Effect of Lipid Composition on the Interaction of Liposomes with THP-1-Derived Macrophages.

Recently, liposomal formulations that target macrophages have been used to treat lung diseases. However, the detailed mechanism of the cellular uptake must be elucidated to identify a formulation with excellent cellular uptake efficiency to treat non-tuberculous mycobacterial lung disease. We studied the effect of lipid composition on the cellular uptake of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol (Chol) liposomes with a size of approximately 200 nm into THP-1-derived macrophages. The amount of DPPC/Chol liposomes (80/20 mol%) was greater than that of DPPC/Chol (60/40 mol%) and DPPC/Chol (67/33 mol%) liposomes. The anisotropy of 1,6-diphenyl-1,3,5-hexatriene indicated that the membrane fluidity of the DPPC/Chol (80/20 mol%) liposomes was higher than that of the other two liposomes. DPPC/Chol (80/20 mol%) and DPPC/Chol (67/33 mol%) liposomes were taken up via clathrin- and caveolae-mediated endocytosis and phagocytosis. However, proteins involved in cellular uptake through ligand-receptor interactions were adsorbed to a greater extent on DPPC/Chol (80/20 mol%) liposomes than on DPPC/Chol (67/33 mol%) liposomes. Pretreatment of cells with antibodies against the low-density lipoprotein receptor and scavenger receptor type B1 largely inhibited the uptake efficiency of DPPC/Chol (80/20 mol%) liposomes. Our results indicate that the membrane fluidity of DPPC/Chol liposomes, which is controlled by the Chol ratio, is an important factor in controlling protein adsorption and the subsequent uptake efficiency of liposomes.

Quantitative lipid composition characterization of intact liposomes via 31P nuclear magnetic resonance spectroscopy.

Drug delivery systems (DDS) are important methods to maximize drug efficacy by enabling in vivo accumulation at the target site. Liposomes, which are nanoscale vesicles consisting of lipid bilayers, are widely used for clinical DDS. The lipid composition of an intact liposome is a significant factor that directly affects its characteristics and functions. Thus, it is important to develop quantitative or qualitative analytical methods to characterize the lipid composition. Nuclear magnetic resonance (NMR) of phosphorus (31P) is a particularly sensitive and non-destructive approach because phospholipid components have one 31P nucleus per molecule. Here, we demonstrate quantitative observations of individual phospholipids in intact liposomes via solution 31P-NMR. In addition, the 31P linewidths became narrower if the liposomes contained > 10 mol% of polyethylene glycol-(PEGylated) phospholipids, which also contributed to liposome down-sizing. Down-sizing and PEGylation are important strategies for efficient drug delivery. Hence, 31P-NMR can be used to analyze phospholipids in liposomes and related pharmaceutical preparations for quality control.

Novel tetrahydrofolate-dependent d-serine dehydratase activity of serine hydroxymethyltransferases.

d-Serine plays vital physiological roles in the functional regulation of the mammalian brain, where it is produced from l-serine by serine racemase and degraded by d-amino acid oxidase. In the present study, we identified a new d-serine metabolizing activity of serine hydroxymethyltransferase (SHMT) in bacteria as well as mammals. SHMT is known to catalyze the conversion of l-serine and tetrahydrofolate (THF) to glycine and 5,10-methylenetetrahydrofolate, respectively. In addition, we found that human and Escherichia coli SHMTs have d-serine dehydratase activity, which degrades d-serine to pyruvate and ammonia. We characterized this enzymatic activity along with canonical SHMT activity. Intriguingly, SHMT required THF to catalyze d-serine dehydration and did not exhibit dehydratase activity toward l-serine. Furthermore, SHMT did not use d-serine as a substrate in the canonical hydroxymethyltransferase reaction. The d-serine dehydratase activities of two isozymes of human SHMT were inhibited in the presence of a high concentration of THF, whereas that of E. coli SHMT was increased. The pH and temperature profiles of d-serine dehydratase and serine hydroxymethyltransferase activities of these three SHMTs were partially distinct. The catalytic efficiency (kcat /Km ) of dehydratase activity was lower than that of hydroxymethyltransferase activity. Nevertheless, the d-serine dehydratase activity of SHMT was physiologically important because d-serine inhibited the growth of an SHMT deletion mutant of E. coli, ∆glyA, more than that of the wild-type strain. Collectively, these results suggest that SHMT is involved not only in l- but also in d-serine metabolism through the degradation of d-serine.

An in vitro lipid-mixing assay to investigate the fusion between small extracellular vesicles and endosome.

Small extracellular vesicles (sEVs) such as exosomes can efficiently deliver nucleic acids into the cytosol of recipient cells. However, the molecular mechanism of the subsequent fusion with an endosome is not well understood. In this study, we developed an in vitro lipid-mixing assay using an endosomal-mimicking anionic liposome to investigate the fusion between sEVs and endosomes. We observed that the particle number ratio between the sEVs and the anionic liposomes, the diameter of the liposomes, and the buffer pH were all important for fusion activity. Furthermore, we optimized the liposomal lipid composition and demonstrated that incorporating the anionic lipid bis(monooleoylglycero) phosphate and cholesterol was important for efficient and reliable fusion. Our in vitro assay suggested that a decrease in pH increased the fusion activity. Additionally, it was suggested that this pH-dependent increase in the fusion activity was predominantly due to a change in the sEVs. sEVs possess a larger fusion activity than artificial liposomes that mimic the physicochemical properties of exosomes. These results are consistent with those of previous in vivo studies, supporting the physiological relevance of our system. This study provides an important platform for further research to clarify the molecular mechanisms of fusion between sEVs and endosomes.

Characterization of human cystathionine γ-lyase enzyme activities toward d-amino acids.

Various d-amino acids play important physiological roles in mammals, but the pathways of their production remain unknown except for d-serine, which is generated by serine racemase. Previously, we found that Escherichia coli cystathionine ß-lyase possesses amino acid racemase activity in addition to ß-lyase activity. In the present work, we evaluated the enzymatic activities of human cystathionine γ-lyase, which shares a relatively high amino acid sequence identity with cystathionine ß-lyase. The enzyme did not show racemase activity toward various amino acids including alanine and lyase and dehydratase activities were highest toward l-cystathionine and l-homoserine, respectively. The enzyme also showed weak activity toward l-cysteine and l-serine but no activity toward d-amino acids. Intriguingly, the pH and temperature profiles of lyase activity were distinct from those of dehydratase activity. Catalytic efficiency was higher for lyase activity than for dehydratase activity.

Analysis of the interaction of cyclosporine congeners with cell membrane models.

Owing to the relatively high molecular weight of macrocyclic peptides, investigation of the cellular uptake mechanism is required for the efficient design of macrocyclic peptides as potential drugs. We have previously reported, using HPLC, that cyclosporine A, a model macrocyclic peptide, and its congeners B, C, and D had different lipophilicity despite differing by only one amino acid. In the present study, we investigated how this difference in lipophilicity affected the interaction of the congeners with cell membranes. The circular dichroism spectra showed that the secondary structures were similar between the four congeners even at high temperature. The molar ellipticity of the four congeners in the presence of liposomes, as a cell membrane model, differed, and cyclosporines D and A showed lower molar ellipticity, while cyclosporine C exhibited higher molar ellipticity. Fluorescent spectra analysis using Laurdan indicated that liposome hydration was decreased in the presence of the cyclosporines, especially cyclosporines D and A. HPLC analysis also quantitatively showed that the amount of cyclosporine molecules internalized in HpG2 cells was the largest for cyclosporine D. We determined, using spectroscopy and HPLC, that the intensity of the interaction of the congeners with cell membranes was overall correlated with the lipophilicity derived from the side chains of each congener. Our results will contribute to the design of new macrocyclic peptides with favorable drug properties.

Current Status and Challenges of Analytical Methods for Evaluation of Size and Surface Modification of Nanoparticle-Based Drug Formulations.

The present review discusses the current status and difficulties of the analytical methods used to evaluate size and surface modifications of nanoparticle-based pharmaceutical products (NPs) such as liposomal drugs and new SARS-CoV-2 vaccines. We identified the challenges in the development of methods for (1) measurement of a wide range of solid-state NPs, (2) evaluation of the sizes of polydisperse NPs, and (3) measurement of non-spherical NPs. Although a few methods have been established to analyze surface modifications of NPs, the feasibility of their application to NPs is unknown. The present review also examined the trends in standardization required to validate the size and surface measurements of NPs. It was determined that there is a lack of available reference materials and it is difficult to select appropriate ones for modified NP surface characterization. Research and development are in progress on innovative surface-modified NP-based cancer and gene therapies targeting cells, tissues, and organs. Next-generation nanomedicine should compile studies on the practice and standardization of the measurement methods for NPs to design surface modifications and ensure the quality of NPs.

Identification of a novel d-amino acid aminotransferase involved in d-glutamate biosynthetic pathways in the hyperthermophile Thermotoga maritima.

The hyperthermophilic bacterium Thermotoga maritima has an atypical peptidoglycan that contains d-lysine alongside the usual d-alanine and d-glutamate. We previously identified a lysine racemase involved in d-lysine biosynthesis, and this enzyme also possesses alanine racemase activity. However, T. maritima has neither alanine racemase nor glutamate racemase enzymes; hence, the precise biosynthetic pathways of d-alanine and d-glutamate remain unclear in T. maritima. In the present study, we identified and characterized a novel d-amino acid aminotransferase (TM0831) in T. maritima. TM0831 exhibited aminotransferase activity towards 23 d-amino acids, but did not display activity towards l-amino acids. It displayed high specific activities towards d-homoserine and d-glutamine as amino donors. The most preferred acceptor was 2-oxoglutarate, followed by glyoxylate. Additionally, TM0831 displayed racemase activity towards four amino acids including aspartate and glutamate. Catalytic efficiency (kcat /Km ) for aminotransferase activity was higher than for racemase activity, and pH profiles were distinct between these two activities. To evaluate the functions of TM0831, we constructed a TTHA1643 (encoding glutamate racemase)-deficient Thermus thermophilus strain (∆TTHA1643) and integrated the TM0831 gene into the genome of ∆TTHA1643. The growth of this TM0831-integrated strain was promoted compared with ∆TTHA1643 and was restored to almost the same level as that of the wild-type strain. These results suggest that TM0831 is involved in d-glutamate production. TM0831 is a novel d-amino acid aminotransferase with racemase activity that is involved in the production of d-amino acids in T. maritima.

Effect of hydrophobic moment on membrane interaction and cell penetration of apolipoprotein E-derived arginine-rich amphipathic α-helical peptides.

We previously developed an amphipathic arginine-rich peptide, A2-17, which has high ability to directly penetrate across cell membranes. To understand the mechanism of the efficient cell-penetrating ability of the A2-17 peptide, we designed three structural isomers of A2-17 having different values of the hydrophobic moment and compared their membrane interaction and direct cell penetration. Confocal fluorescence microscopy revealed that cell penetration efficiency of peptides tends to increase with their hydrophobic moment, in which A2-17 L14R/R15L, an A2-17 isomer with the highest hydrophobic moment, predominantly remains on plasma cell membranes. Consistently, Trp fluorescence analysis indicated the deepest insertion of A2-17 L14R/R15L into lipid membranes among all A2-17 isomers. Electrophysiological analysis showed that the duration and charge flux of peptide-induced pores in lipid membranes were prominent for A2-17 L14R/R15L, indicating the formation of stable membrane pores. Indeed, the A2-17 L14R/R15L peptide exhibited the strongest membrane damage to CHO-K1 cells. Atomic force microscopy quantitatively defined the peptide-induced membrane perturbation as the decrease in the stiffness of lipid vesicles, which was correlated with the hydrophobic moment of all A2-17 isomers. These results indicate that optimal membrane perturbation by amphipathic A2-17 peptide is critical for its efficient penetration into cells without inducing stabilized membrane pores.

Enhancing the Endocytosis of Phosphatidylserine-Containing Liposomes through Tim4 by Modulation of Membrane Fluidity.

Phosphatidylserine (PS) is a unique lipid that is recognized by the endogenetic receptor, T-cell immunoglobulin mucin protein 4 (Tim4), and PS-containing liposomes have potential use in therapeutic applications. We prepared PS-containing liposomes of various lipid compositions and examined how lipid membrane fluidity affects PS recognition by Tim4 and the resulting endocytosis efficiency into Hela cells. Surface plasmon resonance and laurdan studies showed that increasing lipid membrane fluidity increased the stability of the PS-Tim4 interaction but hampered the entry of liposomes into cells. These results show that endocytosis efficiency is determined by balancing opposing forces induced by membrane fluidity. We found that inclusion of the zwitterionic helper lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, into liposomes ensured efficient cellular internalization because the presence of this lipid provides an ideal balance of lipid fluidity and Tim4 affinity. The results showed that PS recognition by Tim4 and the resulting endocytosis efficiency can be maximized by modulating the membrane fluidity of liposomes by selecting a zwitterionic helper lipid. This study improves our understanding of how to rationally optimize nanotechnology for targeted drug delivery.

Acetylornithine aminotransferase TM1785 performs multiple functions in the hyperthermophile Thermotoga maritima.

The hyperthermophilic bacterium Thermotoga maritima peptidoglycan contains unusual d-lysine alongside typical d-alanine and d-glutamate. We previously identified lysine racemase and threonine dehydratase, but knowledge of d-amino acid metabolism remains limited. Herein, we identified and characterized T. maritima acetylornithine aminotransferase TM1785. The enzyme was most active towards acetyl-l-ornithine, but also utilized l-glutamate, l-ornithine and acetyl-l-lysine as amino donors, and 2-oxoglutarate was the preferred amino acceptor. TM1785 also displayed racemase activity towards four amino acids and lyase activity towards l-cysteine, but no dehydratase activity towards l-serine, l-threonine or corresponding d-amino acids. Catalytic efficiency (kcat /Km ) was highest for aminotransferase activity and lowest for racemase activity. TM1785 is a novel acetylornithine aminotransferase associated with l-arginine biosynthesis that possesses two additional distinct activities.

Effect of Sample Concentration on Nanoparticle Tracking Analysis of Small Extracellular Vesicles and Liposomes Mimicking the Physicochemical Properties of Exosomes.

For quantitative analysis, data should be obtained at a sample concentration that is within the range of linearity. We examined the effect of sample concentration on nanoparticle tracking analysis (NTA) of small extracellular vesicles (sEVs), including exosomes, by comparing NTA results of sEVs with those obtained for polystyrene nanoparticles (PSN) and liposomes, which mimic lipid composition and physicochemical properties of exosomes. Initially, NTA of PSN at different concentrations was performed and the particle sizes determined were validated by dynamic light scattering. The major peak maxima for PSN mixtures of different sizes at the higher particle numbers were similar, with some fluctuation of the minor peak maxima observed at the lower particle number, which was also observed for sEVs. Sample concentration is critical for obtaining reproducible data for liposomes and exosomes and increasing the sample concentration caused an increase in data variability because of particle interactions. The inter-day repeatability of particles sizes and concentration for sEVs were 7.47 and 4.51%, respectively. Analysis of the linearity range revealed that this was narrower for sEVs when compared with that of liposomes. Owing to the use of liposomes that mimic the lipid composition and physicochemical properties of exosomes and proteinase-treated sEVs, it was demonstrated that these different analytical results could be possibly caused by the protein corona of sEVs. Consideration of the sample concentration and linearity range is important for obtaining reproducible and reliable data of sEVs.

Glyoxylate reductase/hydroxypyruvate reductase regulates the free d-aspartate level in mammalian cells.

Multiple d-amino acids are present in mammalian cells, and these compounds have distinctive physiological functions. Among the free d-amino acids identified in mammals, d-aspartate plays critical roles in the neuroendocrine and endocrine systems, as well as in the central nervous system. Mammalian cells have the molecular apparatus necessary to take up, degrade, synthesize, and release d-aspartate. In particular, d-aspartate is degraded by d-aspartate oxidase (DDO), a peroxisome-localized enzyme that catalyzes the oxidative deamination of d-aspartate to generate oxaloacetate, hydrogen peroxide, and ammonia. However, little is known about the molecular mechanisms underlying d-aspartate homeostasis in cells. In this study, we established a cell line that overexpresses cytoplasm-localized DDO; this cell line cannot survive in the presence of high concentrations of d-aspartate, presumably because high levels of toxic hydrogen peroxide are produced by metabolism of abundant d-aspartate by DDO in the cytoplasm, where hydrogen peroxide cannot be removed due to the absence of catalase. Next, we transfected these cells with a complementary DNA library derived from the human brain and screened for clones that affected d-aspartate metabolism and improved cell survival, even when the cells were challenged with high concentrations of d-aspartate. The screen identified a clone of glyoxylate reductase/hydroxypyruvate reductase (GRHPR). Moreover, the GRHPR metabolites glyoxylate and hydroxypyruvate inhibited the enzymatic activity of DDO. Furthermore, we evaluated the effects of GRHPR and peroxisome-localized DDO on d- and l-aspartate levels in cultured mammalian cells. Our findings show that GRHPR contributes to the homeostasis of these amino acids in mammalian cells.

Identification and biochemical characterization of threonine dehydratase from the hyperthermophile Thermotoga maritima.

The peptidoglycan of the hyperthermophile Thermotoga maritima contains an unusual component, D-lysine (D-Lys), in addition to the typical D-alanine (D-Ala) and D-glutamate (D-Glu). In a previous study, we identified a Lys racemase that is presumably associated with D-Lys biosynthesis. However, our understanding of D-amino acid metabolism in T. maritima and other bacteria remains limited, although D-amino acids in the peptidoglycan are crucial for preserving bacterial cell structure and resistance to environmental threats. Herein, we characterized enzymatic and structural properties of TM0356 that shares a high amino acid sequence identity with serine (Ser) racemase. The results revealed that TM0356 forms a tetramer with each subunit containing a pyridoxal 5'-phosphate as a cofactor. The enzyme did not exhibit racemase activity toward various amino acids including Ser, and dehydratase activity was highest toward L-threonine (L-Thr). It also acted on L-Ser and L-allo-Thr, but not on the corresponding D-amino acids. The catalytic mechanism did not follow typical Michaelis-Menten kinetics; it displayed a sigmoidal dependence on substrate concentration, with highest catalytic efficiency (kcat/K0.5) toward L-Thr. Interestingly, dehydratase activity was insensitive to allosteric regulators L-valine and L-isoleucine (L-Ile) at low concentrations, while these L-amino acids are inhibitors at high concentrations. Thus, TM0356 is a biosynthetic Thr dehydratase responsible for the conversion of L-Thr to α-ketobutyrate and ammonia, which is presumably involved in the first step of the biosynthesis of L-Ile.

Acidic pH-induced changes in lipid nanoparticle membrane packing.

To enable the release of the encapsulated nucleic acids into the cytosol of targeted cells, the interaction of lipid nanoparticles (LNPs) with endosomes is critical. We investigated changes in the physicochemical properties of LNPs containing ionizable cationic lipids that were induced by acidic pH, which reflects the conditions in the maturation of endosomes. We prepared a LNP containing an ionizable cationic lipid. The laurdan generalized polarization values, which are related to the hydration degree of the lipid membrane interface and are often used as an indicator of membrane packing, decreased with a decrease in pH value, showing that the membrane packing was decreased under acidic conditions. Furthermore, the pH-induced variation increased with an increasing percentage of ionizable cationic lipids in the LNPs. These results indicated that electrostatic repulsion between lipid molecules at acidic pH decreased the packing density of the lipids in the LNP membrane. Reducing the order of lipids could be a trigger to form a non-bilayer structure and allow fusion of the LNPs with the membrane of maturing endosomes in an acidic environment. The LNPs were used to incorporate and transport small interfering RNA (siRNA) into cells for knockdown of the expression of ß-galactosidase. The knockdown efficiency of siRNA encapsulated in LNPs tended to increase with the ratio of KC2. These results, which demonstrate the underlying phenomena for the fusion of membranes, will help clarify the mechanism of the release of encapsulated nucleic acids.

Detection of material-derived differences in the stiffness of egg yolk phosphatidylcholine-containing liposomes using atomic force microscopy.

Naturally sourced phospholipids are used in many liposomal pharmaceuticals. The present report describes a method to detect the effects of different egg yolk phosphatidylcholines (EPCs) on liposomal physicochemical properties. Five EPC-containing liposomes were prepared using five different EPCs obtained from different suppliers. There was no significant difference in purity between each EPC. The stiffness of the liposomes was examined via atomic force microscopy (AFM) in relation to the liposomal membrane permeability coefficient of encapsulated calcein after gel filtration, which is indicative of liposomal stability including the release of a hydrophilic drug from a liposome. Although the size of the liposome and the encapsulation efficiency of calcein did not significantly change with the type of EPC used, the liposome stiffness was found to vary depending on the EPC used, and liposomes with a similar stiffness were found to show a similar membrane permeability to calcein. Our results indicate the usefulness of stiffness measurement, using AFM as the analytical method, to detect material-derived differences in EPC-containing liposomes that affect drug release from the liposomes. Because drug release is one of the most important liposomal functions, combining this method with other analytical methods could be useful in selecting material for the development and quality control of EPC-containing liposomes.

Physicochemical Characterization of Liposomes That Mimic the Lipid Composition of Exosomes for Effective Intracellular Trafficking.

Exosomes mediate communication between cells in the body by the incorporation and transfer of biological materials. To design an artificial liposome, which would mimic the lipid composition and physicochemical characteristics of naturally occurring exosomes, we first studied the physicochemical properties of exosomes secreted from HepG2 cells. The exosome stiffness obtained by atomic force microscopy was moderate. Some liposomes were then fabricated to mimic the representative reported lipid composition of exosomes. Their physicochemical properties and cellular internalization efficiencies were investigated to optimize the cellular internalization efficiency of the liposomes. A favorable internalization efficiency was obtained by incubating HeLa cells with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol (Chol)/1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) (40/40/20 mol %) liposomes, which have a similar stiffness and zeta potential to exosomes. A dramatic increase in internalization efficiency was demonstrated by adding DOPS to simple DSPC/Chol liposomes. We found that DOPS had a more desirable effect on cellular internalization than its saturated lipid counterpart, 1,2-distearoyl-sn-glycero-3-phospho-l-serine. Furthermore, it was shown that the phosphatidylserine-binding protein, T-cell immunoglobulin mucin protein 4, was largely involved in the intracellular transfer of DSPC/Chol/DOPS liposomes. Thus, DOPS was a key lipid to provide the appropriate stiffness, zeta potential, and membrane surface affinity of the resulting liposome. Our results may help develop efficient drug carriers aiming to internalize active substances into cells.

Robust Nanoparticle Morphology and Size Analysis by Atomic Force Microscopy for Standardization.

Because of the complexity of nanomedicines, analysis of their morphology and size has attracted considerable attention both from researchers and regulatory agencies. The atomic force microscope (AFM) has emerged as a powerful tool because it can provide detailed morphological characteristics of nanoparticles both in the air and in aqueous medium. However, to our knowledge, AFM methods for nanomedicines have yet to be standardized or be listed in any pharmacopeias. To assess the applicability of standardization of AFM, in this study, we aimed to identify robust conditions for assessing the morphology and size of nanoparticles based on a polystyrene nanoparticle certified reference material standard. The spring constant of the cantilever did not affect the size of the nanoparticles but needed to be optimized depending on the measurement conditions. The size analysis method of the obtained images affected the results of the analyzed size values. The results analyzed by cross-sectional line profiling were independent of the measurement conditions and gave similar results to those from dynamic light scattering. It was indicated that approximately 100 particles are required for a representative measurement. Under the optimized conditions, there were no significant inter-instrument differences in the analyzed size values of polystyrene nanoparticles both in air and under aqueous conditions.

d-Serine and d-Alanine Regulate Adaptive Foraging Behavior in Caenorhabditis elegans via the NMDA Receptor.

d-Serine (d-Ser) is a coagonist for NMDA-type glutamate receptors and is thus important for higher brain function. d-Ser is synthesized by serine racemase and degraded by d-amino acid oxidase. However, the significance of these enzymes and the relevant functions of d-amino acids remain unclear. Here, we show that in the nematode Caenorhabditis elegans, the serine racemase homolog SERR-1 and d-amino acid oxidase DAAO-1 control an adaptive foraging behavior. Similar to many organisms, C. elegans immediately initiates local search for food when transferred to a new environment. With prolonged food deprivation, the worms exhibit a long-range dispersal behavior as the adaptive foraging strategy. We found that serr-1 deletion mutants did not display this behavior, whereas daao-1 deletion mutants immediately engaged in long-range dispersal after food removal. A quantitative analysis of d-amino acids indicated that d-Ser and d-alanine (d-Ala) are both synthesized and suppressed during food deprivation. A behavioral pharmacological analysis showed that the long-range dispersal behavior requires NMDA receptor desensitization. Long-term pretreatment with d-Ala, as well as with an NMDA receptor agonist, expanded the area searched by wild-type worms immediately after food removal, whereas pretreatment with d-Ser did not. We propose that d-Ser and d-Ala are endogenous regulators that cooperatively induce the long-range dispersal behavior in C. elegans through actions on the NMDA receptor.SIGNIFICANCE STATEMENT In mammals, d-serine (d-Ser) functions as an important neuromodulator of the NMDA-type glutamate receptor, which regulates higher brain functions. In Caenorhabditis elegans, previous studies failed to clearly define the physiological significance of d-Ser, d-alanine (d-Ala), and their metabolic enzymes. In this study, we found that these d-amino acids and their associated enzymes are active during food deprivation, leading to an adaptive foraging behavior. We also found that this behavior involved NMDA receptor desensitization.

A colorimetric assay method for measuring d-glutamate cyclase activity.

In mammals, metabolism of free d-glutamate is regulated by d-glutamate cyclase (DGLUCY), which reversibly converts d-glutamate to 5-oxo-d-proline and H2O. Metabolism of these d-amino acids by DGLUCY is thought to regulate cardiac function. In this study, we established a simple, accurate, and sensitive colorimetric assay method for measuring DGLUCY activity. To this end, we optimized experimental procedures for derivatizing 5-oxo-d-proline with 2-nitrophenylhydrazine hydrochloride. 5-Oxo-d-proline was derivatized with 2-nitrophenylhydrazine hydrochloride in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as a catalyst to generate the acid hydrazides, whose levels were then determined using a colorimetric method. Under optimized conditions, we examined the sensitivity and accuracy of the colorimetric method and compared our technique with other methods by high-performance liquid chromatography with ultraviolet-visible or fluorescence detection. Moreover, we assessed the suitability of this colorimetric method for measuring DGLUCY activity in biological samples. Our colorimetric method could determine DGLUCY activity with adequate validity and reliability. This method will help to elucidate the relationship among DGLUCY activity, the physiological and pathological roles of d-glutamate and 5-oxo-d-proline, and cardiac function.