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Crustacean phenoloxidase (PO) and hemocyanin (Hc) are classified as type 3 copper proteins. PO catalyzes the oxidation of mono- and di-phenol compounds, which is the rate-limiting step of melanization, while Hc generally functions as a dioxygen-transporting protein in the hemolymph of arthropods. To date, many studies have shown PO activity in Hc, which is inspired by their structural similarity. Here, the source of PO activity in crustaceans was re-examined by purifying Hc and PO exclusively from the hemolymph of kuruma prawn. The conventional procedure for the preparation of arthropod Hc, which includes precipitation of Hc by ultracentrifugation and subsequent purification by size exclusion chromatography, was not able to completely remove hemolymph-type PO from Hc. In contrast, fractionation with 50% saturation of ammonium sulfate and subsequent hydrophobic chromatography yielded sufficiently pure Hc, which contained no detectable PO protein and virtually no PO enzymatic activity. These results indicate that the main source of PO activity in the hemolymph of kuruma prawn is hemolymph-type PO and that the improved purification method of Hc is preferable for evaluating the PO activity of Hc.

The evolution of herbicide resistance in weeds is an example of parallel evolution, through which genes encoding herbicide target proteins are repeatedly represented as evolutionary targets. The number of herbicide target-site genes differs among species, and little is known regarding the effects of duplicate gene copies on the evolution of herbicide resistance. We investigated the evolution of herbicide resistance in Monochoria vaginalis, which carries five copies of sulfonylurea target-site acetolactate synthase (ALS) genes. Suspected resistant populations collected across Japan were investigated for herbicide sensitivity and ALS gene sequences, followed by functional characterisation and ALS gene expression analysis. We identified over 60 resistant populations, all of which carried resistance-conferring amino acid substitutions exclusively in MvALS1 or MvALS3. All MvALS4 alleles carried a loss-of-function mutation. Although the enzymatic properties of ALS encoded by these genes were not markedly different, the expression of MvALS1 and MvALS3 was prominently higher among all ALS genes. The higher expression of MvALS1 and MvALS3 is the driving force of the biased representation of genes during the evolution of herbicide resistance in M. vaginalis. Our findings highlight that gene expression is a key factor in creating evolutionary hotspots.

Tyrosinase (Ty) and catechol oxidase (CO) are members of type-3 copper enzymes. While Ty catalyzes both phenolase and catecholase reactions, CO catalyzes only the latter reaction. In the present study, Ty was found to catalyze the catecholase reaction, but hardly the phenolase reaction in the presence of the metallochaperon called "caddie protein (Cad)". The ability of the substrates to dissociate the motif shielding the active-site pocket seems to contribute critically to the substrate specificity of Ty. In addition, a mutation at the N191 residue, which forms a hydrogen bond with a water molecule near the active center, decreased the inherent ratio of phenolase versus catecholase activity. Unlike the wild-type complex, reaction intermediates were not observed when the catalytic reaction toward the Y98 residue of Cad was progressed in the crystalline state. The increased basicity of the water molecule may be necessary to inhibit the proton transfer from the conjugate acid to a hydroxide ion bridging the two copper ions. The deprotonation of the substrate hydroxyl by the bridging hydroxide seems to be significant for the efficient catalytic cycle of the phenolase reaction.

Secretory and membrane proteins synthesized in the endoplasmic reticulum (ER) are folded with intramolecular disulphide bonds, viz. oxidative folding, catalysed by the protein disulphide isomerase (PDI) family proteins. Here, we identified a novel soybean PDI family protein, GmPDIL6. GmPDIL6 has a single thioredoxin-domain with a putative N-terminal signal peptide and an active centre (CKHC). Recombinant GmPDIL6 forms various oligomers binding iron. Oligomers with or without iron binding and monomers exhibited a dithiol oxidase activity level comparable to those of other soybean PDI family proteins. However, they displayed no disulphide reductase and extremely low oxidative refolding activity. Interestingly, GmPDIL6 was mainly expressed in the cotyledon during synthesis of seed storage proteins and GmPDIL6 mRNA was up-regulated under ER stress. GmPDIL6 may play a role in the formation of disulphide bonds in nascent proteins for oxidative folding in the ER.

Ferritin, a ubiquitous iron storage protein, has a crucial role in innate immunity in arthropods, which have no adaptive immune system. Arthropods are thought to have two types of ferritin molecules: the secreted type and the cytosolic type. Here, we present the first crystal structure of ferritin from crustacean, kuruma prawn (Marsupenaeus japonicus), at 1.16 A resolution. This shrimp ferritin (MjFer) is the cytosolic type, and its structure shows well-conserved ferritin fold composed of a 4-helix bundle that assembles into a cage-like 24-mer. The structure of MjFer was more similar to those of human and vertebrate ferritins than to that of the secreted-type arthropod ferritin from an insect. MjFer possesses both a ferroxidase site and a nucleation site, which are the main characteristics of vertebrate H and L chain ferritins, respectively. The first crystal structure of crustacean ferritin, MjFer, has exceptionally high quality that provides the detailed structural information of metal moving pathway in ferritin.

Phenoloxidases (POs) play a crucial role in melanization of crustaceans. There are at least two types of POs characterized in crustaceans: the conventional type (POα here) that is expressed in hemocytes and POβ, a secreted protein synthesized in the hepatopancreas. We investigated the source of PO activity in the hemolymph of a lobster and determined the kinetic parameters of mono- and di-PO activities. In the lobster hemolymph, POα, which formed a hexamer similar to both POβ and hemocyanin, contributed to PO activity, whereas the amount of POβ was low. Kinetic analyses using purified prophenoloxidase of crustaceans showed that lobster POα has a higher rate constant, while shrimp POβ has higher specificity in both mono- and di-PO reactions, when tyramine and dopamine were employed as substrates. There should be at least two types of PO molecules in crustacean hemolymph, but the dominant PO molecule type varies among species.

Highlights • In patients with refractory constipation, it is necessary to care in mind that some patients require surgery. • Single incision laparoscopic surgery (SILS) is regarded as the major advance in minimally invasive surgery. • Total colectomy by SILS is very useful for the patients with intestinal neuronal dysplasia-type B., Introduction Intestinal neuronal dysplasia type B (IND-B) is an infrequent disease of the submucosal plexus of intestine manifesting chronic intestinal obstruction or severe chronic constipation. IND is rarely reported in adult patients. Presentation of a case The present study reports on the case of a 36 year-old woman suffering from longstanding chronic constipation and who was diagnosed with severe constipation in more than 20 years. Although she began to take a large amount of stimulant laxatives, such as “senna” and “bisacodyl”, constipation symptoms did not improve, she was admitted to our hospital. It was diagnosed with refractory constipation of the medication treatment-resistance, total colectomy with ileorectal anastomosis by single incision laparoscopic surgery (SILS) was performed. The final pathological diagnosis was IND-B. Discussion Refractory constipation after medical treatment is often seen in young generation. SILS has benefits of better cosmesis, reduced morbidity, reduced postoperative pain, and reduced length of hospital stay. Conclusion For the patients with refractory constipation associated with neuropathy such as IND, total colectomy by SILS was very effective.

In the endoplasmic reticulum (ER), ER oxidoreductin 1 (ERO1) catalyzes intramolecular disulfide-bond formation within its substrates in coordination with protein-disulfide isomerase (PDI) and related enzymes. However, the molecular mechanisms that regulate the ERO1–PDI system in plants are unknown. Reduction of the regulatory disulfide bonds of the ERO1 from soybean, GmERO1a, is catalyzed by enzymes in five classes of PDI family proteins. Here, using recombinant proteins, vacuum-ultraviolet circular dichroism spectroscopy, biochemical and protein refolding assays, and quantitative immunoblotting, we found that GmERO1a activity is regulated by reduction of intramolecular disulfide bonds involving Cys-121 and Cys-146, which are located in a disordered region, similarly to their locations in human ERO1. Moreover, a GmERO1a variant in which Cys-121 and Cys-146 were replaced with Ala residues exhibited hyperactive oxidation. Soybean PDI family proteins differed in their ability to regulate GmERO1a. Unlike yeast and human ERO1s, for which PDI is the preferred substrate, GmERO1a directly transferred disulfide bonds to the specific active center of members of five classes of PDI family proteins. Of these proteins, GmPDIS-1, GmPDIS-2, GmPDIM, and GmPDIL7 (which are group II PDI family proteins) failed to catalyze effective oxidative folding of substrate RNase A when there was an unregulated supply of disulfide bonds from the C121A/C146A hyperactive mutant GmERO1a, because of its low disulfide-bond isomerization activity. We conclude that regulation of plant ERO1 activity is particularly important for effective oxidative protein folding by group II PDI family proteins.

Most proteins produced in the endoplasmic reticulum (ER) of eukaryotic cells fold via disulfide formation (oxidative folding). Oxidative folding is catalyzed by protein disulfide isomerase (PDI) and PDI-related ER protein thiol disulfide oxidoreductases (ER oxidoreductases). In yeast and mammals, ER oxidoreductin-1s (Ero1s) supply oxidizing equivalent to the active centers of PDI. In this study, we expressed recombinant soybean Ero1 (GmERO1a) and found that GmERO1a oxidized multiple soybean ER oxidoreductases, in contrast to mammalian Ero1s having a high specificity for PDI. One of these ER oxidoreductases, GmPDIM, associated in vivo and in vitro with GmPDIL-2, was unable to be oxidized by GmERO1a. We therefore pursued the possible cooperative oxidative folding by GmPDIM, GmERO1a, and GmPDIL-2 in vitro and found that GmPDIL-2 synergistically accelerated oxidative refolding. In this process, GmERO1a preferentially oxidized the active center in the A': domain among the A: , A': , and B: domains of GmPDIM. A disulfide bond introduced into the active center of the A': domain of GmPDIM was shown to be transferred to the active center of the A: domain of GmPDIM and the A: domain of GmPDIM directly oxidized the active centers of both the A: or A': domain of GmPDIL-2. Therefore, we propose that the relay of an oxidizing equivalent from one ER oxidoreductase to another may play an essential role in cooperative oxidative folding by multiple ER oxidoreductases in plants.

Hemocyanin (Hc) and phenoloxidase (PO) are members of the type 3 copper protein family. Although arthropod Hc and PO exhibit similar three-dimensional structures of the copper-containing active site, Hc functions as an oxygen transport protein, showing minimal or no phenoloxidase activity. Here, we present the crystal structure of the oxy form of Hc from Panulirus japonicus (PjHc) at 1.58 A resolution. The structure of the di-copper active site of PjHc was found to be almost identical to that of PO. Although conserved amino acids and the water molecule crucial for the enzymatic activity were observed in PjHc at almost the same positions as those in PO, PjHc showed no enzymatic activity under our experimental conditions. One striking difference between PjHc and arthropod PO was the presence of a “blocker residue” near the binuclear copper site of PjHc. This blocker residue comprised a phenylalanine residue tightly stacked with an imidazole ring of a CuA coordinated histidine and hindered substrates from accessing the active site. Our results suggest that the blocker residue is also a determining factor of the catalytic activity of type 3 copper proteins.

Ferritin, a multimeric iron storage protein distributed in almost all living kingdoms, has been highlighted recently as a nutritional iron source in plant-derived foodstuffs, because ferritin iron is suggested to have high bioavailability. In soybean seeds, ferritin contributes largely to the net iron contents. Here, the oligomeric states and iron contents of soybean ferritin during food processing (especially tofu gel formation) were analyzed. Ferritin was purified from tofu gel as an iron-containing oligomer (approximately 1000 Fe atoms per oligomer), which was composed of two types of subunits similar to the native soybean seed ferritin. Circular dichroism spectra also showed no differences in α-helical structure between native soybean ferritin and tofu ferritin. The present data demonstrate that ferritin was stable during the heat treatment (boiling procedure) in food processing, although partial denaturation was observed at temperatures higher than 80 °C.

Iron deficiency anemia (IDA) is the most common and widespread nutritional disorder in the world, so it is crucial to explore a safe and efficient functional factor for iron supplement. Fortunately, ferritin as a class of naturally occurring iron storage proteins can accommodate 4500 iron atoms within its inner cavity. Thus, it seems to be a suitable candidate as novel, utilizable forms of iron supplementary for populations with a low iron status. Additionally, ferritin also can be used as a vehicle for bioactive compounds delivery after the remove of the iron. This chapter focuses on recent progress in structure, function, and nutrition of ferritin.

Iron is one of the essential trace elements for humans. In this study, the iron contents in fresh, dried, and toasted nori (Pyropia yezoensis) were analyzed. The mean iron content of fresh, dried, and toasted nori were 19.0, 22.6, and 26.2 mg/100 g (dry weight), respectively. These values were superior to other food of plant origin. Furthermore, most of the iron in nori was maintained during processing, such as washing, drying, and toasting. Then, the form of iron in fresh, dried, and toasted nori was analyzed. As a result, an iron storage protein ferritin contributed to iron storage in raw and dried nori, although the precise rate of its contribution is yet to be determined, while ferritin protein cage was degraded in the toasted nori. It is the first report that verified the ferritin contribution to iron storage in such edible macroalgae with commercial importance.

Soybean 7S globulin, known as β-conglycinin, has been shown to regulate human plasma cholesterol and triglyceride levels. Furthermore, the α′ subunit of β-conglycinin has specifically been shown to possess low-density lipoprotein (LDL)-cholesterol-lowering activity. Therefore, accumulation of the α′ subunit of β-conglycinin in rice seeds could lead to the production of new functional rice that could promote human health. Herein, we used the low-glutelin rice mutant ‘Koshihikari’ (var. a123) and suppressed its glutelins and prolamins, the major seed storage proteins of rice, by RNA interference. The accumulation levels of the α′ subunit in the lines with suppressed glutelin and prolamin levels were >20 mg in 1 g of rice seeds, which is considerably higher than those in previous studies. Oral administration of the transgenic rice containing the α′ subunit exhibited a hypocholesterolemic activity in rats; the serum total cholesterol and LDL cholesterol levels were significantly reduced when compared to those of the control rice (var. a123). The cholesterol-lowering action by transgenic rice accumulating the α′ subunit induces a significant increase in fecal bile acid excretion and a tendency to increase in fecal cholesterol excretion. This is the first report that transgenic rice exhibits a hypocholesterolemic activity in rats in vivo by using the β-conglycinin α′ subunit.

Most proteins synthesized in the endoplasmic reticulum (ER) possess intramolecular and intermolecular disulfide bonds, which play an important role in the conformational stability and function of proteins. Hence, eukaryotic cells contain protein disulfide bond formation pathways such as the protein disulfide isomerase (PDI)-ER oxidoreductin 1 (Ero1) system in the ER lumen. In this study, we identified soybean PDIL7 (GmPDIL7), a novel soybean ER membrane-bound PDI family protein, and determined its enzymatic properties. GmPDIL7 has a putative N-terminal signal sequence, a thioredoxin domain with an active center motif (CGHC), and a putative C-terminal transmembrane region. Likewise, we demonstrated that GmPDIL7 is ubiquitously expressed in soybean tissues and is localized in the ER membrane. Furthermore, GmPDIL7 associated with other soybean PDI family proteins in vivo and GmPDIL7 mRNA was slightly upregulated under ER stress. The redox potential of recombinant GmPDIL7 expressed in Escherichia coli was −187 mV, indicating that GmPDIL7 could oxidize unfolded proteins. GmPDIL7 exhibited a dithiol oxidase activity level that was similar to other soybean PDI family proteins. However, the oxidative refolding activity of GmPDIL7 was lower than other soybean PDI family proteins. GmPDIL7 was well oxidized by GmERO1. Taken together, our results indicated that GmPDIL7 primarily plays a role as a supplier of disulfide bonds in nascent proteins for oxidative folding on the ER membrane. Database The nucleotide sequence data for the GmPDIL7 cDNA are available in the DNA Data Bank of Japan (DDBJ) databases under the accession numbers LC158001. Enzyme Protein disulfide isomerase: EC 5.3.4.1

Erythropoiesis requires large amounts of iron for hemoglobin synthesis, which is mainly provided by macrophages and the intestines in a transferrin (Tf)-bound form. Bone marrow erythroblasts incorporate Tf through endocytosis, which is mediated by transferrin receptor 1 (TFR1). Recently, human TFR1, aside from its role as a Tf receptor, was also found to be a receptor for the H-subunit of ferritin (FTH). In humans, hematopoietic erythroid precursor cells express high levels of TFR1 and specifically take up the FTH homopolymer (H-ferritin). H-ferritin inhibits the formation of burst forming unit-erythroid colonies in vitro. TFR2, which is also a Tf receptor, is predominantly expressed in hepatocytes and erythroid precursor cells. In the liver, TFR2 forms a complex with HFE, a hereditary hemochromatosis-associated protein, and acts as an iron sensor. In mice, hepatocyte-specific knockout of the TFR2 gene has been shown to cause systemic iron-overload with decreased expression of hepcidin, the central regulator of iron homeostasis. In erythroid cells, TFR2 forms a complex with the erythropoietin receptor and facilitates its trafficking to the cell membrane. Moreover, hematopoietic cell-specific knockout of the TFR2 gene causes microcytic erythrocytosis in mice. This review focuses on the molecular evolution and functions of these TFRs and their ligands.

Iron is an essential element for virtually all kingdoms of life, and especially for primary producers in ocean ecosystems. To date, the molecular mechanism of iron utilization by macroalgae remains largely unknown. To elucidate the strategy of iron acquisition and storage in macroalgae, we focused on the function of the iron storage protein ferritin in the sea lettuce, Ulva pertusa, which has abundant iron content. Judging from the primary structure, U. pertusa ferritin (UpFer) can be classified as a land-plant-type ferritin, which is usually found in plastids. The gene of UpFer was expressed in the peripheral, central and rhizoid parts. Western blot analysis showed that UpFER was present and functioned in processed 26- and 22-kDa forms. Furthermore, recombinant UpFER had iron incorporation activity comparable to other ferritins. These results suggest that ferritin also functions as an iron storage protein as in unicellular algae and land plants.

Plant ferritin is a naturally occurring heteropolymer in plastids, where Fe2+ is oxidatively deposited into the protein. However, the effect of this process on the coexistence of DNA and plant ferritin in the plastids is unknown. To investigate this effect, we built a system in which various plant ferritins and DNA coexist, followed by treatment with ferrous ions under aerobic conditions. Interestingly, naturally occurring soybean seed ferritin (SSF), a heteropolymer with an H-1/H-2 ratio of 1 to 1 in the apo form, completely protected DNA from oxidative damage during iron oxidative deposition into protein, and a similar result was obtained with its recombinant form, but not with its homopolymeric counterparts, apo rH-1 and apo rH-2. We demonstrate that the difference in DNA protection between heteropolymeric and homopolymeric plant ferritins stems from their different strategies to control iron chemistry during the above oxidative process. For example, the detoxification reaction occurs only in the presence of apo heteropolymeric SSF (hSSF), thereby preventing the production of hydroxyl radicals. In contrast, hydroxyl radicals are apparently generated via the Fenton reaction when apo rH-1 or rH-2 is used instead of apo hSSF. Thus, a combination of H-1 and H-2 subunits in hSSF seems to impart a unique DNA-protective function to the protein, which was previously unrecognized. This new finding advances our understanding of the structure and function of ferritin and of the widespread occurrence of heteropolymeric plant ferritin in nature.

Plant ferritins have some unique structural and functional features. Most of these features can be related to the plant-specific “extension peptide” (EP), which exists in the N-terminus of the mature region of a plant ferritin. Recent crystallographic analysis of a plant ferritin revealed the structure of the EP, however, two points remain unclear: (i) whether the structures of well-conserved EP of plant ferritins are common in all plants, and (ii) whether the EP truly contributes to the shell stability of the plant ferritin oligomer. To clarify these matters, we have cloned a green-plant-type ferritin cDNA from a green alga, Ulva pertusa, and investigated its crystal structure. Ulva pertusa ferritin (UpFER) has a plant-ferritin-specific extension peptide composed of 28 amino acid residues. In the crystal structure of UpFER, the EP lay on and interacted with the neighboring threefold symmetry-related subunit. The amino acid residues involved in the interaction were very highly conserved among plant ferritins. The EPs masked the hydrophobic pockets on the ferritin shell surface by lying on them, and this made the ferritin oligomer more hydrophilic. Furthermore, differential scanning calorimetric analysis of the native and its EP-deletion mutant suggested that the EP contributed to the thermal stability of the plant ferritin shell. Thus, the shell stability and surface hydrophobicity of plant ferritin were controlled by the presence or absence of the plant-ferritin-specific EP. This regulation can account for those processes such as shell stability, degradation, and association of plant ferritin, which are significantly related to iron utilization in plants.

Melanization is one of the major immune responses in arthropods. Prophenoloxidases (proPOs) catalyze the oxidation of mono- or o-diphenols, a reaction that is the key initial step of melanin formation. Well-characterized proPOs from crustaceans are synthesized in haemocytes and are released into plasma in response to microbial attack. However, PO activity does exist in the plasma of haemolymph without pathogenic infections. Here, we demonstrate that a novel type of proPO contributes to such PO activity in the plasma fraction of haemolymph of crustaceans. The novel enzyme, which was purified from the plasma of the kuruma prawn (Marsupenaeus japonicus), possessed strong and specific monophenol and o-diphenol oxidation activity compared with that of known haemocyte-type proPO. Amino acid sequence analyses indicated that this enzyme was distinct from the known proPO. The cDNA sequence and deduced amino acid sequence of this enzyme has a putative binuclear copper center, and showed approximately 30% and 20% identity with the primary structures of reported proPO and haemocyanin sequences of the kuruma prawn, respectively. Reverse transcription PCR analysis showed that this enzyme was synthesized in the hepatopancreas rather than in haemocytes. Although the primary structure and enzymatic properties of this novel enzyme suggested that it is a phenoloxidase, its biogenesis, tissue distribution, and oligomeric state resemble those of haemocyanin, which belongs to the same protein family (type III copper protein). This novel proPO enzyme may share a role with the already characterized version, itself a major component of the innate immune system in crustaceans.

*† These authors contributed equally to this work. SUMMARY Chitinases are a class of ubiquitous proteins that are widely distributed in plants. Defense is the major natural role for chitinases, primarily against fungal pathogens. Little is known regarding their non-defensive roles in seeds. In this study, a new class III chitinase from pomegranate seeds (pomegranate seed chitinase, PSC) was isolated and purified to homogeneity. The native state of PSC is a monomer with a molecular weight of approximately 30 kDa. This chitinase naturally binds calcium ions with high capacity and low affinity, suggesting that PSC is a calcium storage protein. Consistent with this idea, its amino acid sequence (inferred from cDNA) is rich in acidic amino acid residues, especially Asp, similar to reported calcium storage proteins. The presence of calcium considerably improves the stability of the protein but has little effect on its enzymatic activity. Transmission electron microscopy analyses indicate that, similar to phytoferritin, this enzyme is widely distributed in the stroma of amyloplasts of the embryonic cells, suggesting that amyloplasts in seeds could serve as an alternative plastid for calcium storage. Indeed, the transmission electron microscopy results showed that, within the embryonic cells, calcium ions are mainly distributed in the stroma of the amyloplasts, consistent with a role for PSC in calcium storage. Thus, the plant appears to have evolved a new plastid for calcium storage in seeds. During seed germination, the content of this enzyme decreases with time, suggesting that it is involved in the germination process.

Naturally occurring phytoferritin is a heteropolymer consisting of two different H-type subunits, H-1 and H-2. Prior to this study, however, the function of the two subunits in oxidative deposition of iron in ferritin was unknown. The data show that, upon aerobic addition of 48-200 Fe(2+)/shell to apoferritin, iron oxidation occurs only at the diiron ferroxidase center of recombinant H1 (rH-1). In addition to the diiron ferroxidase mechanism, such oxidation is catalyzed by the extension peptide (a specific domain found in phytoferritin) of rH-2, because the H-1 subunit is able to remove Fe(3+) from the center to the inner cavity better than the H-2 subunit. These findings support the idea that the H-1 and H-2 subunits play different roles in iron mineralization in protein. Interestingly, at medium iron loading (200 irons/shell), wild-type (WT) soybean seed ferritin (SSF) exhibits a stronger activity in catalyzing iron oxidation (1.10 ± 0.13 μm iron/subunit/s) than rH-1 (0.59 ± 0.07 μm iron/subunit/s) and rH-2 (0.48 ± 0.04 μm iron/subunit/s), demonstrating that a synergistic interaction exists between the H-1 and H-2 subunits in SSF during iron mineralization. Such synergistic interaction becomes considerably stronger at high iron loading (400 irons/shell) as indicated by the observation that the iron oxidation activity of WT SSF is ∼10 times larger than those of rH-1 and rH-2. This helps elucidate the widespread occurrence of heteropolymeric ferritins in plants.

Iron in phytoferritin from legume seeds is required for seedling germination and early growth. However, the mechanism by which phytoferritin regulates its iron complement to these physiological processes remains unknown. In the present study, protein degradation is found to occur in purified SSF (soya bean seed ferritin) (consisting of H-1 and H-2 subunits) during storage, consistent with previous results that such degradation also occurs during seedling germination. In contrast, no degradation is observed with animal ferritin under identical conditions, suggesting that SSF autodegradation might be due to the EP (extension peptide) on the exterior surface of the protein, a specific domain found only in phytoferritin. Indeed, EP-deleted SSF becomes stable, confirming the above hypothesis. Further support comes from a protease activity assay showing that EP-1 (corresponding to the EP of the H-1 subunit) exhibits significant serine protease-like activity, whereas the activity of EP-2 (corresponding to the EP of the H-2 subunit) is much weaker. Consistent with the observation above, rH-1 (recombinant H-1 ferritin) is prone to degradation, whereas its analogue, rH-2, becomes very stable under identical conditions. This demonstrates that SSF degradation mainly originates from the serine protease-like activity of EP-1. Associated with EP degradation is a considerable increase in the rate of iron release from SSF induced by ascorbate in the amyloplast (pH range, 5.8–6.1). Thus phytoferritin may have facilitated the evolution of the specific domain to control its iron complement in response to cell iron need in the seedling stage.

The polyguanine-rich DNA sequences commonly found at telomeres and in rDNA arrays have been shown to assemble into structures known as G quadruplexes, or G4 DNA, stabilized by base-stacked G quartets, an arrangement of four hydrogen-bonded guanines. G4 DNA structures are resistant to the many helicases and nucleases that process intermediates arising in the course of DNA replication and repair. The lagging strand DNA replication protein, Dna2, has demonstrated a unique localization to telomeres and a role in de novo telomere biogenesis, prompting us to study the activities of Dna2 on G4 DNA-containing substrates. We find that yeast Dna2 binds with 25-fold higher affinity to G4 DNA formed from yeast telomere repeats than to single-stranded DNA of the same sequence. Human Dna2 also binds G4 DNAs. The helicase activities of both yeast and human Dna2 are effective in unwinding G4 DNAs. On the other hand, the nuclease activities of both yeast and human Dna2 are attenuated by the formation of G4 DNA, with the extent of inhibition depending on the topology of the G4 structure. This inhibition can be overcome by replication protein A. Replication protein A is known to stimulate the 5′- to 3′-nuclease activity of Dna2; however, we go on to show that this same protein inhibits the 3′- to 5′-exo/endonuclease activity of Dna2. These observations are discussed in terms of possible roles for Dna2 in resolving G4 secondary structures that arise during Okazaki fragment processing and telomere lengthening.

Renal cell carcinoma (RCC) is relatively resistant to chemotherapy and radiotherapy. Recent advances in drug development are providing novel agents for the treatment of RCC, but the effects are still minimal. In addition, there is an urgent need to identify diagnostic markers for RCC. In this report, to discover potential diagnostic markers and therapeutic targets, we subjected RCC samples to a quantitative proteomic analysis utilizing 2-nitrobenzenesulfenyl (NBS) reagent. Proteins were extracted from RCC and adjacent normal tissue, obtained surgically from patients, and labeled with NBS reagent containing six (12)C or (13)C. This was followed by trypsin digestion and the enrichment of labeled peptides. Samples were then subjected to analysis by MALDI-TOF MS. NBS-labeled peptides with a 6 Da difference were identified by MS/MS. Thirty-four proteins were upregulated in more than 60% of the patients of which some were previously known, and some were novel. The identity of a few proteins was confirmed by Western blotting and quantitative real time RT-PCR. The results suggest that NBS-based quantitative proteomic analysis is useful for discovering diagnostic markers and therapeutic targets for RCC.

In recent years, a lot of 3D character dance animation movies are created by amateur users using 3DCG animation editing tools (e.g. MikuMikuDance). Whereas, most of them are created manually. Then automatic facial animation system for dancing character will be useful to create dance movies and visualize impressions effectively. Therefore, we address the challenging theme to estimate dancing character's emotions (we call "dance emotion"). In previous work considering music features, DiPaola et al. [2006] proposed music-driven emotionally expressive face system. To detect the mood of the input music, they used a hierarchical framework (Thayer model), and achieved to generate facial animation that matches music emotion. However, their model can't express subtleties of emotion between two emotions because input music divided into few moods sharply using Gaussian mixture model. In addition, they decide more detailed moods based on the psychological rules that uses score information, so they requires MIDI data. In this paper, we propose "dance emotion model" to visualize dancing character's emotion as facial expression. Our model is built by the coordinate information frame by frame on the emotional space through perceptional experiment using music and dance motion database without MIDI data. Moreover, by considering the displacement on the emotional space, we can express not only a certain emotion but also subtleties of emotions. As the result, our system got a higher accuracy comparing with the previous work. We can create the facial expression result soon by inputting audio data and synchronized motion. It is shown the utility through the comparison with previous work in Figure 1.

Background The major wheat seed proteins are storage proteins that are synthesized in the rough endoplasmic reticulum (ER) of starchy endosperm cells. Many of these proteins have intra- and intermolecular disulfide bonds. In eukaryotes, the formation of most intramolecular disulfide bonds in the ER is thought to be catalyzed by protein disulfide isomerase (PDI) family proteins. The cDNAs that encode eight groups of bread wheat (Triticum aestivum L.) PDI family proteins have been cloned, and their expression levels in developing wheat grains have been determined. The purpose of the present study was to characterize the enzymatic properties of the wheat PDI family proteins and clarify their expression patterns in wheat caryopses. Results PDI family cDNAs, which are categorized into group I (TaPDIL1Aα, TaPDIL1Aβ, TaPDIL1Aγ, TaPDIL1Aδ, and TaPDIL1B), group II (TaPDIL2), group III (TaPDIL3A), group IV (TaPDIL4D), and group V (TaPDIL5A), were cloned. The expression levels of recombinant TaPDIL1Aα, TaPDIL1B, TaPDIL2, TaPDIL3A, TaPDIL4D, and TaPDIL5A in Escherichia coli were established from the cloned cDNAs. All recombinant proteins were expressed in soluble forms and purified. Aside from TaPDIL3A, the recombinant proteins exhibited oxidative refolding activity on reduced and denatured ribonuclease A. Five groups of PDI family proteins were distributed throughout wheat caryopses, and expression levels of these proteins were higher during grain filling than in the late stage of maturing. Localization of these proteins in the ER was confirmed by fluorescent immunostaining of the immature caryopses. In mature grains, the five groups of PDI family proteins remained in the aleurone cells and the protein matrix of the starchy endosperm. Conclusions High expression of PDI family proteins during grain filling in the starchy endosperm suggest that these proteins play an important role in forming intramolecular disulfide bonds in seed storage proteins. In addition, these PDI family proteins that remain in the aleurone layers of mature grains likely assist in folding newly synthesized hydrolytic enzymes during germination. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0460-2) contains supplementary material, which is available to authorized users.

Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body's iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin.

Granulomatosis with polyangiitis (GPA) sometimes involves the eye orbit; however, choroidal involvements in GPA had been rarely reported. We report a rare case presenting with a choroidal mass in an 83-year-old Japanese woman who presented with left eye pain. Diagnostic biopsy revealed necrotizing vasculitis with infiltrates of inflammatory cells. Diagnosis was localized granulomatosis with polyangiitis. Combined treatments with corticosteroid plus azathioprine resolved the choroidal mass region. Although treatment with corticosteroid and immunosuppressive agents improves the prognosis of the disease, ocular morbidity is still well recognized. Clinicians should consider a differential diagnosis of GPA in patients with inflammatory choroidal tumors.

Chitinase hydrolyzes the β-1,4-glycosidic bond in chitin. In higher plants, this enzyme has been regarded as a pathogenesis-related protein. Recently, we identified a class III chitinase, which functions as a calcium storage protein in pomegranate (Punica granatum) seed (PSC, pomegranate seed chitinase). Here, we solved a crystal structure of PSC at 1.6 Å resolution. Although its overall structure, including the structure of catalytic site and non-proline cis-peptides, was closely similar to those of other class III chitinases, PSC had some unique structural characteristics. First, there were some metal-binding sites with coordinated water molecules on the surface of PSC. Second, many unconserved aspartate residues were present in the PSC sequence which rendered the surface of PSC negatively charged. This acidic electrostatic property is in contrast to that of hevamine, well-characterized plant class III chitinase, which has rather a positively charged surface. Thus, the crystal structure provides a clue for metal association property of PSC.

The Dna2 protein is a multifunctional enzyme with 5'-3' DNA helicase, DNA-dependent ATPase, 3' exo/endonuclease, and 5' exo/endonuclease. The enzyme is highly specific for structures containing single-stranded flaps adjacent to duplex regions. We report here two novel activities of both the yeast and human Dna2 helicase/nuclease protein: single strand annealing and ATP-independent strand exchange on short duplexes. These activities are independent of ATPase/helicase and nuclease activities in that mutations eliminating either nuclease or ATPase/helicase do not inhibit strand annealing or strand exchange. ATP inhibits strand exchange. A model rationalizing the multiple catalytic functions of Dna2 and leading to its coordination with other enzymes in processing single-stranded flaps during DNA replication and repair is presented.

T2 tobacco lines overexpressing soybean ferritin in the plastids (+TPs) or apoplasm (AFs) under the regulation of CaMV 35S promoter were grown on MS nutrient solution. After 1 month growth, statuses of six major divalent‐metals (Ca, Cu, Fe, Mg, Mn, and Zn) were measured in leaves and roots. Both +TPs and AFs showed enhanced growth (max. 1.7×) in leaves than the control line. The Fe contents in leaves of +TPs and AFs were significantly larger (1.9–2.8×) than that of the control line. The other metal contents in leaves of +TPs and AFs were almost the same as or less than those of the control line. In contrast to the result of leaves, the growth enhancement in roots was not clear in +TPs, but in AFs. Also, some of the non‐ferrous metal contents in roots of +TPs and AFs were dramatically increased compared with those of the control line (Mn, 1.9–10.4×; Zn, 1.6–2.3×), whereas the differences in content of Fe, Cu, Ca, and Mg were insignificant. These results demonstrated that the ferritin overexpressio...

Most plant seeds contain 11S globulins as major storage proteins for their nutrition. Soybean glycinin belongs to the 11S globulin family and consists of five kinds of subunits. We determined the crystal structure of a homohexamer of the glycinin A3B4 subunit at 2.1-Å resolution. The crystal structure shows that the hexamer has 32-point group symmetry formed by face-to-face stacking of two trimers. The interface buries the highly conserved interchain disulfide. Based on the structure, we propose that an ingenious face-to-face mechanism controls the hexamer formation of the 11S globulin by movement of a mobile disordered region to the side of the trimer after posttranslational processing. Electrostatic analysis of the faces suggests that the interchain disulfide-containing face has high positive potential at acidic pH, which induces dissociation of the hexamer into trimers that may be susceptible to proteinases after seed imbibition. This dissociation might result in the degradation and mobilization of 11S globulins as storage proteins in embryos during germination and seedling growth.

Background: MCM and Cdc45 are required for the initiation and elongation stages of eukaryotic DNA replication. Recent studies show that a purified Mcm4/6/7 complex has DNA helicase activity. However, the biochemical function of the MCM complex and Cdc45 bound to chromatin has not been elucidated. Results: We have examined the biochemical properties of MCM proteins bound to chromatin fractions using Xenopus egg extracts. Immunoprecipitation of MCM proteins extracted under denaturing conditions reveals that all six subunits of MCM and Cdc45 form a tight complex following the initiation of DNA replication, and that both CDK activity and Cdc45 are essential for the complex formation. Chromatin immunoprecipitation of MCM proteins and Cdc45 shows that a complex containing MCM and Cdc45 has a DNA helicase activity which is dependent on CDK activity and Cdc45 in the extracts. Furthermore, both the complex and the helicase activity are resistant to treatment with phosphatase and high salt. Conclusions: Following the initiation of DNA replication, a tight MCM–Cdc45 complex is formed on chromatin and its formation is closely correlated with the DNA helicase activity of chromatin immunoprecipitates containing MCM and Cdc45. We propose that the tight MCM–Cdc45 complex functions as a replicative DNA helicase in vivo.

Expression of the Ids2 (iron deficiency-specific clone 2) gene was compared between native barley and heterogenous tobacco regarding spatial specificity and iron-deficiency responsiveness. The results of Northern blot and in situ hybridization analyses demonstrated that the Ids2 expression in barley was specific to the endodermis and/or pericycle and in the cortex of roots, and responded exactly to iron starvation. Analysis of the GUS activity regulated by eight 5’-deletion clones of the Ids2 promoter in tobacco exhibited that every clone could promote root specific and iron-deficiency responsive expression. Aspects of the spatial specificity and iron-deficiency responsiveness (e.g. sensitivity, stability and returnability) in tobacco were also similar to those in barley, although a few variations were recognized. It is probable that even the shortest Ids2 promoter region in this experiment (-272/-47) contains the iron-deficiency-inducible and the root-specific cis-element(s), which is recognized by two different types of iron-acquisition strategy plants, barley and tobacco.

We have recently identified a new class III chitinase from pomegranate seeds (PSC). Interestingly, this new chitinase naturally binds calcium ions with high capacity and low affinity, suggesting that PSC is a Ca-storage protein. Analysis of the amino acid sequence showed that this enzyme is rich in acidic amino acid residues, especially Asp, which are responsible for calcium binding. Different from other known chitinases, PSC is located in the stroma of amyloplasts in pomegranate seeds. Transmission electron microscopy (TEM) analysis indicated that the embryonic cells of pomegranate seeds are rich in calcium ions, most of which are distributed in the stroma and the starch granule of the amyloplasts, consistent with the above idea that PSC is involved in calcium storage, a newly non-defensive function.

Phenoloxidase (PO), which is classified as a type 3 copper protein, catalyzes the hydroxylation of monophenol to o-diphenol and subsequent oxidation to the corresponding o-quinone. The geometry and coordination environment of the active site of the arthropod PO are very similar to those of the arthropod hemocyanin (Hc). However, unlike the POs, Hc is an oxygen carrier in crustaceans, and does not possess PO activity in general. Recently, we identified a new type of proPO from a crustacean and designated it proPOβ. This enzyme has many characteristics that are rather similar to those of Hc, such as its maturation, localization, and oligomeric state. Here, we determined the crystal structure of proPOβ prepared from the hemolymph of kuruma prawns (Marsupenaeus japonicus) at 1.8-A resolution. M. japonicus proPOβ forms a homohexamer rather similar to that of arthropod Hc. The geometry of the active copper site in proPOβ is nearly identical to that of arthropod Hc. Furthermore, the well-characterized ‘place-holder’ phenylalanine is present (Phe72). However, the accessibility to the active site differs in several ways. First, another phenylalanine, which shields the active site by interacting with a copper-coordinated histidine in crustacean Hc, is replaced by valine in the proPOβ structure. Second, two tyrosines, Tyr208 and Tyr209, both of which are absent in Hc, show the alternative conformations and form a pathway providing access to the reaction center. Thus, the present crystal structure clarifies the similarities and differences in the activity of two closely related proteins, PO and Hc. Database Structural data are available in the RSCB protein data bank under the accession number 3WKY. Structured digital abstract proPObeta and proPObeta bind by x-ray crystallography (View interaction)

DNA polymerase ɛ (Polɛ) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA polymerases, DNA polymerases α and δ (Polα and δ), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Polɛ in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Polɛ is indeed required for chromosomal DNA replication. In Polɛ-depleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Polα are loaded onto the chromatin template in Polɛ-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Polɛ, along with Polα and Polδ, is necessary for coordinated chromosomal DNA replication in eukaryotic cells.