Chemo-Enzymatic Synthesis of Isomeric I-branched Polylactosamines Using Traceless Blocking Groups.

Poly-N-acetyl lactosamines (polyLacNAc) are common structural motifs of N- and O-linked glycan, glycosphingolipids and human milk oligosaccharides. They can be branched by the addition of ß1,6-linked N-acetyl-glucosamine (GlcNAc) moieties to internal galactoside (Gal) residues by the I-branching enzyme beta-1,6-N-acetylglucosaminyltransferase 2 (GCNT2). I-branching has been implicated in many biological processes and is also associated with various diseases such as cancer progression. Currently, there is a lack of methods that can install, in a regioselective manner, I-branches and allows the preparation of isomeric poly-LacNAc derivatives. Here, we described a chemo-enzymatic strategy that addresses this deficiency and is based on the enzymatic assembly of an oligo-LacNAc chain that at specific positions is modified by a GlcNTFA moiety. Replacement of the trifluoroacetyl (TFA) moiety by tert-butyloxycarbonyl (Boc) gives compounds in which the galactoside at the proximal site is blocked from modification by GCNT2. After elaboration of the antennae, the Boc group can be removed, and the resulting amine acetylated to give natural I-branched structures. It is also shown that fucosides can function as a traceless blocking group that can provide complementary I-branched structures from a single precursor. The methodology made it possible to synthesize a library of polyLacNAc chains having various topologies.

Dietary flavonoid myricetin 3-O-galactoside suppresses α-melanocyte stimulating hormone-induced melanogenesis in B16F10 melanoma cells by regulating PKA and ERK1/2 activation.

Melanogenesis is the process where skin pigment melanin is produced through tyrosinase activity. Overproduction of melanin causes skin disorders such as freckles, spots, and hyperpigmentation. Myricetin 3-O-galactoside (M3G) is a dietary flavonoid with reported bioactivities. M3G was isolated from Limonium tetragonum and its anti-melanogenic properties were investigated in α-melanocyte stimulating hormone-stimulated B16F10 melanoma cells. The in vitro anti-melanogenic capacity of M3G was confirmed by inhibited tyrosinase and melanin production. M3G-mediated suppression of melanogenic proteins, tyrosinase, microphthalmia-associated transcription factor (MITF), and tyrosinase-related proteins (TRP)-1 and TRP-2, were confirmed by mRNA and protein levels, analyzed by RT-qPCR and Western blot, respectively. Furthermore, M3G suppressed Wnt signaling through the inhibition of PKA phosphorylation. M3G also suppressed the consequent phosphorylation of CREB and nuclear levels of MITF. Analysis of MAPK activation further revealed that M3G increased the activation of ERK1/2 while p38 and JNK activation remained unaffected. Results showed that M3G suppressed melanogenesis in B16F10 cells by decreasing tyrosinase production and therefore inhibiting melanin formation. A possible action mechanism was the suppression of CREB activation and upregulation of ERK phosphorylation which might cause the decreased nuclear levels of MITF. In conclusion, M3G was suggested to be a potential nutraceutical with anti-melanogenic properties.

Iridoid- and flavonoid-enriched fractions of Cornus sanguinea and Cornus mas exert antioxidant and anti-inflammatory effects and inhibit key enzymes in the treatment of metabolic disorders.

Background: Berry fruits are recognized as a "superfood" due to their high content of bioactive compounds and health benefits. Scope and approach: Herein, extracts of Cornus sanguinea and Cornus mas fresh and dried fruits obtained by different extraction procedures (ethanolic and hydroalcoholic maceration, ultrasound-assisted extraction, and Soxhlet apparatus) were analysed using liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-QTOF-MS) and compared to identify the main healthy compounds and their impact on the inhibition of key enzymes (pancreatic lipase, α-glucosidase, and α-amylase) associated with metabolic disorders. The antioxidant activity and inhibition of nitric oxide (NO) and NF-κB pathway were also investigated. Key findings and conclusions: Flavonoids, iridoids, and phenolic acids were the main classes of identified compounds. Herein, kaempferol 3-O-galactoside, kaempferol 3-O-glucoside, quercetin, quercetin 3-O-xyloside, and myricetin 3-O-galactoside were detected for the first time in C. sanguinea. Remarkable antioxidant effects and promising α-glucosidase and lipase inhibitory activity were observed with extracts obtained by hydroalcoholic maceration of both Cornus dried fruits. Consequently, these extracts were subjected to fractionation using Amberlite XAD-16 resin. The most promising biological activities, which are attributed to the presence of some flavonoids and iridoids, were detected with the C. sanguinea fractions, in particular SD2(II). The results of this study offer new insights into the potential development of functional foods, nutraceuticals, and food supplements using the Cornus species.

Chaperonin GroEL hydrolyses ortho-nitrophenyl ß-galactoside.

We serendipitously found that chaperonin GroEL can hydrolyze ortho-nitrophenyl ß-galactoside (ONPG), a well-known substrate of the enzyme ß-galactosidase. The ONPG hydrolysis by GroEL follows typical enzyme kinetics. Our experiments and molecular docking studies suggest ONPG binding at the ATP binding site of GroEL.

Quercetin 3-O-Galactoside Isolated from Limonium tetragonum Inhibits Melanogenesis by Regulating PKA/MITF Signaling and ERK Activation.

Quercetin 3-O-galactoside (Q3G) is a common dietary flavanol that has been shown to possess several bioactivities, including anti-melanogenesis. However, how Q3G exerts its anti-melanogenic effect has not been studied. The current study, therefore aimed to investigate the anti-melanogenesis potential of Q3G and elucidate the underlying action mechanism in α-melanocyte-stimulating hormone (α-MSH)-induced hyperpigmentation model of B16F10 murine melanoma cells. Results showed that α-MSH stimulation significantly increased tyrosinase (TYR) and melanin production, which were significantly downregulated by Q3G treatment. The treatment with Q3G suppressed the transcriptional and protein expressions of melanogenesis-related enzymes TYR, tyrosinase related protein-1 (TRP-1), and TRP-2, along with the melanogenic transcription factor microphthalmia-associated transcription factor (MITF) in B16F10 cells. It was shown that Q3G downregulated MITF expression and suppressed its transcriptional activity by inhibiting the cAMP-dependent protein kinase A (PKA)-mediated activation of CREB and GSK3ß. In addition, MAPK-regulated MITF activation signaling was also involved in the inhibition of melanin production by Q3G. The results suggest that the anti-melanogenic properties of Q3G rationalize further studies in vivo to confirm its action mechanism and consequent utilization as a cosmetic ingredient against hyperpigmentation.

ZnI2-Mediated ß-Galactosylation of C2-Ether-Type Donor.

Conventional glycosylation with galactosyl donors having C-2 benzyl (Bn) ether-type functionality often leads to anomeric mixtures, due to the anomeric and steric effects that stabilize the 1,2-cis-α- and 1,2-trans-ß-glycosides, respectively. Herein we report a versatile ZnI2-directed ß-galactosylation approach employing a 4,6-O-tethered and 2-O-Bn galactosyl donor for the stereoselective and efficient synthesis of ß-O-galactosides. With a broad substrate scope, the reaction tolerates a wide range of functional groups and complex molecular architectures, providing stereocontrolled ß-galactosides in moderate to excellent yields. The practicality of this transformation is demonstrated through the synthesis of a tetrasaccharide arabinogalactan fragment with high stereoselectivity.

Identification and Quantification of Anthocyanin and Catechin Compounds in Purple Tea Leaves and Flakes.

Tea is the first most popular beverage worldwide and is available in several selections such as black (fully oxidized), Oolong (partially oxidized) and green (non-oxidized), in addition to purple tea, an emerging variety derived from the same tea plant (Camellia sinensis). This study investigated purple tea leaves (non-oxidized) and flakes (water extractable) to thoroughly identify their composition of anthocyanins and catechins and to study the effect of a water extraction process on their compositional properties in comparison with green tea. Anthocyanin and catechin compounds were separated and quantified using UPLC, and their identity was confirmed using LC-MS/MS in positive and negative ionization modes. Delphinidin was the principal anthocyaninidin in purple tea, while cyanidin came in second. The major anthocyanin pigments in purple tea were delphinidin-coumaroyl-hexoside followed by delphinidin-3-galactoside and cyanidin-coumaroyl-hexoside. The water extraction process resulted in substantial reductions in anthocyanins in purple tea flakes. There were no anthocyanin compounds detected in green tea samples. Both purple and green tea types were rich in catechins, with green tea containing higher concentrations than purple tea. The main catechin in purple or green tea was epigallocatechin gallate (EGCG) followed by either epicatechin gallate (ECG) or epigallocatechin (EGC), subject to tea type. The extraction process increased the concentration of catechins in both purple and green tea flakes. The results suggest that purple tea holds promise in making healthy brews, natural colorants and antioxidants and/or functional ingredients for beverages, cosmetics and healthcare industries due to its high content of anthocyanins and catechins.

Far-red Fluorescent Senescence-associated ß-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry.

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-ß-Gal). Typically, the SA-ß-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-ß-Gal substrates, including C12-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C12-FDG is a well-documented probe for SA-ß-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-ß-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling. Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.

Galectin-3 Is a Natural Binding Ligand of MCAM (CD146, MUC18) in Melanoma Cells and Their Interaction Promotes Melanoma Progression.

Melanoma cell adhesion molecule (MCAM, CD146, MUC18) is a heavily glycosylated transmembrane protein and a marker of melanoma metastasis. It is expressed in advanced primary melanoma and metastasis but rarely in benign naevi or normal melanocytes. More and more evidence has shown that activation of the MCAM on cell surface plays a vital role in melanoma progression and metastasis. However, the natural MCAM binding ligand that initiates MCAM activation in melanoma so far remains elusive. This study revealed that galectin-3, a galactoside-binding protein that is commonly overexpressed in many cancers including melanoma, is naturally associated with MCAM on the surface of both skin and uveal melanoma cells. Binding of galectin-3 to MCAM, via O-linked glycans on the MCAM, induces MCAM dimerization and clustering on cell surface and subsequent activation of downstream AKT signalling. This leads to the increases of a number of important steps in melanoma progression of cell proliferation, adhesion, migration, and invasion. Thus, galectin-3 is a natural binding ligand of MCAM in melanoma, and their interaction activates MCAM and promotes MCAM-mediated melanoma progression. Targeting the galectin-3-MCAM interaction may potentially be a useful therapeutic strategy for melanoma treatment.

Composition and antioxidant activity of anthocyanins from Aronia melanocarpa extracted using an ultrasonic-microwave-assisted natural deep eutectic solvent extraction method.

A time-saving, efficient, and environmentally friendly ultrasonic-microwave-assisted natural deep eutectic solvent (UMAE-NADES) extraction method was developed for the extraction of anthocyanins from Aronia melanocarpa. Eight different natural eutectic solvents were screened initially, and choline chloride-glycerol was selected as the extraction solvent. The extraction conditions were optimized using the response surface methodology, and the extraction rate of anthocyanins was higher than those achieved using the traditional ethanol method, natural deep eutectic solvent extraction method, and ultrasonic-microwave-assisted ethanol method. Six anthocyanins, including cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, cyanidin-3-O-arabinoside, cyanidin-3-O-xyloside, cyanidin-3,5-O-dihexoside, and the dimer of cyanidin-hexoside were identified and extracted at a purity of 448.873 mg/g using high performance liquid chromatography-mass spectrometry (HPLC-MS). The compounds extracted using UMAE-NADES had higher antioxidant capacities than those extracted by the other three methods. The UMAE-NADES demonstrated significant efficiency toward the extraction of bioactive substances and has potential utility in the food and pharmaceutical industries.

Flavonoids from Rhododendron nivale Hook. f delay aging via modulation of gut microbiota and glutathione metabolism.

BACKGROUND: Rhododendron nivale Hook. f (R.n), one of the four Manna Stash used in Tibetan medicine to delay aging, possesses anti-aging pharmacological activity. However, which R.n ingredients contain anti-aging properties and the underlying mechanisms involved are unclear. HYPOTHESIS/PURPOSE: Based on interactions between gut microbiota and natural medicines and the important role of gut microbiota in anti-aging, the study investigated the hypothesis that R.n possesses anti-aging properties and the interaction of gut microbiota with R.n is responsible for its anti-aging effects. STUDY DESIGN: The primary active ingredients of R.n and their target function and pathway enrichment were explored. An aging mouse model was used to clarify the underlying anti-aging mechanisms of R.n. METHODS: Chromatography, spectroscopy, nuclear magnetic technology, and pharmacology were used to reveal the major active ingredients of ethanol extract residues of R.n (RNEA). The target function and pathway enrichment of these active ingredients were explored. Plasma metabolomics coupled with intestinal flora evaluation and bioinformatics analysis was used to clarify the underlying anti-aging mechanisms of RNEA. RESULTS: Myricetin-3-ß-D-xylopyranoside, hyperin, goospetin-8-methyl ether 3-ß-D-galactoside, and diplomorphanin B were separated and identified from RNEA. The network pharmacology study revealed that the active ingredients' target function and pathway enrichment focused mainly on the glutathione antioxidant system. In a D-galactose-induced mouse model of aging, RNEA was shown to possess suitable anti-aging pharmacological activity, as indicated by the amelioration of memory loss and weakened superoxide dismutase and glutathione peroxidase activities. Plasma metabolomics coupled with intestinal flora examination and bioinformatics analysis revealed that RNEA could regulate the expression of glutathione-related enzymes and ameliorate D-galactose-induced imbalances in methionine, glycine, and serine, and betaine and galactose metabolism. The results showed that RNEA reshaped the disordered intestinal flora and mitigated the D-galactose-mediated decline in glutathione oxidase expression, further confirming that the anti-aging effect of RNEA was closely related to regulation of the glutathione antioxidant system. CONCLUSION: RNEA, consisting of myricetin-3-ß-D-xylopyranoside, hyperin, goospetin-8-methyl ether 3-ß-D-galactoside, and diplomorphanin B, possesses anti-aging activity. The anti-aging effect of RNEA might be due to reshaping intestinal flora homeostasis, increasing the expression of glutathione peroxidase 4 in the intestines and liver, enhancing glutathione peroxidase activity, and reinforcing the glutathione antioxidant system.

Integrative Analysis of Metabolomics and Transcriptomics Reveals Molecular Mechanisms of Anthocyanin Metabolism in the Zikui Tea Plant (Camellia sinensis cv. Zikui).

In this study, we performed an association analysis of metabolomics and transcriptomics to reveal the anthocyanin biosynthesis mechanism in a new purple-leaf tea cultivar Zikui (Camellia sinensis cv. Zikui) (ZK). Three glycosylated anthocyanins were identified, including petunidin 3-O-glucoside, cyanidin 3-O-galactoside, and cyanidin 3-O-glucoside, and their contents were the highest in ZK leaves at 15 days. This is the first report on petunidin 3-O-glucoside in purple-leaf tea. Integrated analysis of the transcriptome and metabolome identified eleven dependent transcription factors, among which CsMYB90 had strong correlations with petunidin 3-O-glucoside, cyanidin 3-O-galactoside, and cyanidin 3-O-glucoside (PCC > 0.8). Furthermore, we also identified key correlated structural genes, including two positively correlated F3'H (flavonoid-3'-hydroxylase) genes, two positively correlated ANS (anthocyanin synthase) genes, and three negatively correlated PPO (polyphenol oxidase) genes. Overexpression of CsMYB90 in tobacco resulted in dark-purple transgenic calluses. These results showed that the increased accumulation of three anthocyanins in ZK may promote purple-leaf coloration because of changes in the expression levels of genes, including CsMYB90, F3'Hs, ANSs, and PPOs. These findings reveal new insight into the molecular mechanism of anthocyanin biosynthesis in purple-leaf tea plants and provide a series of candidate genes for the breeding of anthocyanin-rich cultivars.

A Simple Quantitative Assay for Measuring ß-Galactosidase Activity Using X-Gal in Yeast-Based Interaction Analyses.

Yeast-based interaction assays to determine protein-protein and protein-nucleic acid interactions commonly rely on the reconstitution of chimeric transcription factors that activate the expression of target reporter genes. The enzyme ß-galactosidase (ß-gal), coded by the LacZ gene of Escherichia coli, is a widely used reporter in yeast systems, and its expression is commonly assessed by evaluating its activity. X-gal (5-bromo-4-chloro-3-indolyl-ß-d-galactopyranoside) is an inexpensive and sensitive substrate of ß-gal, whose hydrolysis results in an intensely blue colored and easily detectable end product, 5,5'-dibromo-4,4'-dichloro-indigo. The insoluble nature of this end product, however, makes X-gal-based assays unsuitable for direct spectrophotometric absorbance quantification. As such, the use of X-gal is mostly restricted to solid-support approaches, such as colony lift or agar plate assays, which often only provide a qualitative readout. In this article, we describe a quantitative solid-phase X-gal assay to measure protein-protein interaction strength in yeast cells using a simple and low-cost experimental setup. We have optimized multiple aspects of the assay, namely sample preparation, reaction time, and quantification method, for speed and consistency. By integrating the use of a freely available ImageJ-based plugin, we have further standardized the assay for reliability and reproducibility. This improved quantitative X-gal assay can be performed in a standard molecular biology lab without the need for any specialized equipment other than an inexpensive and widely accessible smartphone camera. To exemplify the protocol, we provide detailed step-by-step instructions to perform a quantitative X-gal assay to assess the interaction between two Arabidopsis thaliana proteins, SUPPRESSOR OF PHYA-105 1 (SPA1) and PRODUCTION OF ANTHOCYANIN PIGMENT 2 (PAP2). To demonstrate the sensitivity of our assay in detecting weaker interactions, we also compare the results with a liquid-phase assay that uses ONPG (ortho-nitrophenyl-ß-galactopyranoside) as a substrate for ß-gal. The quantitative X-gal assay described here can easily be adapted for high-throughout interaction studies and protein domain mapping, even in yeast strains with low levels of LacZ expression. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of competent yeast cells and transformation Alternate Protocol 1: In-house preparation of yeast competent cells for use in lithium acetate (LiAc)-mediated yeast transformation Support Protocol: Long-term storage and revival of frozen yeast strain stocks Basic Protocol 2: Measuring ß-galactosidase activity via the quantitative X-gal assay Alternate Protocol 2: Quantification of interaction strength using liquid ONPG assay.

Fungal Secondary Metabolite Exophillic Acid Selectively Inhibits the Entry of Hepatitis B and D Viruses.

Current anti-hepatitis B virus (HBV) drugs are suppressive but not curative for HBV infection, so there is considerable demand for the development of new anti-HBV agents. In this study, we found that fungus-derived exophillic acid inhibits HBV infection with a 50% maximal inhibitory concentration (IC50) of 1.1 µM and a 50% cytotoxic concentration (CC50) of >30 µM in primary human hepatocytes. Exophillic acid inhibited preS1-mediated viral attachment to cells but did not affect intracellular HBV replication. Exophillic acid appears to target the host cells to reduce their susceptibility to viral attachment rather than acting on the viral particles. We found that exophillic acid interacted with the HBV receptor, sodium taurocholate cotransporting polypeptide (NTCP). Exophillic acid impaired the uptake of bile acid, the original function of NTCP. Consistent with our hypothesis that it affects NTCP, exophillic acid inhibited infection with HBV and hepatitis D virus (HDV), but not that of hepatitis C virus. Moreover, exophillic acid showed a pan-genotypic anti-HBV effect. We thus identified the anti-HBV/HDV activity of exophillic acid and revealed its mode of action. Exophillic acid is expected to be a potential new lead compound for the development of antiviral agents.

Cyanidin 3-O-ß-Galactoside Alleviated Cognitive Impairment in Mice by Regulating Brain Energy Metabolism During Aging.

Metabolic disorder, which commonly happens among senile people worldwide, is an important sign of aging. The early symptoms of neurodegenerative diseases include a decrease in energy metabolism and mitochondrial dysfunction. Comparably, early dietary intervention may be more effective in preventing or delaying brain aging, owing to its role in regulating metabolism. Polyphenol intake has shown its potential in preventing Alzheimer's disease. However, whether there are close connections between polyphenols and the energy metabolism of the brain during aging remains unclear. This study sought to evaluate whether cyanidin 3-O-ß-galactoside from black chokeberry (Aronia melanocarpa (Michx.) Elliott) has positive effects on energy metabolism, as well as cognitive function in aging mice. Intragastrical administration of cyanidin 3-O-ß-galactoside (25 and 50 mg/kg/day) for 8 weeks effectively alleviated the decline in brain glucose uptake (decline rate 18.29% versus 1.05%, 7.63%) of aging mice. Moreover, cyanidin 3-O-ß-galactoside also alleviated neuronal damage in the hippocampus (number of neurons 212.33 ± 16.19 versus 285.33 ± 29.53, 301.67 ± 10.07; p < 0.05) and cortex (number of neurons 82.00 ± 4.58 versus 111.67 ± 6.51, 112.00 ± 1.00; p < 0.05). Furthermore, cyanidin 3-O-ß-galactoside reduced ß-amyloid load in the brain and significantly increased the crossing-platform number (0.92 ± 1.11 versus 1.83 ± 0.68, 2.08 ± 0.58; p < 0.05) in the Morris water maze test. We further determined that protein kinase B (AKT) might be the target of cyanidin 3-O-ß-galactoside, which played a beneficial role in controlling the energy metabolism of the brain. These results suggested that early intervention of anthocyanins could promote neuroprotection under the challenge of brain energy metabolism.

Investigations on the binding specificity of ß-galactoside analogues with human galectin-1 using molecular dynamics simulations.

Galectin-1 (Gal-1) is the first member of galectin family, which has a carbohydrate recognition domain, specifically binds towards ß-galactoside containing oligosaccharides. Owing its association with carbohydrates, Gal-1 is involved in many biological processes such as cell signaling, adhesion and pathological pathways such as metastasis, apoptosis and increased tumour cell survival. The development of ß-galactoside based inhibitors would help to control the Gal-1 expression. In the current study, we carried out molecular dynamics (MD) simulations to examine the structural and dynamic behaviour Gal-1-thiodigalactoside (TDG), Gal-1-lactobionic acid (LBA) and Gal-1-beta-(1→6)-galactobiose (G16G) complexes. The analysis of glycosidic torsional angles revealed that ß-galactoside analogues TDG and LBA have a single binding mode (BM1) whereas G16G has two binding modes (BM1 and BM2) for interacting with Gal-1 protein. We have computed the binding free energies for the complexes Gal-1-TDG, Gal-1-LBA and Gal-1-G16G using MM/PBSA and are -6.45, -6.22 and -3.08 kcal/mol, respectively. This trend agrees well with experiments that the binding of Gal-1 with TDG is stronger than LBA. Further analysis revealed that the interactions due to direct and water-mediated hydrogen bonds play a significant role to the structural stability of the complexes. The result obtained from this study is useful to formulate a set of rules and derive pharmacophore-based features for designing inhibitors against galectin-1.Communicated by Ramaswamy H. Sarma.

Mechanism Analysis of Antiangiogenic d-Isofloridoside from Marine Edible Red algae Laurencia undulata in HUVEC and HT1080 cell.

Laurencia undulata, as one of the most biologically active species in the genus Laurencia, is an edible folk herb red algae. Among them, d-isofloridoside (DIF, 940.68 Da) is isolated from Laurencia undulata, which has antioxidant and matrix metalloproteinases (MMP) inhibitory activities. However, its mechanism of action on tumor angiogenesis has not yet been reported. In this study, we have studied the mechanism of DIF on tumor metastasis and angiogenesis in HT1080 cell and human vascular endothelial cell (HUVEC). The results show that DIF can reduce the activity of MMP-2/9, and can inhibit the expression of hypoxia-inducible factor-1α (HIF-1α) by regulating the downstream PI3K/AKT and mitogen-activated protein kinases (MAPK) pathways, thereby down-regulating the production of vascular endothelial growth factor (VEGF) in CoCl2-induced HT1080 cell. In addition, DIF can inhibit the activation of VEGF receptor (VEGFR-2), regulate downstream PI3K/AKT, MAPK, nuclear factor-kappa B (NF-κB) signal pathways, activate apoptosis, and thus down-regulate the production of platelet-derived growth factor (PDGF) in VEGF-induced HUVEC. In conclusion, our research shows that DIF has the potential to develop into a tumor-preventing functional food and tumor angiogenesis inhibitor, and it can provide theoretical guidance for the high-value comprehensive utilization of edible red algae Laurencia undulata.

Benzimidazole-galactosides bind selectively to the Galectin-8 N-Terminal domain: Structure-based design and optimisation.

We have obtained the X-ray crystal structure of the galectin-8 N-terminal domain (galectin-8N) with a previously reported quinoline-galactoside ligand at a resolution of 1.6 Å. Based on this X-ray structure, a collection of galactosides derivatised at O3 with triazole, benzimidazole, benzothiazole, and benzoxazole moieties were designed and synthesised. This led to the discovery of a 3-O-(N-methylbenzimidazolylmethyl)-galactoside with a Kd of 1.8 µM for galectin-8N, the most potent selective synthetic galectin-8N ligand to date. Molecular dynamics simulations showed that benzimidazole-galactoside derivatives bind the non-conserved amino acid Gln47, accounting for the higher selectivity for galectin-8N. Galectin-8 is a carbohydrate-binding protein that plays a key role in pathological lymphangiogenesis, modulation of the immune system, and autophagy. Thus, the benzimidazole-derivatised galactosides represent promising compounds for studies of the pathological implications of galectin-8, as well as a starting point for the development of anti-tumour and anti-inflammatory therapeutics targeting galectin-8.

Distinguishing Galactoside Isomers with Mass Spectrometry and Gas-Phase Infrared Spectroscopy.

Sequencing glycans is demanding due to their structural diversity. Compared to mammalian glycans, bacterial glycans pose a steeper challenge because they are constructed from a larger pool of monosaccharide building blocks, including pyranose and furanose isomers. Though mammalian glycans incorporate only the pyranose form of galactose (Galp), many pathogens, including Mycobacterium tuberculosis and Klebsiella pneumoniae, contain galactofuranose (Galf) residues in their cell envelope. Thus, glycan sequencing would benefit from methods to distinguish between pyranose and furanose isomers of different anomeric configurations. We used infrared multiple photon dissociation (IRMPD) spectroscopy with mass spectrometry (MS-IR) to differentiate between pyranose- and furanose-linked galactose residues. These targets pose a challenge for MS-IR because the saccharides lack basic groups, and galactofuranose residues are highly flexible. We postulated cationic groups that could complex through hydrogen bonding would offer a solution. Here, we present the first MS-IR analysis of hexose ammonium adducts. We compared their IR fingerprints with those of lithium adducts. We determined the diagnostic MS-IR signatures of the α- and ß-anomers of galactose in furanose and pyranose forms. We also showed these signatures could be applied to disaccharides to assign galactose ring size. Our findings highlight the utility of MS-IR for analyzing the unique substructures that occur in bacterial glycans.

Curcumin-Galactomannoside Complex inhibits the Proliferation of Human Cervical Cancer Cells: Possible Role in Cell Cycle Arrest and Apoptosis.

BACKGROUND: Cervical cancer is the most common cancer and has the highest morbidity rate of gynaecological malignancies in women worldwide. So, the development of effective anti-cancer agents to treat this condition is vital. Considering the recent interest in free (unconjugated) curcuminoids delivery, the present study investigated the efficacy of a novel food-grade, free-curcuminoids (curcumin-galactomannoside complex; CGM) on cervical cancer cells (HeLa) of human origin. In this study, we examined the anticancer potential of CGM as well as its effects on the cell cycle and the apoptosis of HeLa cancer cell. METHODS: Determination of anti-proliferative and apoptosis validation of CGM on HeLa cells was performed by 3-(4,5-Dimethylthiazol-2-yl)-2, 5,-diphenyltetrazolium bromide (MTT), acridine orange/propidium iodide and annexin-V-fluorescein isothiocyanate assays. Measurement of Reactive Oxygen Species (ROS) production, Caspase activities and protein expression experiments were performed to investigate the potential mechanisms of action in the apoptotic process. RESULTS: The cytotoxic assays revealed that the CGM showed inhibition of cell survival and exhibited high cytotoxic activity against HeLa cells at 25 µg/mL. Further studies on morphological changes were done in CGM-treated cervical cancer cells contributing to apoptosis. Flow cytometry analysis with Annexin V-FITC and PI staining precisely indicated that CGM induced apoptosis in HeLa cell lines at 25 µg/mL. By the supplementation of CGM showed an increase in Bax and cleaved caspase-8 protein in HeLa cells after 48 h exposure. CONCLUSION: The evidence obtained from this study suggests that CGM is a potent and promising natural formulation against cervical cancer cells via induction of apoptosis through ROS mediated mitochondrial damage in HeLa cells. Hence, CGM could be further explored as a potential lead in treating cancer.
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