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Journal of Biomolecular NMR Mar 2024Reducing sugars can spontaneously react with free amines in protein side chains leading to posttranslational modifications (PTMs) called glycation. In contrast to...
Reducing sugars can spontaneously react with free amines in protein side chains leading to posttranslational modifications (PTMs) called glycation. In contrast to glycosylation, glycation is a non-enzymatic modification with consequences on the overall charge, solubility, aggregation susceptibility and functionality of a protein. Glycation is a critical quality attribute of therapeutic monoclonal antibodies. In addition to glucose, also disaccharides like maltose can form glycation products. We present here a detailed NMR analysis of the Amadori product formed between proteins and maltose. For better comparison, data collection was done under denaturing conditions using 7 M urea-d in DO. The here presented correlation patterns serve as a signature and can be used to identify maltose-based glycation in any protein that can be denatured. In addition to the model protein BSA, which can be readily glycated, we present data of the biotherapeutic abatacept containing maltose in its formulation buffer. With this contribution, we demonstrate that NMR spectroscopy is an independent method for detecting maltose-based glycation, that is suited for cross-validation with other methods.
Topics: Maillard Reaction; Maltose; Nuclear Magnetic Resonance, Biomolecular; Proteins; Magnetic Resonance Spectroscopy
PubMed: 38114873
DOI: 10.1007/s10858-023-00432-5 -
Foods (Basel, Switzerland) Sep 2023Malto-oligosaccharides (MOSs) from starch conversion is advantageous for food and pharmaceutical applications. In this study, an efficient malto-oligosaccharide-forming...
Malto-oligosaccharides (MOSs) from starch conversion is advantageous for food and pharmaceutical applications. In this study, an efficient malto-oligosaccharide-forming α-amylase AmyCf was identified from myxobacter sp. strain CF23. AmyCf is composed of 417 amino acids with N-terminal 41 amino acids as the signal peptide, and conserved glycoside hydrolase family 13 (GH13) catalytic module and predicted C-terminal domain with β-sheet structure are also identified. Phylogenetic and functional analysis demonstrated that AmyCf is a novel member of GH13_6 subfamily. The special activity of AmyCf toward soluble starch and raw wheat starch is 9249 U/mg and 11 U/mg, respectively. AmyCf has broad substrate specificity toward different types of starches without requiring Ca. Under ideal circumstances of 60 °C and pH 7.0, AmyCf hydrolyzes gelatinized starch into maltose and maltotriose and maltotetraose as the main hydrolytic products with more than 80% purity, while maltose and maltotriose are mainly produced from the hydrolysis of raw wheat starch with more than 95% purity. The potential applicability of AmyCf in starch processing is highlighted by its capacity to convert gelatinized starch and raw starch granules into MOSs. This enzymatic conversion technique shows promise for the low-temperature enzymatic conversion of raw starch.
PubMed: 37761198
DOI: 10.3390/foods12183487 -
Journal of Traditional and... Jul 2023Brown algae () extract (DP) presented high inhibitory potential against α-amylase. The present study aims to isolate, purify and evaluate the antihyperglycemic and...
BACKGROUND AND AIMS
Brown algae () extract (DP) presented high inhibitory potential against α-amylase. The present study aims to isolate, purify and evaluate the antihyperglycemic and anti-type 2 diabetic activities of marine hydroquinone from DP.
METHODS
Marine hydroquinones were isolated using silica gel, HPLC, and NMR spectroscopy was used to identify compound 1 and compound 2 as zonarol and isozonarol, respectively. The anti-hyperglycemic and anti-type 2 diabetic activities of zonarol were investigated by assay (α-amylase, α-glucosidase), Lineweaver-Burk plot and Type 2 diabetes mellitus model (T2DM) mice induced by streptozotocin (STZ).
RESULT
Zonarol had the highest content and the strongest inhibitory activity against α-glucosidase (IC value of 6.03 mg L) and α-amylase (IC value of 19.29 mg L) in a competitive inhibition and mix-type manner, respectively. The maltose and starch loading tests revealed that zonarol significantly reduced postprandial glycemia after 30 min loading (9.12 and 8.12 mg/dL, respectively), compared to normal (11.37 and 12.37 mg/dL, respectively). Zonarol exhibited pancreatic islet cell rejuvenation, as evidenced by increased pancreatic islet mass, and hence helps in the restoration of insulin levels and therefore improves the glucose metabolism in STZ-induced diabetic mice. Zonarol treatment in T2DM elevated abundant levels of main SCFAs (propionate, butyrate, and valeric acid), which are closely related to glucose metabolism homeostasis.
CONCLUSION
Our finding indicates that zonarol could be used as a food supplement to treat hyperglycemia and diabetes.
PubMed: 37396160
DOI: 10.1016/j.jtcme.2023.03.007 -
The Journal of Biological Chemistry Sep 2023Reductive dehalogenases are corrinoid and iron-sulfur cluster-containing enzymes that catalyze the reductive removal of a halogen atom. The oxygen-sensitive and...
Reductive dehalogenases are corrinoid and iron-sulfur cluster-containing enzymes that catalyze the reductive removal of a halogen atom. The oxygen-sensitive and membrane-associated nature of the respiratory reductive dehalogenases has hindered their detailed kinetic study. In contrast, the evolutionarily related catabolic reductive dehalogenases are oxygen tolerant, with those that are naturally fused to a reductase domain with similarity to phthalate dioxygenase presenting attractive targets for further study. We present efficient heterologous expression of a self-sufficient catabolic reductive dehalogenase from Jhaorihella thermophila in Escherichia coli. Combining the use of maltose-binding protein as a solubility-enhancing tag with the btuCEDFB cobalamin uptake system affords up to 40% cobalamin occupancy and a full complement of iron-sulfur clusters. The enzyme is able to efficiently perform NADPH-dependent dehalogenation of brominated and iodinated phenolic compounds, including the flame retardant tetrabromobisphenol, under both anaerobic and aerobic conditions. NADPH consumption is tightly coupled to product formation. Surprisingly, corresponding chlorinated compounds only act as competitive inhibitors. Electron paramagnetic resonance spectroscopy reveals loss of the Co(II) signal observed in the resting state of the enzyme under steady-state conditions, suggesting accumulation of Co(I)/(III) species prior to the rate-limiting step. In vivo reductive debromination activity is readily observed, and when the enzyme is expressed in E. coli strain W, supports growth on 3-bromo-4-hydroxyphenylacetic as a sole carbon source. This demonstrates the potential for catabolic reductive dehalogenases for future application in bioremediation.
Topics: Escherichia coli; NADP; Oxygen; Vitamin B 12; Phenols; Electron Spin Resonance Spectroscopy; Hydrolases; Rhodobacteraceae; Protein Structure, Tertiary; Models, Molecular; Maltose-Binding Proteins; Recombinant Fusion Proteins; Coenzymes
PubMed: 37495113
DOI: 10.1016/j.jbc.2023.105086 -
ACS Infectious Diseases Apr 2024Tuberculosis (TB), caused by (Mtb), is the leading cause of death worldwide by infectious disease. Treatment of Mtb infection requires a six-month course of multiple...
Tuberculosis (TB), caused by (Mtb), is the leading cause of death worldwide by infectious disease. Treatment of Mtb infection requires a six-month course of multiple antibiotics, an extremely challenging regimen necessitated by Mtb's ability to form drug-tolerant persister cells. Mtb persister formation is dependent on the trehalose catalytic shift, a stress-responsive metabolic remodeling mechanism in which the disaccharide trehalose is liberated from cell surface glycolipids and repurposed as an internal carbon source to meet energy and redox demands. Here, using a biofilm-persister model, metabolomics, and cryo-electron microscopy (EM), we found that azidodeoxy- and aminodeoxy-d-trehalose analogues block the Mtb trehalose catalytic shift through inhibition of trehalose synthase TreS (Rv0126), which catalyzes the isomerization of trehalose to maltose. Out of a focused eight-member compound panel constructed by chemoenzymatic synthesis, the natural product 2-trehalosamine exhibited the highest potency and significantly potentiated first- and second-line TB drugs in broth culture and macrophage infection assays. We also report the first structure of TreS bound to a substrate analogue inhibitor, obtained via cryo-EM, which revealed conformational changes likely essential for catalysis and inhibitor binding that can potentially be exploited for future therapeutic development. Our results demonstrate that inhibition of the trehalose catalytic shift is a viable strategy to target Mtb persisters and advance trehalose analogues as tools and potential adjunctive therapeutics for investigating and targeting mycobacterial persistence.
Topics: Humans; Mycobacterium tuberculosis; Trehalose; Cryoelectron Microscopy; Tuberculosis; Catalysis
PubMed: 38485491
DOI: 10.1021/acsinfecdis.4c00138 -
Water content, transition temperature and fragility influence protection and anhydrobiotic capacity.BioRxiv : the Preprint Server For... Nov 2023Water is essential for metabolism and all life processes. Despite this, many organisms distributed across the kingdoms of life survive near-complete desiccation or...
Water is essential for metabolism and all life processes. Despite this, many organisms distributed across the kingdoms of life survive near-complete desiccation or anhydrobiosis (Greek for "life without water"). Increased intracellular viscosity, leading to the formation of a vitrified state is necessary, but not sufficient, for survival while dry. What properties of a vitrified system make it desiccation-tolerant or -sensitive are unknown. We have analyzed 18 different vitrified systems, composed of one of three protective disaccharides (trehalose, sucrose, or maltose) and varying amounts of glycerol, quantifying their enzyme-protective capacity and their material properties in a dry state. We find that protection conferred by mixtures containing maltose correlates strongly with increased water content, increased glass-transition temperature, and reduced glass former fragility, while the protection of glasses formed with sucrose correlates with increased glass transition temperature and the protection conferred by trehalose glasses correlates with reduced glass former fragility. Thus, different vitrified sugars confer protection through distinct material properties. Extending on this, we have examined the material properties of a dry desiccation tolerant and intolerant life stage from three different organisms. In all cases, the dried desiccation tolerant life stage of an organism had an increased glass transition temperature relative to its dried desiccation intolerant life stage, and this trend is also seen in all three organisms when considering reduced glass former fragility. These results suggest that while drying of different protective sugars results in vitrified systems with distinct material properties that correlate with their enzyme-protective capacity, in nature organismal desiccation tolerance relies on a combination of these properties. This study advances our understanding of how protective and non-protective glasses differ in terms of material properties that promote anhydrobiosis. This knowledge presents avenues to develop novel stabilization technologies for pharmaceuticals that currently rely on the cold-chain.
PubMed: 38014150
DOI: 10.1101/2023.06.30.547256 -
Food Science & Nutrition Oct 2023Water kefir is a non-dairy probiotic beverage. It is obtained by fermentation of water kefir grains with a sugar solution. This study aims to determine the properties of...
Water kefir is a non-dairy probiotic beverage. It is obtained by fermentation of water kefir grains with a sugar solution. This study aims to determine the properties of water kefir beverages obtained by fermentation of mandarin and persimmon-containing water for 42 h. According to microbiological results, both fruits containing water samples provided a high number of lactic acid bacteria and yeasts. Moreover, after fermentation, pH, Brix, and dry matter content did not significantly differ. On the other hand, fructose, maltose, and acetic acid contents of mandarin water kefir are significantly higher than persimmon water kefir ( < .05). Persimmon water kefir had higher total phenolic contents, twice as much as mandarin water kefir ( < .05). Both water kefirs had good color properties. The organoleptic acceptability of the fruit water kefirs was promising.
PubMed: 37823165
DOI: 10.1002/fsn3.3561 -
Heliyon Oct 2023High-quality and genuine honey is crucial to provide consumers with natural honey and prevent any potential health issues. This study aimed to examine the quality of...
High-quality and genuine honey is crucial to provide consumers with natural honey and prevent any potential health issues. This study aimed to examine the quality of commercial honey available in the Addis Ababa market. A total of 30 honey samples were randomly collected from eight sub-cities of Addis Ababa city. Both High-Performance Liquid Chromatography (HPLC) and UV-Vis spectroscopic methods were used to determine 12 physicochemical and three antioxidant activity parameters in the honey samples according to internationally recognized standards. The findings of this study showed that the hydroxymethylfurfural (HMF), free acidity, and ash content of all commercial honey samples conformed to honey standards. However, except for honey samples collected from processors (19.48 ± 0.4 %) and retail outlets (20.49 ± 0.13 %), all other commercial honey samples failed to meet the moisture content criteria (≤21 %). Proline levels in honey samples taken from the street (67.1 ± 0.52 mg/kg) were also found to be below the required standard. The commercial honey samples contained fructose, glucose, sucrose, and maltose within a range of 33.85 ± 0.65 to 48.61 ± 0.51 %, 33.07 ± 1.58 to 44.3 ± 0.82 %, 0.91 ± 0.05 to 6.23 ± 2.49 %, and 0.51 ± 0.14 to 2.4 ± 0.44 %, respectively. Furthermore, honey samples from market areas showed good Total Phenolic Content (TPC), Total Flavonoid Content (TFC), and antioxidant activity. Overall, the results revealed that all physicochemical parameters, except for proline, moisture, and sucrose content, complied with approved standards (Codex Alimentarius, European Union (EU), and Ethiopia Standard Agency (ESA). Accordingly, it is recommended that stakeholders receive regular training on how to manage honey quality issues and detect adulteration techniques to prevent contaminated honey from reaching the markets.
PubMed: 37860501
DOI: 10.1016/j.heliyon.2023.e20830 -
Parasites & Vectors Sep 2023The emergence and spread of drug resistance in Trichomonas vaginalis parasites has become an important concern in trichomoniasis treatment. Fast and reliable growth...
BACKGROUND
The emergence and spread of drug resistance in Trichomonas vaginalis parasites has become an important concern in trichomoniasis treatment. Fast and reliable growth assessment is critical for validating in vitro drug susceptibility and high-throughput screening of newly developed drugs.
METHODS
Modified media without yeast extract were evaluated for their ability to support the growth of T. vaginalis parasites. The potential of the nucleic acid-binding dye SYBR Green I for detecting T. vaginalis drug resistance was characterized, and seeding parasite concentration and incubation time were optimized. The fluorescence assay based on SYBR Green I was further validated in four T. vaginalis isolates with different susceptibilities to the antibiotics metronidazole, tinidazole, ornidazole and secnidazole, and compared with the traditional method that detects minimum lethal concentrations (MLCs).
RESULTS
A modified medium consisting of RPMI 1640 and Tryptone Plus as replacements for yeast extract and tryptone, respectively, in traditional trypticase-yeast extract-maltose (TYM) medium exhibited similar performance as TYM medium in maintaining T. vaginalis growth, while it showed much lower background fluorescent signals. The T. vaginalis SYBR Green I-based fluorescence (TSF) drug assay was found to have to satisfy one of two conditions to demonstrate the 50% inhibitory concentration of metronidazole for the sensitive isolate TV-334: (i) a seeding density of 3 × 10 parasites/ml and an incubation time of 48 h; or (ii) a seeding density of 1 × 10 parasites/ml and an incubation time of 72 h. Subsequent validation experiments revealed that the 48-h incubation/3 × 10 parasites/ml seeding density condition had a greater sensitivity to detect drug resistance than the 72-h condition. The TSF assay also exhibited high efficiency in identifying parasite drug resistance, as evidenced by its strong correlation with the standard MLC assay results (P = 0.003).
CONCLUSIONS
This study presents a robust TSF assay that has the potential to facilitate high-throughput, automated in vitro anti-trichomoniasis susceptibility testing for drug resistance monitoring and drug development. In comparison to the standard MLC method, this assay offers the advantages of reduced labor and elimination of subjective examination.
Topics: Animals; Trichomonas vaginalis; Drug Evaluation, Preclinical; Metronidazole; Trichomonas Infections
PubMed: 37723582
DOI: 10.1186/s13071-023-05919-6 -
ACS Omega Nov 2023Nanoparticle (NP) surfaces act as the interface as they interact with living systems and play a critical role in defining their cellular response. The nature of these...
Nanoparticle (NP) surfaces act as the interface as they interact with living systems and play a critical role in defining their cellular response. The nature of these interactions should be well understood to design safer and more effective NPs to be used in a wide range of biomedical applications. At the moment, it is not clear how a subtle change in surface chemistry will affect an NP's behavior in a biological system. Thus, understanding the role of such a small change is critical and may allow one to fine-tune a biological response. In this study, the cellular response to -OH orientation differences generated on gold glyconanoparticles, which are recently considered promising therapeutic agents as they mimic a glycocalyx, is investigated. As model molecules, glucose and mannose (C2 epimer) as monosaccharides and lactose and maltose (galactose and glucose as free units, C4 epimer) as disaccharides were chosen to monitor the cellular response in A549, BEAS-2b, and MDA-MB-231 cells through cellular uptake, cytotoxicity, and cell cycle progression. The three cell lines gave various and remarkable cellular responses to the same subtle -OH differences on gold glyconanoparticles, and it is determined that not only -OH orientation differences but also the number of saccharides on gold glyconanoparticles affect the cellular response. It was shown that mannose (C2 epimer to glucose) was significant with the promise of being a therapeutic agent for lung cancer therapy, whereas the toxicological profile of MDA-MB-231 cells was affected by AuNPs-glucose the most. This study demonstrates that clearly small chemical alterations on a NP surface can result in a significant cellular response. It can be concluded that the -OH orientation at the second and fourth carbon of a carbohydrate ring has a critical role in designing and engineering novel gold glyconanoparticles (consisting of monolayer mono- or disaccharides) for a specific cancer therapy.
PubMed: 38024762
DOI: 10.1021/acsomega.3c05920