Molecular characteristics of tissue-bound semicarbazide-sensitive amine oxidase (SSAO) in guinea pig tissues

Various mammalian tissues contain a tissue-bound amine oxidizing enzyme distinct from mitochondrial outer membrane enzyme, monoamine oxidase (MAO, EC 1.4.3.4), termed semicarbazide-sensitive amine oxidase (SSAO, EC 1.4.3.6). An increase in SSAO activity was found in patients suffering from vascular disorders such as diabetes and diabetic complications. It has previously been shown that 2-bromoethylamine (2-BEA) is a potent, and selective suicidal inhibitor of tissue-bound SSAO. The aim of this study was to investigate the interaction of this suicidal SSAO inhibitor with the tissue-bound enzyme in guinea pig lung, kidney, stomach, and heart homogenates. The conditions necessary for this inhibitor to titrate the concentrations of this enzyme were also determined. 2-BEA appears to interact with SSAO, as reported previously for this enzyme from different sources, in a manner consistent with an irreversible, "suicide" reaction. Because of this property, 2-BEA could be used to titrate the concentrations of SSAO active centers in these tissues under the appropriate conditions employed. Although some possible non-specific binding of the inhibitor to sites other than the active center of the enzyme, metabolism of this inhibitor and/or presence of enzyme subtypes was hypothesized, the molecular characteristics of SSAO in these tissues (Km, Vmax values, enzyme efficiencies, approximate enzyme concentrations, and molecular turnover numbers) towards the substrate kynuramine (0.1 mM) at pH 7.4 and 37 degrees C have been estimated.     

Mouse model of Prinzmetal angina by disruption of the inward rectifier Kir6.1

The inwardly rectifying K(+) channel Kir6.1 forms K(+) channels by coupling with a sulfonylurea receptor in reconstituted systems, but the physiological roles of Kir6.1-containing K(+) channels have not been determined. We report here that mice lacking the gene encoding Kir6.1 (known as Kcnj8) have a high rate of sudden death associated with spontaneous ST elevation followed by atrioventricular block as seen on an electrocardiogram. The K(+) channel opener pinacidil did not induce K(+) currents in vascular smooth-muscle cells of Kir6.1-null mice, and there was no vasodilation response to pinacidil. The administration of methylergometrine, a vasoconstrictive agent, elicited ST elevation followed by cardiac death in Kir6.1-null mice but not in wild-type mice, indicating a phenotype characterized by hypercontractility of coronary arteries and resembling Prinzmetal (or variant) angina in humans. The Kir6.1-containing K(+) channel is critical in the regulation of vascular tonus, especially in the coronary arteries, and its disruption may cause Prinzmetal angina.     

Functional vanilloid receptors in cultured normal human epidermal keratinocytes

Vanilloid receptor subtype 1, VR1, is an ion channel that serves as a polymodal detector of pain-producing chemicals such as capsaicin and protons in primary afferent neurons. Here we showed that both capsaicin and acidification produced elevations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured human epidermal keratinocytes. The capsaicin- and acidification-evoked increases in [Ca(2+)](i) were inhibited by capsazepine, an antagonist to VR1. VR1-like immunoreactivity was observed in the cells. These findings suggest that functional VR1-like protein is present and functions as a sensor against noxious chemical stimuli, such as capsaicin or acidification, in epidermal keratinocytes.     

Dimer structure of magainin 2 bound to phospholipid vesicles

Magainin 2 from African clawed frog Xenopus laevis is an antimicrobial peptide with broad spectra and action mechanisms considered to permeabilize bacterial membranes. CD, vibration, and solid-state NMR spectroscopies indicate the peptide adopts an alpha-helical conformation on binding to phospholipid bilayers, and its micelle-bound conformation, being monomeric and alpha-helical, is well detailed. We showed, however, that the peptide dimerizes on binding to phospholipid bilayers. This difference in the conformation and aggregation state between micelle- and bilayer-bound states prompted us to analyze the conformation of an equipotent analog of magainin 2 (F5Y,F16W magainin 2) bound to phosphatidylcholine vesicles using transferred nuclear Overhauser enhancement (TRNOE) spectroscopy. While observed medium-range TRNOE cross peaks were characteristic of alpha-helix, many long-range cross peaks were not compatible with the peptide's monomeric state. Simulated annealing calculations generated dimer structures indicating (1) two peptide molecules have a largely helical conformation in antiparallel orientation forming a short coiled-coil structure, (2) residues 4-20 are well converged and residues 9-20 are in an alpha-helical conformation, and (3) the interface of the two peptide molecules is formed by well-defined side chains of hydrophobic residues. Finally, determined structures are compatible with numerous investigations examining magainin-phospholipid interactions.     

Intraspecific variation in the status of ant symbiosis on a myrmecophyte,<Emphasis Type="Italic"> Macaranga bancana,</Emphasis> between primary and secondary forests in Borneo

A tree species, Macaranga bancana , distributed in South East Asian tropics has a mutualistic relationship with specific symbiotic ant species, which defend the plant from herbivores. To examine the intraspecific variation in the status of the ant-plant symbiosis among microhabitats of different light conditions, we investigated the species composition of nesting ants and the herbivory damage on M. bancana saplings by field observations and sampling in primary and secondary forests in Sarawak. In addition, the effectiveness of non-ant (physical and chemical) defenses were estimated by feeding the larvae of a polyphagous lepidopteran with M. bancana leaves from saplings in the two types of forests. All saplings in the primary forest were colonized by two Crematogaster ant species that had been known to be the obligate symbionts of M. bancana, while in the secondary forest, about half of the saplings were occupied by several ant species that were not obligate symbionts. There was little herbivory damage on saplings colonized by the two Crematogaster symbiont ants in both forest types, while the saplings colonized by the other ant species suffered a 10–60% loss of leaf area. Larval mortality of the polyphagous lepidopteran Spodoptera litura was significantly higher when larvae fed on leaves of M. bancana saplings in the secondary forest than when fed on leaves of M. bancana saplings in the primary forest. These results suggest that the symbiosis between ants and M. bancana is looser and the non-ant-defenses are stronger in secondary forests, where light is more intense, than in primary forests.     

Continuous culture of novel mitochondrial cells lacking nuclei

We isolated stable cell lines, designated as mitochondrial cells, from cybrids obtained by fusing mitochondria-less HeLa cells with platelets from patients with Leigh syndrome, a subtype of mitochondrial encephalomyopathy. The cells contain a pathogenic point mutation, T9176C, in the mitochondrial DNA. Hematoxylin-eosin staining, confocal fluorescent microscopy and flow cytometry in fixed or living cells showed that the majority of these mitochondrial cells lack nuclear DNA and nuclei, but contain active mitochondria. Despite the absence of nuclear DNA, these cells can be continuously generated in culture. Therefore, it is likely that they arise from the minority of cells which possess a nucleus.     

The in vivo roles of STEF/Tiam1, Rac1 and JNK in cortical neuronal migration

The coordinated migration of neurons is a pivotal step for functional architectural formation of the mammalian brain. To elucidate its molecular mechanism, gene transfer by means of in utero electroporation was applied in the developing murine brain, revealing the crucial roles of Rac1, its activators, STEF/Tiam1, and its downstream molecule, c-Jun N-terminal kinase (JNK), in the cerebral cortex. Functional repression of these molecules resulted in inhibition of radial migration of neurons without affecting their proper differentiation. Interestingly, distinct morphological phenotypes were observed; suppression of Rac1 activity caused loss of the leading process, whereas repression of JNK activity did not, suggesting the complexity of the signaling cascade. In cultured neurons from the intermediate zone, activated JNK was detected along microtubules in the processes. Application of a JNK inhibitor caused irregular morphology and increased stable microtubules in processes, and decreased phosphorylation of microtubule associated protein 1B, raising a possibility of the involvement of JNK in controlling tubulin dynamics in migrating neurons. Our data thus provide important clues for understanding the intracellullar signaling machinery for cortical neuronal migration.     

Impact of whitefish on an enclosure ecosystem in a shallow eutrophic lake: changes in nutrient concentrations,phytoplankton and zoobenthos

Large bag-type (75 m³) and tube-type (105 m³) enclosures were set up in the shallow eutrophic Lake Suwa and were each stocked with exotic planktivorous whitefish (Coregonus lavaretus maraena). The release of whitefish caused the increase in nutrient concentration in the tube-type enclosure whereas no such increase was observed in the bag-type enclosure. Bottom sediment seemed to be an important source of chironomid food for whitefish. The proportion of phytoplankton measuring<10μm and 20–40μm, which respectively corresponded toOchromonas spp. andCryptomonas sp., were lower in the fish enclosures than in the control, which might have been caused by high grazing pressure by rotifers. The predation by whitefish might have affected the species composition of phytoplankton through reducing copepod predation on rotifers, not through reducing the densities of cladocerans which directly feed on phytoplankton as many investigators have reported. The phytoplankton biomass was not affected much by the release of fish. Possible reasons are that the increase in density of rotifers reduced the biomass of available phytoplankton and also that inedible Cyanophyceae were in the decreasing phase of their seasonal succession and could not increase successfully in spite of elevated nutrient levels.