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Social dominance, in which an individual asserts control over others or benefits most after social conflict, has an influence on social behaviour. However, the mechanisms mediating social dominance remain unclear. Social dominance within social groups determines the distribution of rewards such as food and access to mating partners, which can act as reinforcers. In this study, we used the water competition test to determine whether mice were dominant or subordinate. It has been previously reported that mice heterozygous for a missense mutation in Grin1 (Grin1Rgsc174 ) showed altered social behaviour, with increased locomotor activity, novelty seeking and anxiety. However, social dominance in these mice has not been previously investigated. We subjected Grin1Rgsc174/+ mice to the water competition test using IntelliCage and observed that Grin1 influences competitive dominance. We found that Grin1Rgsc174/+ mice exhibited social subordination characterised by decreased corner visit frequency and occupancy time at the beginning of the task. However, Grin1Rgsc174/+ mice retained increased basal activity and exploring behaviour under a group-housed environment. Our findings suggested that Grin1 plays an important role in determining social dominance.

We describe the bioluminescence of a genetically engineered Escherichia coli harboring a recombined plasmid with a catalase gene promoter fused lux gene cluster, responsible for the generation of photons closely associated with respiratory inhibition, with the aim of applying it for cyanide sensing. This E. coli construct was favorably utilized for the microplate assay of cyanide by leveraging the microenvironment of the biocompatible alginate. The brightness of the bioluminescence, induced by cyanide stimulation of the respiration causative of the production of hydrogen peroxide, positively correlates with its concentration. Moreover, visualization of cyanide with a consumer digital camera, ranging in concentration from about 0.01 mg CN·L-1 in the alginate sol to around 100 mg CN·L-1 in its gel, was attained.

Diphenyl (α-aminoalkyl)phosphonates act as mechanism-based inhibitors against serine proteases by forming a covalent bond with the hydroxy group of the active center Ser residue. Because the covalent bond was found to be broken and replaced by 2-pyridinaldoxime methiodide (2PAM), we employed a peptidyl derivative bearing diphenyl 1-amino-2-phenylethylphosphonate moiety (Phe(p) (OPh)2 ) to target the active site of chymotrypsin and to selectively anchor to Lys175 in the vicinity of the active site. Previously, it was reported that the configuration of the α-carbon of phosphorus in diphenyl (α-aminoalkyl)phosphonates affects the inactivation reaction of serine proteases, i.e., the (R)-enantiomeric diphenyl phosphonate is comparable to l-amino acids and it effectively reacts with serine proteases, whereas the (S)-enantiomeric form does not. In this study, we evaluated the stereochemical effect of the phosphonate moiety on the selective chemical modification. Epimeric dipeptidyl derivatives, Ala-(R or S)-Phe(p) (OPh)2 , were prepared by separation with RP-HPLC. A tripeptidyl (R)-epimer (Ala-Ala-(R)-Phe(p) (OPh)2 ) exhibited a more potent inactivation ability against chymotrypsin than the (S)-epimer. The enzyme inactivated by the (R)-epimer was more effectively reactivated with 2PAM than the enzyme inactivated by the (S)-epimer. Finally, N-succinimidyl (NHS) active ester derivatives, NHS-Suc-Ala-Ala- (R or S)-Phe(p) (OPh)2 , were prepared, and we evaluated their action when modifying Lys175 in chymotrypsin. We demonstrated that the epimeric NHS derivative that possessed the diphenyl phosphonate moiety with the (R)-configuration effectively modified Lys175 in chymotrypsin, whereas that with the (S)-configuration did not. These results demonstrate the utility of peptidyl derivatives that bear an optically active diphenyl phosphonate moiety as affinity labeling probes in protein bioconjugation. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 521-530, 2016.

Tripeptidyl peptidase (TPP) is an exopeptidase that sequentially hydrolyzes tripeptides from the N-terminus of oligopeptides or polypeptides. We performed screening for isolating novel TPP-producing microorganisms from soil samples. TPP activity was observed in the culture supernatant of Streptomyces herbaricolor TY-21 by using Ala-Ala-Phe-p-nitroanilide (pNA) as the substrate. TPP from the culture supernatant was purified to approximately 790-fold. It was shown to cleave oxidized insulin B-chain, thereby with releasing tripeptide units, but not the N-terminal-protected peptide, Cbz-Ala-Ala-Phe-pNA. The TPP gene, designated tpp, was isolated from a partial genomic DNA library of S. herbaricolor TY-21. The TPP gene consisted of 1488 bp, and encoded a 133-amino acid pre-pro-peptide and a 362-amino acid mature enzyme containing conserved amino acid residues (Asp-36, His-77, and Ser-282) similar to the catalytic residues in subtilisin. TY-21 TPP belonged to the peptidase S8A family in the MEROPS database. The mature TY-21 TPP showed approximately 49% identity with tripeptidyl peptidase subtilisin-like (TPP S) from Streptomyces lividans strain 66.

We have visualized redox and structural changes in the mitochondria of yeast Saccharomyces cerevisiae as a eukaryotic cell model using a genetically encoded yellow fluorescent protein (Y1-Yellow) and conventional fluorescence microscopy. Y1-Yellow originating from a yellow emitting luminous bacterium Aliivibrio sifiae Y1 was fused with a mitochondria-targeted sequence (mt-sequence). Y1-Yellow fluorescence arising only from the mitochondrial site and the color of yellow fluorescence could be easily differentiated from cellular autofluorescence and from that of conventional probes. Y1-Yellow expressing S. cerevisiae made the yellow fluorescence conspicuous at the mitochondrial site in response to reactive oxygen species (ROS) transiently derived in the wake of pretreatment with hydrogen peroxide. Based on our observation with Y1-Yellow fluorescence, we also showed that mitochondria rearrange to form a cluster structure surrounding chromosomal DNA via respiratory inhibition by cyanide, followed by the generation of ROS. In contrast, uptake of an uncoupler of oxidative phosphorylation is not responsible for mitochondrial rearrangement. These results indicate the utility of Y1-Yellow for visualization of mitochondrial vitality and morphology in living cells.

To examine the mechanisms of diabetes-enhanced inflammation, ear inflammation was induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in streptozotocin (STZ)-injected diabetic and control mice. The inflammatory response was determined from ear thickness and histology. The mRNA expression of several inflammation-related genes 8, 24 and 32 h after TPA treatment was determined by quantitative real-time RT-PCR. Ear thickness did not differ between the two groups at 8 h, but was greater in the diabetic mice than control mice at 24 and 32 h (late phase). STZ-diabetic conditions variously affected TPA-induced gene expression. The changes 8 h after TPA treatment probably reflected transcriptional regulation, and the genes were divided into three groups, up-regulated (IL-6, MCP-1, HO-1 and SOCS3), unregulated (IL-1beta, TNF-alpha and IL-10) and down-regulated (RANTES) genes. TPA-induced gene expression of cytokines, except for RANTES, peaked at 8 h and significantly declined in the late phase in control mice, while the expression of IL-1beta and TNF-alpha did not decline in the late phase in the diabetic mice. This result indicated the destabilization process for these mRNA, a type of post-transcriptional regulation, to be impaired under STZ-induced diabetic conditions; however, TPA-induced gene and protein expression of TTP, an RNA-binding protein involved in mRNA decay, were adversely enhanced in the diabetic mice. These findings suggested that STZ-induced diabetes affected the transcriptional and post-transcriptional control of TPA-induced inflammation, and greater mRNA levels of IL-1beta and TNF-alpha in the late phase were probably responsible for the diabetes-enhanced inflammation.

To establish a covalent chromatography system for purification of naturally occurring chymotrypsin-like serine proteases, a diphenyl 1-amino-2-phenylethylphosphonate derivative bearing Gly-Gly-Gly as a spacer was immobilized on the Sepharose 4FF gel. In this system, bovine chymotrypsin was selectively bound to the phosphonate immobilized on the gel and then released by the action of 2-pyridinealdoxime methiodide as the reactivated enzyme. Based on the study results for selective binding and reactivation conditions, chymotrypsin-like proteases from pancreatin (hog pancreas) were rapidly and highly purified within three hours. Keywords

Background: Amyloid-β (Aβ) degradation in brains of Alzheimer disease patients is a crucial focus for the clarification of disease pathogenesis. Nevertheless, the mechanisms underlying Aβ degradation in the human brain remain unclear. Objective: This study aimed to quantify the levels of small C-terminal Aβ fragments generated upon Aβ degradation in human cerebrospinal fluid (CSF). Methods: A fraction containing small peptides was isolated and purified from human CSF by high-pressure liquid chromatography. Degradation products of Aβ C termini were identified and measured by liquid chromatography-tandem mass spectrometry. The C-terminal fragments of Aβ in the conditioned medium of cultured cells transfected with the Swedish variant of βAPP (sw βAPP) were analyzed. These fragments in brains of PS1 I213T knock-in transgenic mice, overexpressing sw βAPP, were also analyzed. Results: The peptide fragments GGVV and GVV, produced by the cleavage of Aβ40, were identified in human CSF as well as in the brains of the transgenic mice and in the conditioned medium of the cultured cells. Relative to Aβ40 levels, GGVV and GVV levels were 7.6 ± 0.81 and 1.5 ± 0.18%, respectively, in human CSF. Levels of the GGVV fragment did not increase by the introduction of genes encoding neprilysin and insulin-degrading enzyme to the cultured cells. Conclusion: Our results indicate that a substantial amount of Aβ40 in human brains is degraded via a neprilysin- or insulin-degrading enzyme-independent pathway.

Oligopeptidase B (OPB; EC 3.4.21.83) from 2 Gram-negative bacteria, Stenotrophomonas maltophilia (Stm) and Serratia marcescens (Sem), and the Gram-positive bacterium Rhodococcus erythropolis (Re) were cloned and characterized to clarify their activities and substrate specificities using peptidyl-MCA substrates containing Arg or Lys. The cloned enzymes, Stm, Sem and ReOPBs, in addition to Escherichia coli OPB (EcOPB) were expressed using a pET expression system. Although the Stm and SemOPBs share 45% sequence identity to each other and up to 60% identity with respect to their catalytic domains, their activities towards MCA substrates were quite different. StmOPB is approximately 100-500 times more active than SemOPB and 3-30 times more active than EcOPB. The activity of ReOPB is comparable to that of StmOPB and it shares 40% and 36% identity to StmOPB and SemOPB, respectively. Some features of Stm, Re and EcOPBs are similar to those of previously cloned OPBs, which were also strongly inhibited by substrates, but SemOPB differs from all other OPBs in that it is not inhibited by substrates; even substrates containing double arginine at 35μM did not inhibit SemOPB. On the other hand, the same substrates at only 5μM inhibited the activity of the Stm, Re, and EcOPB. This phenomenon was not observed with substrates containing single or double lysine., Biological and Pharmaceutical Bulletin, 35(11), pp.2010-2016; 2012

This study was conducted to investigate whether or not a food substitute (Dr. BAANs(R)) containing three bioactive components L-arginine, omega-3 polyunsaturated fatty acid, and ribonucleic acid, supplied orally to 15 overweight patients, may have efficacy to prevent or improve the metabolic syndrome of these patients. To provide supporting data for this clinical study, the in vivo fatty acid metabolism of obese mice was analyzed using (125)I labeled 15-(p-iodophenyl)-9-methylpentadecanoic acid (9MPA) in the tissues' lipid pool. After 3 months of intervention, the results showed that there were improvements observed in liver functions, lipid profiles and metabolic syndrome marker. Significant differences were also found in the values of blood pressure, body weight, percentage of body fat, and body mass index. In the animal study, the tissue uptake of (125)I-9MPA at 10 min after injection was higher in obese mice than in the control mice and the treatment with Dr. BAANs(R) in obese mice decreased the uptake significantly. The final product metabolite of p-iodophenylacetic acid in obese mice was increased significantly by the treatment. In conclusion, this food substitute may have a beneficial effect for the prevention or improvement of metabolic syndrome.

A new inhibitor, H-Ala-Ile-pyrrolidin-2-yl boronic acid, was developed as an inhibitor against prolyl tripeptidyl aminopeptidase with a K(i) value of 88.1 nM. The structure of the prolyl tripeptidyl aminopeptidase complexed with the inhibitor (enzyme-inhibitor complex) was determined at 2.2 A resolution. The inhibitor was bound to the active site through a covalent bond between Ser603 and the boron atom of the inhibitor. This structure should closely mimic the structure of the reaction intermediate between the enzyme and substrate. We previously proposed that two glutamate residues, Glu205 and Glu636, are involved in the recognition of substrates. In order to clarify the function of these glutamate residues in substrate recognition, three mutant enzymes, E205A, E205Q, and E636A were generated by site-directed mutagenesis. The E205A mutant was expressed as an inclusion body. The E205Q mutant was expressed in soluble form, but no activity was detected. Here, the structures of the E636A mutant and its complex with the inhibitor were determined. The inhibitor was located at almost the same position as in the wild-type enzyme-inhibitor complex. The amino group of the inhibitor interacted with Glu205 and the main-chain carbonyl group of Gln203. In addition, a water molecule in the place of Glu636 of the wild-type enzyme interacted with the amino group of the inhibitor. This water molecule was located near the position of Glu636 in the wild-type and formed a hydrogen bond with Gln203. The k(cat)/K(M) values of the E636A mutant toward the two substrates used were smaller than those of the wild-type by two orders of magnitude. The K(i) value of our inhibitor for the E636A mutant was 48.8 microM, which was 554-fold higher than that against the wild-type enzyme. Consequently, it was concluded that Glu205 and Glu636 are significant residues for the N-terminal recognition of a substrate., Journal of molecular biology, 375(3), pp.708-719; 2008

Kumamolisin-As is an acid collagenase with a subtilisin-like fold. Its active site contains a unique catalytic triad, Ser278-Glu78-Asp82, and a putative transition-state stabilizing residue, Asp164. In this study, the mutants D164N and E78H/D164N were engineered in order to replace parts of the catalytic machinery of kumamolisin-As with the residues found in the equivalent positions in subtilisin. Unlike the wild-type and D164N proenzymes, which undergo instantaneous processing to produce their 37-kDa mature forms, the expressed E78H/D164N proenzyme exists as an equilibrated mixture of the nicked and intact forms of the precursor. X-ray crystallographic structures of the mature forms of the two mutants showed that, in each of them, the catalytic Ser278 makes direct hydrogen bonds with the side chain of Asn164. In addition, His78 of the double mutant is distant from Ser278 and Asp82, and the catalytic triad no longer exists. Consistent with these structural alterations around the active site, these mutants showed only low catalytic activity (relative k(cat) at pH 4.0 1.3% for D164N and 0.0001% for E78H/D164N). pH-dependent kinetic studies showed that the single D164N substitution did not significantly alter the logk(cat) vs. pH and log(k(cat)/Km) vs. pH profiles of the enzyme. In contrast, the double mutation resulted in a dramatic switch of the logk(cat) vs. pH profile to one that was consistent with catalysis by means of the Ser278-His78 dyad and Asn164, which may also account for the observed ligation/cleavage equilibrium of the precursor of E78H/D164N. These results corroborate the mechanistic importance of the glutamate-mediated catalytic triad and oxyanion-stabilizing aspartic acid residue for low-pH peptidase activity of the enzyme.

VimelysinisauniquemetalloproteinasefromVibriosp.T1800exhibitinghighactivityatlow temperature and high stability in organic solvents such as ethanol. A 1,821 bp openreading frame of the vimelysin gene encoded 607 amino acid residues consisting of anN-terminal pro-region, a mature enzyme, and a C-terminal pro-region. The matureenzyme region showed 80%, 57% and 35% sequence identity with the mature forms ofvibriolysin from V. vulnificus, pseudolysin from Pseudomonas aeruginosa, and thermo-lysin from Bacillus thermoproteolyticus, respectively. The catalytic residues and zinc-bindingmotifsofmetalloproteinasesarewellconservedinvimelysin.ThevimelysingenewasexpressedinE.coliJM109cellsandtherecombinantenzymewaspurifiedasa38-kDamature form from cell-free extracts. The purified recombinant enzyme is indistinguish-ablefromtheenzymepurifieddirectlyfromVibrio.Toobtainmutantsexhibitinghigherstabilityinorganicsolvents,randommutationswereintroducedbyerror-pronePCRand600transformantswerescreened.TheN123Dmutantexhibitstwotimeshigherstabilityin organic solvents than the wild-type enzyme. A plausible mechanism for the stabilityof the N123D mutant in organic solvents was discussed based on homology models ofvimelysin and the N123D mutant.Key words: alcohol resistance, metalloproteinase, pseudolysin, random mutagenesis,vimelysin.Microbial proteinases have many unique characteristics.For instance, Achromobactor I proteinase (1, 2) andStaphylococcus aureus V8 proteinase (3, 4) are unique intheir strict substrate specificities. Thermolysin fromBacillus thermoproteolyticus (5) exhibits high thermal sta-bility and proteinase from alkalophilic Bacillus (6) exhibitsmaximum activity under extremely alkaline conditions.In order to utilize it as an additive in detergents or forpeptide synthesis, we succeeded in isolating Vibrio sp.T1800 from marine bacteria, which produce a proteinaseexhibiting high stability in organic solvents such asethanol (7, 8). The purified metalloproteinase, namedvimelysin, has a molecular mass of 38 kDa and exhibitshigher stability in organic solvents than thermolysin, arepresentative metalloproteinase from microorganisms.Vimelysin showed 40% activity compared to that of a con-trol in the presence of 10% ethanol, whereas thermolysinshowed only 5% activity under the same conditions (7, 8).As for substrate specificity, vimelysin preferred Phe atthe P1

For site-selective chemical modification in the vicinity of the active site of chymotrypsin (Csin), a peptide derivative 1 bearing a diphenyl 1-amino-2-phenylethylphosphonate moiety for targeting t...

Kumamolisin, an extracellular proteinase derived from an acido/thermophilic Bacillus, belongs to the sedolisin family of endopeptidases characterized by a subtilisin-like fold and a Ser-Glu-Asp catalytic triad. In kumamolisin, the Asp82 carboxylate hydrogen bonds to Glu32-Trp129, which might act as a proton sink stabilizing the catalytic residues. The 1.2/1.3 A crystal structures of the Glu32→Ala and Trp129→Ala mutants show that both mutations affect the active-site conformation, causing a 95% activity decrease. In addition, the 1.2 A crystal structure of the Ser278→Ala mutant of pro-kumamolisin was determined. The prodomain exhibits a half-β sandwich core docking to the catalytic domain similarly as the equivalent subtilisin prodomains in their catalytic-domain complexes. This pro-kumamolisin structure displays, for the first time, the uncleaved linker segment running across the active site and connecting the prodomain with the properly folded catalytic domain. The structure strongly points to an initial intramolecular activation cleavage in subtilases, as presumed for pro-subtilisin and pro-furin.

Kumamolisin-As (previously called ScpA) is the first known example of a collagenase from the sedolisin family (MEROPS S53). This enzyme is active at low pH and in elevated temperatures. In this study that used x-ray crystallographic and biochemical methods, we investigated the structural basis of the preference of this enzyme for collagen and the importance of a glutamate residue in the unique catalytic triad (Ser(278)-Glu(78)-Asp(82)) for enzymatic activity. Crystal structures of the uninhibited enzyme and its complex with a covalently bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence of a narrow S2 pocket and a groove that encompasses the active site and is rich in negative charges. Limited endoproteolysis studies of bovine type-I collagen as well as kinetic studies using peptide libraries randomized at P1 and P1', showed very strong preference for arginine at the P1 position, which correlated very well with the presence of a negatively charged residue in the S1 pocket of the enzyme. All of these features, together with those predicted through comparisons with fiddler crab collagenase, a serine peptidase, rationalize the enzyme's preference for collagen. A comparison of the Arrhenius plots of the activities of kumamolisin-As with either collagen or peptides as substrates suggests that collagen should be relaxed before proteolysis can occur. The E78H mutant, in which the catalytic triad was engineered to resemble that of subtilisin, showed only 0.01% activity of the wild-type enzyme, and its structure revealed that Ser(278), His(78), and Asp(82) do not interact with each other; thus, the canonical catalytic triad is disrupted.

The molecular structure of the pepstatin-insensitive carboxyl peptidase from Scytalidium lignicolum , formerly known as scytalidopepsin B, was solved by multiple isomorphous replacement phasing methods and refined to an R factor of 0.230 ( R free = 0.246) at 2.1-Å resolution. In addition to the structure of the unbound peptidase, the structure of a product complex of cleaved angiotensin II bound in the active site of the enzyme was also determined. We propose the name scytalidocarboxyl peptidase B (SCP-B) for this enzyme. On the basis of conserved, catalytic residues identified at the active site, we suggest the name Eqolisin for the enzyme family. The previously uninvestigated SCP-B fold is that of a β-sandwich; each sheet has seven antiparallel strands. A tripeptide product, Ala-Ile-His, bound in the active site of SCP-B has allowed for identification of the catalytic residues and the residues in subsites S1, S2, and S3, which are important for substrate binding. The most likely hydrolytic mechanism involves nucleophilic attack of a general base (Glu-136)-activated water (OH - ) on the si -face of the scissile peptide carbonylcarbon atom to form a tetrahedral intermediate. Electrophilic assistance and oxyanion stabilization is provided by the side-chain amide of Gln-53. Protonation of the leaving-group nitrogen is accomplished by the general acid function of the protonated carboxyl group of Glu-136.

High-resolution crystallographic analysis of a complex of the serine-carboxyl proteinase sedolisin with pseudo-iodotyrostatin revealed two molecules of this inhibitor bound in the active site of the enzyme, marking subsites from S3 to S3 ′ . The mode of binding represents two products of the proteolytic reaction. Substrate specificity of sedolisin was investigated using peptide libraries and a new peptide substrate for sedolisin, MCA–Lys–Pro–Pro–Leu–Glu#Tyr–Arg–Leu–Gly–Lys(DNP)–Gly, was synthesized based on the results of the enzymatic and crystallographic studies and was shown to be efficiently cleaved by the enzyme. The kinetic parameters for the substrate, measured by the increase in fluorescence upon relief of quenching, were: k cat =73±5 s −1 , K m =0.12±0.011 μM, and k cat / K m =608±85 s −1 μM −1 .

In order to reduce the protein content of wastewater, photosynthetic bacteria producing proteinases were screened from wastewater of various sources and stocked in culture. An isolated strain, KDDS1, was identified as Rubrivivax gelatinosus, a purple nonsulfur bacterium that secretes proteinase under micro-aerobic conditions under light at 35 degrees C. Molecular weight of the purified enzyme was estimated to be 32.5 kDa. The enzyme showed the highest activity at 45 degrees C and pH 9.6, and the activity was completely inhibited by phenylmethyl sulfonyl fluoride (PMSF), but not by EDTA. The amino-terminal 24 amino acid sequence of the enzyme showed about 50% identity to those of serine proteinases from Pseudoalteromonas piscicida strain O-7 and Burkholderia pseudomallei. Thus, the enzyme from Rvi. gelatinosus KDDS1 was thought to be a serine-type proteinase. This was the first serine proteinase characterized from photosynthetic bacteria.

In order to operate high-Tc SQUID magnetometers in an environment with no or weak magnetic shielding, we incorporated slots along the wide (3 mm) grain boundary junction in the pickup coil and the flux dam of direct-coupled SQUID magnetometers. It was confirmed that the slots formed along the wide grain boundary junction suppressed vortex motion and prevented a large increase in low-frequency noise. When we applied external fields above a threshold value of the flux dam after zero-field cooling, the flux-locked-loop output fluctuated, and the low-frequency field noise Bn increased but became stable after a time. The relaxation time after field application was longer for higher fields. A slight increase in the low-frequency noise remained, which was attributed to the fluctuation of the flux trapped in the slots having grain boundaries. This paper was presented at the 8th International Superconductive Electronics Conference, Osaka, Japan, 19–22 June 2001.

Crystal structures of the serine-carboxyl proteinase from Pseudomonas sp. 101 (PSCP), complexed with a number of inhibitors, have been solved and refined at high- to atomic-level resolution. All of these inhibitors (tyrostatin, pseudo-tyrostatin, AcIPF, AcIAF, and chymostatin, as well as previously studied iodotyrostatin and pseudo-iodotyrostatin) make covalent bonds to the active site Ser287 through their aldehyde moieties, while their side chains occupy subsites S1-S4 of the enzyme. The mode of binding of the inhibitors is almost identical for their P1 and P2 side chains, while significant differences are observed for P3 and P4 (if present). Kinetic parameters for the binding of these nanomolar inhibitors to PSCP have been established and correlated with the observed mode of binding. The preferences of this enzyme for a larger side chain in P2 as well as Tyr or Phe in P1 are explained by the size, shape, and characteristics of the S2 and S1 regions of the protein structure, respectively. Networks of hydrogen bonds involving glutamic and aspartic acids have been analyzed for the atomic-resolution structure of the native enzyme. PSCP contains a calcium-binding site that consists of Asp328, Asp348, three amide carbonyl groups, and a water molecule, in almost perfect octahedral coordination. The presence of Ca(2+) cation is necessary for the activity of the enzyme.

We fabricated directly coupled dc SQUID magnetometers which had a mesh structure from YBa 2 Cu 3 O 7− x thin films deposited on SrTiO 3 bicrystal substrates. A pickup coil had two flux dams of grain boundary junction (GBJ) near the SQUID. In order to suppress the flux motion in the flux dams, we formed 5 μm wide strip lines and slots across the GB of the flux dams. The flux noise was measured after applying static magnetic field in a magnetically shielded room. The flux-locked-loop output was stable and the increase of low-frequency noise was suppressed up to an applied field of 40 μT. It is suggested that slotted flux dams are useful to suppress the low-frequency noise in the change of applied field.

The pressure dependence of the activity and spectroscopic properties of four carboxyl proteinases were investigated. Two were pepstatin-sensitive carboxyl proteinases (porcine pepsin and proteinase A from baker's yeast) and two were pepstatin-insensitive carboxyl proteinases (from Pseudomonas sp. 101 (pseudomonapepsin; PCP) and Xanthomonas sp. T-22 (xanthomonapepsin; XCP)). The specificity constant [k(cat)/K(m(app))] of PCP and XCP for a synthetic peptide substrate showed only a slight decrease with increasing pressure, whereas pepsin and proteinase A showed substantial disactivation at higher pressures. The calculated apparent activation volume (Delta V((k(cat)/(K(m)) was about 1, 3, 13, and 14 mL.mol(-1) for PCP, XCP, pepsin, and proteinase A, respectively. The hydrolysis of acid-denatured myoglobin by the four carboxyl proteinases was only slightly affected by high pressure (except for proteinase A at 400 MPa), in contrast to the results for the peptide hydrolysis. In fact, PCP, XCP, and proteinase A actually showed slightly higher degradations of acid-denatured myoglobin at higher pressures. The residual activities of these enzymes after the incubation at high pressures implied a pressure-induced stabilization towards autolysis. The changes in the fourth derivative near-UV absorbance spectrum of the four enzymes in aqueous solution were measured at various pressures from 0.1 to 400 MPa. Upon an increase in pressure, the peaks from PCP and XCP red-shifted slightly, whereas pepsin and proteinase A blue-shifted substantially, thus indicating a more polar environment. The intrinsic fluorescence also decreased upon increasing pressure. However, the change for XCP was rather small, but the change for the other three was very large. The changes in the peak wavelength for pepsin and proteinase A were characteristic, and also indicated a more polar environment under high pressure. An analysis by the center of spectra mass (CSM) gave the Delta G and Delta V of transition as 9.8 kJ x mol(-1) and -24 mL x mol(-1) (pepsin) and 11.7 kJ x mol(-1) and -43 mL x mol(-1) (proteinase A), respectively, by assuming a simple two-state transition. The circular dichroism (CD) showed relatively small changes after 1-h incubations at 400 MPa, indicating that the secondary structures were largely maintained.

A novel type of 5-oxoprolinase was found in a cell extract of strain N-38A, which was later identified as Alcaligenes faecalis . The enzyme in the cell extract was purified to a homogeneous state with a yield of 16.6%. The molecular weight of the purified enzyme was estimated to be 47,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, suggesting that the enzyme is a monomeric protein. The enzyme specifically catalyzed a decyclization of l -pyroglutamate without hydrolyzing ATP and also without any requirements for metal ions such as Mg 2+ and K + . The optimal pH for the decyclization was 7.4. The reaction was reversible. The equilibrium constant of the reaction, K eq = [ l -glutamate]/[ l -pyroglutamate], was evaluated to be approximately 0.035, which indicates that the reaction tends to form l -pyroglutamate. The amino-terminal amino acid sequence of the enzyme was H-Glu-Pro-Arg-Leu-Asp-Thr-Ser-Gln-Leu-Tyr-Ala-Asp-Val-His-Phe-. No protein with a similar sequence was found in the DNASIS database. Based on these data, it was strongly suggested that the enzyme described here is a novel type of 5-oxoprolinase.

We detected a prolyl endopeptidase (EC 3.4.21.26) in the cell-free extract of Pseudomonas sp. KU-22. The enzyme was purified approximately 2,000-fold by a series of column chromatographies. The molecular weight and isoelectric point of the purified enzyme were estimated to be 76,000 and pH 4.9, respectively. The enzyme was most active at 45°C and pH 8.0 with benzyloxycarbonyl- l -alanyl- l -alanyl- l -proline p -nitroanilide (Z-Ala-Ala-Pro-NphNO 2 ) as a substrate. Significant inhibition of the enzyme by DFP and prolyl endopeptidase-specific inhibitors was observed. Some properties noted, particularly pH stability, amino acid composition, and pI of the enzyme, are clearly different from those of other bacterial prolyl endopeptidases reported to date, indicating that the enzyme is a member of the microbial prolyl endopeptidase family.

Muraymycin analogues with a lipophilic substituent were synthesized using an Ugi four-component assemblage. This approach provides ready access to a range of analogues simply by altering the aldehyde component. The impact of the lipophilic substituent on the antibacterial activity was very large, and analogues 7b−e and 8b−e exhibited good activity against a range of Gram-positive bacterial pathogens including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. This study also showed that the accessory urea-dipeptide motif contributes to MraY inhibitory and antibacterial activity. The knowledge obtained from our structure−activity relationship study of muraymycins provides further direction toward the design of potent MraY inhibitors. This study has set the stage for the generation of novel antibacterial “lead” compounds based on muraymycins.

Background Robo1, Robo2 and Rig-1 (Robo3), members of the Robo protein family, are candidate receptors for the chemorepellents Slit and are known to play a crucial role in commissural axon guidance in the spinal cord. However, their roles at other axial levels remain unknown. Here we examine expression of Robo proteins by cerebellofugal (CF) commissural axons in the rostral hindbrain and investigate their roles in CF axon pathfinding by analysing Robo knockout mice. Results We analysed the expression of Robo proteins by CF axons originating from deep cerebellar neurons in rodent embryos, focusing on developmental stages of their midline crossing and post-crossing navigation. At the stage of CF axon midline crossing, mRNAs of Robo1 and Robo2 are expressed in the nuclear transitory zone of the cerebellum, where the primordium of the deep cerebellar nuclei are located, supporting the notion that CF axons express Robo1 and Robo2. Indeed, immunohistochemical analysis of CF axons labelled by electroporation to deep cerebellar nuclei neurons indicates that Robo1 protein, and possibly also Robo2 protein, is expressed by CF axons crossing the midline. However, weak or no expression of these proteins is found on the longitudinal portion of CF axons. In Robo1/2 double knockout mice, many CF axons reach the midline but fail to exit it. We find that CF axons express Rig-1 (Robo3) before they reach the midline but not after the longitudinal turn. Consistent with this in vivo observation, axons elicited from a cerebellar explant in co-culture with a floor plate explant express Rig-1. In Rig-1 deficient mouse embryos, CF axons appear to project ipsilaterally without reaching the midline. Conclusion These results indicate that Robo1, Robo2 or both are required for midline exit of CF axons. In contrast, Rig-1 is required for their approach to the midline. However, post-crossing up-regulation of these proteins, which plays an important role in spinal commissural axon guidance, does not appear to be required for the longitudinal navigation of CF axons after midline crossing. Our results illustrate that although common mechanisms operate for midline crossing at different axial levels, significant variation exists in post-crossing navigation.

The crystal structure of dipeptidyl aminopeptidase IV fromStenotrophomonas maltophiliawas determined at 2.8-Å resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space groupP43212, with unit cell parametersa=b= 105.9 Å andc= 161.9 Å. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal β-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes.Stenotrophomonasdipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In theStenotrophomonasenzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.

A chromosomal DNA of Scytalidium lignicolum was digested with Sau3AI. The digest was self-ligated and amplified by inverse PCR procedure using primers designed based on the nucleotide sequences of up- and down-stream regions of an intron present in the scytalidopepsin B gene. Analysis of the nucleotide sequence of PCR product (700 bp) showed that the enzyme is synthesized as a precursor protein consisting of the prepro- and mature enzyme regions. The deduced amino acid sequence was highly similar to those of aspergillopepsin A and recently reported endothiapepsins B and C, but quite different from those of pepstatin-insensitive bacterial acid proteases and the pepstatin-sensitive aspartic protease family.

An extracellular alkaline proteinase was purified from the culture filtrate of Pimelobacter sp. Z-483. The molecular mass of the purified enzyme was estimated to be approximately 23 kDa by SDS-PAGE and 27 kDa by gel filtration. The optimum pH and temperature for the hydrolysis of casein were approximately 9 and 50°C, respectively. The enzyme activity was strongly inhibited by 1,10-phenanthroline and mercury chloride, but not by EDTA, phosphoramidon and Zincov. The amino terminal sequence of the enzyme was similar to that of an alkaline elastase (MAP1) from Myxococcus xanthus. However, the specificity of the alkaline proteinase in the cleavage of the oxidized insulin B-chain was different from those of the Myxococcus enzyme and animal elastase.

Tripeptidyl peptidase I (TTP-I), also known as CLN2, a member of the family of serine-carboxyl proteinases (S53), plays a crucial role in lysosomal protein degradation and a deficiency in this enzyme leads to fatal neurodegenerative disease. Recombinant human TPP-I and its mutants were analyzed in order to clarify the biochemical role of TPP-I and its mechanism of activity. Ser280, Glu77, and Asp81 were identified as the catalytic residues based on mutational analyses, inhibition studies, and sequence similarities with other family members. TPP-I hydrolyzed most effectively the peptide Ala-Arg-Phe*Nph-Arg-Leu (*, cleavage site) (k(cat)/K(m) = 2.94 microM(-1).s(-1)). The k(cat)/K(m) value for this substrate was 40 times higher than that for Ala-Ala-Phe-MCA. Coupled with other data, these results strongly suggest that the substrate-binding cleft of TPP-I is composed of only six subsites (S(3)-S(3)'). TPP-I prefers bulky and hydrophobic amino acid residues at the P(1) position and Ala, Arg, or Asp at the P(2) position. Hydrophilic interactions at the S(2) subsite are necessary for TPP-I, and this feature is unique among serine-carboxyl proteinases. TPP-I might have evolved from an ancestral gene in order to cleave, in cooperation with cathepsins, useless proteins in the lysosomal compartment.

Sedolisins (serine-carboxyl peptidases) are proteolytic enzymes whose fold resembles that of subtilisin; however, they are considerably larger, with the mature catalytic domains containing approximately 375 amino acids. The defining features of these enzymes are a unique catalytic triad, Ser-Glu-Asp, as well as the presence of an aspartic acid residue in the oxyanion hole. High-resolution crystal structures have now been solved for sedolisin from Pseudomonas sp. 101, as well as for kumamolisin from a thermophilic bacterium, Bacillus novo sp. MN-32. The availability of these crystal structures enabled us to model the structure of mammalian CLN2, an enzyme which, when mutated in humans, leads to a fatal neurodegenerative disease. This review compares the structural and enzymatic properties of this newly defined MEROPS family of peptidases, S53, and introduces their new nomenclature.

Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest.Alicyclobacillus sendaiensisstrain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (kcat, 5.41 s−1;Km, 32 μM) and Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser (kcat, 351 s−1;Km, 214 μM), where the asterisks denote the scissile bonds. The clonedscpAgene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of thescpAgene in theEscherichia colicells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of thescpAgene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.

The gene encoding kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase, was cloned and expressed. (i) Kumamolysin was synthesized as a large precursor consisting of two regions: amino-terminal prepro (188 amino acids) and mature proteins (384 amino acids). (ii) The deduced amino acid sequence of the mature region exhibited high similarity to those of such bacterial pepstatin-insensitive enzymes as Pseudomonas carboxyl proteinase (PSCP; EC 3.4.23.37, identity = 37%), Xanthomonas carboxyl proteinase (XCP; EC 3.4.23.33, identity = 36%), and human CLN2 gene product (identity = 36%), which is related to a fatal neurodegenerative disease. (iii) The presumed catalytic triad, Glu78, Asp82, Ser278 [three-dimensional structure of PSCP: Wlodawer, A. et al. (2001) Nature Struct. Biol., 8, 442-446], was found to be conserved in the amino acid sequence of kumamolysin. (iv) Kumamolysin was inactivated by such aldehyde-type inhibitors as Ac-Ile-Pro-Phe-CHO (K(i) = 0.7 0.14 microM). In PSCP, it has been clarified that these inhibitors form a hemiacetal linkage with the catalytic serine residue and inactivate the enzyme. (v) Mutational analysis of the Ser278 residue revealed that the mutant lost both auto-processing activity and proteolytic activity. These results strongly suggest that kumamolysin has a unique catalytic triad consisting of Glu78, Asp82, and Ser278 residues, as previously observed for PSCP.

Recent developments of high-Tc SQUIDs have enabled high sensitivity magnetometers to be used in wide range of places, such as laboratory and outdoor fields. At the early stage of developing multichannel system for measurement of magnetocardiogram (MCG) in clinical application, we have fabricated a single channel high-Tc SQUID magnetometer system. The system includes a direct-coupled SQUID with slot structure, a simple magnetically shielded room (MSR), and some active compensation electronics for the purpose of reducing various environmental field noises. A novel active noise cancellation was made by using a combination of a normal conducting detection coil that was horizontally wound in the middle height of the MSR, and two compensation coils that were wound at the top and bottom of the MSR. In addition, adaptive noise cancellation was supplemented by means of adaptive digital filter that was implemented in a digital signal processor. A total noise field attenuation of 50–60 dB was attained at 0.5–100 Hz. Low noise signals from the human heart were measured with a high-Tc SQUID in the noise reduced space in the MSR.

An expression and secretion system for scytalidopepsin B, an acid protease from Scytalidium lignicolum, was constructed in yeast. Saccharomyces cerevisiae AH22 was transformed with an yeast-E. coli shuttle vector, pAM82, in which an yeast invertase signal segment and the cDNA encoding the pro- and mature enzyme regions were inserted. The transformant was found to secret a pepstatin-insensitive acid protease, when cultured aerobically in a low phosphate (Pi) medium. Amino terminal amino acid sequencing analysis indicated that the recombinant acid protease was accurately processed and secreted as a mature form.

The gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A was cloned and characterized. The coding region of this gene is 1,299 bp long. The predicted primary protein is composed of 433 amino acid residues, with a 31-amino-acid signal peptide. The mature protein is composed of 402 amino acid residues with a molecular mass of 46,163 Da. The derived amino acid sequence of the enzyme showed no significant sequence similarity to any other proteins reported so far. The 5-oxoprolinase gene was expressed in Escherichia coli by using a regulatory expression system with an isopropyl-β- d -thiogalactopyranoside-inducible tac promoter, and its expression level was approximately 16 mg per liter. The purified enzyme has the same characteristics as the authentic enzyme, except for the amino terminus, which has three additional amino acids. The enzyme was markedly inhibited by p -chloromercuribenzoic acid, EDTA, o -phenanthroline, HgCl 2 , and CuSO 4 . The EDTA-inactivated enzyme was completely restored by the addition of Zn 2+ or Co 2+ . In addition, the enzyme was found to contain 1 g-atom of zinc per mol of protein. These results suggest that the 5-oxoprolinase produced by A. faecalis N-38A is a zinc metalloenzyme.

We designed a double-pickup-coil configuration for direct-coupling-type high-TC SQUID magnetometers, in which two pickup coils are connected directly to one SQUID on a substrate. The SQUID and pickup coils can be arranged symmetrically without covering the bicrystal line on the substrate. The devices, consisting of washer-shaped pickup coils and transmission-line-type SQUIDs made of YBa2Cu3O7-y, were fabricated on 10 x 10 mm SrTiO3 bicrystal substrates, and were driven as magnetometers. The effective areas ranging from 0.32 to 0.42 mm2 were evaluated experimentally. A magnetic-field sensitivity of about 100 fT/√Hz was obtained in white noise region for one of the magnetometers.

We developed a method for the analyzing mutagenic potential of DNA damage based on the oligonucleotide transformation technique in yeast. Using this assay we have analyzed mutagenic specificities of various DNA lesions. In the present study, we analyzed the mutagenic properties of 2-hydroxyadenine and 5-hydroxycytosine in yeast. Oligonucleotides containing 2-hydroxyadenine or 5-hydroxycytosine were used for the transformation. The oligonucleotides showed transforming activities similar to unmodified oligonucleotides. This indicates that no repair systems were working on them. The sequencing data of the transformants showed that 5-hydroxycytosine and 2-hydroxyadenine are read mainly as cytosine and adenine. We will also discuss the mechanism of oligonucleotide transformation and its application to the mutagenesis study.

An inhibitor of Serratia metalloproteinase was found in the cultured broth of strain SI-23, which was identified to be Actinosynnema sp. The inhibitor had a potent inhibitory effect on Serratia metalloproteinase and a weak effect on thermolysin, but no effect on other groups of proteinases. The structure of the inhibitor was deduced to be propioxatin A, reported as an enkephalinase B inhibitor.

A chromosomal DNA fragment of Scytalidium lignicolum that encodes the mature enzyme region of acid protease B (Scytalido-pepsin B), was cloned and its nucleotides sequenced. The fragment contained a 76-bp intron at the middle of the mature enzyme-coding region. The mature enzyme was composed of 206 amino acid residues with a molecular weight of 21,550. There were some discrepancies between the amino acid sequence deduced from these results and that previously established by protein sequencing.

A screening test was undertaken to isolate microorganisms that produced dipeptidyl aminopeptidase. The hydrolytic activity toward alanyl-phenylalanine p-nitroanilide was found in a culture filtrate of a actinomyces strain (WM-23), newly isolated from a soil sample. The enzyme (WM-23 dipeptidyl aminopeptidase) was isolated from the culture filtrate as a homogeneous preparation. The WM-23 enzyme, inhibited by phenylmethylsulfonyl fluoride, may be classified to mammalian dipeptidyl aminopeptidase II. The enzymatic characteristics were investigated.

A direct-coupling-type high-Tc SQUID magnetometer with two pickup loops per SQUID on a substrate has been fabricated. To suppress the flux trapping and vortex entry, the superconducting film of the whole device except a 5-μm-wide transmission-line-shaped SQUID is composed of narrow (5 μm) lines with slots and holes. The threshold field for the flux trapping during field cooling was evaluated to be around 130 μT, a much larger field than the geomagnetic field. Moreover, applying a field after cooling reduced the critical current of the SQUID to zero at 6 μT. And vortex entry into the pickup loop occurred at a field exceeding about 10 μT.

The 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) gene from Escherichia coli HB101 was cloned and expressed in E. coli DH1. The hybrid plasmid pSD1, with a 2.8-kbp insert of chromosomal DNA at the BamHI site of pBR322, was subcloned into pUC19 to construct plasmid pSD3. The entire nucleotide sequence of an inserted PstI-BamHI fragment of plasmid pSD3 was determined by the dideoxy chain-termination method. Within this sequence, the mature enzyme protein-encoding sequence was found to start at a GTG initiation codon and to comprise 765 bp, as judged by comparison with the protein sequence. The deduced amino acid sequence of the enzyme indicated that the molecular weight is 26,778. The transformant of E. coli DH1 harboring pSD3 with a 1.8-kbp fragment showed about 200-fold-higher enzyme activity than the host. The enzyme was purified by a single chromatography step on DEAE-Toyopearl and obtained as crystals, with an activity yield of 39%. The purified enzyme was homogeneous, as judged by sodium dodecyl sulfate gel electrophoresis. The enzyme was most active at pH 8.5 and stable between pH 8 and 9. The enzyme was NAD+ dependent and had a pI of 4.3. The molecular mass was estimated to be 120 kDa by the gel filtration method and 28 kDa by electrophoresis, indicating that the enzyme exists in a tetrameric form.

Tripeptidyl-peptidase I, also known as CLN2, is a member of the family of sedolisins (serine-carboxyl peptidases). In humans, defects in expression of this enzyme lead to a fatal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis. Similar enzymes have been found in the genomic sequences of several species, but neither systematic analyses of their distribution nor modeling of their structures have been previously attempted. We have analyzed the presence of orthologs of human CLN2 in the genomic sequences of a number of eukaryotic species. Enzymes with sequences sharing over 80% identity have been found in the genomes of macaque, mouse, rat, dog, and cow. Closely related, although clearly distinct, enzymes are present in fish (fugu and zebra), as well as in frogs (Xenopus tropicalis). A three-dimensional model of human CLN2 was built based mainly on the homology with Pseudomonas sp. 101 sedolisin. CLN2 is very highly conserved and widely distributed among higher organisms and may play an important role in their life cycles. The model presented here indicates a very open and accessible active site that is almost completely conserved among all known CLN2 enzymes. This result is somehow surprising for a tripeptidase where the presence of a more constrained binding pocket was anticipated. This structural model should be useful in the search for the physiological substrates of these enzymes and in the design of more specific inhibitors of CLN2.

We found a novel type of proteinase, which we named masikinolysin, in the culture filtrate of Streptomyces griseoloalbus strain SN-22. The molecular weight and isoelectric point of the purified enzyme were determined to be 26,000 and 6.4, respectively. The optimum conditions were 45°C at pH 9.0. From the cleavage sites of the insulin B-chain and lysozyme, the enzyme specifically hydrolyzed the carboxyl side of alanine and valine residues.

(1992). A Novel Laccase Inhibitor, N-Hydroxyglycine, Produced by Penicillium citrinum YH-31. Bioscience, Biotechnology, and Biochemistry: Vol. 56, No. 6, pp. 987-988.

(1992). Purification and Characterization of a Novel Type of Chitinase from Vibrio alginolyticusTK-22. Bioscience, Biotechnology, and Biochemistry: Vol. 56, No. 2, pp. 368-369.