Intracellular and extracellular concentrations of Na+ modulate Mg2+ transport in rat ventricular myocytes

Apparent free cytoplasmic concentrations of Mg2+ ([Mg2+]i) and Na+ ([Na+]i) were estimated in rat ventricular myocytes using fluorescent indicators, furaptra (mag-fura-2) for Mg2+ and sodium-binding benzofuran isophthalate for Na+, at 25 degrees C in Ca2+-free conditions. Analysis included corrections for the influence of Na+ on furaptra fluorescence found in vitro and in vivo. The myocytes were loaded with Mg2+ in a solution containing 24 mM Mg2+ either in the presence of 106 mM Na+ plus 1 mM ouabain (Na+ loading) or in the presence of only 1.6 mM Na+ to deplete the cells of Na+ (Na+ depletion). The initial rate of decrease in [Mg2+]i from the Mg2+-loaded cells was estimated in the presence of 140 mM Na+ and 1 mM Mg2+ as an index of the rate of extracellular Na+-dependent Mg2+ efflux. Average [Na+]i, when estimated from sodium-binding benzofuran isophthalate fluorescence in separate experiments, increased from 12 to 31 mM and 47 mM after Na+ loading for 1 and 3 h, respectively, and decreased to approximately 0 mM after 3 h of Na+ depletion. The intracellular Na+ loading significantly reduced the initial rate of decrease in [Mg2+]i, on average, by 40% at 1 h and by 64% at 3 h, suggesting that the Mg2+ efflux was inhibited by intracellular Na+ with 50% inhibition at approximately 40 mM. A reduction of the rate of Mg2+ efflux was also observed when Na+ was introduced into the cells through the amphotericin B-perforated cell membrane (perforated patch-clamp technique) via a patch pipette that contained 130 mM Na+. When the cells were heavily loaded with Na+ with ouabain in combination with intracellular perfusion from the patch pipette containing 130 mM Na+, removal of extracellular Na+ caused an increase in [Mg2+]i, albeit at a very limited rate, which could be interpreted as reversal of the Mg2+ transport, i.e., Mg2+ influx driven by reversed Na+ gradient. Extracellular Na+ dependence of the rate of Mg2+ efflux revealed that the Mg2+ efflux was activated by extracellular Na+ with half-maximal activation at 55 mM. These results contribute to a quantitative characterization of the Na+-Mg2+ exchange in cardiac myocytes.     

Sodium gradient-dependent transport of magnesium in rat ventricular myocytes

Cytoplasmic concentration of Mg²⁺([Mg²⁺]_i) was measured with a fluorescentindicator furaptra in ventricular myocytes enzymatically dissociatedfrom rat hearts (25°C). To study Mg²⁺ transport acrossthe cell membrane, cells were treated with ionomycin inCa²⁺-free (0.1 mM EGTA) and high-Mg²⁺ (10 mM)conditions to facilitate passive Mg²⁺ influx. Rate of riseof [Mg²⁺]_i due to the net Mg²⁺influx was significantly smaller in the presence of 130 mMextracellular Na⁺ than in its absence. We also tested theextracellular Na⁺ dependence of the net Mg²⁺efflux from cells loaded with Mg²⁺. After[Mg²⁺]_i was raised by ionomycin and highMg²⁺ to the level 0.5-0.6 mM above the basal value(~0.7 mM), washout of ionomycin and lowering extracellular[Mg²⁺] to 1.2 mM caused rapid decline of[Mg²⁺]_i in the presence of 140 mMNa⁺. This net efflux of Mg²⁺ was completelyinhibited by withdrawal of extracellular Na⁺ and waslargely attenuated by imipramine, a known inhibitor of Na⁺/Mg²⁺ exchange, with 50% inhibition at 79 µM. The relation between the rate of net Mg²⁺ efflux andextracellular Na⁺ concentration([Na⁺]_o) had a Hill coefficient of 2 and[Na⁺]_o at half-maximal rate of 82 mM. Theseresults demonstrate the presence of Na⁺ gradient-dependentMg²⁺ transport, which is consistent withNa⁺/Mg²⁺ exchange, in cardiac myocytes.

     

Monophyletic Origin of the Order Chiroptera and Its Phylogenetic Position Among Mammalia, as Inferred from the Complete Sequence of the Mitochondrial DNA of a Japanese Megabat, the Ryukyu Flying Fox (Pteropus dasymallus)

Complete sequences of mitochondrial DNA (mtDNA) are useful for the reconstruction of phylogenetic trees of mammals and, in particular, for inferring higher-order relationships in mammals. In this study, we determined the complete sequence (16,705 bp) of the mtDNA of a Japanese megabat, the Ryukyu flying fox (Pteropus dasymallus). We analyzed this sequence phylogenetically by comparing it with the complete sequence of mtDNAs of 35 mammals in an effort to reevaluate the enigmatic relationship between Megachiroptera and Microchiroptera and the relationships between them and other mammals. Maximum-likelihood analysis of 12 concatenated mitochondrial proteins from 36 mammals strongly suggested the monophyly of the order Chiroptera and its close relationship to Fereuungulata (Carnivora + Perissodactyla + Cetartiodactyla). We estimated that megabats and microbats diverged approximately 58 MyrBP and discussed the origin and early evolution of Chiroptera based on our findings. Received: 28 January 2000 / Accepted: 30 June 2000     

Versatility of the accessory C proteins of Sendai virus: contribution to virus assembly as an additional role

The P/C mRNA of Sendai virus (SeV) encodes a nested set of accessory proteins, C', C, Y1, and Y2, referred to collectively as C proteins, using the +1 frame relative to the open reading frame of phospho (P) protein and initiation codons at different positions. The C proteins appear to be basically nonstructural proteins as they are found abundantly in infected cells but greatly underrepresented in the virions. We previously created a 4C(-) SeV, which expresses none of the four C proteins, and concluded that the C proteins are categorically nonessential gene products but greatly contribute to viral full replication and infectivity (A. Kurotani et al., Genes Cells 3:111-124, 1998). Here, we further characterized the 4C(-) virus multiplication in cultured cells. The viral protein and mRNA synthesis was enhanced with the mutant virus relative to the parental wild-type (WT) SeV. However, the viral yields were greatly reduced. In addition, the 4C(-) virions appeared to be highly anomalous in size, shape, and sedimentation profile in a sucrose gradient and exhibited the ratios of infectivity to hemagglutination units significantly lower than those of the WT. In the WT infected cells, C proteins appeared to colocalize almost perfectly with the matrix (M) proteins, pretty well with an external envelope glycoprotein (hemagglutinin-neuraminidase [HN]), and very poorly with the internal P protein. In the absence of C proteins, there was a significant delay of the incorporation of M protein and both of the envelope proteins, HN and fusion (F) proteins, into progeny virions. These results strongly suggest that the accessory and basically nonstructural C proteins are critically required in the SeV assembly process. This role of C proteins was further found to be independent of their recently discovered function to counteract the antiviral action of interferon-alpha/beta. SeV C proteins thus appear to be quite versatile.     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> is a useful transporter for introducing T-DNA of the binary plasmid into the chrysanthemum, <Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Kitamura,genome

Agrobacterium rhizogenes was used for efficient transformation of chrysanthemum. Two types of Agrobacterium, A. rhizogenes (A-13) and A. tumefaciens (LBA4404), which harbor pIG121-Hm, were employed for infection. In the A. rhizogenes-infected explants, hairy roots were not observed on any tested medium or culture condition. When explants were cultured on shoot induction medium, calli were formed at the cutting edge within 4–6 weeks of culture, and shoot primordia were observed on the callus surface after 2 weeks of callus formation. Consequently, with gus introduction, a significantly higher transformation rate was observed for A. rhizogenes (6.0%) compared with A. tumefaciens (3.3%). However, only 0.6% of the frequency of rol insertion was exhibited in A. rhizogenes mediation. These results indicate that A. rhizogenes effectively introduces T-DNA of the binary plasmid into the chrysanthemum genome by introducing Ri T-DNA at a low frequency. It also indicates that the system is a useful alternative for the transformation of chrysanthemum.     

Accelerated sulfur cycle in coastal marine sediment beneath areas of intensive shellfish aquaculture

Prokaryotes in marine sediments taken from two neighboring semi-enclosed bays (the Yamada and Kamaishi bays) at the Sanriku coast in Japan were investigated by the culture-independent molecular phylogenetic approach coupled with chemical and activity analyses. These two bays were chosen in terms of their similar hydrogeological and chemical characteristics but different usage modes; the Yamada bay has been used for intensive shellfish aquaculture, while the Kamaishi bay has a commercial port and is not used for aquaculture. Substantial differences were found in the phylogenetic composition of 16S rRNA gene clone libraries constructed for the Yamada and Kamaishi sediments. In the Yamada library, phylotypes affiliated with delta-Proteobacteria were the most abundant, and those affiliated with gamma-Proteobacteria were the second-most abundant. In contrast, the Kamaishi library was occupied by phylotypes affiliated with Planctomycetes, gamma-Proteobacteria, delta-Proteobacteria, and Crenarchaeota. In the gamma-Proteobacteria, many Yamada phylotypes were related to free-living and symbiotic sulfur oxidizers, whereas the Kamaishi phylotype was related to the genus Pseudomonas. These results allowed us to hypothesize that sulfate-reducing and sulfur-oxidizing bacteria have become abundant in the Yamada sediment. This hypothesis was supported by quantitative competitive PCR (qcPCR) with group-specific primers. The qcPCR also suggested that organisms closely related to Desulfotalea in the Desulfobulbaceae were the major sulfate-reducing bacteria in these sediments. In addition, potential sulfate reduction and sulfur oxidation rates in the sediment samples were determined, indicating that the sulfur cycle has become active in the Yamada sediment beneath the areas of intensive shellfish aquaculture.     

Ubiquitination of alpha-synuclein

Filamentous alpha-synuclein depositions are the defining hallmarks of a subset of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. We previously reported that alpha-synuclein in those brains are extensively phosphorylated at Ser129 [Fujiwara et al. (2002) Nat. Cell Biol. 4, 160-164] and also partially ubiquitinated [Hasegawa et al. (2002) J. Biol. Chem. 277, 49071-49076]. Here, we investigate ubiquitination of alpha-synuclein in vitro and in vivo and report the ubiquitination sites and the effects of familial PD-linked mutations, phosphorylation, and fibril formation on ubiquitination. Protein-sequence analysis revealed that Lys21, Lys23, Lys32, and Lys34 within the repeats in the amino-terminal half are liable to ubiquitination in vitro. A site-directed mutagensis study confirmed that these are the major ubiquitination sites. A53T and A30P mutations had no significant effect on ubiquitination. Similarly, phosphorylation of alpha-synuclein at Ser129 did not affect ubiquitination. Notably, we show that assembled, filamentous alpha-synuclein is less ubiquitinated than the soluble form and that the major ubiquitination sites are localized to Lys6, Lys10, and Lys12 at the amino-terminal region of filamentous alpha-synuclein. Furthermore, we successfully detected ubiquitination of alpha-synuclein in 293T cells by cotransfection with alpha-synuclein and ubiquitin. The in vivo ubiquitination sites were found to be identical to those in filamentous alpha-synuclein. PD-linked mutations and phosphorylation at Ser129 had no effects on ubiquitination of alpha-synuclein in vivo. These data may have implications for the mechanisms of the formation of alpha-synuclein deposits in alpha-synucleinopathy brains.