DNA methylation and the opposing NMDAR dysfunction in schizophrenia and major depression disorders: a converging model for the therapeutic effects of psychedelic compounds in the treatment of psychiatric illness.

Psychedelic compounds are being increasingly explored as a potential therapeutic option for treating several psychiatric conditions, despite relatively little being known about their mechanism of action. One such possible mechanism, DNA methylation, is a process of epigenetic regulation that changes gene expression via chemical modification of nitrogenous bases. DNA methylation has been implicated in the pathophysiology of several psychiatric conditions, including schizophrenia (SZ) and major depressive disorder (MDD). In this review, we propose alterations to DNA methylation as a converging model for the therapeutic effects of psychedelic compounds, highlighting the N-methyl D-aspartate receptor (NMDAR), a crucial mediator of synaptic plasticity with known dysfunction in both diseases, as an example and anchoring point. We review the established evidence relating aberrant DNA methylation to NMDAR dysfunction in SZ and MDD and provide a model asserting that psychedelic substances may act through an epigenetic mechanism to provide therapeutic effects in the context of these disorders.

Acute Symptomatic Seizures and Risk of Seizure Recurrence in Patients with Anti-NMDAR, Anti-LGI1, and Anti-GABABR Encephalitis.

AIMS: To analyze the clinical characteristics of acute symptomatic seizures and predict the risk factors for seizure recurrence in patients with anti-N-methyl-D-aspartate receptor (NMDAR), anti-leucine-rich glioma-inactivated 1 (LGI1), and anti-gamma-aminobutyric acid B receptor (GABABR) encephalitis. METHODS: In this retrospective study, we included hospitalized patients who had been diagnosed with anti-NMDAR, anti-LGI1, and anti-GABABR encephalitis between November 2014 and April 2021. Binary logistic regression analysis was performed to identify the potential risk factors for seizure recurrence. RESULTS: In total, 262 patients with anti-NMDAR, anti-LGI1, and anti-GABABR encephalitis were included, 197 (75.2%) of whom presented with acute symptomatic seizures. During follow-up, 42 patients exhibited seizure recurrence. In anti-NMDAR encephalitis, frontal lobe abnormality on brain magnetic resonance imaging, delayed immunotherapy, early seizures, and focal motor onset were associated with seizure recurrence. CONCLUSIONS: Acute symptomatic seizure is a common clinical feature observed in patients with anti-NMDAR, anti-LGI1, and anti-GABABR encephalitis, with 50% of patients presenting with seizures as an initial symptom. The prognosis of patients with acute symptomatic seizures can be improved after receiving immunotherapy. Nevertheless, a minority of patients will experience seizure recurrence; therefore, restarting immunotherapy is recommended.

Nicotine downregulates miR-375-3p via neurotrophic tyrosine receptor kinase 2 to enhance the malignant behaviors of laryngopharyngeal squamous epithelial cells.

Nicotine exposure from smoking constitutes a significant global public health concern. Furthermore, smoking represents a pivotal risk factor for head and neck squamous cell carcinoma (HNSCC). However, the influence of nicotine on HNSCC remains relatively underexplored. Our aim was to unravel the molecular mechanisms that underlie the effect of nicotine on the metastatic cascade of HNSCC. In this study, we discovered a significant association between smoking and HNSCC metastasis and prognosis. Nicotine significantly enhanced HNSCC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro. Analysis of TCGA-HNSCC and FDEENT-HNSCC cohorts revealed reduced miR-375-3p levels in HNSCC tumor tissues, particularly among current smokers. Additionally, miR-375-3p level was strongly correlated with both lymph node metastasis and tumor stage. By downregulating miR-375-3p, nicotine promotes HNSCC cell metastasis in vitro and hematogenous metastatic capacity in vivo. Utilizing transcriptomic sequencing, molecular docking, dual-luciferase reporter assay, and fluorescence in situ hybridization (FISH), we demonstrated that miR-375-3p specifically binds to 3' untranslated region (3'UTR) of NTRK2 mRNA. Thus, this study uncovers a novel nicotine-induced mechanism involving miR-375-3p-mediated NTRK2 targeting, which promotes HNSCC metastasis. These findings have implications for improving the prognosis of patients with HNSCC, especially in smokers.

Lymphomatosis cerebri with coexistent anti-N-methyl-D-aspartate receptor antibody: A case report.

Diagnosis of lymphomatosis cerebri (LC) is usually delayed because of its rarity and the need for pathological confirmation. The association of LC with humoral immunity has scarcely been reported. Herein, we present a woman with a 2-week history of dizziness and gait ataxia, followed by diplopia, altered mental status, and spasticity of all limbs. Magnetic resonance imaging (MRI) of the brain showed multifocal lesions involving bilateral subcortical white matter, deep gray structures, and brainstem. Oligoclonal bands and anti-N-methyl-D-aspartate receptor (NMDAR) antibodies were present in cerebrospinal fluid (CSF) twice. She was initially treated with methylprednisolone but still worsening. A stereotactic brain biopsy confirmed the diagnosis of LC. This is a report on the distinctive coexistence of the rare CNS lymphoma variant and the anti-NMDAR antibody.

Conservation and coevolution determine evolvability of different classes of disordered residues in human intrinsically disordered proteins.

Structure, function, and evolution are interdependent properties of proteins. Diversity of protein functions arising from structural variations is a potential driving force behind protein evolvability. Intrinsically disordered proteins or regions (IDPs or IDRs) lack well-defined structure under normal physiological conditions, yet, they are highly functional. Increased occurrence of IDPs in eukaryotes compared to prokaryotes indicates strong correlation of protein evolution and disorderedness. IDPs generally have higher evolution rate compared to globular proteins. Structural pliability allows IDPs to accommodate multiple mutations without affecting their functional potential. Nevertheless, how evolutionary signals vary between different classes of disordered residues (DRs) in IDPs is poorly understood. This study addresses variation of evolutionary behavior in terms of residue conservation and intra-protein coevolution among structural and functional classes of DRs in IDPs. Analyses are performed on 579 human IDPs, which are classified based on length of IDRs, interacting partners and functional classes. We find short IDRs are less conserved than long IDRs or full IDPs. Functional classes which require flexibility and specificity to perform their activity comparatively evolve slower than others. Disorder promoting amino acids evolve faster than order promoting amino acids. Pro, Gly, Ile, and Phe have unique coevolving nature which further emphasizes on their roles in IDPs. This study sheds light on evolutionary footprints in different classes of DRs from human IDPs and enhances our understanding of the structural and functional potential of IDPs.

A Tar aspartate receptor and Rubisco-like protein substitute biotin in the growth of rhizobial strains.

Biotin is a key cofactor of metabolic carboxylases, although many rhizobial strains are biotin auxotrophs. When some of these strains were serially subcultured in minimal medium, they showed diminished growth and increased excretion of metabolites. The addition of biotin, or genetic complementation with biotin synthesis genes resulted in full growth of Rhizobium etli CFN42 and Rhizobium phaseoli CIAT652 strains. Half of rhizobial genomes did not show genes for biotin biosynthesis, but three-quarters had genes for biotin transport. Some strains had genes for an avidin homologue (rhizavidin), a protein with high affinity for biotin but an unknown role in bacteria. A CFN42-derived rhizavidin mutant showed a sharper growth decrease in subcultures, revealing a role in biotin storage. In the search of biotin-independent growth of subcultures, CFN42 and CIAT652 strains with excess aeration showed optimal growth, as they also did, unexpectedly, with the addition of aspartic acid analogues α- and N-methyl aspartate. Aspartate analogues can be sensed by the chemotaxis aspartate receptor Tar. A tar homologue was identified and its mutants showed no growth recovery with aspartate analogues, indicating requirement of the Tar receptor in such a phenotype. Additionally, tar mutants did not recover full growth with excess aeration. A Rubisco-like protein was found to be necessary for growth as the corresponding mutants showed no recovery either with high aeration or aspartate analogues; also, diminished carboxylation was observed. Taken together, our results indicate a route of biotin-independent growth in rhizobial strains that included oxygen, a Tar receptor and a previously uncharacterized Rubisco-like protein.

At least three xenon binding sites in the glycine binding domain of the N-methyl D-aspartate receptor.

Xenon can produce general anesthesia. Its main protein target is the N-methyl-D-aspartate receptor, a ionotropic channel playing a pivotal role in the function of the central nervous system. The molecular mechanisms allowing this noble gas to have such a specific effect remain obscure, probably as a consequence of the lack of structural data at the atomic level of detail. As a result of five independent molecular dynamics simulations, three different binding sites were found for xenon in the glycine binding domain of the N-methyl-D-aspartate receptor, the xenon binding constant being of the order of 2 108 s-1⋅M-1. On the other hand, the absolute binding free energy of xenon in these sites ranges between -3 and -14 kJ⋅mole-1. Noteworthy, it depends significantly upon the protein conformer chosen for performing the calculation, suggesting that larger values could be obtained, if other conformers were considered. These three sites are next to each other, one of them being next to the glycine site. This could noteworthy explain why the F758W and F758Y mutations can prevent competitive inhibition by xenon without affecting glycine binding.

Antibodies against N-Methyl D-Aspartate Receptor in Psychotic Disorders: A Systematic Review.

OBJECTIVE: The objective of this study was to provide comprehensive evidence synthesis including all available up-to-date data about the prevalence of N-methyl D-aspartate receptor (NMDAR) antibodies (ABs) in psychotic patients in order to evaluate the clinical relevance of ABs as well as to specify potential explanations of the heterogeneity of the findings and determine areas for further research. METHODS: A literature search was conducted using the PubMed/Medline, Web of Knowledge, and Scopus databases. RESULTS: Forty-seven studies and 4 systematic reviews (including 2 meta-analyses) were included in the present review. Studies that used cell-based assays (CBAs) provided heterogeneous results on AB prevalence, obviously depending on the type of detection assay and sample characteristics. Improvement of AB detection methods is necessary to determine the real prevalence of ABs across different groups of patients and healthy people. Live CBAs seem to have better sensitivity but probably poorer specificity than fixed CBAs. Moreover, some links between AB-positive status and acute symptoms are possible. A small amount of data on immunotherapy in AB-positive patients raises the possibility of its effectiveness but obviously require further research. CONCLUSIONS: NMDAR ABs are definitely present in a subset of psychotic patients. NMDAR ABs might shape psychosis and underlie some symptoms, and immunotherapy might be regarded as a treatment option for patients failing to respond to other therapies.

Reactive Oxygen Species Contributes to Type 2 Diabetic Neuropathic Pain via the Thioredoxin-Interacting Protein-NOD-Like Receptor Protein 3- N -Methyl-D-Aspartic Acid Receptor 2B Pathway.

BACKGROUND: The number of patients with diabetic neuropathic pain (DNP) continues to increase, but available treatments are limited. This study aimed to examine the influence of reactive oxygen species (ROS)-thioredoxin-interacting protein (TXNIP)-NOD-like receptor protein 3 (NLRP3)- N -methyl-D-aspartic acid receptor 2B (NR2B) pathway on type 2 DNP. METHODS: Male Sprague-Dawley rats were fed with a high-fat and high-sugar diet for 8 weeks. Then, rats were intraperitoneally injected with streptozotocin (STZ, 35 mg/kg) to induce type 2 diabetes mellitus in rats. Diabetic rats with <85% of their basic levels in mechanical withdrawal threshold and thermal withdrawal latency were classified as DNP rats on day 14 after STZ injection. DNP rats were treated with ROS scavenger N-tert-Butyl-α-phenylnitrone (PBN, 100 mg·kg -1 ·d -1 ) or TXNIP small interfering ribonucleic acid (10 µg/d) once daily for 14 days. The level of ROS, protein levels of NLRP3, TXNIP, cysteinyl aspartate-specific proteinase-1 (caspase-1), interleukin-1ß (IL-1ß), NR2B phosphorylation at Tyr1472 (p-NR2B), total NR2B (t-NR2B), and distribution of NLRP3 in the spinal cord were examined. In vitro experiments, BV2 cells and PC12 cells were individually cultured and cocultured in a high-glucose environment (35 mmol/L D-glucose). The level of ROS and protein levels of NLRP3, TXNIP, caspase-1, and IL-1ß in BV2 cells, and p-NR2B, t-NR2B in PC12 cells were detected. The level of ROS was detected by the flow cytometry approach. The protein levels were detected by the Western blot technique. The location of NLRP3 was observed by immunofluorescent staining. The interaction between TXNIP and NLRP3 was detected by coimmunoprecipitation assay. RESULTS: The level of spinal ROS increased in DNP rats. The mechanical allodynia and thermal hyperalgesia of DNP rats were alleviated after systemic administration of PBN. This administration decreased protein levels of NLRP3, TXNIP, caspase-1, IL-1ß, and p-NR2B and the coupling of TXNIP to NLRP3 in spinal cords of DNP rats. Furthermore, knockdown of spinal TXNIP alleviated nociceptive hypersensitivity and decreased protein levels of NLRP3, TXNIP, caspase-1, IL-1ß, and p-NR2B in DNP rats. The level of ROS and protein levels of NLRP3, TXNIP, caspase-1, IL-1ß, the coupling of TXNIP to NLRP3, and the IL-1ß secretion increased in BV2 cells, and the protein expression of p-NR2B increased in cocultured PC12 cells in a high-glucose environment. All of these in vitro effects were significantly blocked after treatment of PBN. CONCLUSIONS: Our findings suggest that spinal ROS can contribute to type 2 DNP through TXNIP-NLRP3-NR2B pathway.

Phenol-Benzoxazolone bioisosteres: Synthesis and biological evaluation of tricyclic GluN2B-selective N-methyl- d-aspartate receptor antagonists.

Tricyclic tetrahydrooxazolo[4,5-h]-[3]benzazepin-9-ols 22 were designed as phenol bioisosteres of tetrahydro-3-benzazepine-1,7-diols. Key features of the synthesis are the introduction of the trifluoromethylsulfonyl and allyl protective groups at the heterocyclic N-atoms. Two methods were developed to convert the triflyl-protected ketone 16 into tricyclic alcohols 21 bearing various N-substituents. According to the first method, trifluoromethanesulfinate was removed by K2 CO3 . Following the selective reduction of the imino moiety of 17 with NaBH(OAc)3 afforded the aminoketone 18, which was reductively alkylated and reduced. According to the second method, both the imine and the ketone of the iminoketone 17 were reduced with NaBH4 to yield the aminoalcohol 20, which was alkylated or reductively alkylated to form tertiary amines 21f-21r. In the last step, the allyl protective group of 21 was removed with RhCl3 and HCl to obtain oxazolones 22. In receptor binding studies using [3 H]ifenprodil as radioligand ketone, 22m showed the highest GluN2B affinity (Ki = 88 nM). However, a reduced affinity toward GluN2B subunit-containing N-methyl- d-aspartate (NMDA) receptors was observed for oxazolones 22 compared to bioisosteric 3-benzazepine-1,7-diols. High selectivity of 22m for the ifenprodil binding site of GluN2B-NMDA receptors over the 1-(1-phenylcyclohexyl)piperidine binding site and σ2 receptors was observed, but only negligible selectivity over σ1 receptors. In two-electrode voltage clamp experiments, the 4-phenylbutyl derivative 22d (Ki = 422 nM) demonstrated 80% inhibition of ion flux at a concentration of 1 µM. The differences in GluN2B affinity and inhibitory activity are explained by docking studies. In conclusion, 22d is regarded as a novel scaffold of highly potent GluN1/GluN2B antagonists.

N-methyl d-aspartate receptor hypofunction reduces visual contextual integration.

Visual cognition is finely tuned to the elements in a scene but also relies on contextual integration to improve visual detection and discrimination. This integration is impaired in patients with schizophrenia. Studying impairments in contextual integration may lead to biomarkers of schizophrenia, tools to monitor disease progression, and, in animal models, insight into the underlying neural deficits. We developed a nonhuman primate model to test the hypothesis that hypofunction of the N-methyl d-aspartate receptor (NMDAR) impairs contextual integration. Two male rhesus macaques (Macaca mulatta) were trained to indicate which of two patterns on the screen had the highest contrast. One of these patterns appeared in isolation, and the other was surrounded by a high-contrast pattern. In humans, this high-contrast context is known to lead to an underestimation of contrast. This so-called Chubb illusion is thought to result from surround suppression, a key contextual integration mechanism. To test the involvement of NMDAR in this process, we compared animals' perceptual bias with and without intramuscular injections of a subanesthetic dose of the NMDAR antagonist ketamine. In the absence of ketamine, the animals reported a Chubb illusion - matching reports in healthy humans. Hence, monkeys - just like humans - perform visual contextual integration. This reaffirms the importance of nonhuman primates to help understand visual cognition. Injection of ketamine significantly reduced the strength of the illusion and thus impaired contextual integration. This supports the hypothesis that NMDAR hypofunction plays a causal role in specific behavioral impairments observed in schizophrenia.

Lithium Enhances the GABAergic Synaptic Activities on the Hypothalamic Preoptic Area (hPOA) Neurons.

Lithium (Li+) salt is widely used as a therapeutic agent for treating neurological and psychiatric disorders. Despite its therapeutic effects on neurological and psychiatric disorders, it can also disturb the neuroendocrine axis in patients under lithium therapy. The hypothalamic area contains GABAergic and glutamatergic neurons and their receptors, which regulate various hypothalamic functions such as the release of neurohormones, control circadian activities. At the neuronal level, several neurotransmitter systems are modulated by lithium exposure. However, the effect of Li+ on hypothalamic neuron excitability and the precise action mechanism involved in such an effect have not been fully understood yet. Therefore, Li+ action on hypothalamic neurons was investigated using a whole-cell patch-clamp technique. In hypothalamic neurons, Li+ increased the GABAergic synaptic activities via action potential independent presynaptic mechanisms. Next, concentration-dependent replacement of Na+ by Li+ in artificial cerebrospinal fluid increased frequencies of GABAergic miniature inhibitory postsynaptic currents without altering their amplitudes. Li+ perfusion induced inward currents in the majority of hypothalamic neurons independent of amino-acids receptor activation. These results suggests that Li+ treatment can directly affect the hypothalamic region of the brain and regulate the release of various neurohormones involved in synchronizing the neuroendocrine axis.

Expression patterns of L-amino acid receptors in the murine STC-1 enteroendocrine cell line.

Regulation of gut function depends on the detection and response to luminal contents. Luminal L-amino acids (L-AA) are detected by several receptors including metabotropic glutamate receptors 1 and 4 (mGluR1 and mGluR4), calcium-sensing receptor (CaSR), GPRC family C group 6 subtype A receptor (GPRC6A) and umami taste receptor heterodimer T1R1/T1R3. Here, we show that murine mucosal homogenates and STC-1 cells, a murine enteroendocrine cell line, express mRNA for all L-AA receptors. Immunohistochemical analysis demonstrated the presence of all L-AA receptors on STC-1 with CaSR being most commonly expressed and T1R1 least expressed (35% versus 15% of cells); mGluRs and GPRC6a were intermediate (~ 20% of cells). Regarding coexpression of L-AA receptors, the mGluRs and T1R1 were similarly coexpressed with CaSR (10-12% of cells) whereas GPRC6a was coexpressed least (7% of cells). mGluR1 was coexpressed with GPRC6a in 11% of cells whereas coexpression between other receptors was less (2-8% of cells). CaSR and mGluR1 were coexpressed with glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) in 20-25% of cells whereas T1R1 and GPRC6a were coexpressed with GLP-1 and PYY less (8-12% of cells). Only mGluR4 showed differential coexpression with GLP-1 (13%) and PYY (21%). L-Phenylalanine (10 mM) caused a 3-fold increase in GLP-1 release, which was strongly inhibited by siRNA to CaSR indicating functional coupling of CaSR to GLP-1 release. The results suggest that not all STC-1 cells express (and coexpress) L-AA receptors to the same extent and that the pattern of response likely depends on the pattern of expression of L-AA receptors.

Flexible Hinges in Bacterial Chemoreceptors.

Transmembrane bacterial chemoreceptors are extended, rod-shaped homodimers with ligand-binding sites at one end and interaction sites for signaling complex formation and histidine kinase control at the other. There are atomic-resolution structures of chemoreceptor fragments but not of intact, membrane-inserted receptors. Electron tomography of in vivo signaling complex arrays lack distinct densities for chemoreceptor rods away from the well-ordered base plate region, implying structural heterogeneity. We used negative staining, transmission electron microscopy, and image analysis to characterize the molecular shapes of intact homodimers of the Escherichia coli aspartate receptor Tar rendered functional by insertion into nanodisc-provided E. coli lipid bilayers. Single-particle analysis plus tomography of particles in a three-dimensional matrix revealed two bend loci in the chemoreceptor cytoplasmic domain, (i) a short, two-strand gap between the membrane-proximal, four-helix-bundle HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemoreceptors, and phosphatases) domain and the membrane-distal, four-helix coiled coil and (ii) aligned glycines in the extended, four-helix coiled coil, the position of a bend noted in the previous X-ray structure of a receptor fragment. Our images showed HAMP bends from 0° to ∼13° and glycine bends from 0° to ∼20°, suggesting that the loci are flexible hinges. Variable hinge bending explains indistinct densities for receptor rods outside the base plate region in subvolume averages of chemotaxis arrays. Bending at flexible hinges was not correlated with the chemoreceptor signaling state. However, our analyses showed that chemoreceptor bending avoided what would otherwise be steric clashes between neighboring receptors that would block the formation of core signaling complexes and chemoreceptor arrays.IMPORTANCE This work provides new information about the shape of transmembrane bacterial chemoreceptors, crucial components in the molecular machinery of bacterial chemotaxis. We found that intact, lipid-bilayer-inserted, and thus functional homodimers of the Escherichia coli chemoreceptor Tar exhibited bends at two flexible hinges along their ∼200-Å, rod-like, cytoplasmic domains. One hinge was at the short, two-strand gap between the membrane-proximal, four-helix-bundle HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemoreceptors, and phosphatases) domain and the membrane-distal, four-helix coiled coil. The other hinge was at aligned glycines in the extended, four-helix coiled coil, where a bend had been identified in the X-ray structure of a chemoreceptor fragment. Our analyses showed that flexible hinge bending avoided structural clashes in chemotaxis core complexes and their arrays.

Low-Intensity Ultrasound Decreases Ischemia-Induced Edema by Inhibiting N-Methyl-d-Aspartic Acid Receptors.

BACKGROUND: We have previously shown that low-intensity ultrasound (LIUS), a noninvasive mechanical stimulus, inhibits brain edema formation induced by oxygen and glucose deprivation (OGD) or treatment with glutamate, a mediator of OGD-induced edema, in acute rat hippocampal slice model in vitro. METHODS: In this study, we treated the rat hippocampal slices with N-methyl-d-aspartic acid (NMDA) or (S)-3,5-dihydroxyphenylglycine (DHPG) to determine whether these different glutamate receptor agonists induce edema. The hippocampal slices were then either sonicated with LIUS or treated with N-methyl-d-aspartic acid receptor (NMDAR) antagonists, namely, MK-801 and ketamine, and observed their effects on edema formation. RESULTS: We observed that treatment with NMDA, an agonist of ionotropic glutamate receptors, induced brain edema at similar degrees compared with that induced by OGD. However, treatment with DHPG, an agonist of metabotropic glutamate receptors, did not significantly induce brain edema. Treatment with the NMDAR antagonists MK-801 or ketamine efficiently prevented brain edema formation by both OGD and NMDA in a concentration-dependent manner. N-Methyl-d-aspartic acid-induced brain edema was alleviated by LIUS in an intensity-dependent manner when ultrasound was administered at 30, 50, or 100 mW/cm2 for 20 minutes before the induction of the edema. Furthermore, LIUS reduced OGD- and NMDA-induced phosphorylation of NMDARs at Y1325. CONCLUSION: These results suggest that LIUS can inhibit OGD- or NMDA-induced NMDAR activation by preventing NMDAR phosphorylation, thereby reducing a subsequent brain edema formation. The mechanisms by which LIUS inhibits NMDAR phosphorylation need further investigation.

Methionine and valine activate the mammalian target of rapamycin complex 1 pathway through heterodimeric amino acid taste receptor (TAS1R1/TAS1R3) and intracellular Ca2+ in bovine mammary epithelial cells.

Amino acids play a key role in regulating milk protein synthesis partly through activation of the mammalian target of rapamycin (mTOR) signaling pathway. However, the involvement of extracellular AA sensing receptors in this process is not well understood. In nonruminants, it is well established that the AA taste 1 receptor member 1/3 (TAS1R1/TAS1R3) heterodimer contributes to the sensing of most l-AA. Whether this receptor is functional in bovine mammary cells is unknown. The objective of this study was to determine essential AA signaling through TAS1R1/TAS1R3 and their roles in regulating mTOR signaling pathway and casein mRNA abundance in primary bovine mammary epithelial cells and the Mac-T cell line. The bovine mammary epithelial cells were stimulated with complete Dulbecco's modified Eagle's medium (+EAA), medium without EAA (-EAA), or medium supplemented with only 1 of the 10 essential AA, respectively. The nonessential AA levels were the same across all treatments. Small interference RNA targeting TAS1R1 were designed and transfected into bovine primary mammary epithelial cells (bPMEC). Supplementation of a complete mixture of essential AA or Arg, Val, Leu, His, Phe, Met, and Ile individually led to greater mTOR phosphorylation. Phosphorylation of ribosomal protein S6 kinase ß-1 was greater in the presence of Val, Leu, Trp, Met, and Ile. Valine, Leu, Met, and Ile led to greater eIF4E-binding protein 1 phosphorylation. Although +EAA and a few individual AA tested induced increases in intracellular calcium, Met and Val were the most potent. Knockdown of TAS1R1 decreased intracellular calcium in bPMEC cultured with both Val and Met. Phosphorylation of mTOR, ribosomal protein S6 kinase ß-1, and eIF4E-binding protein 1 was lower when TAS1R1 was knocked-down in bPMEC supplemented with Val and Met. In addition, small interference RNA silencing of TAS1R1 resulted in lower ß-casein (CSN2) abundance. The TAS1R1/TAS1R3 receptor may sense extracellular AA and activate mTOR signaling in bovine mammary cells, likely by elevating intracellular calcium concentration. This mechanism appears to have a role in Met- and Val-induced changes in CSN2 mRNA abundance. Further in vivo studies will have to be performed to assess the relevance of this mechanism in the mammary gland.

Overexpression of the leucine-rich receptor-like kinase gene LRK2 increases drought tolerance and tiller number in rice.

Drought represents a key limiting factor of global crop distribution. Receptor-like kinases play major roles in plant development and defence responses against stresses such as drought. In this study, LRK2, which encodes a leucine-rich receptor-like kinase, was cloned and characterized and found to be localized on the plasma membrane in rice. Promoter-GUS analysis revealed strong expression in tiller buds, roots, nodes and anthers. Transgenic plants overexpressing LRK2 exhibited enhanced tolerance to drought stress due to an increased number of lateral roots compared with the wild type at the vegetative stage. Moreover, ectopic expression of LRK2 seedlings resulted in increased tiller development. Yeast two-hybrid screening and bimolecular fluorescence complementation (BiFC) indicated a possible interaction between LRK2 and elongation factor 1 alpha (OsEF1A) in vitro. These results suggest that LRK2 functions as a positive regulator of the drought stress response and tiller development via increased branch development in rice. These findings will aid our understanding of branch regulation in other grasses and support improvements in rice genetics.

Reduced dietary protein level influences the free amino acid and gene expression profiles of selected amino acid transceptors in skeletal muscle of growing pigs.

This study was conducted to evaluate the effect of reduced dietary protein level on growth performance, muscle mass weight, free amino acids (FAA) and gene expression profile of selected amino acid transceptors in different fibre type of skeletal muscle tissues (longissimus dorsi, psoas major, biceps femoris) of growing pigs. A total of 18 cross-bred growing pigs (Large White × Landrace × Duroc) with initial body weight (9.57 ± 0.67 kg) were assigned into three dietary treatments: 20% crude protein (CP) diet (normal recommended, NP), 17% CP diet (low protein, LP) and 14% CP diet (very low protein, VLP). The results indicated improved feed-to-gain ratio was obtained for pigs fed LP and NP diets (p < 0.01), while the pigs fed VLP diet showed the worst growth performance (p < 0.01). There was no significant difference in the weights of longissimus dorsi and psoas major muscle between LP and NP groups (p > 0.05). Majority of the determined FAA concentration of LP group were greater than or equal to those of NP group in both longissimus dorsi and psoas major muscle (p < 0.01). Further, the mRNA expression levels of sodium-coupled neutral amino acid transceptor 2, L-type amino acid transceptor 1 and proton-assisted amino acid transceptors 2 were higher in skeletal muscle tissue in LP group compared to those of the pigs fed NP or VLP diet. These results suggested that reduced dietary protein level (3 points of percentage less than recommended level) would upregulate the mRNA expression of amino acid transceptors to enhance the absorption of FAA in skeletal muscle of growing pigs. There seems to be a relationship between response of AA transceptors to the dietary protein level in skeletal muscle tissue of different fibre type. To illustrate the underlying mechanisms will be beneficial to animal nutrition.

Structural Analysis of the Ligand-Binding Domain of the Aspartate Receptor Tar from Escherichia coli.

The Escherichia coli cell-surface aspartate receptor Tar mediates bacterial chemotaxis toward an attractant, aspartate (Asp), and away from a repellent, Ni(2+). These signals are transmitted from the extracellular region of Tar to the cytoplasmic region via the transmembrane domain. The mechanism by which extracellular signals are transmitted into the cell through conformational changes in Tar is predicted to involve a piston displacement of one of the α4 helices of the homodimer. To understand the molecular mechanisms underlying the induction of Tar activity by an attractant, the three-dimensional structures of the E. coli Tar periplasmic domain with and without bound aspartate, Asp-Tar and apo-Tar, respectively, were determined. Of the two ligand-binding sites, only one site was occupied, and it clearly showed the electron density of an aspartate. The slight changes in conformation and the electrostatic surface potential around the aspartate-binding site were observed. In addition, the presence of an aspartate stabilized residues Phe-150' and Arg-73. A pistonlike displacement of helix α4b' was also induced by aspartate binding as predicted by the piston model. Taken together, these small changes might be related to the induction of Tar activity and might disturb binding of the second aspartate to the second binding site in E. coli.