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Journal of Microbiology and... May 2024Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for...
Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for large-scale biosynthesis. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of enzymatic systems for large-scale. is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of for efficient glucosylation of flavonoids using apigenin as a model flavonoid, with the heterologous expression of a promiscuous glycosyltransferase, YdhE from and the endogenous overexpression of genes encoding UDP-glucose pyrophosphorylase and encoding phosphoglucomutase involved in the synthesis of UDP-glucose to create a cell factory system capable of efficiently glucosylation apigenin with a high yield of glucosides production. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin-4'-O-β-glucoside) at 25°C, and 0.6 mM of APG2 (apigenin-7-O-β-glucoside), 1.7 mM of APG3 (apigenin-4',7-O-β-diglucoside) and 2.1 mM of APG4 (apigenin-4',5-O-β-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin and 37°C. The cost-effective developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars.
Topics: Corynebacterium glutamicum; Apigenin; Metabolic Engineering; Glucosides; Glycosylation; Bacillus licheniformis; Uridine Diphosphate Glucose; Bacterial Proteins; UTP-Glucose-1-Phosphate Uridylyltransferase; Glycosyltransferases
PubMed: 38563097
DOI: 10.4014/jmb.2401.01017 -
Journal of Agricultural and Food... Apr 2024Many species of the Urticaceae family are important cultivated fiber plants that are known for their economic and industrial values. However, their secondary metabolite...
Many species of the Urticaceae family are important cultivated fiber plants that are known for their economic and industrial values. However, their secondary metabolite profiles and associated biosynthetic mechanisms have not been well-studied. Using as a model, we conducted widely targeted metabolomics, which revealed 523 secondary metabolites, including a unique accumulation of flavonol glycosides in bulblet. Through full-length transcriptomic and RNA-seq analyses, the related genes in the flavonoid biosynthesis pathway were identified. Finally, weighted gene correlation network analysis and functional characterization revealed four LbUGTs, including LbUGT78AE1, LbUGT72CT1, LbUGT71BX1, and LbUGT71BX2, can catalyze the glycosylation of flavonol aglycones (kaempferol, myricetin, gossypetin, and quercetagetin) using UDP-Gal and UDP-Glu as the sugar donors. LbUGT78AE1 and LbUGT72CT1 showed substrate promiscuity, whereas LbUGT71BX1 and LbUGT71BX2 exhibited different substrate and sugar donor selectivity. These results provide a genetic resource for studying in the Urticaceae family, as well as key enzymes responsible for the metabolism of valuable flavonoid glycosides.
Topics: Glycosides; Glycosyltransferases; Flavonoids; Flavonols; Plants; Uridine Diphosphate; Gene Expression Profiling; Urticaceae; Sugars
PubMed: 38557049
DOI: 10.1021/acs.jafc.4c00488 -
Journal of Ethnopharmacology Jun 2024Aristolochic acids (AAs) are naturally occurring nitro phenanthrene carboxylic acids primarily found in plants of the Aristolochiaceae family. Aristolochic acid D (AAD)...
ETHNOPHARMACOLOGICAL RELEVANCE
Aristolochic acids (AAs) are naturally occurring nitro phenanthrene carboxylic acids primarily found in plants of the Aristolochiaceae family. Aristolochic acid D (AAD) is a major constituent in the roots and rhizomes of the Chinese herb Xixin (the roots and rhizomes of Asarum heterotropoides F. Schmidt), which is a key material for preparing a suite of marketed Chinese medicines. Structurally, AAD is nearly identical to the nephrotoxic aristolochic acid I (AAI), with an additional phenolic group at the C-6 site. Although the nephrotoxicity and metabolic pathways of AAI have been well-investigated, the metabolic pathway(s) of AAD in humans and the influence of AAD metabolism on its nephrotoxicity has not been investigated yet.
AIM OF THE STUDY
To identify the major metabolites of AAD in human tissues and to characterize AAD O-glucuronidation kinetics in different enzyme sources, as well as to explore the influence of AAD O-glucuronidation on its nephrotoxicity.
MATERIALS AND METHODS
The O-glucuronide of AAD was biosynthesized and its chemical structure was fully characterized by both H-NMR and C-NMR. Reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses were conducted to assess the crucial enzymes involved in AAD O-glucuronidation in humans. Docking simulations were performed to mimic the catalytic conformations of AAD in human UDP-glucuronosyltransferases (UGTs), while the predicted binding energies and distances between the deprotonated C-6 phenolic group of AAD and the glucuronyl moiety of UDPGA in each tested human UGT isoenzyme were measured. The mitochondrial membrane potentials (MMP) and reactive oxygen species (ROS) levels in HK-2 cells treated with either AAI, or AAD, or AAD O-glucuronide were tested, to elucidate the impact of O-glucuronidation on the nephrotoxicity of AAD.
RESULTS
AAD could be rapidly metabolized in human liver and intestinal microsomes (HLM and HIM, respectively) to form a mono-glucuronide, which was purified and fully characterized as AAD-6-O-β-D-glucuronide (AADG) by NMR. UGT1A1 was the predominant enzyme responsible for AAD-6-O-glucuronidation, while UGT1A9 contributed to a lesser extent. AAD-6-O-glucuronidation in HLM, HIM, UGT1A1 and UGT1A9 followed Michaelis-Menten kinetics, with the K values of 4.27 μM, 9.05 μM, 3.87 μM, and 7.00 μM, respectively. Docking simulations suggested that AAD was accessible to the catalytic cavity of UGT1A1 or UGT1A9 and formed catalytic conformations. Further investigations showed that both AAI and AAD could trigger the elevated intracellular ROS levels and induce mitochondrial dysfunction and in HK-2 cells, but AADG was hardly to trigger ROS accumulation and mitochondrial dysfunction.
CONCLUSION
Collectively, UGT1A-catalyzed AAD 6-O-glucuronidation represents a crucial detoxification pathway of this naturally occurring AAI analogs in humans, which is very different from that of AAI.
Topics: Humans; Aristolochic Acids; Glucuronides; Microsomes, Liver; Reactive Oxygen Species; Glucuronosyltransferase; Kinetics; Catalysis; Mitochondrial Diseases; Uridine Diphosphate
PubMed: 38548118
DOI: 10.1016/j.jep.2024.118116 -
Science (New York, N.Y.) Mar 2024Cellular purines, particularly adenosine 5'-triphosphate (ATP), fuel many metabolic reactions, but less is known about the direct effects of pyrimidines on cellular...
Cellular purines, particularly adenosine 5'-triphosphate (ATP), fuel many metabolic reactions, but less is known about the direct effects of pyrimidines on cellular metabolism. We found that pyrimidines, but not purines, maintain pyruvate oxidation and the tricarboxylic citric acid (TCA) cycle by regulating pyruvate dehydrogenase (PDH) activity. PDH activity requires sufficient substrates and cofactors, including thiamine pyrophosphate (TPP). Depletion of cellular pyrimidines decreased TPP synthesis, a reaction carried out by TPP kinase 1 (TPK1), which reportedly uses ATP to phosphorylate thiamine (vitamin B1). We found that uridine 5'-triphosphate (UTP) acts as the preferred substrate for TPK1, enabling cellular TPP synthesis, PDH activity, TCA-cycle activity, lipogenesis, and adipocyte differentiation. Thus, UTP is required for vitamin B1 utilization to maintain pyruvate oxidation and lipogenesis.
Topics: Adenosine Triphosphate; Lipogenesis; Pyrimidines; Pyruvates; Thiamine; Thiamine Pyrophosphate; Uridine Triphosphate; Oxidation-Reduction; Citric Acid Cycle; Protein Kinases; Humans; HeLa Cells; Pyruvate Dehydrogenase Complex
PubMed: 38547260
DOI: 10.1126/science.adh2771 -
Marine Drugs Feb 2024Floridoside is a galactosyl-glycerol compound that acts to supply UDP-galactose and functions as an organic osmolyte in response to salinity in Rhodophyta....
Floridoside is a galactosyl-glycerol compound that acts to supply UDP-galactose and functions as an organic osmolyte in response to salinity in Rhodophyta. Significantly, the UDP-galactose pool is shared for sulfated cell wall galactan synthesis, and, in turn, affected by thallus development alongside carposporogenesis induced by volatile growth regulators, such as ethylene and methyl jasmonate, in the red seaweed . In this study, we monitored changes in the floridoside reservoir through gene expression controlling both the galactose pool and glyceride pool under different reproductive stages of and we considered changing salinity conditions. Floridoside synthesis was followed by expression analysis of () as UDP-galactose is obtained from UDP-glucose and glucose-1P, and through gene expression as degradation of floridoside occurs through the cleavage of galactosyl residues. Meanwhile, glycerol 3-phosphate is connected with the galactoglyceride biosynthetic pathway by glycerol 3-phosphate dehydrogenase (G3PD), monogalactosyl diacylglyceride synthase (MGDGS), and digalactosyl diacylglyceride synthase (DGDGS). The results of our study confirm that low transcripts are correlated with thalli softness to locate reproductive structures, as well as constricting the synthesis of UDP-hexoses for galactan backbone synthesis in the presence of two volatile regulators and methionine. Meanwhile, modulates expression according to cystocarp maturation, and we found high transcripts in late development stages, as occurred in the presence of methyljasmonate, compared to early stages in ethylene. Regarding the acylglyceride pool, the upregulation of , , and gene expression in treated with MEJA supports lipid remodeling, as high levels of transcripts for and provide membrane stability during late development stages of cystocarps. Similar behavior is assumed in three naturally collected thalli development stages-namely, fertile, fertilized, and fertile-under 65 psu salinity conditions. Low transcripts for and high for are reported in infertile and fertilized thalli, which is the opposite to high transcripts for and low for encountered in fertile thalli within visible cystocarps compared to each of their corresponding stages in 35 psu. No significant changes are reported for and . It is concluded that cystocarp and thallus development stages affect galactose and glycerides pools with interwoven effects on cell wall polysaccharides.
Topics: Seaweed; Glycerol; Galactose; alpha-Galactosidase; Rhodophyta; Galactans; Glucose; Uridine Diphosphate; Cyclopentanes; Glycerophosphates; Oxylipins
PubMed: 38535456
DOI: 10.3390/md22030115 -
Protein Science : a Publication of the... Apr 2024The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the...
The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway cytoplasmic step is responsible for the phosphoenolpyruvate (PEP)-UNAG catalytic reaction, forming UNAG enolpyruvate and inorganic phosphate. Reportedly, UDP-N-acetylmuramic acid (UNAM) binds tightly to MurA forming a dormant UNAM-PEP-MurA complex and acting as a MurA feedback inhibitor. MurA inhibitors are complex, owing to competitive binding interactions with PEP, UNAM, and UNAG at the MurA active site. We used computational methods to explore UNAM and UNAG binding. UNAM showed stronger hydrogen-bond interactions with the Arg120 and Arg91 residues, which help to stabilize the closed conformation of MurA, than UNAG. Binding free energy calculations using end-point computational methods showed that UNAM has a higher binding affinity than UNAG, when PEP is attached to Cys115. The unbinding process, simulated using τ-random acceleration molecular dynamics, showed that UNAM has a longer relative residence time than UNAG, which is related to several complex dissociation pathways, each with multiple intermediate metastable states. This prevents the loop from opening and exposing the Arg120 residue to accommodate UNAG and potential new ligands. Moreover, we demonstrate the importance of Cys115-linked PEP in closed-state loop stabilization. We provide a basis for evaluating novel UNAM analogues as potential MurA inhibitors. PUBLIC SIGNIFICANCE: MurA is a critical enzyme involved in bacterial cell wall biosynthesis and is involved in antibiotic resistance development. UNAM can remain in the target protein's active site for an extended time compared to its natural substrate, UNAG. The prolonged interaction of this highly stable complex known as the 'dormant complex' comprises UNAM-PEP-MurA and offers insights into antibiotic development, providing potential options against drug-resistant bacteria and advancing our understanding of microbial biology.
Topics: Molecular Dynamics Simulation; Peptidoglycan; Alkyl and Aryl Transferases; Anti-Bacterial Agents; Uridine Diphosphate; Muramic Acids
PubMed: 38532715
DOI: 10.1002/pro.4969 -
Zhurnal Nevrologii I Psikhiatrii Imeni... 2024Asthenia, asthenic syndrome, asthenic condition, asthenic reaction, asthenic disorders are terms that describe the state of «impotence». Fatigue that occurs against...
Asthenia, asthenic syndrome, asthenic condition, asthenic reaction, asthenic disorders are terms that describe the state of «impotence». Fatigue that occurs against the background of habitual physical or intellectual stress for a person, and persists after rest, is asthenia. For people of the older age group, the term senile asthenia syndrome is used. Asthenia manifests itself with increased fatigue and exhaustion, mood instability, increased irritability, sleep disorders. Asthenic conditions manifest themselves along with a decrease in physical activity, increased cognitive and mental fatigue. Asthenic syndrome (AS) are considered as an integral part of cardiovascular diseases (CVD), as one of the manifestations of cerebrovascular pathology. Senile asthenia syndrome (SAS) is a geriatric syndrome characterized by an age-associated decrease in the physiological reserve and functions of many body systems, including cognitive functions. One of the drugs that has a positive effect on the severity of AS and improves the state of cognitive functions is the domestic drug Recognan (citicoline). The effectiveness of Recognan in the treatment of AS in patients with CVD, SAS, and post-COVID asthenia has been shown. It is recommended to prescribe Recognan orally at 500 mg / day for 30 days. Recognan has a nootropic and antiasthenic effect.
Topics: Male; Humans; Aged; Asthenia; Syndrome; Cognition Disorders; Cytidine Diphosphate Choline; Frailty; Cardiovascular Diseases
PubMed: 38529864
DOI: 10.17116/jnevro202412403157 -
A recombinant fungal photolyase autonomously enters human cell nuclei to fix UV-induced DNA lesions.Biotechnology Letters Jun 2024Solar ultraviolet radiations induced DNA damages in human skin cells with cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PPs) as the most frequent...
Solar ultraviolet radiations induced DNA damages in human skin cells with cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PPs) as the most frequent lesions. CPDs are repaired much slower than 6-4PPs by the nucleotide excision repair pathway, which are thus the major lesions that interfere with key cellular processes and give rise to gene mutations, possibly resulting in skin cancer. In prokaryotes and multicellular eukaryotes other than placental mammals, CPDs can be rapidly repaired by CPD photolyases in one simple enzymatic reaction using the energy of blue light. In this study, we aim to construct recombinant CPD photolyases that can autonomously enter human cell nuclei to fix UV-induced CPDs. A fly cell penetration peptide and a viral nucleus localization signal peptide were recombined with a fungal CPD photolyase to construct a recombinant protein. This engineered CPD photolyase autonomously crosses cytoplasm and nuclear membrane of human cell nuclei, which then efficiently photo-repairs UV-induced CPD lesions in the genomic DNA. This further protects the cells by increasing SOD activity, and decreasing cellular ROSs, malondialdehyde and apoptosis.
Topics: Humans; Deoxyribodipyrimidine Photo-Lyase; Ultraviolet Rays; Cell Nucleus; DNA Damage; DNA Repair; Recombinant Proteins; Pyrimidine Dimers; Fungal Proteins
PubMed: 38523200
DOI: 10.1007/s10529-024-03474-3 -
The Korean Journal of Gastroenterology... Mar 2024This study compared the effectiveness and safety of glecaprevir/pibrentasvir (GLE/PIB) and sofosbuvir/ledipasvir (SOF/LDV) in real-life clinical practice.
BACKGROUND/AIMS
This study compared the effectiveness and safety of glecaprevir/pibrentasvir (GLE/PIB) and sofosbuvir/ledipasvir (SOF/LDV) in real-life clinical practice.
METHODS
The data from genotype 1 or 2 chronic hepatitis C patients treated with GLE/PIB or sofosbuvir + ribavirin or SOF/LDV in South Korea were collected retrospectively. The analysis included the treatment completion rate, sustained virologic response at 12 weeks (SVR12) test rate, treatment effectiveness, and adverse events.
RESULTS
Seven hundred and eighty-two patients with genotype 1 or 2 chronic hepatitis C who were treated with GLE/PIB (n=575) or SOF/LDV (n=207) were included in this retrospective study. The baseline demographic and clinical characteristics revealed significant statistical differences in age, genotype, ascites, liver cirrhosis, and hepatocellular carcinoma between the GLE/PIB and SOF/LDV groups. Twenty-two patients did not complete the treatment protocol. The treatment completion rate was high for both regimens without statistical significance (97.7% vs. 95.7%, p=0.08). The overall SVR12 of intention-to-treat analysis was 81.2% vs. 80.7% without statistical significance (p=0.87). The overall SVR12 of per protocol analysis was 98.7% vs. 100% without statistical significance (p=0.14). Six patients treated with GLE/PIB experienced treatment failure. They were all male, genotype 2, and showed a negative hepatitis C virus RNA level at the end of treatment. Two patients treated with GLE/PIB stopped medication because of fever and abdominal discomfort.
CONCLUSIONS
Both regimens had similar treatment completion rates, effectiveness, and safety profiles. Therefore, the SOF/LDV regimen can also be considered a viable DAA for the treatment of patients with genotype 1 or 2 chronic hepatitis C.
Topics: Humans; Male; Sofosbuvir; Antiviral Agents; Hepatitis C, Chronic; Hepacivirus; Retrospective Studies; Treatment Outcome; Liver Neoplasms; Genotype; Drug Therapy, Combination; Sulfonamides; Pyrrolidines; Fluorenes; Quinoxalines; Aminoisobutyric Acids; Benzimidazoles; Lactams, Macrocyclic; Cyclopropanes; Leucine; Proline
PubMed: 38522854
DOI: 10.4166/kjg.2023.141 -
International Journal of Infectious... Jun 2024Yellow fever (YF) is a potentially lethal viral hemorrhagic fever that can be prevented with the 17D live attenuated YF vaccine. However, this vaccination can cause...
Yellow fever (YF) is a potentially lethal viral hemorrhagic fever that can be prevented with the 17D live attenuated YF vaccine. However, this vaccination can cause severe adverse reactions including vaccine-associated YF. Here, we describe the case of a 32-year-old female who was permanently immunosuppressed with an anti-CD20 antibody due to multiple sclerosis. Following YF vaccination, the patient developed a variety of symptoms such as febrile temperatures, muscle and joint pain, headaches, and dysuria. A vaccine-associated YF with viremia was diagnosed. To avoid a potentially severe course of the disease, sofosbuvir was used as antiviral treatment followed by the resolution of symptoms and serological response. As travelers with chronic diseases and immunosuppression will increasingly engage in long distance travel, this case demonstrates the importance of assessing patient history prior to the administration of live vaccines and points towards a possible therapeutic approach in those suffering from vaccine-associated YF.
Topics: Adult; Female; Humans; Antiviral Agents; Immunocompromised Host; Rituximab; Sofosbuvir; Yellow Fever; Yellow Fever Vaccine; Antigens, CD20; Multiple Sclerosis
PubMed: 38521450
DOI: 10.1016/j.ijid.2024.107017