decreased the tensile strength but sharply increased the elongation.