-
Biology of Reproduction Oct 2022Roles of fructose in elongating ovine conceptuses are poorly understood, despite it being the major hexose sugar in fetal fluids and plasma throughout gestation....
Roles of fructose in elongating ovine conceptuses are poorly understood, despite it being the major hexose sugar in fetal fluids and plasma throughout gestation. Therefore, we determined if elongating ovine conceptuses utilize fructose via metabolic pathways for survival and development. Immunohistochemical analyses revealed that trophectoderm and extra-embryonic endoderm express ketohexokinase and aldolase B during the peri-implantation period of pregnancy for conversion of fructose into fructose-1-phosphate for entry into glycolysis and related metabolic pathways. Conceptus homogenates were cultured with 14C-labeled glucose and/or fructose under oxygenated and hypoxic conditions to assess contributions of glucose and fructose to the pentose cycle (PC), tricarboxylic acid cycle, glycoproteins, and lipid synthesis. Results indicated that both glucose and fructose contributed carbons to each of these pathways, except for lipid synthesis, and metabolized to pyruvate and lactate, with lactate being the primary product of glycolysis under oxygenated and hypoxic conditions. We also found that (1) conceptuses preferentially oxidized glucose over fructose (P < 0.05); (2) incorporation of fructose and glucose at 4 mM each into the PC by Day 16 conceptus homogenates was similar in the presence or absence of glucose, but incorporation of glucose into the PC was enhanced by the presence of fructose (P < 0.05); (3) incorporation of fructose into the PC in the absence of glucose was greater under oxygenated conditions (P < 0.01); and (4) incorporation of glucose into the PC under oxygenated conditions was greater in the presence of fructose (P = 0.05). These results indicate that fructose is an important metabolic substrate for ovine conceptuses.
Topics: Animals; Female; Fructokinases; Fructose; Fructose-Bisphosphate Aldolase; Glucose; Lactates; Lipids; Pentoses; Pregnancy; Pyruvates; Sheep; Sheep, Domestic
PubMed: 35835585
DOI: 10.1093/biolre/ioac144 -
Molecules (Basel, Switzerland) Oct 2020In the last three decades, oligonucleotides have been extensively investigated as probes, molecular ligands and even catalysts within therapeutic and diagnostic... (Review)
Review
In the last three decades, oligonucleotides have been extensively investigated as probes, molecular ligands and even catalysts within therapeutic and diagnostic applications. The narrow chemical repertoire of natural nucleic acids, however, imposes restrictions on the functional scope of oligonucleotides. Initial efforts to overcome this deficiency in chemical diversity included conservative modifications to the sugar-phosphate backbone or the pendant base groups and resulted in enhanced in vivo performance. More importantly, later work involving other modifications led to the realization of new functional characteristics beyond initial intended therapeutic and diagnostic prospects. These results have inspired the exploration of increasingly exotic chemistries highly divergent from the canonical nucleic acid chemical structure that possess unnatural physiochemical properties. In this review, the authors highlight recent developments in modified oligonucleotides and the thrust towards designing novel nucleic acid-based ligands and catalysts with specifically engineered functions inaccessible to natural oligonucleotides.
Topics: Animals; Aptamers, Nucleotide; Base Pairing; Carbohydrates; Catalysis; Enzymes; Gene Editing; Humans; Ligands; Nucleic Acids; Oligonucleotides; Oligonucleotides, Antisense; Pentoses
PubMed: 33066073
DOI: 10.3390/molecules25204659 -
International Journal of Biological... Dec 2022The ultrasonic-assisted extraction of polysaccharides from Camellia fascicularis (PCF) was optimized using response surface methodology. After separation and...
The ultrasonic-assisted extraction of polysaccharides from Camellia fascicularis (PCF) was optimized using response surface methodology. After separation and purification with DEAE-52 cellulose and Sephadex G-200 glucan gel columns, the purified polysaccharide components of PCFa-1 and PCFc-1 were analyzed for their structural characterization, antioxidant and anti-tumor activities in vitro. The results indicated that liquid to material ratio of 42 mL/g, ultrasonic time of 53 min, ultrasonic temperature of 73 °C, and ultrasonic power of 215 W were the optimum extraction conditions for PCF with maximum yields (4.05 %). PCFa-1 and PCFc-1 contained 5.88 % and 9.58 % uronic acid content, with 7.53 and 108.91 kDa of average molecular weights, respectively. The PCFa-1 was mainly constituted of galactose, arabinose, and glucose, while PCFc-1 was primarily composed of arabinose, glucose, galacturonic acid, and rhamnose. Fourier transform infrared spectra revealed that PCFa-1 and PCFc-1 contained typical polysaccharide bands. Scanning electron microscopy showed that the surface of PCFa-1 and PCFc-1 were irregular and clumpy structures. Nuclear magnetic resonance showed that PCFa-1 and PCFc-1 were mainly α-glycosidic bond conformation. Furthermore, the PCFc-1 showed better antioxidant capacities than PCFa-1 against hydroxyl, DPPH, and ABTS radicals and exhibited more potent toxicity on A549 and HepG2 cells. These research results suggested that PCF, especially PCFc-1, possesses great potential as natural antioxidants and anti-tumor drugs.
Topics: Antioxidants; Camellia; Arabinose; Polysaccharides; Glucose
PubMed: 36152704
DOI: 10.1016/j.ijbiomac.2022.09.176 -
EcoSal Plus Dec 2022Very few labs have had the good fortune to have been able to focus for more than 50 years on a relatively narrow research topic and to be in a field in which both... (Review)
Review
Very few labs have had the good fortune to have been able to focus for more than 50 years on a relatively narrow research topic and to be in a field in which both basic knowledge and the research technology and methods have progressed as rapidly as they have in molecular biology. My research group, first at Brandeis University and then at Johns Hopkins University, has had this opportunity. In this review, therefore, I will describe largely the work from my laboratory that has spanned this period and which was carried out by 40 plus graduate students, several postdoctoral associates, my technician, and me. In addition to presenting the scientific findings or results, I will place many of the topics in scientific context and, because we needed to develop a good many of the experimental methods behind our findings, I will also describe some of these methods and their importance. Also included will be occasional comments on how the research community or my research group functioned. Because a wide variety of approaches were used throughout our work, no ideal organization of this review is apparent. Therefore, I have chosen to use a hybrid structure in which there are six sections. Within each of the sections, experiments and findings will be described roughly in chronological order. Frequent cross references between parts and sections will be made because some findings and experimental approaches could logically have been described in more than one place.
Topics: Humans; Arabinose; Learning; Technology; Operon
PubMed: 36519894
DOI: 10.1128/ecosalplus.ESP-0012-2021 -
PloS One 2022Proteoglycan glycosaminoglycan (GAG) chains are attached to a serine residue in the protein through a linkage series of sugars, the first of which is xylose. Xylosides...
Proteoglycan glycosaminoglycan (GAG) chains are attached to a serine residue in the protein through a linkage series of sugars, the first of which is xylose. Xylosides are chemicals which compete with the xylose at the enzyme xylosyl transferase to prevent the attachment of GAG chains to proteins. These compounds have been employed at concentrations in the millimolar range as tools to study the role of GAG chains in proteoglycan function. In the course of our studies with xylosides, we conducted a dose-response curve for xyloside actions on neural cells. To our surprise, we found that concentrations of xylosides in the nanomolar to micromolar range had major effects on cell morphology of hippocampal neurons as well as of Neuro2a cells, affecting both actin and tubulin cytoskeletal dynamics. Such effects/morphological changes were not observed with higher xyloside concentrations. We found a dose-dependent alteration of GAG secretion by Neuro2a cells; however, concentrations of xylosides which were effective in altering neuronal morphology did not cause a large change in the rate of GAG chain secretion. In contrast, both low and high concentrations of xylosides altered HS and CS composition. RNAseq of treated cells demonstrated alterations in gene expression only after treatment with millimolar concentration of xylosides that had no effect on cell morphology. These observations support a novel action of xylosides on neuronal cells.
Topics: Glycosaminoglycans; Glycosides; Proteoglycans; Xylose
PubMed: 35763520
DOI: 10.1371/journal.pone.0269972 -
Applied Microbiology and Biotechnology Jun 2022Understanding the mechanisms involved in tolerance to inhibitors is the first step in developing robust yeasts for industrial second-generation ethanol (E2G) production....
Understanding the mechanisms involved in tolerance to inhibitors is the first step in developing robust yeasts for industrial second-generation ethanol (E2G) production. Here, we used ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) and MetaboAnalyst 4.0 for analysis of MS data to examine the changes in the metabolic profile of the yeast Spathaspora passalidarum during early fermentation of hemicellulosic hydrolysates containing high or low levels of inhibitors (referred to as control hydrolysate or CH and strategy hydrolysate or SH, respectively). During fermentation of SH, the maximum ethanol production was 16 g L with a yield of 0.28 g g and productivity of 0.22 g L h, whereas maximum ethanol production in CH fermentation was 1.74 g L with a yield of 0.11 g g and productivity of 0.01 g L h. The high level of inhibitors in CH induced complex physiological and biochemical responses related to stress tolerance in S. passalidarum. This yeast converted compounds with aldehyde groups (hydroxymethylfurfural, furfural, 4-hydroxybenzaldehyde, syringaldehyde, and vanillin) into less toxic compounds, and inhibitors were found to reduce cell viability and ethanol production. Intracellularly, high levels of inhibitors altered the energy homeostasis and redox balance, resulting in lower levels of ATP and NADPH, while that of glycolytic, pentose phosphate, and tricarboxylic acid (TCA) cycle pathways were the most affected, being the catabolism of glucogenic amino acids, the main cellular response to inhibitor-induced stress. This metabolomic investigation reveals interesting targets for metabolic engineering of ethanologenic yeast strains tolerant against multiple inhibitors for E2G production. KEY POINTS: • Inhibitors in the hydrolysates affected the yeast's redox balance and energy status. • Inhibitors altered the glycolytic, pentose phosphate, TCA cycle and amino acid pathways. • S. passalidarum converted aldehyde groups into less toxic compounds.
Topics: Ethanol; Fermentation; Phosphates; Polysaccharides; Saccharomyces cerevisiae; Saccharomycetales; Tandem Mass Spectrometry; Xylose
PubMed: 35622124
DOI: 10.1007/s00253-022-11987-y -
Microbial Cell Factories Aug 2023R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R....
R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R. toruloides grows on several pentose sugars and alcohols, further engineering of the native pathway is required for efficient conversion of biomass-derived sugars to higher value bioproducts. A previous high-throughput study inferred that R. toruloides possesses a non-canonical L-arabinose and D-xylose metabolism proceeding through D-arabitol and D-ribulose. In this study, we present a combination of genetic and metabolite data that refine and extend that model. Chiral separations definitively illustrate that D-arabitol is the enantiomer that accumulates under pentose metabolism. Deletion of putative D-arabitol-2-dehydrogenase (RTO4_9990) results in > 75% conversion of D-xylose to D-arabitol, and is growth-complemented on pentoses by heterologous xylulose kinase expression. Deletion of putative D-ribulose kinase (RTO4_14368) arrests all growth on any pentose tested. Analysis of several pentose dehydrogenase mutants elucidates a complex pathway with multiple enzymes mediating multiple different reactions in differing combinations, from which we also inferred a putative L-ribulose utilization pathway. Our results suggest that we have identified enzymes responsible for the majority of pathway flux, with additional unknown enzymes providing accessory activity at multiple steps. Further biochemical characterization of the enzymes described here will enable a more complete and quantitative understanding of R. toruloides pentose metabolism. These findings add to a growing understanding of the diversity and complexity of microbial pentose metabolism.
Topics: Xylose; Arabinose; Pentoses
PubMed: 37537595
DOI: 10.1186/s12934-023-02126-x -
Microbiology Spectrum Oct 2022Maintaining the health of seafarers is a difficult task during long-term voyages. Little is known about the corresponding changes in the gut microbiome-host interaction....
Maintaining the health of seafarers is a difficult task during long-term voyages. Little is known about the corresponding changes in the gut microbiome-host interaction. This study recruited 30 seafarers undertaking a 6-month voyage and analyzed their gut microbiota using 16S rRNA gene sequencing. Fecal untargeted metabolomics analysis was performed using liquid chromatography-mass spectrometry. Significant changes in the composition of the gut microbiota and an increased ratio of / at the end (day 180) of the 6-month voyage, relative to the start (day 0), were observed. At the genus level, the abundances of and were significantly increased, while the abundance of was decreased. Predicted microbial functional analysis revealed significant decreases in folate biosynthesis and biotin metabolism. Furthermore, 20 differential metabolites within six differentially enriched human metabolic pathways (including arginine biosynthesis, lysine degradation, phenylalanine metabolism, sphingolipid metabolism, pentose and glucuronate interconversions, and glycine, serine, and threonine metabolism) were identified by comparing the fecal metabolites at day 0 and day 180. Spearman correlation analysis revealed close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might affect specific human metabolic pathways. This study adopted a multi-omics approach and provides potential targets for maintaining the health of seafarers during long-term voyages. These findings are worthy of more in-depth exploration in future studies. Maintaining the health of seafarers undertaking long-term voyages is a difficult task. Apart from the alterations in the gut microbiome and fecal metabolites after a long-term voyage, our study also revealed that 20 differential metabolites within six differentially enriched human metabolic pathways are worthy of attention. Moreover, we found close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might impact specific human metabolic pathways. Accordingly, preventative measures, such as adjusting the gut microbiota by decreasing potential pathobionts or increasing potential probiotics as well as offsetting the decrease in B vitamins and beneficial metabolites (e.g., d-glucuronic acid and citrulline) via dietary adjustment or nutritional supplements, might improve the health of seafarers during long-term sea voyages. These findings provide valuable clues about gut microbiome-host interactions and propose potential targets for maintaining the health of seafarers engaged in long-term sea voyages.
Topics: Humans; Gastrointestinal Microbiome; RNA, Ribosomal, 16S; Vitamin B Complex; Citrulline; Biotin; Lysine; Metabolomics; Feces; Pentoses; Glucuronates; Glycine; Glucuronic Acid; Serine; Phenylalanine; Sphingolipids; Threonine; Arginine; Folic Acid
PubMed: 36197290
DOI: 10.1128/spectrum.01899-22 -
Organic & Biomolecular Chemistry Apr 2020S-Glycosides are important tools for the elucidation of specific protein-carbohydrate interactions and can significantly aid structural and functional studies of...
S-Glycosides are important tools for the elucidation of specific protein-carbohydrate interactions and can significantly aid structural and functional studies of carbohydrate-active enzymes, as they are often inert or act as enzyme inhibitors. In this context, this work focuses on the introduction of an S-linkage into arabinoxylan oligosaccharides (AXs) in order to obtain a small collection of synthetic tools for the study of AXs degrading enzymes. The key step for the introduction of the S-glycosidic linkage involved anomeric thiol S-alkylation of an orthogonally protected l-arabinopyranoside triflate. The resulting S-linked disaccharide was subsequently employed in a series of glycosylation reactions to obtain a selectively protected tetrasaccharide. This could be further elaborated through chemoselective deprotection and glycosylation reactions to introduce branching l-arabinofuranosides.
Topics: Arabinose; Cross-Linking Reagents; Disaccharides; Glycosides; Glycosylation; Oligosaccharides; Sulfhydryl Compounds; Xylans
PubMed: 32206767
DOI: 10.1039/d0ob00470g -
Bioresource Technology May 2020Alpha-L-arabinofuranoside arabinofuranohydrolase (ARA), more commonly known as alpha-L-arabinofuranosidase (E.C. number 3.2.1.55), is a hydrolytic enzyme, catalyzing the... (Review)
Review
Alpha-L-arabinofuranoside arabinofuranohydrolase (ARA), more commonly known as alpha-L-arabinofuranosidase (E.C. number 3.2.1.55), is a hydrolytic enzyme, catalyzing the cleavage of alpha-L-arabinose by acting on the non-reducing ends of alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)- and/or (1,5)-linked arabinoxylans and arabinogalactans. ARA functions as debranching enzyme removing arabinose substituents from arabinoxylan and arabinoxylooligomers, thereby, boosting the hydrolysis of arabinoxylan fraction of hemicellulose and improving bioconversion of lignocellulosic biomass. Previously, comprehensive information on this enzyme has not been reviewed thoroughly. Therefore, the main aim of this review is to highlight the important properties of this interesting enzyme, microorganisms used for its production, and enhanced production using genetic engineering approach. An account on synergism with other biomass hydrolyzing enzymes and various industrial applications of this enzyme has also been provided along with an outlook on further research and development.
Topics: Arabinose; Biomass; Glycoside Hydrolases; Hydrolysis; Substrate Specificity; Xylans
PubMed: 32089440
DOI: 10.1016/j.biortech.2020.123019