Possible role of a taurine transporter in the deep-sea mussel Bathymodiolus septemdierum in adaptation to hydrothermal vents

Various invertebrates inhabiting hydrothermal vents possess sulfur-oxidizing bacteria in their tissues; however, the mechanisms by which toxic sulfides are delivered to these endosymbionts remain unknown. Recently, detoxification of sulfides using thiotaurine, a sulfur-containing amino acid, has been suggested. In this study, we propose the involvement of a taurine transporter in sulfide detoxification in the deep-sea mussel Bathymodiolus septemdierum by demonstrating: (i) the abundance of its mRNA in the gill; (ii) its activity under a wide range of salinities; (iii) its low Michaelis constant value in taurine transportation; and (iv) its affinity for thiotaurine and the thiotaurine precursor, hypotaurine.     

Pleomorphic configuration of the trimeric capsid proteins of Rice dwarf virus that allows formation of both the outer capsid and tubular crystals

In the double-shelled capsid of Phytoreovirus, the outer capsid attaches firmly to the 3-fold axes of the T = 1 core. It then forms a T = 13 lattice via lateral interactions among the P8 trimers (Wu et al., 2000, Virology 271, 18-25). Purified P8 molecules also assemble into hexagonal monolayers as well as tubular crystals. To explore the mechanisms of formation of these structures, the configurations of P8 trimers were compared and verified in particles of Rice dwarf virus and in tubular crystals (tubes) whose structure was determined by cryoelectron microscopy using helical reconstruction technique. Remarkable variations in intertrimer contacts were observed in the tubes and in the surface lattice of Rice dwarf virus capsid. Superposition of the atomic structure of P8 trimers in the structures analyzed by cryoelectron microscopy allowed us to identify groups of specific and stable interactions, some of which were preserved in the tubes and the quasi-equivalent T = 13 icosahedral lattice of the virion's shell. The flexible nature of the binding between P8 trimers, created via electrostatic interactions that hold radially inward, appears to allow the outer-capsid P8 trimers to envelop the ragged surface of the core, forming the double shell of an intact viral particle.     

Identification of amino acid residues of matrix metalloproteinase-7 essential for binding to cholesterol sulfate

Matrix metalloproteinase-7 (MMP-7; matrilysin) induces homotypic adhesion of colon cancer cells by cleaving cell surface protein(s) and enhances their metastatic potential. Our previous study (Yamamoto, K., Higashi, S., Kioi, M., Tsunezumi, J., Honke, K., and Miyazaki, K. (2006) J. Biol. Chem. 281, 9170-9180) demonstrated that binding of MMP-7 to cell surface cholesterol sulfate (CS) is essential for the cell membrane-associated proteolytic action of the protease. To determine the region of MMP-7 essential for binding to CS, we constructed chimeric proteases consisting of various parts of MMP-7 and those of the catalytic domain of MMP-2; the latter protease does not have an affinity for CS. Studies of these chimeric proteases and other mutants of MMP-7 revealed that Ile29, Arg33, Arg51, and Trp55, in the internal sequence, and the C-terminal three residues corresponding to residues 171-173 of MMP-7 are essential for binding to CS. An MMP-7 mutant, which had the internal 4 residues at positions 29, 33, 51, and 55 of MMP-7 replaced with the corresponding residues of MMP-2 and the C-terminal 3 residues deleted, had essentially no affinity for CS. This mutant and wild-type MMP-7 showed similar proteolytic activity toward fibronectin, whereas the mutant lacked the ability to induce the colon cancer cell aggregation. In the three-dimensional structure of MMP-7, the residues essential for binding to CS are located on the molecular surface in the opposite side of the catalytic cleft of the protease. Therefore, it is assumed that the active site of MMP-7 bound to cell surface is directed outside. We speculate that the direction of the cell-bound MMP-7 makes it feasible for the protease to cleave its substrates on cell surface.     

Identification of amino acid residues of the matrix metalloproteinase-2 essential for its selective inhibition by beta-amyloid precursor protein-derived inhibitor

The extracellular domain of beta-amyloid precursor protein (APP) contains an inhibitor against matrix metalloproteinase-2 (MMP-2, gelatinase A). Our previous study ( Higashi, S. and Miyazaki, K. (2003) J Biol Chem 278, 14020-14028 ) demonstrated that the inhibitor is localized within the ISYGN-DALMP sequence of APP, and a synthetic decapeptide containing this sequence (named APP-derived inhibitory peptide, APP-IP) selectively inhibits the activity of MMP-2. To determine the region of interaction that correlates with the selective inhibition, we constructed various MMP-2 mutants. An MMP-2 mutant, which had the hemopexin-like domain and three fibronectin-like type II domains of MMP-2 deleted, and native MMP-2 showed similar affinities for APP-IP, suggesting that only the catalytic domain of MMP-2 is essential for the interaction. Studies of chimeric proteases, consisting of various parts of the MMP-2 catalytic domain and those of MMP-7 (matrilysin) or MMP-9 (gelatinase B), further revealed that Ala(88) and Gly(94) in the non-prime side and Tyr(145) and Thr(146) in the prime side of the substrate-binding cleft of MMP-2 contribute separately to the selective inhibition. Replacement of the amino acid residue at position 94 of a chimeric MMP mutant affected its interaction with the C-terminal Pro(10) of APP-IP, whereas that of residues 145-148 affected the interaction with Tyr(3) of the inhibitor, suggesting that the N to C direction of APP-IP relative to the substrate-binding cleft of MMP is analogous to that of propeptide in proMMP, and opposite to that of substrate. When the APP-IP sequence was added to the N terminus of the catalytic domain of MMP-2, the activity of the protease was intramolecularly inhibited. We speculate that the direction of interaction makes the active site-bound APP-IP resistant to cleavage, thereby supporting the inhibitory action of the peptide inhibitor.     

Characterization of the traD operon of naphthalene-catabolic plasmid NAH7: a host-range modifier in conjugative transfer

Pseudomonas putida G7 carries a naphthalene-catabolic and self-transmissible plasmid, NAH7, which belongs to the IncP-9 incompatibility group. Adjacent to the putative origin of conjugative transfer (oriT) of NAH7 are three genes, traD, traE, and traF, whose functions and roles in conjugation were previously unclear. These three genes were transcribed monocistronically and thus were designated the traD operon. Mutation of the three genes in the traD operon resulted in 10- to 10⁵-fold decreases in the transfer frequencies of the plasmids from Pseudomonas to Pseudomonas and Escherichia coli and from E. coli to E. coli. On the other hand, the traD operon was essential for the transfer of NAH7 from E. coli to Pseudomonas strains. These results indicated that the traD operon is a host-range modifier in the conjugative transfer of NAH7. The TraD, TraE, and TraF proteins were localized in the cytoplasm, periplasm, and membrane, respectively, in strain G7 cells. Our use of a bacterial two-hybrid assay system showed that TraE interacted in vivo with other essential components for conjugative transfer, including TraB (coupling protein), TraC (relaxase), and MpfH (a channel subunit in the mating pair formation system).     

Histopathological and ultrastructural features of Koi herpesvirus (KHV)-infected carp Cyprinus carpio, and the morphology and morphogenesis of KHV

Epizootics of Koi herpesvirus (KHV) cause mass mortalities in koi carp and common carp worldwide. We used a newly developed 'per-gill infection' procedure with live KHV, and then conducted detailed histopathological and ultrastructural studies of KHV-infected cells including an examination of the morphology and morphogenesis of KHV. The primary target of KHV was respiratory epithelial cells of the gill lamellae, and release of virions from infected epithelial cells resulted in a systemic infection affecting the kidney, spleen, heart, brain and liver. The pathognomonic feature of infected cells was the formation of intranuclear inclusion bodies with marginal hyperchromatosis in the nucleus. Within the nucleus, assembly of capsids and nucleocapsids and an increase in filamentous nucleoproteins were evident. Enveloped nucleocapsids budded from the inner nuclear membrane into the perinuclear space. De-enveloped nucleocapsids were translocated in the cytoplasm to be embedded within inclusion bodies where tegumentation of the nucleocapsid occurred. Enveloped virions that had budded into intracytoplasmic vesicles and virions located extracellularly were composed of an electron-dense core, surrounded in turn by the capsid, the tegument and finally an envelope with projections. The morphology and morphogenesis of KHV were the same as those of viruses within the family Herpesviridae.     

Do spatial effects appear at low dilution rate in chemostat?

The chemostat theory on two species competition has shown that the dilution rate where transition of dominance occurs – transition-dilution rate – is independent of limiting-nutrient concentration. However, we obtained the experimental data indicating that the transition-dilution rate changed with variations in limiting-ammonium concentrations, using the chemostat mixed-culture of the cyanobacterium Microcystis novacekii and the green alga Scenedesmus quadricauda. The transition-dilution rate was dependent on the concentration of limiting ammonium in the influx culture medium. We tried to simulate the experimental results. Though the dilution rate has been considered independent of nutrient concentration, we introduce the effective dilution rate that depends on nutrient concentration (ammonium concentration in this study). A hyperbolic Monod-type function is used to represent the effective dilution rate for each species. The maximum dilution rate of the function is set to be the mechanical dilution rate (nominal dilution rate) of the chemostat culture. The calculation shows that the nominal transition-dilution rate where transition of dominance occur decreases with increased concentration. This simulation is well consistent with our experimental data. These results may suggest that the species-specificity of limiting nutrients, here nitrogen. Or they may imply that the depreciation of nitrogen becomes critical when both dilution rate and concentration are very low, especially for the green algae. In the latter case, spatial effects are induced internally in the ecosystem.     

Pyrroloquinoline quinone-dependent dehydrogenases from Ketogulonicigenium vulgare catalyze the direct conversion of L-sorbosone to L-ascorbic acid

A novel enzyme, L-sorbosone dehydrogenase 1 (SNDH1), which directly converts L-sorbosone to L-ascorbic acid (L-AA), was isolated from Ketogulonicigenium vulgare DSM 4025 and characterized. This enzyme was a homooligomer of 75-kDa subunits containing pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups. Two isozymes of SNDH, SNDH2 consisting of 75-kDa and 55-kDa subunits and SNDH3 consisting of 55-kDa subunits, were also purified from the bacterium. All of the SNDHs produced L-AA, as well as 2-keto-L-gulonic acid (2KGA), from L-sorbosone, suggesting that tautomerization of L-sorbosone causes the dual conversion by SNDHs. The sndH gene coding for SNDH1 was isolated and analyzed. The N-terminal four-fifths of the SNDH amino acid sequence exhibited 40% identity to the sequence of a soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. The C-terminal one-fifth of the sequence exhibited similarity to a c-type cytochrome with a heme-binding motif. A lysate of Escherichia coli cells expressing sndH exhibited SNDH activity in the presence of PQQ and CaCl2. Gene disruption analysis of K. vulgare indicated that all of the SNDH proteins are encoded by the sndH gene. The 55-kDa subunit was derived from the 75-kDa subunit, as indicated by cleavage of the C-terminal domain in the bacterial cells.     

Decreases of calcium and phosphorus in monkey cardiac walls with development and aging

To elucidate compositional changes of the cardiac walls with development and aging, the authors investigated changes of elements in the atrial and ventricular walls of monkeys. The left and right atrial walls, left and right ventricular walls, and interatrial and interventricular septa were resected from the subjects. The subjects consisted of 17 rhesus and 13 Japanese monkeys, ranging in age from 10 d to 33 yr. The element content of the cardiac walls was analyzed by inductively coupled plasma-atomic emission spectrometry. The Ca and P contents decreased in all of the left and right atrial and ventricular walls, interatrial septa, and interventricular septa with development, whereas the S and Mg contents decreased in the left and right ventricular walls with development. Regarding the relationships among elements, significant direct correlations were found among Ca, P, Mg, and Zn in all of the left and right atrial walls, left and right ventricular walls, and interatrial and interventricular septa, with some exceptions. As Ca decresed in the cardic walls, P, Mg, and Zn decreased simultaneously in the cardiac walls. The mass ratio of Ca/P decreased gradually with Ca decrease in both the atrial and ventricular walls, but it was not constant.