Specific inhibitory effects of exogenous d-Aspartate on the proliferation of intestinal epithelial cells.

d-amino acids have been actively examined since improved analytical techniques revealed their presence in animal bodies. Although D-Asp was identified in mammals earlier than D-Ser, research on D-Asp has lagged behind that on D-Ser, mainly because the target protein of D-Asp remains unknown. To date, the only reported functions of D-Asp are its roles in reproduction and suggested neuromodulatory functions. Since d-amino acids are also present in food, it is important to clarify their effects on gastrointestinal epithelial cells, which are always contacted after ingestion. Therefore, the present study examined the effects of d-amino acids on gastrointestinal tract basal cells. The effects of 11 types of amino acids (Ala, Arg, Asn, Asp, Gln, Glu, Leu, Lys, Pro, Ser, and Val) on the proliferation of three types of gastrointestinal epithelial cells (HGC-27, IEC-6, and Caco-2) were assessed. Although the proliferation of HGC-27 and Caco-2 was not affected by any of the 11 types of L- and d-amino acids, D-Asp inhibited the proliferation of IEC-6, derived from small intestinal epithelial cells, in concentration- and exposure time-dependent manners. The present study also examined uptake transporters, metabolic enzymes, and insulin signaling pathways; however, the mechanisms underlying the inhibitory effects of D-Asp on the proliferation of IEC-6 were not elucidated. A more detailed understanding of these mechanisms may lead to the development of pharmaceuticals as main drugs or formulation materials. Further studies are warranted on the physiological effects of d-amino acids, including D-Asp.

Neuroprotective effect of whey protein hydrolysate containing leucine-aspartate-isoleucine-glutamine-lysine on HT22 cells in hydrogen peroxide-induced oxidative stress.

This study aimed to investigate the neuroprotective effects of whey protein hydrolysate (WPH) containing the pentapeptide leucine-aspartate-isoleucine-glutamine-lysine (LDIQK). Whey protein hydrolysate (50, 100, and 200 µg/mL) demonstrated the ability to restore the viability of HT22 cells subjected to 300 µM hydrogen peroxide (H2O2)-induced oxidative stress. Furthermore, at a concentration of 200 µg/mL, it significantly reduced the increase in reactive oxygen species production and calcium ion (Ca2+) influx induced by H2O2 by 46.1% and 46.2%, respectively. Similarly, the hydrolysate significantly decreased the levels of p-tau, a hallmark of tauopathy, and BCL2 associated X (BAX), a proapoptosis factor, while increasing the protein levels of choline acetyltransferase (ChAT), an enzyme involved in acetylcholine synthesis, brain-derived neurotrophic factor (BDNF), a nerve growth factor, and B-cell lymphoma 2 (BCL2, an antiapoptotic factor. Furthermore, it increased nuclear factor erythroid 2-related factor 2 (Nrf2)-hemoxygenase-1(HO-1) signaling, which is associated with the antioxidant response, while reducing the activation of mitogen-activated protein kinase (MAPK) signaling pathway components, namely phosphor-extracellular signal-regulated kinases (p-ERK), phosphor-c-Jun N-terminal kinases (p-JNK), and p-p38. Column chromatography and tandem mass spectrometry analysis identified LDIQK as a compound with neuroprotective effects in WPH; it inhibited Ca2+ influx and regulated the BAX/BCL2 ratio. Collectively, WPH containing LDIQK demonstrated neuroprotective effects against H2O2-induced neuronal cell damage, suggesting that WPH or its active peptide, LDIQK, may serve as a potential edible agent for improving cognitive dysfunction.

Anticandidal Effect of New Imidazole Derivatives Over Aspartic Protease Inhibition.

Candidiasis is one of the most serious microbial infections in the world. One of the main virulence factors for Candida albicans is the crucial secretion of aspartic proteases (Saps). Saps are hydrolytic enzymes that play a major role in many fungal pathophysiological processes as well as in many levels of the associations between the fungus and its host. In this work, we report on the synthesis, characterization, and anti-candida agent evaluation of a family of 13 imidazolidine-based aspartate protease inhibitors. In vitro and in silico enzyme inhibition studies have confirmed these compounds' ability to inhibit fungal aspartate protease. Based on the molecular mechanistic value scores from molecular docking and MD simulations, we selected the top compounds 5b (binding energy -13.90 kcal/mol) and 5m (binding energy -12.94 kcal/mol) from among 5a-l based on the molecular mechanistic value scores from molecular docking and MD simulations for use in in vitro validations. In the results, imidazolidine derivatives showed strong aspartic protease inhibition activity. In conclusion, compounds 5b and 5m were found as potent anti-candida agents and screened for further pre-clinical and clinical validations.

Synthesis and Anti-Liver Fibrosis Research of Aspartic Acid Derivatives.

Liver fibrosis plays an important role in the progression of liver disease, but there is a severe shortage of direct and efficacious pharmaceutical clinical interventions. Literature research indicates that aspartic acid exhibits hepatoprotective properties. In this paper, 32 target compounds were designed and synthesized utilizing aspartic acid as the lead compound, of which 22 were new compounds not reported in the literature. These compounds were screened for their inhibitory effects on the COL1A1 promoter to assess in vitro anti-liver fibrosis activity and summarized structure-activity relationships. Four compounds exhibited superior potency with inhibition rates ranging from 66.72% to 97.44%, substantially higher than EGCG (36.46 ± 4.64%) and L-Asp (11.33 ± 0.35%). In an LPS-induced inflammation model of LX-2 cells, both 41 and 8a could inhibit the activation of LX-2 cells, reducing the expression of COL1A1, fibronectin, and α-SMA. Upon further investigation, 41 and 8a ameliorated liver fibrosis by inhibiting the IKKß-NF-κB signaling pathway to alleviate inflammatory response. Overall, the study evaluated the anti-liver fibrosis effects of aspartic acid derivatives, identified the potency of 41, and conducted a preliminary exploration of mechanisms, laying the foundation for the discovery of novel anti-liver fibrosis agents.

FN1 mediated activation of aspartate metabolism promotes the progression of triple-negative and luminal a breast cancer.

BACKGROUND: Breast cancer (BC) is regarded as one of the most common cancers diagnosed among the female population and has an extremely high mortality rate. It is known that Fibronectin 1 (FN1) drives the occurrence and development of a variety of cancers through metabolic reprogramming. Aspartic acid is considered to be an important substrate for nucleotide synthesis. However, the regulatory mechanism between FN1 and aspartate metabolism is currently unclear. METHODS: We used RNA sequencing (RNA seq) and liquid chromatography-mass spectrometry to analyze the tumor tissues and paracancerous tissues of patients. MCF7 and MDA-MB-231 cells were used to explore the effects of FN1-regulated aspartic acid metabolism on cell survival, invasion, migration and tumor growth. We used PCR, Western blot, immunocytochemistry and immunofluorescence techniques to study it. RESULTS: We found that FN1 was highly expressed in tumor tissues, especially in Lumina A and TNBC subtypes, and was associated with poor prognosis. In vivo and in vitro experiments showed that silencing FN1 inhibits the activation of the YAP1/Hippo pathway by enhancing YAP1 phosphorylation, down-regulates SLC1A3-mediated aspartate uptake and utilization by tumor cells, inhibits BC cell proliferation, invasion and migration, and promotes apoptosis. In addition, inhibition of FN1 combined with the YAP1 inhibitor or SLC1A3 inhibitor can effectively inhibit tumor growth, of which inhibition of FN1 combined with the YAP1 inhibitor is more effective. CONCLUSION: Targeting the "FN1/YAP1/SLC1A3/Aspartate metabolism" regulatory axis provides a new target for BC diagnosis and treatment. This study also revealed that intratumoral metabolic heterogeneity plays an important role in the progression of different subtypes of breast cancer.

Mutation of aspartic acid 262 on estrogen receptor abrogates estradiol signaling pathway.

OBJECTIVE: ERα (estrogen receptor alpha) exerts nuclear genomic actions and membrane-initiated non-genomic effects. The mutation of aspartic acid into alanine in vitro revealed the critical role of aspartic acid 258 (corresponding to mouse amino acid site 262) of ERα for non-nuclear function. Our previous in vitro study revealed that this mutation blocked estrogen's non-genomic effects on vascular endothelial H2S release. Here, we studied the in vivo role of the aspartic acid 262 of ERα in the reproductive system and in the vascular tissue. APPROACH AND RESULTS: We generated a mouse model harboring a point mutation of the murine counterpart of this aspartic acid into alanine (ERαD262A). Our results showed that the ERαD262A females are fertile with standard hormonal serum levels, but the uterine development and responded with estrogen and follicular development are disrupted. In line with our previous study, we found that the rapid dilation of the aorta was abrogated in ERαD262A mice. In contrast to the previously reported R264-ERα mice, the classical estrogen genomic effector SP1/NOS3/AP1 and the nongenomic effectors p-eNOs, p-AKT, and p-ERK were disturbed in the ERαD262A aorta. Besides, the serum H2S concentration was decreased in ERαD262A mice. Together, ERαD262A mice showed compromised both genomic and non-genomic actions in response to E2. CONCLUSIONS: These data showed that aspartic acid 262 of ERα are important for both genomic and non-genomic effects of E2. Our data provide a theoretical basis for further selecting an effective non-genomic mouse model and provide a new direction for developing estrogen non-genomic effect inhibitors.

Impact of dietary supplementation with N-carbamoyl-aspartic acid on serum metabolites and intestinal microflora of sows.

BACKGROUND: N-Carbamoyl-aspartic acid (NCA) is a critical precursor for de novo biosynthesis of pyrimidine nucleotides. To investigate the cumulative effects of maternal supplementation with NCA on the productive performance, serum metabolites and intestinal microbiota of sows, 40 pregnant sows (∼day 80) were assigned into two groups: (1) the control (CON) and (2) treatment (NCA, 50 g t-1 NCA). RESULTS: Results showed that piglets from the NCA group had heavier birth weight than those in the CON group (P < 0.05). In addition, maternal supplementation with NCA decreased the backfat loss of sows during lactation (P < 0.05). Furthermore,16S-rRNA sequencing results revealed that maternal NCA supplementation decreased the abundance of Cellulosilyticum, Fournierella, Anaerovibrio, and Oribacterium genera of sows during late pregnancy (P < 0.05). Similarly, on the 14th day of lactation, maternal supplementation with NCA reduced the diversity of fecal microbes of sows as evidenced by significantly lower observed species, Chao1, and Ace indexes, and decreased the abundance of Lachnospire, Faecalibacterium, and Anaerovorax genera, while enriched the abundance of Catenisphaera (P < 0.05). Untargeted metabolomics showed that a total of 48 differentially abundant biomarkers were identified, which were mainly involved in metabolic pathways of arginine/proline metabolism, phenylalanine/tyrosine metabolism, and fatty acid biosynthesis, etc. CONCLUSION: Overall, the results indicated that NCA supplementation regulated intestinal microbial composition of sows and serum differential metabolites related to arginine, proline, phenylalanine, tyrosine, and fatty acids metabolism that may contribute to regulating the backfat loss of sows, and the birth weight and diarrhea rate of piglets. © 2022 Society of Chemical Industry.

Aspergillomarasmine A inhibits metallo-ß-lactamases by selectively sequestering Zn2.

Class B metallo-ß-lactamases (MBLs) are Zn2+-dependent enzymes that catalyze the hydrolysis of ß-lactam antibiotics to confer resistance in bacteria. Several problematic groups of MBLs belong to subclass B1, including the binuclear New Delhi MBL (NDM), Verona integrin-encoded MBL, and imipenemase-type enzymes, which are responsible for widespread antibiotic resistance. Aspergillomarasmine A (AMA) is a natural aminopolycarboxylic acid that functions as an effective inhibitor of class B1 MBLs. The precise mechanism of action of AMA is not thoroughly understood, but it is known to inactivate MBLs by removing one catalytic Zn2+ cofactor. We investigated the kinetics of MBL inactivation in detail and report that AMA is a selective Zn2+ scavenger that indirectly inactivates NDM-1 by encouraging the dissociation of a metal cofactor. To further investigate the mechanism in living bacteria, we used an active site probe and showed that AMA causes the loss of a Zn2+ ion from a low-affinity binding site of NDM-1. Zn2+-depleted NDM-1 is rapidly degraded, contributing to the efficacy of AMA as a ß-lactam potentiator. However, MBLs with higher metal affinity and stability such as NDM-6 and imipenemase-7 exhibit greater tolerance to AMA. These results indicate that the mechanism of AMA is broadly applicable to diverse Zn2+ chelators and highlight that leveraging Zn2+ availability can influence the survival of MBL-producing bacteria when they are exposed to ß-lactam antibiotics.

Long noncoding RNA OVAAL enhances nucleotide synthesis through pyruvate carboxylase to promote 5-fluorouracil resistance in gastric cancer.

5-Fluorouracil (5-FU) is widely used in gastric cancer treatment, yet 5-FU resistance remains an important clinical challenge. We established a model based on five long noncoding RNAs (lncRNA) to effectively assess the prognosis of gastric cancer patients; among them, lncRNA OVAAL was markedly upregulated in gastric cancer and associated with poor prognosis and 5-FU resistance. In vitro and in vivo assays confirmed that OVAAL promoted proliferation and 5-FU resistance of gastric cancer cells. Mechanistically, OVAAL bound with pyruvate carboxylase (PC) and stabilized PC from HSC70/CHIP-mediated ubiquitination and degradation. OVAAL knockdown reduced intracellular levels of oxaloacetate and aspartate, and the subsequent pyrimidine synthesis, which could be rescued by PC overexpression. Moreover, OVAAL knockdown increased sensitivity to 5-FU treatment, which could be reversed by PC overexpression or repletion of oxaloacetate, aspartate, or uridine. OVAAL overexpression enhanced pyrimidine synthesis to promote proliferation and 5-FU resistance of gastric cancer cells, which could be abolished by PC knockdown. Thus, OVAAL promoted gastric cancer cell proliferation and induced 5-FU resistance by enhancing pyrimidine biosynthesis to antagonize 5-FU induced thymidylate synthase dysfunction. Targeting OVAAL-mediated nucleotide metabolic reprograming would be a promising strategy to overcome chemoresistance in gastric cancer.

Metabolic Remodeling Promotes Cardiac Hypertrophy by Directing Glucose to Aspartate Biosynthesis.

RATIONALE: Hypertrophied hearts switch from mainly using fatty acids (FAs) to an increased reliance on glucose for energy production. It has been shown that preserving FA oxidation (FAO) prevents the pathological shift of substrate preference, preserves cardiac function and energetics, and reduces cardiomyocyte hypertrophy during cardiac stresses. However, it remains elusive whether substrate metabolism regulates cardiomyocyte hypertrophy directly or via a secondary effect of improving cardiac energetics. OBJECTIVE: The goal of this study was to determine the mechanisms of how preservation of FAO prevents the hypertrophic growth of cardiomyocytes. METHODS AND RESULTS: We cultured adult rat cardiomyocytes in a medium containing glucose and mixed-chain FAs and induced pathological hypertrophy by phenylephrine. Phenylephrine-induced hypertrophy was associated with increased glucose consumption and higher intracellular aspartate levels, resulting in increased synthesis of nucleotides, RNA, and proteins. These changes could be prevented by increasing FAO via deletion of ACC2 (acetyl-CoA-carboxylase 2) in phenylephrine-stimulated cardiomyocytes and in pressure overload-induced cardiac hypertrophy in vivo. Furthermore, aspartate supplementation was sufficient to reverse the antihypertrophic effect of ACC2 deletion demonstrating a causal role of elevated aspartate level in cardiomyocyte hypertrophy. 15N and 13C stable isotope tracing revealed that glucose but not glutamine contributed to increased biosynthesis of aspartate, which supplied nitrogen for nucleotide synthesis during cardiomyocyte hypertrophy. CONCLUSIONS: Our data show that increased glucose consumption is required to support aspartate synthesis that drives the increase of biomass during cardiac hypertrophy. Preservation of FAO prevents the shift of metabolic flux into the anabolic pathway and maintains catabolic metabolism for energy production, thus preventing cardiac hypertrophy and improving myocardial energetics.

The pncA gene mutations of Mycobacterium tuberculosis in multidrug-resistant tuberculosis.

The pncA gene encodes pyrazinamidase enzyme which converts drug pyrazinamide to active form pyrazinoic acid, but mutations in this gene can prevent enzyme activity which leads to pyrazinamide resistance. The cross-sectional study was carried out during 2016-2017 for 12 months. The purpose of the study was to detect mutation at codon 12 and codon 85 in the pncA gene in local multidrug-resistant tuberculosis (MDR-TB) patients by developing a simple molecular test so that disease could be detected timely in the local population. DNA extracted from sputum-cultured samples from MDR-TB patients and subjected to semi-multiplex allele-specific PCR by using self-designed primers against the pncA gene. Among 75 samples, 53 samples were subjected to molecular analysis based on purified DNA quantity and quality. The primers produced 250 and 480 bp fragments, indicating the mutations at codon 12 (aspartate to alanine) and codon 85 (leucine to proline) respectively. MDR-TB was more common in the age group 21-40 years. Fifty-seven percent of samples (n = 30) were found positive for pncA mutations, whereas 43% of samples (n = 23) showed negative results. Thirteen percent of samples (n = 4) had mutations at codon 12 in which aspartate was converted to alanine, and they produced an amplified product of 480 bp. Eighty-seven percent of samples (n = 26) had mutations at codon 85 in which leucine was converted to proline and amplified product size was 250 bp. The mutations were simple nucleotide substitutions. The prevalence of mutations in which leucine was substituted by proline was higher than the mutations in which aspartate was substituted by alanine. A high prevalence of substitution mutation (CTG → CCG; leucine to proline) was detected in MDR-TB cases. Earlier detection of MDR-TB via an effective molecular diagnostic method can control the MDR tuberculosis spread in the population.

Milk metabolome reveals pyrimidine and its degradation products as the discriminant markers of different corn silage-based nutritional strategies.

The purpose of this study was to evaluate the effect of 6 different feeding systems (based on corn silage as the main ingredient) on the chemical composition of milk and to highlight the potential of untargeted metabolomics to find discriminant marker compounds of different nutritional strategies. Interestingly, the multivariate statistical analysis discriminated milk samples mainly according to the high-moisture ear corn (HMC) included in the diet formulation. Overall, the most discriminant compounds, identified as a function of the HMC, belonged to AA (10 compounds), peptides (71 compounds), pyrimidines (38 compounds), purines (15 compounds), and pyridines (14 compounds). The discriminant milk metabolites were found to significantly explain the metabolic pathways of pyrimidines and vitamin B6. Interestingly, pathway analyses revealed that the inclusion of HMC in the diet formulation strongly affected the pyrimidine metabolism in milk, determining a significant up-accumulation of pyrimidine degradation products, such as 3-ureidopropionic acid, 3-ureidoisobutyric acid, and 3-aminoisobutyric acid. Also, some pyrimidine intermediates (such as l-aspartic acid, N-carbamoyl-l-aspartic acid, and orotic acid) were found to possess a high discrimination degree. Additionally, our findings suggested that the inclusion of alfalfa silage in the diet formulation was potentially correlated with the vitamin B6 metabolism in milk, being 4-pyridoxic acid (a pyridoxal phosphate degradation product) the most significant and up-accumulated compound. Taken together, the accumulation trends of different marker compounds revealed that both pyrimidine intermediates and degradation products are potential marker compounds of HMC-based diets, likely involving a complex metabolism of microbial nitrogen based on total splanchnic fluxes from the rumen to mammary gland in dairy cows. Also, our findings highlight the potential of untargeted metabolomics in both foodomics and foodomics-based studies involving dairy products.

Toxicity effects of chlorantraniliprole in zebrafish (Danio rerio) involving in liver function and metabolic phenotype.

Chlorantraniliprole (CAP), a representative bisamide insecticide, is widely used in rice fields around the world, posing potential toxicity risks to aquatic organisms. In this study, we examined the effects of exposure to CAP on growth and metabolic phenotype of zebrafish (Danio rerio) and oxidative stress and apoptosis in the liver of zebrafish (Danio rerio). First, we identified that CAP had a low bioaccumulation in zebrafish. Subsequently, growth phenotype analysis revealed that CAP could significantly increase liver weight and liver index in zebrafish. In addition, we found that CAP exposure could cause significant changes in indicators of oxidative stress, resulting in a significant increase in the content of malondialdehyde (MDA), causing oxidative stress in the liver of zebrafish. Meanwhile, the expression levels of apoptosis-related genes were also significantly changed and apoptosis was promoted in the liver of zebrafish with CAP exposure. Importantly, the results of metabolomics analysis shown that CAP exposure could significantly disrupt the metabolic phenotype of zebrafish, interfering with multiple metabolic pathways, mainly including valine, leucine and isoleucine biosynthesis and degradation, alanine, aspartate and glutamate metabolism and d-glutamine and D-glutamate metabolism. Last but not least, correlation analysis identified strong links between changes in liver function involving oxidative stress and apoptosis and changes in metabolic phenotype of zebrafish following CAP exposure. In brief, these results indicate that potential environmental risks of CAP to aquatic organisms should receive more attention.

In Silico Identification of Potential Insect Peptides against Biofilm-Producing Staphylococcus aureus.

Biofilm-producing Staphylococcus aureus (SA) strains are frequently found in medical environments, from surgical/ wound sites, medical devices. These biofilms reduce the efficacy of applied antibiotics during the treatment of several infections, such as cystic fibrosis, endocarditis, or urinary tract infections. Thus, the development of potential therapeutic agents to destroy the extra protective biofilm layers or to inhibit the biofilm-producing enzymes is urgently needed. Advanced and cost-effective bioinformatics tools are advantageous in locating and speeding up the selection of antibiofilm candidates. Based on the potential drug characteristics, we have selected one-hundred thirty-three antibacterial peptides derived from insects to assess for their antibiofilm potency via molecular docking against five putative biofilm formation and regulated target enzymes: the staphylococcal accessory regulator A or SarA (PDB ID: 2FRH), 4,4'-diapophytoene synthase or CrtM (PDB ID: 2ZCQ), clumping factor A or ClfA (PDB ID: 1N67) and serine-aspartate repeat protein C or SdrC (PDB ID: 6LXH) and sortase A or SrtA (PDB ID: 1T2W) of SA bacterium. In this study, molecular docking was performed using HPEPDOCK and HDOCK servers, and molecular interactions were examined using BIOVIA Discovery Studio Visualizer-2019. The docking score (kcal/mol) range of five promising antibiofilm peptides against five targets was recorded as follows: diptericin A (-215.52 to -303.31), defensin (-201.11 to -301.92), imcroporin (-212.08 to -287.64), mucroporin (-228.72 to -286.76), apidaecin II (-203.90 to -280.20). Among these five, imcroporin and mucroporin were 13 % each, while defensin contained only 1 % of positive net charged residues (Arg+Lys) projected through ProtParam and NetWheels tools. Similarly, imcroporin, mucroporin and apidaecin II were 50 %, while defensin carried 21.05 % of hydrophobic residues predicted by the tool PEPTIDE. 2.0. Most of the peptides exhibited potential characteristics to inhibit S. aureus-biofilm formation via disrupting the cell membrane and cytoplasmic integrity. In summary, the proposed hypothesis can be considered a cost-effective platform for selecting the most promising bioactive drug candidates within a limited timeframe with a greater chance of success in experimental and clinical studies.

Uncovering the Metabolic Origin of Aspartate for Tumor Growth Using an Integrated Molecular Deactivator.

Reprogrammed glucose metabolism is vital for cancer cells, but aspartate, an intermediate metabolic product, is the limiting factor for cancer cell proliferation. However, due to the complexity of metabolic pathways, it remains unclear whether glucose is the primary source of endogenous aspartate. Here, we report the design of an innovative molecular deactivator, based on a multifunctional upconversion nanoprobe, to explore the link between glucose and aspartate. This molecular deactivator mainly works in the acidic, hypoxic tumor microenvironment and deactivates multiple types of glucose transporters on cancer cell membranes upon illumination at 980 nm. Cancer cell proliferation in vivo is strongly inhibited by blocking glucose transporters. Our experimental data confirm that the cellular synthesis of aspartate for tumor growth is glucose-dependent. This work also demonstrates the untapped potential of molecularly engineered upconversion nanoprobes for discovering hidden metabolic pathways and improving therapeutic efficacy of conventional antitumor drugs.

Testing the Role of Glutamate NMDA Receptors in Peripheral Trigeminal Nociception Implicated in Migraine Pain.

The pro-nociceptive role of glutamate in the CNS in migraine pathophysiology is well established. Glutamate, released from trigeminal afferents, activates second order nociceptive neurons in the brainstem. However, the function of peripheral glutamate receptors in the trigeminovascular system suggested as the origin site for migraine pain, is less known. In the current project, we used calcium imaging and patch clamp recordings from trigeminal ganglion (TG) neurons, immunolabelling, CGRP assay and direct electrophysiological recordings from rat meningeal afferents to investigate the role of glutamate in trigeminal nociception. Glutamate, aspartate, and, to a lesser extent, NMDA under free-magnesium conditions, evoked calcium transients in a fraction of isolated TG neurons, indicating functional expression of NMDA receptors. The fraction of NMDA sensitive neurons was increased by the migraine mediator CGRP. NMDA also activated slowly desensitizing currents in 37% of TG neurons. However, neither glutamate nor NMDA changed the level of extracellular CGRP. TG neurons expressed both GluN2A and GluN2B subunits of NMDA receptors. In addition, after removal of magnesium, NMDA activated persistent spiking activity in a fraction of trigeminal nerve fibers in meninges. Thus, glutamate activates NMDA receptors in somas of TG neurons and their meningeal nerve terminals in magnesium-dependent manner. These findings suggest that peripherally released glutamate can promote excitation of meningeal afferents implicated in generation of migraine pain in conditions of inherited or acquired reduced magnesium blockage of NMDA channels and support the usage of magnesium supplements in migraine.

Synthesis of Tetrasubstituted Phosphorus Analogs of Aspartic Acid as Antiproliferative Agents.

An efficient general method for the synthesis of a wide family of α-aminophosphonate analogs of aspartic acid bearing tetrasubstituted carbons is reported through an aza-Reformatsky reaction of α-iminophosphonates, generated from α-aminophosphonates, in an umpolung process. In addition, the α-aminophosphonate substrates showed in vitro cytotoxicity, inhibiting the growth of carcinoma human tumor cell lines A549 (carcinomic human alveolar basal epithelial cell) and SKOV3 (human ovarian carcinoma). In view of the possibilities in the diversity of the substituents that offer the synthetic methodology, an extensive profile structure-activity is presented, measuring IC50 values up to 0.34 µM in the A549 and 9.8 µM in SKOV3 cell lines.

Fish proteins as potential precursors of taste-active compounds: an in silico study.

BACKGROUND: Fish protein is a good source of amino acids and peptides with sensory properties. Theoretically, the type of protein affects the taste quality of the protein hydrolysates. To better use fish protein in the food ingredients industry, an in silico approach was adopted to evaluate the potential of fish protein to release taste-active compounds. RESULTS: Six types of protein from seven commercial fishes were screened from the Uniprot knowledge base. The results showed that a remarkable number of umami fragments presented in myosin and parvalbumin (PB), such as glutamic acid (Glu), aspartic acid (Asp), and Asp- and Glu- containing peptides, whereas sweet amino acids and bitter peptides (e.g., Pro- and Gly- containing peptides) were mainly found in collagen (CGI) in all fish samples. After the in silico proteolysis by papain, a difference in the profile of taste-active fragments was observed among the six types of proteins. Amino acids were the main hydrolysis products of these proteins, especially umami, sweet, and bitter amino acids, significantly contributing to the taste formation of protein hydrolysates. Besides, the myosin and CGI hydrolysates were abundant in taste active peptides both in types and quantities. CONCLUSION: Myosin is a promising protein source for producing umami fragments, and CGI seems to be a good precursor of sweet and bitter fragments. Different types of protein have an essential effect on the taste of protein hydrolysates. © 2022 Society of Chemical Industry.

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.

A mathematical model for Escherichia coli chemotaxis to competing stimuli.

Chemotactic bacteria sense and respond to temporal and spatial gradients of chemical cues in their surroundings. This phenomenon plays a critical role in many microbial processes such as groundwater bioremediation, microbially enhanced oil recovery, nitrogen fixation in legumes, and pathogenesis of the disease. Chemical heterogeneity in these natural systems may produce numerous competing signals from various directions. Predicting the migration behavior of bacterial populations under such conditions is necessary for designing effective treatment schemes. In this study, experimental studies and mathematical models are reported for the chemotactic response of Escherichia coli to a combination of attractant (α-methylaspartate) and repellent (NiCl2 ), which bind to the same transmembrane receptor complex. The model describes the binding of chemoeffectors and phosphorylation of the kinase in the signal transduction mechanism. Chemotactic parameters of E. coli (signaling efficiency σ , stimuli sensitivity coefficient γ , and repellent sensitivity coefficient κ ) were determined by fitting the model with experimental results for individual stimuli. Interestingly, our model naturally identifies NiCl2 as a repellent for κ>1 . The model is capable of describing quantitatively the response to the individual attractant and repellent, and correctly predicts the change in direction of bacterial population migration for competing stimuli with a twofold increase in repellent concentration.