Semi-rational design of L-amino acid deaminase for production of pyruvate and d-alanine by Escherichia coli whole-cell biocatalyst

In our previous study, one-step pyruvate and d-alanine production from d,l-alanine by a whole-cell biocatalyst Escherichia coli expressing l-amino acid deaminase (Pm1) derived from Proteus mirabilis was investigated. However, due to the low catalytic efficiency of Pm1, the pyruvate titer was relatively low. Here, semi-rational design based on site-directed saturation mutagenesis was carried out to improve the catalytic efficiency of Pm1. A novel high-throughput screening (HTS) method for pyruvate based on 2,4-dinitrophenylhydrazine indicator was then established. The catalytic efficiency (k_cat/K_m) of the mutant V437I screened out by this method was 1.88 times higher than wild type. Next, to improve the growth of the engineered strain BLK07, the genes encoding for Xpk and Fbp were integrated into its genome to construct non-oxidative glycolysis (NOG) pathway. Finally, the CRISPR/Cas9 system was used to integrate the N6-pm1-V437I gene into the genome of BLK07. Pyruvic acid titer of the plasmid-free strain reached 42.20 g/L with an l-alanine conversion rate of 77.62% and a d-alanine resolution of 82.4%. This work would accelerate the industrial production of pyruvate and d-alanine by biocatalysis, and the HTS method established here could be used to screen other Pm1 mutants with high pyruvate titers.

     

Sleep Disturbance and Alzheimer’s Disease: The Glial Connection

Poor quality and quantity of sleep are very common in elderly people throughout the world. Growing evidence has suggested that sleep disturbances could accelerate the process of neurodegeneration. Recent reports have shown a positive correlation between sleep deprivation and amyloid-β (Aβ)/tau aggregation in the brain of Alzheimer’s patients. Glial cells have long been implicated in the progression of Alzheimer’s disease (AD) and recent findings have also suggested their role in regulating sleep homeostasis. However, how glial cells control the sleep–wake balance and exactly how disturbed sleep may act as a trigger for Alzheimer’s or other neurological disorders have recently gotten attention. In an attempt to connect the dots, the present review has highlighted the role of glia-derived sleep regulatory molecules in AD pathogenesis.

Graphical Abstract

Role of glia in sleep disturbance and Alzheimer’s progression.

     

Calpain Inhibitors as Potential Therapeutic Modulators in Neurodegenerative Diseases

It is considered a significant challenge to understand the neuronal cell death mechanisms with a suitable cure for neurodegenerative disorders in the coming years. Calpains are one of the best-considered “cysteine proteases activated” in brain disorders. Calpain is an important marker and mediator in the pathophysiology of neurodegeneration. Calpain activation being the essential neurodegenerative factor causing apoptotic machinery activation, it is crucial to develop reliable and effective approaches to prevent calpain-mediated apoptosis in degenerating neurons. It has been recently seen that the “inhibition of calpain activation” has appeared as a possible therapeutic target for managing neurodegenerative diseases. A systematic literature review of PubMed, Medline, Bentham, Scopus, and EMBASE (Elsevier) databases was conducted. The present article reviews the basic pathobiology and role of selective calpain inhibitors used in various neurodegenerative diseases as a therapeutic target.

Graphical Abstract

     

Hypoxia-Inducible Factor-1α (HIF-1α) Inhibition Impairs Retinoic Acid-Induced Differentiation in SH-SY5Y Neuroblastoma Cells,Leading to Reduced Neurite Length and Diminished Gene Expression Related to Cell Differentiation

Neuroblastoma is the most common extracranial solid tumour in childhood, originated from cells of the neural crest during the development of the Sympathetic Nervous System. Retinoids are vitamin-A derived differentiating agents utilised to avoid disease resurgence in high-risk neuroblastoma treatment. Several studies indicate that hypoxia—a common feature of the tumoural environment—is a key player in cell differentiation and proliferation. Hypoxia leads to the accumulation of the hypoxia-inducible factor-1α (HIF-1α). This work aims to investigate the effects of the selective inhibition of HIF-1α on the differentiation induced by retinoic acid in human neuroblastoma cells from the SH-SY5Y lineage to clarify its role in cell differentiation. Our results indicate that HIF-1α inhibition impairs RA-induced differentiation by reducing neuron-like phenotype and diminished immunolabeling and expression of differentiation markers.

Graphic Abstract

HIF1A is involved in Retinoic Acid (RA) induced differentiation in SH-SY5Y neuroblastoma cells. siRNA HIF1A gene silencing leads to a weaker response to RA, demonstrated by changes in the neuro-like phenotype and diminished expression of differentiation markers.

     

Comparative evaluation of Aspergillus niger strains for endogenous pectin-depolymerization capacity and suitability for d-galacturonic acid production

Pectinaceous agricultural residues rich in d-galacturonic acid (d-GalA), such as sugar beet pulp, are considered as promising feedstocks for waste-to-value conversions. Aspergillus niger is known for its strong pectinolytic activity. However, while specialized strains for production of citric acid or proteins are well characterized, this is not the case for the production of pectinases. We, therefore, systematically compared the pectinolytic capabilities of six A. niger strains (ATCC 1015, ATCC 11414, NRRL 3122, CBS 513.88, NRRL 3, and N402) using controlled batch cultivations in stirred-tank bioreactors. A. niger ATCC 11414 showed the highest polygalacturonase activity, specific protein secretion, and a suitable morphology. Furthermore, d-GalA release from sugar beet pulp was 75% higher compared to the standard lab strain A. niger N402. Our study, therefore, presents a robust initial strain selection to guide future process improvement of d-GalA production from agricultural residues and identifies a high-performance base strain for further genetic optimizations.

     

Effects of sex and estrous cycle on the brain and plasma arginine metabolic profile in rats

l-arginine is a versatile amino acid with a number of bioactive metabolites. Increasing evidence implicates altered arginine metabolism in the aging and neurodegenerative processes. The present study, for the first time, determined the effects of sex and estrous cycle on the brain and blood (plasma) arginine metabolic profile in naïve rats. Female rats displayed significantly lower levels of l-arginine in the frontal cortex and three sub-regions of the hippocampus when compared to male rats. Moreover, female rats had significantly higher levels of l-arginine and γ-aminobutyric acid, but lower levels of l-ornithine, agmatine and putrescine, in plasma relative to male rats. The observed sex difference in brain l-arginine appeared to be independent of the enzymes involved in its metabolism, de novo synthesis and blood-to-brain transport (cationic acid transporter 1 protein expression at least), as well as circulating l-arginine. While the estrous cycle did not affect l-arginine and its metabolites in the brain, there were estrous cycle phase-dependent changes in plasma l-arginine. These findings demonstrate the sex difference in brain l-arginine in the estrous cycle-independent manner. Since peripheral blood has been increasingly used to identify biomarkers of brain pathology, the influences of sex and estrous cycle on blood arginine metabolic profile need attention when experimental research involves female rodents.

     

The role of GDP-l-galactose phosphorylase in the control of ascorbate biosynthesis

The enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff–Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization. All transgenic lines expressing functional SW protein green fluorescent protein fusions driven by their endogenous promoters showed a high accumulation of the fusion proteins, with the exception of those lines expressing GDP-l-galactose phosphorylase (GGP) protein, which had very low abundance. Transient expression of individual or combinations of SW pathway enzymes in N. benthamiana only increased ascorbate concentration if GGP was included. Although we did not detect direct interaction between the different enzymes of the pathway using yeast-two hybrid analysis, consecutive SW enzymes, as well as the first and last enzymes (GMP and l-GalDH) associated in coimmunoprecipitation studies. This association was supported by gel filtration chromatography, showing the presence of SW proteins in high-molecular weight fractions. Finally, metabolic control analysis incorporating known kinetic characteristics showed that previously reported feedback repression at the GGP step, combined with its relatively low abundance, confers a high-flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.

Metabolic engineering, genetic analysis, and functional mutant complementation identify GDP-l-galactose phosphorylase as the main control point in ascorbate biosynthesis in green tissues.     

Anti-inflammatory effect of l-cysteine (a semi-essential amino acid) on 5-FU-induced oral mucositis in hamsters

Oral mucositis is an inflammation of the oral mucosa mainly resulting from the cytotoxic effect of 5-fluorouracil (5-FU). The literature shows anti-inflammatory action of l-cysteine (l-cys) involving hydrogen sulfide (H₂S). In view of these properties, we investigate the effect of l-cys in oral mucositis induced by 5-FU in hamsters. The animals were divided into the following groups: saline 0.9%, mechanical trauma, 5-FU 60–40 mg/kg, l-cys 10/40 mg and NaHS 27 µg/kg. 5-FU was administered on days 1st to 2nd; 4th day excoriations were made on the mucosa; 5th–6th received l-cys and NaHS. For data analysis, histological analyses, mast cell count, inflammatory and antioxidants markers, and immunohistochemistry (cyclooxygenase-2(COX-2)/inducible nitric oxide synthase (iNOs)/H₂S) were performed. Results showed that l-cys decreased levels of inflammatory markers, mast cells, levels of COX-2, iNOS and increased levels of antioxidants markers and H₂S when compared to the group 5-FU (p < 0.005). It is suggested that l-cys increases the H₂S production with anti-inflammatory action in the 5-FU lesion.

     

Synthesis of novel sugar derived aziridines,as starting materials giving access to sugar amino acid derivatives

d-Erythrosyl aziridines were obtained from d-erythrosyl triazoles either by photolysis or through diazirine intermediates. These were found to undergo rich, high yielding chemistry by reaction with protic acids (HCl, BiI₃/H₂O and trifluoroacetic acid) leading to two types of furanoid sugar α-amino acids, and polyhydroxylprolines. Based on experimental evidence, reaction mechanisms have been proposed for the syntheses.

     

Effect of l-Carnosine as adjunctive therapy in the management of children with autism spectrum disorder: a randomized controlled study

l-Carnosine is an amino acid that acts as an anti-oxidant, anti-toxic and neuroprotective agent. There is a paucity of data about the effectiveness of l-Carnosine in the management of autism spectrum disorder (ASD) in children. This study aimed at investigating the effectiveness of l-Carnosine as adjunctive therapy in the management of ASD. This was a randomized controlled trial. Children aged 3–6 years with a diagnosis of mild to moderate ASD were assigned to standard care arm (occupational and speech therapy) and intervention care arm (l-Carnosine, 10–15 mg/kg in 2 divided doses) plus standard care treatment. The children were assessed at the baseline and the end of 2 months for the scores of Childhood Autism Rating Scale, Second Edition—Standard Version (CARS2-ST), Autism Treatment Evaluation Checklist (ATEC), BEARS sleep screening tool and 6-item Gastrointestinal Severity Index (6-GSI). Of the sixty-seven children enrolled, sixty-three children had completed the study. No statistically significant difference (p > 0.05) was observed for any of the outcome measures assessed. Supplementation of l-Carnosine did not improve the total score of CARS2-ST, ATEC, BEARS sleep screening tool and 6-GSI scores of children with ASD. Further investigations are needed with more objective assessments to critically validate the effectiveness of l-Carnosine on ASD children for more decisive results.

     

A conservative distribution of tridomain NDP-heptose synthetases in actinobacteria

Heptoses are important structural components of Gram-negative bacterium cell wall and participate in bacterial colonization, infection, and immune recognition. Current knowledge of NDP-heptose originating from d-sedoheptulose 7-phosphate in Grampositive bacterium remains limited. Here, in silico analysis suggested that the special tridomain NDP-heptose synthetases with isomerase, kinase, and nucleotidyltransferase activities are conservatively distributed in Actinobacteria class of Gram-positive bacterium. Enzymatical characterization of the tridomain proteins from different strains showed that they are involved in ADP-d-glycero-β-d-manno-heptose biosynthesis despite the unexpected discovery of kinase activities deficient in some proteins. The presence of three types of NDP-heptose synthetases in Gram-positive bacterium suggests that it is also a rich source of heptoses and the heptose moieties may play important roles in vivo. Our work updates the understanding of NDP-heptose biosynthesis in Gram-positive bacterium and lays a solid foundation for further physiological function explorations.

     

Physicochemical properties of l- and dl-valine: first-principles calculations

At present, physicochemical properties of amino acid molecular crystals are of the utmost interest. The compounds where molecules have different chirality are the focus of particular interest. This paper, presents a study on the structural and electronic properties of crystalline l- and dl-valine within the framework of density functional theory including van der Waals interactions. The results of this study showed that electronic properties of the two forms of valine are similar at zero pressure. Pressure leads to different responses in these crystals which is manifested as various deformations of molecules. The pressure effect on the infrared spectra and distribution of electron density of l- and dl-valine has been studied.

     

Colorimetric recognition of aromatic amino acid enantiomers by gluconic acid-capped gold nanoparticles

In this work, we prepared gold nanoparticles (AuNPs) by employing gluconic acid (GlcA) as reducing-cum-stabilizing agent. The proposed GlcA-AuNPs successfully worked as a colorimetric sensor for visual chiral recognition of aromatic amino acid enantiomers, namely tyrosine (d/l-Tyr), phenylalanine (d/l-Phe), and tryptophan (d/l-Trp). After adding L-types to GlcA-AuNPs solution, the color of the mixture changed from red to purple (or gray), while no obvious color change occurred on the addition of D-types. The effect can be detected by naked eyes. The particles have been characterized by transmission electron microscopy, Fourier-transform infrared spectroscopy, zeta potential, the dynamic light scattering analysis as well as UV–Vis spectroscopy. This assay can be used to determine the enantiomeric excess of l-Trp in the range from 0 to + 100%. The method has advantages in simplicity, sensitivity, fast response, and low cost.

     

First enzymological characterization of selenocysteine β-lyase from a lactic acid bacterium,Leuconostoc mesenteroides

We succeeded in expressing selenocysteine β-lyase (SCL) from a lactic acid bacterium, Leuconostoc mesenteroides LK-151 (Lm-SCL), in the soluble fractions of Escherichia coli Rosetta (DE3) using a novel expression vector of pET21malb constructed by ourselves that has both maltose binding protein (MBP)- and 6?×?His-tag. Lm-SCL acted on l-selenocysteine, l-cysteine, and l-cysteine sulfinic acid but showed a high preference for l-selenocysteine. The k_cat and k_cat/K_m values of Lm-SCL were determined to be 108 (min^?1) and 42.0 (min^?1?mM^?1), respectively, and this was enough catalytic efficiency to suggest that Lm-SCL might also be involved in supplying elemental selenium from l-selenocysteine to selenoproteins like other SCLs. The optimum temperature and optimum pH of Lm-SCL were determined to be 37 °C and pH 6.5, respectively. Lm-SCL was stable at 37–45 °C and pH 6.5–7.5. Lm-SCL was completely inhibited by the addition of hydroxylamine, semicarbazide, and iodoacetic acid. The enzyme activity of Lm-SCL was decreased in the presence of various metal ions, especially Cu²⁺. The quaternary structure of Lm-SCL is a homodimer with a subunit molecular mass of 47.5 kDa. The similarity of the primary structure of Lm-SCL to other SCLs from Citrobacter freundii, Escherichia coli, humans, or mouse was calculated to be 47.0, 48.0, 12.5, or 24.0%, respectively. Unlike Ec-SCL, our mutational and molecular docking simulation studies revealed that C362 of Lm-SCL might also catalyze the deselenation of l-selenocysteine in addition to the desulfuration of l-cysteine.

     

A New Preparation of 2,3,6-Tri-O-Methyl d-Galactose

d-galactose was incompletely methylated with methyl sulphate and sodium hydroxide, and two trimethylgalactoses were chromatographically separated from the products. Gas-liquid chromatographic examination, periodate oxidation and melting points of them or their suitable derivatives showed that one of them was 2,3,6-tri-O-methyl d-galactose, and the other was presumed to be 2,4,6-tri-O-methyl d-galactose, For confirmation of 2,3,6- tri-O-methyl d-galactose, 2,3-di-O-methyl l-threose and its aldonophenylhydrazide were prepared from 2,3-di-O-methyl l-arabinose as authentic sample.     

Simultaneous determination of l-tryptophan impurities in meat products

L-tryptophan has been used as a feed additive for swine and poultry and as a nutrient supplement for humans. However, some impurities in l-tryptophan have been reported as causative components of eosinophilia-myalgia syndrome. Therefore, from a safety perspective, it is important to analyze meat samples for these impurities. This study aims to develop an analytical method for the simultaneous detection of l-tryptophan impurities in meat products using LC–MS/MS. Among the various impurities, detection methods for (S)-2-amino-3-(5-hydroxy-1H-indol-3-yl)propanoic acid (5-hydroxytryptophan) (HTP), 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (MTCA), 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo-[2,3-b]-indole-2-carboxylic acid (PIC), and 1,1′-ethylidenebistryptophan (EBT) and 2-(3-indoylmethyl)-l-tryptophan (IMT) were developed. The developed method allowed simultaneous determination of these four impurities in 5 min. No interferences from the matrix were observed, and the method showed good sensitivity to each analyte. The method detection limit and limit of quantification in meat matrices were below 11.2 and 35.7 μg/kg, respectively.

     

Pectin Isolated from the Midrib of Leaves of Nicotiana tabacum

A pectin isolated from tobacco midrib contained residues of d-galacturonic acid (83.7%), L-rhamnose (2.2%), l-arabinose (2.4%) and d-galactose (11.2%) and small amounts of d-xylose and d-glucose. Methylation analysis of the pectin gave 2, 3, 5-tri- and 2, 3-di-O-methyl-l-arabinose, 3, 4-di- and 3-O-methyl-l-rhamnose and 2, 3, 6-tri-O-methyl-d-galactose. Reduction with lithium aluminum hydride of the permethylated pectin gave mainly 2, 3-di-O-methyl-d-galactose and the above methylated sugars. Partial acid hydrolysis gave homologous series of β-(1 → 4)-linked oligosaccharides up to pentaose of d-galactopyranosyl residues, and 2-O-(α-d-galactopyranosyluronic acid)-l-rhamnose, and di- and tri-saccharides of α-(1 → 4)-linked d-galactopyranosyluronic acid residues.

These results suggest that the tobacco pectin has a backbone consisting of α-(1 → 4)-linked d-galactopyranosyluronic acid residues which is interspersed with 2-linked l-rhamnopyranosyl residues. Some of the l-rhamnopyranosyl residues carry substituents on C-4. The pectin has long chain moieties of β-(1 → 4)-linked d-galactopyranosy] residues.     

Synthesis of Some New α- and β-(l→2) Linked Disaccharides Containing L-Quinovose (6-Deoxy L-Glucose)

Hepta-O-acetyl-2-0-β-l-quinovopyranosyl-α-d-glucose (VI) and hepta-O-acetyl-2-O-α-l-quinovopyranosyl-β-d-gIucose (VIII) were prepared by the coupling of 2,3,4-tri-O-acetyl-α-l-quinovopyranosyl bromide (IV) with l,3,4,6-tetra-O-acetyl-α-D-glucose (V) in the presence of mercuric cyanide and mercuric bromide in absolute acetonitrile.

Similarly, hepta-O-acetyW-O-α-l-quinovopyranosyl-α-d-galactose (X) and hepta-O-acetyl-2-O-β-L-quinovopyranosyl-α-d-galactose (XI) were prepared by the reaction of IV with 1,3,4,6-tetra-O-acetyl-α-d-galactose (IX).

Removal of the protecting groups of VI, VIII, X and XI afforded the corresponding disaccharides. On treatment with hydrogen bromide, VI, VIII, X and XI gave the corresponding acetobromo derivatives.     

Topiramate Improves Neuroblast Differentiation of Hippocampal Dentate Gyrus in the <Emphasis Type="SmallCaps">d</Emphasis>-Galactose-Induced Aging Mice via Its Antioxidant Effects

Some anticonvulsant drugs are associated with cognitive ability in patients; Topiramate (TPM) is well known as an effective anticonvulsant agent applied in clinical settings. However, the effect of TPM on the cognitive function is rarely studied. In this study, we aimed to observe the effects of TPM on cell proliferation and neuronal differentiation in the dentate gyrus (DG) of the d-galactose-induced aging mice by Ki-67 and doublecortin (DCX) immunohistochemistry. The study is divided into four groups including control, d-galactose-treated group, 25 and 50 mg/kg TPM-treated plus d-galactose-treated groups. We found, 50 mg/kg (not 25 mg/kg) TPM treatment significantly increased the numbers of Ki-67⁺ cells and DCX immunoreactivity, and improved neuroblast injury induced by d-galactose treatment. In addition, we also found that decreased immunoreactivities and protein levels of antioxidants including superoxide dismutase and catalase induced by d-galactose treatment were significantly recovered by 50 mg/kg TPM treatment in the mice hippocampal DG (P < 0.05). In conclusion, our present results indicate that TPM can ameliorate neuroblast damage and promote cell proliferation and neuroblast differentiation in the hippocampal DG via increasing SODs and catalase levels in the d-galactose mice.