Design,Synthesis and Bioactivity of N-Glycosyl-N′-(5-substituted phenyl-2-furoyl) Hydrazide Derivatives

Condensation products of 5-substituted phenyl-2-furoyl hydrazide with different monosaccharides d-glucose, d-galactose,d-mannose, d-fucose and d-arabinose were prepared. The anomerization and cyclic-acyclic isomers were investigated by ¹H NMR spectroscopy. The results showed that, except for the d-glucose derivatives, which were in the presence of β-anomeric forms, all derivatives were in an acyclic Schiff base form. Their antifungal and antitumor activities were studied. The bioassay results indicated that some title compounds showed superior effects over the commercial positive controls.     

l-Cystathionine Inhibits the Mitochondria-Mediated Macrophage Apoptosis Induced by Oxidized Low Density Lipoprotein

This study was designed to investigate the regulatory role of l-cystathionine in human macrophage apoptosis induced by oxidized low density lipoprotein (ox-LDL) and its possible mechanisms. THP-1 cells were induced with phorbol 12-myristate 13-acetate (PMA) and differentiated into macrophages. Macrophages were incubated with ox-LDL after pretreatment with l-cystathionine. Superoxide anion, apoptosis, mitochondrial membrane potential, and mitochondrial permeability transition pore (MPTP) opening were examined. Caspase-9 activities and expression of cleaved caspase-3 were measured. The results showed that compared with control group, ox-LDL treatment significantly promoted superoxide anion generation, release of cytochrome c (cytc) from mitochondrion into cytoplasm, caspase-9 activities, cleavage of caspase-3, and cell apoptosis, in addition to reduced mitochondrial membrane potential as well as increased MPTP opening. However, 0.3 and 1.0 mmol/L l-cystathionine significantly reduced superoxide anion generation, increased mitochondrial membrane potential, and markedly decreased MPTP opening in ox-LDL + l-cystathionine macrophages. Moreover, compared to ox-LDL treated-cells, release of cytc from mitochondrion into cytoplasm, caspase-9 activities, cleavage of caspase-3, and apoptosis levels in l-cystathionine pretreated cells were profoundly attenuated. Taken together, our results suggested that l-cystathionine could antagonize mitochondria-mediated human macrophage apoptosis induced by ox-LDL via inhibition of cytc release and caspase activation.     

Localization of D-��-Aspartyl Residue-Containing Proteins in Various Tissues

Prior to the emergence of life, it is believed that only l-amino acids were selected for formation of protein and that d-amino acids were eliminated on the primitive Earth. Whilst homochirality is essential for life, the occurrence of proteins containing d-β-aspartyl (Asp) residues in various tissues from elderly subjects has been reported recently. Here, we demonstrate the presence of a d-β-Asp-containing protein in the cardiac muscle of heart, blood vessels of the lung, chief cells of the stomach, longitudinal and circular muscle of the stomach, small intestine and large intestine. Since the d-β-Asp residue occurs through a succinimide intermediate, this isomer may potentially be generated in proteins more easily than initially thought. Formation of the d-β-Asp residue in proteins may be related to stress.     

Oxidation Stress as a Mechanism of Aging in Human Erythrocytes: Protective Effect of Quercetin

Aging is a multi-factorial process developing through a complex net of interactions between biological and cellular mechanisms and it involves oxidative stress (OS) as well as protein glycation. The aim of the present work was to verify the protective role of Quercetin (Q), a polyphenolic flavonoid compound, in a d-Galactose (d-Gal)-induced model of aging in human erythrocytes. The anion-exchange capability through the Band 3 protein (B3p) measured by the rate constant of the SO₄²⁻ uptake, thiobarbituric acid reactive substances (TBARS) levels—a marker of lipid peroxidation—total sulfhydryl (-SH) groups, glycated hemoglobin (A1c), and a reduced glutathione/oxidized glutathione (GSH-GSSG) ratio were determined following the exposure of erythrocytes to 100 mM d-Gal for 24 h, with or without pre-incubation with 10 µM Q. The results confirmed that d-Gal activated OS pathways in human erythrocytes, affecting both membrane lipids and proteins, as denoted by increased TBARS levels and decreased total sulfhydryl groups, respectively. In addition, d-Gal led to an acceleration of the rate constant of the SO₄²⁻ uptake through the B3p. Both the alteration of the B3p function and oxidative damage have been improved by pre-treatment with Q, which preferentially ameliorated lipid peroxidation rather than protein oxidation. Moreover, Q prevented glycated A1c formation, while no protective effect on the endogenous antioxidant system (GSH-GSSG) was observed. These findings suggest that the B3p could be a novel potential target of antioxidant treatments to counteract aging-related disturbances. Further studies are needed to confirm the possible role of Q in pharmacological strategies against aging.     

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.     

Solvation,Cancer Cell Photoinactivation and the Interaction of Chlorin Photosensitizers with a Potential Passive Carrier Non-Ionic Surfactant Tween 80

Cancer and drug-resistant superinfections are common and serious problems afflicting millions worldwide. Photodynamic therapy (PDT) is a successful and clinically approved modality used for the management of many neoplastic and nonmalignant diseases. The combination of the light-activated molecules, so-called photosensitizers (PSs), with an appropriate carrier, is proved to enhance PDT efficacy both in vitro and in vivo. In this paper, we focus on the solvation of several potential chlorin PSs in the 1-octanol/phosphate saline buffer biphasic system, their interaction with non-ionic surfactant Tween 80 and photoinactivation of cancer cells. The chlorin conjugates containing d-galactose and l-arginine fragments are found to have a much stronger affinity towards a lipid-like environment compared to ionic chlorins and form molecular complexes with Tween 80 micelles in water with two modes of binding. The charged macrocyclic PSs are located in the periphery of surfactant micelles near hydrophilic head groups, whereas the d-galactose and l-arginine conjugates are deeper incorporated into the micelle structure occupying positions around the first carbon atoms of the hydrophobic surfactant residue. Our results indicate that both PSs have a pronounced affinity toward the lipid-like environment, leading to their preferential binding to low-density lipoproteins. This and the conjugation of chlorin e₆ with the tumor-targeting molecules are found to enhance their accumulation in cancer cells and PDT efficacy.     

Biochemical Characterization of Pyranose Oxidase from Streptomyces canus—Towards a Better Understanding of Pyranose Oxidase Homologues in Bacteria

Pyranose oxidase (POx, glucose 2-oxidase; EC 1.1.3.10, pyranose:oxygen 2-oxidoreductase) is an FAD-dependent oxidoreductase and a member of the auxiliary activity (AA) enzymes (subfamily AA3_4) in the CAZy database. Despite the general interest in fungal POxs, only a few bacterial POxs have been studied so far. Here, we report the biochemical characterization of a POx from Streptomyces canus (ScPOx), the sequence of which is positioned in a separate, hitherto unexplored clade of the POx phylogenetic tree. Kinetic analyses revealed that ScPOx uses monosaccharide sugars (such as d-glucose, d-xylose, d-galactose) as its electron-donor substrates, albeit with low catalytic efficiencies. Interestingly, various C- and O-glycosides (such as puerarin) were oxidized by ScPOx as well. Some of these glycosides are characteristic substrates for the recently described FAD-dependent C-glycoside 3-oxidase from Microbacterium trichothecenolyticum. Here, we show that FAD-dependent C-glycoside 3-oxidases and pyranose oxidases are enzymes belonging to the same sequence space.     

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.     

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.     

Synthesis and Properties of Alkyl <Emphasis Type="Italic">β</Emphasis>-<Emphasis Type="SmallCaps">d</Emphasis>-Galactopyranoside

A series of alkyl β-d-galactopyranosides were prepared by the trichloroacetimidate method with d-galactose and alcohols with different chain lengths as raw materials. Their solubility, surface tension, emulsification, foaming, wettability, thermotropic liquid crystalline properties, and thermal stability were investigated. Alkyl β-d-galactopyranosides are soluble in water and ethanol, and the solubility decreases with increasing alkyl chain length. Decyl β-d-galactopyranoside was insoluble in water, but soluble in ethanol. Dissolution of alkyl β-d-galactopyranoside in water is an endothermic process with dissolution enthalpies greater than zero. Nonyl β-d-galactopyranoside had an excellent emulsifying property, better foaming ability and the best foam stability. The CMC values of alkyl β-d-galactopyranosides decrease with increasing of alkyl chain length. Alkyl β-d-galactopyranosides are thermally stable up to 270 °C. Alkyl β-d-galactopyranosides show the distinctive optical texture of a thermotropic liquid crystal smectic A type phase. Decyl β-d-galactopyranoside showed the strongest wettability.     

Structure and Antitumor and Immunomodulatory Activities of a Water-Soluble Polysaccharide from Dimocarpus longan Pulp

A new water-soluble polysaccharide (longan polysaccharide 1 (LP1)) was extracted and successfully purified from Dimocarpus longan pulp via diethylaminoethyl (DEAE)-cellulose anion-exchange and Sephacryl S-300 HR gel chromatography. The chemical structure was determined using Infrared (IR), gas chromatography (GC) and nuclear magnetic resonance (NMR) analysis. The results indicated that the molecular weight of the sample was 1.1 × 10⁵ Da. Monosaccharide composition analysis revealed that LP1 was composed of Glc, GalA, Ara and Gal in a molar ratio of 5.39:1.04:0.74:0.21. Structural analysis indicated that LP1 consisted of a backbone of →4)-α-d-Glcp-(1→4)-α-d-GalpA-(1→4)-α-d-Glcp-(1→4)-β-d-Glcp-(1→ units with poly saccharide side chains composed of →2)-β-d-Fruf-(1→2)-l-sorbose-(1→ attached to the O-6 position of the α-d-Glcp residues. In vitro experiments indicated that LP1 had significantly high antitumor activity against SKOV3 and HO8910 tumor cells, with inhibition percentages of 40% and 50%, respectively. In addition, LP1 significantly stimulated the production of the cytokine interferon-γ (IFN-γ), increased the activity of murine macrophages and enhanced B- and T-lymphocyte proliferation. The results of this study demonstrate that LP1 has potential applications as a natural antitumor agent with immunomodulatory activity.     

Phytochemical and Antioxidant Profile of the Medicinal Plant Melia azedarach Subjected to Water Deficit Conditions

Environmental stress triggered by climate change can alter the plant’s metabolite profile, which affects its physiology and performance. This is particularly important in medicinal species because their economic value depends on the richness of their phytocompounds. We aimed to characterize how water deficit modulated the medicinal species Melia azedarach’s lipophilic profile and antioxidant status. Young plants were exposed to water deficit for 20 days, and lipophilic metabolite profile and the antioxidant capacity were evaluated. Leaves of M. azedarach are rich in important fatty acids and oleamide. Water deficit increased the radical scavenging capacity, total phenol, flavonoids, and catechol pools, and the accumulation of β-sitosterol, myo-inositol, succinic acid, sucrose, d-glucose and derivatives, d-psicofuranose, d-(+)-fructofuranose, and the fatty acids stearic, α-linolenic, linoleic and palmitic acids. These responses are relevant to protecting the plant against climate change-related stress and also increase the nutritional and antioxidant quality of M. azedarach leaves.     

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.     

New Triterpenoids with Cytotoxic Activity from Actinidia Valvata

Two new triterpenoids, 30-O-β-d-glucopyranosyloxy-2α,3α,24-trihydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (1) and 2α,3β,3,30-tetrahydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (2) were isolated from roots of Actinidia valvata Dunn. Their structures were elucidated by means of extensive spectroscopic studies. Both these two new compounds showed moderate cytotoxic activity in vitro against BEL-7402 and SMMC-7721 tumor cell line.     

Hydrogen Bonding Drives Helical Chirality via 10-Membered Rings in Dipeptide Conjugates of Ferrocene-1,1′-Diamine

Considering the enormous importance of protein turns as participants in various biological events, such as protein–protein interactions, great efforts have been made to develop their conformationally and proteolytically stable mimetics. Ferrocene-1,1′-diamine was previously shown to nucleate the stable turn structures in peptides prepared by conjugation with Ala (III) and Ala–Pro (VI). Here, we prepared the homochiral conjugates of ferrocene-1,1′-diamine with l-/d-Phe (32/35), l-/d-Val (33/36), and l-/d-Leu (34/37) to investigate (1) whether the organometallic template induces the turn structure upon conjugation with amino acids, and (2) whether the bulky or branched side chains of Phe, Val, and Leu affect hydrogen bonding. Detailed spectroscopic (IR, NMR, CD), X-ray, and DFT studies revealed the presence of two simultaneous 10-membered interstrand hydrogen bonds, i.e., two simultaneous β-turns in goal compounds. A preliminary biological evaluation of d-Leu conjugate 37 showed its modest potential to induce cell cycle arrest in the G0/G1 phase in the HeLa cell line but these results need further investigation.     

Crosstalk between Delta Opioid Receptor and Nerve Growth Factor Signaling Modulates Neuroprotection and Differentiation in Rodent Cell Models

Both opioid signaling and neurotrophic factor signaling have played an important role in neuroprotection and differentiation in the nervous system. Little is known about whether the crosstalk between these two signaling pathways will affect neuroprotection and differentiation. Previously, we found that nerve growth factor (NGF) could induce expression of the delta opioid receptor gene (Oprd1, dor), mainly through PI3K/Akt/NF-κB signaling in PC12h cells. In this study, using two NGF-responsive rodent cell model systems, PC12h cells and F11 cells, we found the delta opioid neuropeptide [d-Ala², d-Leu⁵] enkephalin (DADLE)-mediated neuroprotective effect could be blocked by pharmacological reagents: the delta opioid antagonist naltrindole, PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, MAPK inhibitor PD98059, and Trk inhibitor K252a, respectively. Western blot analysis revealed that DADLE activated both the PI3K/Akt and MAPK pathways in the two cell lines. siRNA Oprd1 gene knockdown experiment showed that the upregulation of NGF mRNA level was inhibited with concomitant inhibition of the survival effects of DADLE in the both cell models. siRNA Oprd1 gene knockdown also attenuated the DADLE-mediated neurite outgrowth in PC12h cells as well as phosphorylation of MAPK and Akt in PC12h and F11 cells, respectively. These data together strongly suggest that delta opioid peptide DADLE acts through the NGF-induced functional G protein-coupled Oprd1 to provide its neuroprotective and differentiating effects at least in part by regulating survival and differentiating MAPK and PI3K/Akt signaling pathways in NGF-responsive rodent neuronal cells.     

Structural Determination and Genetic Identification of the O-Antigen from an Escherichia coli Strain,LL004, Representing a Novel Serogroup

The O-antigen is the outermost component of the lipopolysaccharide layer in Gram-negative bacteria, and the variation of O-antigen structure provides the basis for bacterial serological diversity. Here, we determined the O-antigen structure of an Escherichia coli strain, LL004, which is totally different from all of the E. coli serogroups. The tetrasaccharide repeating unit was determined as →4)-β-d-Galp-(1→3)-β-d-GlcpNAc6OAc(~70%)-(1→3)-β-d-GalpA-(1→3)-β-d-GalpNAc-(1→ with monosaccharide analysis and NMR spectra. We also characterized the O-antigen gene cluster of LL004, and sequence analysis showed that it correlated well with the O-antigen structure. Deletion and complementation testing further confirmed its role in O-antigen biosynthesis, and indicated that the O-antigen of LL004 is assembled via the Wzx/Wzy dependent pathway. Our findings, in combination, suggest that LL004 should represent a novel serogroup of E. coli.     

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.