Steric hindrance and structural flexibility shape the functional properties of a guanine-rich oligonucleotide.

Ligand/protein molecular recognition involves a dynamic process, whereby both partners require a degree of structural plasticity to regulate the binding/unbinding event. Here, we present the characterization of the interaction between a highly dynamic G-rich oligonucleotide, M08s-1, and its target protein, human α-thrombin. M08s-1 is the most active anticoagulant aptamer selected thus far. Circular dichroism and gel electrophoresis analyses indicate that both intramolecular and intermolecular G-quadruplex structures are populated in solution. The presence of thrombin stabilises the antiparallel intramolecular chair-like G-quadruplex conformation, that provides by far the main contribution to the biological activity of the aptamer. The crystal structure of the thrombin-oligonucleotide complex reveals that M08s-1 adopts a kinked structural organization formed by a G-quadruplex domain and a long duplex module, linked by a stretch of five purine bases. The quadruplex motif hooks the exosite I region of thrombin and the duplex region is folded towards the surface of the protein. This structural feature, which has never been observed in other anti-exosite I aptamers with a shorter duplex motif, hinders the approach of a protein substrate to the active site region and may well explain the significant increase in the anticoagulant activity of M08s-1 compared to the other anti-exosite I aptamers.

Lactic acid induces HSPA1A expression through ERK1/2 activation.

Heat shock protein (HSP) A1A protects cells from various stressors. The concentrated liquid of the traditional Japanese rice black vinegar Kurozu increased HSPA1A expression in normal rat liver RLN-10 cells. Lactic acid, the primary component of concentrated Kurozu, induced HSPA1A expression in a concentration-dependent manner. Induction with 4 m m lactic acid increased HSPA1A expression by three times compared with that in the absence of lactic acid. The induction was inhibited by staurosporine or a selective MEK1/2 inhibitor (SL327). The phosphorylation of ERK1/2 was increased by lactic acid. These results suggest that lactic acid induces HSPA1A expression by activating ERK1/2. As well as lactate, 3,5-dihydroxybenzoic acid (DHBA), a ligand for G protein-coupled receptor 81 (GPR81), also induced HSPA1A at lower concentrations than lactate. The increased effect of DHBA on HSPA1A expression as compared with lactate may be related to the higher affinity of DHBA for GPR81 than of lactate.

Development of cyclic peptides with potent in vivo osteogenic activity through RaPID-based affinity maturation.

Osteoporosis is caused by a disequilibrium between bone resorption and bone formation. Therapeutics for osteoporosis can be divided into antiresorptives that suppress bone resorption and anabolics which increase bone formation. Currently, the only anabolic treatment options are parathyroid hormone mimetics or an anti-sclerostin monoclonal antibody. With the current global increases in demographics at risk for osteoporosis, development of therapeutics that elicit anabolic activity through alternative mechanisms is imperative. Blockade of the PlexinB1 and Semaphorin4D interaction on osteoblasts has been shown to be a promising mechanism to increase bone formation. Here we report the discovery of cyclic peptides by a novel RaPID (Random nonstandard Peptides Integrated Discovery) system-based affinity maturation methodology that generated the peptide PB1m6A9 which binds with high affinity to both human and mouse PlexinB1. The chemically dimerized peptide, PB1d6A9, showed potent inhibition of PlexinB1 signaling in mouse primary osteoblast cultures, resulting in significant enhancement of bone formation even compared to non-Semaphorin4D-treated controls. This high anabolic activity was also observed in vivo when the lipidated PB1d6A9 (PB1d6A9-Pal) was intravenously administered once weekly to ovariectomized mice, leading to complete rescue of bone loss. The potent osteogenic properties of this peptide shows great promise as an addition to the current anabolic treatment options for bone diseases such as osteoporosis.

Discovery of a Highly Specific Anti-methotrexate (MTX) DNA Aptamer for Antibody-Independent MTX Detection.

High-dose methotrexate (MTX) therapy is used to treat a wide variety of cancers such as leukemia and lymphoma, while the resulting high blood concentration of MTX faces a risk of life-threatening side effects, so it is essential to monitor the concentration carefully. Currently, the MTX concentration is measured using antibody-based kits in a clinical setting; however, the heterogeneity and batch-to-batch variation of antibodies potentially compromise the detection limit. Here, we developed MTX detection systems with chemically synthesizable homogeneous oligonucleotides. Microbead-assisted capillary electrophoresis (MACE)-SELEX against MTX successfully identified MSmt7 with a similar level of specificity to anti-MTX antibodies within three rounds. The 3'-end of MSmt7 was coupled to a peroxidase-like hemin-DNAzyme to construct a bifunctional oligonucleotide for MTX sensing, where MTX in 50% human serum was detected with a limit of detection (LoD) of 118 nM. Furthermore, amplifying the DNAzyme region with rolling circle amplification significantly improved the sensitivity with an LoD of 290 pM. Presented oligonucleotide-based MTX detection systems will pave the way for antibody-independent MTX detection with reliability and less cost in the laboratory and the clinic.

Evaluation of G-quartet-forming deoxyguanines in antiparallel G-quadruplexes using optical spectroscopy and deoxyguanine-to-deoxythymidine scanning.

Due to the dynamic conformations of G-quadruplex structures (G4), determining the guanines that form G4 in a guanine-rich sequence is elusive. Here, we report a method for identifying deoxyguanines (dGs) forming antiparallel G4 by optical spectroscopy. The method, referred to as dG-to-deoxythymidine (dT) scanning, compares the spectra between a wild type and a single nucleobase dG-to-dT mutant at all dG positions. The most strongly involved dGs to form antiparallel G4 in the two model sequences were estimated using dG-to-dT scanning by circular dichroism (CD) and UV-Vis melting curve. This simple and robust method will facilitate understanding de novo antiparallel G4.

One-Pot In Vitro Ribosomal Synthesis of Macrocyclic Depsipeptides.

Here, we report a method for the one-pot ribosomal synthesis of macrocyclic depsipeptides. This method is based on a Ser-Pro-Cys-Gly (SPCG) motif discovered by in vitro selection of peptides for the function of self-acylation in the presence of a thioester acyl donor, which forms an O-acyl isopeptide bond via intramolecular S-to-O acyl transfer. Ribosomal synthesis of linear peptides containing the SPCG motif and a backbone "acyl donor" thioester at a downstream position results in spontaneous conversion to the corresponding cyclic depsipeptides (CDPs) in a nearly independent manner of ring size and sequence context. Mutational analysis of the SPCG motif revealed that the P and G residues are dispensable to some extent, but the arrangement of residues in SXCX is crucial for efficient acyl transfer, e.g., CPSG is much less efficient. Finally, one-pot ribosomal synthesis of macrocyclic depsipeptides with various ring sizes and sequences has been demonstrated. This synthetic method can facilitate the ribosomal construction of highly diverse CDP libraries for the discovery of de novo bioactive CDPs.

Site-Specific Nonenzymatic Peptide S/O-Glutamylation Reveals the Extent of Substrate Promiscuity in Glutamate Elimination Domains.

Formation of dehydroalanine and dehydrobutyrine residues via tRNA-dependent dehydration of serine and threonine is a key post-translational modification in the biosynthesis of lanthipeptide and thiopeptide RiPPs. The dehydration process involves two reactions, wherein the O-glutamyl Ser/Thr intermediate, accessed by a dedicated enzyme utilizing Glu-tRNAGlu as the acyl donor, is recognized by the second enzyme, referred to as the glutamate elimination domain (ED), which catalyzes the eponymous reaction yielding a dehydroamino acid. Many details of ED catalysis remain unexplored because the scope of available substrates for testing is limited to those that the upstream enzymes can furnish. Here, we report two complementary strategies for direct, nonenzymatic access to diverse ED substrates. We establish that a thiol-thioester exchange reaction between a Cys-containing peptide and an α thioester of glutamic acid leads an S-glutamylated intermediate which can act as a substrate for EDs. Furthermore, we show that the native O-glutamylated substrates can be accessible from S-glutamylated peptides upon a site-specific S-to-O acyl transfer reaction. Combined with flexible in vitro translation utilized for rapid peptide production, these chemistries enabled us to dissect the substrate recognition requirements of three known EDs. Our results establish that EDs are uniquely promiscuous enzymes capable of acting on substrates with arbitrary amino acid sequences and performing retro-Michael reaction beyond the canonical glutamate elimination. To facilitate substrate recruitment, EDs apparently engage in nonspecific hydrophobic interactions with their substrates. Altogether, our results establish the substrate scope of EDs and provide clues to their catalysis.

Accelerated Discovery of Potent Bioactive anti-TNFα Aptamers by Microbead-Assisted Capillary Electrophoresis (MACE)-SELEX.

Dysregulation of tumor necrosis factor-α (TNFα), a pro-inflammatory cytokine, causes several diseases, making it an important therapeutic target. Here, we identified a novel DNA aptamer against human TNFα using in vitro selection, which included a high exclusion pressure process against non-binding and weak binders through microbead-assisted capillary electrophoresis (MACE) in only three rounds. Among the 15 most enriched aptamers, Apt14 exhibited the highest inhibitory activity for the interaction between TNFα and its cognate receptor in mouse L929 cells. For further improving the bioactivity of the aptamer, dimerization programed by hybridization was evaluated, resulting in the Apt14 dimer exhibited a twofold higher binding affinity and stronger inhibition compared to the monomer counterpart. Rapid identification of bioactive aptamers using MACE in combination with facile dimerization by hybridization accelerates the discovery of novel bioactive aptamers, paving the way toward replacing current monoclonal antibody therapy with the less expensive and non-immunogenic aptamer therapy.

Facile Synthesis of Dimeric Thioether-Macrocyclic Peptides with Antibody-like Affinity against Plexin-B1.

Macrocyclic peptides have gained increasing attention due to their ease of discovery through various in vitro display platforms as well as their potential in possessing favorable properties of both small molecule and antibody drug classes. It is well-known that the avidity achieved through the bivalent binding mode of antibodies gives rise to their slow dissociation rates and thus high potency as drug molecules. Here, we report the synthesis of dimeric thioether-macrocyclic peptides through a branched synthesis approach allowing for synthesis of dimeric peptides in a comparable number of steps as monomers and tunability of linker lengths from 30 to 200 Å. Applying this method to synthesize dimers of a model PlexinB1-binding macrocyclic peptide showed close to 300-fold increases in their apparent binding affinity, bringing the KD down from 8 nM to 30 pM as well as affording improved biological activities when compared to their monomeric counterparts. These enhancements demonstrate that this is a simple synthetic strategy to harness the benefits of bivalence that antibodies naturally possess.

In vivo programming of endogenous antibodies via oral administration of adaptor ligands.

Vaccination is a reliable method of prophylaxis and a crucial measure for public health. However, the majority of vaccines cannot be administered orally due to their degradation in the harsh gut environment or inability to cross the GI tract. In this study, we report the first proof-of-concept study of orally producible chemically programmed antibodies via specific conjugation of adaptor ligands to endogenous antibodies, in vivo. Pre-immuniztion with 2,4-dinitrophenyl (DNP), or the reactive hapten, 1,3-diketone (DK), or a novel reactive hapten, vinyl sulfone (VS) in mice, followed by oral administration of adaptor ligands composed of the hapten and biotin to the pre-immunized mice resulted in successful in vivo formation of the biotin-hapten-antibody complexes within 2h. Pharmacokinetic evaluations revealed that apparent serum concentrations of programmed antibodies were up to 144nM and that the serum half-lives reached up to 34.4h. These findings show promise for the future development of orally bioavailable drug-hapten-antibody complexes asa strategy to quickly and easily modulate immune targets for aggressive pathogens as well as cancer.

Synthesis of fused tricyclic peptides using a reprogrammed translation system and chemical modification.

Here we report a unique method of ribosomally synthesizing fused tricyclic peptides. Flexizyme-assisted in vitro translation of a linear peptide with the N-terminal chloroacetyl group and four downstream cysteines followed by the addition of 1,3,5-tris(bromomethyl)benzene results in selective production of the fused tricyclic peptide. This technology can be used for the ribosomal synthesis of fused tricyclic peptide libraries for the in vitro selection of bioactive peptides with tricyclic topology.

Helical peptide-foldamers having a chiral five-membered ring amino acid with two azido functional groups.

A chiral five-membered ring α,α-disubstituted α-amino acid (R,R)-Ac5c(dN3) having two azido functional groups has been designed and synthesized. The cyclic amino acid (R,R)-Ac5c(dN3) could be efficiently converted into several cyclic amino acids with various two 1,2,3-triazole functional groups. (R,R)-Ac5c(dN3) homochiral peptides (up to hexapeptide) and (R,R)-Ac5c(dN3)-containing l-Leu-based peptides were prepared, and their conversion of azido functional groups into triazole groups was completed. The preferred conformation of oligomers, before and after the "click reaction", together with the azido gauche effect of amino acid residues were studied using FT-IR absorption, CD, (1)H NMR, and X-ray crystallographic analysis. The cyclic amino acid (R,R)-Ac5c(dN3) could be used as a helical conformation controlling residue and also has a versatile functionalizing site in its oligopeptides.

Flavor assessment of a lactic fermented vinegar described in Japanese books from the Edo period (1603-1867).

Aims: Rice vinegar is a traditional fermented seasoning in Japan, and its production remained unchanged for over 800 years until the Edo period. However, based on the available information regarding rice vinegar production methods from this period and the results of reproduction experiments, we speculated that unlike the modern-day acetic fermented vinegar, rice vinegar produced during the Edo period was lactic fermented. Main methods: To verify this assumption, we analyzed the flavor components of Honcho, a lactic fermented product prepared using a method described in books, including "Honchoshokkan" from the Edo period, by capillary electrophoresis/time-of-flight mass spectrometry, high-performance liquid chromatography, gas chromatography mass spectrometry, and taste sensor analysis. Sensory evaluation was also conducted to assess validation as a seasoning. Results: Honcho contains 2 % lactic acid, which gives it its acidity, and small amounts of other nonvolatile acids, but significantly lower levels of acetic acid (0.188 ± 0.015 g/100 mL, p < 0.01). It contains more than double the free amino acids of Kurozu, a modern rice vinegar, and more glutamic acid. Boiling to remove ethanol from yeast fermentation concentrated the free amino acids 1.5 times. Sensor taste analysis showed Honcho had weaker acidity but stronger umami taste than commercial rice vinegar. The volatile compounds related to acetic acid fermentation were significantly different between Honcho and Kurozu. Boiling increased Honcho's acidity, mainly through non-volatile acids. Significance: These findings provide evidence to indicate that Honcho was an acidic seasoning for heat-cooking, which is uncommon in Japanese cuisine today and is mentioned in Edo period books. This seasoning contains many amino acids, implying that it adds umami flavor, not only the sourness of modern vinegar.

Development of a two-dimensional LC-MS/MS system for the determination of proline and 4-hydroxyproline enantiomers in biological and food samples.

A two-dimensional LC-MS/MS system has been developed for the enantioselective determination of proline (Pro), cis-4-hydroxyproline (cis-4-Hyp) and trans-4-hydroxyproline (trans-4-Hyp) in a variety of biological samples. The amino acids were pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the NBD-derivatives were separated by a reversed-phase column (Singularity RP18) as their D plus L mixtures in the first dimension. The collected target fractions were then introduced into the second dimension where the enantiomers were separated by a Pirkle-type enantioselective column (Singularity CSP-001S) and determined by a tandem mass spectrometer (Triple Quad™ 5500). The method was validated by the standard amino acids and also by human plasma, and sufficient results were obtained for the calibration, precision and accuracy. The method was applied to human plasma and urine, bivalve tissues and fermented food/beverages. D-Pro was widely found in the human physiological fluids, bivalves and several fermented products. Although trans-4-D-Hyp was not found in all the tested samples, cis-4-D-Hyp was present in human urine and tissues of the ark shell, and further studies focusing on the origin and physiological significance of these D-enantiomers are expected.

Facile and stabile linkages through tyrosine: bioconjugation strategies with the tyrosine-click reaction.

The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivatives in peptide and protein chemistry, we synthesized PTAD derivatives possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivatives and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective trifunctionalization of proteins is readily achieved. In particular cases, we noted that PTAD decomposition resulted in formation of a putative isocyanate byproduct that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addition of a small amount of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene glycol) chains onto proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen, whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated products. Finally, we applied the tyrosine click reaction to create a novel antibody-drug conjugate. For this purpose, we synthesized a PTAD derivative linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the potential of this reaction in creating protein conjugates with small molecules. The tyrosine click linkage demonstrated stability to extremes of pH, temperature, and exposure to human blood plasma indicating that this linkage is significantly more robust than maleimide-type linkages that are commonly employed in bioconjugations. These studies support the broad utility of this reaction in the chemoselective modification of small molecules, peptides, and proteins under mild aqueous conditions over a broad pH range using a wide variety of biologically acceptable buffers such as phosphate buffered saline (PBS) and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) buffers as well as others and mixed buffered compositions.

A neutralizable dimeric anti-thrombin aptamer with potent anticoagulant activity in mice.

Heparin-induced thrombocytopenia (HIT) is a complication caused by administration of the anticoagulant heparin. Although the number of patients with HIT has drastically increased because of coronavirus disease 2019 (COVID-19), the currently used thrombin inhibitors for HIT therapy do not have antidotes to arrest the severe bleeding that occurs as a side effect; therefore, establishment of safer treatments for HIT patients is imperative. Here, we devised a potent thrombin inhibitor based on bivalent aptamers with a higher safety profile via combination with the antidote. Using an anti-thrombin DNA aptamer M08s-1 as a promising anticoagulant, its homodimer and heterodimer with TBA29 linked by a conformationally flexible linker or a rigid duplex linker were designed. The dimerized M08s-1-based aptamers had about 100-fold increased binding affinity to human and mouse thrombin compared with the monomer counterparts. Administration of these bivalent aptamers into mice revealed that the anticoagulant activity of the dimers significantly surpassed that of an approved drug for HIT treatment, argatroban. Moreover, adding protamine sulfate as an antidote against the most potent bivalent aptamer completely suppressed the anticoagulant activity of the dimer. Emerging potent and neutralizable anticoagulant aptamers will be promising candidates for HIT treatment with a higher safety profile.

Formylbenzene diazonium hexafluorophosphate reagent for tyrosine-selective modification of proteins and the introduction of a bioorthogonal aldehyde.

4-Formylbenzene diazonium hexafluorophosphate (FBDP) is a novel bench-stable crystalline diazonium salt that reacts selectively with tyrosine to install a bioorthogonal aldehyde functionality. Model studies with N-acyl-tyrosine methylamide allowed us to identify conditions optimal for tyrosine ligation reactions with small peptides and proteins. FBDP-based conjugation was used for the facile introduction of small molecule tags, poly(ethylene glycol) chains (PEGylation), and functional small molecules onto model proteins and to label the surface of living cells.

Inner residues of macrothiolactone in autoinducer peptides I/IV circumvent spontaneous S-to-O acyl transfer to the upstream serine residue.

Autoinducing peptides I and IV (AIP-I/IV) are naturally occurring cyclic thiodepsipeptides (CTPs) bearing a Ser-Thr-Cys-Asp/Tyr (STC[D/Y]) tetrapeptide motif, where the Cys thiol (HSC) in the side-chain is linked to the Met C-terminal carboxylic acid (MCOOH) to form 5-residue macrothiolactones,-SC(D/Y)FIMCO-. We have recently reported that CTPs containing SX1CX2 motifs spontaneously undergo macrolactonization to yield cyclic depsipeptides (CDPs) by an unprecedented rapid S-to-O acyl transfer to the upstream Ser hydroxyl group. Interestingly, even though the STC[D/Y] motif in AIP-I/IV is a member of the SX1CX2 motif family, it maintains the CTP form. This suggests that AIP-I/IV have a structural or chemical motive for avoiding such an S-to-O acyl transfer, thus retaining the CTP form intact. Here we have used genetic code reprogramming to ribosomally synthesize various AIP-I analogs and studied what the determinant is to control the formation of CTP vs. CDP products. The study revealed that a Gly substitution of the inner Asp/Tyr or Met residues in the thiolactone drastically alters the resistance to the promotion of the S-to-O acyl transfer, giving the corresponding CDP product. This suggests that the steric hindrances originating from the α-substituted sidechain in these two amino acids in the AIP-I/IV thiolactone likely play a critical role in controlling the resistance against macrolactone rearrangement to the upstream Ser residue.

Conformational studies on peptides containing α,α-disubstituted α-amino acids: chiral cyclic α,α-disubstituted α-amino acid as an α-helical inducer.

Four types of α,α-disubstituted amino acids {i.e., α-aminoisobutyric acid (Aib), 1-aminocyclopentanecarboxylic acid (Ac(5)c), (3S,4S)-1-amino-(3,4-dimethoxy)cyclopentanecarboxylic acid [(S,S)-Ac(5)c(dOM)] and its enantiomer (R,R)-Ac(5)c(dOM)} were introduced into l-leucine-based hexapeptides and nonapeptides. The dominant conformations of eight peptides: Cbz-(L-Leu-L-Leu-dAA)(2)-OMe [dAA = 1: Aib; 2: Ac(5)c; 3: (S,S)-Ac(5)c(dOM); 4: (R,R)-Ac(5)c(dOM)] and Boc-(L-Leu-L-Leu-dAA)(3)-OMe [dAA = 5: Aib; 6: Ac(5)c; 7: (S,S)-Ac(5)c(dOM); 8: (R,R)-Ac(5)c(dOM)], were investigated by IR, CD spectra and X-ray crystallographic analysis. The CD spectra revealed that Aib hexapeptide 1 and Ac(5)c hexapeptide 2 formed right-handed (P) 3(10)-helices, while Ac(5)c(dOM) hexapeptides 3 and 4 formed a mixture of (P) 3(10)- and α-helices. The Aib nonapeptide 5 formed a (P) 3(10)-helix, the Ac(5)c nonapeptide 6 formed a mixture of (P) 3(10)- and α-helices, and the Ac(5)c(dOM) nonapeptides 7 and 8 formed (P) α-helices. X-Ray crystallographic analysis revealed that the Aib hexapeptide 1 formed a (P) 3(10)-helix, while (S,S)-Ac(5)c(dOM) hexapeptide 3 formed a (P) α-helix. In addition, the Ac(5)c nonapeptide 6 and (R,R)-Ac(5)c(dOM) nonapeptide 8 formed (P) α-helices. The Aib and achiral Ac(5)c residues have the propensity to form 3(10)-helices in short peptides, whereas the chiral Ac(5)c(dOM) residues have a penchant for forming α-helices.

Protective effects of Kurozu and Kurozu Moromimatsu on dextran sulfate sodium-induced experimental colitis.

BACKGROUND: Kurozu, a traditional Japanese black vinegar made from unpolished rice, and Kurozu Moromimatsu (Kurozu-M), its sediment, are both consumed in Japan as health foods or supplements. However, it is not known whether they have anti-colitis activity. AIMS: We examined the protective effects of Kurozu and Kurozu-M in an animal model of dextran sulfate sodium (DSS)-induced colitis. METHODS: DSS-induced colitis was induced in C57 black 6 mice by orally administering 3.5% DSS solution for 12 days. The control group received basal CE-2 diet (n = 10), the Kurozu group received CE-2 containing Kurozu (n = 10), the Kurozu-M group received CE-2 containing Kurozu-M (n = 10), and the acetic acid group received CE-2 containing acetic acid (n = 10), starting a week before DSS administration. Changes of body weight and bloody stool frequency were monitored. At 12 days after DSS administration, mice were killed for pathological examination and measurement of nitrotyrosine levels in rectal tissues. RESULTS: Kurozu significantly inhibited body weight loss during 6-12 days after DSS administration and reduced bloody stool frequency during 2-12 days, and also significantly decreased nitrotyrosine levels at 12 days, compared to the control group. Kurozu-M significantly inhibited body weight loss during 6-8 days after DSS administration and reduced bloody stool frequency during 2-12 days, but tissue nitrotyrosine level was not significantly different from the control. Acetic acid had no ameliorating effect on DSS-induced colitis compared to the control group. CONCLUSIONS: Kurozu and Kurozu-M have protective effects against DSS-induced colitis. Kurozu has anti-oxidative and anti-nitration activity.