Intense impact of IL-1ß expressing inflammatory macrophages in acute aortic dissection.

There is no treatment for acute aortic dissection (AAD) targeting inflammatory cells. We aimed to identify the new therapeutic targets associated with inflammatory cells. We characterized the specific distribution of myeloid cells of both human type A AAD samples and a murine AAD model generated using angiotensin II (ANGII) and ß-aminopropionitrile (BAPN) by single-cell RNA sequencing (scRNA-seq). We also examined the effect of an anti-interleukin-1ß (IL-1ß) antibody in the murine AAD model. IL1B+ inflammatory macrophages and classical monocytes were increased in human AAD samples. Trajectory analysis demonstrated that IL1B+ inflammatory macrophages differentiated from S100A8/9/12+ classical monocytes uniquely observed in the aorta of AAD. We found increased infiltration of neutrophils and monocytes with the expression of inflammatory cytokines in the aorta and accumulation of inflammatory macrophages before the onset of macroscopic AAD in the murine AAD model. In blocking experiments using an anti-IL-1ß antibody, it improved survival of murine AAD model by preventing elastin degradation. We observed the accumulation of inflammatory macrophages expressing IL-1ß in both human AAD samples and in a murine AAD model. Anti-IL-1ß antibody could improve the mortality rate in mice, suggesting that it may be a treatment option for AAD.

Chronic stress alters lipid mediator profiles associated with immune-related gene expressions and cell compositions in mouse bone marrow and spleen.

Despite the importance of lipid mediators in stress and depression and their link to inflammation, the influence of stress on these mediators and their role in inflammation is not fully understood. This study used RNA-seq, LC-MS/MS, and flow cytometry analyses in a mouse model subjected to chronic social defeat stress to explore the effects of acute and chronic stress on lipid mediators, gene expression, and cell population in the bone marrow and spleen. In the bone marrow, chronic stress induced a sustained transition from lymphoid to myeloid cells, accompanied by corresponding changes in gene expression. This change was associated with decreased levels of 15-deoxy-d12,14-prostaglandin J2, a lipid mediator that inhibits inflammation. In the spleen, chronic stress also induced a lymphoid-to-myeloid transition, albeit transiently, alongside gene expression changes indicative of extramedullary hematopoiesis. These changes were linked to lower levels of 12-HEPE and resolvins, both critical for inhibiting and resolving inflammation. Our findings highlight the significant role of anti-inflammatory and pro-resolving lipid mediators in the immune responses induced by chronic stress in the bone marrow and spleen. This study paves the way for understanding how these lipid mediators contribute to the immune mechanisms of stress and depression.

N-Acetylcysteine Suppresses Microglial Inflammation and Induces Mortality Dose-Dependently via Tumor Necrosis Factor-α Signaling.

N-acetylcysteine (NAC) is an antioxidant that prevents tumor necrosis factor (TNF)-α-induced cell death, but it also acts as a pro-oxidant, promoting reactive oxygen species independent apoptosis. Although there is plausible preclinical evidence for the use of NAC in the treatment of psychiatric disorders, deleterious side effects are still of concern. Microglia, key innate immune cells in the brain, play an important role in inflammation in psychiatric disorders. This study aimed to investigate the beneficial and deleterious effects of NAC on microglia and stress-induced behavior abnormalities in mice, and its association with microglial TNF-α and nitric oxide (NO) production. The microglial cell line MG6 was stimulated by Escherichia coli lipopolysaccharide (LPS) using NAC at varying concentrations for 24 h. NAC inhibited LPS-induced TNF-α and NO synthesis, whereas high concentrations (≥30 mM) caused MG6 mortality. Intraperitoneal injections of NAC did not ameliorate stress-induced behavioral abnormalities in mice, but high-doses induced microglial mortality. Furthermore, NAC-induced mortality was alleviated in microglial TNF-α-deficient mice and human primary M2 microglia. Our findings provide ample evidence for the use of NAC as a modulating agent of inflammation in the brain. The risk of side effects from NAC on TNF-α remains unclear and merits further mechanistic investigations.

Rice peptide with amino acid substitution inhibits biofilm formation by Porphyromonas gingivalis and Fusobacterium nucleatum.

OBJECTIVE: Rice peptide has antibacterial properties that have been tested in planktonic bacterial culture. However, bacteria form biofilm at disease sites and are resistant to antibacterial agents. The aim of this study was to clarify the mechanisms of action of rice peptide and its amino acid substitution against periodontopathic bacteria and their antibiofilm effects. DESIGN: Porphyromonas gingivalis and Fusobacterium nucleatum were treated with AmyI-1-18 rice peptide or its arginine-substituted analog, G12R, under anaerobic conditions. The amount of biofilm was evaluated by crystal violet staining. The integrity of the bacteria cytoplasmic membrane was studied in a propidium iodide (PI) stain assay and transmission electron microscopy (TEM). RESULTS: Both AmyI-1-18 and G12R inhibited biofilm formation of P. gingivalis and F. nucleatum; in particular, G12R inhibited F. nucleatum at lower concentrations. However, neither peptide eradicated established biofilms significantly. According to the minimum inhibitory concentration and minimum bactericidal concentration against P. gingivalis, AmyI-1-18 has bacteriostatic properties and G12R has bactericidal activity, and both peptides showed bactericidal activity against F. nucleatum. PI staining and TEM analysis indicated that membrane disruption by G12R was enhanced, which suggests that the replacement amino acid reinforced the electostatic interaction between the peptide and bacteria by increase of cationic charge and α-helix content. CONCLUSIONS: Rice peptide inhibited biofilm formation of P. gingivalis and F. nucleatum, and bactericidal activity via membrane destruction was enhanced by amino acid substitution.

Crystal structure of rice defensin OsAFP1 and molecular insight into lipid-binding.

Defensins are antibacterial peptides that function in the innate immune system. OsAFP1, a defensin identified from Oryza sativa (rice), exhibits antimicrobial activity against rice pathogens. Intriguingly, OsAFP1 was also shown to demonstrate potent antifungal activity against the human pathogenic fungus Candida albicans by inducing apoptosis in target cells, suggesting that OsAFP1 represents a potential new antibiotic candidate; however, further analyses, particularly at the structural level, are required to elucidate the mechanistic underpinnings of OsAFP1 antifungal activity. Here, we determined the three-dimensional structure of OsAFP1 using X-ray crystallography. OsAFP1 features the cysteine-stabilized αß structure highly conserved in plant defensins and presents a dimeric structure that appears necessary for antifungal activity. Superimposition of the OsAFP1 structure with that of Nicotiana alata NaD1 complexed with phosphatidic acid indicated that the target molecule is likely trapped between the S2-S3 loops of each OsAFP1 dimer. In lipid-binding analyses performed using nitrocellulose membranes immobilized with various membrane lipid components, OsAFP1 was found to bind to phosphatidylinositols (PIPs) harboring phosphate groups, particularly PI(3)P. These results indicate that OsAFP1 exerts antifungal activity by binding to PI(3)P contained in the C. albicans cell membrane, thereby applying cellular stress and inducing apoptosis. Furthermore, the OsAFP1 structure and site-specific-mutation analyses revealed that Arg1, His2, Leu4, Arg9, and Phe10 play critical roles in OsAFP1 dimer formation. Thus, our study provides novel insights into the antifungal mechanism of OsAFP1.

Identification and characterization of multifunctional cationic peptides from enzymatic hydrolysates of soybean proteins.

In this study, we used the commercial soybean protein hydrolysate Hinute-DC6 as a novel starting material from which to purify and identify multifunctional cationic peptides. After fractionation, Hinute-DC6 was separated into 20 fractions with varying isoelectric points (pI) by ampholyte-free isoelectric focusing (autofocusing). Subsequently, we purified and identified the cationic peptides from fractions 19 and 20, which had pI values greater than 12, using reversed-phase high-performance liquid chromatography and matrix-assisted laser/desorption ionization-time-of-flight mass spectrometry. Of the 83 cationic peptides identified, 14 had high pI values and net charges greater than +2, and were chemically synthesized and assayed for various bioactivities, including hemolytic, antimicrobial, lipopolysaccharide (LPS)-neutralizing, and angiogenic activities. None of the 14 cationic peptides tested exhibited hemolytic activity toward mammalian red blood cells at concentrations up to 1000 µM. Five of the cationic peptides exhibited antimicrobial activities against at least one of four human-pathogenic microorganisms tested. In addition, in chromogenic LPS-neutralizing assays using Limulus amebocyte lysates, the 50% effective concentrations of these 14 peptides were between 0.069 and 5.2 µM. Tube-formation assays in human umbilical vein endothelial cells showed that each of the 14 cationic peptides exhibited significant angiogenic activities at 10 µM, with values similar to those of the positive control LL-37. Our results demonstrate that the 14 identified cationic peptides have multiple functions with negligible hemolytic activity. These data indicate that the cationic peptides isolated from Hinute-DC6 and fractions containing these cationic peptides have the potential to be used as multifunctional ingredients for healthcare applications.

Identification of cationic peptides derived from low protein rice by-products and evaluation of their multifunctional activities.

Low protein rice (LPR) by-products were used as a source of novel multifunctional cationic peptides. The LPR by-products were separated by ampholyte-free isoelectric focusing (autofocusing) into 20 fractions containing peptides with different isoelectric points (pIs). Subsequently, the antimicrobial activity of each fraction was evaluated against four pathogenic microorganisms. In addition, the cationic peptides from fractions exhibiting antimicrobial activity were purified using reversed-phase high-performance liquid chromatography and identified using matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. Of the 11 cationic peptides identified, five peptides with pI values greater than 9.31 and net charges greater than +2 were chemically synthesized for multiple functionalities, including antimicrobial, lipopolysaccharide (LPS)-neutralizing, and angiogenic activities. Among these five cationic peptides, only LPR-KRK, which had a net charge of +9, exhibited antimicrobial activity against three of the four pathogenic microorganisms tested. Chromogenic LPS-neutralizing assays using Limulus amebocyte lysate showed that the 50% effective concentrations of these five peptides were between 0.11 and 3.09 µM. Tube-formation assays using human umbilical vein endothelial cells showed that all five peptides exhibited significant angiogenic activity at 1 µM and 10 µM, while none exhibited hemolytic activity toward mammalian red blood cells at concentrations up to 500 µM. Our results demonstrate that these five cationic peptides exhibit multiple biological functionalities with little or no hemolytic activity. Thus, fractions containing cationic peptides obtained from LPR by-products have the potential to be used as dietary supplements and functional ingredients in food products.

Social defeat stress-specific increase in c-Fos expression in the extended amygdala in mice: Involvement of dopamine D1 receptor in the medial prefrontal cortex.

We recently reported that dopamine D1 receptor in the medial prefrontal cortex (mPFC) is activated by subthreshold social defeat stress and suppresses the induction of depressive-like behavior in mice. However, which mPFC projection(s) mediates this antidepressant-like effect remains poorly understood. Here we show that social defeat stress specifically increased c-Fos expression, a marker for neuronal activity, in distinct brain regions involved in emotional regulation, relative to novelty-induced exploration. Among these brain areas, D1 knockdown in the mPFC decreased social defeat stress-induced c-Fos expression in the interstitial nucleus of the posterior limb of the anterior commissure (IPAC), a subregion of the extended amygdala. Using retrograde adeno-associated virus vectors and transgenic mice expressing Cre recombinase under the D1 promoter, we also found that D1-expressing deep-layer pyramidal neurons in the mPFC send direct projections to the IPAC. These findings indicate that social defeat stress specifically activates neurons in distinct brain areas, among which the IPAC is regulated by dopamine D1 receptor in the mPFC perhaps through direct projections. Thus, this study provides hints toward identifying neural circuits that underlie antidepressant-like effects of stress-induced dopamine D1 receptor signaling in the mPFC.

Wound healing activity and mechanism of action of antimicrobial and lipopolysaccharide-neutralizing peptides from enzymatic hydrolysates of rice bran proteins.

In our previous study, we identified multifunctional cationic peptides from enzymatic hydrolysates of rice bran proteins (RBPs) that have antimicrobial and lipopolysaccharide-neutralizing activities. In this study, we investigated the potential of the peptides RBP-LRR, RBP-EKL, and RBP-SSF to promote proliferation, angiogenesis (tube formation), and migration in human umbilical vein endothelial cells (HUVECs). To determine mechanisms of wound healing actions, angiogenic and migration-promoting activities of these peptides were evaluated following pretreatments of HUVECs with specific inhibitors. In these experiments, the cationic peptides RBP-LRR, RBP-EKL, and RBP-SSF induced cell proliferation at low concentrations of 0.1 µM or 1 µM. Moreover, the three cationic peptides had angiogenic activities at concentrations more than 1 µM in tube formation assays, and their effects were similar to those of LL-37. Subsequent scratch migration assays exhibited that RBP-LRR, RBP-EKL, and RBP-SSF promote wound closure at optimum concentrations of 10, 10, and 0.1 µM, respectively. In further studies, we performed tube formation assays using HUVECs pretreated with SU5416, which inhibits vascular endothelial growth factor (VEGF) receptors, and suggested the possibility that the three cationic peptides induce angiogenesis by activating VEGF receptors. In corresponding scratch migration assays using HUVECs, pretreatment with the proliferation inhibitor mitomycin C did not alter the effects of RBP-LRR and RBP-EKL, and significant contribution to wound closure were mediated by cell migration regardless of proliferation rates. In contrast, RBP-SSF contributed to wound closure exclusively by promoting cell proliferation. The present data indicate that RBP-LRR, RBP-EKL, and RBP-SSF are candidates for use as wound healing agents.

Identification and characterization of multifunctional cationic peptides from traditional Japanese fermented soybean Natto extracts.

In this study, we investigated the lipopolysaccharide (LPS)-neutralizing and angiogenic activities of cationic peptides derived from the traditional Japanese fermented product Natto, which is made by fermenting cooked soybeans using Bacillus subtilis. Initially, we prepared 20 fractions of Natto extracts with various isoelectric points (pI's) using ampholyte-free isoelectric focusing (autofocusing). Cationic peptides were then purified from fractions 19 and 20, whose pH values were greater than 12, using reversed-phase high-performance liquid chromatography, and were identified using matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. Among the 13 identified cationic peptides, seven (KFNKYGR, FPFPRPPHQK, GQSSRPQDRHQK, QRFDQRSPQ, ERQFPFPRPPHQK, GEIPRPRPRPQHPE, and EQPRPIPFPRPQPR) had pI's greater than 9.5, positive net charges, and differing molecular weights. These peptides were then chemically synthesized and applied to chromogenic LPS-neutralizing assays using Limulus amebocyte lysates, and 50% effective (neutralizing) concentrations of 2.6-5.5 µM were demonstrated. In addition, tube formation assays in human umbilical vein endothelial cells revealed angiogenic activities for all but one (GEIPRPRPRPQHPE) of these seven cationic peptides, with increases in relative tube lengths of 23-31% in the presence of peptides at 10 µM. Subsequent experiments showed negligible hemolytic activity of these peptides at concentrations of up to 500 µM in mammalian red blood cells. Collectively, these data demonstrate that six cationic peptides from Natto extracts, with the exception of GEIPRPRPRPQHPE, have LPS-neutralizing and angiogenic activities but do not induce hemolysis.

Peptides from rice endosperm protein restrain periodontal bone loss in mouse model of periodontitis.

OBJECTIVE: Food-derived peptides have been reported to exhibit antibacterial activity against periodontal pathogenic bacteria. However, no effect has been shown on inflammation and bone resorption in periodontal pathology. The overall objective of the current study was to investigate how rice peptides influence biological defense mechanisms against periodontitis-induced inflammatory bone loss, and identify their novel functions as a potential anti-inflammatory drug. DESIGN: The expression of inflammatory and osteoclast-related molecules was examined in mouse macrophage-derived RAW 264.7 cell cultures using qPCR. Subsequently, the effect of these peptides on inflammatory bone loss in mouse periodontitis was examined using a mouse model of tooth ligation. Briefly, periodontal bone loss was induced for 7 days in mice by ligating the maxillary second molar and leaving the contralateral tooth un-ligated (baseline control). The mice were microinjected daily with the peptide in the gingiva until the day before euthanization. One week after the ligation, TRAP-positive multinucleated cells (MNCs) were enumerated from five random coronal sections of the ligated sites in each mouse. RESULTS: Rice peptides REP9 and REP11 significantly inhibited transcription activity of inflammatory and osteoclast-related molecules. Local treatment with the rice peptides, in mice subjected to ligature-induced periodontitis, inhibited inflammatory bone loss, explaining the decreased numbers of osteoclasts in bone tissue sections. CONCLUSION: Therefore, these data suggested that the rice peptides possess a protective effect against periodontitis.

Cationic peptides from enzymatic hydrolysates of soybean proteins exhibit LPS-neutralizing and angiogenic activities.

In this study, we prepared fractions containing multifunctional cationic peptides by separating the commercial soybean protein hydrolysate Hinute-AM into 20 fractions. These fractions contained peptides with various isoelectric points (pI), as indicated by ampholyte-free isoelectric focusing (autofocusing). Thus, we purified and identified the cationic peptides from fractions 19 and 20, which had pH values greater than 10, using reversed-phase high-performance liquid chromatography and matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. Among 19 identified cationic peptides, NKNAKPPSPR, PGKKNAIV, KSGPGMSPR, NVSKPPRVV, RKVGAGGRKPLG, and LPCVIGGVPKRV had high pI values and were included as chemically synthesized peptides in assays of various functions, including lipopolysaccharide (LPS)-neutralizing and angiogenic activities. Chromogenic LPS-neutralizing assays using Limulus amebocyte lysates showed that 50% effective concentrations of these six peptides were between 1.63 and 2.65 µM, and were higher than that (0.12 µM) of polymyxin B. Moreover, in tube-formation assays in human umbilical vein endothelial cells, all of the six cationic peptides except LPCVIGGVPKRV exhibited angiogenic activities similar to those of the positive control LL-37. In addition, the six identified cationic peptides had no hemolytic activity at concentrations up to 500 µM in mammalian red blood cells. Our results demonstrate that five of the identified cationic peptides, excluding LPCVIGGVPKRV, have multiple functions and little or no hemolytic activity. These data indicate that fractions containing cationic peptides from Hinute-AM have the potential to be used as dietary supplements and functional ingredients in food products.

[Two-staged Operation for Bilateral Giant Bullae Successfully Performed with Veno-venous Extracorporeal Membrane Oxygenation( V-V ECMO) for First Surgery].

57-year-old man with bilateral giant lung bullae was admitted to our hospital for dyspnea on exertion as his chief complaint. The size and shape of right lung bullae were bigger and more complicated than those of left lung bullae. He underwent 2-staged bullectomy by video-assisted thoracoscopic surgery (VATS). Left lung bullectomy was expected to be more safely done, and was performed with veno-venous extracorporeal membrane oxygenation( V-V ECMO) prior to right side without any complications. The right lung bullectomy was performed 6 months after 1st operation without V-V ECMO. These reoperations were necessary due to postoperative air leak, however, his respiratory condition was well maintained by his left lung which had already been remarkably improved.

The PBII gene of the human salivary proline-rich protein P-B produces another protein, Q504X8, with an opiorphin homolog, QRGPR.

OBJECTIVES: The NCBI gene database and human-transcriptome database for alternative splicing were used to determine the expression of mRNAs for P-B (SMR3B) and variant form of P-B. The translational product from the former mRNA was identified as the protein named P-B, whereas that from the latter has not yet been elucidated. In the present study, we investigated the expression of P-B and its variant form at the protein level. DESIGN: To identify the variant protein of P-B, (1) cationic proteins with a higher isoelectric point in human pooled whole saliva were purified by a two dimensional liquid chromatography; (2) the peptide fragments generated from the in-solution of all proteins digested with trypsin separated and analyzed by MALDI-TOF-MS; and (3) the presence or absence of P-B in individual saliva was examined by 15% SDS-PAGE. RESULTS: The peptide sequences (I37PPPYSCTPNMNNCSR52, C53HHHHKRHHYPCNYCFCYPK72, R59HHYPCNYCFCYPK72 and H60HYPCNYCFCYPK72) present in the variant protein of P-B were identified. The peptide sequence (G6PYPPGPLAPPQPFGPGFVPPPPPPPYGPGR36) in P-B (or the variant) and sequence (I37PPPPPAPYGPGIFPPPPPQP57) in P-B were identified. The sum of the sequences identified indicated a 91.23% sequence identity for P-B and 79.76% for the variant. There were cases in which P-B existed in individual saliva, but there were cases in which it did not exist in individual saliva. CONCLUSIONS: The variant protein is produced by excising a non-canonical intron (CC-AC pair) from the 3'-noncoding sequence of the PBII gene. Both P-B and the variant are subject to proteolysis in the oral cavity.

Identification and characterization of multifunctional cationic and amphipathic peptides from soybean proteins.

In this study, we identified and chemically synthesized three cationic and amphipathic peptides (Glycinin-17, BCAS-16, and BCBS-11) from soybean proteins. These peptides had high isoelectric points, high positive net charges, and included multiple hydrophobic amino acids. Subsequently, we identified multiple functions of these peptides, including antimicrobial, lipopolysaccharide-neutralizing, and angiogenic activities, and examined their cytotoxic activities against mammalian red blood cells. Glycinin-17, BCAS-16, and BCBS-11 exhibited antimicrobial activity against Porphyromonas gingivalis and Candida albicans whereas Glycinin-17 did not possess antimicrobial effects on Propionibacterium acnes and Streptococcus mutans. Membrane-depolarization assays and flow cytometric analyses showed that the antimicrobial properties of Glycinin-17, BCAS-16, and BCBS-11 against P. gingivalis, P. acnes, and S. mutans were dependent on membrane-disrupting potential. In contrast, major antimicrobial activities of these peptides against C. albicans were dependent on interactions with targets other than cell membranes. Furthermore, chromogenic Limulus amebocyte lysate assays showed that 50% effective concentrations (EC50 , 0.12-0.31 µM) of these three peptides neutralize LPS with similar potency (EC50 : 0.11 µM) to that of polymyxin B. Moreover, tube-formation assays in human umbilical vein endothelial cells showed similar angiogenic activities of the three peptides as that following treatment with LL-37. Although BCAS-16 exhibited hemolytic activity, the rate of hemolysis for Glycinin-17 and BCBS-11 in the presence of 500-µM Glycinin-17 and BCBS-11 was less than 2%. These results demonstrate that cationic and amphipathic peptides from soybean proteins, particularly Glycinin-17 and BCBS-11, have potential as multifunctional ingredients for healthcare applications.

Effects of arginine and leucine substitutions on anti-endotoxic activities and mechanisms of action of cationic and amphipathic antimicrobial octadecapeptide from rice α-amylase.

Previously, we showed that the antimicrobial cationic and amphipathic octadecapeptide AmyI-1-18 from rice α-amylase (AmyI-1) inhibited the endotoxic activity of lipopolysaccharide (LPS) from Escherichia coli. In addition, we demonstrated that several AmyI-1-18 analogs containing arginine or leucine substitutions, which were designed on the basis of the helical wheel projection of AmyI-1-18, exhibited higher antimicrobial activity against human pathogenic microorganisms than AmyI-1-18. In the present study, anti-inflammatory (anti-endotoxic) activities of five AmyI-1-18 analogs containing arginine or leucine substitutions were investigated. Two single arginine-substituted and two single leucine-substituted AmyI-1-18 analogs inhibited the production of LPS-induced nitric oxide in mouse macrophages (RAW264) more effectively than AmyI-1-18. These data indicate that enhanced cationic and hydrophobic properties of AmyI-1-18 are associated with improved anti-endotoxic activity. In subsequent chromogenic Limulus amebocyte lysate assays, 50% inhibitory concentrations (IC50 ) of the three AmyI-1-18 analogs (G12R, D15R, and E9L) were 0.11-0.13 µm, indicating higher anti-endotoxic activity than that of AmyI-1-18 (IC50, 0.22 µm), and specific LPS binding activity. In agreement, surface plasmon resonance analyses confirmed direct LPS binding of three AmyI-1-18 analogs. In addition, AmyI-1-18 analogs exhibited little or no cytotoxic activity against RAW264 cells, indicating that enhancements of anti-inflammatory and LPS-neutralizing activities following replacement of arginine or leucine did not result in significant increases in cytotoxicity. This study shows that the arginine-substituted and leucine-substituted AmyI-1-18 analogs with improved anti-endotoxic and antimicrobial activities have clinical potential as dual-function host defense agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Rice Bran Protein as a Potent Source of Antimelanogenic Peptides with Tyrosinase Inhibitory Activity.

Rice (Oryza sativa) is consumed as a staple food globally, and rice bran, the byproduct, is an unused biomass that is ultimately discarded as waste. Thus, in the present study, a technique for producing tyrosinase inhibitory peptides from rice bran protein (RBP) was developed. Simultaneous treatment of RBP with chymotrypsin and trypsin produced numerous peptides. Subsequently, six tyrosinase inhibitory peptides were isolated from the hydrolysate fractions in a multistep purification protocol, and their amino acid sequences were determined. Three of these peptides had a C-terminal tyrosine residue and exhibited significant inhibitory effects against tyrosinase-mediated monophenolase reactions. Furthermore, peptide CT-2 (Leu-Gln-Pro-Ser-His-Tyr) potently inhibited melanogenesis in mouse B16 melanoma cells without causing cytotoxicity, suggesting the potential of CT-2 as an agent for melanin-related skin disorder treatment. The present data indicate that RBP is a potent source of tyrosinase inhibitory peptides and that simultaneous treatment of RBP with chymotrypsin and trypsin efficiently produces these peptides.

Inhibitory effects of a novel cationic dodecapeptide [CL(14-25)] derived from cyanate lyase of rice on endotoxic activities of LPSs from Escherichia coli and periodontopathic Aggregatibacter actinomycetemcomitans.

OBJECTIVE: CL(14-25), a dodecapeptide of cyanate lyase from rice, is a novel cationic α-helical antimicrobial peptide. In this study, we examined inhibitory ability of CL(14-25) against endotoxic activities of lipopolysaccharides (LPSs) from Escherichia coli and periodontal pathogenic Aggregatibacter actinomycetemcomitans. METHODS: Endotoxin-neutralizing activity of CL(14-25) was evaluated by inhibition to induction of cytokine and nitric oxide in human aortic endothelial cells (HAECs) and RAW264 mouse macrophage cells, respectively. Protective effect of CL(14-25) was determined in mice against lethal toxicity of LPS. RESULTS: IL-6 in HAECs was induced by stimulation with LPS preparations of A. actinomycetemcomitans and E. coli tested in this study, and addition of CL(14-25) to the medium caused inhibition of their induction in a dose-dependent manner. CL(14-25) inhibited NO induction in RAW264 cells by a smooth type LPS of E. coli O55:B5 and an Rc type LPS of E. coli J5 as well as lipid A of E. coli R515 in a dose-dependent manner. Simultaneous injection of E. coli O55:B5 LPS and CL(14-25) in BALB/c mice resulted in prevention of lethal toxicity of the former. The results of a Limulus amebocyte lysate assay and surface plasmon resonance analysis of interaction between CL(14-25) and E. coli LPS or lipid A showed that CL(14-25) specifically binds to a lipid A moiety of LPS. CONCLUSION: The results of present study suggest that CL(14-25) has a potential to be used as a nutraceutical agent for periodontal therapy.

Effect of a mouthrinse containing rice peptide CL(14-25) on early dental plaque regrowth: a randomized crossover pilot study.

BACKGROUND: We aimed to evaluate clinically the effect of mouthrinse containing a rice peptide on early dental plaque regrowth. METHODS: The study was designed as a double-masked, two-group crossover randomized pilot trial, involving 10 periodontally healthy volunteers. After receiving a professional tooth cleaning at baseline, over the next 3 days each participant refrained from all oral hygiene measures and had two daily rinses with 20 ml of the test mouthrinse containing 0.4 % rice peptide CL(14-25) or placebo rinse. At the end of each experimental period, plaque score was assessed using the modified Volpe's method, and the participants filled out a questionnaire. Each participant underwent a 7-day washout period followed by a second allocation. The plaque score was the primary outcome of the study and participant perception was the secondary outcome. RESULTS: No adverse effects were observed in the participants during the study. Clinically, the mean plaque score of the examined teeth was significantly lower in the test group (2.44 ± 0.74, CI: 1.91-2.96) than the placebo group (2.65 ± 0.63, CI: 2.20-3.10) (P < 0.05). When analyzed according to the type of teeth, a significantly lower score of the premolars/molars was observed in the test group (2.39 ± 0.68, CI: 2.08-2.71) than that in the placebo group (2.66 ± 0.58, CI: 2.39-2.93) (P < 0.05). CONCLUSIONS: The mouthrinse containing 0.4 % rice peptide CL(14-25) was effective in reducing the early regrowth of dental plaque. However, clinical relevance of this efficacy needs to be validated in a future large-scale study. TRIAL REGISTRATION: UMIN Clinical Trials Registry (UMIN-CTR) R000014000. Date of formal registration: November 1, 2013.

Contribution of cationic amino acids toward the inhibition of Arg-specific cysteine proteinase (Arg-gingipain) by the antimicrobial dodecapeptide, CL(14-25), from rice protein.

CL(14-25), a dodecapeptide, exhibits antimicrobial activity against Porphyromonas gingivalis with the 50% growth-inhibitory concentration (IC50 ) value of 145 µM, and arginine-specific gingipain (Rgp)-inhibitory activity. Kinetic analysis revealed that CL(14-25) is a mixed-type inhibitor, with inhibition constants (Ki and Ki ' values) of 1.4 × 10(-6) M and 4.3 × 10(-6) M, respectively. To elucidate the contributions of four cationic amino acid residues at the N- and C-termini of CL(14-25) toward Rgp-inhibitory activity, we investigated the Rgp-inhibitory activities of truncated and alanine-substituted analogs of CL(14-25). Rgp-inhibitory activities significantly decreased by truncated analogs, CL(15-25) and CL(16-25), whereas those of CL(14-24) and CL(14-23) were almost as high as that of CL(14-25). Rgp-inhibitory activities of alanine-substituted analogs, CL(R14A) and CL(R14A, R15A) also significantly decreased, whereas those of CL(K25A) and CL(R24A, K25A) were higher than that of CL(14-25). These results suggest that the arginine residue at position 15 substantially contributes to the Rgp-inhibitory activity and that the arginine residue at position 14 plays important roles in exerting Rgp-inhibitory activity. In this study, we demonstrated that CL(K25A) was a potent, dual function, peptide inhibitor candidate, exhibiting Rgp-inhibitory activity with Ki and Ki ' of 9.6 × 10(-7) M and 1.9 × 10(-6) M, respectively, and antimicrobial activity against P. gingivalis with an IC50 value of 51 µM.