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Journal of Dairy Science Mar 2020The mammary gland undergoes distinct periods of growth, development, and secretory activity. During bovine lactation, a gradual decrease in the number of mammary...
The mammary gland undergoes distinct periods of growth, development, and secretory activity. During bovine lactation, a gradual decrease in the number of mammary epithelial cells largely accounts for the decline in milk production with advancing lactation. The net decline in cell number (approx. 50%) is due to cell death but is simultaneously accompanied by cell renewal. Although the rate of cell proliferation is slow, by the end of lactation most cells in the gland were formed after calving. Typically milking is terminated when cows are in the final 2 mo of pregnancy. This causes regenerative involution, wherein extensive cell replacement and mammary growth occurs. We hypothesized that replacement of senescent secretory cells and progenitor cells during the dry period increases milk yield in the next lactation. Analysis of global gene expression revealed networks and canonical pathways during regenerative involution that support cell turnover and mammary growth, and reflect oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress. Immune responses consistent with influx of neutrophils, macrophages, and lymphocytes, and processes that support mammary differentiation and lactogenesis were also evident. Data also suggest that replication of stem and progenitor cells occurs during the dry period. Relying on long-term retention of bromodeoxyuridine-labeled DNA, we identified putative bovine mammary stem cells. These label-retaining epithelial cells (LREC) are in low abundance within mammary epithelium (<1%), predominantly estrogen receptor-negative, and localized in a basal or suprabasal layer of the epithelium. Analyses of gene expression in laser-microdissected LREC are consistent with the concept that LREC represent stem cells and progenitor cells, which differ in properties and location within the epithelial layer. We identified potential markers for these cells and have increased their number by infusing xanthosine through the teat canal of prepubertal heifers. Altering population dynamics of mammary stem and progenitor cells during the mammary cycle may be a means to increase efficiency of milk production.
Topics: Animals; Bromodeoxyuridine; Cattle; Cell Count; Cell Differentiation; Cell Proliferation; Epithelial Cells; Epithelium; Female; Lactation; Mammary Glands, Animal; Milk; Population Dynamics; Pregnancy; Ribonucleosides; Stem Cells; Xanthines
PubMed: 31704023
DOI: 10.3168/jds.2019-17241 -
Acta Crystallographica. Section C,... May 2020The positional change of nitrogen-7 of the RNA constituent guanosine to the bridgehead position-5 leads to the base-modified nucleoside 5-aza-7-deazaguanosine. Contrary...
The positional change of nitrogen-7 of the RNA constituent guanosine to the bridgehead position-5 leads to the base-modified nucleoside 5-aza-7-deazaguanosine. Contrary to guanosine, this molecule cannot form Hoogsteen base pairs and the Watson-Crick proton donor site N3-H becomes a proton-acceptor site. This causes changes in nucleobase recognition in nucleic acids and has been used to construct stable `all-purine' DNA and DNA with silver-mediated base pairs. The present work reports the single-crystal X-ray structure of 7-iodo-5-aza-7-deazaguanosine, CHINO (1). The iodinated nucleoside shows an anti conformation at the glycosylic bond and an N conformation (O4'-endo) for the ribose moiety, with an antiperiplanar orientation of the 5'-hydroxy group. Crystal packing is controlled by interactions between nucleobase and sugar moieties. The 7-iodo substituent forms a contact to oxygen-2' of the ribose moiety. Self-pairing of the nucleobases does not take place. A Hirshfeld surface analysis of 1 highlights the contacts of the nucleobase and sugar moiety (O-H...O and N-H...O). The concept of pK-value differences to evaluate base-pair stability was applied to purine-purine base pairing and stable base pairs were predicted for the construction of `all-purine' RNA. Furthermore, the 7-iodo substituent of 1 was functionalized with benzofuran to detect motional constraints by fluorescence spectroscopy.
Topics: Base Pairing; Crystallography, X-Ray; DNA; Guanosine; Molecular Conformation; Nucleic Acids; Purines; Ribonucleosides; Silver
PubMed: 32367834
DOI: 10.1107/S2053229620004684 -
Methods (San Diego, Calif.) Oct 2012miRNAs are short (20-23 nt) RNAs that are loaded into proteins of the Argonaute (AGO) family and guide them to partially complementary target sites on mRNAs, resulting...
miRNAs are short (20-23 nt) RNAs that are loaded into proteins of the Argonaute (AGO) family and guide them to partially complementary target sites on mRNAs, resulting in mRNA destabilization and/or translational repression. It is estimated that about 60% of the mammalian genes are potentially regulated by miRNAs, and therefore methods for experimental miRNA target determination have become valuable tools for the characterization of posttranscriptional gene regulation. Here we present a step-by-step protocol and guidelines for the computational analysis for the large-scale identification of miRNA target sites in cultured cells by photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation (PAR-CLIP) of AGO proteins.
Topics: Animals; Argonaute Proteins; Computational Biology; Gene Expression Regulation; Genome; Mice; MicroRNAs; RNA, Messenger; Ribonucleosides
PubMed: 22926237
DOI: 10.1016/j.ymeth.2012.08.006 -
Biochemistry Jun 2021We report the initial characterization of the α-ribazole (α-R) kinase enzyme of (CblS), which converts α-R to α-R-phosphate (α-RP) during the synthesis of...
We report the initial characterization of the α-ribazole (α-R) kinase enzyme of (CblS), which converts α-R to α-R-phosphate (α-RP) during the synthesis of cobamides. We implemented a continuous spectrophotometric assay to obtain kinetic parameters for several potential substrates and to study the specificity of the enzyme for α-N-linked ribosides. The apparent values for α-R and ATP were 358 and 297 μM, respectively. We also report methods for synthesizing and quantifying non-commercially available α-ribosides and β-ribazole (β-R). Purified CblS activated α-R and other α-ribosides, including α-adenosine (α-Ado). CblS did not phosphorylate β-N-linked glycosides like β-adenosine or β-R. Expression of in a subsp. sv Typhimurium LT2 () strain lacking the nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyl transferase (CobT) enzyme resulted in the activation of various benzimidazole α-ribosides, and the synthesis of benzimidazolyl cobamides to levels that supported robust growth. Notably, α-Ado did not support growth under similar conditions, in spite of the fact that CblS phosphorylated α-Ado . When α-Ado was provided at a very high concentration, growth was observed. This result suggested that in α-Ado transport may be inefficient. We conclude that CblS has specificity for α-N-glycosidic bonds, but not for the base in α-ribosides.
Topics: Bacterial Proteins; Enzyme Assays; Geobacillus; Kinetics; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Purine-Nucleoside Phosphorylase; Ribonucleosides; Salmonella; Substrate Specificity
PubMed: 34105957
DOI: 10.1021/acs.biochem.1c00119 -
The Plant Cell Jan 2023In nucleotide metabolism, nucleoside kinases recycle nucleosides into nucleotides-a process called nucleoside salvage. Nucleoside kinases for adenosine, uridine, and...
In nucleotide metabolism, nucleoside kinases recycle nucleosides into nucleotides-a process called nucleoside salvage. Nucleoside kinases for adenosine, uridine, and cytidine have been characterized from many organisms, but kinases for inosine and guanosine salvage are not yet known in eukaryotes and only a few such enzymes have been described from bacteria. Here we identified Arabidopsis thaliana PLASTID NUCLEOSIDE KINASE 1 (PNK1), an enzyme highly conserved in plants and green algae belonging to the Phosphofructokinase B family. We demonstrate that PNK1 from A. thaliana is located in plastids and catalyzes the phosphorylation of inosine, 5-aminoimidazole-4-carboxamide-1-β-d-ribose (AICA ribonucleoside), and uridine but not guanosine in vitro, and is involved in inosine salvage in vivo. PNK1 mutation leads to increased flux into purine nucleotide catabolism and, especially in the context of defective uridine degradation, to over-accumulation of uridine and UTP as well as growth depression. The data suggest that PNK1 is involved in feedback regulation of purine nucleotide biosynthesis and possibly also pyrimidine nucleotide biosynthesis. We additionally report that cold stress leads to accumulation of purine nucleotides, probably by inducing nucleotide biosynthesis, but that this adjustment of nucleotide homeostasis to environmental conditions is not controlled by PNK1.
Topics: Inosine; Nucleosides; Nucleotides; Purine Nucleotides; Uridine
PubMed: 36342213
DOI: 10.1093/plcell/koac320 -
Bioscience Reports Dec 2017Loss-of-function of barley mildew locus o () confers durable broad-spectrum penetration resistance to the barley powdery mildew pathogen, f. sp. (). Given the...
Loss-of-function of barley mildew locus o () confers durable broad-spectrum penetration resistance to the barley powdery mildew pathogen, f. sp. (). Given the importance of mutants in agriculture, surprisingly few molecular components have been identified to be required for this type of resistance in barley. With the aim to identify novel cellular factors contributing to -based resistance, we devised a pharmacological inhibitor screen. Of the 41 rationally chosen compounds tested, five caused a partial suppression of resistance in barley, indicated by increased levels of host cell entry. These chemicals comprise brefeldin A (BFA), 2',3'-dideoxyadenosine (DDA), 2-deoxy-d-glucose, spermidine, and 1-aminobenzotriazole. Further inhibitor analysis corroborated a key role for both anterograde and retrograde endomembrane trafficking in resistance. In addition, all four ribonucleosides, some ribonucleoside derivatives, two of the five nucleobases (guanine and uracil), some guanine derivatives as well as various polyamines partially suppress resistance in barley via yet unknown mechanisms. Most of the chemicals identified to be effective in partially relieving resistance in barley also to some extent compromised powdery mildew resistance in an double mutant. In summary, our study identified novel suppressors of resistance that may serve as valuable probes to unravel further the molecular processes underlying this unusual type of disease resistance.
Topics: Agriculture; Agrochemicals; Brefeldin A; DDT; Deoxyglucose; Disease Resistance; Hordeum; Plant Proteins; Ribonucleosides; Spermidine; Triazoles
PubMed: 29127104
DOI: 10.1042/BSR20171389 -
Chemical Research in Toxicology Mar 2019Reactive nitrogen species (RNS) are produced during infection and inflammation, and the effects of these agents on proteins, DNA, and lipids are well recognized. In...
Reactive nitrogen species (RNS) are produced during infection and inflammation, and the effects of these agents on proteins, DNA, and lipids are well recognized. In contrast, the effects of RNS damaged metabolites are less appreciated. 5-Amino-3-β-(d-ribofuranosyl)-3 H-imidazo-[4,5- d][1,3]oxazine-7-one (oxanosine) and its nucleotides are products of guanosine nitrosation. Here we demonstrate that oxanosine monophosphate (OxMP) is a potent reversible competitive inhibitor of IMPDH. The value of K varies from 50 to 340 nM among IMPDHs from five different organisms. UV spectroscopy and X-ray crystallography indicate that OxMP forms a ring-opened covalent adduct with the active site Cys (E-OxMP*). Unlike the covalent intermediate of the normal catalytic reaction, E-OxMP* does not hydrolyze, but instead recyclizes to OxMP. IMPDH inhibitors block proliferation and can induce apoptosis, so the inhibition of IMPDH by OxMP presents another potential mechanism for RNS toxicity.
Topics: Enzyme Inhibitors; Humans; IMP Dehydrogenase; Molecular Structure; Phosphates; Ribonucleosides
PubMed: 30746940
DOI: 10.1021/acs.chemrestox.8b00342 -
Translational Research : the Journal of... Apr 2020Seasonal influenza viruses cause major morbidity and mortality worldwide, threatening in particular older adults and the immunocompromised. Two classes of influenza...
Seasonal influenza viruses cause major morbidity and mortality worldwide, threatening in particular older adults and the immunocompromised. Two classes of influenza therapeutics dominate current disease management, but both are compromised by pre-existing or rapidly emerging viral resistance. We have recently reported a novel ribonucleoside analog clinical candidate, EIDD-2801, that combines potent antiviral efficacy in ferrets and human airway epithelium cultures with a high barrier against viral escape. In this study, we established fundamental EIDD-2801 efficacy paradigms against pandemic and seasonal influenza A virus (IAV) strains in ferrets that can be used to inform exposure targets and treatment regimens. Based on reduction of shed virus titers, alleviation of clinical signs, and lowered virus burden in upper and lower respiratory tract tissues, lowest efficacious oral dose concentrations of EIDD-2801, given twice daily, were 2.3 and 7 mg/kg of body weight against seasonal and pandemic IAVs, respectively. The latest opportunity for initiation of efficacious treatment was 36 hours after infection of ferrets. Administered in 12-hour intervals, three 7 mg/kg doses of EIDD-2801 were sufficient for maximal therapeutic benefit against a pandemic IAV and significantly shortened the time to resolution of clinical signs. Ferrets infected with pandemic IAV and treated following the minimally efficacious EIDD-2801 regimen demonstrated significantly less shed virus and inflammatory cellular infiltrates in nasal lavages, but mounted a robust humoral antiviral response after recovery that was indistinguishable from that of vehicle-treated animals. These results provide an experimental basis in a human disease-relevant influenza animal model for clinical testing of EIDD-2801.
Topics: Animals; Antiviral Agents; Cytidine; Disease Models, Animal; Dogs; Dose-Response Relationship, Drug; Drug Resistance, Viral; Ferrets; HEK293 Cells; Humans; Hydroxylamines; Influenza A virus; Influenza, Human; Madin Darby Canine Kidney Cells; Mutation; Ribonucleosides
PubMed: 31945316
DOI: 10.1016/j.trsl.2019.12.002 -
Journal of Medicinal Chemistry Apr 2015The conversion of selected β-D-2,6-diaminopurine nucleosides (DAPNs) to their phosphoramidate prodrug (PD) substantially blocks the conversion to the G-analog allowing...
β-D-2'-C-Methyl-2,6-diaminopurine Ribonucleoside Phosphoramidates are Potent and Selective Inhibitors of Hepatitis C Virus (HCV) and Are Bioconverted Intracellularly to Bioactive 2,6-Diaminopurine and Guanosine 5'-Triphosphate Forms.
The conversion of selected β-D-2,6-diaminopurine nucleosides (DAPNs) to their phosphoramidate prodrug (PD) substantially blocks the conversion to the G-analog allowing for the generation of two bioactive nucleoside triphosphates (NTPs) in human hepatocytes. A variety of 2'-C-methyl DAPN-PDs were prepared and evaluated for inhibition of HCV viral replication in Huh-7 cells, cytotoxicity in various cell lines, and cellular pharmacology in both Huh-7 and primary human liver cells. The DAPN-PDs were pan-genotypic, effective against various HCV resistant mutants, and resistant variants could not be selected. 2'-C-Me-DAPN-TP and 2'-C-Me-GTP were chain terminators for genotype 1b HCV-pol, and single nucleotide incorporation assays revealed that 2'-C-Me-DAPN-TP was incorporated opposite U. No cytotoxicity was observed with our DAPN-PD when tested up to 50 μM. A novel, DAPN-PD, 15c, has been selected for further evaluation because of its good virologic and toxicologic profile and its ability to deliver two active metabolites, potentially simplifying HCV treatment.
Topics: 2-Aminopurine; Amides; Antiviral Agents; Cell Line; Cells, Cultured; Guanosine Triphosphate; Hepacivirus; Hepatitis C; Humans; Methylation; Phosphoric Acids; Prodrugs; Ribonucleosides
PubMed: 25849312
DOI: 10.1021/jm501874e -
Journal of Pharmacological Sciences 2012We previously reported that higher therapeutic effects were obtained in rheumatoid arthritis (RA) patients and RA model animals when the dosing-times of methotrexate and...
We previously reported that higher therapeutic effects were obtained in rheumatoid arthritis (RA) patients and RA model animals when the dosing-times of methotrexate and tacrolimus were chosen according to the 24-h rhythms of the inflammatory response. Mizoribine (MZR) is an immunosuppressive agent and is used against RA in the same manner as methotrexate and tacrolimus. In this study, we examined whether a dosing-time dependency of the therapeutic effect of MZR could be detected in collagen-induced arthritis (CIA) rats. To measure C-reactive protein (CRP) and tumor necrosis factor (TNF)-α levels, blood was collected from CIA rats at different times. MZR was administered at two different dosing-times based on these findings and its effects and toxicity were examined. CRP and TNF-α concentrations in blood showed significant 24-h rhythms. The exacerbation of arthritis and excessive increase in leukocytes in CIA rats were markedly lower in the group treated with MZR at the dark phase than those of the group treated with MZR at the light phase. These findings suggest that the therapeutic index of RA therapy may be improved by administering MZR at a time in the day when the inflammatory reaction begins to activate.
Topics: Animals; Arthritis, Experimental; Collagen; Drug Chronotherapy; Immunosuppressive Agents; Rats; Rats, Inbred Lew; Ribonucleosides
PubMed: 23018897
DOI: 10.1254/jphs.12059fp