Sugar Nucleosides Research Articles

Concise Affinity-Based Purification of Ligated mRNA for Structure-Activity Relationship Studies of Nucleosugar Modification Patterns.

Position-specific nucleoside sugar modifications have been shown to improve the translational activity and stability of chemically synthesized mRNA. For pharmaceutical applications of chemically modified mRNAs, a rapid purification methodology is imperative to identify the optimal modification pattern. However, while the chemical synthesis of mRNAs can be accomplished by splint ligation of oligonucleotide fragments, the current purification method for ligated mRNAs based on denaturing polyacrylamide gel electrophoresis tends to be time consuming. In this study, we developed a two-step affinity purification method for rapid sample preparation. In this method, ligated mRNA is captured by oligo dT magnetic beads and streptavidin magnetic beads with 3'-biotinylated oligo DNA, which are complementary to the 3'-poly(A) and 5' terminal sequences of the target mRNA, respectively. Therefore, the target mRNA can be isolated from a complex mixture of splint ligations. Using this method, six sugar-modified mRNAs were simultaneously purified, and the translational activities of these mRNAs were evaluated immediately after purification. The results demonstrate that this methodology is suitable for the rapid preparation of various chemically synthesized mRNAs to identify their optimal modification patterns.

Analyzing the temporal response mechanisms of the vascular bundles formation of grafted cucumber to different light intensity modes: A joint transcriptomic and metabolomic approach

In plant grafting, the reconnection of vascular bundles between the rootstock and scion not only establishes functional xylem and phloem connections, but also signifies the completion of graft healing. In our previous studies focused on early-stage morphology and physiology of grafted seedlings, we have demonstrated that utilizing light intensities of 50-100-150 μmol/(m2·s) during days 1-3, 4-6, and 7-9 optimizes cucumber/pumpkin graft healing. To further explore the underlying mechanisms, we investigate light intensity regimes: 50-100-100 μmol/(m2·s) and 50-100-150 μmol/(m2·s) during the same time frames (days 1-3, 4-6, and 7-9 after grafting), denoted as CK and T treatments. Initially, we observe histological characteristics of vascular bundle formation under these conditions, followed by the analyses of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione peroxidase (GSH-Px) enzyme activities, ascorbic acid (AsA) content in the antioxidant system, and the sequencing of transcriptome and metabolome. It is found that grafted seedlings under the T treatment achieve faster vascular bundle reconnection and shorter healing process compared to those under the CK treatment. Enzyme activities (POD, SOD, GSH-Px) increase over time under both light regimes, while CAT activity and AsA content decrease. Notably, by the 9th day, the T treatment (T9d) exhibits higher SOD, POD and CAT activities than the CK treatment (CK9d). Our findings indicate that increasing light intensity during the days 7-9 after grafting can mitigate oxidative damage at the grafting site and benefit vascular bundle reconnection. Transcriptome and metabolome analyses show the up-regulations of Cinnamaldehyde in the phenylpropanoid biosynthesis pathway, the PHGDH gene and L-Homocysteine in the cysteine and methionine metabolism pathway under T treatment in the days 7-9 after grafting. These enhance the antioxidant enzyme activities and reduce the oxidative damage to the grafting site. Additionally, GAE-related genes, and metabolites D-Xylose and Undecaprenyl phosphatealpha-L-Ara4FN in the amino sugar and nucleotide sugar metabolism pathways are also up-regulated under T treatment. The ethylene responsive factor (ERF) transcription factor CsaRAP2.3 positively regulates Undecaprenyl phosphatealpha-L-Ara4FN expression, promoting cell wall synthesis and therefore accelerating vascular bundle reconnection. In summary, during the vascular bundle formation period of cucumber graft healing, both light intensity modes coordinate regulation of phenylpropanoid biosynthesis, cysteine and methionine metabolism, amino sugar and nucleoside sugar metabolism, and ERF transcription factors, promoting graft healing from both stress resistance and cell wall synthesis. Furthermore, building upon the light intensity applied during the callus formation phase, increasing the light intensity during the vascular bundle formation period can significantly enhance cell wall synthesis, thus expediting the graft healing process. Nonetheless, the generalizability of these research findings to other grafting techniques and species requires further investigation.

Curcumin inhibits growth of Porphyromonas gingivalis by arrest of bacterial dipeptidyl peptidase activity

ABSTRACT Background Curcumin is a multi-functional polyphenol with anti-bacterial and anti-inflammatory effects and may have potential for treatment of periodontal diseases. The present study was conducted to examine the molecular basis of the anti-bacterial effect of curcumin against Porphyromonas gingivalis using metabolome analysis. Materials and Methods P. gingivalis were incubated with 10 µg/mL curcumin, and then metabolites were analyzed with CE-TOF/MS. Expression levels of sigma factors were also evaluated using RT-PCR assays. The activities of dipeptidyl peptidases (DPPs) were assessed by examining the degradation reactions of MCA-labeled peptides. Results The relative amounts of various glycogenic amino acids were significantly decreased when P. gingivalis was incubated with curcumin. Furthermore, the metabolites on the amino acid degradation pathway, including high-energy compounds such as ATP, various intermediate metabolites of RNA/DNA synthesis, nucleoside sugars and amino sugars were also decreased. Additionally, the expression levels of sigma-54 and sigma-70 were significantly decreased, and the same results as noted following nutrient starvation. Curcumin also significantly suppressed the activities of some DPPs, while the human DPP-4 inhibitors markedly inhibited the growth of P. gingivalis and activities of the DPPs. Conclusions Curcumin suppresses the growth of P. gingivalis by inhibiting DPPs and also interferes with nucleic acid synthesis and central metabolic pathways, beginning with amino acid metabolism.

Transcriptomic response of Pinus massoniana to infection stress from the pine wood nematode Bursaphelenchus xylophilus

The pinewood nematode (PWN) Bursaphelenchus xylophilus is a forestry quarantine pest and causes an extremely dangerous forest disease that is spreading worldwide. Due to the complex pathogenic factors of pine wood nematode disease, the pathogenesis is still unknown. B. xylophilus ultimately invades a host and causes death. However, little is known about the defence-regulating process of host pine after infection by B. xylophilus at the molecular level. Therefore, we wanted to understand how Pinus massoniana regulates its response to invasion by B. xylophilus. P. massoniana were artificially inoculated with B. xylophilus solution, while those without B. xylophilus solution were used as controls. P. massoniana inoculated with B. xylophilus solution for 0 h, 6 h, 24 h, and 120 h was subjected to high-throughput sequencing to obtain transcriptome data. At various time points (0 h, 6 h, 24 h, 120 h), gene transcription was measured in P. massoniana inoculated with PWN. At different time points, P. massoniana gene transcription differed significantly, with a response to early invasion by PWN. According to Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, P. massoniana response to PWN invasion involves a wide range of genes, including plant hormone signal transformation, flavonoid biosynthesis, amino sugar and nucleoside sugar metabolism, and MAPK signalling pathways. Among them, inoculation for 120 hours had the greatest impact on differential genes. Subsequently, weighted gene coexpression network analysis (WGCNA) was used to analyse transcriptional regulation of P. massoniana after PWN infection. The results showed that the core gene module of P. massoniana responding to PWN was “MEmagenta”, enriched in oxidative phosphorylation, amino sugar and nucleotide sugar metabolism, and the MAPK signalling pathway. MYB family transcription factors with the highest number of changes between infected and healthy pine trees accounted for 20.4% of the total differentially expressed transcription factors. To conclude, this study contributes to our understanding of the molecular mechanism of initial PWN infection of P. massoniana. Moreover, it provides some important background information on PWN pathogenic mechanisms.

Comprehensive analysis of 33 human cancers reveals clinical implications and immunotherapeutic value of the solute carrier family 35 member A2.

Solute carrier family 35 member A2 (SLC35A2), which belongs to the SLC35 solute carrier family of human nucleoside sugar transporters, has shown regulatory roles in various tumors and neoplasms. However, the function of SLC35A2 across human cancers remains to be systematically assessed. Insights into the prediction ability of SLC35A2 in clinical practice and immunotherapy response remains limited. We obtained the gene expression and protein levels of SLC35A2 in a variety of tumors from Molecular Taxonomy of Breast Cancer International Consortium, The Cancer Genome Atlas, Gene Expression Omnibus, Chinese Glioma Genome Atlas, and Human Protein Atlas databases. The SLC35A2 level was validated by immunohistochemistry. The predictive value for prognosis was evaluated by Kaplan-Meier survival and Cox regression analyses. Correlations between SLC35A2 expression and DNA methylation, genetic alterations, tumor mutation burden (TMB), microsatellite instability (MSI), and tumor microenvironment were performed using Spearman's correlation analysis. The possible downstream pathways of SLC35A2 in different human cancers were explored using gene set variation analysis. The potential role of SLC35A2 in the tumor immune microenvironment was evaluated via EPIC, CIBERSORT, MCP-counter, CIBERSORT-ABS, quanTIseq, TIMER, and xCell algorithms. The difference in the immunotherapeutic response of SLC35A2 under different expression conditions was evaluated by the tumor immune dysfunction and exclusion (TIDE) score as well as four independent immunotherapy cohorts, which includes patients with bladder urothelial carcinoma (BLCA, N = 299), non-small cell lung cancer (NSCLC, N = 72 and N = 36) and skin cutaneous melanoma (SKCM, N = 25). Potential drugs were identified using the CellMiner database and molecular docking. SLC35A2 exhibited abnormally high or low expression in 23 cancers and was significantly associated with the prognosis. In various cancers, SLC35A2 expression and mammalian target of rapamycin complex 1 signaling were positively correlated. Multiple algorithmic immune infiltration analyses suggested an inverse relation between SLC35A2 expression and infiltrating immune cells, which includes CD4+T cells, CD8+T cells, B cells, and natural killer cells (NK) in various tumors. Furthermore, SLC35A2 expression was significantly correlated with pan-cancer immune checkpoints, TMB, MSI, and TIDE genes. SLC35A2 showed significant predictive value for the immunotherapy response of patients with diverse cancers. Two drugs, vismodegib and abiraterone, were identified, and the free binding energy of cytochrome P17 with abiraterone was higher than that of SLC35A2 with abiraterone. Our study revealed that SLC35A2 is upregulated in 20 types of cancer, including lung adenocarcinoma (LUAD), breast invasive carcinoma (BRCA), colon adenocarcinoma (COAD), and lung squamous cell carcinoma (LUSC). The upregulated SLC35A2 in five cancer types indicates a poor prognosis. Furthermore, there was a positive correlation between the overexpression of SLC35A2 and reduced lymphocyte infiltration in 13 cancer types, including BRCA and COAD. Based on data from several clinical trials, patients with LUAD, LUSC, SKCM, and BLCA who exhibited high SLC35A2 expression may experience improved immunotherapy response. Therefore, SLC35A2 could be considered a potential predictive biomarker for the prognosis and immunotherapy efficacy of various tumors. Our study provides a theoretical basis for further investigating its prognostic and therapeutic potentials.

Effects of Different Environmental Variables on the Ingestion of Microcystis aeruginosa by Moina mongolica

How to control the frequent occurrence of cyanobacteria, especially the outbreak of toxin-producing Microcystis aeruginosa, has been a subject of constant research. This investigation focused on the effect of Moina mongolica on restricting M. aeruginosa blooms under different variables (temperature, light intensity, and salinity) and its growth at the molecular level. The results of batch experiments showed that the range of M. mongolica feeding rates was from 4.02 ± 0.81 × 103~182.23 ± 5.37 × 103 cells/ind·h in the whole experiment, where the highest feeding rates of larva M. mongolica and adult M. mongolica were 133.21 ± 5.24 × 103 vs. 182.23 ± 5.37 × 103 cells/ind·h at 30 °C, 85.88 ± 0.44 × 103 vs. 143.15 ± 14.07 × 103 cells/ind·h at 3000 lx and 88.18 ± 0.32 × 103 vs. 84.49 ± 4.95 × 103 cells/ind·h at 0‰ salinity, respectively. The results of transcriptomics further demonstrated that the response of M. mongolica to M. aeruginosa toxicity was caused by the downregulation of relevant functional genes (cell components, cell processes, metabolic processes, and protein complexes) and related signaling pathways (apoptosis, phagosome, lysosome, ribosome, oxidative phosphorylation, amino and nucleoside sugar metabolism, and PPAR signaling pathways). The findings show that M. mongolica can be released to low-salinity lakes and coastal areas (the subtropic and temperate zones) to prevent and inhibit M. aeruginosa blooms in the early summer phase. Additionally, the results achieved by the investigation will provide the relevant technology for inhibiting cyanobacteria blooms because M. mongolica even resists the produced toxin by M. aeruginosa.

Transcriptomics and Metabolomics Analysis of Sclerotium rolfsii Fermented with Differential Carbon Sources.

Scleroglucan is obtained from Sclerotium rolfsii and is widely used in many fields. In this study, transcriptomics combined with metabolomics were used to study the global metabolites and gene changes. The results of the joint analysis showed that the DEGs (differentially expressed genes) and DEMs (differentially expressed metabolites) of SEPS_48 (fermented with sucrose as a carbon source for 48 h) and GEPS_48 (fermented with glucose as a carbon source for 48 h) comparison groups were mainly related to cell metabolism, focusing on carbohydrate metabolism, amino acid metabolism, and amino sugar and nucleoside sugar metabolism. We therefore hypothesized that the significant differences in these metabolic processes were responsible for the differences in properties. Moreover, the joint analysis provides a scientific theoretical basis for fungal polysaccharides biosynthesis and provides new insights into the effects of carbon sources on the production. As an excellent bioenergy and biological product, scleroglucan can be better applied in different fields, such as the food industry.

Linking Microbial Functional Gene Abundance and Daqu Extracellular Enzyme Activity: Implications for Carbon Metabolism during Fermentation.

Daqu is the starter of Baijiu, it provides the microbes and enzymes necessary for fermentation. Studies have already established carbohydrate metabolism as the primary functional module in Daqu fermentation. The present study investigated the changes in microbial functions and the relationship between carbohydrate metabolism-related functional genes and extracellular enzyme activity during the Daqu fermentation. Amplicon sequencing identified 38 bacterial and 10 fungal phyla in Daqu samples, while shotgun metagenomic sequencing classified and annotated 40.66% of the individual features, of which 40.48% were prokaryotes. KEGG annotation showed that the pathways related to metabolites were less in the early fermentation stage, but higher in the middle and late stages. The functional genes related to pyruvate metabolism, glyoxylate and dicarboxylate metabolism, and propanoate metabolism were relatively high in the early and late stages of fermentation, while that for start and cross metabolism was relatively low. The study also found that amino sugar and nucleoside sugar metabolism were dominant in the middle stage of fermentation. Finally, the correlation network analysis showed that amylase activity positively correlated with many carbon metabolism-related pathways, while liquefaction activity negatively correlated with these pathways. In conclusion, the present study provides a theoretical basis for improving and stabilizing the quality of Daqu.

Genetic evidence that uptake of the fluorescent analog 2NBDG occurs independently of known glucose transporters.

The fluorescent derivative of glucose, 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]-D-glucose (2NBDG), is a widely used surrogate reagent to visualize glucose uptake in live cells at single cell resolution. Using CRISPR-Cas9 gene editing in 5TGM1 myeloma cells, we demonstrate that ablation of the glucose transporter gene Slc2a1 abrogates radioactive glucose uptake but has no effect on the magnitude or kinetics of 2NBDG import. Extracellular 2NBDG, but not NBD-fructose was transported by primary plasma cells into the cytoplasm suggesting a specific mechanism that is unlinked from glucose import and that of chemically similar compounds. Neither excess glucose nor pharmacological inhibition of GLUT1 impacted 2NBDG uptake in myeloma cells or primary splenocytes. Genetic ablation of other expressed hexose transporters individually or in combination with one another also had no impact on 2NBDG uptake. Ablation of the genes in the Slc29 and Slc35 families of nucleoside and nucleoside sugar transporters also failed to impact 2NBDG import. Thus, cellular uptake of 2NBDG is not necessarily a faithful indicator of glucose transport and is promoted by an unknown mechanism.

Comprehensive expression analysis reveals upregulated LUZP2 in prostate cancer tissues

BackgroundLeucine zipper protein 2 (LUZP2) is a vital gene encoding leucine zipper protein. It is of great importance in the incidence and progress of several human cancers. However, little is known about the role and clinical effects of LUZP2 in prostate cancer (PCa). Therefore, it is crucial to unravel the clinicopathological value of LUZP2 in PCa. In all, 1467 PCa and 549 non-prostate cancer (non-PCa) prostate samples were collected from mRNA chip and RNA-sequencing datasets. The protein levels of LUZP2 were verified in 91 prostate gland tissues by in-house immunohistochemistry (IHC). The standardized mean difference (SMD) was calculated to analyze LUZP2 expression. Survival analysis was also conducted to explore the prognostic significance of LUZP2 in PCa. R software was employed to identify the upregulated differently expressed genes (up-DEGs) and coexpressed genes (CEGs) of LUZP2. Additionally, we explored the prospective molecular mechanism of CEGs of LUZP2 through GO and KEGG pathway analyses. ResultsCompared with non-PCa, LUZP2 showed predominantly higher expression in PCa (SMD = 1.05, AUC = 0.88). IHC indicated the protein expression level of LUZP2 was consistently upregulated in PCa tissues (SMD = 2.23, 95%CI: 1.67–2.79). LUZP2 upregulation had an AUC of 0.88 (95%CI: 0.85–0.90) to distinguish PCa from non-PCa tissues. KEGG pathway analysis showed that the pathways of amino sugar and nucleoside sugar metabolism were chiefly enriched with the LUZP2 CEGs in PCa. Conclusion:LUZP2 upregulation might play a promoting function in the occurrence of PCa.How to cite: Li S-H, Yang Y-P, He R-Q, et al. Comprehensive expression analysis reveals upregulated LUZP2 in prostate cancer tissues. Electron J Biotechnol 2022;59. https://doi.org/10.1016/j.ejbt.2022.06.001

Cryptic Phosphorylation-Mediated Divergent Biosynthesis of High-Carbon Sugar Nucleoside Antifungals.

Establishing a general biosynthetic scheme for natural products is critical for a broader understanding of natural product biosynthesis and the structural prediction of metabolites based on genome sequence information. High-carbon sugar nucleoside antimicrobials are an underexplored class of natural products with unique structures and important biological activities. Recent studies on C6 sugar nucleoside antifungal natural products, such as nikkomycins and polyoxins, revealed a novel biosynthetic mechanism involving cryptic phosphorylation. However, the generality of this biosynthetic mechanism remained unexplored. We here report in vitro characterization of the biosynthesis of a C7 sugar nucleoside antifungal, malayamycin A. Our results demonstrate that the malayamycin biosynthetic enzymes specifically accept 2'-phosphorylated biosynthetic intermediates, suggesting that cryptic phosphorylation-mediated biosynthesis is conserved beyond C6 sugar nucleosides. Furthermore, the results suggest a generalizable divergent biosynthetic mechanism for high-carbon sugar nucleoside antifungals. In this model, C6 and C7 sugar nucleoside biosyntheses proceed via a common C8 sugar nucleoside precursor, and the sugar size is determined using the functions of α-ketoglutarate (α-KG)-dependent dioxygenases (NikI/PolD for C6 sugar nucleosides and MalI for C7 sugar nucleosides). These results provide an important guidance for the future genome-mining discovery of high-carbon sugar nucleoside antimicrobials.

Unveiling the regioselective synthesis of antiviral 5-isoxazol-5-yl-2´-deoxyuridines from the perspective of a molecular electron density theory

The regioselective synthesis of a potent antiviral sugar nucleoside isoxazole analogue in the [3+2] cycloaddition (32CA) reaction of acetonitrile- -N-oxide (ANO) and acetyl-protected 5-ethynyl-2?-deoxyuridine (EDU) has been studied at the MPWB1K/6-311G(d,p) level within perspective of the molecular electron density theory (MEDT). From an electron localization function (ELF) analysis, ANO is classified as a zwitterionic species devoid of any pseudoradical or carbenoid centre. The ortho regioisomer is energetically preferred over the meta one by the activation enthalpy of 21.7?24.3 kJ mol-1, suggesting complete regioselectivity in agreement with the experiment. The activation enthalpy increases from 53.9 kJ mol-1 in the gas phase to 71.5 kJ mol-1 in water, suggesting more facile reaction in low polar solvents. The minimal global electron density transfer (GEDT) at the TSs suggests non-polar character and the formation of new covalent bonds has not been started at the located TSs, showing non-covalent intermolecular interactions from an atoms-in- -molecules (AIM) study and in the independent gradient model (IGM) isosurfaces. The AIM analysis shows more accumulation of electron density at the C?C interacting region relative to the C?O one, and earlier C?C bond formation is predicted from a bonding evolution theory (BET) study.

Convertible and Constrained Nucleotides: The 2'-Deoxyribose 5'-C-Functionalization Approach, a French Touch.

Many strategies have been developed to modulate the biological or biotechnical properties of oligonucleotides by introducing new chemical functionalities or by enhancing their affinity and specificity while restricting their conformational space. Among them, we review our approach consisting of modifications of the 5’-C-position of the nucleoside sugar. This allows the introduction of an additional chemical handle at any position on the nucleotide chain without disturbing the Watson–Crick base-pairing. We show that 5’-C bromo or propargyl convertible nucleotides (CvN) are accessible in pure diastereoisomeric form, either for nucleophilic displacement or for CuAAC conjugation. Alternatively, the 5’-carbon can be connected in a stereo-controlled manner to the phosphate moiety of the nucleotide chain to generate conformationally constrained nucleotides (CNA). These allow the precise control of the sugar/phosphate backbone torsional angles. The consequent modulation of the nucleic acid shape induces outstanding stabilization properties of duplex or hairpin structures in accordance with the preorganization concept. Some biological applications of these distorted oligonucleotides are also described. Effectively, the convertible and the constrained approaches have been merged to create constrained and convertible nucleotides (C2NA) providing unique tools to functionalize and stabilize nucleic acids.

Biodegradation and physiological response mechanism of Bacillus aryabhattai to cyclotetramethylenete-tranitramine (HMX) contamination

This study aims to reveal the biodegradation and interaction mechanism of cyclotetramethylenete-tranitramine (HMX) by a newly isolated bacteria. In this study, a bacterial strain (Bacillus aryabhattai) with high efficiency for HMX degradation was used as the test organism to analyze the changes in growth status, cell function, and mineral metabolism following exposure to different stress concentrations (0 and 5 mg L−1) of HMX. Non-targeted metabonomics was used to reveal the metabolic response of this strain to HMX stress. The results showed that when the HMX concentration was 5 mg L−1, the removal rate of HMX within 24 h of inoculation with Bacillus aryabhatta was as high as 90.5%, the OD600 turbidity was 1.024, and the BOD5 was 225 mg L−1. Scanning electron microscope (SEM) images showed that the morphology of bacteria was not obvious Variety, Fourier transform infrared spectroscopy (FTIR) showed that the cell surface –OH functional groups drifted, and ICP-MS showed that the cell mineral element metabolism was disturbed. Non-targeted metabonomics showed that HMX induced the differential expression of 254 metabolites (133 upregulated and 221 downregulated). The main differentially expressed metabolites during HMX stress were lipids and lipid-like molecules, and the most significantly affected metabolic pathway was purine metabolism. At the same time, the primary metabolic network of bacteria was disordered. These results confirmed that Bacillus aryabhattai has a high tolerance to HMX and can efficiently degrade HMX. The degradation mechanism involves the extracellular decomposition of HMX and transformation of the degradation products into intracellular purines, amino sugars, and nucleoside sugars that then participate in cell metabolism.

Synthesis of Natural Product-Like Tricyclic Higher-Carbon Sugar Nucleosides

Because of the structural novelty and interesting biological profiles, the synthesis of higher-carbon sugars has been highly pursued. In this work, we first synthesized a series of structurally novel bis-uracil containing tricyclic higher-carbon sugar nucleosides (4a–e) using D-xylose as the starting material and the classical Vorbruggen glycosylation as the key synthetic step. The yields of the target compound were good. Unfortunately, despite the presence of pharmaceutically relevant uracil fragment, compounds 4a–e were inactive against the proliferation of several cancer cell lines (EC109, EC9706, PC-3, and MGC-803). Whether and how 4a–e functioned as anticancer agents would be further studied in our laboratory.

Keratan sulfate-based glycomimetics using Langerin as a target for COPD: lessons from studies on Fut8 and core fucose.

Glycosylation represents one of the most abundant posttranslational modification of proteins. Glycosylation products are diverse and are regulated by the cooperative action of various glycosyltransferases, glycosidases, substrates thereof: nucleoside sugars and their transporters, and chaperons. In this article, we focus on a glycosyltransferase, α1,6-fucosyltransferase (Fut8) and its product, the core fucose structure on N-glycans, and summarize the potential protective functions of this structure against emphysema and chronic obstructive pulmonary disease (COPD). Studies of FUT8 and its enzymatic product, core fucose, are becoming an emerging area of interest in various fields of research including inflammation, cancer and therapeutics. This article discusses what we can learn from studies of Fut8 and core fucose by using knockout mice or in vitro studies that were conducted by our group as well as other groups. We also include a discussion of the potential protective functions of the keratan sulfate (KS) disaccharide, namely L4, against emphysema and COPD as a glycomimetic. Glycomimetics using glycan analogs is one of the more promising therapeutics that compensate for the usual therapeutic strategy that involves targeting the genome and the proteome. These typical glycans using KS derivatives as glycomimetics, will likely become a clue to the development of novel and effective therapeutic strategies.

Stereochemistry of internucleoside phosphorus atom affects sugar pucker and acid hydrolysis of N3′-P5′ thio-phosphoramidates

Investigation of the sugar pucker of the two diastereomers of dinucleotide N3′-P5′-thio-phosphoramidates TNPSTNH2 and TNPSTOH shows that the Sp isomer adopts a more C3′-endo (North) sugar ring configuration than the Rp counterpart. In contrast, P-stereochemistry of oligonucleotide phosphorothioate (O3′-P5′) compounds has no effect on the nucleoside sugar puckering. This difference is also reflected in the different rate of acid hydrolysis for the two isomers. Thus, the Rp stereoisomer with less prevalent C3′-endo configuration has an acid hydrolysis rate constant ~50% higher than that of the Sp molecule. The TNPOTNH2 and TNPOTOH dinucleotides are hydrolyzed an order of magnitude faster than TNPSTNH2 and TNPSTOH, respectively. In addition, dinucleotides with the terminal 3′-OH group are hydrolyzed two times faster than their 3′-NH2 counterparts.

Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker

Ribonucleotides (rNTPs) are predicted to be incorporated into the genome at a rate of up to 3 million times per cell division, making rNTPs the most common non-standard nucleotide in the human genome. Typically, misinserted ribonucleotides are repaired by the ribonucleotide excision repair (RER) pathway, which is initiated by RNase H2 cleavage. However, rNTPs are susceptible to spontaneous depurination generating abasic ribonucleotides (rAPs), which are unable to be processed by RNase H2. Additionally, rAPs have been found in nascent RNA and coupled to R-loops. Recent work identified that base excision repair (BER) protein AP-Endonuclease 1 (APE1) is responsible for the initial processing of rAPs embedded in DNA and in R-loops. APE1 is a well characterized AP endonuclease that cleaves 5′ of abasic sites, but its ability to cleave at rAPs remains poorly understood. Here, we utilize enzyme kinetics, X-ray crystallography, and molecular dynamics simulations to provide insight into rAP processing by APE1. Enzyme kinetics were used to determine pre-steady-state rates of APE1 cleavage on DNA substrates containing rAP, revealing a decrease in activity compared to cleavage at a canonical deoxy-AP substrate. Using X-ray crystallography, we identified novel contacts between the rAP and the APE1 active site. We demonstrate that the rAP sugar pucker is accommodated in the active site in a C3′-endo conformation, influencing its position and contributing to a decrease in activity compared to the deoxy-AP site. Together, this work provides molecular level insights into rAP processing by APE1 and advances our understanding of ribonucleotide processing within genomic DNA.

Pyridoxal-5′-phosphate-dependent alkyl transfer in nucleoside antibiotic biosynthesis

Several nucleoside antibiotics are structurally characterized by a 5′′-amino-5′′-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside, (5′S,6′S)-5′-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origins of which have yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR-GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-l-methionine (AdoMet) as the direct precursor of the N-alkylamine, but unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5′-phophosate (PLP)-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to utilizing a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway.

Metabolic Pathway Profiling in Intracellular and Extracellular Environments of Streptococcus thermophilus During pH-Controlled Batch Fermentations.

Elucidating the metabolite profiles during the growth of Streptococcus thermophilus is beneficial for understanding its growth characteristics. The changes in the intracellular and extracellular concentrations of carbohydrates, nucleotides, amino sugars, nucleoside sugars, fatty acids, and amino acids, as well as their metabolites over time, were investigated by metabolomics technology. Most metabolites of nucleotides were highly accumulated in the intracellular environment after the mid-exponential phase. Increases in the intracellular unsaturated fatty acids and N-acetyl-glucosamine and N-acetyl-muramoate recycling provided potential evidence that cell envelope remodeling occurred after the mid-exponential phase. At the later fermentation stages, potentially functional metabolite produced by glycine was highly accumulated in the intracellular environment. Additionally, potential toxic metabolites produced by phenylalanine and tyrosine could not be excreted into the extracellular environment in a timely basis. The accumulation of large amounts of these metabolites might be the primary cause of the overconsumption of amino acids and influence the growth of S. thermophilus.