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Plant Communications Apr 2024Cytokinins are a type of mobile phytohormone that regulate plant growth, development, and environmental adaptability. The major cytokinin species include isopentenyl... (Review)
Review
Cytokinins are a type of mobile phytohormone that regulate plant growth, development, and environmental adaptability. The major cytokinin species include isopentenyl adenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and dihydrozeatin (DZ). The spatial distributions of different cytokinin species in different organelles, cells, tissues, and organs are primarily shaped by biosynthesis via isopentenyltransferases (IPT), cytochrome P450 monooxygenase, and 5'-ribonucleotide phosphohydrolase, and by conjugation or catabolism via glycosyltransferase or cytokinin oxidase/dehydrogenase (CKX). Cytokinins bind to histidine receptor kinases (HKs) in the endoplasmic reticulum (ER) or plasma membrane (PM) and relay signals to response regulators (RRs) in the nucleus by shuttle proteins known as histidine phosphotransfer proteins (HPs). The movements of cytokinins from sites of biosynthesis to signal perception sites usually require long-distance, intercellular, and intracellular transport. In the past decade, ATP-binding cassette (ABC) transporters, purine permeases (PUP), AZA-GUANINE RESISTANT (AZG) transporters, equilibrative nucleoside transporters (ENT), and Sugars Will Eventually be Exported Transporters (SWEET) have been characterized as involved in cytokinin transport processes. This review begins by introducing the spatial distributions of various cytokinins and the subcellular localizations of the proteins involved in cytokinin metabolism and signaling. Highlights focus on an inventory of the characterized transporters involved in cytokinin compartmentalization, including long-distance, intercellular, and intracellular transport, and the regulation of spatial distributions of cytokinins by environmental cues. Future directions for cytokinin research are also discussed.
PubMed: 38689499
DOI: 10.1016/j.xplc.2024.100936 -
European Biophysics Journal : EBJ Oct 20238-azaguanine is a triazolopyrimidine nucleobase analog possessing potent antibacterial and antitumor activities, and it has been implicated as a lead molecule in cancer...
8-azaguanine is a triazolopyrimidine nucleobase analog possessing potent antibacterial and antitumor activities, and it has been implicated as a lead molecule in cancer and malaria therapy. Its intrinsic fluorescence properties can be utilized for monitoring its interactions with biological polymers like proteins or nucleic acids. In order to better understand these interactions, it is important to know the tautomeric equilibrium of this compound. In this work, the tautomeric equilibrium of all natural neutral and anionic compound forms (except highly improbable imino-enol tautomers) as well as their methyl derivatives and ribosides was revealed by quantum chemistry methods. It was shown that, as expected, tautomers protonated at positions 1 and 9 dominate neutral forms both in gas phase and in aqueous solution. 8-azaguanines methylated at any position of the triazole ring are protonated at position 1. The computed vertical absorption and emission energies are in very good agreement with the experimental data. They confirm the validity of the assumption that replacing the proton with the methyl group does not significantly change the positions of absorption and fluorescence peaks.
Topics: Azaguanine; Spectrum Analysis; Protons; Proteins; Quantum Theory
PubMed: 37507591
DOI: 10.1007/s00249-023-01672-x -
JHEP Reports : Innovation in Hepatology Mar 2023Chronic hepatitis delta is the most severe form of chronic viral hepatitis and is associated with faster progression towards cirrhosis, liver decompensation, and...
BACKGROUND & AIMS
Chronic hepatitis delta is the most severe form of chronic viral hepatitis and is associated with faster progression towards cirrhosis, liver decompensation, and hepatocellular carcinoma. Hepatitis delta virus (HDV)'s tight dependency on hepatitis B virus and the host cell machinery for its life cycle limits the development of direct-acting antivirals. Thus, we aimed to identify compounds that could block HDV replication by targeting its antigenomic ribozyme.
METHODS
We generated stable Huh7 human hepatoma cells expressing a reporter gene (Gaussia luciferase) either downstream (Gluc-2xRz) or upstream (2xRz-Gluc) of two HDV antigenomic ribozyme sequences. We performed high-throughput screening of three small molecule libraries. The secreted luciferase was measured as a readout of ribozyme inhibition upon addition of the molecules. Each plate was considered valid when the Z factor was >0.4. Specificity and toxicity evaluations were performed for the hits with a Z-score >5 and half-maximal inhibitory concentration was calculated by performing a dose-response experiment.
RESULTS
A dose-dependent induction of luciferase expression was detected in Gluc-2xRz-transfected cells incubated with the antisense morpholino, suggesting that the catalytic activity of the ribozyme cloned downstream of the reporter gene was efficiently inhibited. Among the 6,644 compounds screened, we identified four compounds that showed a specific inhibitory effect on the HDV antigenomic ribozyme in Gluc-2xRz cells, . three histone deacetylase inhibitors and the purine analogue 8-azaguanine. The latter also significantly decreased HDV replication (by 40%) in differentiated HepaRG cells six days post infection.
CONCLUSION
Using a novel cell culture model, we identified four small molecules active against the antigenomic HDV ribozyme. These results may provide insights into the structural requirements of molecules designed for the potent and specific inhibition of HDV replication.
IMPACT AND IMPLICATIONS
Chronic hepatitis delta is the most severe form of chronic viral hepatitis and is associated with faster progression towards cirrhosis, liver decompensation, and the development of hepatocellular carcinoma. Despite the current development of several new compounds, there is still a need for efficient antiviral treatments specifically targeting hepatitis delta virus (HDV). This work describes a novel cell culture model that allows for the high-throughput screening of compounds able to inhibit HDV ribozymes. We identified four small molecules active against the antigenomic HDV ribozyme (the ribozyme involved in the early step of HDV replication), with the strongest activity shown by 8-azaguanine, a purine analogue. Our data may provide insights into the structural requirements of molecules designed to inhibit HDV.
PubMed: 36704052
DOI: 10.1016/j.jhepr.2022.100652 -
Frontiers in Chemistry 2022DNA-encoded libraries are a prime technology for target-based small molecule screening. Native DNA used as genetic compound barcode is chemically vulnerable under many...
DNA-encoded libraries are a prime technology for target-based small molecule screening. Native DNA used as genetic compound barcode is chemically vulnerable under many reaction conditions. DNA barcodes that are composed of pyrimidine nucleobases, 7-deazaadenine, and 7-deaza-8-azaguanine have been investigated for their suitability for encoded chemistry both experimentally and computationally. These four-letter barcodes were readily ligated by T4 ligation, amplifiable by Taq polymerase, and the resultant amplicons were correctly sequenced. Chemical stability profiling showed a superior chemical stability compared to native DNA, though higher susceptibility to depurination than a three-letter code based on pyrimidine DNA and 7-deazaadenine.
PubMed: 35755251
DOI: 10.3389/fchem.2022.894563 -
Life Sciences Jan 2022Sjögren's syndrome (SjS) is an autoimmune disease with a strong genetic association. To date, no vaccine or therapeutic agent exists to cure SjS, and patients must rely...
BACKGROUND
Sjögren's syndrome (SjS) is an autoimmune disease with a strong genetic association. To date, no vaccine or therapeutic agent exists to cure SjS, and patients must rely on lifelong therapies to treat symptoms. Human leukocyte antigens (HLA) are primary susceptibility loci that form the genetic basis for many autoimmune diseases, including SjS. In this study, we sought to determine whether blocking MHC class II IAg antigen presentation in the NOD mouse would alleviate SjS by preventing the recognition of autoantigens by pathogenic T cells.
METHODS
Mapping of the antigenic epitopes of Ro60 autoantigen to IAg of the NOD mice was performed using structural modeling and in-vitro stimulation. Tetraazatricyclo-dodecane (TATD) and 8-Azaguanine (8-Aza) were previously identified as potential binders to IA of the NOD mice using in silico drug screening. Mice were treated with 20mgs/kg via IP every day five days/week for 23 weeks. Disease profiling was conducted.
FINDINGS
Specific peptides of Ro60 autoantigen were identified to bind to IAg and stimulated splenocytes of the NOD mice. Treating NOD mice with TATD or 8-Azaguanine alleviated SjS symptoms by improving salivary and lacrimal gland secretory function, decreasing the levels of autoantibodies, and reducing the severity of lymphocytic infiltration in the salivary and lacrimal glands.
INTERPRETATION
This study presents a novel therapeutic approach for SjS by identifying small molecules capable of inhibiting T cell response via antigen-specific presentation.
FUNDING
CQN is supported financially in part by PHS grants AI130561, DE026450, and DE028544 from the National Institutes of Health.
Topics: Alkanes; Animals; Antigen Presentation; Antimetabolites, Antineoplastic; Azaguanine; Female; Histocompatibility Antigens Class II; Mice; Mice, Inbred NOD; Polycyclic Compounds; Sjogren's Syndrome
PubMed: 34843735
DOI: 10.1016/j.lfs.2021.120182 -
International Journal of Clinical and... 2021In recent years, studies have shown that , as an oncogene, is involved in progression of cancers. However, its relationship with prognosis in glioma patients is rarely...
In recent years, studies have shown that , as an oncogene, is involved in progression of cancers. However, its relationship with prognosis in glioma patients is rarely reported. Our purpose was to explore the role of in glioma. Based on 1814 glioma samples from multiple databases such as The Cancer Genome Atlas (TCGA), The Chinese Glioma Genome Atlas (CGGA), and The Gene Expression Omnibus (GEO), we use a variety of bioinformatics methods to verify the mechanism of action of in glioma from mRNA to protein, from appearance to mechanism analysis, from clinical features to prognosis. Then, the connectivity map (CMap) tool was used to predict drugs that inhibit the expression of . First, we found is highly expressed in glioma at mRNA and protein levels. Second, is an independent risk factor in prognosis and has suitable clinical diagnostic value in glioma. It was also positively correlated with World Health Organization (WHO) grade, age, and histology, and negatively correlated with mutation and codeletion. Third, base excision, cell cycle, and mismatch repair pathway were activated by in glioma. We predict small molecules to inhibit such as 8-azaguanine, gw8510, 6-thioguanosine, and ursodeoxycholic acid. This study is the first comprehensive analysis of , revealing a relationship between this novel oncogene, clinical characteristics of patients with glioma, and a mechanism leading to poor prognosis. It also provides a biomarker for diagnosis and treatment of glioma and reveal the pathologic progress of glioma at the genetic level.
PubMed: 34646412
DOI: No ID Found -
Technology in Cancer Research &... 2021Human umbilical endothelial cells (HUVECs) have been proved to be an effective whole-cell vaccine inhibiting tumor angiogenesis. In this study, we fused HUVECs with...
PURPOSE
Human umbilical endothelial cells (HUVECs) have been proved to be an effective whole-cell vaccine inhibiting tumor angiogenesis. In this study, we fused HUVECs with human lung adenocarcinoma cells A549 s, aiming at preparing lung cancer vaccine to achieve dual effects of anti-tumor angiogenesis and specific immunity to tumor cells.
METHODS
A549 cells were induced by ethyl methane sulfonate (EMS) and 8-azaguanine (8-AG) to get hypoxanthine guanine phosphoribosyl transferase (HGPRT) auxotrophic A549 cells. Then Fused HGPRT auxotrophic A549 cells with primary HUVEC cells by combining electrofusion with polyethylene glycol (PEG). Afterward the fusion cells were screened by HAT and HT selective medium and sorted by flow cell sorter to obtain high-purity HUVEC-A549 cells. Finally, HUVEC-A549 cells were identified by karyotype analysis and western blotting.
RESULTS
The fusion efficiency of HUVEC-A549 cells prepared by combining electrofusion with polyethylene glycol (PEG) was significantly higher than that of electrofusion and PEG (43.0% vs 17.60% vs 2.71%, < 0.05). After screened by HAT and HT selective medium and sorted by flow cell sorter, the proportion of HUVEC-A549 cells can count for 71.2% ± 3.2%. The mode of chromosomes in HUVEC-A549 cells was 68, and the chromosome was triploid. VE-cadherin and platelet endothelial cell adhesion molecule-1 (CD31) were highly expressed in HUVECs and HUVEC-A549 cells, but not in A549 cells.
CONCLUSIONS
These results indicate that HUVEC-A549 cells retain the biological characteristics of human umbilical vein endothelial cells and A549 cells. It can be used in the experimental study of lung cancer cell vaccine.
Topics: A549 Cells; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Cell Fusion; Human Umbilical Vein Endothelial Cells; Humans; Immunotherapy; Karyotype; Lung Neoplasms; Neovascularization, Pathologic; Polyethylene Glycols
PubMed: 34318732
DOI: 10.1177/15330338211034260 -
Biomolecules Apr 2021A comparative study of the possibilities of using ribokinase → phosphopentomutase → nucleoside phosphorylase cascades in the synthesis of modified nucleosides was...
A comparative study of the possibilities of using ribokinase → phosphopentomutase → nucleoside phosphorylase cascades in the synthesis of modified nucleosides was carried out. Recombinant phosphopentomutase from HB27 was obtained for the first time: a strain producing a soluble form of the enzyme was created, and a method for its isolation and chromatographic purification was developed. It was shown that cascade syntheses of modified nucleosides can be carried out both by the mesophilic and thermophilic routes from D-pentoses: ribose, 2-deoxyribose, arabinose, xylose, and 2-deoxy-2-fluoroarabinose. The efficiency of 2-chloradenine nucleoside synthesis decreases in the following order: Rib (92), dRib (74), Ara (66), F-Ara (8), and Xyl (2%) in 30 min for mesophilic enzymes. For thermophilic enzymes: Rib (76), dRib (62), Ara (32), F-Ara (<1), and Xyl (2%) in 30 min. Upon incubation of the reaction mixtures for a day, the amounts of 2-chloroadenine riboside (thermophilic cascade), 2-deoxyribosides (both cascades), and arabinoside (mesophilic cascade) decreased roughly by half. The conversion of the base to 2-fluoroarabinosides and xylosides continued to increase in both cases and reached 20-40%. Four nucleosides were quantitatively produced by a cascade of enzymes from D-ribose and D-arabinose. The ribosides of 8-azaguanine (thermophilic cascade) and allopurinol (mesophilic cascade) were synthesized. For the first time, D-arabinosides of 2-chloro-6-methoxypurine and 2-fluoro-6-methoxypurine were synthesized using the mesophilic cascade. Despite the relatively small difference in temperatures when performing the cascade reactions (50 and 80 °C), the rate of product formation in the reactions with enzymes was significantly higher. enzymes also provided a higher content of the target products in the reaction mixture. Therefore, they are more appropriate for use in the polyenzymatic synthesis of modified nucleosides.
Topics: Bacterial Proteins; Escherichia coli; Nucleosides; Pentoses; Pentosyltransferases; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Thermus thermophilus
PubMed: 33923608
DOI: 10.3390/biom11040586 -
Frontiers in Bioengineering and... 20208-Azaguanine (1) is a special 1,2,3-triazole containing natural product that possesses potent antibacterial and antitumor activities. In the present study, the entire...
8-Azaguanine (1) is a special 1,2,3-triazole containing natural product that possesses potent antibacterial and antitumor activities. In the present study, the entire 8-azaguanine biosynthetic gene cluster was located from CGMCC4.1633. Targeted gene disruption, heterologous expression analysis, and feeding experiments identified crucial genes for 8-azaguanine production. Moreover, we characterized the structure of two novel metabolites, analyzed NO (or reactive nitrogen species) related genes 8-azgA/B and radical SAM enzyme homologous 8-AzgG, and verified the non-enzymatic ring formation reaction of 8-azaguanine 1,2,3-triazole. All of the data and presumptions provide insight into the timing and mechanism of the enzymatic and non-enzymatic pathway that produce 8-azaguanine-type 1,2,3-triazole.
PubMed: 33251204
DOI: 10.3389/fbioe.2020.603514 -
High-Throughput Screening Assay to Identify Small Molecule Inhibitors of Marburg Virus VP40 Protein.ACS Infectious Diseases Oct 2020Marburg virus (MARV) causes sporadic outbreaks of severe disease with high case fatality rates in humans. To date, neither therapeutics nor prophylactic approaches have...
Marburg virus (MARV) causes sporadic outbreaks of severe disease with high case fatality rates in humans. To date, neither therapeutics nor prophylactic approaches have been approved for MARV disease. The MARV matrix protein VP40 (mVP40) plays central roles in virus assembly and budding. mVP40 also inhibits interferon signaling by inhibiting the function of Janus kinase 1. This suppression of host antiviral defenses likely contributes to MARV virulence and therefore is a potential therapeutic target. We developed and optimized a cell-based high-throughput screening (HTS) assay in 384-well format to measure mVP40 interferon (IFN) antagonist function such that inhibitors could be identified. We performed a pilot screen of 1280 bioactive compounds and identified 3 hits, azaguanine-8, tosufloxacin hydrochloride, and linezolid, with scores > 3 and no significant cytotoxicity. Of these, azaguanine-8 inhibited MARV growth at noncytotoxic concentrations. These data demonstrate the suitability of the HTS mVP40 assay for drug discovery and suggest potential directions for anti-MARV therapeutic development.
Topics: Animals; High-Throughput Screening Assays; Humans; Interferons; Marburg Virus Disease; Marburgvirus; Virus Assembly
PubMed: 32870648
DOI: 10.1021/acsinfecdis.0c00512