Purification,Biochemical Characterization,and Cloning of Phospholipase D from <Emphasis Type="Italic">Streptomyces racemochromogenes</Emphasis> Strain 10-3

We previously isolated Streptomyces racemochromogenes strain 10-3, which produces a phospholipase D (PLD) with high transphosphatidylation activity. Here, we purified and cloned the PLD (PLD103) from the strain. PLD103 exerted the highest hydrolytic activity at a slightly alkaline pH, which is in contrast to the majority of known Streptomyces PLDs that have a slightly acidic optimum pH. PLD103 shares only 71–76% amino acid sequence identity with other Streptomyces PLDs that have a slightly acidic optimum pH; thus, the diversity in the primary structure might explain the discrepancy observed in the optimum pH. The purified PLD displayed high transphosphatidylation activity in the presence of glycerol, l-serine, and 2-aminoethanol hydrochloride with a conversion rate of 82–97% in a simple one-phase system, which was comparable to the rate of other Streptomyces PLDs in a complicated biphasic system.     

Structure of the summer under fast ice microbial community near Syowa Station,eastern Antarctica

Temporal variations in the microbial community structure of plankton, which is composed of autotrophic and heterotrophic pico-, nano- and microplankton, were investigated during the austral summer of 2005/2006 under fast ice near Syowa Station, eastern Antarctica. Autotrophic algal populations were composed almost entirely of diatoms followed by phytoflagellates such as autotrophic dinoflagellates and cryptophytes. Among the microbial community, heterotrophic biomass was dominated by heterotrophic dinoflagellates and naked ciliates and finally exceeded autotrophic biomass. Qualitative microscopic analysis revealed that heterotrophic dinoflagellates were ingesting large number of diatoms. Synchronizing fluctuation of naked ciliates with phytoflagellates suggested a predator–prey relationship between them. Our results suggest that the pelagic food webs under the extensive ice-covered areas in coastal Antarctic regions are not short but complex.     

Distinct behavior of claudin-3 and -4 around lactation period in mammary alveolus in mice

Tight junctions (TJs) are the most apical junctional complexes and restrict the fluid flux through the paracellular pathway. In the mammary glands, the tightness of TJs occurs shortly after parturition to prevent the leakage of milk components from the lumen and the loosening of TJs is induced immediately after weaning. Claudins are transmembrane proteins, and their composition at the apical-most regions determines the permeability of TJs. In this study, we investigated the localization and expression patterns of claudin-3 and -4 in the mammary glands around the lactation period because it is unclear how claudins construct mammary TJs in the apical-most regions. Our results showed that claudin-3 and -4 change not only their level of expression but also their localization in the processes of parturition, lactation, and weaning. Claudin-3 was concentrated in the apical-most regions during lactation, whereas claudin-4 gradually decreased at the beginning of lactation and increased drastically immediately after weaning. The qualitative change of claudin-3 was also identified by western blotting analysis as an additional band around the lactation period. In addition, parts of the mammary epithelial cells showed intensive positive reactions to claudin-4 in the lateral membrane and cytoplasm after weaning, concurrently with the involution mammary glands. These results indicate that claudin-3 in the apical-most regions maintains the impermeable TJs during lactation, and claudin-4 contributes to the permeability changes of TJs immediately after parturition and weaning.     

Structure-activity relationship studies of sphingosine-1-phosphate receptor agonists with N-cinnamyl-β-alanine moiety

Structure-activity relationship of sphingosine-1-phosphate receptor agonist was examined. In terms of reducing the flexibility of molecule, hit compound 1 was modified to improve S1P₁ agonistic activity as well as selectivity over S1P₃ agonistic activity. Novel S1P agonists with cinnamyl scaffold or 1,2,5,6-tetrahydropyridine scaffold were identified.     

Therapeutic effects of novel sphingosine-1-phosphate receptor agonist W-061 in murine DSS colitis

Although IL-17 is a pro-inflammatory cytokine reportedly involved in various autoimmune inflammatory disorders, its role remains unclear in murine models of colitis. Acute colitis was induced by 2.5% dextran sodium sulfate (DSS) treatment for 5 days. A novel sphingosine-1-phosphate receptor agonist W-061, a prototype of ONO-4641, was orally administered daily, and histopathological analysis was performed on the colon. The number of lymphocytes and their cytokine production were also evaluated in spleen, mesenteric lymph node, Peyer's patch and lamina propria of the colon. Daily administration of W-061 resulted in improvement of DSS-induced colitis, and significantly reduced the number of CD4+ T cells in the colonic lamina propria. Numbers of both Th17 and Th1 cells were reduced by W-061 treatment. W-061, however, had no influence on the number of Treg cells in lamina propria. Thus, Th17 and Th1 cells in lamina propria were thought to be the key subsets in the pathogenesis of DSS-induced colitis. In conclusion, W-061 may be a novel therapeutic strategy to ameliorate acute aggravation of inflammatory bowel diseases.     

Involvement of heme regulatory motif in heme-mediated ubiquitination and degradation of IRP2

Iron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.     

Isoform-specific phosphorylation of metabotropic glutamate receptor 5 by protein kinase C (PKC) blocks Ca2+ oscillation and oscillatory translocation of Ca2+-dependent PKC

Prolonged activation of metabotropic glutamate receptor 5a (mGluR5a) causes synchronized oscillations in intracellular calcium, inositol 1,4,5-trisphosphate production, and protein kinase C (PKC) activation. Additionally, mGluR5 stimulation elicited cyclical translocations of myristoylated alanine-rich protein kinase C substrate, which were opposite to that of gammaPKC (i.e. from plasma membrane to cytosol) and dependent on PKC activity, indicating that myristoylated alanine-rich protein kinase C substrate is repetitively phosphorylated by oscillating gammaPKC on the plasma membrane. Mutation of mGluR5 Thr(840) to aspartate abolished the oscillation of gammaPKC, but the mutation to alanine (T840A) did not. Cotransfection of gammaPKC with betaIIPKC, another Ca2+-dependent PKC, resulted in synchronous oscillatory translocation of both classical PKCs. In contrast, cotransfection of deltaPKC, a Ca2+-independent PKC, abolished the oscillations of both gammaPKC and inositol 1,4,5-trisphosphate. Regulation of the oscillations was dependent on deltaPKC kinase activity but not on gammaPKC. Furthermore, the T840A-mGluR5-mediated oscillations were not blocked by the deltaPKC overexpression. These results revealed that activation of mGluR5 causes translocation of both gammaPKC and deltaPKC to the plasma membrane. deltaPKC, but not gammaPKC, phosphorylates mGluR5 Thr(840), leading to the blockade of both Ca2+ oscillations and gammaPKC cycling. This subtype-specific targeting proposes the molecular basis of the multiple functions of PKC.     

Steric effects of isoleucine 107 on heme reorientation reaction in human myoglobin

Structural factors to regulate the heme reorientation reaction in myoglobin were examined and we found that the side chain at position 107 (Ile107), which is located between the 2-vinyl and 3-methyl groups of heme, forms a kinetic barrier for the heme rotation about the alpha-gamma axis. The phenylalanine-substituted mutant showed an extremely slow heme reorientation rate, compared to that of the wild-type protein, while replacement by the decreased side chain, valine, at position 107 accelerated the reorientation reaction. Considering that the spectroscopic data show only minor structural changes in the heme environments of the Ile107 mutants, the side chain at position 107 sterically interacts with the heme peripheral groups in the activation state for the heme reorientation, which supports the intramolecular mechanism that the heme rotates about the alpha-gamma axis without leaving the "protein cage."     

Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm <Emphasis Type="Italic">Bombyx mori</Emphasis>

To construct an efficient system for the production of recombinant proteins in silkworm (Bombyx mori), we investigated the promoter activity of the silkworm sericin 1, 2, and 3 genes (Ser1, Ser2, and Ser3) using a GAL4/UAS binary gene expression system in transgenic silkworm. The promoter activity of the upstream region of Ser1 was strong, yielding high expression of an enhanced green fluorescent protein (EGFP) transgene in the middle and posterior regions of the middle silk gland (MSG) after day 2 of the fifth instar. The Ser3 upstream region exhibited moderate promoter activity in the anterior MSG, but the Ser2 upstream region did not exhibit any promoter activity. Since the strongest promoter activity was observed for Ser1, we devised a system for the production of recombinant proteins using a GAL4–Ser1 promoter construct (Ser1-GAL4). Transgenic silkworms harboring both the Ser1-GAL4 construct and the previously reported upstream activating sequence (UAS)–EGFP construct, which contains the TATA box region of the Drosophila hsp70 gene, yielded approximately 100 μg EGFP per larva. When we then analyzed the TATA box region, signal peptide, and intron sequences for their effects on production from the UAS-EGFP construct, we found that the optimization of these sequences effectively increased production to an average of 500 μg EGFP protein per transgenic larva. We conclude that this binary system is a useful tool for the mass production of recombinant proteins of biomedical and pharmaceutical interest in silkworm.     

Chain length effects of oligo(L-lysine)-shelled dendrimers on interaction with DNA

The interaction of novel oligo(L-lysine)-shelled dendrimers (G3-PLL) with DNA was studied by means of circular dichroism spectroscopy, dynamic light scattering, and melting behavior of double-stranded DNA. G3-PLLs having various oligo(L-lysine) (PLL) segment (n = 5-40) were successfully synthesized by graft-polymerization of L-lysine NCA initiated with amino groups at the 3rd-generation poly(amidoamine) dendrimer surface. The ionization property of the newly prepared G3-PLLs were first examined. The evaluated pK(a) values (8.3-8.5) for G3-PLLs were found to be significantly lower than those (9.2-9.4) for the corresponding linear PLLs, probably due to alignment of PLL segments on the three-dimensional core surface. The binding experiments of G3-PLLs to DNA showed that double-stranded DNAs were fairly strongly bound to G3-PLLs primarily through electrostatic interactions. In addition, G3-PLLs served as a DNA cross-linker. A longer PLL-containing G3-PLL was found to interact with DNA more effectively than a shorter one.