L-Canavanine, a Root Exudate From Hairy Vetch (Vicia villosa) Drastically Affecting the Soil Microbial Community and Metabolite Pathways

L-Canavanine, a conditionally essential non-proteinogenic amino acid analog to L-arginine, plays important roles in cell division, wound healing, immune function, the release of hormones, and a precursor for the synthesis of nitric oxide (NO). In this report, we found that the L-canavanine is released into the soil from the roots of hairy vetch (Vicia villosa) and declines several weeks after growth, while it was absent in bulk proxy. Hairy vetch root was able to exudate L-canavanine in both pots and in vitro conditions in an agar-based medium. The content of the L-canavanine in pots and agar conditions was higher than the field condition. It was also observed that the addition of L-canavanine significantly altered the microbial community composition and diversity in soil. Firmicutes and Actinobacteria became more abundant in the soil after the application of L-canavanine. In contrast, Proteobacteria and Acidobacteria populations were decreased by higher L-canavanine concentration (500 nmol/g soil). Prediction of the soil metabolic pathways using PICRUSt2 estimated that the L-arginine degradation pathway was enriched 1.3-fold when L-canavanine was added to the soil. Results indicated that carbon metabolism-related pathways were altered and the degradation of nitrogen-rich compounds (i.e., amino acids) enriched. The findings of this research showed that secretion of the allelochemical L-canavanine from the root of hairy vetch may alter the soil microbial community and soil metabolite pathways to increase the survival chance of hairy vetch seedlings. This is the first report that L-canavanine acts as an allelochemical that affects the biodiversity of soil microbial community.

Preclinical evaluation of new α-radionuclide therapy targeting LAT1: 2-[211At]astato-α-methyl-L-phenylalanine in tumor-bearing model

PURPOSE: Targeted α-radionuclide therapy has growing attention as a promising therapy for refractory cancers. However, the application is limited to certain types of cancer. Since L-type amino acids transporter 1 (LAT1) is highly expressed in various human cancers, we prepared LAT1-selective α-emitting amino acid analog, 2-[211At]astato-α-methyl-L-phenylalanine ([211At]-2-AAMP), and evaluated its potential as a therapeutic agent. METHODS: [211At]-2-AAMP was prepared from the stannyl precursor. Stability of [211At]-2-AAMP was evaluated by both in vitro and in vivo. In vitro studies using LAT1 expressing human ovary cancer cell line, SKOV3, were performed for evaluating cellular uptake and cytotoxicity of [211At]-2-AAMP. Biodistribution and therapeutic studies in SKOV3 bearing mice were performed after intravenous injection of [211At]-2-AAMP. RESULTS: [211At]-2-AAMP was stable in murine plasma in vitro and excreted into urine as intact. Cellular uptake of [211At]-2-AAMP was inhibited by treatment with LAT1-selective inhibitor. After 24 hours of incubation, [211At]-2-AAMP suppressed clonogenic growth at 10 kBq/ml, and induced cell death and DNA double strand break at 25 kBq/ml. When injected to mice, [211At]-2-AAMP exhibited the peak accumulation in the tumor at 30 min postinjection, and the radioactivity levels in the tumor retained up to 60 min. The majority of the radioactivity was rapidly eliminated from the body into the urine as an intact form immediately after injection. [211At]-2-AAMP significantly improved the survival of mice (P<0.05) without serious side effects. CONCLUSION: [211At]-2-AAMP showed α-radiation-dependent cellular growth inhibition after taking up via LAT1. Furthermore, [211At]-2-AAMP provided a beneficial effect on survival in vivo. These findings suggest that [211At]-2-AAMP might be useful for treatment of LAT1-positive cancer.

Revealing nascent proteomics in signaling pathways and cell differentiation

Regulation of gene expression at the level of protein synthesis is a crucial element in driving how the genetic landscape is expressed. However, we are still limited in technologies that can quantitatively capture the immediate proteomic changes that allow cells to respond to specific stimuli. Here, we present a method to capture and identify nascent proteomes in situ across different cell types without disturbing normal growth conditions, using O-propargyl-puromycin (OPP). Cell-permeable OPP rapidly labels nascent elongating polypeptides, which are subsequently conjugated to biotin-azide, using click chemistry, and captured with streptavidin beads, followed by digestion and analysis, using liquid chromatography–tandem mass spectrometry. Our technique of OPP-mediated identification (OPP-ID) allows detection of widespread proteomic changes within a short 2-hour pulse of OPP. We illustrate our technique by recapitulating alterations of proteomic networks induced by a potent mammalian target of rapamycin inhibitor, MLN128. In addition, by employing OPP-ID, we identify more than 2,100 proteins and uncover distinct protein networks underlying early erythroid progenitor and differentiation states not amenable to alternative approaches such as amino acid analog labeling. We present OPP-ID as a method to quantitatively identify nascent proteomes across an array of biological contexts while preserving the subtleties directing signaling in the native cellular environment.

Rebamipide, an Amino Acid Analog of 2(1H)-Quinolinone, Inhibits the Formation of Human Osteoclasts

Objectives. Drug repositioning or drug reprofiling (DR) has recently been growing in importance. DR has a significant advantage over traditional drug development because the repositioned drug has already passed toxicity tests; its safety is known, and the risk of adverse toxicology is reduced. In the current study, we investigated the role of rebamipide, a mucosa-protecting agent, with recently reported anti-inflammatory function, in human osteoclastogenesis. Methods. Peripheral blood mononuclear cells (PBMCs) were cultured in the presence of M-CSF and sRANKL. Osteoclast formation was evaluated by immunohistological staining for CD51/61 (vitronectin receptors). Osteoclast formation, in the presence or absence of rebamipide (0, 1, and 3 mM), was observed by time-lapse photography and actin ring formation. The number of absorption sites and area of absorption were calculated using Osteologic™ plates. Pit formation was studied by 3D-SEM. Results. Rebamipide inhibited human osteoclast formation at 3 mM, a pharmacological concentration, and inhibited resorbing activity dose-dependently. Rebamipide induced the degradation of actin rings in mature osteoclasts. This mechanism may involve inhibiting the osteoclast fusion pathway through reducing the expression of DC-specific transmembrane protein (DC-STAMP). Conclusions. The present study suggests that rebamipide would be useful as a novel agent for osteoporosis and rheumatoid arthritis.

Effect of fluorophenylalanine on indole-3-acetic acid oxidation in Avena coleoptiles

Effects of the amino acid analog D,L-p-fluorophenylalanine on auxin oxidizing activity from <i>Avena saliva</i> cv. Clintford have been examined. Incubation of etiolated <i>Avena</i> coleoptile apices with D,L-p-fluorophenylalanine, previously reported to promote elongation and depress phenolic metabolism, lowers auxin oxidizing activity. Temperature and pH profiles demonstrated that depression of oxidizing activity by D,L-p-fluorophenylalanine occurs over a relatively wide range. Incubation of coleoptiles in media containing L-phenylalanine alone or L-phenylalanine combined with D,L-p-fluorophenylalanine result in oxidizing activities which are, respectively, slightly higher than or restored to control levels. Thus, dark incubation of coleoptiles with D,L-p-fluorophenylalanine results in auxin oxidizing activity which differs from that of control and other treatment groups. Oxidizing activity from D,L-p-fluorophenylalanine pretreated coleoptiles has a Michaelis constant (70 µM) and a maximum velocity (0.200) which are consistently lower than the control constants. These findings provide further clarification of the effects of D,L-p-fluorophenylalanine, on coleoptile elongation and auxin oxidizing activity in etiolated <i>Avena</i> coleoptiles.