Genetic regulation and manipulation of nicotine biosynthesis in tobacco: strategies to eliminate addictive alkaloids.

Tobacco (Nicotiana tabacum L.) is a widely cultivated crop of the genus Nicotiana. Due to the highly addictive nature of tobacco products, tobacco smoking remains the leading cause of preventable death and disease. There is therefore a critical need to develop tobacco varieties with reduced or non-addictive nicotine levels. Nicotine and related pyridine alkaloids biosynthesized in the roots of tobacco plants are transported to the leaves, where they are stored in vacuoles as a defense against predators. Jasmonate, a defense-related plant hormone, plays a crucial signaling role in activating transcriptional regulators that coordinate the expression of downstream metabolic and transport genes involved in nicotine production. In recent years, substantial progress has been made in molecular and genomics research, revealing many metabolic and regulatory genes involved in nicotine biosynthesis. These advances have enabled us to develop tobacco plants with low or ultra-low nicotine levels through various methodologies, such as mutational breeding, genetic engineering, and genome editing. We review the recent progress on genetic manipulation of nicotine production in tobacco, which serves as an excellent example of plant metabolic engineering with profound social implications.

Step-by-step instructions for detecting whirlpool sign in adnexal torsion.

Objective: Adnexal torsion is a common gynecological emergency whose prompt diagnosis is essential because a delay may lead to ovarian dysfunction. Although the whirlpool sign is reliable for diagnosing ovarian cyst torsion, technical difficulties hinder its use by sonographers. Here we developed a systematic approach to visualizing this sign by focusing on the fact that torsion arises from the space between the uterus and the pelvic wall. One must determine the origin of the torsion via transverse imaging of the uterus and follow the twisted ligaments to the ovarian cyst. Patients and Methods: Two women aged 56 (Case 1) and 28 years (Case 2) visited our hospital with lower abdominal pain. Transvaginal ultrasonography showed a 7-cm right ovarian cyst in Case 1 and a 5-cm cyst in the Douglas pouch in Case 2; normal bilateral ovaries and the whirlpool sign were detected in both cases. Under laparoscopic guidance in Cases 1 and 2, an ovarian cyst and a paraovarian cyst were confirmed and removed. Results: Our step-by-step method allowed us to identify the whirlpool sign and confirm adnexal torsion, leading to prompt surgery in both cases. Conclusion: Using a systematic procedure helps less experienced practitioners detect the whirlpool sign.

Identification of a regiospecific S-oxygenase for the production of marasmin in traditional medicinal plant Tulbaghia violacea.

Marasmin [S-(methylthiomethyl)-L-cysteine-4-oxide] is a pharmaceutically valuable sulfur-containing compound produced by the traditional medicinal plant, Tulbaghia violacea. Here, we report the identification of an S-oxygenase, TvMAS1, that produces marasmin from its corresponding sulfide, S-(methylthiomethyl)-L-cysteine. The amino acid sequence of TvMAS1 showed high sequence similarity to known flavin-containing S-oxygenating monooxygenases in plants. Recombinant TvMAS1 catalyzed regiospecific S-oxygenation at S4 of S-(methylthiomethyl)-L-cysteine to yield marasmin, with an apparent K m value of 0.55 mM. TvMAS1 mRNA accumulated with S-(methylthiomethyl)-L-cysteine and marasmin in various organs of T. violacea. Our findings suggest that TvMAS1 catalyzes the S-oxygenation reaction during the last step of marasmin biosynthesis in T. violacea.

Natural and induced variations in transcriptional regulator genes result in low-nicotine phenotypes in tobacco.

In tobacco, the homologous ETHYLENE RESPONSE FACTOR (ERF) transcription factors ERF199 and ERF189 coordinate the transcription of multiple metabolic genes involved in nicotine biosynthesis. Natural alleles at the NIC1 and NIC2 loci greatly affect alkaloid accumulation and overlap with ERF199 and ERF189 in the tobacco genome, respectively. In this study, we identified several low-nicotine tobacco varieties lacking ERF199 or ERF189 from a tobacco germplasm collection. We characterized the sequence of these new nic1 and nic2 alleles, as well as the previously defined alleles nic1-1 and nic2-1. Moreover, we examined the influence of different nic alleles on alkaloid contents and expression levels of genes related to nicotine biosynthesis. We also demonstrated that the deletion of a distal genomic region attenuates ERF199 expression, resulting in a moderately negative effect on the alkaloid phenotype. Our study provides new insights into the regulation of nicotine biosynthesis and novel genetic resources to breed low-nicotine tobacco.

Tandem Mass Spectrum Similarity-Based Network Analysis Using 13C-Labeled and Non-labeled Metabolome Data to Identify the Biosynthetic Pathway of the Blood Pressure-Lowering Asparagus Metabolite Asparaptine A.

The biosynthetic pathway of asparaptine, a naturally occurring inhibitor of angiotensin-converting enzyme (ACE) in vitro, is largely unknown in Asparagus officinalis. To determine which metabolites are involved in the pathway, we performed tandem mass spectrum similarity-based metabolome network analysis using 13C-labeled and non-labeled valine-fed asparagus calluses. We revealed that S-(2-carboxy-n-propyl)-cysteine as an intermediate and two new metabolites as asparaptine analogues, lysine- and histidine-type conjugates, are involved in the pathway. Asparaptine was therefore renamed asparaptine A (arginine type), and the two analogues were named asparaptines B (lysine type) and C (histidine type). Oral feeding of asparaptine A to a hypertensive mouse breed showed that this metabolite lowers both the blood pressure and heart rate within 2 h and the effect of asparaptine A wears off after 2 days. These results suggest that asparaptine A may not only have effects as an ACE inhibitor but also have ß-antagonistic effects.

Retrograde sulfur flow from glucosinolates to cysteine in Arabidopsis thaliana.

Specialized (secondary) metabolic pathways in plants have long been considered one-way routes of leading primary metabolite precursors to bioactive end products. Conversely, endogenous degradation of such "end" products in plant tissues has been observed following environmental stimuli, including nutrition stress. Therefore, it is of general interest whether specialized metabolites can be reintegrated into primary metabolism to recover the invested resources, especially in the case of nitrogen- or sulfur-rich compounds. Here, we demonstrate that endogenous glucosinolates (GLs), a class of sulfur-rich plant metabolites, are exploited as a sulfur source by the reallocation of sulfur atoms to primary metabolites such as cysteine in Arabidopsis thaliana Tracer experiments using 34S- or deuterium-labeled GLs depicted the catabolic processing of GL breakdown products in which sulfur is mobilized from the thioglucoside group in GL molecules, potentially accompanied by the release of the sulfate group. Moreover, we reveal that beta-glucosidases BGLU28 and BGLU30 are the major myrosinases that initiate sulfur reallocation by hydrolyzing particular GL species, conferring sulfur deficiency tolerance in A. thaliana, especially during early development. The results delineate the physiological function of GL as a sulfur reservoir, in addition to their well-known functions as defense chemicals. Overall, our findings demonstrate the bidirectional interaction between primary and specialized metabolism, which enhances our understanding of the underlying metabolic mechanisms via which plants adapt to their environments.

Reduction of formaldehyde emission from urea-formaldehyde resin with a small quantity of graphene oxide.

Graphene oxide (GO) has theoretically been identified as a candidate for adsorbing formaldehyde molecules. However, whether GO can actually serve as a scavenger for formaldehyde resin adhesives must be experimentally verified due to the complex interaction between GO and formaldehyde molecules in the presence of resin, the competition between the formaldehyde emission rate and its adsorption rate on the scavenger, and other complications. From the results from this study we experimentally demonstrate that GO synthesised by the improved Hummers' method is a powerful scavenger for a urea-formaldehyde (UF) resin. We investigate the effect of the added amount of GO on the formaldehyde emission from UF resin. The emission from the UF/GO composite resin is 0.22 ± 0.03 mg L-1, which is an 81.5% reduction compared to that of the control UF resin when adding 0.20 wt% GO into the UF resin. However, adding higher amounts of GO (more than 0.20 wt%) increases the formaldehyde emission and the emission approaches that of pure UF resin (1.19 ± 0.36 mg L-1). This is likely due to the more acidic pH of the composite, which may lead to a faster curing reaction of the UF resin and acceleration of the emission.

Circulating steroids and mood disorders in patients with polycystic ovary syndrome.

Aberrant androgen metabolism is a characteristic feature of polycystic ovary syndrome (PCOS). Various androgens as well as their precursors and metabolites can accumulate in the blood of PCOS patients. Although these steroids include neuroactive steroids, such as allopregnanolone and androstenedione (Δ4A), it remains unknown whether altered blood steroid levels contribute to the high risk of mood disorders in PCOS. In this study, we measured blood levels of 11 steroids in 25 PCOS patients using liquid chromatography-tandem mass spectrometry and chemiluminescent enzyme immunoassay, and assessed the psychological status of these patients using the Hospital Anxiety and Depression Scale (HADS) questionnaire. We also examined age and the degree of metabolic abnormalities of each patient. Steroid values of the patients were compared to our previous data from 31 eumenorrheic women. As a result, 20 patients exhibited aberrant blood levels of one or more of the 11 tested steroids. In most cases, Δ4A and allopregnanolone levels were within or close to the reference ranges. Levels of four steroids were negatively correlated with patients' age, while no correlation was observed between steroid values and metabolic conditions. Seven patients showed high HADS scores. HADS scores were correlated with blood Δ4A levels even after stratifying by body mass indexes, but not with the levels of other steroids or clinical data. These results indicate that the high frequency of anxiety and depression in PCOS patients cannot be ascribed to altered blood levels of a specific steroid, although there may be a weak association between circulating Δ4A levels and psychological conditions of the patients.

Recellularization of decellularized cancellous bone scaffolds using low-temperature cell seeding.

Artificial in vitro blood production has been presented by recent literature as a necessary and achievable aim. In order to obtain the required hematopoietic stem cells (HSCs) proliferation and differentiation for mature blood cell production, studies have been conducted on using either cytokine-rich conditions or co-culturing with other cells. Alternatively, three-dimensional (3D) cell culture environments (such as tissue scaffolds) have been shown to affect cell morphology, proliferation and differentiation. Therefore, we investigated decellularized cancellous bones (DCBs), which provide 3D structure and natural extracellular matrix, as a scaffold for preserving and growing HSC niches in vitro. Additionally, we optimized a cell seeding method using mesenchymal stem cells as supporting cells. We discovered that, although adhering only to the top of DCBs when seeded at 37 °C, mesenchymal stem cells adhered to the inside of the scaffold at 4 °C, indicating that the seeding temperature is important to control the adherence ability of stem cells. This, in turn, was revealed to be important for HSC cell seeding on 3D extracellular matrix, and provides the required cell methodology to use DCBs as a great scaffold for blood cell production.

Solution structure of the zinc finger domain of human RNF144A ubiquitin ligase.

RNF144A is involved in protein ubiquitination and functions as an ubiquitin-protein ligase (E3) via its RING finger domain (RNF144A RING). RNF144A is associated with degradation of heat-shock protein family A member 2 (HSPA2), which leads to the suppression of breast cancer cell proliferation. In this study, the solution structure of RNF144A RING was determined using nuclear magnetic resonance. Moreover, using a metallochromic indicator, we spectrophotometrically determined the stoichiometry of zinc ions and elucidated that RNF144A RING binds two zinc atoms. This structural analysis provided the position and range of the active site of RNF144A RING at the atomic level, which contributes to the creation of artificial RING fingers having the specific ubiquitin-conjugating enzyme (E2)-binding capability.

A simple scoring method to predict augmented renal clearance in haematologic malignancies.

WHAT IS KNOWN AND OBJECTIVE: Augmented renal clearance (ARC; hyperfiltration with over 130 mL/min/1.73 m2 of creatinine clearance (CLcr )) commonly occurs in critically ill patients. Recent reports indicate that ARC also occurs in haematologic malignancies. However, the risk factors for ARC in haematologic malignancies remain unknown, and there is no established method to predict ARC in haematologic malignancies. Our objective was to explore the risk factors for ARC retrospectively and develop a scoring method to predict ARC. METHODS: A single-centre, retrospective, observational cohort study was conducted at the Sendai Medical Center (Sendai, Japan); 133 patients (April 2017-March 2019) and 41 patients (April-November 2019) with haematopoietic tumours who were administered vancomycin were enrolled in the analysis and validation cohorts, respectively. To define ARC, we calculated the vancomycin serum concentration when CLcr  = 130 mL/min/1.73 m2 using a one-compartment model. Patients with ARC were defined as those whose actual concentration of vancomycin remained lower than the calculated concentration. Using the analysis cohort, we explored risk factors of ARC and developed a scoring method to predict ARC in haematologic malignancies. The reproducibility of the scoring system was demonstrated using the validation cohort. RESULTS AND DISCUSSION: Through multivariate analysis, young age (P < .001), leukaemia (P = .001) and low serum creatinine (P < .001) were identified as risk factors. According to this result, we established the ARC detection method: age ≤ 50 years = 3 points, 50 years < age ≤65 years = 1 point, leukaemia = 2 points, low SCr = 2 points; patients scoring ≥ 5 points represent the ARC high-risk group. Using this scoring system, we could detect ARC with a sensitivity and specificity of 60.0% and 89.7% in the analysis cohort and 90.0% and 90.9% in the validation cohort, respectively. WHAT IS NEW AND CONCLUSION: Our scoring method could predict ARC in haematologic malignancies and is useful as a simple screening tool for ARC.

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.

Transcriptome analysis of Pueraria candollei var. mirifica for gene discovery in the biosyntheses of isoflavones and miroestrol.

BACKGROUND: Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. RESULTS: Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein. CONCLUSIONS: Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica.

Endometrial preparation methods for frozen-thawed embryo transfer are associated with altered risks of hypertensive disorders of pregnancy, placenta accreta, and gestational diabetes mellitus.

STUDY QUESTION: What were the risks with regard to the pregnancy outcomes of patients who conceived by frozen-thawed embryo transfer (FET) during a hormone replacement cycle (HRC-FET)? SUMMARY ANSWER: The patients who conceived by HRC-FET had increased risks of hypertensive disorders of pregnancy (HDP) and placenta accreta and a reduced risk of gestational diabetes mellitus (GDM) in comparison to those who conceived by FET during a natural ovulatory cycle (NC-FET). WHAT IS KNOWN ALREADY: Previous studies have shown that pregnancy and live-birth rates after HRC-FET and NC-FET are comparable. Little has been clarified regarding the association between endometrium preparation and other pregnancy outcomes. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study of patients who conceived after HRC-FET and those who conceived after NC-FET was performed based on the Japanese assisted reproductive technology registry in 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: The pregnancy outcomes were compared between NC-FET (n = 29 760) and HRC-FET (n = 75 474) cycles. Multiple logistic regression analyses were performed to investigate the potential confounding factors. MAIN RESULTS AND THE ROLE OF CHANCE: The pregnancy rate (32.1% vs 36.1%) and the live birth rate among pregnancies (67.1% vs 71.9%) in HRC-FET cycles were significantly lower than those in NC-FET cycles. A multiple logistic regression analysis showed that pregnancies after HRC-FET had increased odds of HDPs [adjusted odds ratio, 1.43; 95% confidence interval (CI), 1.14-1.80] and placenta accreta (adjusted odds ratio, 6.91; 95% CI, 2.87-16.66) and decreased odds for GDM (adjusted odds ratio, 0.52; 95% CI, 0.40-0.68) in comparison to pregnancies after NC-FET. LIMITATIONS, REASONS FOR CAUTION: Our study was retrospective in nature, and some cases were excluded due to missing data. The implication of bias and residual confounding factors such as body mass index, alcohol consumption, and smoking habits should be considered in other observational studies. WIDER IMPLICATIONS OF THE FINDINGS: Pregnancies following HRC-FET are associated with higher risks of HDPs and placenta accreta and a lower risk of GDM. The association between the endometrium preparation method and obstetrical complication merits further attention. STUDY FUNDING/COMPETING INTEREST(S): No funding was obtained for this work. The authors declare no conflicts of interest in association with the present study. TRIAL REGISTRATION NUMBER: Not applicable.

S-Alk(en)ylcysteine sulfoxides in the genus Allium: proposed biosynthesis, chemical conversion, and bioactivities.

S-Alk(en)ylcysteine sulfoxides are sulfur-containing natural products characteristic of the genus Allium. Both the flavor and medicinal properties of Allium plants are attributed to a wide variety of sulfur-containing compounds that are generated from S-alk(en)ylcysteine sulfoxides. Previous radiotracer experiments proposed that S-alk(en)ylcysteine sulfoxides are biosynthesized from glutathione. The recent identification of γ-glutamyl transpeptidases and a flavin-containing S-oxygenase involved in the biosynthesis of S-allylcysteine sulfoxide (alliin) in garlic (Allium sativum) provided insights into the reaction order of deglutamylation and S-oxygenation together with the localization of the biosynthesis, although the rest of the enzymes in the pathway still await discovery. In intact plants, S-alk(en)ylcysteine sulfoxides are stored in the cytosol of storage mesophyll cells. During tissue damage, the vacuolar enzyme alliinase contacts and hydrolyzes S-alk(en)ylcysteine sulfoxides to produce the corresponding sulfenic acids, which are further converted into various sulfur-containing bioactive compounds mainly via spontaneous reactions. The formed sulfur-containing compounds exhibit bioactivities related to pathogen defense, the prevention and alleviation of cancer and cardiovascular diseases, and neuroprotection. This review summarizes the current understanding of the occurrence, biosynthesis, and alliinase-triggered chemical conversion of S-alk(en)ylcysteine sulfoxides in Allium plants as well as the impact of S-alk(en)ylcysteine sulfoxides and their derivatives on medicinal, food, and agricultural sciences.

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.

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.