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The Super-Kamiokande detector is a ring imaging Cherenkov detector for neutrino physics and proton-decay search and consists of 50 000 tons of pure water equipped with about 13 000 photo-multipliers. The old front-end electronics and online system running for more than one decade were all upgraded in September, 2008 and the data acquisition was started successfully. The new front-end electronics is based on a charge to time converter and a multi-hit Time to Digital converter. TCP/IP based readout channel is implemented to handle large amounts of data. In the new data acquisition scheme, the hardware event-trigger for the data reduction is replaced by processing all the hits in the online farm, so that we are able to lower the threshold of the detection energy for solar neutrino and analyze consecutive events whose time interval is too long to detect in the previous system. To make the new online system to be capable of processing larger dataflow of up to 470 MB/s, we utilize Gigabit and 10-Gigabit Ethernet technologies and distribute the load over Linux PCs to handle a large amount of data. In this paper, we will describe the design and performance of the new system in the commissioning. [ABSTRACT FROM AUTHOR]

The nucleation density and crystallinity of diamond prepared in an electron cyclotron resonance plasma by applying a negative substrate bias have been studied as a function of bias run time. The mean ion energy onto the substrate was set at approximately 30 and 50 eV by controlling the bias voltage. For short bias times of 10–60 min, the nucleation density increased with time and the highest nucleation density (107–108 cm-2) and crystallinity were obtained at 60 min of biasing. For long bias times of 60–150 min, the nucleation density decreased with time accompanied by deterioration of the crystallinity, presumably due to a reduction of the effective substrate bias voltage caused by the delamination of the deposited amorphous carbon film. The incubation period for nucleation was estimated between 10 and 30 min, which was interpreted as the time to produce the amorphous carbon hydrogenated matrix characterized by relatively ordered structures with respect to carbon sp2 phase. [Copyright &y& Elsevier]

CEL-III is a hemolytic lectin purified from the marine invertebrate Cucumaria echinata. New expression system of CEL-III was constructed, and the recombinant thioredoxin-fused CEL-III (Trx-CEL-III) showed strong hemolytic and carbohydrate-binding activity as same as authentic CEL-III. Mutation analysis of Trx-CEL-III suggested that carbohydrate binding to subdomain 1α and 2β of CEL-III might be important for the hemolytic activity., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Two kinds of Kunitz-type protease inhibitors, AKPI1 and AKPI2, were purified from Apios americana tubers by four steps of column chromatographies and their cDNA cloning was performed. AKPI1 cDNA consist of 809 nucleotides, and the matured protein had 190 amino acids with 20,594 Da. AKPI2 cDNA consist of 794 nucleotides, and the matured protein had 177 amino acids with 19,336 Da. P1 site of AKPI2 was Leu88, suggested the target enzyme was chymotrypsin. On the other hand, Gly85-Ile86-Ser87 was positioned around P1 site of AKTI1. Sequence analysis suggested that two forms (single-chain and two-chain form) of AKPI2 protein were present in the tubers. Recombinant AKPI2 expressed by E.coli system showed inhibitory activity toward serine proteases and heat stability. The Ki values toward chymotrypsin and trypsin were 4 × 10 -7 M and 6 × 10 -6 M, respectively. Abbreviations: AAL: Apios americana lectin; AATI: Apios americana Bowman-Birk type trypsin inhibitor; ACE: angiotensin-converting enzyme; IPTG: isopropyl-β-D-thio-galactopyranoside; Ki : inhibition constant; KPIs: Kunitz-type protease inhibitors; L-BAPA: Benzoyl-L-arginine p -nitroanilide monohydrochloride; L-BTPA: Benzoyl-L-tyrosine p -nitroanilide; PFLNA: Pyr-Phe-Leu- p -nitroanilide; RP-HPLC: reverse-phase high-performance liquid chromatography; RT-PCR: reverse transcription-polymerase chain reaction; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SLIC: sequence and ligation independent cloning; STANA: N-Succinyl-Ala-Ala-Ala- p -nitroanilide; SHR: spontaneously hypertensive rats; TFA: trifluoroacetic acid; UTR: untranslated region.

Auxin is thought to be an important factor in the induction of galls by galling insects. We have previously shown that both galling and nongalling insects synthesize indole-3-acetic acid (IAA) from tryptophan (Trp) via two intermediates, indole-3-acetaldoxime (IAOx) and indole-3-acetaldehyde (IAAld). In this study, we isolated an enzyme that catalyzes the last step "IAAld → IAA" from a silk-gland extract of Bombyx mori. The enzyme, designated "BmIAO1", contains two 2Fe-2S iron-sulfur-cluster-binding domains, an FAD-binding domain, and a molybdopterin-binding domain, which are conserved in aldehyde oxidases. BmIAO1 causes the nonenzymatic conversion of Trp to IAAld and the enzymatic conversion of IAOx to IAA, suggesting that BmIAO1 alone is responsible for IAA production in B. mori. However, a detailed comparison of pure BmIAO1 and the crude silk-gland extract suggested the presence of other enzymes involved in IAA production from Trp. Abbreviations: BA: benzoic acid; CE: collision energy; CXP: collision cell exit potential; DP: declustering potential; IAA: indole-3-acetic acid; IBI1: IAA biosynthetic inhibitor-1; IAAld: indole-3-acetaldehyde; ICA: indole-3-carboxylic acid; IAOx: indole-3-acetaldoxime; IEtOH: indole-3-ethanol; LC-MS/MS: liquid chromatography-tandem mass spectrometry; Trp: tryptophan.

Background/aim: The aim of this study was to assess the appropriateness of pulmonary metastasectomy (MT), with a focus on the primary organ., Patients and Methods: The pathological status of the primary organ, outcome of the MT, disease-free interval, and overall survival were assessed., Results: The primary organ was the most significant prognostic factor analyzed, with a relative risk of 4.6 (95% confidence interval: 1.69-12.56, p=0.003). Patients with colorectal carcinoma had a better survival than those with another primary organ (p=0.003). The hazard ratios by primary organ in comparison to colorectal carcinoma were 3.2 for head and neck carcinoma, 3.5 for soft tissue sarcoma, 8.3 for hepatocellular carcinoma, and 8.9 for urinary carcinoma., Conclusion: Colorectal carcinoma is associated with a greater survival benefit than cancer of other primary organs. Colorectal carcinoma cases should be more aggressively considered for MT than other primary organ cases., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

In the course of our study of the biosynthetic pathway of auxin, a class of phytohormones, in insects, we proposed the biosynthetic pathway tryptophan (Trp)→indole-3-acetaldoxime (IAOx)→indole-3-acetadehyde (IAAld)→indole-3-acetic acid (IAA). In this study, we identified two branches in the metabolic pathways in the silkworm, possibly affecting the efficiency of IAA production: Trp→indole-3-pyruvic acid→indole-3-lactic acid and IAAld→indole-3-ethanol. We also determined the apparent conversion activities (2.05×10 -7 UmL -1 for Trp→IAA, 1.30×10 -5 UmL -1 for IAOx→IAA, and 3.91×10 -1 UmL -1 for IAAld→IAA), which explain why IAOx and IAAld are barely detectable as either endogenous compounds or metabolites of their precursors. The failure to detect IAAld, even in the presence of an inhibitor of the conversion IAAld→IAA, is explained by a switch in the conversion from IAAld→IAA to IAAld→IEtOH., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Lotus ( Nelumbo nucifera Gaertn.) seed proteome has been the focus of our studies, and we have recently established the first proteome dataset for its mature seed endosperm. The current study unravels the immature endosperm, as well as the embryo proteome, to provide a comprehensive dataset of the lotus seed proteins and a comparison between the mature and immature endosperm tissues across the seed's development. One-dimensional gel electrophoresis (SDS-PAGE) linked with tandem mass spectrometry provided a protein inventory of the immature endosperm (122 non-redundant proteins) and embryo (141 non-redundant proteins) tissues. Comparing with the previous mature endosperm dataset (66 non-redundant proteins), a total of 206 non-redundant proteins were identified across all three tissues of the lotus seed. Results revealed some significant differences in proteome composition between the three lotus seed tissues, most notably between the mature endosperm and its immature developmental stage shifting the proteins from nutrient production to nutrient storage.

Nelumbo nucifera (Gaertn.) or lotus, is an aquatic plant native to India, and presently consumed as food mainly in China and Japan. Lotus is also widely used in Indian and Chinese traditional medicine. Extracts from different parts of the lotus plant have been reported to show diverse biological activities-antioxidant, free radical scavenging, anti-inflammatory, and immunomodulatory. Despite this, little work has been done in isolating and identifying proteins responsible for these activities, or yet importantly to establish a lotus proteome. The aim of our group is to develop a proteome catalog of the lotus plant, starting with its seed, the nutrient rich food source. In this present study, the seed endosperm-most abundant in proteins, and main nutrient storage tissue-was targeted for protein extraction by testing five different extraction protocols, followed by their proteomic analyses using complementary 1DE and 2DE approaches in conjunction with MS/MS. The inventory of 66 nonredundant proteins obtained by 1DE-MS and the 30 obtained by 2DE-MS provides the first catalog of the lotus seed endosperm, where the most abundant protein functions were in categories of metabolic activities related to carbohydrate metabolism and nutrient storage., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An Apios americana lectin (AAL) and a lectin-like protein (AALP) were purified from tubers by chromatography on Butyl-Cellulofine, ovomucoid-Cellulofine, and DEAE-Cellulofine columns. AAL showed strong hemagglutinating activity toward chicken and goose erythrocytes, but AALP showed no such activity toward any of the erythrocytes tested. The hemagglutinating activity of AAL was not inhibited by mono- or disaccharides, but was inhibited by glycoproteins, such as asialofetuin and ovomucoid, suggesting that AAL is an oligosaccharide-specific lectin. The cDNAs of AAL and AALP consist of 1,093 and 1,104 nucleotides and encode proteins of 302 and 274 amino acid residues, respectively. Both amino acid sequences showed high similarity to known legume lectins, and those of their amino acids involved in carbohydrate and metal binding were conserved.

CEL-III is a hemolytic lectin purified from the marine invertebrate Cucumaria echinata. Previous research has indictated that CEL-III is composed of several isoforms. Here we identified five CEL-III isolectin genes, designated CEL-III-L1, CEL-III-L2, CEL-III-S1, CEL-III-S2, and CEL-III-LS1, by cDNA cloning. The deduced amino acid sequences suggested they shared 94.0-99.8% identical residues. Among the amino acid residues involved in carbohydrate binding, the His residue, which contributes to stacking with sugar, in subdomain 1α was replaced by Tyr in CEL-III-L2. The recombinant proteins were expressed in Escherichia coli or insect cells. rCEL-III-L2 showed higher hemolytic activity than those of the other isolectins. Furthermore, an apparent oligomer band of rCEL-III-L2 was detected on erythrocyte membranes, although the other isolectins showed smear bands. These results suggest that Tyr36 of CEL-III-L2 is important for the expression of hemolytic activity and oligomerization.

The T2K experiment observes indications of ν(μ) → ν(e) appearance in data accumulated with 1.43×10(20) protons on target. Six events pass all selection criteria at the far detector. In a three-flavor neutrino oscillation scenario with |Δm(23)(2)| = 2.4×10(-3)  eV(2), sin(2)2θ(23) = 1 and sin(2)2θ(13) = 0, the expected number of such events is 1.5±0.3(syst). Under this hypothesis, the probability to observe six or more candidate events is 7×10(-3), equivalent to 2.5σ significance. At 90% C.L., the data are consistent with 0.03(0.04) < sin(2)2θ(13) < 0.28(0.34) for δ(CP) = 0 and a normal (inverted) hierarchy.

A Manduca sexta (tobacco hornworm) cysteine protease inhibitor, MsCPI, purified from larval hemolymph has an apparent molecular mass of 11.5 kDa, whereas the size of the mRNA is very large (∼9 kilobases). MsCPI cDNA consists of a 9,273 nucleotides that encode a polypeptide of 2,676 amino acids, which includes nine tandemly repeated MsCPI domains, four cystatin-like domains and one procathepsin F-like domain. The procathepsin F-like domain protein was expressed in Escherichia coli and processed to its active mature form by incubation with pepsin. The mature enzyme hydrolyzed Z-Leu-Arg-MCA, Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA rapidly, whereas hydrolysis of Suc-Leu-Tyr-MCA and Z-Arg-Arg-MCA was very slow. The protease was strongly inhibited by MsCPI, egg-white cystatin and sunflower cystatin with K(i) values in the nanomolar range. When the MsCPI tandem protein linked to two MsCPI domains was treated with proteases, it was degraded by the cathepsin F-like protease. However, tryptic digestion converted the MsCPI tandem protein to an active inhibitory form. These data support the hypothesis that the mature MsCPI protein is produced from the MsCPI precursor protein by trypsin-like proteases. The resulting mature MsCPI protein probably plays a role in the regulation of the activity of endogenous cysteine proteases., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The steady-state and time-resolved fluorescence spectroscopy is one of the most powerful method to detect and analyze subtle conformation change and interaction between peptide elements in protein. Phytocystatin Scb isolated from sunflower seeds includes a single Trp residue at position 85. In an attempt to investigate the interaction of the N-terminal region of Scb with the first and second hairpin loops by fluorescence spectroscopy of Trp residue, two Scb mutants in which single Trp locates at position 52 and 58, respectively, and their N-terminal removed mutants were generated. The N-terminal truncation changed the fluorescence decay kinetics of Trp52 from the triple exponential to double. Furthermore, the time-resolved fluorescence anisotropy residue indicated that the segmental motion of Trp52 was significantly enhanced by its N-terminal truncation. In contrast, Trp58 and Trp85 had little influence. The N-terminal successive truncations of Scb and its mutants resulted in the weaken inhibitors to papain. These results suggested that the N-terminal region of Scb interacts with the peptide segment preceding the first hairpin loop, thereby stabilizing the conformation of the hairpin loop structure.

Royal jelly (RJ) is an exclusive food for queen honey bee (Apis mellifera L.) that is synthesized and secreted by young worker bees. RJ is also widely used in medical products, cosmetics, and as health foods. However, little is known about RJ functionality and the total protein components, although recent research is attempting to unravel the RJ proteome. We have embarked on a detailed investigation of the RJ proteome, using a modified protein extraction protocol and two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with tandem mass spectrometry. Simultaneously, we examined total soluble protein from RJ collected at 24, 48, and 72 h after honey bee larvae deposition twice (in two flower blooming seasons), to check differences, if any, in RJ proteome therein. Both 1- and 2-D gels stained with silver nitrate revealed similar protein profiles among these three time points. However, we observed a clear difference in two bands (ca. MW of 55 and 75 kDa) on 1-D gel between the first and the second collection of RJ. A similar difference was also observed in the 2-D gel. Except for this difference, the protein profiles were similar at the 3 time points. As the RJ from 48 (or sometimes 72) is commercially used, we selected the RJ sample at 48 h for detailed analysis with the first collection. 1-DGE identified 90 and 15 proteins from the first and second selection, respectively; in total, 47 nonredundant proteins were identified. 2-DGE identified 105 proteins comprising 14 nonredundant proteins. In total, 52 nonredundant proteins were identified in this study, and other than the major royal jelly protein family and some other previously identified proteins, 42 novel proteins were identified. Furthermore, we also report potentially post-translationally modified (phosphorylation and glycosylation) RJ proteins based on the Pro-Q diamond/emerald phosphoprotein/glycoprotein gel stains; MRJP 2p and 7p were suggested as potential phosphoproteins. The 2-DGE data were integrated to develop a 2-D gel reference map, and all data are accessible through RJ proteomics portal (http://foodfunc.agr.ibaraki.ac.jp/RJP.html).

Mushroom can be defined as a macrofungus with a distinctive fruiting body. Mushrooms of class Basidiomycete are primarily wood degradation fungi, but serve as food and a part of traditional medicine used by humans. Although their life cycle is fairly well-established, the information on the molecular components, especially proteins are very limited. Here, we report proteomics analysis of two edible mushrooms (fruiting bodies) Sparassis crispa and Hericium erinaceum using one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) based complementary proteomics approaches. 1-DGE coupled with liquid chromatography and mass spectrometry identified 77 (60 nonredundant proteins) and 121 (88 nonredundant proteins) proteins from S. crispa and H. erinaceum, respectively. 2-DGE analysis revealed 480 and 570 protein spots stained with colloidal coomassie brilliant blue in S. crispa and H. erinaceum, respectively. Of the 71 and 115 selected protein spots from S. crispa and H. erinaceum 2D gel blots on polyvinyldifluoride (PVDF) membranes, respectively, 29 and 35 nonredundant proteins were identified by N-terminal amino acid sequencing. Identified nonredundant proteins from 1- or 2-DGE belonged to 19 functional categories. Twenty-one proteins were found common in both S. crispa and H. erinaceum proteomes, including 14-3-3 protein and septin. Together this study provides evidence for the presence of a large number of functionally diverse proteins, expressed in the fruiting body of two economically important mushrooms, S. crispa and H. erinaceum. Data obtained from 1-DGE and 2-DGE analyses is accessible through the mushroom proteomics portal http://foodfunc.agr.ibaraki.ac.jp/mushprot.html.

Manduca sexta is an excellent insect model for studying insect physiology, including hemolymph proteins. Larvae stages of this insect are highly damaging to tobacco leaves causing a drastic decrease in crop yield. Investigation on the larval biology should help in controlling its destructive potential, thus increasing crop yields. The hemolymph is the source of its immunity to disease and environmental factors, which invariably involves protein components. To better understand the physiology of M. sexta and the protein components expressed during its life cycle, two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with N-terminal amino acid sequencing and liquid chromatography-mass spectrometry, were employed to analyze the fifth instar larvae hemolymph proteins. These proteomics approaches together identified 123 proteins, which constituted a total of 58 nonredundant proteins and belonged to 10 functional categories. Defense (49%), transport and metabolism (15%), storage (9%), and metamorphosis (7%) categories were highly represented accounting for 80% of the identified proteins. Besides identification of previously reported proteins, 18 novel proteins were identified, which include the lipoprotein-releasing system transmembrane protein lolC, 50S ribosomal protein L24, inducible serine protease inhibitor 1, imaginal disk growth factor, protein disulfide-isomerase-like protein ERp57, etc. The 2-DGE data were integrated to develop a 2-D gel reference map. Data obtained from 1-DGE and 2-DGE analyses are accessible through the M. sexta proteomics portal ( http://foodfunc.agr.ibaraki.ac.jp/mansehemoprot.html). Together, this study provides evidence for the presence of a large number of functionally diverse protein families in the hemolymph of M. sexta. These proteins correlate well with the fifth instar stage, the transition from larvae to pupae.

An Apios americana trypsin inhibitor, AATI, was purified from Apios tubers by chromatography on DEAE Cellulofine A-500 and Sephadex G-50. The molecular mass of AATI was determined to be 6,437 Da by matrix-assisted laser desorption and ionization time-of-flight mass spectrometer (MALDI-TOF-MS). It showed strong inhibitory activity toward serine proteases, and the inhibition constants toward trypsin and chymotrypsin were 3.0 x 10(-9) M and 1.0 x 10(-6) M respectively. The inhibitory activity was not affected by heating at 80 degrees C for 2 h or by incubation at a wide range of pH values, suggesting that AATI has remarkable heat-stability and pH-stability. AATI cDNA consists of 552 nucleotides, and includes an open reading frame encoding a protein of 116 amino acids. The results of N-terminal amino acid sequencing of AATI and MALDI-TOF-MS analysis suggested that the deduced amino acid sequence had 50 and seven extra amino acids at the N- and C-termini respectively. Thus the mature AATI protein consists of 59 amino acid residues. Comparison of the amino acid sequence with those of the trypsin inhibitors from plants suggests that AATI belongs to the Bowman-Birk family and that it contains two possible reactive sites toward trypsin at Lys62 and Arg88.

CEL-III is a Ca(2+)-dependent hemolytic lectin, isolated from the marine invertebrate Cucumaria echinata. The three-dimensional structure of CEL-III/GalNAc and CEL-III/methyl alpha-galactoside complexes was solved by x-ray crystallographic analysis. In these complexes, five carbohydrate molecules were found to be bound to two carbohydrate-binding domains (domains 1 and 2) located in the N-terminal 2/3 portion of the polypeptide and that contained beta-trefoil folds similar to ricin B-chain. The 3-OH and 4-OH of bound carbohydrate molecules were coordinated with Ca(2+) located at the subdomains 1alpha, 1gamma, 2alpha, 2beta, and 2gamma, simultaneously forming hydrogen bond networks with nearby amino acid side chains, which is similar to carbohydrate binding in C-type lectins. The binding of carbohydrates was further stabilized by aromatic amino acid residues, such as tyrosine and tryptophan, through a stacking interaction with the hydrophobic face of carbohydrates. The importance of amino acid residues in the carbohydrate-binding sites was confirmed by the mutational analyses. The orientation of bound GalNAc and methyl alpha-galactoside was similar to the galactose moiety of lactose bound to the carbohydrate-binding site of the ricin B-chain, although the ricin B-chain does not require Ca(2+) ions for carbohydrate binding. The binding of the carbohydrates induced local structural changes in carbohydrate-binding sites in subdomains 2alpha and 2beta. Binding of GalNAc also induced a slight change in the main chain structure of domain 3, which could be related to the conformational change upon binding of specific carbohydrates to induce oligomerization of the protein.

The midgut environment of anopheline mosquitoes plays an important role in the development of the malaria parasite. Using genetic manipulation of anopheline mosquitoes to change the environment in the mosquito midgut may inhibit development of the malaria parasite, thus blocking malaria transmission. Here we generate transgenic Anopheles stephensi mosquitoes that express the C-type lectin CEL-III from the sea cucumber, Cucumaria echinata, in a midgut-specific manner. CEL-III has strong and rapid hemolytic activity toward human and rat erythrocytes in the presence of serum. Importantly, CEL-III binds to ookinetes, leading to strong inhibition of ookinete formation in vitro with an IC(50) of 15 nM. Thus, CEL-III exhibits not only hemolytic activity but also cytotoxicity toward ookinetes. In these transgenic mosquitoes, sporogonic development of Plasmodium berghei is severely impaired. Moderate, but significant inhibition was found against Plasmodium falciparum. To our knowledge, this is the first demonstration of stably engineered anophelines that affect the Plasmodium transmission dynamics of human malaria. Although our laboratory-based research does not have immediate applications to block natural malaria transmission, these findings have significant implications for the generation of refractory mosquitoes to all species of human Plasmodium and elucidation of mosquito-parasite interactions.

A cysteine protease inhibitor (CPI) with an apparent molecular mass of 11.5kDa was purified from larval hemolymph of the tobacco hornworm (Manduca sexta) by gel filtration on Sephadex G-50 followed by hydrophobic and ion-exchange column chromatographies. The purified cysteine proteinase inhibitor, denoted as MsCPI, strongly inhibited the plant cysteine protease, papain, with a K(i) value of 5.5 x 10(-9)M. Nucleotide sequence analysis of a partial cDNA encoding MsCPI indicated that MsCPI consists of 105 amino acid residues in a sequence that is similar to sarcocystatin A from Sarcophaga peregrina. However, northern blotting and PCR analyses using the specific primers of MsCPI suggested that the mRNA encoding MsCPI had a size of more than 12 kilobases, which included at least six tandemly repeated MsCPI segments. MsCPI was expressed in Escherichia coli and the recombinant protein effectively inhibited cysteine proteases from plants as well as from animals such as cathepsins B (K(i), 6.8 nM), H (3.0 nM), and L (0.87 nM). There was no inhibition exhibited toward trypsin, chymotrypsin, subtilisin, pepsin or themolysin.

Plant Kunitz-type protease inhibitors contain a conserved Asn residue in the N-terminal region. To investigate the role of Asn residue in protease inhibitory activities, Erythrina variegata trypsin inhibitor a (ETIa), E. variegata chymotrypsin inhibitor (ECI), and their mutants, ETIa-N12A and ECI-N13A, were used. Both mutants exhibit weaker inhibitory activities toward their cognate proteases than the wild-type proteins and were readily cleaved at reactive sites. Furthermore, kinetic analysis of the interactions of the mutated proteins with their cognate proteases by surface plasmon resonance (SPR) measurement indicated that replacements of the Asn residue mainly affected dissociation rate constants. The conserved Asn residues of Kunitz-type inhibitors play an important role in exhibiting effective inhibitory activity by stabilizing the structures of the primary binding loop and protease-inhibitor complex.

We previously cloned two distinct cDNA clones, NGR1 and NGR3, encoding S-like ribonucleases (RNases) induced by wounding and tobacco mosaic virus (TMV) infection, respectively, in Nicotiana glutinosa leaves. To gain insight into the regulatory mechanism of the RNase genes, we analyzed nucleotide sequences of the genes ngr1 (4.1 kbp) and ngr3 (5.3 kbp), containing their structural genes as well as 5'-flanking regions. The ngr1 gene is organized in three exons with two intervening introns, and ngr3 has four exons interrupted by three introns. Primer extension analyses localized single transcription initiation sites at -32 and -99 upstream of the translation initiation codons ATG in the genes ngr1 and ngr3, respectively. The beta-glucuronidase (GUS) reporter gene analysis with serial 5'-deletion mutants as well as a gel shift assay defined the wound-responsive region at residues -509 to -288 in gene ngr1 and a TMV-responsive region at the residues -401 to -174 in ngr3, respectively. Sequence search using PLACE and PlantCARE data bases showed that a wound-responsive element: the WUN-motif, occurs within the wound-responsive region in ngr1, while ngr3 contains several potential cis-regulating elements, such as the elicitor responsiveness element: the W-box, a TMV responsive element: GT1, and the WUN-motif at positions between -401 and -174. These findings suggested that some of these cis-elements may be involved in inducible expressions of ngr1 and ngr3. Furthermore, the gel shift assay suggested that the dissociation of protein factor(s) upon TMV-infection from the regulatory region may cause an inducible expression of ngr3.

CEL-III is a Ca(2+)-dependent, galactose/N-acetylgalactosamine (GalNAc)-specific lectin isolated from the marine invertebrate Cucumaria echinata. This lectin exhibits strong hemolytic activity and cytotoxicity through pore formation in target cell membranes. The amino acid sequence of CEL-III revealed the N-terminal two-thirds to have homology to the B-chains of ricin and abrin, which are galactose-specific plant toxic lectins; the C-terminal one-third shows no homology to any known proteins. To examine the carbohydrate-binding ability of the N-terminal region of CEL-III, the protein comprising Pyr1-Phe283 was expressed in Escherichia coli cells. The expressed protein showed both the ability to bind to a GalNAc-immobilized column as well as hemagglutinating activity for rabbit erythrocytes, confirming that the N-terminal region has binding activity for specific carbohydrates. Since the C-terminal region could not be expressed in E. coli cells, a fragment containing this region was produced by limited proteolysis of the native protein by trypsin. The resulting C-terminal 15 kDa fragment of CEL-III exhibited a tendency to self-associate, forming an oligomer. When mixed with erythrocytes, the oligomer of the C-terminal fragment caused hemagglutination, probably due to hydrophobic interaction with cell membranes, while the monomeric fragment did not. Chymotryptic digestion of the preformed CEL-III oligomer induced upon lactose binding also yielded an oligomer of the C-terminal fragment comprising six molecules of the 16 kDa fragment. These results suggest that after binding to cell surface carbohydrate chains, CEL-III oligomerizes through C-terminal domains, leading to the formation of ion-permeable pores by hydrophobic interaction with the cell membrane.

Ribonuclease P (RNase P) is an endonuclease responsible for generating the 5(') end of matured tRNA molecules. A homology search of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 genome database revealed that the four genes, PH1481, PH1601, PH1771, and PH1877, have a significant homology to those encoding RNase P protein subunits, hpop5, Rpp21, Rpp29, and Rpp30, of human, respectively. These genes were expressed in Escherichia coli cells, and the resulting proteins Ph1481p, Ph1601p, Ph1771p, and Ph1877p were purified to apparent homogeneity in a set of column chromatographies. The four proteins were characterized in terms of their capability to bind the cognate RNase P RNA from P. horikoshii. All four proteins exhibited the binding activity to the RNase P RNA. In vitro reconstitution of four putative RNase P proteins with the in vitro transcripted P. horikoshii RNase P RNA revealed that three proteins Ph1481p, Ph1601p, and Ph1771p, and RNase P RNA are minimal components for the RNase P activity. However, addition of the fourth protein Ph1877p strongly stimulated enzymatic activity, indicating that all four proteins and RNase P RNA are essential for optimal RNase P activity. The present data will pave the way for the elucidation of the reaction mechanism for archaeal as well as eukaryotic RNase P.

Ribosomal protein L2 is a primary 23S rRNA binding protein in the large ribosomal subunit. We examined the contribution of the N- and C-terminal regions of Bacillus stearothermophilus L2 (BstL2) to the 23S rRNA binding activity. The mutant desN, in which the N-terminal 59 residues of BstL2 were deleted, bound to the 23S rRNA fragment to the same extent as wild type BstL2, but the mutation desC, in which the C-terminal 74 amino acid residues were deleted, abolished the binding activity. These observations indicated that the C-terminal region is involved in 23S rRNA binding. Subsequent deletion analysis of the C-terminal region found that the C-terminal 70 amino acids are required for efficient 23S rRNA binding by BstL2. Furthermore, the surface plasmon resonance analysis indicated that successive truncations of the C-terminal residues increased the dissociation rate constants, while they had little influence on association rate constants. The result indicated that reduced affinities of the C-terminal deletion mutants were due only to higher dissociation rate constants, suggesting that the C-terminal region primarily functions by stabilizing the protein L2-23S rRNA complex.

Ribosomal protein L5, a 5S rRNA binding protein in the large subunit, is composed of a five-stranded antiparallel beta-sheet and four alpha-helices, and folds in a way that is topologically similar to the ribonucleprotein (RNP) domain [Nakashima et al., RNA 7, 692-701, 20011. The crystal structure of ribosomal protein L5 (BstL5) from Bacillus stearothermophilus suggests that a concave surface formed by an anti-parallel beta-sheet and long loop structures are strongly involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurred at beta-strands and loop structures in BstL5. The mutation of Lys33 at the beta 1-strand caused a significant reduction in 5S rRNA binding. In addition, the Arg92, Phe122, and Glu134 mutations on the beta2-strand, the alpha3-beta4 loop, and the beta4-beta5 loop, respectively, resulted in a moderate decrease in the 5S rRNA binding affinity. In contrast, mutation of the conserved residue Pro65 at the beta2-strand had little effect on the 5S rRNA binding activity. These results, taken together with previous results, identified Lys33, Asn37, Gln63, and Thr90 on the beta-sheet structure, and Phe77 at the beta2-beta3 loop as critical residues for the 5S rRNA binding. The contribution of these amino acids to 5S rRNA binding was further quantitatively evaluated by surface plasmon resonance (SPR) analysis by the use of BIAcore. The results showed that the amino acids on the beta-sheet structure are required to decrease the dissociation rate constant for the BstL5-5S rRNA complex, while those on the loops are to increase the association rate constant for the BstL5-5S rRNA interaction.

Novel types of proteinase inhibitors with multi-inhibitory activities were generated by replacement of phytocystatin domains in sunflower multi-cystatin (SMC) by the serine proteinase inhibitor BGIT from bitter gourd seeds. Two chimeric inhibitors SMC-T3 and SMC-T23, in which the third domain in SMC and the second and third domains in SMC were replaced by BGIT, acquired trypsin inhibitory activity (Ki: 1.46 x 10(-7) M and 1.75 x 10(-7) M), retaining inhibitory activity toward papain (Ki: 4.5 x 10(-8) M and 1.52 x 10(-7) M), respectively. We compared the chimeric inhibitors and the recombinant SMC (r-SMC) in relation to their effects on the growth of larval Spodoptera exigua. When the second instar larvae were reared on a diet containing rSMC, SMC-T3, or SMC-T23 for ten days, a significant reduction in weight gain was observed. Mean weights for rSMC, SMC-T3, and SMC-T23 were 43 mg, 32 mg, and 43 mg, respectively, as compared with that (60 mg) for the absence of the inhibitor. In contrast, BGIT had little effect on the growth of the S. exigua larvae. This result indicated that the chimeric inhibitor SMC-T3 with two phytocystatin domains and one serine proteinase inhibitor domain is an efficient inhibitor of proteinases in the S. exigua larvae. Therefore, this novel type of proteinase inhibitor with multi-inhibitory activities may represent a promising protein for successful application to a transgenic plant with insect resistance.

CEL-III, a galactose/N-acetylgalactosamine (Gal/GalNAc)-specific lectin purified from a marine invertebrate, Cucumaria echinata, has a strong hemolytic activity, especially toward human and rabbit erythrocytes in the presence of Ca2+. We evaluated the role of Ca2+ in hemagglutinating and hemolytic activities of CEL-III. We found that Ca2+ is closely associated with both activities of CEL-III. The fluorescence spectra of CEL-III upon binding to Ca2+ were measured. The result showed a structural change of CEL-III in the presence of Ca2+. The structural change of CEL-III upon Ca2+ binding was further demonstrated by stabilization against urea denaturation and by insusceptibility to protease digestions. CEL-III was completely unfolded at a low concentration of 2 M urea, while CEL-III complexed with Ca2+ was stable in 6 M urea. As for protease digestions, CEL-III monomer and oligomer were readily digested by trypsin, chymotrypsin, and papain in the absence of Ca2+, while they were insusceptible to the three proteases in the presence of Ca2+. The papain digestion of the decalcified oligomer produced a large C-terminal peptide, suggestting that the C-terminal region of CEL-III may participate in oligomerization of CEL-III as a core domain.

Sunflower cystatin a (Sca) is distinguished from other phytocystatins by its lack of the N-terminal about 20 amino acids, resulting in the absence of the evolutionarily conserved Gly residue. The cDNA encoding Sca was amplified by PCR methods. The cDNA consists of 520 nucleotides and includes an open reading frame encoding a polypeptide of 98 amino acids. Comparison of the deduced amino acid sequence with the Sca protein sequence indicated that the deduced sequence has an extra 15 amino acids and one amino acid at the N- and C-termini, respectively. This result suggests that Sca is synthesized as a preprotein (preSca) and proteolytic cleavages at peptide bonds may give rise to the mature Sca. To address this assumption and also to investigate the significance of the N-terminal extension sequence to Sca for inhibitory activity, a recombinant pre-Sca (rpre-Sca), in which the N-terminal extension was fused to the matured Sca, and a recombinant matured Sca (rSca) were overproduced in Escherichia coli cells. Incubation of the rpre-Sca with a seed extract resulted in a mobility by SDS-PAGE that was the same as rSca, demonstrating a proteolytic cleavage by endogenous proteinases. The rSca and rpre-Sca proteins were further characterized with respect to inhibitory activity and sensorgrams of the interaction with papain. The result showed that rpre-Sca had stronger inhibitory activity than rSca, and that the increased activity toward papain was due to a lower dissociation rate constant. This finding indicates that the N-terminal region of rpre-Sca increases the inhibitory activity by stabilizing the rpre-Sca and papain complex.

Two cysteine proteinase inhibitors, cystatins Sca and Scb, were previously isolated from sunflower seeds [Kouzuma et al. J. Biochem. 119 (1996) 1106-1113]. A cDNA clone encoding a novel phytocystatin with three repetitive cystatin domains was isolated from a cDNA library of sunflower seeds using the Sca cDNA fragment as a hybridization probe. The cDNA insert comprises 1,093 bp and encodes 282 amino acid residues. The deduced amino acid sequences of the domains are highly similar to each other (66-81%), sharing 65-90% identical residues with Sca. The cDNA was expressed in Escherichia coli cells, and then the recombinant sunflower multicystatin (SMC) was purified and its inhibitory activity toward papain was examined. SMC exhibited strong inhibitory activity toward papain, with a stoichiometry of 1:3, indicating that each cystatin domain independently functions as a potent cysteine proteinase inhibitor. Proteolysis of SMC with Asn-specific proteinase suggested that post-translational processing by an Asn-specific proteinase may give rise to mature Sca-like phytocystatins.

CEL-III, a galactose/N-acetylgalactosamine (Gal/GalNAc) specific lectin purified from a marine invertebrate Cucumaria echinata has a strong hemolytic activity especially toward human and rabbit erythrocytes. We determined the primary structure of the CEL-III by examining the amino acid sequences of the protein and the nucleotide sequence of the cDNA. The cDNA encoding CEL-III has 1823 nucleotides and an open reading frame of 1296 nucleotides. CEL-III is composed of 432 amino acid residues with a M(r) of 47¿ omitted¿457 and has six internal tandem repeats, each with of 40-50 amino acids, comprising the N-terminal two-thirds of the molecule. Similar repeats are found in the B-chains of cytotoxic plant lectins, such as ricin and abrin, where six repetitive sequences extend throughout the molecules. A hydropathy plot predicts hydrophobic segments in the C-terminal region of CEL-III. These findings suggest that the N-terminal region of CEL-III plays an important role in binding to carbohydrate receptors on the target cell membranes, an event which triggers an intermolecular hydrophobic interaction of the C-terminal region, the result being oligomerization of CEL-III to lead to pore-formation in erythrocyte membrane.

We previously demonstrated that amino acid residues Gln62 (P3), Phe63 (P2), Leu64 (P1), and Phe67 (P3') in the primary binding loop of Erythrina variegata chymotrypsin inhibitor (ECI), a member of the Kunitz inhibitor family, are involved in its strong inhibitory activity toward chymotrypsin [Iwanaga et al. (1998) J. Biochem. 124, 663-669]. To determine whether or not these four amino acid residues predominantly contribute to the strong inhibitory activity of ECI, they were simultaneously replaced by Ala. The results showed that a quadruple mutant, Q62A/F63A/L64A/F67A, retained considerable inhibitory activity (Ki, 5.6 x 10(-7) M), indicating that in addition to the side chains of these four amino acid residues, the backbone structure of the primary binding loop in ECI is essential for the inhibitory activity toward chymotrypsin. Two chimeric proteins, in which the primary binding loops of ECI and ETIa were exchanged: an isoinhibitor from E. variegata with lower chymotrypsin inhibitory activity, were constructed to determine whether the backbone structure of the primary binding loop of ECI was formed by the amino acid residues therein, or through an interaction between the primary binding loop and the residual structure designated as the "scaffold." A chimeric protein, ECI/ETIa, composed of the primary binding loop of ECI and the scaffold of ETIa showed weaker inhibitory activity (Ki, 1.3 x 10(-6) M) than ECI (Ki, 9.8 x 10(-8) M). In contrast, a chimera, ETIa/ECI, comprising the primary binding loop of ETIa and the scaffold of ECI inhibited chymotrypsin more strongly (Ki, 5.7 x 10(-7) M) than ETIa (Ki, 1.3 x 10(-6) M). These results indicate that the intramolecular interaction between the primary binding loop and the scaffold of ECI plays an important role in the strong inhibitory activity toward chymotrypsin. Furthermore, surface plasmon resonance analysis revealed that the side chains on the primary binding loop of ECI contribute to both an increase in the association rate constant (kon) and a decrease in the dissociation rate constant (koff) for the ECI-chymotrypsin interaction, whereas the backbone structure of the primary binding loop mainly contributes to a decrease in the dissociation rate constant.

Sunflower cystatin Scb differs from other phytocystatins in that it is a highly basic protein with a pI value of 9.6 and includes six additional amino acids (Arg30-Leu-Gln-Arg-Thr34, Thr37) in the middle region as compared with other phytocystatins [Kouzuma et al. (1996) J. Biochem. 119, 1106-1113]. We identified and sequenced a complete cDNA encoding the Scb; the cDNA of Scb consists of 645 nucleotides and includes an open reading frame encoding a polypeptide of 123 amino acids. On the basis of these findings, Scb appears to be synthesized as a prepeptide consisting of a signal sequence of 22 amino acids and a mature protein of 101 amino acids. A recombinant Scb (rScb) was produced by expression in Escherichia coli and purified by gel filtration on Sephacryl S-200 followed by ion-exchange column chromatography on a S-Sepharose column. rScb exhibited almost the same inhibitory activity toward papain as the authentic Scb did, but its inhibition profile toward cathepsins B, L, and H was slightly different. Scb mutant proteins, in which selected N-terminal residues or the additional amino acids were deleted, were subsequently constructed and characterized with respect to their inhibitory activities toward papain. The result revealed that the additional sequence (Arg30-Leu-Gln-Arg-Thr34) in Scb is not essential for papain-inhibitory activity, while the N-terminal amino acids (Ile1-Pro2) as well as the N-terminal glycine residues Gly3 and/or Gly4 play an important role in manifesting the inhibitory activity toward papain.

Erythrina variegata trypsin inhibitors designated ETIa and ETIb belong to the Kunitz family trypsin inhibitor, but ETIa is unique in its ability to inhibit tissue-type plasminogen activator, while ETIb is not. The cDNA clone encoding ETIb was isolated from the seed cDNA library constructed in the lambda phage lambda gt11. The ETIb cDNA insert consists of 765 bp, including an open reading frame of 606 pb from ATG to TGA codons. The deduced amino acid sequence consists of 202 amino acids, having the signal peptides of 22 amino acids in the N-terminus and 2 amino acids in C-terminus. The cDNA fragment encoding the mature form of ETIb was introduced into an expression vector, pET-22b, and expressed in Escherichia coli BL21 (ED3) in a functional form. Furthermore, the ETIb mutant bP61R/F62L, in which Pro61 and Phe62 in ETIb were changed to the corresponding amino acid residues Arg and Leu, respectively, as in ETIa, was constructed, and its inhibitory potency toward tPA was assayed. This mutant showed significant tPA inhibitory activity, albeit less than ETIa. The result demonstrates that the Arg61 and Leu62 residues in ETIa are important in inhibiting tPA, and also suggest that beside these two residues, the other amino acid(s) or other structural element may be involved in interaction of ETIa with tPA.

Erythrina variegata trypsin inhibitor ETIa belongs to the Kunitz inhibitor family, but is unique in its ability to bind and inhibit tissue-type plasminogen activator (tPA). A cDNA clone encoding ETIa was isolated from the lambda gt11 cDNA library using specific antiserum as a probe and characterized by nucleotide sequencing. The cloned ETIa cDNA consists of 762 nucleotides and includes an open reading frame encoding a polypeptide of 198 amino acids. Comparison of the deduced protein sequence and the determined protein sequence indicated the presence of two signal peptides composed of 24 and 2 amino acids at the N- and C-termini, respectively. The cDNA encoding mature ETIa was amplified by polymerase chain reaction (PCR), ligated into the expression vector pET-22b, and expressed in Escherichia coli BL21(DE3). The recombinant ETIa (rETIa) was expressed in E. coli as inclusion bodies; it was purified to homogeneity by gel filtration on Sephadex G-75. The rETIa exhibited almost the same inhibitory activity toward trypsin and tPA as ETIa. Six mutants, in which the amino acids Arg61, Leu62, Arg63, and Ala65 were replaced by Pro, Phe, Leu/Asp, and Tyr, respectively, were constructed by site-specific mutagenesis and expressed in E. coli. The site-specific mutation of Arg63 to Leu (aR63L) or Asp (aR63D) in ETIa resulted in abolition of the inhibitory activities toward both trypsin and tPA. The mutants aR61P and aL62F showed significantly reduced tPA-inhibitory activity, and furthermore the double mutant aR61P/L62F lacked tPA-inhibitory activity, despite retaining the trypsin-inhibitory activity. In contrast, the mutant aA65Y exhibited tPA-inhibitory activity to the same extent as rETIa. This result suggests that Arg61 and Leu62 in ETIa, in addition to Arg63, may play an important role in the interaction with tPA.

The Erythrina variegata Kunitz family trypsin inhibitors, ETIa and ETIb, prolonged the activated partial thromboplastin time (APTT) and also the prothrombin time (PT) of human plasma, but the Kunitz family chymotrypsin inhibitor, ECI, and Bowman-Birk family inhibitor, EBI, from E. variegata hardly prolonged these times. Trypsin inhibitors ETIa and ETIb inhibited the amidolytic activity of factor Xa, and ETIb but not ETIA inhibited plasma kallikrein. Neither ETIa nor ETIb exhibited any inhibitory activity toward beta-factor XIIa and thrombin. Furthermore, trypsin inhibitors ETIa and ETIb inhibited plasmin, a serine proteinase in the fibrinolytic system, whereas ECI and EBI did not. These results indicate that Erythrina Kunitz proteinase inhibitors possess different potency toward serine proteinases in the blood coagulation and fibrinolytic systems, in spite of their high similarity in amino acid sequence.

Two proteinaceous cysteine proteinase inhibitors (cystatins) referred to as Sca and Scb were purified to homogeneity from the seeds of sunflower (Heliantas annuus) by gel filtration on Sephadex G-75 followed by a series of ion-exchange column chromatographies and reverse-phase HPLC (RP-HPLC). The isoelectric points (pI) of Sca and Scb were estimated to be 5.6 and 9.6, respectively. The inhibitory potencies of these two cystatins were examined with cysteine proteinases from various sources, such as plants, mammals, and bacteria. Papain was strongly inhibited by both Sca and Scb with Ki values of 5.6 x 10(-9) and 1.7 x 10(-10) M, respectively. Sca and Scb were also found to be potent inhibitors of ficin (Ki values of 1.9 x 10(-6) and 2.8 x 10(-9) M, respectively). Rat cathepsin H was inhibited strongly by Scb and slightly by Sca. Although rat cathepsins B and L were significantly inhibited by Scb, they were scarcely affected by Sca. Neither Sca nor Scb inhibited Arg-gingipain, an arginine-specific cysteine proteinase of Porphyromonas gingivalis. The complete amino acid sequences of the two inhibitors were determined by protein chemical methods. The proteins Sca and Scb consist of 83 and 101 amino acid residues with M(r) of 9,330 and 11,187, respectively, and there are identical residues at 34 positions in the two sequences, that is at 42% of the residues compared. Comparison of their sequences with those of other cystatins revealed that Sca shares 59-73% identical residues with other phytocystatins, while Scb shows less identity to other phytocystatins, sharing only 28-38% identical residues. Furthermore, only 20-27% of the residues of both cystatins, Sca and Scb, are identical to those of the animal cystatins.

A Bowman-Birk family proteinase inhibitor (EBI) was isolated from the seeds of Erythrina variegata. The protein was purified by ion-exchange column chromatography on DEAE-cellulose followed by gel filtration on Sephadex G-75. The stoichiometry with trypsin was estimated to be 1:1, while that with chymotrypsin was not obvious, as determined from the titration patterns of its inhibitory activities. The complete amino acid sequence of EBI was determined by sequencing tryptic and chymotryptic peptides. The EBI protein consists of 61 amino acid residues, which is the shortest among the Bowman-Birk family inhibitors sequenced to date, and has a M(r) of 6,689. Comparison of this sequence with those of other leguminous Bowman-Birk family inhibitors revealed that EBI could be classified as a group II inhibitor, showing the best homology (67%) to the Bowman-Birk proteinase inhibitor from soybeans.

The amino acids of the chymotrypsin inhibitor (ECI) from the Erythrina variegata seeds have been sequenced. The sequence was solved by analysis of peptides derived from the protein by enzymatic digestions with trypsin and Staphylococcus aureus V8 proteinase, as well as by chemical cleavage with o-iodosobenzoic acid. The ECI consists of 179 amino acid residues with a pyroglutamic acid as the N-terminal residue and has a calculated molecular weight of 19,791. Comparison of this sequence with the sequences of the two trypsin inhibitors, ETIa and ETIb, from the E. variegata seeds shows that about 60% of the residues of ECI are identical to those of ETIa and ETIb and that the reactive sites, Arg63, in ETIa and ETIb change to Leu64 in ECI.

The Kunitz-type trypsin inhibitors, ETIa and ETIb, and chymotrypsin inhibitor ECI were isolated from the seeds of Erythrina variegata. The proteins were extracted from a defatted meal of seeds with 10 mM phosphate buffer, pH 7.2, containing 0.15 M NaCl, and purified by DEAE-cellulose and Q-Sepharose column chromatographies. The stoichiometry of trypsin inhibitors with trypsin was estimated to be 1:1, while that of chymotrypsin inhibitor with chymotrypsin was 1:2, judging from the titration patterns of their inhibitory activities. The complete amino acids of the two trypsin inhibitors were sequenced by protein chemical methods. The proteins ETIa and ETIb consist of 172 and 176 amino acid residues and have M(r) 19,242 and M(r) 19,783, respectively, and share 112 identical amino acid residues, which is 65% identity. They show structural features characteristic of the Kunitz-type trypsin inhibitor (i.e., identical residues at about 45% with soybean trypsin inhibitor STI). Furthermore, the trypsin inhibitors show a significant homology to the storage proteins, sporamin, in sweet potato and the taste-modifying protein, miraculin, in miracle fruit, having about 30% identical residues.