Isolation, characterization and cDNA cloning of a one-lobed transferrin from the ascidian Halocynthia roretzi

Transferrin was isolated from plasma of the ascidian Halocynthia roretzi by ion-exchange chromatography. The molecular weight of the plasma transferrin was determined to be 52K by SDS-polyacrylamide gel electrophoresis and gel filtration. Ascidian plasma transferrin was found to bind one mole of iron ion per mole of protein. The reductive S-pyridylethylated transferrin was subjected to Edman degradation analysis for determination of the N-terminal amino acid sequence, and it was also subjected to proteolytic fragmentation to yield peptide fragments, whose amino acid sequences were determined by Edman degradation analysis. Using the above amino acid sequences, a cDNA clone (1880 base pairs) encoding a protein of 372 amino acids containing a signal peptide of 21 amino acids was isolated from an H. roretzi hepatopancreas cDNA library. The reduced amino acid sequence contains the same sequences of the peptide fragments. A comparison of the amino acid sequence of ascidian transferrin with those of other members of the transferrin family revealed that the ascidian transferrin is composed of only the N-terminal lobe of two-lobed vertebrate transferrins. Thus, a one-lobed transferrin is present in the ascidian H. roretzi.     

Involvement of specialized DNA polymerases in mutagenesis by 8-hydroxy-dGTP in human cells

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2′-deoxyguanosine 5′-triphosphate (8-OH-dGTP), was examined using human 293T cells. Shuttle plasmid DNA containing the supF gene was first transfected into the cells, and then 8-OH-dGTP was introduced by means of osmotic pressure. The DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T → C:G substitution mutations in the cells. The knock-downs of DNA polymerases η and ζ, and REV1 by siRNAs reduced the A:T → C:G substitution mutations, suggesting that these DNA polymerases are involved in the misincorporation of 8-OH-dGTP opposite A in human cells. In contrast, the knock-down of DNA polymerase ι did not affect the 8-OH-dGTP-induced mutations. The decrease in the induced mutation frequency was more evident by double knock-downs of DNA pols η plus ζ and REV1 plus DNA pol ζ (but not by that of DNA pol η plus REV1), suggesting that REV1-DNA pol η and DNA pol ζ work in different steps. These results indicate that specialized DNA polymerases are involved in the mutagenesis induced by the oxidized dGTP.     

Inhibitory specificity change of the ovomucoid third domain of the silver pheasant upon introduction of an engineered Cys14-Cys39 bond

The ovomucoid third domain from silver pheasant (OMSVP3), a typical Kazal-type inhibitor, strongly inhibits different serine proteases of various specificities, i.e., chymotrypsin, Streptomyces griseus protease, subtilisin, and elastase. Structural studies have suggested that conformational flexibility in the reactive site loop of the free inhibitor may be related to broad specificity of the ovomucoid. On the basis of the structural homology between OMSVP3 and ascidian trypsin inhibitor (ATI), which has a cystine-stabilized alpha-helical (CSH) motif in the sequence, we prepared the disulfide variant of OMSVP3, introducing an engineered disulfide bond between positions 14 and 39 near the reactive site (Met18-Glu19) by site-directed mutagenesis. The disulfide variant P14C/N39C retained potent inhibitory activities toward alpha-chymotrypsin (CHT) and S. griseus proteases A and B (SGPA and SGPB), while this variant lost most of its inhibitory activity toward porcine pancreatic elastase (PPE). We determined the solution structure of P14C/N39C, as well as that of wild-type OMSVP3, by two-dimensional nuclear magnetic resonance (2D NMR) methods and compared their structures to elucidate the structural basis of the inhibitory specificity change. For the molecular core consisting of a central alpha-helix and a three-stranded antiparallel beta-sheet, essentially no structural difference was detected between the two (pairwise rmsd value = 0.41 A). In contrast to this, a significant difference was detected in the loop from Cys8 to Thr17, where in P14C/N39C it has drawn approximately 4 A nearer the central helix to form the engineered Cys14-Cys39 bond. Concomitantly, the Tyr11-Pro12 cis-peptide linkage, which is highly conserved in ovomucoid third domains, was isomerized to the trans configuration. Such structural change in the loop near the reactive site may possibly affect the inhibitory specificity of P14C/N39C for the corresponding proteases.     

Promotive effects of far-infrared ray on full-thickness skin wound healing in rats

The biological effects of far-infrared ray (FIR) on whole organisms remain poorly understood. The aim of our study was to investigate not only the hyperthermic effect of the FIR irradiation, but also the biological effects of FIR on wound healing. To evaluate the effect of FIR on a skin wound site, the speed of full-thickness skin wound healing was compared among groups with and without FIR using a rat model. We measured the skin wound area, skin blood flow, and skin temperature before and during FIR irradiation, and we performed histological inspection. Wound healing was significantly more rapid with than without FIR. Skin blood flow and skin temperature did not change significantly before or during FIR irradiation. Histological findings revealed greater collagen regeneration and infiltration of fibroblasts that expressed transforming growth factor-beta1 (TGF-beta1) in wounds in the FIR group than in the group without FIR. Stimulation of the secretion of TGF-beta1 or the activation of fibroblasts may be considered as a possible mechanisms for the promotive effect of FIR on wound healing independent of skin blood flow and skin temperature.     

N-Terminal modifications of the 19S regulatory particle subunits of the yeast proteasome

The yeast (Saccharomyces cerevisiae) contains three N-acetyltransferases, NatA, NatB, and NatC, each of which acetylates proteins with different N-terminal regions. The 19S regulatory particle of the yeast 26S proteasome consists of 17 subunits, 12 of which are N-terminally modified. By using nat1, nat3, and mak3 deletion mutants, we found that 8 subunits, Rpt4, Rpt5, Rpt6, Rpn2, Rpn3, Rpn5, Rpn6, and Rpn8, were NatA substrates, and that 2 subunits, Rpt3 and Rpn11, were NatB substrates. Mass spectrometric analysis revealed that the initiator Met of Rpt2 precursor polypeptide was processed and a part of the mature Rpt2 was N-myristoylated. The crude extracts from the normal strain and the nat1 deletion mutant were similar in chymotrypsin-like activity in the presence of ATP in vitro and in the accumulation level of the 26S proteasome. These characteristics were different from those of the 20S proteasome: the chymotrypsin-like activity and accumulation level of 20S proteasome were appreciably higher from the nat1 deletion mutant than from the normal strain.     

Cell-cycle dependent dynamic change of 26S proteasome distribution in tobacco BY-2 cells

The 26S proteasome is known to play pivotal roles in cell-cycle progression in various eukaryotic cells; however, little is known about its role in higher plants. Here we report that the subcellular distribution of the 26S proteasome is dynamically changed in a cell-cycle dependent manner in tobacco BY-2 cells as determined by immunostaining with anti-Rpn10 (a regulatory PA700 subunit) and anti-20S catalytic proteasome antibodies. The 26S proteasome was found to localize not only in nuclear envelopes and mitotic spindles but also in preprophase bands (PPBs) and phragmoplasts appearing in G(2) and M phases, respectively. MG132, a proteasome inhibitor, exclusively caused cell-cycle arrest not only at the metaphase but also the early stage of PPB formation at the G(2) phase and the collapse of the phragmoplast, which seems to be closely related to proteasome distribution in the cells.     

Zymosan induces production of superoxide anions by hemocytes of the solitary ascidian Halocynthia roretzi

Reactive oxygen intermediates (ROIs), including superoxide anions and hydrogen peroxide, are generated by phagocytes in invertebrates, as well as in vertebrates. To understand the molecular mechanisms underlying the generation of ROIs by hemocytes of the solitary ascidian Halocynthia roretzi, we established a method of measuring ROIs using luminol-dependent chemiluminescence (LDCL). LDCL analyses revealed that both zymosan and phorbol myristate acetate (PMA), but not lipopolysaccharide, beta1,3-glucan, or formylpeptide, induced the generation of ROIs by H. roretzi hemocytes. The zymosan-induced LDCL was markedly inhibited by the addition of superoxide dismutase (SOD) or H. roretzi plasma. A calcium-chelating reagent, BAPTA-AM, completely inhibited the zymosan-induced LDCL. On the other hand, the PMA-induced LDCL was only slightly inhibited by the addition of SOD or BAPTA-AM. Spectroscopic analysis at a low temperature revealed that H. roretzi hemocytes had absorption spectra specific for type b cytochrome, a component of the NADPH oxidase complex in mammalian phagocytes. These results strongly suggest that H. roretzi hemocytes generate superoxide anions upon phagocytosis and that intracellular calcium ions and possibly an NADPH oxidase complex are involved in their generation by H. roretzi hemocytes.     

Mutagenic effects of 2-hydroxy-dATP on replication in a HeLa extract: induction of substitution and deletion mutations

The mutagenicity of an oxidized form of dATP, 2-hydroxydeoxyadenosine 5′-triphosphate (2-OH-dATP), was examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. 2-OH-dATP induced mutations in a dose-dependent manner and elicited substitution and deletion mutations. Of the substitutions, a G·C→A·T transition including a tandem (CC→TT) mutation was mainly observed. This result agrees with our previous observation that mammalian DNA polymerase α misincorporates the oxidized nucleotide opposite C, but is in contrast to the finding that 2-OH-dATP elicits G·C→T·A transversions in Escherichia coli. This type of mutation was also elicited, but to a lesser extent. Interestingly, the mutagenicity of 2-OH-dATP was enhanced in the presence of 2-hydroxydeoxyadenosine 5′-diphosphate, an inhibitor of the MTH1 protein, suggesting that this protein functions in the hydrolysis of 2-OH-dATP in the replication reaction mixture, and probably in living cells. These results indicate that 2-OH-dATP is mutagenic and that its mutagenicity is suppressed by the MTH1 protein in mammalian cells.     

ISG15 modification of Ubc13 suppresses its ubiquitin-conjugating activity

ISG15 is one of the interferon-stimulated genes and is classified as a ubiquitin-like protein. Upon interferon stimuli, ISG15 is upregulated and becomes conjugated to various cellular proteins (ISGylation). Several target proteins for ISGylation have recently been identified, but the biological consequence of protein ISGylation remains unclear. In the course of our study to identify components of the ISGylation system, we found that Ubc13, an E2 enzyme for ubiquitin conjugation, is covalently modified with ISG15. To determine the meaning of ISGylation of Ubc13, we isolated ISG15-modified Ubc13 protein and compared its ubiquitin-conjugating activity with that of an unmodified one. We found that ISGylation of Ubc13 suppresses its ability to form a thioester intermediate with ubiquitin.