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The superfamily of acid proteases has two catalytic aspartates for proteolysis of their peptide substrates. Here, we show a minimal structural scaffold, the structural catalytic core (SCC), which is conserved within each family of acid proteases, but varies between families, and thus can serve as a structural marker of four individual protease families. The SCC is a dimer of several structural blocks, such as the DD-link, D-loop, and G-loop, around two catalytic aspartates in each protease subunit or an individual chain. A dimer made of two (D-loop + DD-link) structural elements makes a DD-zone, and the D-loop + G-loop combination makes a psi-loop. These structural markers are useful for protein comparison, structure identification, protein family separation, and protein engineering. [ABSTRACT FROM AUTHOR]

A comprehensive study was performed on the supramolecular ordering and optical properties of thin nanostructured glycerohydrogel sol-gel plates based on chitosan L- and D-aspartate and their individual components in the X-ray, UV, visible, and IR ranges. Our comparative analysis of chiroptical characteristics, optical collimated transmittance, the average cosine of the scattering angle, microrelief and surface asymmetry, and the level of structuring shows a significant influence of the wavelength range of electromagnetic radiation and the enantiomeric form of aspartic acid on the functional characteristics of the sol-gel materials. At the macrolevel of the supramolecular organization, a complex topography of the surface layer and a dense amorphous–crystalline ordering of polymeric substances were revealed, while at the nanolevel, there were two forms of voluminous scattering domains: nanospheres with diameters of 60–120 nm (L-) and 45–55 nm (D-), anisometric particles of lengths within ~100–160 (L-) and ~85–125 nm (D-), and widths within ~10–20 (L-) and ~20–30 nm (D-). The effect of optical clearing on glass coated with a thin layer of chitosan L-(D-)aspartate in the near-UV region was discovered (observed for the first time for chitosan-based materials). The resulting nanocomposite shape-stable glycerohydrogels seem promising for sensorics and photonics. [ABSTRACT FROM AUTHOR]

Symbiosis receptor-like kinase SYMRK is required for root nodule symbiosis between legume plants and nitrogen-fixing bacteria. To understand symbiotic signaling from SYMRK, we determined the crystal structure to 1.95 Å and mapped the phosphorylation sites onto the intracellular domain. We identified four serine residues in a conserved "alpha-I" motif, located on the border between the kinase core domain and the flexible C-terminal tail, that, when phosphorylated, drives organogenesis. Substituting the four serines with alanines abolished symbiotic signaling, while substituting them with phosphorylation-mimicking aspartates induced the formation of spontaneous nodules in the absence of bacteria. These findings show that the signaling pathway controlling root nodule organogenesis is mediated by SYMRK phosphorylation, which may help when engineering this trait into non-legume plants. [ABSTRACT FROM AUTHOR]

Interactions in enzymes between catalytic and neighboring amino acids and how these interactions facilitate catalysis are examined. In examples from both natural and designed enzymes, it is shown that increases in catalytic rates may be achieved through elongation of the buffer range of the catalytic residues; such perturbations in the protonation equilibria are, in turn, achieved through enhanced coupling of the protonation equilibria of the active ionizable residues with those of other ionizable residues. The strongest coupling between protonation states for a pair of residues that deprotonate to form an anion (or a pair that accept a proton to form a cation) is achieved when the difference in the intrinsic pKas of the two residues is approximately within 1 pH unit. Thus, catalytic aspartates and glutamates are often coupled to nearby acidic residues. For an anion-forming residue coupled to a cation-forming residue, the elongated buffer range is achieved when the intrinsic pKa of the anion-forming residue is higher than the intrinsic pKa of the (conjugate acid of the) cation-forming residue. Therefore, the high pKa, anion-forming residues tyrosine and cysteine make good coupling partners for catalytic lysine residues. For the anion–cation pairs, the optimum difference in intrinsic pKas is a function of the energy of interaction between the residues. For the energy of interaction ε expressed in units of (ln 10)RT, the optimum difference in intrinsic pKas is within ∼1 pH unit of ε. [ABSTRACT FROM AUTHOR]

Nitric oxide (•NO), a diffusible free radical, is an intercellular messenger, playing a crucial role in several key brain physiological processes, including in neurovascular coupling (NVC). In the brain, glutamatergic activation of the neuronal nitric oxide synthase (nNOS) enzyme constitutes its main synthesis pathway. However, when oxygen (O2) supply is compromised, such as in stroke, ischemia, and aging, such •NO production pathway may be seriously impaired. In this context, evidence suggests that, as already observed in the gastric compartment, the reduction of nitrite by dietary compounds (such as ascorbate and polyphenols) or by specific enzymes may occur in the brain, constituting an important rescuing or complementary mechanism of •NO production. Here, using microsensors selective for •NO, we show that nitrite enhanced the •NO production in a concentration-dependent manner and in the presence of ascorbate evoked by N-methyl-D-aspartate (NMDA) and glutamate stimulation of rat hippocampal slices. Additionally, nitrite potentiated the •NO production induced by oxygen-glucose deprivation (OGD). Overall, these observations support the notion of a redox interaction of ascorbate with nitrite yielding •NO upon neuronal glutamatergic activation and given the critical role of NO as the direct mediator of neurovascular coupling may represents a key physiological mechanism by which •NO production for cerebral blood flow (CBF) responses to neuronal activation is sustained under hypoxic/acidic conditions in the brain. [ABSTRACT FROM AUTHOR]

The crystal structure of NH4[Zn(cma)(H2O)2]·H2O (cma3– = N-carboxymethylaspartate(3–)) is determined by single crystal X-ray structure analysis. The orthorombic crystals (P212121, a = 7.7901(4) Å, b = 11.2368(4) Å, c = 13.2048(5) Å, α = β = γ = 90°, Z = 4) were obtained from the reaction mixture in the form of racemic conglomerate. The single crystal X-ray structure analysis revealed the maximum deviation of bond angles around the Zn atom from an ideal octahedral geometry 14.09° with ∑ = 67.23° and Θ = 236.69°. Intermolecular interactions are based mainly on a moderate N–H⋯O and O–H⋯O hydrogen bonds. The structure shares similar structural features with other structures containing aspartates and their derivatives as a ligands. The results of using different HAR methods based on semi-empirical (B3LYP) and non-empirical (PBE0) global hybrid GGA DFT functionals were compared. The crystal structure of NH4[Zn(cma)(H2O)2]·H2O (cma3– = N-carboxymethylaspartate(3–)) is determined by single crystal X-ray structure analysis. The orthorombic crystals (P212121, a = 7.7901(4) Å, b = 11.2368(4) Å, c = 13.2048(5) Å, α = β = γ = 90°, Z = 4) were obtained from the reaction mixture in the form of racemic conglomerate. [ABSTRACT FROM AUTHOR]

Compared to folded proteins, the sequences of intrinsically disordered proteins (IDPs) are enriched in polar and charged amino acids. Glutamate is one of the most enriched amino acids in IDPs, while the chemically similar amino acid aspartate is less enriched. So far, the underlying functional differences between glutamates and aspartates in IDPs remain poorly understood. In this study, we examine the differential effects of aspartate and glutamates in IDPs by comparing the function and conformational ensemble of glutamate and aspartate variants of the disordered protein Dss1, using a range of assays, including interaction studies, nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and molecular dynamics simulation. First, we analyze the sequences of the rapidly growing database of experimentally verified IDPs (DisProt) and show that glutamate enrichment is not caused by a taxonomy bias in IDPs. From analyses of local and global structural properties as well as cell growth and protein-protein interactions using a model acidic IDP from yeast and three Glu/Asp variants, we find that while the Glu/Asp variants support similar function and global dimensions, the variants differ in their binding affinities and population of local transient structural elements. We speculate that these local structural differences may play roles in functional diversity, where glutamates can support increased helicity, important for folding and binding, while aspartates support extended structures and form helical caps, as well as playing more relevant roles in, e.g., transactivation domains and ion-binding. [ABSTRACT FROM AUTHOR]

Salinity is the most variable ecological parameter in the lagoon with daily and seasonal variations . The changes in growth, serum biochemistry and antioxidant enzymes activities of Cardisoma armatum were examined during 84-day exposure to five different sublethal salinities (0, 5, 10, 15 and 20 ppt). At the start of the trial, the crab initial average body weight was not significantly different (P > 0.05). The highest weight gain was recorded in 15 ppt (47.40±1.01 g), followed by 20 ppt, 10 ppt and 5 ppt with no significant differences between them. At the end of 84 days experiment, the crab exhibited the lowest body weight growth (48.29 %) at 0 ppt and the highest (81.04±1.08%) at 10 ppt. The 10 ppt treatment had the highest specific growth rates (0.31±0.11 %/day), followed by 5 ppt, 20 ppt, 15 ppt, and then 0 ppt treatments. Aspartate and alanine aminotransferases significantly decreased after 0 ppt, but mean serum protein value increased with salinity increase. Greater activities of catalase, superoxide dismutase and malondialdehyde were recorded in 5-20 ppt. All the antioxidant enzyme activities (with the exception of glutathione) showed significant differences. Thus, deviation from the brackish water condition adversely affects the growth and immune functions of the gecarcinid, resulting in population decline in reclaimed wetlands. [ABSTRACT FROM AUTHOR]

Copper(I) is an essential metal for all life forms. Though Cu(II) is the most abundant and stable state, its reduction to Cu(I) via an unclear mechanism is prerequisite for its bioutilization. In eukaryotes, the copper transporter-1 (CTR1) is the primary highaffinity copper importer, although its mechanism and role in Cu(II) reduction remain uncharacterized. Here we show that extracellular amino-terminus of human CTR1 contains two methionine-histidine clusters and neighboring aspartates that distinctly bind Cu(I) and Cu(II) preceding its import. We determined that hCTR1 localizes at the basolateral membrane of polarized MDCK-II cells and that its endocytosis to Common-Recycling-Endosomes is regulated by reduction of Cu(II) to Cu(I) and subsequent Cu(I) coordination by the methionine cluster. Wedemonstrate the transient binding of both Cu(II) and Cu(I) during the reduction process is facilitated by aspartates that also act as another crucial determinant of hCTR1 endocytosis. Mutating the first Methionine cluster (7Met-Gly-Met9) and Asp13 abrogated copper uptake and endocytosis upon copper treatment. This phenotype could be reverted by treating the cells with reduced and nonreoxidizable Cu(I). We show that histidine clusters, on other hand, bind Cu(II) and are crucial for hCTR1 functioning at limiting copper. Finally, we show that two Nterminal His-Met-Asp clusters exhibit functional complementarity, as the second cluster is sufficient to preserve copperinduced CTR1 endocytosis upon complete deletion of the first cluster. We propose a novel and detailed mechanism by which the two His-Met-Asp residues of hCTR1 amino-terminus not only bind copper, but also maintain its reduced state, crucial for intracellular uptake. [ABSTRACT FROM AUTHOR]

Dicarboxylic amino acid-based surfactants (N-dodecyl derivatives of -aminomalonate, -aspartate, and -glutamate) in combination with hexadecyltrimethylammonium bromide (HTAB) form a variety of aggregates. Composition and concentration-dependent mixtures exhibit liquid crystal, gel, precipitate, and clear isotropic phases. Liquid crystalline patterns, formed by surfactant mixtures, were identified by polarizing optical microscopy. FE-SEM studies reveal the existence of surface morphologies of different mixed aggregates. Phase transition and associated weight loss were found to depend on the composition where thermotropic behaviours were revealed through combined differential scanning calorimetry and thermogravimetric studies. Systems comprising more than 60 mol% HTAB demonstrate shear-thinning behaviour. Gels cause insignificant toxicity to human peripheral lymphocytes and irritation to bare mouse skin; they do not display the symptoms of cutaneous irritation, neutrophilic invasion, and inflammation (erythema, edema, and skin thinning) as evidenced by cumulative irritancy index score. Gels also exhibit substantial antibacterial effects on Staphylococcus aureus, a potent causative agent of skin and soft tissue infections, suggesting its possible application as a vehicle for topical dermatological drug delivery. [ABSTRACT FROM AUTHOR]

Background. With an increase in antibiotic resistance in microorganisms, increasing the effectiveness of antibiotic therapy has become a crucial task for medical and pharmaceutical sciences. One possible way to increase the effectiveness of antibiotic therapy is to monitor biologically active substances like amino acids for their capability of increasing the activity of antibiotics. This research aimed to study the effects of tryptophan and zinc aspartate on the activity of doxycycline in relation to E. coli and P. aeruginosa. Material and methods. The in vitro serial dilution method was used to determine the minimum inhibitory concentration (MIC) of doxycycline independently and in combination with tryptophan and zinc aspartate, for E. coli and P. aeruginosa. Results. Tryptophan and zinc aspartate at a concentration of 2 μg/ml significantly increased the antibiotic activity of doxycycline against E. coli and P. aeruginosa (p<0.0001). Conclusions. Biologically active substances, tryptophan and zinc aspartate have a modulating effect on the antibiotic sensitivity of microorganisms towards antibacterial drugs like doxycycline. [ABSTRACT FROM AUTHOR]

Corneal innervation plays a major role in the pathobiology of diabetic corneal disease. However, innervation impact has mainly been investigated in the context of diabetic epitheliopathy and wound healing. Further studies are warranted in the corneal stroma-nerve interactions. This study unravels the nerve influence on corneal stroma metabolism. Corneal stromal cells were isolated from healthy (HCFs) and diabetes mellitus (Type1DM and Type2 DM) donors. Cells were cultured on polycarbonate membranes, stimulated by stable Vitamin C, and stroma-only and stroma-nerve co-cultures were investigated for metabolic alterations. Innervated compared to stroma-only constructs exhibited significant alterations in pyrimidine, glycerol phosphate shuttle, electron transport chain and glycolysis. The most highly altered metabolites between healthy and T1DMs innervated were phosphatidylethanolamine biosynthesis, and pyrimidine, methionine, aspartate metabolism. Healthy and T2DMs main pathways included aspartate, glycerol phosphate shuttle, electron transport chain, and gluconeogenesis. The metabolic impact on T1DMs and T2DMs was pyrimidine, purine, aspartate, and methionine. Interestingly, the glucose-6-phosphate and oxaloacetate was higher in T2DMs compared to T1DMs. Our in vitro co-culture model allows the examination of key metabolic pathways corresponding to corneal innervation in the diabetic stroma. These novel findings can pave the way for future studies to fully understand the metabolic distinctions in the diabetic cornea. [ABSTRACT FROM AUTHOR]

It is well-established that the secondary active transporters GltTk and GltPh catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified GltTk, and derived the rate equation for a mechanism in which two sodium ions bind before and another after aspartate. Re-analysis of existing data on GltPh using this equation allowed for determination of the turnover number (0.14 s−1), without the need for error-prone protein quantification. To overcome the complication that purified transporters may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding the kinetic analysis, we employed a rapid method using synthetic nanobodies to inactivate one population. Oppositely oriented GltTk proteins showed the same transport kinetics, consistent with the use of an identical gating element on both sides of the membrane. Our work underlines the value of bona fide transport experiments to reveal mechanistic features of Na+-aspartate symport that cannot be observed in detergent solution. Combined with previous pre-equilibrium binding studies, a full kinetic mechanism of structurally characterized aspartate transporters of the SLC1A family is now emerging. Trinco et al. measure aspartate uptake rates in proteoliposomes containing purified prokaryotic Na+-coupled aspartate transporter GltTk. To overcome limitation of protein orientation, they use synthetic nanobody that blocks transporters from outside and reveal mechanistic features of Na+-aspartate symport that cannot be observed in detergent solution. [ABSTRACT FROM AUTHOR]

Ischemic stroke has become an important cause of death and disability for residents worldwide. At present, the main treatment methods are thrombolysis and embolectomy. However, a large number of patients miss the window period of thrombolysis and embolectomy or have other conditions that are not suitable for thrombolysis. Therefore, cerebral protective drugs or bioactive substances are needed to protect neural tissue and improve the prognosis. However, there is still a lack of strong and effective drugs or bioactive substances for selection. Osteopontin (OPN) is a phosphorylated glycoprotein that plays an important role in cell adhesion, differentiation, survival and repair by binding to cellular integrin receptors and inducing signal transmission. Numerous studies have shown that osteopontin can protect brain tissue and reduce brain injury caused by ischemic stroke by various means such as promoting angiogenesis, and promoting neural stem cell migration in the pathogenesis of ischemic stroke. [ABSTRACT FROM AUTHOR]

Cells have two purine pathways that synthesize adenine and guanine ribonucleotides from phosphoribose via inosylate. A chemical hybrid between adenine and guanine, 2-aminoadenine (Z), replaces adenine in the DNA of the cyanobacterial virus S-2L. We show that S-2L and Vibrio phage PhiVC8 encode a third purine pathway catalyzed by PurZ, a distant paralog of succinoadenylate synthase (PurA), the enzyme condensing aspartate and inosylate in the adenine pathway. PurZ condenses aspartate with deoxyguanylate into dSMP (N6-succino-2-amino-2′-deoxyadenylate), which undergoes defumarylation and phosphorylation to give dZTP (2-amino-2′-deoxyadenosine-5′-triphosphate), a substrate for the phage DNA polymerase. Crystallography and phylogenetics analyses indicate a close relationship between phage PurZ and archaeal PurA enzymes. Our work elucidates the biocatalytic innovation that remodeled a DNA building block beyond canonical molecular biology. [ABSTRACT FROM AUTHOR]

Seizures are common in patients with gliomas; however, the mechanisms of epileptogenesis in gliomas have not been fully understood. This study hypothesized that analyzing quantified metabolites using magnetic resonance spectroscopy (MRS) might provide novel insights to better understand the epileptogenesis in gliomas, and specific metabolites might be indicators of preoperative seizures in gliomas. We retrospectively investigated patient information (gender, age at diagnosis of tumor, their survival time) and tumor information (location, histology, genetic features, and metabolites according to MRS) in patients with gliomas. The data were correlated with the incidence of seizure and analyzed statistically. Of 146 adult supratentorial gliomas, isocitrate dehydrogenase (IDH) mutant tumors significantly indicated higher incidence of preoperative seizures than IDH wild-type gliomas. However, MRS study indicated that glutamate concentration in IDH wild-type gliomas was higher than that in IDH mutant gliomas. Glutamate was not associated with high frequency of preoperative seizures in patients with gliomas. Instead, increased total N-acetyl-l-aspartate (tNAA) was significantly associated with them. Moreover, multivariable analysis indicated that increased level of tNAA was an independent predictor of preoperative seizures. According to MRS analysis, tNAA, rather than glutamate, might be a useful to detect preoperative seizures in patient with supratentorial gliomas. [ABSTRACT FROM AUTHOR]

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d-Aspartate (d-Asp) and d-serine (d-Ser) have been proposed to promote early-phase LTP in vitro and to enhance spatial memory in vivo. Here, we investigated the behavioural effects of chronic consumption of d-Asp and d-Ser on spatial learning of mice together with the expression of NMDA receptors. We also studied the alterations of neurogenesis by morphometric analysis of bromo-deoxyuridine incorporating and doublecortin expressing cells in the hippocampus. Our results specify a time period (3–4 h post-training), within which the animals exposed to d-Asp (but not d-Ser) show a more stable memory during retrieval. The cognitive improvement is due to elimination of transient bouts of destabilization and reconsolidation of memory, rather than to enhanced acquisition. d-Asp also protracted reversal learning probably due to reduced plasticity. Expression of GluN1 and GluN2A subunits was elevated in the hippocampus of d-Asp (but not d-Ser) treated mice. d-Asp or d-Ser did not alter the proliferation of neuronal progenitor cells in the hippocampus. The observed learning-related changes evoked by d-Asp are unlikely to be due to enhanced proliferation and recruitment of new neurones. Rather, they are likely associated with an upregulation of NMDA receptors, as well as a reorganization of receptor subunit assemblies in existing hippocampal/dentate neurons. [ABSTRACT FROM AUTHOR]

The serine protease Tk‐subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis possesses three insertion loops (IS1‐IS3) on its surface, as compared to its mesophilic counterparts. Although IS1 and IS2 are required for maturation of Tk‐subtilisin at high temperatures, the role of IS3 remains unknown. Here, CD spectroscopy revealed that IS3 deletion arrested Tk‐subtilisin folding at an intermediate state, in which the central nucleus was formed, but the subsequent folding propagation into terminal subdomains did not occur. Alanine substitution of the aspartate residue in IS3 disturbed the intraloop hydrogen‐bonding network, as evidenced by crystallographic analysis, resulting in compromised folding at high temperatures. Taking into account the high conservation of IS3 across hyperthermophilic homologues, we propose that the presence of IS3 is important for folding of hyperthermophilic subtilisins in high‐temperature environments. [ABSTRACT FROM AUTHOR]

Aspartate transcarbamoylase (ATC), an essential enzyme for de novo pyrimidine biosynthesis, is uniquely regulated in plants by feedback inhibition of uridine 5-monophosphate (UMP). Despite its importance in plant growth, the structure of this UMP-controlled ATC and the regulatory mechanism remain unknown. Here, we report the crystal structures of Arabidopsis ATC trimer free and bound to UMP, complexed to a transition-state analog or bearing a mutation that turns the enzyme insensitive to UMP. We found that UMP binds and blocks the ATC active site, directly competing with the binding of the substrates. We also prove that UMP recognition relies on a loop exclusively conserved in plants that is also responsible for the sequential firing of the active sites. In this work, we describe unique regulatory and catalytic properties of plant ATCs that could be exploited to modulate de novo pyrimidine synthesis and plant growth. Aspartate transcarbamoylase acts in de novo pyrimidine biosynthesis and in plants is regulated by feedback inhibition via uridine 5-monophosphate (UMP). Here Bellin et al. describe the structural basis for this feedback inhibition, showing that UMP blocks the active site by binding to a plant specific UMP recognition loop. [ABSTRACT FROM AUTHOR]

The essential human O-linked β- N -acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme responsible for modifying thousands of intracellular proteins with the monosaccharide O-GlcNAc. This unique modification plays crucial roles in human health and disease, but the substrate recognition of OGT remains poorly understood. Intriguingly, the only human enzyme reported to remove this modification, O-GlcNAcase (OGA), is O-GlcNAc modified. Here, we exploited a GlcNAc electrophilic probe (GEP1A) to rapidly screen OGT mutants in a fluorescence assay that can discriminate between altered OGT-sugar and -protein substrate binding to help elucidate the binding mode of OGT toward OGA protein substrate. Since OGT tetratricopeptide repeat (TPR) domain plays a key role in OGT-OGA binding, we screened 30 OGT TPR mutants, which revealed 15 "ladder like" asparagine or aspartate residues spanning TPRs 3–7 and 10–13.5 that affect OGA O-GlcNAcylation. By applying a truncated OGA construct, we found that OGA's N-terminal region or pseudo histone acetyltransferase domain is not required for its O-GlcNAcylation, suggesting OGT functionally interacts with OGA through its catalytic and/or stalk domains. This work represents the first effort to systemically investigate each OGT TPR and our findings will facilitate the development of new strategies to investigate the role of substrate-specific O-GlcNAcylation. Unlabelled Image • GlcNAc electrophilic probe in-gel fluorescence assay was applied to screen 30 OGT mutants against OGA protein substrate. • D152 and ten Asn-ladder residues spanning OGT TPRs 3–13.5 facilitate O-GlcNAcylation of OGA. • Four Asp-ladder residues closer to the OGT active site hamper OGT-OGA interaction. • OGA HAT domain and N-terminal region are dispensable for its O-GlcNAcylation. [ABSTRACT FROM AUTHOR]

Traboulsi syndrome is a rare autosomal recessive genetic disorder. The present study aimed to identify the pathogenic variants in the ASPH gene responsible for a rare and unique presentation of Traboulsi syndrome associated with cardiac disorder. DNA was isolated from the blood samples from 3 clinically diagnosed Traboulsi syndrome patients (n = 3) after obtaining a prior-informed consent. All three had classical ocular and facial dysmorphic features, and two of them also had associated cardiac problems. Mutation screening was performed for the exons of ASPH gene by Sanger sequencing in these patients and 350 controls. Sequence data analysis was performed using Seqscape and insilico protein analysis by SIFT, PyMOL, and Dynamut softwares. A novel homozygous variant(c.1853 T > A) in exon 21 was identified by Sanger sequencing in two of the three cases while a known pathogenic variant in exon 25 was identified in the third proband. The novel nonsense variant in exon 21 results in a premature truncation of gene resulting in a protein of 543 amino acids. This variant is not reported in ExAC, dbSNP and 1000 genome databases. Both the patients harboring this novel variant, had a unique presentation of Traboulsi syndrome with cardiac dysfunction. In silico analysis predicted the mutation to affect the calcium-binding activity of the gene which might explain the associated cardiac dysfunction in these two patients. The novel pathogenic mutation displayed a perfect genotype-phenotype correlation in two probands of Traboulsi syndrome with cardiac dysfunction. [ABSTRACT FROM AUTHOR]

Autophagy catabolizes cellular constituents to promote survival during nutrient deprivation. Yet, a metabolic comprehension of this recycling operation, despite its crucial importance, remains incomplete. Here, we uncover a specific metabolic function of autophagy that exquisitely adjusts cellular metabolism according to nitrogen availability in the budding yeast Saccharomyces cerevisiae. Autophagy enables metabolic plasticity to promote glutamate and aspartate synthesis, which empowers nitrogen-starved cells to replenish their nitrogen currency and sustain macromolecule synthesis. Our findings provide critical insights into the metabolic basis by which autophagy recycles cellular components and may also have important implications in understanding the role of autophagy in diseases such as cancer. Autophagy is known to promote cellular survival upon starvation, although how recycled components fit into cellular metabolism has not been well established. Here, the authors show in yeast that autophagy tunes cellular metabolism based on nitrogen availability via glutamate and aspartate synthesis. [ABSTRACT FROM AUTHOR]

Caspase (or cysteinyl-aspartate specific proteases) enzymes play important roles in apoptosis and inflammation, and the non-identical but overlapping specificity profiles (that is, cleavage recognition sequence) direct cells to different fates. Although all caspases prefer aspartate at the P1 position of the substrate, the caspase-6 subfamily shows preference for valine at the P4 position, while caspase-3 shows preference for aspartate. In comparison with human caspases, caspase-3a from zebrafish has relaxed specificity and demonstrates equal selection for either valine or aspartate at the P4 position. In the context of the caspase-3 conformational landscape, we show that changes in hydrogen bonding near the S3 subsite affect selection of the P4 amino acid. Swapping specificity with caspase-6 requires accessing new conformational space, where each landscape results in optimal binding of DxxD (caspase-3) or VxxD (caspase-6) substrate and simultaneously disfavors binding of the other substrate. Within the context of the caspase-3 conformational landscape, substitutions near the active site result in nearly equal activity against DxxD and VxxD by disrupting a hydrogen bonding network in the substrate binding pocket. The converse substitutions in zebrafish caspase-3a result in increased selection for P4 aspartate over valine. Overall, the data show that the shift in specificity that results in a dual function protease, as in zebrafish caspase-3a, requires fewer amino acid substitutions compared with those required to access new conformational space for swapping substrate specificity, such as between caspases-3 and -6. [ABSTRACT FROM AUTHOR]

The proliferation and differentiation of rhizomes are crucial for the propagation of Cymbidium species. We systematically assessed the effects of different concentrations of 20 amino acids on the proliferation and differentiation of C. goeringii rhizomes. Rhizome proliferation rates were significantly higher in media with 2.0 mmol·L-1 cysteine, 0.5 mmol·L-1 arginine, 0.2 mmol·L-1 asparagine, 1.0 mmol·L-1 proline, and 0.5 mmol·L-1 lysine compared with those in the control. Additionally, 1.0 mmol·L-1 tyrosine, 0.5 mmol·L-1 asparagine, and 0.2 mmol·L-1 aspartate were beneficial for rhizome differentiation. Furthermore, two combinations of amino acids, 0.5 mmol·L-1 arginine + 1.0 mmol·L-1 proline and 0.5 mmol·L-1 arginine + 2.0 mmol·L-1 cysteine, resulted in proliferation rates of 3.05 and 3.01, respectively, after 60 days. The highest differentiation rate (5.39 after 60 days) was observed in media with 0.5 mmol·L-1 asparagine + 0.2 mmol·L-1 aspartate. This study demonstrated that certain combinations of amino acids can effectively promote the proliferation and differentiation of rhizomes during the rapid propagation of C. goeringii. [ABSTRACT FROM AUTHOR]

• N-acetyl aspartate is an important measure of neuronal metabolic health. • This meta-analysis synthesises results from 182 studies of this chemical in psychosis. • N-acetyl aspartate levels are reduced in the hippocampus in high risk of psychosis. • In first episode psychosis, levels are lower in the frontal cortex and thalamus. • In chronic schizophrenia, levels are lower in many cortical and white matter areas. N-acetyl-aspartate (NAA) is a readily measured marker of neuronal metabolism. Previous analyses in schizophrenia have shown NAA levels are low in frontal, temporal and thalamic regions, but may be underpowered to detect effects in other regions, in high-risk states and in first episode psychosis. We searched for magnetic resonance spectroscopy studies comparing NAA in chronic schizophrenia, first episode psychosis and high risk of psychosis to controls. 182 studies were included and meta-analysed using a random-effects model for each region and illness stage. NAA levels were significantly lower than controls in the frontal lobe [Hedge's g = -0.36, p < 0.001], hippocampus [-0.52, p < 0.001], temporal lobe [-0.35, p = 0.031], thalamus [-0.32, p = 0.012] and parietal lobe [-0.25, p = 0.028] in chronic schizophrenia, and lower than controls in the frontal lobe [-0.26, p = 0.002], anterior cingulate cortex [-0.24, p = 0.016] and thalamus [-0.28, p = 0.028] in first episode psychosis. NAA was lower in high-risk of psychosis in the hippocampus [-0.20, p = 0.049]. In schizophrenia, NAA alterations appear to begin in hippocampus, frontal cortex and thalamus, and extend later to many other regions. [ABSTRACT FROM AUTHOR]

This study was conducted to design an electrochemical sensor for detection of l‐glutamate (L‐Glu) and Aspartate (Asp), which contribute largely to the umami taste of beef. Using N, N'‐diphenylthiourea (DPTU), polypyrrole (PPy), and polyvinyl chloride (PVC), a composite electrode (DPTU/PVC/PPy/Pt) was prepared for rapidly electrochemical detection of l‐Glu and Asp. The surface morphology of the synthesized functionalized electrode was characterized by Field Emission Scanning Electron Microscopy (FESEM). Potentials of umami amino acids accounted for 97.8%, while six interferential amino acids existed. The linear correlation between the content of l‐Glu and Asp in beef broth was studied under different treatment conditions. The sensor compared with an amino acid analyzer well detected the contents of l‐Glu and Asp in beef broths, with a coefficient of 0.991 in Pearson correlation analysis and an accuracy of 88.9%. The proposed electrochemical sensor showed good concurrence with previously reported methods and was effectively employed for the quantification of l‐ Glu and Asp in beef. Practical Application: The sensor exhibits the good performance with high stability and high accuracy. And it is a potential sensing platform with good reproducibility, making the proposed method suitable and reliable for routine analysis of L‐Glu and Asp in beef. This method was proved promising for quantitative detecting amino acids in beef. [ABSTRACT FROM AUTHOR]

HIV-1 Env glycoprotein gp160 exists as a trimer of heterodimers on the viral surface. In most structures of the soluble ectodomain of trimeric HIV-1 Envelope glycoprotein, the region from 512-517 of the fusion peptide and 547- 568 of the N- heptad repeat is disordered. We used aspartate scanning mutagenesis of Subtype B, JRFL Env as an alternate method to probe residue burial in the context of cleaved, cell surface expressed Env, as buried residues should be intolerant to substitution with Asp. The data are inconsistent with a fully disordered 547-568 stretch as residues 548, 549, 550, 555, 556, 559, 562, 566-569 are all sensitive to Asp substitution. In the fusion peptide region, residues 513 and 515 were also sensitive to Asp substitution, suggesting that the fusion peptide may not be fully exposed in native Env.gp41 is metastable in the context of native trimer. Introduction of Asp at residues that are exposed in the pre-fusion state but buried in the post-fusion state is expected to destabilize the post-fusion state and any intermediate states where the residue is buried. We therefore performed sCD4 induced gp120 shedding experiments to identify Asp mutants at residues 551, 554-559, 561-567, 569 that could prevent gp120 shedding. We also observed similar mutational effects on shedding for equivalent mutants in the context of Clade C Env from isolate, 4-2J.41. These substitutions can potentially be used to stabilize native like trimer derivatives that are used as HIV-1 vaccine immunogens. [ABSTRACT FROM AUTHOR]

Copyright of Food Research & Development is the property of Food Research & Development Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The phytohormone cytokinin influences many aspects of plant growth and development, several of which also involve the cellular process of autophagy, including leaf senescence, nutrient remobilization, and developmental transitions. The Arabidopsis type-A response regulators (type-A ARR) are negative regulators of cytokinin signaling that are transcriptionally induced in response to cytokinin. Here, we describe a mechanistic link between cytokinin signaling and autophagy, demonstrating that plants modulate cytokinin sensitivity through autophagic regulation of type-A ARR proteins. Type-A ARR proteins were degraded by autophagy in an AUTOPHAGY-RELATED (ATG)5-dependent manner, and this degradation is promoted by phosphorylation on a conserved aspartate in the receiver domain of the type-A ARRs. EXO70D family members interacted with type-A ARR proteins, likely in a phosphorylation-dependent manner, and recruited them to autophagosomes via interaction of the EXO70D AIM with the core autophagy protein, ATG8. Consistently, loss-of-function exo70D1,2,3 mutants exhibited compromised targeting of type-A ARRs to autophagic vesicles, have elevated levels of type-A ARR proteins, and are hyposensitive to cytokinin. Disruption of both type-A ARRs and EXO70D1,2,3 compromised survival in carbon-deficient conditions, suggesting interaction between autophagy and cytokinin responsiveness in response to stress. These results indicate that the EXO70D proteins act as selective autophagy receptors to target type-A ARR cargos for autophagic degradation, demonstrating modulation of cytokinin signaling by selective autophagy. [ABSTRACT FROM AUTHOR]

Escherichia coli NhaA is a prototypical sodium-proton antiporter responsible for maintaining cellular ion and volume homeostasis by exchanging two protons for one sodium ion; despite two decades of research, the transport mechanism of NhaA remains poorly understood. Recent crystal structure and computational studies suggested Lys300 as a second proton-binding site; however, functional measurements of several K300 mutants demonstrated electrogenic transport, thereby casting doubt on the role of Lys300. To address the controversy, we carried out state-of-the-art continuous constant pH molecular dynamics simulations of NhaA mutants K300A, K300R, K300Q/D163N, and K300Q/D163N/D133A. Simulations suggested that K300 mutants maintain the electrogenic transport by utilizing an alternative proton-binding residue Asp133. Surprisingly, while Asp133 is solely responsible for binding the second proton in K300R, Asp133 and Asp163 jointly bind the second proton in K300A, and Asp133 and Asp164 jointly bind two protons in K300Q/D163N. Intriguingly, the coupling between Asp133 and Asp163 or Asp164 is enabled through the proton-coupled hydrogen-bonding network at the flexible intersection of two disrupted helices. These data resolve the controversy and highlight the intricacy of the compensatory transport mechanism of NhaA mutants. Alternative proton-binding site and proton sharing between distant aspartates may represent important general mechanisms of proton-coupled transport in secondary active transporters. [ABSTRACT FROM AUTHOR]

Forisomes are protein bodies known exclusively from sieve elements of legumes. Forisomes contribute to the regulation of phloem transport due to their unique Ca2+-controlled, reversible swelling. The assembly of forisomes from sieve element occlusion (SEO) protein monomers in developing sieve elements and the mechanism(s) of Ca2+-dependent forisome contractility are poorly understood because the amino acid sequences of SEO proteins lack conventional protein–protein interaction and Ca2+-binding motifs. We selected amino acids potentially responsible for forisome-specific functions by analyzing SEO protein sequences in comparison to those of the widely distributed SEO-related (SEOR), or SEOR proteins. SEOR proteins resemble SEO proteins closely but lack any Ca2+ responsiveness. We exchanged identified candidate residues by directed mutagenesis of the Medicago truncatula SEO1 gene, expressed the mutated genes in yeast (Saccharomyces cerevisiae) and studied the structural and functional phenotypes of the forisome-like bodies that formed in the transgenic cells. We identified three aspartate residues critical for Ca2+ responsiveness and two more that were required for forisome-like bodies to assemble. The phenotypes observed further suggested that Ca2+-controlled and pH-inducible swelling effects in forisome-like bodies proceeded by different yet interacting mechanisms. Finally, we observed a previously unknown surface striation in native forisomes and in recombinant forisome-like bodies that could serve as an indicator of successful forisome assembly. To conclude, this study defines a promising path to the elucidation of the so-far elusive molecular mechanisms of forisome assembly and Ca2+-dependent contractility. [ABSTRACT FROM AUTHOR]

A hallmark of the catalytically essential Walker B motif of P-loop NTPases is the presence of an acidic residue (aspartate/glutamate) for efficient Mg2+ coordination. Although the Walker B motif has been identified in well-studied examples of P-loop NTPases, its identity is ambiguous in many families, for example, in the prokaryotic small Ras-like GTPase family of MglA. MglA, belonging to TRAFAC class of P-loop NTPases, possesses a threonine at the position equivalent to Walker B aspartate in eukaryotic Ras-like GTPases. To resolve the identity of the Walker B residue in MglA, we carried out a comprehensive analysis of Mg2+ coordination on P-loop NTPase structures. Atoms in the octahedral coordination of Mg2+ and their interactions comprise a network including water molecules, Walker A, Walker B and switch motifs of P-loop NTPases. Based on the conserved geometry of Mg2+ coordination, we confirm that a conserved aspartate functions as the Walker B residue of MglA, and validate it through mutagenesis and biochemical characterization. Location of the newly identified aspartate is spatially equivalent to the Walker B residue of the ASCE division of P-loop NTPases. Furthermore, similar to the allosteric regulation of the Walker B aspartate conformation in MglA, we identify protein families in which large conformational changes involving Walker B motif potentially function as allosteric regulators. The study unravels conserved features of Mg2+ coordination among divergent families of P-loop NTPases, especially between ancient Ras-like GTPases and ASCE family of ATPases. The conserved geometric features provide a foundation for design of nucleotide-hydrolyzing enzymes. Unlabelled Image • We report the discovery of a novel motif related to Walker B in the prokaryotic family of small Ras-like GTPases. • MglA and a few members of the ASCE family possess a threonine that orients the Walker B aspartate. • Conformational changes in Walker B residues are involved in allosteric regulation. • Unifying features among widely divergent members of P-loop NTPases are highlighted. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Potent inhibitors of an essential microbial enzyme have been shown to be effective growth inhibitors of Candida albicans, a pathogenic fungus. C. albicans is the main cause of oropharyngeal candidiasis, and also causes invasive fungal infections, including systemic sepsis, leading to serious complications in immunocompromised patients. As the rates of drug‐resistant fungal infections continue to rise novel antifungal treatments are desperately needed. The enzyme aspartate semialdehyde dehydrogenase (ASADH) is critical for the functioning of the aspartate biosynthetic pathway in microbes and plants. Because the aspartate pathway is absent in humans, ASADH has the potential to be a promising new target for antifungal research. Deleting the asd gene encoding for ASADH significantly decreases the survival of C. albicans, establishing this enzyme as essential for this organism. Previously developed ASADH inhibitors were tested against several strains of C. albicans to measure their possible therapeutic impact. The more potent inhibitors show a good correlation between enzyme inhibitor potency and fungal growth inhibition. Growth curves generated by incubating different C. albicans strains with varying enzyme inhibitor levels show significant slowing of fungal growth by these inhibitors against each of these strains, similar to the effect observed with a clinical antifungal drug. The most effective inhibitors also demonstrated relatively low cytotoxicity against a human epithelial cell line. Taken together, these results establish that the ASADH enzyme is a promising new target for further development as a novel antifungal treatment against C. albicans and related fungal species. [ABSTRACT FROM AUTHOR]

Aspartate transcarbamoylase (ATCase) is a key enzyme which regulates and catalyzes the second step of de novo pyrimidine synthesis in all organisms. Escherichia coli ATCase is a prototypic enzyme regulated by both product feedback and substrate cooperativity, whereas human ATCase is a potential anticancer target. Through structural and biochemical analyses, we revealed that R167/130's loop region in ATCase serves as a gatekeeper for the active site, playing a new and unappreciated regulatory role in the catalytic cycle of ATCase. Based on virtual compound screening simultaneously targeting the new regulatory region and active site of human ATCase, two compounds were identified to exhibit strong inhibition of ATCase activity, proliferation of multiple cancer cell lines, and growth of xenograft tumors. Our work has not only revealed a previously unknown regulatory region of ATCase that helps uncover the catalytic and regulatory mechanism of ATCase, but also successfully guided the identification of new ATCase inhibitors for anticancer drug development using a dual‐targeting strategy. Database: Structure data are available in Protein Data Bank under the accession numbers: 6KJ7 (G166P ecATCase), 6KJ8 (G166P ecATCase‐holo), 6KJ9 (G128/130A ecATCase), and 6KJA (G128/130A ecATCase‐holo). [ABSTRACT FROM AUTHOR]

Background: Ankylosing spondylitis (AS) is a common chronic inflammatory arthritis, causing lasting back pain with progressive loss of spinal mobility. However, the exact pathogenesis of AS remains unclear. We aim to use the metabolomics analysis to characterize the metabolic profile of AS, in order to better understand the pathogenesis of AS and monitor disease activity and progression. Methods: The ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-TQ-MS) was used for investigating the serum amino acid metabolomic profiling of 30 AS patients, in comparison with 32 rheumatoid arthritis (RA) patients and 30 healthy controls, combined with multivariate statistical analysis. Metabolite association analysis with disease activity was performed using generalized linear regression. The metabolic pathway analysis for the important metabolites was performed using MetPA and the metabolic network was constructed. Results: A total of 29 amino acids and biogenic amines were detected in all participants by UPLC-TQ-MS. It showed significant amino acid differences between the AS/RA patients and control subjects. Additionally, 4-hydroxy-L-proline, alanine, γ-aminobutyric acid, glutamine, and taurine were identified as candidate markers shared by AS/RA groups. Specifically, lysine, proline, serine, and alanine were found correlated with disease activity of AS. Furthermore, the most significant metabolic pathway identified were alanine, aspartate, and glutamate metabolism, arginine and proline metabolism, aminoacyl tRNA biosynthesis and glycine, serine, and threonine metabolism. Conclusions: These preliminary results demonstrate that UPLC-TQ-MS analysis method is a powerful tool to identify metabolite profiles of AS. Research in identified disease activity–associated metabolites and biological pathways may provide assistance for clinical diagnosis and pathological mechanism of AS. Key Points • There are perturbations of serum amino acid metabolism in AS, compared with RA and healthy controls, determined by UPLC-TQ-MS. • Metabolomics pathway is used to analysis for the differential metabolites of AS. • The altered serum amino acid could monitor disease activity of AS. [ABSTRACT FROM AUTHOR]

• In silico prediction identified target RNA editing sites of two PPR proteins. • MEF46 is an E+ subclass PPR and required for RNA editing at nad5 -1958. • MEF47 is an E subclass PPR and essential for two sites, nad3 -64 and ccmC -614. • The target of MEF46 is a target of DYW2 as well, while those of MEF47 are not. • These results support the functional connection between E+ subclass PPRs and DYW2. Terrestrial plants have C-to-U RNA editing in the transcripts of plastids and mitochondria. Target specificity for more than several hundred editing sites are governed by PLS (P PR, L ong and S hort) class Pentatricopeptide repeat (PPR) proteins with additional C-terminal domains. Half of these PPR proteins have DYW (Aspartate (D), Tyrosine (Y) and Tryptophan (W)) domains, which most likely harbour cytidine deaminase activity. The other half of them, E subclass and E+ subclass proteins, contain no or only a part of the DYW domain. Missing DYW domains in the E and E+ subclass PPR proteins are likely to be complemented by other DYW containing proteins. All target sites of so far characterized E+ subclass PPR proteins show defects in dyw2 mutants, suggesting that the DYW2 protein complements the missing DYW domains in the E+ subclass PPR proteins. Here we report two novel RNA editing factors, MEF46 and MEF47, which belong to E+ and E subclass, respectively. The defective editing site in mef46, nad5 -1958 , overlaps with the affected sites in dyw2 mutants, while that in mef47 , nad3 -64 and ccmC -614 do not, further supporting the specific functional connection between E+ subclass PPR proteins and DYW2. [ABSTRACT FROM AUTHOR]

This study tested the hypothesis that amino acids are oxidized at higher rates than glucose and palmitate for ATP production in tissues of largemouth bass (LMB, a carnivorous fish). Slices (10 to 50 mg) of liver, proximal intestine, kidney, and skeletal muscle isolated from LMB were incubated at 26 °C for 2 h in oxygenated Krebs–Henseleit bicarbonate buffer (pH 7.4, with 5 mM d-glucose) containing either d-[U-14C]glucose, 2 mM l-alanine plus l-[U-14C]alanine, 2 mM l-aspartate plus l-[U-14C]aspartate, 2 mM l-glutamate plus l-[U-14C]glutamate, 2 mM l-glutamine plus l-[U-14C]glutamine, 2 mM l-leucine plus l-[U-14C]leucine, or 2 mM palmitate plus [U-14C]palmitate. In parallel experiments, tissues were incubated with a [U-14C]-labeled tracer and a mixture of unlabeled substrates [alanine, aspartate, glutamate, glutamine, leucine, and palmitate (2 mM each) plus 5 mM glucose]. 14CO2 was collected to calculate the rates of substrate oxidation. In separate experiments, O2 consumption by each tissue was measured in the presence of individual or a mixture of substrates. The activities of key metabolic enzymes were also measured. Results indicated that the liver and skeletal muscle had a limited ability to oxidize glucose and palmitate to CO2 for ATP production in the presence of individual or a mixture of substrates due to low activities of carnitine palmitoyltransferase-I, hexokinase and pyruvate dehydrogenase. In the presence of individual substrates, each amino acid was actively oxidized by all the tissues. In the presence of a mixture of substrates, glutamine and glutamate were the major metabolic fuels in the proximal intestine and kidney, as glutamine for the liver and aspartate for skeletal muscle. All the tissues had high activities of glutaminase, glutamate dehydrogenase, and transaminases. At the same extracellular concentration of amino acids (2 mM) in a mixture of energy substrates, glutamine was the major metabolic fuel for the liver of the LMB, glutamine and glutamate for the proximal intestine and kidneys, and aspartate for the skeletal muscle. Glutamine plus glutamate plus aspartate generated 60–70% of ATP in LMB tissues. [ABSTRACT FROM AUTHOR]

Copyright of Maintenance & Reliability / Eksploatacja i Niezawodność is the property of Polish Scientific & Technical Society Consumables, Polish Maintenance Society and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Plasmepsins are a group of diverse aspartic proteases in the malaria parasite Plasmodium. Their functions are strikingly multifaceted, ranging from hemoglobin degradation to secretory organelle protein processing for egress, invasion, and effector export. Some, particularly the digestive vacuole plasmepsins, have been extensively characterized, whereas others, such as the transmission-stage plasmepsins, are minimally understood. Some (e.g. plasmepsin V) have exquisite cleavage sequence specificity; others are fairly promiscuous. Some have canonical pepsin-like aspartic protease features, whereas others have unusual attributes, including the nepenthesin loop of plasmepsinVand a histidine in place of a catalytic aspartate in plasmepsin III. We have learned much about the functioning of these enzymes, but more remains to be discovered about their cellular roles and even their mechanisms of action. Their importance in many key aspects of parasite biology makes them intriguing targets for antimalarial chemotherapy. Further consideration of their characteristics suggests that some are more viable drug targets than others. Indeed, inhibitors of invasion and egress offer hope for a desperately needed new drug to combat this nefarious organism. [ABSTRACT FROM AUTHOR]

The European corn borer, Ostrinia nubilalis Hbn., is a pest Lepidopteran species whose larvae overwinter by entering diapause, gradually becoming cold-hardy. To investigate metabolic changes during cold hardening, activities of four metabolic enzymes – citrate synthase (CS), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in whole-body homogenates of pupae, non-diapausing and diapausing larvae acclimated to 5 °C, −3 °C and −16 °C. The highest CS activity was detected in non-diapausing larvae, reflecting active development, while the highest in vitro LDH activity was recorded in diapausing larvae at temperatures close to 0 °C, evidencing a metabolic switch towards anaerobic metabolism. However, in-gel LDH activity showed that production of pyruvate from lactate is triggered by sub-zero temperatures. The activities of both aminotransferases were highest in non-diapausing larvae. Our findings suggest that during diapause and cold hardening the aminotransferases catalyse production of L-alanine, an important cryoprotectant, and L-aspartate, which is closely tied to both transamination reactions and Krebs cycle. The results of this study indicate that, during diapause, the activity of metabolic enzymes is synchronized with exogenous factors, such as temperatures close to 0 °C. These findings support the notion that diapause is metabolically plastic and vibrant, rather than simply a passive, resting state. [ABSTRACT FROM AUTHOR]

Anionic species of aspartic acid, Asp−, having a zwitterionic backbone and a deprotonated side chain, appears to be a good example for analyzing dipole‐ion and ion pair interactions. Density functional theory calculations were herein performed to investigate the low energy conformers of Asp− embedded in a dielectric continuum modeling an aqueous environment, through a scan of the potential energy as a function of the side chain (χ1, χ2) torsion angles. The most energetically favorable conformers having g+g− and g−g+ side chain orientations are found to be stabilized by charge‐enhanced intramolecular H‐bonding involving the positively charged (NH3+) and the two negatively charged (COO−) groups. These conformers were further used to analyze Asp− + nW clusters (W: water, n = 1 or 3), and Asp−/Asp− pair formation. COO− groups were found to be the most attractive sites for hosting a water molecule (binding energy: −6.0 ± 1.5 kcal/mol), compared to NH3+ groups (binding energy: −4.7 ± 1.1 kcal/mol). Energy separation between g+g− and g−g+ conformers increases upon explicit hydration. Asp−/Asp− ion pairs, stabilized by the interaction between the NH3+ group of a partner and the COO− group of the other, shows a quite constant binding energy (−8.1 ± 0.2 kcal/mol), whatever the pair type, and the relative orientation of the two interacting partners. This study suggests a first step to achieve a more realistic image of intermolecular interactions in aqueous environment, especially upon increasing concentration. It can also be considered as a preliminary attempt to assess the interactions of the Lys+...Asp−/Glu− ion pairs stabilizing intra‐ and interchain interactions in proteins. [ABSTRACT FROM AUTHOR]

Following nerve stimulation, there are two distinct phases of Ca2+-dependent neurotransmitter release: a fast, synchronous release phase, and a prolonged, asynchronous release phase. Each of these phases is tightly regulated and mediated by distinct mechanisms. Synaptotagmin 1 is the major Ca2+ sensor that triggers fast, synchronous neurotransmitter release upon Ca2+ binding by its C2A and C2B domains. It has also been implicated in the inhibition of asynchronous neurotransmitter release, as blocking Ca2+ binding by the C2A domain of synaptotagmin 1 results in increased asynchronous release. However, the mutation used to block Ca2+ binding in the previous experiments (aspartate to asparagine mutations, sytD-N) had the unintended side effect of mimicking Ca2+ binding, raising the possibility that the increase in asynchronous release was directly caused by ostensibly constitutive Ca2+ binding. Thus, rather than modulating an asynchronous sensor, sytD-N may be mimicking one. To directly test the C2A inhibition hypothesis, we utilized an alternate C2A mutation that we designed to block Ca2+ binding without mimicking it (an aspartate to glutamate mutation, sytD-E). Analysis of both the original sytD-N mutation and our alternate sytD-E mutation at the Drosophila neuromuscular junction showed differential effects on asynchronous release, as well as on synchronous release and the frequency of spontaneous release. Importantly, we found that asynchronous release is not increased in the sytD-E mutant. Thus, our work provides new mechanistic insight into synaptotagmin 1 function during Ca2+-evoked synaptic transmission and demonstrates that Ca2+ binding by the C2A domain of synaptotagmin 1 does not inhibit asynchronous neurotransmitter release in vivo. [ABSTRACT FROM AUTHOR]

Glutamine (Gln) is converted to excitatory (glutamate, aspartate) and inhibitory (γ-amino butyric acid) amino acid neurotransmitters in brain, and is a source of energy during glucose deprivation. Current research utilized an Analytical Quality by Design approach to optimize levels and combinations of critical gas pressure (sheath, auxiliary, sweep) and temperature (ion transfer tube, vaporizer) parameters for high-sensitivity mass spectrometric quantification of brain tissue glutamine. A Design of Experiments (DOE) matrix for evaluation of relationships between these multiple independent variables and a singular response variable, e.g. glutamine chromatogram area, was developed by statistical response surface methodology using central composite design. A second-order polynomial equation was generated to identify and predict singular versus combinatory effects of synergistic and antagonistic factors on chromatograph area. Predicted versus found outcomes overlapped, with enhanced area associated with the latter. DOE methodology was subsequently used to evaluate liquid chromatographic variable effects, e.g. flow rate, column temperature, and mobile phase composition on the response variable. Results demonstrate that combinatory AQbD-guided mass spectrometric/liquid chromatographic optimization significantly enhanced analytical sensitivity for Gln, thus enabling down-sized brain tissue sample volume procurement for quantification of this critical amino acid. [ABSTRACT FROM AUTHOR]

The present study aimed to identify and compare the microbial signatures between periodontally healthy and periodontitis subjects using 454 sequences of 16S rRNA genes. Subgingival plaque samples were collected from ten periodontally healthy subjects and ten matched chronic periodontitis patients. Bacterial DNA was extracted and next-generation sequencing of 16S rRNA genes was performed. The microbial composition differed between healthy subjects and periodontitis patients at all phylogenetic levels. Particularly, 16 species, including Lautropia mirabilis and Neisseria subflava predominated in healthy subjects, whereas nine species, including Porphyromonas gingivalis and Filifactor alocis predominated in periodontitis. UniFrac, a principal coordinate and network analysis, confirmed distinct community profiles in healthy subjects and periodontitis patients. Using predicted function profiling, pathways involved in phenylpropanoid, GPI-anchor biosynthesis, and metabolism of alanine, arginine, aspartate, butanoate, cyanoamino acid, fatty acid, glutamate, methane, proline, and vitamin B6 were significantly over-represented in periodontitis patients. These results highlight the oral microbiota alterations in microbial composition in periodontitis and suggest the genes and metabolic pathways associated with health and periodontitis. Our findings help to further elucidate microbial composition and interactions in health and periodontitis. [ABSTRACT FROM AUTHOR]

Introduction: Chronic alcohol consumption has been observed to be associated with a range of cognitive impairments that impact on treatment management. In this spectroscopy study, we examined the association of N-Acetyl Aspartate (NAA), a marker of neuronal integrity, and cognitive impairment in alcohol dependent patients. Method: Using in vivo proton magnetic resonance spectroscopy (1H-MRS), we examined brain metabolite levels in 31 alcohol dependent individuals. 1H-MRS from the parietal lobe were analyzed to yield absolute concentrations of NAA. Alcohol history, neurocognitive function including Clock Drawing Test (CDT), Mini Mental State Exam (MMSE), Trial Making Test (TMT) and Balloon Analogue Risk Task (BART) were also assessed. Covariates included concurrent medication, age and recent alcohol consumption. Results: There were statistically significant bivariate associations between NAA and the variables age, CDT and BART (r = −.45, P = 01; r = −.53, P =.01; r =.49, P =.02) respectively) but there were no statistically significant associations with other measures of cognitive function. Controlling for age, concurrent medication and recent alcohol consumption, multiple linear regression revealed a negative association between parietal NAA (Model: F = 6.96, R2 =.66, P =.001) and CDT scores (B = −.35, P =.03), a positive association with BART scores (B =.47, P =.02). Conclusion: These results demonstrate that in alcohol dependent patients lower NAA/Cr is associated with reduced cognitive functioning and increased risk-taking. [ABSTRACT FROM AUTHOR]

Background/Aims: Still in 1999 the first hints were published for the pharmacoresistant Cav2.3 calcium channel to be involved in the generation of epileptic seizures, as transcripts of alpha1E (Cav2.3) and alpha1G (Cav3.1) are changed in the brain of genetic absence epilepsy rats from Strasbourg (GAERS). Consecutively, the seizure susceptibility of mice lacking Cav2.3 was analyzed in great detail by using 4-aminopyridine, pentylene-tetrazol, N-methyl-D-aspartate and kainic acid to induce experimentally convulsive seizures. Further, γ-hydroxybutyrolactone was used for the induction of non-convulsive absence seizures. For all substances tested, Cav2.3- competent mice differed from their knockout counterparts in the sense that for convulsive seizures the deletion of the pharmacoresistant channel was beneficial for the outcome during experimentally induced seizures [1]. The antiepileptic drug lamotrigine reduces seizure activity in Cav2.3-competent but increases it in Cav2.3-deficient mice. In vivo, Cav2.3 must be under tight control by endogenous trace metal cations (Zn2+ and Cu2+). The dyshomeostasis of either of them, especially of Cu2+, may alter the regulation of Cav2.3 severely and its activity for Ca2+ conductance, and thus may change hippocampal and neocortical signaling to hypoor hyperexcitation. Methods: To investigate by telemetric EEG recordings the mechanism of generating hyperexcitation by kainate, mice were tested for their sensitivity of changes in neuronal (intracerebroventricular) concentrations of the trace metal cation Zn2+. As the bloodbrain barrier limits the distribution of bioavailable Zn2+ or Cu2+ into the brain, we administered micromolar Zn2+ ions intracerebroventricularly in the presence of 1 mM histidine as carrier and compared the effects on behavior and EEG activity in both genotypes. Results: Kainate seizures are more severe in Cav2.3-competent mice than in KO mice and histidine lessens seizure severity in competent but not in Cav2.3-deficient mice. Surprisingly, Zn2+ plus histidine resembles the kainate only control with more seizure severity in Cav2.3-competent than in deficient mice. Conclusion: Cav2.3 represents one important Zn2+-sensitive target, which is useful for modulating convulsive seizures. [ABSTRACT FROM AUTHOR]

D-Aspartate, aspartate racemase activity, and D-aspartate oxidase activity were detected in tissues from several types of starfish. Aspartate racemase activity in male testes of Patiria pectinifera was significantly elevated in the summer months of the breeding season compared with spring months. We also compared aspartate racemase activity with the gonad index and found that activity in individuals with a gonad index ≥6% was four-fold higher than that of individuals with a gonad index <6%. The ratio of the D-form of aspartate to total aspartate was approximately 25% in testes with a gonad index <6% and this increased to approximately 40% in testes with a gonad index ≥6%. However, such changes were not observed in female ovaries. Administration of D-aspartate into male starfish caused testicular growth. These results indicate the possible involvement of aspartate racemase and D-aspartate in testicular maturation in echinoderm starfish. Changes in the ratio of D-aspartate to total aspartate and in aspartate racemase activity during maturation of the starfish testis. [ABSTRACT FROM AUTHOR]