Antibacterial and Antibiofilm Effects of Allelopathic Compounds Identified in Medicago sativa L. Seedling Exudate against Escherichia coli.

In this study, the allelopathic properties of Medicago sativa L. (alfalfa) seedling exudates on the germination of seeds of various species were investigated. The compounds responsible for the allelopathic effects of alfalfa were identified and characterized by employing liquid chromatography ion mobility high-resolution mass spectrometry. Crude exudates inhibited the germination of seeds of all various plant species tested. Overall, nine compounds in alfalfa were identified and quantified. The most predominant compounds were a hyperoside representing a flavonoid glucoside, the non-proteinogenic amino acid canavanine, and two dipeptides, identified as H-Glu-Tyr-OH and H-Phe-Glu-OH. The latter corresponds to the first finding that dipeptides are exuded from alfalfa seedlings. In addition, the antibacterial and antibiofilm activities of alfalfa exudate and its identified compounds were elucidated. Both hyperoside and canavanine revealed the best antibacterial activity with minimum inhibitory concentration (MIC) values that ranged from 8 to 32 and 32 to 256 µg/mL, respectively. Regarding the antibiofilm action, hyperoside and canavanine caused a decline in the percentage of E. coli isolates that possessed a strong and moderate biofilm-forming potential from 68.42% to 21.05% and 31.58%, respectively. Studies on their inhibiting effects exhibit that these major substances are predominantly responsible for the allelopathic and antimicrobial effects of the crude exudates.

Using canavanine resistance to measure mutation rates in Schizosaccharomyces pombe.

We constructed a panel of S. pombe strains expressing DNA polymerase ε variants associated with cancer, specifically POLES297F, POLEV411L, POLEL424V, POLES459F, and used these to compare mutation rates determined by canavanine resistance with other selective methods. Canavanine-resistance mutation rates are broadly similar to those seen with reversion of the ade-485 mutation to adenine prototrophy, but lower than 5-fluoroorotic acid (FOA)-resistance rates (inactivation of ura4+ or ura5+ genes). Inactivation of several genes has been associated with canavanine resistance in S. pombe but surprisingly whole genome sequencing showed that 8/8 spontaneous canavanine-resistant mutants have an R175C mutation in the any1/arn1 gene. This gene encodes an α-arrestin-like protein involved in mediating Pub1 ubiquitylation of target proteins, and the phenotypic resistance to canavanine by this single mutation is similar to that shown by the original "can1-1" strain, which also has the any1R175C mutation. Some of the spontaneous mutants have additional mutations in arginine transporters, suggesting that this may marginally increase resistance to canavanine. The any1R175C strain showed internalisation of the Cat1 arginine transporter as previously reported, explaining the canavanine-resistance phenotype.

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test.

Continuous culture systems allow for the controlled growth of microorganisms over a long period of time. Here, we develop a novel test for mutagenicity that involves growing yeast in continuous culture systems exposed to low levels of mutagen for a period of approximately 20 days. In contrast, most microorganism-based tests for mutagenicity expose the potential mutagen to the biological reporter at a high concentration of mutagen for a short period of time. Our test improves upon the sensitivity of the well-established Ames test by at least 20-fold for each of two mutagens that act by different mechanisms (the intercalator ethidium bromide and alkylating agent methyl methanesulfonate). To conduct the tests, cultures were grown in small, inexpensive continuous culture systems in media containing (potential) mutagen, and the resulting mutagenicity of the added compound was assessed via two methods: a canavanine-based plate assay and whole genome sequencing. In the canavanine-based plate assay, we were able to detect a clear relationship between the amount of mutagen and the number of canavanine-resistant mutant colonies over a period of one to three weeks of exposure. Whole genome sequencing of yeast grown in continuous culture systems exposed to methyl methanesulfonate demonstrated that quantification of mutations is possible by identifying the number of unique variants across each strain. However, this method had lower sensitivity than the plate-based assay and failed to distinguish the different concentrations of mutagen. In conclusion, we propose that yeast grown in continuous culture systems can provide an improved and more sensitive test for mutagenicity.

Dual role of ER stress in response to metabolic co-targeting and radiosensitivity in head and neck cancer cells.

Arginine deprivation therapy (ADT) is a new metabolic targeting approach with high therapeutic potential for various solid cancers. Combination of ADT with low doses of the natural arginine analog canavanine effectively sensitizes malignant cells to irradiation. However, the molecular mechanisms determining the sensitivity of intrinsically non-auxotrophic cancers to arginine deficiency are still poorly understood. We here show for the first time that arginine deficiency is accompanied by global metabolic changes and protein/membrane breakdown, and results in the induction of specific, more or less pronounced (severe vs. mild) ER stress responses in head and neck squamous cell carcinoma (HNSCC) cells that differ in their intrinsic ADT sensitivity. Combination of ADT with canavanine triggered catastrophic ER stress via the eIF2α-ATF4(GADD34)-CHOP pathway, thereby inducing apoptosis; the same signaling arm was irrelevant in ADT-related radiosensitization. The particular strong supra-additive effect of ADT, canavanine and irradiation in both intrinsically more and less sensitive cancer cells supports the rational of ER stress pathways as novel target for improving multi-modal metabolic anti-cancer therapy.

Combinatory Treatment of Canavanine and Arginine Deprivation Efficiently Targets Human Glioblastoma Cells via Pleiotropic Mechanisms.

Glioblastomas are the most frequent and aggressive form of primary brain tumors with no efficient cure. However, they often exhibit specific metabolic shifts that include deficiency in the biosynthesis of and dependence on certain exogenous amino acids. Here, we evaluated, in vitro, a novel combinatory antiglioblastoma approach based on arginine deprivation and canavanine, an arginine analogue of plant origin, using two human glioblastoma cell models, U251MG and U87MG. The combinatory treatment profoundly affected cell viability, morphology, motility and adhesion, destabilizing the cytoskeleton and mitochondrial network, and induced apoptotic cell death. Importantly, the effects were selective toward glioblastoma cells, as they were not pronounced for primary rat glial cells. At the molecular level, canavanine inhibited prosurvival kinases such as FAK, Akt and AMPK. Its effects on protein synthesis and stress response pathways were more complex and dependent on exposure time. We directly observed canavanine incorporation into nascent proteins by using quantitative proteomics. Although canavanine in the absence of arginine readily incorporated into polypeptides, no motif preference for such incorporation was observed. Our findings provide a strong rationale for further developing the proposed modality based on canavanine and arginine deprivation as a potential antiglioblastoma metabolic therapy independent of the blood-brain barrier.

Yeast-model-based study identified myosin- and calcium-dependent calmodulin signalling as a potential target for drug intervention in chorea-acanthocytosis.

Chorea-acanthocytosis (ChAc) is a rare neurodegenerative disease associated with mutations in the human VPS13A gene. The mechanism of ChAc pathogenesis is unclear. A simple yeast model was used to investigate the function of the single yeast VSP13 orthologue, Vps13. Vps13, like human VPS13A, is involved in vesicular protein transport, actin cytoskeleton organisation and phospholipid metabolism. A newly identified phenotype of the vps13Δ mutant, sodium dodecyl sulphate (SDS) hypersensitivity, was used to screen a yeast genomic library for multicopy suppressors. A fragment of the MYO3 gene, encoding Myo3-N (the N-terminal part of myosin, a protein involved in the actin cytoskeleton and in endocytosis), was isolated. Myo3-N protein contains a motor head domain and a linker. The linker contains IQ motifs that mediate the binding of calmodulin, a negative regulator of myosin function. Amino acid substitutions that disrupt the interaction of Myo3-N with calmodulin resulted in the loss of vps13Δ suppression. Production of Myo3-N downregulated the activity of calcineurin, a protein phosphatase regulated by calmodulin, and alleviated some defects in early endocytosis events. Importantly, ethylene glycol tetraacetic acid (EGTA), which sequesters calcium and thus downregulates calmodulin and calcineurin, was a potent suppressor of vps13Δ. We propose that Myo3-N acts by sequestering calmodulin, downregulating calcineurin and increasing activity of Myo3, which is involved in endocytosis and, together with Osh2/3 proteins, functions in endoplasmic reticulum-plasma membrane contact sites. These results show that defects associated with vps13Δ could be overcome, and point to a functional connection between Vps13 and calcium signalling as a possible target for chemical intervention in ChAc. Yeast ChAc models may uncover the underlying pathological mechanisms, and may also serve as a platform for drug testing.This article has an associated First Person interview with the first author of the paper.

ß-N-Methylamino-L-Alanine Toxicity in PC12: Excitotoxicity vs. Misincorporation.

The implication of ß-N-methylamino-L-alanine (BMAA) in the development of neurodegenerative diseases worldwide has led to several investigations of the mechanism, or mechanisms, of toxicity of this cyanobacterially produced amino acid. The primary mechanism of toxicity that was identified is excitotoxicity, with a second possible mechanism, the misincorporation of BMAA into the primary protein structure and consequent cell damage, having been more recently reported. However, studies on excitotoxicity and misincorporation have been conducted independently and there are therefore no data available on the relative contribution of each of these mechanisms to the total toxicity of BMAA. The rat pheochromocytoma cell line PC12 is an ideal model for a study of this type, as glutamate receptor expression is modified by cell differentiation, which can be affected by exposure to nerve growth factor. In this study, the PC12 cell line was evaluated as a model to study BMAA toxicity via the two proposed mechanisms: excitotoxicity and protein misincorporation. BMAA and canavanine treatment of cultures of PC12 were evaluated for depolarization of the mitochondrial membrane. In canavanine-treated cultures, this was evident after 9 days of treatment and was attributed to the primary mechanism of canavanine toxicity, protein misincorporation. However, no membrane depolarization was observed for BMAA-treated cultures even after 21 days of continuous treatment at 500 µM. Short-term exposure to both BMAA and canavanine resulted in a slight increase in necrosis in undifferentiated cells that was prevented in canavanine-treated cultures by co-incubation with arginine, but not in BMAA-treated cultures by co-incubation with serine. A slight increase in apoptosis was observed in undifferentiated cells treated with either BMAA or glutamate, and ROS production increased in glutamate-treated cells. However, the excitotoxicity was less pronounced than reported in previous studies with neuronal cells. In contrast, apoptosis was greatly increased in both BMAA- and glutamate-treated cells after differentiation and resulting mGluR1 increase, indicating that excitotoxicity is the main, if not only, mechanism of toxicity in PC12.

L-Canavanine Potentiates Cytotoxicity of Chemotherapeutic Drugs in Human Breast Cancer Cells.

BACKGROUND: Due to the high level of argininosuccinate synthase (ASS), a key enzyme for the formation of arginine from citrulline, human breast cancers are often resistant to arginine deprivation therapy. An antimetabolite, Lcanavanine (L-CAV), can be incorporated into proteins in the place of arginine, disturbing protein conformation and leading to cellular death. OBJECTIVES: This study was designed to investigate the potential of L-CAV to enhance the toxicity of chemotherapeutic drugs in the human breast cancer cell line MCF-7, and determine the most favorable drug combination to exert synergistic interaction in the presence or absence of arginine in the medium. METHODS: Combination experiment based on the median-effect principle and mass-action law was conducted using constant ratios of cytotoxic agents as developed by Chou (2006). RESULTS: We observed that L-CAV enhanced the toxicity of cisplatin (CIS) and vinblastine (VIN) in MCF-7, even in the presence of L-ARG. On the other hand, L-CAV potentiated the toxicity of doxorubicin (DOX), paclitaxel (PTX), 5- fluoruracil (5-FU), and amphotericin-B (AMP-B) in cells grown in arginine deprived media. CONCLUSION: We conclude that the combination of L-CAV with CIS or VIN can potentiate the toxicity for breast cancer cells. Thus this report presents a new possibility for treating human breast cancers known to be resistant to arginine deprivation. This initial study requires further investigation in in vivo experiments and exploration of the molecular mechanism of cellular response in human breast cancer.

Toxicity of canavanine in tomato (Solanum lycopersicum L.) roots is due to alterations in RNS, ROS and auxin levels.

Canavanine (CAN) is non-proteinogenic aminoacid and a structural analog of arginine (Arg). Naturally, CAN occurs in legumes e.g. jack bean and is considered as a strong allelochemical. As a selective inhibitor of inducible nitric oxide synthase in mammalians, it could act as a modifier of nitric oxide (NO) concentration in plants. Modifications in the content of endogenous reactive nitrogen species (RNS) and reactive oxygen species (ROS) influence root structure and architecture, being also under hormonal control. The aim of the work was to investigate regulation of root growth in tomato (Solanum lycopersicum L. cv. Malinowy Ozarowski) seedling by application of CAN at concentration (10 and 50 µM) leading to 50% or 100% restriction of root elongation. CAN at higher concentration led to slight DNA fragmentation, increased total RNA and protein level. Decline in total respiration rate after CAN supplementation was not associated with enhanced membrane permeability. Malformations in root morphology (shorter and thicker roots, limited number of lateral roots) were accompanied by modification in NO and ONOO(-) localization; determined mainly in peridermal cells and some border cells. Although, CAN resulted in low RNS production, addition of exogenous NO by usage of NO donors did not reverse its negative effect, nor recovery effect was detected after roots imbibition in Arg. To build up a comprehensive view on mode of action of CAN as root growth inhibitor, it was shown an elevated level of auxin. To summarize, we demonstrated several secondary mode of action of CAN, indicating its toxicity in plants linked to restriction in RNS formation accompanied by simultaneous overaccumulation of ROS.

Effects of the combined arginase and canavanine treatment on leukemic cells in vitro and in vivo.

It was previously demonstrated in in vitro experiments that canavanine (Cav), a natural toxic arginine analogue of plant origin, is a promising candidate for augmenting the antineoplastic effects of arginine starvation. We demonstrated herein that recombinant human arginase, an arginine degrading enzyme, abrogated growth and significantly increased Cav cytotoxicity toward cultured L1210 murine leukemic cells. Cav co-treatment further reduced cells viability in a time-dependent manner and significantly promoted apoptosis induction. In the pilot study we also evaluated for the first time the potential toxicity of the combined arginine deprivation and Cav treatment in healthy mice. Administration of Cav alone or in combination with pegylated cobalt-containing human arginase (Co-hARG) did not evoke any apparent toxic effects in these animals, with no significant behavioural and survival changes after several weeks of the treatment. The therapeutic effects of the combination of Co-hARG and Cav were provisionally evaluated on the highly aggressive murine L1210 leukemia, which is semi-sensitive to arginine deprivation as a monotreatment. Combination of two drugs did not result in significant prolongation of the survival of leukemia-bearing mice. Thus, we have shown that the proposed combinational treatment is rather non-toxic for the animals. It has to be further evaluated in animal studies with alternative tumor models and/or drug doses and treatment modalities.

Arginine deprivation induces endoplasmic reticulum stress in human solid cancer cells.

Deprivation for the single amino acid arginine is a rapidly developing metabolic anticancer therapy, which allows growth control in a number of highly malignant tumors. Here we report that one of the responses of human solid cancer cells to arginine starvation is the induction of prolonged endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Systematic study of two colorectal carcinoma HCT-116 and HT29, glioblastoma U251 MG and ovarian carcinoma SKOV3 cell lines revealed, however, that the ER stress triggered by the absence of arginine does not result in massive apoptosis despite a profound upregulation of the proapoptotic gene CHOP. Instead, Akt- and MAPK-dependent pathways were activated which may counteract proapoptotic signaling. Treatment with DMSO as a disaggregating agent or with cycloheximide to block protein synthesis reduced ER stress evoked by arginine deprivation. On the other hand, ER stress and apoptosis induction in arginine-starved cells could be critically augmented by the arginine analog of plant origin canavanine, but not by the classic ER stress inducer tunicamycin. Our data suggest that canavanine treatment applied under the lack of arginine may enhance the efficacy of arginine deprivation-based anticancer therapy.

Cytotoxic potentiation of vinblastine and paclitaxel by L-canavanine in human cervical cancer and hepatocellular carcinoma cells.

BACKGROUND: The non-protein amino acid L-canavanine (L-CAV), found in several plants of the family Fabaceae is an antimetabolite which shows anticancer activity due to its ability to be incorporated into protein in the place of its analogue, L-arginine (L-ARG), leading to the alteration of the 3D conformation of newly synthesised proteins and usually a loss of their function. PURPOSE: In this study, the ability of L-CAV to potentiate the cytotoxicity of microtubule- targeting drugs used in the chemotherapy of cancer, vinblastine (VIN) and paclitaxel (PTX) was evaluated. MATERIAL AND METHODS: The following cancer cells grown in arginine-rich and arginine-free media were employed: HeLa, Hep G2 and SK-HEP-1. Drug combination experiment used a method based on the median-effect principle and mass-action law. RESULTS: We observed that L-CAV, which is hardly toxic alone, potentiated the cytotoxicity of VIN and PTX in HeLa and hepatocellular carcinoma cells. CONCLUSION: This is the first study showing the cytotoxic potentiation of microtubule-targeting drugs by L-CAV. The mechanism of synergy and animal studies need to be investigated further to see whether L-CAV might become an adjuvant in cancer treatment.

CHIP Knockdown Reduced Heat Shock Response and Protein Quality Control Capacity in Lens Epithelial Cells.

Protein quality control (PQC) systems, including molecular chaperones and ubiquitin-proteasome pathway (UPP), plays an important role in maintaining intracellular protein homeostasis. Carboxyl terminus of Hsc70- interacting protein (CHIP) links the chaperone and UPPs, thus contributing to the repair or removal of damaged proteins. Over-expression of CHIP had previously been used to protect cells from environmental stress. In order to gain a more physiologic mechanism of the advantage conferred by CHIP, we induced a CHIP knockdown and monitored the ability of cells to cope with environmental stress. To knockdown CHIP, the human lens epithelial cell line HLE B3 was transfected with lentiviral particles that encode a CHIP short hairpin RNA (shRNA) or negative control lentiviral particles. Stable CHIP-knock down cells (KD) and negative control cells (NC) were selected with puromycin. After exposure to heat shock stress, there was no change observed in the expression of Hsp90. In contrast, Hsp70 levels increased significantly in NC cells but less so in KD cells. Hsp27 levels also increased after heat shock, but only in NC cells. Protein ubiquitination was reduced when CHIP was knocked down. CHIP knockdown reduced the ability to clear aggregation proteins. When same levels of aggregation-prone RFP-mutant crystallin fusion protein, RFP/V76D-γD, was expressed, there was ~9- fold more aggregates in KD cells as compared to that observed in NC cells. Furthermore, KD cells were more sensitive to toxicity of amino acid analog canavanine as compared to NC cells. Together, these data indicate that CHIP is required for PQC and that CHIP knockdown diminished cellular PQC capacity in lens cells.

Canavanine activates imidazoline I-2 receptors to reduce hyperglycemia in type 1-like diabetic rats.

Canavanine is a guanidinium derivative that has the basic structure of a ligand for the imidazoline receptor (I-R). Furthermore, canavanine is found in an herb that has been shown to improve diabetic disorders. Thus, the present study was designed to investigate the anti-hyperglycemic action of canavanine in rats with streptozotocin (STZ)-induced type 1-like diabetes. Canavanine decreased hyperglycemia in the STZ-induced diabetic rats, and this action was blocked by the antagonist specific to imidazoline I-2 receptors (I-2R), BU224, in a dose-dependent manner. Additionally, canavanine increased the plasma ß-endorphin level, as measured using enzyme-linked immunosorbent assay (ELISA), and this increase was also blocked by BU224 in the same manner. Moreover, amiloride at a dose sufficient to block I-2AR attenuated the actions of canavanine, including the increased ß-endorphin level and the antihyperglycemic effect. Otherwise, canavanine increased the radioactive glucose uptake into skeletal muscles isolated from the diabetic rats. Furthermore, canavanine increased the phosphorylation of AMPK measured using Western blot analysis in these isolated skeletal muscles in a dose-dependent manner. Additionally, the insulin sensitivity of the diabetic rats was markedly increased by canavanine, and this action was also blocked by BU224. Overall, canavanine is capable of activating imidazoline I-2R; I-2AR is linked to an increase in the plasma level of ß-endorphin, and I-2BR is related to effects on the glucose uptake by skeletal muscle that reduces hyperglycemia in type 1-like diabetic rats. Therefore, canavanine can be developed as effective agent to treat the diabetic disorders in the future.

Canavanine increases glucose uptake in C2 C12 cells through the activation of imidazoline I-2B receptors.

Canavanine is a guanidinium derivative that contains the basic structure of the ligand(s) of imidazoline receptor (I-R). Canavanine has been reported to activate the imidazoline I-3 receptor (I-3R) both in vivo and in vitro. Additionally, the activation of the imidazoline I-2B receptor (I-2BR) by guanidinium derivatives may increase glucose uptake. Therefore, the effect of canavanine on the I-2BR was investigated in the present study. Glucose uptake into cultured C2 C12 cells was determined using the radio-ligated tracer 2-[(14) C]-deoxy-glucose. The changes in 5' AMP-activated protein kinase (AMPK) expression were also identified using Western blotting analysis. The canavanine-induced glucose uptake was inhibited in a dose-dependent manner by BU224 (0.01-1 µmol/L), which is a specific I-2BR antagonist, in the C2 C12 cells. Additionally, the canavanine-stimulated AMPK phosphorylation and glucose transporter (GLUT4) expression were also sensitive to BU224 inhibition in the C2 C12 cells. Moreover, both canavanine-stimulated glucose uptake and AMPK phosphorylation were attenuated by high concentrations of amiloride (1-2 µmol/L), which is another established I-2BR inhibitor, in a dose-dependent manner in C2 C12 cells. Additionally, compound C abolished the canavanine-induced glucose uptake and AMPK phosphorylation at a concentration (0.1 µmol/L) sufficient to inhibit AMPK. In conclusion, these data demonstrated that canavanine has an ability to activate I-2BR through the AMPK pathway to increase glucose uptake, which indicates I-2BR as a new target for diabetic therapy.

Exposure to arginine analog canavanine induces aberrant mitochondrial translation products, mitoribosome stalling, and instability of the mitochondrial proteome.

Impairment of mitochondrial protein homeostasis disrupts mitochondrial function and causes human diseases and aging, but the molecular mechanisms of protein synthesis and quality control in mammalian mitochondria are not fully understood. Here we demonstrate in human cells that misincorporation of an arginine analog, canavanine, during mitochondrial protein synthesis, induced aberrant translation products and destabilized the mtDNA-encoded proteome, leading to loss of mitochondrial respiratory chain complexes. Furthermore, in the presence of a high concentration of canavanine, mitoribosome stalling could be demonstrated. The stalling did not, however, occur at arginine codons, but downstream of those codons. In particular, two adjacent arginines induced the most prominent downstream stalling effect, with the distance between the arginine codons and the stalling peak corresponding roughly to the length of the ribosomal exit tunnel. These results suggest that misincorporated canavanine disrupted the proper folding of the hydrophobic nascent polypeptides within the exit tunnel or while being inserted into the inner mitochondrial membrane. The canavanine treatment provides a model system for studying the consequences of mitoribosome stalling and the responses to misfolded proteins exiting the mitochondrial ribosome.

Canavanine induces insulin release via activation of imidazoline I3 receptors.

The aim of the present study was to identify the effect of canavanine on the imidazoline receptor because canavanine is a guanidinium derivative that has a similar structure to imidazoline receptor ligands. Transfected Chinese hamster ovary-K1 cells expressing imidazoline receptors (nischarin (NISCH)-CHO-K1 cells) were used to elucidate the direct effects of canavanine on imidazoline receptors. In addition, the imidazoline I3 receptor has been implicated in stimulation of insulin secretion from pancreatic ß-cells. Wistar rats were used to investigate the effects of canavanine (0.1, 1 and 2.5 mg/kg, i.v.) on insulin secretion. In addition the a specific I3 receptor antagonist KU14R (4 or 8 mg/kg, i.v.) was used to block I3 receptors. Canavanine decreased blood glucose by increasing plasma insulin in rats. In addition, canavanine increased calcium influx into NISCH-CHO-K1 cells in a manner similar to agmatine, the endogenous ligand of imidazoline receptors. Moreover, KU12R dose-dependently attenuated canavanine-induced insulin secretion in HIT-T15 pancreatic ß-cells and in the plasma of rats. The data suggest that canavanine is an agonist of I3 receptors both in vivo and in vitro. Thus, canavanine would be a useful tool in imidazoline receptor research.

Inhibitory effects of sword bean extract on alveolar bone resorption induced in rats by Porphyromonas gingivalis infection.

BACKGROUND: The domesticated legume, Canavalia gladiata (commonly called the sword bean), is known to contain canavanine. The fruit is used in Chinese and Japanese herbal medicine for treating the discharge of pus, but its pharmacological mechanisms are still unclear. OBJECTIVES: This study examined the effect of sword bean extract (SBE) on (i) oral bacteria and human oral epithelial cells in vitro, and (ii) the initiation and progression of experimental Porphyromonas gingivalis-induced alveolar bone resorption in rats. MATERIAL AND METHODS: A high-performance liquid chromatography/ultraviolet method was applied to quantitate canavanine in SBE. By assessing oral bacterial growth, we estimated the minimum inhibitory concentration and minimum bactericidal concentration of SBE, canavanine, chlorhexidine gluconate (CHX) solution. The cytotoxicity of SBE, canavanine, CHX, leupeptin and cystatin for KB cells was determined using a trypan blue assay. The effects of SBE, canavanine, leupeptin and cystatin on Arg-gingipain (Rgp) and Lys-gingipain (Kgp) were evaluated by colorimetric assay using synthetic substrates. To examine its effects on P. gingivalis-associated periodontal tissue breakdown, SBE was orally administered to P. gingivalis-infected rats. RESULT: Sword bean extract contained 6.4% canavanine. SBE and canavanine inhibited the growth of P. gingivalis and Fusobacterium nucleatum. The cytotoxicity of SBE, canavanine and cystatin on KB cells was significantly lower than that of CHX. Inhibition of Rgp with SBE was comparable to that with leupeptin, a known Rgp inhibitor, and inhibition of Kgp with SBE was significantly higher than that with leupeptin at 500 µg/mL ( p < 0.05). P. gingivalis-induced alveolar bone resorption was significantly suppressed by administration of SBE, with bone levels remaining comparable to non-infected animals ( p < 0.05). CONCLUSION: The present study suggests that SBE might be effective against P. gingivalis-associated alveolar bone resorption.

Exclusive induction of G:C to A:T transitions by 3-azido-1,2-propanediol in yeast.

Sodium azide is a strong mutagen which has been successfully employed in mutation breeding of crop plants. In biological systems, it is metabolized to azidoalanine, but further bioactivation to a putative ultimate mutagen as well as the nature of the induced DNA modifications leading to mutations remain elusive. In this study, mutations induced in the CAN1 gene of yeast Saccharomyces cerevisiae by the representative mutagen 3-azido-1,2-propanediol (azidoglycerol, AZG) have been sequenced. Analysis of the forward mutation spectrum to canavanine resistance revealed that AZG induced nearly exclusively G:C to A:T transitions. AZG also induced reversions to tryptophan prototrophy by base-pair substitutions in a dose-dependent manner. This unusual mutational specificity may be shared by other organic azido compounds.

Screening for long-lived genes identifies Oga1, a guanine-quadruplex associated protein that affects the chronological lifespan of the fission yeast Schizosaccharomyces pombe.

Schizosaccharomyces pombe and Saccharomyces cerevisiae are excellent model organisms to study lifespan. We conducted screening to identify novel genes that, when overexpressed, extended the chronological lifespan of fission yeast. We identified seven genes, among which we focused on SPBC16A3.08c. The gene product showed similarity to Ylr150w of S. cerevisiae, which has affinity for guanine-quadruplex nucleic acids (G4). The SPBC16A3.08c product associated with G4 in vitro and complemented the phenotype of an S. cerevisiae Ylr150w deletion mutant. From these results, we proposed that SPBC16A3.08c encoded for a functional homolog of Ylr150w, which we designated ortholog of G4-associated protein (oga1 (+)). oga1 (+) overexpression extended the chronological lifespan and also decreased mating efficiency and caused both high and low temperature-sensitive growth. Deleting oga1 (+) resulted in caffeine-sensitive and canavanine-resistant phenotypes. Based on these results, we discuss the function of Oga1 on the chronological lifespan of fission yeast.