Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in l-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport—NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885—were also expressed at significantly higher levels in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, l-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production.     

Vasorelaxant and Antioxidant Activities of Spilanthes acmella Murr.

This study reports the effect of Spilanthes acmella Murr. extracts on phenylephrine-induced contraction of rat thoracic aorta as well as their antioxidant activity. Results show that the extracts exert maximal vasorelaxations in a dose-dependent manner, but their effects are less than acetylcholine-induced nitric oxide (NO) vasorelaxation. Significant reduction of vasorelaxations is observed in both N^G-nitro-l-arginine methyl ester (l-NAME) and indomethacin (INDO). In the presence of l-NAME plus INDO, synergistic effects are observed, leading to loss of vasorelaxation of both acetylcholine and the extracts. Similarly, the vasorelaxations of the extracts are completely abolished upon the removal of endothelial cells. This demonstrates that the extracts exhibit vasorelaxation via partially endothelium-induced NO and prostacyclin in a dose-dependent manner. Significantly, the ethyl acetate extract exerts immediate vasorelaxation (ED₅₀ 76.1 ng/mL) and is the most potent antioxidant (DPPH assay). The chloroform extract shows the highest vasorelaxation and antioxidation (SOD assay). These reveal a potential source of vasodilators and antioxidants.     

Tryptophanase-Catalyzed l-Tryptophan Synthesis from d-Serine in the Presence of Diammonium Hydrogen Phosphate

Tryptophanase, an enzyme with extreme absolute stereospecificity for optically active stereoisomers, catalyzes the synthesis of l-tryptophan from l-serine and indole through a β-substitution mechanism of the ping-pong type, and has no activity on d-serine. We previously reported that tryptophanase changed its stereospecificity to degrade d-tryptophan in highly concentrated diammonium hydrogen phosphate, (NH₄)₂HPO₄ solution. The present study provided the same stereospecific change seen in the d-tryptophan degradation reaction also occurs in tryptophan synthesis from d-serine. Tryptophanase became active to d-serine to synthesize l-tryptophan in the presence of diammonium hydrogen phosphate. This reaction has never been reported before. d-serine seems to undergo β-replacement via an enzyme-bonded α-aminoacylate intermediate to yield l-tryptophan.     

Asymmetric Dimethylarginine in Chronic Obstructive Pulmonary Disease (ADMA in COPD)

l-Arginine metabolism including the nitric oxide (NO) synthase and arginase pathways is important in the maintenance of airways function. We have previously reported that accumulation of asymmetric dimethylarginine (ADMA) in airways, resulting in changes in l-arginine metabolism, contributes to airways obstruction in asthma and cystic fibrosis. Herein, we assessed l-arginine metabolism in airways of patients with chronic obstructive pulmonary disease (COPD). Lung function testing, measurement of fractional exhaled NO (FeNO) and sputum NO metabolites, as well as quantification of l-arginine metabolites (l-arginine, l-ornithine, l-citrulline, ADMA and symmetric dimethylarginine) using liquid chromatography-mass spectrometry (LC-MS) were performed. Concentrations of l-ornithine, the product of arginase activity, correlated directly with l-arginine and ADMA sputum concentrations. FeNO correlated directly with pre- and post-bronchodilator forced expiratory volume in one second (FEV₁). Sputum arginase activity correlated inversely with total NO metabolite (NO_x) and nitrite concentrations in sputum, and with pre- and post-bronchodilator FEV₁. These findings suggest that ADMA in COPD airways results in a functionally relevant shift of l-arginine breakdown by the NO synthases towards the arginase pathway, which contributes to airway obstruction in these patients.     

Determination of Homoarginine,Arginine, NMMA,ADMA, and SDMA in Biological Samples by HPLC-ESI-Mass Spectrometry

N^G,N^G-dimethyl-l-arginine (ADMA) and N^G-methyl-l-arginine (NMMA) are endogenous inhibitors of nitric oxide synthase (NOS). In contrast, N^G,N′^G-dimethyl-Larginine (SDMA) possesses only a weak inhibitory potency towards neuronal NOS and it is known to limit nitric oxide (NO) production by competing with l-arginine for cellular uptake. The inhibition of NOS is associated with endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. l-Homoarginine (HArg), a structural analog of l-arginine (Arg), is an alternative but less efficient substrate for NOS. Besides, it inhibits arginase, leading to an increased availability of l-arginine for NOS to produce NO. However, its relation with cardiovascular disease remains unclear. To date, several analytical methods for the quantitative determination of Arg, HArg, NMMA, AMDA, and SDMA in biological samples have been described. Here, we present a simple, fast, and accurate HPLC-ESI-MS/MS method which allows both the simultaneous determination and quantification of these compounds without needing derivatization, and the possibility to easily modulate the chromatographic separation between HArg and NMMA (or between SDMA and ADMA). Data on biological samples revealed the feasibility of the method, the minimal sample preparation, and the fast run time which make this method very suitable and accurate for analysis in the basic and clinical settings.     

Adaptive Responses of Citrus grandis Leaves to Copper Toxicity Revealed by RNA-Seq and Physiology

Copper (Cu)-toxic effects on Citrus grandis growth and Cu uptake, as well as gene expression and physiological parameters in leaves were investigated. Using RNA-Seq, 715 upregulated and 573 downregulated genes were identified in leaves of C. grandis seedlings exposed to Cu-toxicity (LCGSEC). Cu-toxicity altered the expression of 52 genes related to cell wall metabolism, thus impairing cell wall metabolism and lowering leaf growth. Cu-toxicity downregulated the expression of photosynthetic electron transport-related genes, thus reducing CO₂ assimilation. Some genes involved in thermal energy dissipation, photorespiration, reactive oxygen species scavenging and cell redox homeostasis and some antioxidants (reduced glutathione, phytochelatins, metallothioneins, l-tryptophan and total phenolics) were upregulated in LCGSEC, but they could not protect LCGSEC from oxidative damage. Several adaptive responses might occur in LCGSEC. LCGSEC displayed both enhanced capacities to maintain homeostasis of Cu via reducing Cu uptake by leaves and preventing release of vacuolar Cu into the cytoplasm, and to improve internal detoxification of Cu by accumulating Cu chelators (lignin, reduced glutathione, phytochelatins, metallothioneins, l-tryptophan and total phenolics). The capacities to maintain both energy homeostasis and Ca homeostasis might be upregulated in LCGSEC. Cu-toxicity increased abscisates (auxins) level, thus stimulating stomatal closure and lowering water loss (enhancing water use efficiency and photosynthesis).     

Mitochondria Related Pathway Is Essential for Polysaccharides Purified from Sparassis crispa Mediated Neuro-Protection against Glutamate-Induced Toxicity in Differentiated PC12 Cells

The present study aims to explore the neuro-protective effects of purified Sparassis crispa polysaccharides against l-glutamic acid (l-Glu)-induced differentiated PC12 (DPC12) cell damages and its underlying mechanisms. The Sparassis crispa water extract was purified by a DEAE-52 cellulose anion exchange column and a Sepharose G-100 column. A fraction with a molecular weight of 75 kDa and a diameter of 88.9 nm, entitled SCWEA, was obtained. SCWEA was identified with a triple helix with (1→3)-linked Rha in the backbone, and (1→2) linkages and (1→6) linkages in the side bone. Our results indicated that the pre-treatment of DPC12 cells with SCWEA prior to l-Glu exposure effectively reversed the reduction on cell viability (by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay) and reduced l-Glu-induced apoptosis (by Hoechst staining). SCWEA decreased the accumulation of intracellular reactive oxygen species, blocked Ca²⁺ influx and prevented depolarization of the mitochondrial membrane potential in DPC12 cells. Furthermore, SCWEA normalized expression of anti-apoptotic proteins in l-Glu-explored DPC12 cells. These results suggested that SCWEA protects against l-Glu-induced neuronal apoptosis in DPC12 cells and may be a promising candidate for treatment against neurodegenerative disease.     

Synthesis and Properties of Poly(l-lactide)-b-poly (l-phenylalanine) Hybrid Copolymers

Hybrid materials constituted by peptides and synthetic polymers have nowadays a great interest since they can combine the properties and functions of each constitutive block, being also possible to modify the final characteristics by using different topologies. Poly(l-lactide-b-l-phenylalanine) copolymers with various block lengths were synthesized by sequential ring-opening polymerization of l-lactide and the N-carboxyanhydride of l-phenylalanine. The resulting block copolymers were characterized by NMR spectrometry, IR spectroscopy, gel permeation chromatography, MALDI-TOF and UV-vis, revealing the successful incorporation of the polyphenylalanine (PPhe) peptide into the previously formed poly(l-lactide) (PLLA) polymer chain. X-ray diffraction and DSC data also suggested that the copolymers were phase-separated in domains containing either crystalline PLLA or PPhe phases. A peculiar thermal behavior was also found by thermogravimetric analysis when polyphenylalanine blocks were incorporated into polylactide.     

Tubular Assembly Formation Induced by Leucine Alignment along the Hydrophobic Helix of Amphiphilic Polypeptides

The introduction of α-helical structure with a specific helix–helix interaction into an amphipathic molecule enables the determination of the molecular packing in the assembly and the morphological control of peptide assemblies. We previously reported that the amphiphilic polypeptide SL12 with a polysarcosine (PSar) hydrophilic chain and hydrophobic α-helix (l-Leu-Aib)₆ involving the LxxxLxxxL sequence, which induces homo-dimerization due to the concave–convex interaction, formed a nanotube with a uniform 80 nm diameter. In this study, we investigated the importance of the LxxxLxxxL sequence for tube formation by comparing amphiphilic polypeptide SL4A4L4 with hydrophobic α-helix (l-Leu-Aib)₂-(l-Ala-Aib)₂-(l-Leu-Aib)₂ and SL12. SL4A4L4 formed spherical vesicles and micelles. The effect of the LxxxLxxxL sequence elongation on tube formation was demonstrated by studying assemblies of PSar-b-(l-Ala-Aib)-(l-Leu-Aib)₆-(l-Ala-Aib) (SA2L12A2) and PSar-b-(l-Leu-Aib)₈ (SL16). SA2L12A2 formed nanotubes with a uniform 123 nm diameter, while SL16 assembled into vesicles. These results showed that LxxxLxxxL is a necessary and sufficient sequence for the self-assembly of nanotubes. Furthermore, we fabricated a double-layer nanotube by combining two kinds of nanotubes with 80 and 120 nm diameters—SL12 and SA2L12A2. When SA2L12A2 self-assembled in SL12 nanotube dispersion, SA2L12A2 initially formed a rolled sheet, the sheet then wrapped the SL12 nanotube, and a double-layer nanotube was obtained.     

Domain Motions and Functionally-Key Residues of l-Alanine Dehydrogenase Revealed by an Elastic Network Model

Mycobacterium tuberculosis l-alanine dehydrogenase (l-MtAlaDH) plays an important role in catalyzing l-alanine to ammonia and pyruvate, which has been considered to be a potential target for tuberculosis treatment. In the present work, the functional domain motions encoded in the structure of l-MtAlaDH were investigated by using the Gaussian network model (GNM) and the anisotropy network model (ANM). The slowest modes for the open-apo and closed-holo structures of the enzyme show that the domain motions have a common hinge axis centered in residues Met133 and Met301. Accompanying the conformational transition, both the 1,4-dihydronicotinamide adenine dinucleotide (NAD)-binding domain (NBD) and the substrate-binding domain (SBD) move in a highly coupled way. The first three slowest modes of ANM exhibit the open-closed, rotation and twist motions of l-MtAlaDH, respectively. The calculation of the fast modes reveals the residues responsible for the stability of the protein, and some of them are involved in the interaction with the ligand. Then, the functionally-important residues relevant to the binding of the ligand were identified by using a thermodynamic method. Our computational results are consistent with the experimental data, which will help us to understand the physical mechanism for the function of l-MtAlaDH.     

Discovery of Benzo[f]indole-4,9-dione Derivatives as New Types of Anti-Inflammatory Agents

Certain benzo[f]indole-4,9-dione derivatives were synthesized and evaluated for their inhibitory effects on superoxide anion generation and neutrophil elastase (NE) release in formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLF)-activated human neutrophils. Results indicated that (Z)-1-benzyl-4-(hydroxyimino)-1H-benzo[f]indol-9(4H)-one (10) showed a potent dual inhibitory effect on NE release and superoxide anion generation with IC₅₀ value of 2.78 and 2.74 μM respectively. The action mechanisms of 10 in human neutrophils were further investigated. Our results showed that compound 10 did not alter fMLF-induced phosphorylation of Src (Src family Y416). Notably, phosphorylation of Akt (S473) and mobilization of [Ca²⁺]_i caused by fMLF was inhibited by compound 10. Further structural optimization of 10 is ongoing.     

Ionophore Ability of Carnosine and Its Trehalose Conjugate Assists Copper Signal in Triggering Brain-Derived Neurotrophic Factor and Vascular Endothelial Growth Factor Activation In Vitro

l-carnosine (β-alanyl-l-histidine) (Car hereafter) is a natural dipeptide widely distributed in mammalian tissues and reaching high concentrations (0.7–2.0 mM) in the brain. The molecular features of the dipeptide underlie the antioxidant, anti-aggregating and metal chelating ability showed in a large number of physiological effects, while the biological mechanisms involved in the protective role found against several diseases cannot be explained on the basis of the above-mentioned properties alone, requiring further research efforts. It has been reported that l-carnosine increases the secretion and expression of various neurotrophic factors and affects copper homeostasis in nervous cells inducing Cu cellular uptake in keeping with the key metal-sensing system. Having in mind this l-carnosine ability, here we report the copper-binding and ionophore ability of l-carnosine to activate tyrosine kinase cascade pathways in PC12 cells and stimulate the expression of BDNF. Furthermore, the study was extended to verify the ability of the dipeptide to favor copper signaling inducing the expression of VEGF. Being aware that the potential protective action of l-carnosine is drastically hampered by its hydrolysis, we also report on the behavior of a conjugate of l-carnosine with trehalose that blocks the carnosinase degradative activity. Overall, our findings describe a copper tuning effect on the ability of l-carnosine and, particularly its conjugate, to activate tyrosine kinase cascade pathways.     

Functional Identification of Proteus mirabilis eptC Gene Encoding a Core Lipopolysaccharide Phosphoethanolamine Transferase

By comparison of the Proteus mirabilis HI4320 genome with known lipopolysaccharide (LPS) phosphoethanolamine transferases, three putative candidates (PMI3040, PMI3576, and PMI3104) were identified. One of them, eptC (PMI3104) was able to modify the LPS of two defined non-polar core LPS mutants of Klebsiella pneumoniae that we use as surrogate substrates. Mass spectrometry and nuclear magnetic resonance showed that eptC directs the incorporation of phosphoethanolamine to the O-6 of l-glycero-d-mano-heptose II. The eptC gene is found in all the P. mirabilis strains analyzed in this study. Putative eptC homologues were found for only two additional genera of the Enterobacteriaceae family, Photobacterium and Providencia. The data obtained in this work supports the role of the eptC (PMI3104) product in the transfer of PEtN to the O-6 of l,d-HepII in P. mirabilis strains.     

New Benzo[c]phenanthridine and Benzenoid Derivatives,and Other Constituents from Zanthoxylum ailanthoides: Effects on Neutrophil Pro-Inflammatory Responses

A new benzo[c]phenanthridine, oxynorchelerythrine (1), and two new benzenoid derivatives, methyl 4-(2-hydroxy-4-methoxy-3-methyl-4-oxobutoxy)benzoate (2) and (E)-methyl 4-(4-((Z)-3-methoxy-3-oxoprop-1-enyl)phenoxy)-2-methylbut-2-enoate (3), have been isolated from the twigs of Zanthoxylum ailanthoides, together with 11 known compounds (4–14). The structures of these new compounds were determined through spectroscopic and MS analyses. Among the isolated compounds, decarine (4), (−)-syringaresinol (6), (+)-episesamin (8), glaberide I (9), (−)-dihydrocubebin (10), and xanthyletin (11) exhibited potent inhibition (IC₅₀ values ≤ 4.79 μg/mL) of superoxide anion generation by human nutrophils in response to N-formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB). Compounds 4, 8, and 11 also inhibited fMLP/CB-induced elastase release with IC₅₀ values ≤ 5.48 μg/mL.     

New Coumarins and Anti-Inflammatory Constituents from the Fruits of Cnidium monnieri

The fruit of Cnidium monnieri is commercially used as healthcare products for the improvement of impotence and skin diseases. Three new coumarins, 3''-O-methylmurraol (1), rel-(1''S,2''S)-1''-O-methylphlojodicarpin (2), and (1''S,2''S)-1''-O-methylvaginol (3), have been isolated from the fruits of C. monnieri, together with 14 known compounds (4–17). The structures of these new compounds were determined through spectroscopic and MS analyses. Compounds 1, 4–12, and 14–17 exhibited inhibition (IC₅₀ ≤ 7.31 µg/mL) of superoxide anion generation by human neutrophils in response to formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB). Compounds 7, 9–11, 15, and 17 inhibited fMLP/CB-induced elastase release with IC₅₀ values ≤7.83 µg/mL. This investigation reveals that bioactive isolates (especially 6, 7, 14, and 17) could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases.     

New Glycosylated Dihydrochalcones Obtained by Biotransformation of 2′-Hydroxy-2-methylchalcone in Cultures of Entomopathogenic Filamentous Fungi

Flavonoids, including chalcones, are more stable and bioavailable in the form of glycosylated and methylated derivatives. The combined chemical and biotechnological methods can be applied to obtain such compounds. In the present study, 2′-hydroxy-2-methylchalcone was synthesized and biotransformed in the cultures of entomopathogenic filamentous fungi Beauveria bassiana KCH J1.5, Isaria fumosorosea KCH J2 and Isaria farinosa KCH J2.6, which have been known for their extensive enzymatic system and ability to perform glycosylation of flavonoids. As a result, five new glycosylated dihydrochalcones were obtained. Biotransformation of 2′-hydroxy-2-methylchalcone by B. bassiana KCH J1.5 resulted in four glycosylated dihydrochalcones: 2′-hydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, 2′,3-dihydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, 2′-hydroxy-2-hydroxymethyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, and 2′,4-dihydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside. In the culture of I. fumosorosea KCH J2 only one product was formed—3-hydroxy-2-methyldihydrochalcone 2′-O-β-d-(4″-O-methyl)-glucopyranoside. Biotransformation performed by I. farinosa KCH J2.6 resulted in the formation of two products: 2′-hydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside and 2′,3-dihydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside. The structures of all obtained products were established based on the NMR spectroscopy. All products mentioned above may be used in further studies as potentially bioactive compounds with improved stability and bioavailability. These compounds can be considered as flavor enhancers and potential sweeteners.     

Structural Basis of Cysteine Ligase MshC Inhibition by Cysteinyl-Sulfonamides

Mycothiol (MSH), the major cellular thiol in Mycobacterium tuberculosis (Mtb), plays an essential role in the resistance of Mtb to various antibiotics and oxidative stresses. MshC catalyzes the ATP-dependent ligation of 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol (GlcN-Ins) with l-cysteine (l-Cys) to form l-Cys-GlcN-Ins, the penultimate step in MSH biosynthesis. The inhibition of MshC is lethal to Mtb. In the present study, five new cysteinyl-sulfonamides were synthesized, and their binding affinity with MshC was evaluated using a thermal shift assay. Two of them bind the target with EC₅₀ values of 219 and 231 µM. Crystal structures of full-length MshC in complex with these two compounds showed that they were bound in the catalytic site of MshC, inducing dramatic conformational changes of the catalytic site compared to the apo form. In particular, the observed closure of the KMSKS loop was not detected in the published cysteinyl-sulfamoyl adenosine-bound structure, the latter likely due to trypsin treatment. Despite the confirmed binding to MshC, the compounds did not suppress Mtb culture growth, which might be explained by the lack of adequate cellular uptake. Taken together, these novel cysteinyl-sulfonamide MshC inhibitors and newly reported full-length apo and ligand-bound MshC structures provide a promising starting point for the further development of novel anti-tubercular drugs targeting MshC.