Single-Cell RNA Sequencing Reveals an Immune Landscape of CD4+ T Cells in Coronary Culprit Plaques With Acute Coronary Syndrome in Humans-Brief Report.

BACKGROUND: Acute coronary syndrome (ACS) involves plaque-related thrombosis, causing primary ischemic cardiomyopathy or lethal arrhythmia. We previously demonstrated a unique immune landscape of myeloid cells in the culprit plaques causing ACS by using single-cell RNA sequencing. Here, we aimed to characterize T cells in a single-cell level, assess clonal expansion of T cells, and find a therapeutic target to prevent ACS. METHODS: We obtained the culprit lesion plaques from 4 patients with chronic coronary syndrome (chronic coronary syndrome plaques) and the culprit lesion plaques from 3 patients with ACS (ACS plaques) who were candidates for percutaneous coronary intervention with directional coronary atherectomy. Live CD45+ immune cells were sorted from each pooled plaque samples and applied to the 10× platform for single-cell RNA sequencing analysis. We also extracted RNA from other 3 ACS plaque samples and conducted unbiased TCR (T-cell receptor) repertoire analysis. RESULTS: CD4+ T cells were divided into 5 distinct clusters: effector, naive, cytotoxic, CCR7+ (C-C chemokine receptor type 7) central memory, and FOXP3 (forkhead box P3)+ regulatory CD4+ T cells. The proportion of central memory CD4+ T cells was higher in the ACS plaques. Correspondingly, dendritic cells also tended to express more HLAs (human leukocyte antigens) and costimulatory molecules in the ACS plaques. The velocity analysis suggested the differentiation flow from central memory CD4+ T cells into effector CD4+ T cells and that from naive CD4+ T cells into central memory CD4+ T cells in the ACS plaques, which were not observed in the chronic coronary syndrome plaques. The bulk repertoire analysis revealed clonal expansion of TCRs in each patient with ACS and suggested that several peptides in the ACS plaques work as antigens and induced clonal expansion of CD4+ T cells. CONCLUSIONS: For the first time, we revealed single cell-level characteristics of CD4+ T cells in patients with ACS. CD4+ T cells could be therapeutic targets of ACS. REGISTRATION: URL: https://upload.umin.ac.jp/cgi-open-bin/icdr_e/ctr_view.cgi?recptno=R000046521; Unique identifier: UMIN000040747.

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.

Microglial subpopulations with distinct transcriptome signatures vary across brain regions in the resting mouse brain.

Microglia are crucial for tissue homeostasis and its disturbance. However, microglial heterogeneity and its relationship with microglial activation in physiological conditions remain elusive. Using single-cell RNA sequencing, we identified microglial subpopulations with distinct transcriptome signatures in the resting brain. The distribution of two major, continuous subpopulations varied across brain regions, especially between cerebral cortices and the hypothalamus. Lipopolysaccharide and chronic social defeat stress, both of which involve the innate immune receptor TLR4, upregulate the marker genes of selective microglial subpopulations. These findings suggest that microglial subpopulations contribute to the heterogeneity of microglial transcriptome and responsiveness within and across brain regions.

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.

Memantine ameliorates the impairment of social behaviors induced by a single social defeat stress as juveniles.

Clinically, juveniles are more sensitive to stress than adults, and exposure to stress as juveniles prolongs psychiatric symptoms and causes treatment resistance. However, the efficacy of antidepressants for juveniles with psychiatric disorders is unknown. In the present study, we investigated whether the expression or development of impaired social behavior was attenuated by memantine, a non-competitive NMDA receptor antagonist. In addition, we clarified the molecular mechanisms related to intracellular signal transduction through NMDA receptors and the ameliorating effect of memantine in mice with impaired social behavior. Acute administration of memantine before the social interaction test, but not before exposure to social defeat stress, attenuated social behavioral impairment. A single social defeat stress increased the phosphorylation of NMDA receptor subunit GluN2A and extracellular-signal-related kinase 1/2 (ERK1/2). Memantine inhibited the increase of phosphorylated GluN2A and ERK1/2 resulting from social interaction behavior. In both GluN2A deficient and pharmacological blockaded mice, social behavioral impairment was not observed in the social interaction test through regulation of ERK1/2 phosphorylation. These findings suggest that memantine ameliorates social behavioral impairment in mice exposed to a single social defeat stress as juveniles by regulating the NMDA receptor and subsequent ERK1/2 signaling activation. Memantine may constitute a novel therapeutic drug for stress-related psychiatric disorders in juveniles with adverse juvenile experiences.

Selective concentration of antimicrobial peptides to heat-treated porous silica gel using adsorption/desorption.

Heat-treated porous silica gel (HT silica gel) previously developed by our group has selectively adsorbed cationic peptides at a pH of 7. Therefore, we focused on the use of antimicrobial peptides (AMPs) as bioactive peptides (BPs). First, 32 AMPs and 32 randomly designed peptides were generated using Fmoc solid synthesis, and their adsorption ratio to HT-silica gel was investigated. Thirty two AMPs showed a relatively higher adsorption ratio of 58.8% compared to that of randomly designed peptides, which was 35.3%. Desorption conditions were investigated using Amyl-1-18 antimicrobial peptides. Next, pepsin hydrolysate from rice endosperm protein (REP) powder was prepared by ourselves. The REP hydrolysate containing dry matter (7.5 mg) was applied to the adsorption/desorption (AD) procedure using HT silica gel to obtain 1.6 mg of AD hydrolysate. When the two hydrolysates were subjected to mass spectrometry, 305 concentrated peptides were obtained. In total, 26 peptides with high content and high enrichment ratios were listed and synthesized. When the antimicrobial activity of these 26 peptides was evaluated using Cutibacterium acnes, five peptides consisting of 12-27 amino acids were identified as novel AMPs. Two of these peptides, which were derived from rice glutelin, showed antimicrobial activity against all four microbes, including Porphyromonas gingivalis, Escherichia coli, and Streptococcus mutans. In the present study, we showed that AMPs could be easily enriched from protein hydrolysate using HT silica gel. The adsorption/desorption procedure using HT silica gel was confirmed to be a useful tool for convenient BP separation.

Early postnatal inhibition of GLAST causes abnormalities of psychobehaviors and neuronal morphology in adult mice.

The importance of glutamate transporters in learning, memory, and emotion remains poorly understood; hence, in the present study, we investigated whether deficiency of pharmacological GLAST in neurodevelopmental processes affects cognitive and/or emotional behaviors in mice. The mice were injected with a glutamate transporter inhibitor, dl-threo-ß-benzyloxyaspartate (dl-TBOA), during the early postnatal period. At 8 weeks of age, they showed impairments in cognitive or emotional behaviors; dysfunction of glutamatergic neurotransmission (increased expressions of GLAST, GLT-1, or GFAP protein, and decreased ability of glutamate release) in the cortex or hippocampus; morphological changes (decreased cell size in the cortex and thickness of the pyramidal neuronal layer of the CA1 area in the hippocampus). Such behavioral and morphological changes were not observed in adult mice injected with dl-TBOA. These results suggest that GLAST plays an important role in the regulation of cognitive and emotional behaviors. Early postnatal glutamatergic facilitation by GLAST dysfunction leads to cognitive and emotional abnormalities due to neurodevelopmental abnormalities such as morphological changes.

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.

Rice Endosperm Protein Administration to Juvenile Mice Regulates Gut Microbiota and Suppresses the Development of High-Fat Diet-Induced Obesity and Related Disorders in Adulthood.

Obesity and related disorders, which are increasing in adults worldwide, are closely linked to childhood diet and are associated with chronic inflammation. Rice endosperm protein (REP) intake during adulthood has been reported to improve lipid metabolism and suppress the progression of diabetic kidney disease in animal models. However, the effects of REP intake during childhood on adulthood health are unclear. Therefore, we used a mouse model to experimentally investigate the preconditioning effects of REP intake during childhood on the development of obesity and related disorders in adulthood. Male C57BL/6J mice were pair-fed a normal-fat diet containing casein or REP during the juvenile period and then a high-fat diet (HFD) containing casein or REP during adulthood. Mice fed REP during the juvenile period showed better body weight, blood pressure, serum lipid profiles, lipopolysaccharide (LPS)-binding protein levels, and glucose tolerance in adulthood than those fed casein during the juvenile period. HFD-induced renal tubulo-glomerular alterations and hepatic microvesicular steatosis were less evident in REP-fed mice than in casein-fed ones. REP intake during the juvenile period improved HFD-induced dysbiosis (i.e., Escherichia genus proliferation and reduced gut microbiota diversity), thereby suppressing endotoxin-related chronic inflammation. Indeed, REP-derived peptides showed antibacterial activity against Escherichia coli, a major producer of LPS. In conclusion, REP supplementation during the juvenile period may regulate the gut microbiota and thus suppress the development of obesity and related disorders in adulthood in mice.

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.

A peptide derived from rice inhibits alveolar bone resorption via suppression of inflammatory cytokine production.

BACKGROUND: Periodontitis is an inflammatory disease that results in alveolar bone resorption due to inflammatory cytokine production induced by bacterial antigens such as lipopolysaccharides (LPS). Here, the preventive effect of the Amyl-1-18 peptide derived from rice in an experimental model of periodontitis and the effect on the anti-inflammatory response were assessed. METHODS: Alveolar bone resorption, gene transcription of proinflammatory cytokines in the gingiva, and the endotoxin level in the oral cavity were evaluated after oral administration of the Amyl-1-18 peptide for 14 days using a ligature-induced periodontitis model in mice. Additionally, murine macrophages were incubated with LPS of Escherichia coli or Porphyromonas gingivalis in the presence of Amyl-1-18 to analyze the suppressive effects of Amyl-1-18 on the cell signaling pathways associated with proinflammatory cytokine production, including inflammasome activities. RESULTS: Oral administration of Amyl-1-18 suppressed alveolar bone resorption and gene transcription of interleukin (il)6 in the gingiva of the periodontitis model, and decreased endotoxin levels in the oral cavity, suggesting modulation of periodontal inflammation by inhibition of endotoxin activities in vivo. Also, Amyl-1-18 suppressed IL-6 production induced by LPS and recombinant IL-1ß in macrophages in vitro but had no effect on inflammasome activity. CONCLUSIONS: The Amyl-1-18 peptide from rice inhibited alveolar bone destruction in mouse periodontitis model via suppressing inflammatory cytokine production induced by LPS. It was suggested that Amyl-1-18 peptide has anti-inflammatory property against LPS, not only by neutralization of LPS and subsequent inhibition of nuclear factor-κB signaling but also by inhibition of the IL-1R-related signaling cascade.

Protein adsorption characteristics of nanoparticle-assembled hollow microspheres of hydroxyapatite and their composites with PLLA microporous membranes.

Nanoparticle-assembled hydroxyapatite (HA) hollow microspheres have a high surface area and are convenient to handle, owing to their characteristic structure. In this study we characterized the protein adsorption of HA hollow microspheres prepared from CaCl2 and K2HPO4 by a water-in-oil-in-water (W/O/W) emulsion method assisted by two surfactants: Span 80 and Tween 20. The HA hollow microspheres adsorbed bovine serum albumin, bovine γ-globulin, equine skeletal muscle myoglobin, and chicken egg white lysozyme in 10 mM sodium phosphate buffer (pH 6.8) in a Langmuir-type adsorption and desorbed the proteins in 800 mM sodium phosphate buffer (pH 6.8). The maximum adsorbed amounts of the HA hollow microspheres were 7.5-9.0 times higher than those of the microrods with a similar size range. The composite membranes of the HA microspheres and the poly(l-lactic acid) (PLLA) microporous membranes exhibited a high adsorption capacity for γ-globulin.

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.