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1
Conrad-Webb, H., and R. A. Butow. "A polymerase switch in the synthesis of rRNA in Saccharomyces cerevisiae." Molecular and Cellular Biology 15, no. 5 (May 1995): 2420–28. http://dx.doi.org/10.1128/mcb.15.5.2420.
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Transcription of ribosomal DNA by RNA polymerase I is believed to be the sole source of the 25S, 18S, and 5.8S rRNAs in wild-type cells of Saccharomyces cerevisiae. Here we present evidence for a switch from RNA polymerase I to RNA polymerase II in the synthesis of a substantial fraction of those rRNAs in respiratory-deficient (petite) cells. The templates for the RNA polymerase II transcripts are largely, if not exclusively, episomal copies of ribosomal DNA arising from homologous recombination events within the ribosomal DNA repeat on chromosome XII. Ribosomal DNA contains a cryptic RNA polymerase II promoter that is activated in petites; it overlaps the RNA polymerase I promoter and produces a transcript equivalent to the 35S precursor rRNA made by RNA polymerase I. Yeast cells that lack RNA polymerase I activity, because of a disruption of the RPA135 gene that encodes subunit II of the enzyme, can survive by using the RNA polymerase II promoter in ribosomal DNA to direct the synthesis of the 35S rRNA precursor. This polymerase switch could provide cells with a mechanism to synthesize rRNA independent of the controls of RNA polymerase I transcription.
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Klein, Beate, and Hartmut Follmann. "Deoxyribonucleotide Biosynthesis in Green Algae. S Phase-Specific Thymidylate Kinase and Unspecific Nucleoside Diphosphate Kinase in Scenedesmus obliquus." Zeitschrift für Naturforschung C 43, no. 5-6 (June 1, 1988): 377–85. http://dx.doi.org/10.1515/znc-1988-5-610.
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NDP kinase and thymidylate kinase are essential for DNA precursor formation in that they phosphorylate the products of de novo deoxyribonucleotide biosynthesis, deoxyribonucleoside 5′-diphosphates and thymidine 5′-monophosphate to the corresponding triphosphates which then serve as DNA polymerase substrates. The two enzymes have been measured in synchronous cultures of the green algae, S. obliquus. Thymidylate kinase exhibits an activity peak at the 11 -12th hour of the 24-hour cell cycle, coinciding with DNA synthesis. Enzyme activity is markedly stimulated in presence of fluorodeoxyuridine in the culture medium. This behaviour of dTMP kinase is very similar to that of three other S phase-specific peak enzymes previously analyzed in synchronous algae, viz. ribonucleotide reductase, thymidylate synthase, and dihydrofolate reductase. In contrast, NDP kinase exhibits high and constant activity through the entire cell cycle. The two kinases have been isolated from cell-free extracts, and separated from each other by chromatography on Blue Sepharose. The peak enzyme, dTMP kinase, has been purified to near homogeneity and its catalytic properties are described; the molecular weight is 56,000. NDP kinase activity is separable into two enzyme fractions, both of molecular weight 100,000 (or higher), which are unspecific with respect to ribonucleotide and deoxyribonucleotide substrates. Characterization and purification of the whole series of deoxyribonucleotide-synthesizing enzymes from one organism provides a basis for in vitro experiments towards reconstitution of an S phase-specific DNA precursor/DNA replication multienzyme aggregate.
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Woolford, C. A., L. B. Daniels, F. J. Park, E. W. Jones, J. N. Van Arsdell, and M. A. Innis. "The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases." Molecular and Cellular Biology 6, no. 7 (July 1986): 2500–2510. http://dx.doi.org/10.1128/mcb.6.7.2500.
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pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.
4
Woolford, C. A., L. B. Daniels, F. J. Park, E. W. Jones, J. N. Van Arsdell, and M. A. Innis. "The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases." Molecular and Cellular Biology 6, no. 7 (July 1986): 2500–2510. http://dx.doi.org/10.1128/mcb.6.7.2500-2510.1986.
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pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.
5
Tiganos, E., and M. B. Herrington. "Kasugamycin inhibition of nonsense suppression by thymine-requiring strains of Escherichia coli K12." Canadian Journal of Microbiology 39, no. 4 (April 1, 1993): 448–50. http://dx.doi.org/10.1139/m93-065.
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Thymine-requiring strains of Escherichia coli suppress nonsense and frame-shift mutations. This appears to occur during translation, suggesting that the lack of activity of an enzyme thymidylate synthase, required for the synthesis of a DNA precursor, alters the fidelity of translation. The aminoglycoside antibiotic kasugamycin, which enhances translational accuracy in vitro, prevents thymine-requiring cells from suppressing. The inhibition of suppression by kasugamycin is not prevented by the introduction of two different kasugamycin-resistance mutations, although the dose required for inhibition increases. These observations support the conclusion that suppression occurs during translation.Key words: suppression, kasugamycin, translation, thymine-requiring.
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Cortes, P., F. Dumler, and N. W. Levin. "Glomerular uracil nucleotide synthesis." American Journal of Physiology-Renal Physiology 255, no. 4 (October 1, 1988): F635—F646. http://dx.doi.org/10.1152/ajprenal.1988.255.4.f635.
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The biosynthesis of basement membrane material requires the sugar derivatives of uridine 5'-triphosphate (UTP) for protein glycosylation. Uridine and orotate utilization for the biosynthesis of uracil ribonucleotides was studied in isolated rat glomeruli incubated in vitro. At a 1 microM concentration total orotate utilization was 9.6 +/- 1.8 pmol.min-1.mg DNA-1 (1 mg DNA approximately 0.175 X 10(6) glomeruli), 51% of the total amount metabolized was used in ribonucleotide formation, and there was a significant UTP accretion. Except at a high initial concentration (50 microM), exogenous uridine failed to increase the UTP pool due to rapid uridine breakdown by a cytosolic phosphorylase. Inhibition of this enzyme with benzylacyclouridine resulted in increased biosynthesis and accretion of UTP, and in a 17-fold higher concentration of uridine, primarily produced from performed sources of nucleosides. Continuous addition of exogenous uridine to maintain its concentration at 1 microM resulted in a total uridine utilization of 550 +/- 30 pmol.min-1.mg DNA-1. Uridine salvage for ribonucleotide biosynthesis was only 3% of the total metabolized. In contrast to uridine, and presumably due to UTP pool compartmentation, orotate incorporation into uridine 5'-diphosphosugars was prominent. The metabolism of exogenous orotate was not decreased by the presence of large amounts of uridine and by an expanded UTP pool. It is concluded that when exogenous orotate is present, it is an important precursor for glomerular uracil ribonucleotide biosynthesis. Due to its rapid rate of catabolism, uridine cannot maintain ribonucleotide biosynthesis at a rate sufficient to result in UTP accretion unless it is provided continuously in substantial quantities.
7
Lai, Lilin, Hongmei Liu, Xiaoyun Wu, and John C. Kappes. "Moloney Murine Leukemia Virus Integrase Protein Augments Viral DNA Synthesis in Infected Cells." Journal of Virology 75, no. 23 (December 1, 2001): 11365–72. http://dx.doi.org/10.1128/jvi.75.23.11365-11372.2001.
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ABSTRACT Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (ΔIN) or 34 C-terminal amino acid residues (Δ34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Δ34 and ΔIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Δ34 and ΔIN mutants intrans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of ΔIN mutant virions could not be complemented with the Δ34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.
8
Fang, Feng, Jason Hoskins, and J. Scott Butler. "5-Fluorouracil Enhances Exosome-Dependent Accumulation of Polyadenylated rRNAs." Molecular and Cellular Biology 24, no. 24 (December 15, 2004): 10766–76. http://dx.doi.org/10.1128/mcb.24.24.10766-10776.2004.
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ABSTRACT The antimetabolite 5-fluorouracil (5FU) is a widely used chemotherapeutic for the treatment of solid tumors. Although 5FU slows DNA synthesis by inhibiting the ability of thymidylate synthetase to produce dTMP, the drug also has significant effects on RNA metabolism. Recent genome-wide assays for 5FU-induced haploinsufficiency in Saccharomyces cerevisiae identified genes encoding components of the RNA processing exosome as potential targets of the drug. In this report, we used DNA microarrays to analyze the effect of 5FU on the yeast transcriptome and found that the drug causes the accumulation of polyadenylated fragments of the 27S rRNA precursor and that defects in the nuclear exoribonuclease Rrp6p enhance this effect. The size distribution of these RNAs and their sensitivity to Rrp6p suggest that they are normally degraded by the nuclear exosome and a 5′-3′ exoribonuclease. Consistent with this hypothesis, 5FU inhibits the growth of RRP6 mutants with defects in the degradation function of the enzyme and it interferes with the degradation of an rRNA precursor. The detection of poly(A)+ pre-RNAs in strains defective in various steps in ribosome biogenesis suggests that the production of poly(A)+ pre-rRNAs may be a general result of defects in rRNA processing. These findings suggest that 5FU inhibits an exosome-dependent surveillance pathway that degrades polyadenylated precursor rRNAs.
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Olsen, W. A., E. Perchellet, and R. L. Malinowski. "Intestinal mucosa in diabetes: synthesis of total proteins and sucrase-isomaltase." American Journal of Physiology-Gastrointestinal and Liver Physiology 250, no. 6 (June 1, 1986): G788—G793. http://dx.doi.org/10.1152/ajpgi.1986.250.6.g788.
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The effects of insulin deficiency on nitrogen metabolism in muscle and liver have been extensively studied with recent in vivo demonstration of impaired protein synthesis in rats with streptozotocin-induced diabetes. Despite the significant contribution of small intestinal mucosa to overall protein metabolism, the effects of insulin deficiency on intestinal protein synthesis have not been completely defined. We studied the effects of streptozotocin-induced diabetes on total protein synthesis by small intestinal mucosa and on synthesis of a single enzyme protein of the enterocyte brush-border membrane sucrase-isomaltase. We used the flooding-dose technique of McNurlan, Tomkins, and Garlick (Biochem. J. 178: 373–379, 1979) to minimize the difficulties of measuring specific radioactivity of precursor phenylalanine and determined incorporation into mucosal proteins and sucrase-isomaltase 20 min after injection of the labeled amino acid. Diabetes did not alter mucosal mass as determined by weight and content of protein and DNA during the 5 days after injection of streptozotocin. Increased rates of sucrase-isomaltase synthesis developed beginning on day 3, and those of total protein developed on day 5. Thus intestinal mucosal protein synthesis is not an insulin-sensitive process.
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Chen, Yuzhi, Wenyun Liu, Donna L. McPhie, Linda Hassinger, and Rachael L. Neve. "APP-BP1 mediates APP-induced apoptosis and DNA synthesis and is increased in Alzheimer's disease brain." Journal of Cell Biology 163, no. 1 (October 13, 2003): 27–33. http://dx.doi.org/10.1083/jcb.200304003.
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APP-BP1, first identified as an amyloid precursor protein (APP) binding protein, is the regulatory subunit of the activating enzyme for the small ubiquitin-like protein NEDD8. We have shown that APP-BP1 drives the S- to M-phase transition in dividing cells, and causes apoptosis in neurons (Chen, Y., D.L. McPhie, J. Hirschberg, and R.L. Neve. 2000. J. Biol. Chem. 275:8929–8935). We now demonstrate that APP-BP1 binds to the COOH-terminal 31 amino acids of APP (C31) and colocalizes with APP in a lipid-enriched fraction called lipid rafts. We show that coexpression of a peptide representing the domain of APP-BP1 that binds to APP, abolishes the ability of overexpressed APP or the V642I mutant of APP to cause neuronal apoptosis and DNA synthesis. A dominant negative mutant of the NEDD8 conjugating enzyme hUbc12, which participates in the ubiquitin-like pathway initiated by APP-BP1, blocks neuronal apoptosis caused by APP, APP(V642I), C31, or overexpression of APP-BP1. Neurons overexpressing APP or APP(V642I) show increased APP-BP1 protein levels in lipid rafts. A similar increase in APP-BP1 in lipid rafts is observed in the Alzheimer's disease brain hippocampus, but not in less-affected areas of Alzheimer's disease brain. This translocation of APP-BP1 to lipid rafts is accompanied by a change in the subcellular localization of the ubiquitin-like protein NEDD8, which is activated by APP-BP1.
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Lane, Andrew N., and Teresa W.-M. Fan. "Regulation of mammalian nucleotide metabolism and biosynthesis." Nucleic Acids Research 43, no. 4 (January 27, 2015): 2466–85. http://dx.doi.org/10.1093/nar/gkv047.
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Abstract Nucleotides are required for a wide variety of biological processes and are constantly synthesized denovo in all cells. When cells proliferate, increased nucleotide synthesis is necessary for DNA replication and for RNA production to support protein synthesis at different stages of the cell cycle, during which these events are regulated at multiple levels. Therefore the synthesis of the precursor nucleotides is also strongly regulated at multiple levels. Nucleotide synthesis is an energy intensive process that uses multiple metabolic pathways across different cell compartments and several sources of carbon and nitrogen. The processes are regulated at the transcription level by a set of master transcription factors but also at the enzyme level by allosteric regulation and feedback inhibition. Here we review the cellular demands of nucleotide biosynthesis, their metabolic pathways and mechanisms of regulation during the cell cycle. The use of stable isotope tracers for delineating the biosynthetic routes of the multiple intersecting pathways and how these are quantitatively controlled under different conditions is also highlighted. Moreover, the importance of nucleotide synthesis for cell viability is discussed and how this may lead to potential new approaches to drug development in diseases such as cancer.
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Kumar, Jagarlamudi Kiran, Sofia Holmgren, Kerstin Hamberg Levedahl, Martin Höglund, Per Venge, and Staffan Eriksson. "AroCell TK 210 ELISA for determination of TK1 protein: age-related reference ranges and comparison with other TK1 assays." BioTechniques 68, no. 6 (June 2020): 334–41. http://dx.doi.org/10.2144/btn-2019-0148.
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Thymidine kinase 1 (TK1) is an enzyme involved in DNA precursor synthesis that has been used as a biomarker for prognosis and monitoring of different malignancies. In this study, we compared two immunoassays for measuring TK1 protein concentrations: the TK 210 ELISA (AroCell AB) and TK1 ELISA from Abcam. Overall, the TK 210 ELISA showed higher sensitivity than the Abcam TK1 ELISA for differentiating hematological malignancies (sensitivity of 0.77 vs 0.45) as well as for distinguishing sera of patients with solid tumors from those of apparently healthy individuals (0.61 vs 0.20). There was no significant difference in the TK1 protein levels determined with the TK 210 ELISA between different age groups from apparently healthy individuals. These results strongly indicate that the AroCell TK 210 ELISA is accurate and sensitive enough to be a valuable tool in cancer management.
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DiCicco-Bloom, E., E. Townes-Anderson, and I. B. Black. "Neuroblast mitosis in dissociated culture: regulation and relationship to differentiation." Journal of Cell Biology 110, no. 6 (June 1, 1990): 2073–86. http://dx.doi.org/10.1083/jcb.110.6.2073.
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Although neuron generation is precisely regulated during ontogeny, little is known about underlying mechanisms. In addition, relationships between precursor proliferation and the apparent sequence of developmental processes, including cell migration, neurite elaboration, transmitter expression and synaptogenesis remain unknown. To address these issues, we used a fully defined neuronal cell culture system derived from embryonic rat sympathetic ganglia (DiCicco-Bloom, E., and I. B. Black. 1988. Proc. Natl. Acad. Sci. USA. 85:4066-4070) in which precursors enter the mitotic cycle. We now find that, in addition to synthesizing DNA, neuroblasts also underwent division in culture, allowing analysis of developmental relationships and mitotic regulation. Our observations indicate that mitotic neuroblasts expressed a wide array of neuron-specific characteristics including extension of neuritic processes with growth cones, elaboration of neurotransmitter enzyme, synthesis and transport of transmitter vesicles and organization of transmitter release sites. These data suggest that neuroblasts in the cell cycle may simultaneously differentiate. Consequently, the apparent sequence of ontogenetic processes is not an immutable, intrinsic neuronal program. How, then, are diverse developmental events coordinated? Our observations indicate that neuroblast mitosis is regulated by a small number of epigenetic factors, including insulin and EGF. Since these signals also influence other processes in developing neurons, epigenetic regulation normally may synchronize diverse ontogenetic events.
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Sobczak, Milena, Katarzyna Kubiak, Magdalena Janicka, Malgorzata Sierant, Barbara Mikolajczyk, and Barbara Nawrot. "Synthesis, physico-chemical and biological properties of DNA and RNA oligonucleotides containing short alkylamino internucleotide bond." Collection of Czechoslovak Chemical Communications 76, no. 12 (2011): 1471–86. http://dx.doi.org/10.1135/cccc2011090.
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The condensation of the 5′-O-DMT-3′-deoxy-3′-aminothymidine with 3′-O-TBDMS-thymidine- 5′-aldehyde, followed by reduction of the resultant imine derivative and removal of tert-butyldimethylsilyl (TBDMS) protecting group, provided a dimer (denoted as TNHT), which is a congener of dithymidine phosphate with the phosphate linkage 3′-O-P(O)(OH)-O-5′ replaced with an amino group (–NH–). After phosphitylation of the 3′-OH group, the dimer TNHT was introduced (by the standard phosphoramidite approach) into a central part of the nonadecathymidylate. This oligomer exhibited lower affinity to the complementary single and double stranded DNA complements as compared to unmodified T19 oligonucleotide. The cleavage of modified oligomer with the snake venom and calf spleen phosphodiesterases was completely suppressed at the site of modification. RNA oligomers containing the TNHT dimer were used for preparation of siRNA molecules directed towards mRNA of BACE1 (beta-site amyloid precursor protein cleaving enzyme). The presence of the TNHT units at the 3′-ends of the RNA strands of the siRNA molecule (the siRNA itself is an effective gene expression inhibitor for BACE1) preserved the gene silencing activity and improved the stability of the modified siRNA in 10% fetal bovine serum.
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Whittingham, Jean L., Juana Carrero-Lerida, James A. Brannigan, Luis M. Ruiz-Perez, Ana P. G. Silva, Mark J. Fogg, Anthony J. Wilkinson, Ian H. Gilbert, Keith S. Wilson, and Dolores González-Pacanowska. "Structural basis for the efficient phosphorylation of AZT-MP (3′-azido-3′-deoxythymidine monophosphate) and dGMP by Plasmodium falciparum type I thymidylate kinase." Biochemical Journal 428, no. 3 (May 27, 2010): 499–509. http://dx.doi.org/10.1042/bj20091880.
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Plasmodium falciparum is the causative agent of malaria, a disease where new drug targets are required due to increasing resistance to current anti-malarials. TMPK (thymidylate kinase) is a good candidate as it is essential for the synthesis of dTTP, a critical precursor of DNA and has been much studied due to its role in prodrug activation and as a drug target. Type I TMPKs, such as the human enzyme, phosphorylate the substrate AZT (3′-azido-3′-deoxythymidine)-MP (monophosphate) inefficiently compared with type II TMPKs (e.g. Escherichia coli TMPK). In the present paper we report that eukaryotic PfTMPK (P. falciparum TMPK) presents sequence features of a type I enzyme yet the kinetic parameters for AZT-MP phosphorylation are similar to those of the highly efficient E. coli enzyme. Structural information shows that this is explained by a different juxtaposition of the P-loop and the azide of AZT-MP. Subsequent formation of the transition state requires no further movement of the PfTMPK P-loop, with no steric conflicts for the azide moiety, allowing efficient phosphate transfer. Likewise, we present results that confirm the ability of the enzyme to uniquely accept dGMP as a substrate and shed light on the basis for its wider substrate specificity. Information resulting from two ternary complexes (dTMP–ADP and AZT-MP–ADP) and a binary complex with the transition state analogue AP5dT [P1-(5′-adenosyl)-P5-(5′-thymidyl) pentaphosphate] all reveal significant differences with the human enzyme, notably in the lid region and in the P-loop which may be exploited in the rational design of Plasmodium-specific TMPK inhibitors with therapeutic potential.
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Dawood, Mahmoud A. O., Mohammed F. El Basuini, Sevdan Yilmaz, Hany M. R. Abdel-Latif, Zulhisyam Abdul Kari, Mohammad Khairul Azhar Abdul Razab, Hamada A. Ahmed, Mahmoud Alagawany, and Mahmoud S. Gewaily. "Selenium Nanoparticles as a Natural Antioxidant and Metabolic Regulator in Aquaculture: A Review." Antioxidants 10, no. 9 (August 27, 2021): 1364. http://dx.doi.org/10.3390/antiox10091364.
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Balanced aquafeed is the key factor for enhancing the productivity of aquatic animals. In this context, aquatic animals require optimal amounts of lipids, proteins, carbohydrates, vitamins, and minerals. The original plant and animals’ ingredients in the basal diets are insufficient to provide aquafeed with suitable amounts of minerals. Concurrently, elements should be incorporated in aquafeed in optimal doses, which differ based on the basal diets’ species, age, size, and composition. Selenium is one of the essential trace elements involved in various metabolic, biological, and physiological functions. Se acts as a precursor for antioxidative enzyme synthesis leading to high total antioxidative capacity. Further, Se can enhance the immune response and the tolerance of aquatic animals to infectious diseases. Several metabolic mechanisms, such as thyroid hormone production, cytokine formation, fecundity, and DNA synthesis, require sufficient Se addition. The recent progress in the nanotechnology industry is also applied in the production of Se nanoparticles. Indeed, Se nanoparticles are elaborated as more soluble and bioavailable than the organic and non-organic forms. In aquaculture, multiple investigations have elaborated the role of Se nanoparticles on the performances and wellbeing of aquatic animals. In this review, the outputs of recent studies associated with the role of Se nanoparticles on aquatic animals’ performances were simplified and presented for more research and development.
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Keller, Ullrich, Manuel Lang, Ivana Crnovcic, Frank Pfennig, and Florian Schauwecker. "The Actinomycin Biosynthetic Gene Cluster of Streptomyces chrysomallus: a Genetic Hall of Mirrors for Synthesis of a Molecule with Mirror Symmetry." Journal of Bacteriology 192, no. 10 (March 19, 2010): 2583–95. http://dx.doi.org/10.1128/jb.01526-09.
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ABSTRACT A gene cluster was identified which contains genes involved in the biosynthesis of actinomycin encompassing 50 kb of contiguous DNA on the chromosome of Streptomyces chrysomallus. It contains 28 genes with biosynthetic functions and is bordered on both sides by IS elements. Unprecedentedly, the cluster consists of two large inverted repeats of 11 and 13 genes, respectively, with four nonribosomal peptide synthetase genes in the middle. Nine genes in each repeat have counterparts in the other, in the same arrangement but in the opposite orientation, suggesting an inverse duplication of one of the arms during the evolution of the gene cluster. All of the genes appear to be organized into operons, each corresponding to a functional section of actinomycin biosynthesis, such as peptide assembly, regulation, resistance, and biosynthesis of the precursor of the actinomycin chromophore 4-methyl-3-hydroxyanthranilic acid (4-MHA). For 4-MHA synthesis, functional analysis revealed genes that encode pathway-specific isoforms of tryptophan dioxygenase, kynurenine formamidase, and hydroxykynureninase, which are distinct from the corresponding enzyme activities of cellular tryptophan catabolism in their regulation and in part in their substrate specificity. Phylogenetic analysis indicates that the pathway-specific tryptophan metabolism in Streptomyces most probably evolved divergently from the normal pathway of tryptophan catabolism to provide an extra or independent supply of building blocks for the synthesis of tryptophan-derived secondary metabolites.
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Kizaki, M., CW Miller, ME Selsted, and HP Koeffler. "Myeloperoxidase (MPO) gene mutation in hereditary MPO deficiency." Blood 83, no. 7 (April 1, 1994): 1935–40. http://dx.doi.org/10.1182/blood.v83.7.1935.1935.
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Abstract Myeloperoxidase (MPO), present in the azurophilic granules of polymorphonuclear leukocytes, is a myeloid enzyme whose synthesis is restricted to promyelocytes. Complete hereditary MPO deficiency affects 1 in 2,000 to 4,000 individuals; however, the genetic cause of this defect is unclear. We have determined the molecular basis of MPO deficiency in one individual (SQ). Granulocytes of SQ had no MPO activity, and had complete absence of mature and precursor MPO protein by Western blotting. Scanning MPO gene structure by Southern blotting detected a novel BgI II fragment in SQ; no other alteration in gross gene structure was detected. We hypothesized that a single base pair mutation formed a new BgI II restriction site, and that this occurred in exon 10 of MPO gene. As predicted, exon 10 from SQ was cleaved by BgI II, but DNA from the normal patients and five other MPO-deficient patients was not cleaved by this enzyme. Direct sequencing of the polymerase chain reaction (PCR) product of exon 10 showed a C to T substitution at codon 569 in exon 10, resulting in arginine (CGG) to tryptophan (TGG) substitution and creating a novel BgI II site. The mutation was homozygous, as shown by both sequencing and Southern blotting, and no other alterations in base sequence were detected. To determine the frequency of this mutation, DNA was collected from 400 normal individuals, and the presence of the mutation was examined by digesting with BgI II after amplifying exon 10 by PCR. No other case with the novel BgI II site was detected, suggesting that this is not a restriction fragment length polymorphism. The rest of the coding region of the MPO gene was sequenced in DNA from SQ, as well as from the five other MPO-deficient individuals and one normal person; no other mutations were found. Our results suggest that a point mutation at codon 569 of MPO gene represents one molecular form of MPO deficiency.
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Kizaki, M., CW Miller, ME Selsted, and HP Koeffler. "Myeloperoxidase (MPO) gene mutation in hereditary MPO deficiency." Blood 83, no. 7 (April 1, 1994): 1935–40. http://dx.doi.org/10.1182/blood.v83.7.1935.bloodjournal8371935.
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Myeloperoxidase (MPO), present in the azurophilic granules of polymorphonuclear leukocytes, is a myeloid enzyme whose synthesis is restricted to promyelocytes. Complete hereditary MPO deficiency affects 1 in 2,000 to 4,000 individuals; however, the genetic cause of this defect is unclear. We have determined the molecular basis of MPO deficiency in one individual (SQ). Granulocytes of SQ had no MPO activity, and had complete absence of mature and precursor MPO protein by Western blotting. Scanning MPO gene structure by Southern blotting detected a novel BgI II fragment in SQ; no other alteration in gross gene structure was detected. We hypothesized that a single base pair mutation formed a new BgI II restriction site, and that this occurred in exon 10 of MPO gene. As predicted, exon 10 from SQ was cleaved by BgI II, but DNA from the normal patients and five other MPO-deficient patients was not cleaved by this enzyme. Direct sequencing of the polymerase chain reaction (PCR) product of exon 10 showed a C to T substitution at codon 569 in exon 10, resulting in arginine (CGG) to tryptophan (TGG) substitution and creating a novel BgI II site. The mutation was homozygous, as shown by both sequencing and Southern blotting, and no other alterations in base sequence were detected. To determine the frequency of this mutation, DNA was collected from 400 normal individuals, and the presence of the mutation was examined by digesting with BgI II after amplifying exon 10 by PCR. No other case with the novel BgI II site was detected, suggesting that this is not a restriction fragment length polymorphism. The rest of the coding region of the MPO gene was sequenced in DNA from SQ, as well as from the five other MPO-deficient individuals and one normal person; no other mutations were found. Our results suggest that a point mutation at codon 569 of MPO gene represents one molecular form of MPO deficiency.
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Basta, Tamara, Yap Boum, Julien Briffotaux, Hubert F. Becker, Isabelle Lamarre-Jouenne, Jean-Christophe Lambry, Stephane Skouloubris, et al. "Mechanistic and structural basis for inhibition of thymidylate synthase ThyX." Open Biology 2, no. 10 (October 2012): 120120. http://dx.doi.org/10.1098/rsob.120120.
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Nature has established two mechanistically and structurally unrelated families of thymidylate synthases that produce de novo thymidylate or dTMP, an essential DNA precursor. Representatives of the alternative flavin-dependent thymidylate synthase family, ThyX, are found in a large number of microbial genomes, but are absent in humans. We have exploited the nucleotide binding pocket of ThyX proteins to identify non-substrate-based tight-binding ThyX inhibitors that inhibited growth of genetically modified Escherichia coli cells dependent on thyX in a manner mimicking a genetic knockout of thymidylate synthase. We also solved the crystal structure of a viral ThyX bound to 2-hydroxy-3-(4-methoxybenzyl)-1,4-naphthoquinone at a resolution of 2.6 Å. This inhibitor was found to bind within the conserved active site of the tetrameric ThyX enzyme, at the interface of two monomers, partially overlapping with the dUMP binding pocket. Our studies provide new chemical tools for investigating the ThyX reaction mechanism and establish a novel mechanistic and structural basis for inhibition of thymidylate synthesis. As essential ThyX proteins are found e.g. in Mycobacterium tuberculosis and Helicobacter pylori , our studies have also potential to pave the way towards the development of new anti-microbial compounds.
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Iwashita, Yuji, Toshio Sakiyama, Mark W. Musch, Mark J. Ropeleski, Hirohito Tsubouchi, and Eugene B. Chang. "Polyamines mediate glutamine-dependent induction of the intestinal epithelial heat shock response." American Journal of Physiology-Gastrointestinal and Liver Physiology 301, no. 1 (July 2011): G181—G187. http://dx.doi.org/10.1152/ajpgi.00054.2011.
Full textAbstract:
Heat shock proteins (Hsps) are highly conserved proteins that play a role in cytoprotection and maintaining intestinal homeostasis. Glutamine is essential for the optimal induction of intestinal epithelial Hsp expression, but its mechanisms of action are incompletely understood. Glutamine is a substrate for polyamine synthesis and stimulates the activity of ornithine decarboxylase (ODC), a key enzyme for polyamine synthesis, in intestinal epithelial cells. Thus we investigated whether polyamines (putrescine, spermidine, or spermine) and their precursor ornithine mediate the induction of Hsp expression in IEC-18 rat intestinal epithelial cells. As previously observed, glutamine was required for heat stress induction of Hsp70 and Hsp25, although it had little effect under basal conditions. Under conditions of glutamine depletion, supplementation of ornithine or polyamines restored the heat-induced expression of Hsp70 and Hsp25. When ODC was inhibited by α-difluoromethylornithine (DFMO), an irreversible ODC inhibitor, the heat stress induction of Hsp70 and Hsp25 was decreased significantly, even in the presence of glutamine. Ornithine, polyamines, and DFMO did not modify the nuclear localization of heat shock transcription factor 1 (HSF-1). However, DFMO dramatically reduced glutamine-dependent HSF-1 binding to an oligonucleotide with heat shock elements (HSE), which was increased by glutamine. In addition, exogenous polyamines recovered the DNA-binding activity. These results indicate that polyamines play a critical role in the glutamine-dependent induction of the intestinal epithelial heat shock response through facilitation of HSF-1 binding to HSE.
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Schmitt, M. E., and D. A. Clayton. "Nuclear RNase MRP is required for correct processing of pre-5.8S rRNA in Saccharomyces cerevisiae." Molecular and Cellular Biology 13, no. 12 (December 1993): 7935–41. http://dx.doi.org/10.1128/mcb.13.12.7935.
Full textAbstract:
RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA from the mitochondrial origin of replication in a manner consistent with a role in priming leading-strand DNA synthesis. Despite the fact that the only known RNA substrate for this enzyme is complementary to mitochondrial DNA, the majority of the RNase MRP activity in a cell is found in the nucleus. The recent characterization of this activity in Saccharomyces cerevisiae and subsequent cloning of the gene coding for the RNA subunit of the yeast enzyme have enabled a genetic approach to the identification of a nuclear role for this ribonuclease. Since the gene for the RNA component of RNase MRP, NME1, is essential in yeast cells and RNase MRP in mammalian cells appears to be localized to nucleoli within the nucleus, we utilized both regulated expression and temperature-conditional mutations of NME1 to assay for a possible effect on rRNA processing. Depletion of the RNA component of the enzyme was accomplished by using the glucose-repressed GAL1 promoter. Shortly after the shift to glucose, the RNA component of the enzyme was found to be depleted severely, and rRNA processing was found to be normal at all sites except the B1 processing site. The B1 site, at the 5' end of the mature 5.8S rRNA, is actually composed of two cleavage sites 7 nucleotides apart. This cleavage normally generates two species of 5.8S rRNA at a ratio of 10:1 (small to large) in most eukaryotes. After RNase MRP depletion, yeast cells were found to have almost exclusively the larger species of 5.8S rRNA. In addition, an aberrant 309-nucleotide precursor that stretched from the A2 to E processing sites of rRNA accumulated in these cells. Temperature-conditional mutations in the RNase MRP RNA gene gave an identical phenotype.Translation in yeast cells depleted of the smaller 5.8S rRNA was found to remain robust, suggesting a possible function for two 5.8S rRNAs in the regulated translation of select messages. These results are consistent with RNase MRP playing a role in a late step of rRNA processing. The data also indicate a requirement for having the smaller form of 5.8S rRNA, and they argue for processing at the B1 position being composed of two separate cleavage events catalyzed by two different activities.
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Schmitt, M. E., and D. A. Clayton. "Nuclear RNase MRP is required for correct processing of pre-5.8S rRNA in Saccharomyces cerevisiae." Molecular and Cellular Biology 13, no. 12 (December 1993): 7935–41. http://dx.doi.org/10.1128/mcb.13.12.7935-7941.1993.
Full textAbstract:
RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA from the mitochondrial origin of replication in a manner consistent with a role in priming leading-strand DNA synthesis. Despite the fact that the only known RNA substrate for this enzyme is complementary to mitochondrial DNA, the majority of the RNase MRP activity in a cell is found in the nucleus. The recent characterization of this activity in Saccharomyces cerevisiae and subsequent cloning of the gene coding for the RNA subunit of the yeast enzyme have enabled a genetic approach to the identification of a nuclear role for this ribonuclease. Since the gene for the RNA component of RNase MRP, NME1, is essential in yeast cells and RNase MRP in mammalian cells appears to be localized to nucleoli within the nucleus, we utilized both regulated expression and temperature-conditional mutations of NME1 to assay for a possible effect on rRNA processing. Depletion of the RNA component of the enzyme was accomplished by using the glucose-repressed GAL1 promoter. Shortly after the shift to glucose, the RNA component of the enzyme was found to be depleted severely, and rRNA processing was found to be normal at all sites except the B1 processing site. The B1 site, at the 5' end of the mature 5.8S rRNA, is actually composed of two cleavage sites 7 nucleotides apart. This cleavage normally generates two species of 5.8S rRNA at a ratio of 10:1 (small to large) in most eukaryotes. After RNase MRP depletion, yeast cells were found to have almost exclusively the larger species of 5.8S rRNA. In addition, an aberrant 309-nucleotide precursor that stretched from the A2 to E processing sites of rRNA accumulated in these cells. Temperature-conditional mutations in the RNase MRP RNA gene gave an identical phenotype.Translation in yeast cells depleted of the smaller 5.8S rRNA was found to remain robust, suggesting a possible function for two 5.8S rRNAs in the regulated translation of select messages. These results are consistent with RNase MRP playing a role in a late step of rRNA processing. The data also indicate a requirement for having the smaller form of 5.8S rRNA, and they argue for processing at the B1 position being composed of two separate cleavage events catalyzed by two different activities.
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Hood, David A. "Mechanisms of exercise-induced mitochondrial biogenesis in skeletal muscleThis paper is one of a selection of papers published in this Special Issue, entitled 14th International Biochemistry of Exercise Conference – Muscles as Molecular and Metabolic Machines, and has undergone the Journal’s usual peer review process." Applied Physiology, Nutrition, and Metabolism 34, no. 3 (June 2009): 465–72. http://dx.doi.org/10.1139/h09-045.
Full textAbstract:
Acute exercise initiates rapid cellular signals, leading to the subsequent activation of proteins that increase gene transcription. The result is a higher level of mRNA expression, often observed during the recovery period following exercise. These molecules are translated into precursor proteins for import into preexisting mitochondria. Once inside the organelle, the protein is processed to its mature form and either activates mitochondrial DNA gene expression, serves as a single subunit enzyme, or is incorporated into multi-subunit complexes of the respiratory chain devoted to electron transport and substrate oxidation. The result of this exercise-induced sequence of events is the expansion of the mitochondrial network within muscle cells and the capacity for aerobic ATP provision. An understanding of the molecular processes involved in this complex pathway of organelle synthesis is important for therapeutic purposes, and is a primary research undertaking in laboratories involved in the study of mitochondrial biogenesis. This pathway in muscle becomes impaired with chronic inactivity and aging, which leads to a reduced muscle aerobic capacity and an increased tendency for mitochondrially mediated apoptosis, a situation that can contribute to muscle atrophy. The resumption, or adoption, of an active lifestyle can ameliorate this metabolic dysfunction, improve endurance, and help maintain muscle mass.
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Barros, Camila D. S., Jomênica B. Livramento, Margaret G. Mouro, Elisa Mieko Suemitsu Higa, Carlos T. Moraes, and Celia Harumi Tengan. "L-Arginine Reduces Nitro-Oxidative Stress in Cultured Cells with Mitochondrial Deficiency." Nutrients 13, no. 2 (February 6, 2021): 534. http://dx.doi.org/10.3390/nu13020534.
Full textAbstract:
L-Arginine (L-ARG) supplementation has been suggested as a therapeutic option in several diseases, including Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like syndrome (MELAS), arguably the most common mitochondrial disease. It is suggested that L-ARG, a nitric oxide (NO) precursor, can restore NO levels in blood vessels, improving cerebral blood flow. However, NO also participates in mitochondrial processes, such as mitochondrial biogenesis, the regulation of the respiratory chain, and oxidative stress. This study investigated the effects of L-ARG on mitochondrial function, nitric oxide synthesis, and nitro-oxidative stress in cell lines harboring the MELAS mitochondrial DNA (mtDNA) mutation (m.3243A>G). We evaluated mitochondrial enzyme activity, mitochondrial mass, NO concentration, and nitro-oxidative stress. Our results showed that m.3243A>G cells had increased NO levels and protein nitration at basal conditions. Treatment with L-ARG did not affect the mitochondrial function and mass but reduced the intracellular NO concentration and nitrated proteins in m.3243A>G cells. The same treatment led to opposite effects in control cells. In conclusion, we showed that the main effect of L-ARG was on protein nitration. Lowering protein nitration is probably involved in the mechanism related to L-ARG supplementation benefits in MELAS patients.
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Puy, Hervé, Karim Zoubida, Lyoumi Said, Lydie M. Da Costa, and Gouya Laurent. "Heme-Related Blood Disorders." Blood 122, no. 21 (November 15, 2013): SCI—18—SCI—18. http://dx.doi.org/10.1182/blood.v122.21.sci-18.sci-18.
Full textAbstract:
Abstract Heme biosynthesis in erythroid cells is intended primarily for the formation of hemoglobin. As in every cell, this synthesis requires a multi-step pathway that involves eight enzymes including the erythroid-specific δ-aminolevulinate synthase (ALAS2, the first regulated enzyme that converts glycine and succinyl CoA into ALA) and the ubiquitous ferrochelatase (FECH, the final enzyme). Heme biosynthesis also requires membrane transporters that are necessary to translocate glycine, precursors of heme, and heme itself between the mitochondria and the cytosol. Defects in normal porphyrin and/or heme synthesis and transport cause four major erythroid inherited disorders, which may or may not be associated with dyserythropoiesis (e.g., sideroblastic, microcytic anemia and/or hemolytic anemia): "X-linked" sideroblastic anemia (XLSA) and X-linked dominant protoporphyria (XLDPP) are two different and opposing disorders but related to altered gene encoding ALAS2 only. Defective activity of this enzyme due to mutations in the ALAS2 gene causes the XLSA phenotype, including microcytic, hypochromic anemia with abundant ringed sideroblasts in the bone marrow. Vice versa, gain-of-function mutations of ALAS2 are responsible of the XLDPP characterized by predominant accumulation of the hydrophobic protoporphyrin (PPIX, the last heme precursor) in the erythrocytes without anemia or sideroblasts. Furthermore, the glycine transporter (SLC25A38) and Glutaredoxin 5 genes are reported to be involved in human non-syndromic sideroblastic anemia. Congenital erythropoietic porphyria (CEP) is the rarest autosomal recessive disorder due to a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. CEP leads to excessive synthesis and accumulation of type I isomers of porphyrins in the reticulocytes, followed by intravascular hemolysis and severe anemia. The ALAS2 gene may act as a modifier gene in CEP patients (Figueras J et al, Blood. 2011;118(6):1443-51). Erythropoietic protoporphyria (EPP) results from a partial deficiency of FECH and leads similarly to XLDPP, to deleterious accumulation of PPIX in erythroid cells. Most EPP patients present intrans to a FECH gene mutation an IVS3-48C hypomorphic allele due to a splice mutation. Abnormal spliced mRNA is degraded which contributes to the lowest FECH enzyme activity and allowed EPP phenotype expression. We have identified an antisense oligonucleotide (ASO) to redirect splicing from cryptic to physiological site and showed that the ASO-based therapy mediates normal splice rescue of FECH mRNA and reduction by 60 percent of PPIX overproduction in primary cultures of EPP erythroid progenitors. Therapeutic approaches to target both ALAS2 inhibition and heme-level reduction may be useful in other erythroid disorders such as thalassemia (where reduced heme biosynthesis was shown to improve the clinical phenotype) or the Diamond-Blackfan anemia (DBA). Indeed, in some DBA patients, an unusual mRNA splicing of heme exporter FLVCR has been found, reminiscent of Flvcr1-deficient mice that develop a DBA-like phenotype with erythroid heme accumulation. Thus, FLVCR may act as a modifier gene for DBA phenotypic variability. Recent advances in understanding the pathogenesis and molecular genetic heterogeneity of heme-related disorders have led to improved diagnosis and treatment. These advances include DNA-based diagnoses for all the porphyrias and some porphyrins and heme transporters, new understanding of the pathogenesis of the erythropoietic disorders, and new and experimental treatments such as chronic erythrocyte transfusions, bone marrow or hematopoietic stem cell transplants, and experimental pharmacologic chaperone and stem cell gene therapies for erythropoietic porphyrias. Disclosures: No relevant conflicts of interest to declare.
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Hegedus, Dwayne D., and George G. Khachatourians. "Isolation and characterization of conditional lethal mutants of Beauveria bassiana." Canadian Journal of Microbiology 40, no. 9 (September 1, 1994): 766–76. http://dx.doi.org/10.1139/m94-121.
Full textAbstract:
Temperature-sensitive mutants of Beauveria bassiana GK2016 were isolated and characterized. Heat-sensitive mutants that grew at 20 °C but not at 30 °C were generated using mutagenesis with ultraviolet light and several rounds of selection. After 2160 colonies from a heat-sensitive mutant enriched population were screened, 11 heat-sensitive strains were isolated for further study. Five mutant strains, HS1, HS2, HS6, HS9, and HS11, were stable and closely resembled the wild-type strain with respect to morphology, growth rate, and enzyme synthesis at 20 °C. Characterization of macromolecular synthesis at 30 °C using a radiolabelled precursor uptake assay indicated that three mutants, HS6, HS9, and HS11, had reduced levels of DNA, RNA, and protein synthesis at the nonpermissive temperature. Mutants affected in cell division and cell wall synthesis were characterized by microscopy. At 30 °C, mutant HS6 was defective in cellular compartmentalization and formed long, branched, aseptate mycelia that fragmented easily. Mutant HS1 was defective in cell wall biosynthesis and at 30 °C ceased to maintain cell wall integrity and lysed. Mutants HS2 and HS9 possessed temperature-sensitive lesions that could not be specified. None of the mutants were directly affected in either nuclear division or mitosis as evidenced by the accumulation of Giemsa-stainable mitotic nuclei at 30 °C. Bioassays conducted with grasshoppers showed the efficacy of the mutant strains, with the exception of HS11, to be comparable to that of the parent strain, at 20 °C. Conversely, at 32 °C the mutant strains were uninfective whereas the parental strain GK2016 was infective. It is our intention to use these strains to examine fundamental aspects of entomopathology by dissecting fungal growth and development in vitro and extending these observations to pathogenesis in insects.Key words: Beauveria bassiana, temperature-sensitive, mutants, characterization, infection.
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Mier, James W., John Przygoda, Mark Allegretta, Peeter A. Poldre, Ruth B. Kundsin, Richard A. Rudders, and Thomas W. North. "Effects of Deoxycytidine on Mycoplasma-Associated Inhibition of Thymidine Incorporation and Growth in Antifolate-Containing Media." International Journal of Immunopathology and Pharmacology 1, no. 2 (May 1988): 123–37. http://dx.doi.org/10.1177/039463208800100205.
Full textAbstract:
Several mycoplasma species markedly inhibit lymphokine- and mitogen-induced 3H-thymidine incorporation in cultured lymphoid cells, but have negligible short-term effects on cellular DNA synthesis as assessed by cytofluorography or by cell counts. The deoxyribonucleotide precursor deoxycytidine (dC) reverses this inhibition, but has little effect on isotope incorporation in uninfected cultures. Human lymphoblastoid leukemia cell lines contaminated with mycoplasma and hypoxanthine guanosine phosphoribosyl transferase (HGPRT)-deficient subclones do not grow in conventional HAT medium, but the unselected parent lines proliferate when dC is included in the culture medium. The beneficial effect of dC on the growth of contaminated cultures in selection medium is amplified by the addition of the cytidine deaminase inhibitor tetrahydrouridine (THU). These observations and corroborating nucleotide pool analysis suggest that dC may exert its beneficial effects on cellular proliferation and isotope utilization by inhibiting a mycoplasma-associated enzyme, thymidine phosphorylase. The data also suggest that the conversion of dC to dU by the cellular enzyme cytidine deaminase reduces the ability of dC to salvage contaminated cultures in the presence of an antifolate. The addition of dC to the culture medium in various 3H-thymidine incorporation assays makes possible the detection of stimulatory lymphokines despite the presence of mycoplasma contamination of the indicator cells. The normalization of nucleotide pools and cellular growth of mycoplasma-infected HGPRT (+) human leukemic cell lines with the addition of dC to HAT selection medium has made possible the use of infected HGPRT-deficient subclones as fusion partners in the generation of T-T hybridomas. Our studies also suggest that the ability of cells to grow in HAT medium only when dC is included is presumptive evidence for mycoplasma infection.
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Ganguly, Shinjini, Aysegul Balyimez, Zaeem Lone, Aimalie Hardaway, Mona Patel, Elai Davicioni, Moshe Chaim Ornstein, et al. "Tumor cell intrinsic androgen biosynthesis by 3β-hydroxy steroid dehydrogenase (HSD3B1) to modulate radiosensitivity in prostate cancer cells." Journal of Clinical Oncology 38, no. 6_suppl (February 20, 2020): 349. http://dx.doi.org/10.1200/jco.2020.38.6_suppl.349.
Full textAbstract:
349 Background: Resistance to ADT is associated with a gain of function mutation in the 3β-HSD enzyme, which catalyzes extragonadal/intratumoral DHT synthesis. As androgen signaling is known to upregulate the DNA damage response (DDR), we investigated whether HSD3B1 genotype modulates DDR and radiosensitivity in PCa. Methods: We stably knocked down HSD3B1 in LNCaP, C42 and VCaP cell lines (which carry the protein stabilizing variant allele) and overexpressed the variant HSD3B1 allele in LAPC4 (harbors a WT allele which readily undergoes degradation). We examined the proliferative and clonogenic capacity of these cells in presence and absence of substrate, DHEA, followed by treatment with IR (400-800 cGy, single fraction). We studied DNA DSB formation and resolution kinetics using γH2AX foci formation in response to radiation. We also measured changes in mRNA expression of DDR response genes pre- and post-radiation. Results: Control shRNA transduced cell lines had increased cell proliferation (p<0.001) and clonogenic survival (2 logs at 800cGy single fraction radiation, p<0.001) in the presence of DHEA compared to HSD3B1 knockdown cells. Variant HSD3B1 cell lines were more radioresistant and exhibited more efficient γH2AX foci resolution at 24 hrs (p <0.05) in a DHEA dependent manner. We observe increased mRNA expression of DDR genes from specific repair networks including non-homologous end joining (PRKDC, XRCC4, XRCC5) and homologous recombination (RAD51, RAD54) in variant HSD3B1 cells. Transcriptional induction of DDR genes following radiation in presence of DHEA was significantly more pronounced in HSD3B1 variant cells, suggesting a more permissive chromatin context. Conclusions: Increased intracellular 3β-HSD drives transcription of NHEJ and HR genes, more rapid resolution of γH2AX foci, and radioresistance in prostate cancer. This work has therapeutic implications related to strategies for combined radiation and androgen directed therapy in localized and metastatic prostate cancer. Prospective validation of treatment strategies combining blockade of adrenal steroid precursor synthesis, ADT, and XRT in high risk disease is warranted.
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Pal, Sharmistha, Jakub Kaplan, Sylwia Stopka, Michael Regan, Benjamin Kann, Nathalie Agar, Charles Stiles, et al. "HGG-38. DE NOVO PYRIMIDINE SYNTHESIS INHIBITION INDUCES REPLICATION CATASTROPHE MEDIATED CELL DEATH IN DIFFUSE MIDLINE GLIOMA." Neuro-Oncology 23, Supplement_1 (June 1, 2021): i25. http://dx.doi.org/10.1093/neuonc/noab090.102.
Full textAbstract:
Abstract Diffuse midline gliomas (DMG) are aggressive and lethal pediatric brain tumors that cannot be cured by conventional therapeutic modalities. Using a genome wide CRISPR screen we identified the de novo pyrimidine biosynthesis pathway as a metaboilic vulnerability in DMGs. BAY2402234 is a small molecule inhibitor of DHODH -a rate liminting enzyme in the de novo pyrimidine biosynthesis pathway. BAY2402234 induces cell death in DMG cells at low nanomolar concentrations while sparing adult glioblastoma cells and normal astrocytes. Further investigations revealed drammatic reduction in cellular UMP pools, the precursor for all pyrimidine nucleotides, after DHODH inhibition, specifically in DMG cells. Cytotoxicity of DHODH inhibition in DMG cells is rescued by exogenous uridine, supporting UMP depletion as the mechanism underlying DMG cell death and also showing that cell death is an “on target” response to BAY2402234. Cell death induced by BAY2402234 is a consequence of replication fork stalling as evident by accumulation of chromatin-bound RPA foci and g-H2AX. Stalled replication forks eventually collapse, resulting in replication catastrophy and apoptosis. Cytotoxic effects of DHODH inhibition are further exacerbated by inhibition of the intra-S checkpoint protein, ATR. Combined treatment of DMG cells with DHODH and ATR inhibitors resulted in enhanced accumulation of chromatin-bound RPA, g-H2AX, replication fork collapse and apoptosis. Importantly, in vivo studies verify that both BAY2402234 (DHODHi), and BAY1895344 (ATRi), cross the blood-brain barrier, accumulate in the brain at therapeutically relevant concentrations, and induce DNA damage in intracranial DMG xenografts in mice. Taken together, our studies have identified DHODH inhibition as a DMG-specific vulnerability resulting in cell death; the mechanism of DHODHi-induced cell death led us to identify combined inhibition of DHODH and ATR as a synergistic therapy against DMG tumors.
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Mousson de camaret, Bénédicte, Jan-Willem Taanman, Sylvie Padet, Maïté Chassagne, Martine Mayençon, Pascale Clerc-Renaud, Ginette Mandon, Marie-Thérèse Zabot, Alain Lachaux, and Dominique Bozon. "Kinetic properties of mutant deoxyguanosine kinase in a case of reversible hepatic mtDNA depletion." Biochemical Journal 402, no. 2 (February 12, 2007): 377–85. http://dx.doi.org/10.1042/bj20060705.
Full textAbstract:
DGUOK [dG (deoxyguanosine) kinase] is one of the two mitochondrial deoxynucleoside salvage pathway enzymes involved in precursor synthesis for mtDNA (mitochondrial DNA) replication. DGUOK is responsible for the initial rate-limiting phosphorylation of the purine deoxynucleosides, using a nucleoside triphosphate as phosphate donor. Mutations in the DGUOK gene are associated with the hepato-specific and hepatocerebral forms of MDS (mtDNA depletion syndrome). We identified two missense mutations (N46S and L266R) in the DGUOK gene of a previously reported child, now 10 years old, who presented with an unusual revertant phenotype of liver MDS. The kinetic properties of normal and mutant DGUOK were studied in mitochondrial preparations from cultured skin fibroblasts, using an optimized methodology. The N46S/L266R DGUOK showed 14 and 10% residual activity as compared with controls with dG and deoxyadenosine as phosphate acceptors respectively. Similar apparent negative co-operativity in the binding of the phosphate acceptors to the wild-type enzyme was found for the mutant. In contrast, abnormal bimodal kinetics were shown with ATP as the phosphate donor, suggesting an impairment of the ATP binding mode at the phosphate donor site. No kinetic behaviours were found for two other patients with splicing defects or premature stop codon. The present study represents the first characterization of the enzymatic kinetic properties of normal and mutant DGUOK in organello and our optimized protocol allowed us to demonstrate a residual activity in skin fibroblast mitochondria from a patient with a revertant phenotype of MDS. The residual DGUOK activity may play a crucial role in the phenotype reversal.
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Kumari, Ashu, Laura A. Richards, and Karen L. MacKenzie. "Downregulation of Dyskerin Impairs Expansion of Normal Hematopoietic Progenitors and Erythroid Colony Formation in the Absence of Apparent Telomere Shortening." Blood 124, no. 21 (December 6, 2014): 1601. http://dx.doi.org/10.1182/blood.v124.21.1601.1601.
Full textAbstract:
Abstract Dyskerin is an RNA binding protein that functions as a core component of the telomerase holoenzyme as well as in ribonuclear protein complexes involved in ribosome biogenesis and RNA processing. Within the telomerase holoenzyme, dyskerin stabilizes the telomerase RNA component (hTR) that functions as a template for synthesis of telomeric DNA at chromosome ends. Telomerase is active in malignant cells as well as normal hematopoietic stem and progenitor cells where it enables long-term cell replication. Mutation of the gene encoding dyskerin (DKC1) is the underlying cause of the inherited disease dyskeratosis congenita (DC), which is characterized by low levels of telomerase activity and shortened telomeres. Bone marrow failure is the most common cause of mortality in DC, however since dyskerin has telomerase-independent roles in fundamental cellular processes, it is not clear whether telomerase insufficiency is the sole cause of hematopoietic dysfunction in this disorder. To gain further insight to the consequences of deregulated expression of dyskerin in haematopoietic cells, we transduced normal human cord blood CD34+ progenitor cells with retroviral vectors encoding two different shRNAs targeting dyskerin. Transduced cells from seven independent experiments were FACS sorted then assayed for expansion and differentiation in vitro, clonogenic potential, telomerase activity and telomere length maintenance. Results from the functional assays showed that depletion of dyskerin impaired cell expansion and compromised the formation of BFU-E colonies. In contrast to BFU-E, CFU-GM colonies did not appear to be altered by dyskerin depletion. Analysis of non-transuduced cells subject to ex vivo expansion along erythroid and myelomonocytic lineages revealed that erythroid precursor cells expressed disproportionately high levels of dyskerin, and correspondingly high telomerase enzyme activity. shRNA-mediated downregulation of dyskerin lowered hTR levels and quenched telomerase activity in the erythroid precursors without altering expression of the telomerase catalytic component, hTERT. These results suggest that upregulation of dyskerin bolsters telomerase activity and renders erythroid precursors vulnerable to dyskerin depletion. However in spite of the reduction in telomerase activity induced by dyskerin shRNA, Southern blot analysis showed that the rate of telomere length shortening was not significantly altered by dyskerin depletion over the time course of these experiments. Together the data show that dyskerin is upregulated during erythroid differentiation, and has an essential function in the erythroid lineage that appears to be independent of its role in telomere length maintenance. Disclosures No relevant conflicts of interest to declare.
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ROCH, Anne-Marie, Gerard QUASH, Yvonne MICHAL, Jacqueline CHANTEPIE, Bernard CHANTEGREL, Christian DESHAYES, Alain DOUTHEAU, and Jacqueline MARVEL. "Altered methional homoeostasis is associated with decreased apoptosis in BAF3 bcl2 murine lymphoid cells." Biochemical Journal 313, no. 3 (February 1, 1996): 973–81. http://dx.doi.org/10.1042/bj3130973.
Full textAbstract:
Methional is a potent inducer of apoptosis in an interleukin 3-dependent murine lymphoid cell line BAF3 b0 when it is added to the culture medium. In these cells transfected with the bcl2 gene, BAF3 bcl2, the apoptotic-inducing activity of methional is dramatically reduced. The addition of disulfiram (an inhibitor of aldehyde dehydrogenase) in order to reduce methional oxidation brought about an increase in apoptosis in BAF3 b0 but not in BAF3 bcl2 cells. In contrast, the addition of quercetin (an inhibitor of aldehyde reductase) in an attempt to diminish methional reduction increased apoptosis in both BAF3 b0 and BAF3 bcl2 cells. The extent of DNA fragmentation in BAF3 bcl2 cells approached that in BAF3 b0 cells in the presence of quercetin and exogenous methional, suggesting a defect in methional biosynthesis in BAF3 bcl2 cells. Direct evidence for this was obtained by measuring labelled methional in cells incubated with the sodium salt of [U-14C]4-methylthio-2-oxobutanoic acid (MTOB), the precursor of methional. The 80% decrease in labelled methional in BAF3 bcl2 compared with BAF3 b0 cells was accompanied by a concomitant rise in the transamination of [14C]MTOB to [14C]methionine in BAF3 bcl2 cells. Inhibition of the transaminase, however, by a synthetic transition-state-type compound, pyridoxal-L-methionine ethyl ester, induced apoptosis in BAF3 b0 but not in BAF3 bcl2 cells, confirming that the defect in BAF3 bcl2 cells was not in the transaminase itself but rather in the oxidative decarboxylation step MTOB →methional. In addition, no evidence was obtained for the synthesis of [14C]malondialdehyde from [14C]methional in BAF3 bcl2 cells. As these cells show no deficiency in their content of reactive oxygen species compared with that of BAF3 b0 cells, they may possess some other defect in the β-hydroxylase enzyme system itself.
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Compagnone, Nathalie A., Peilin Zhang, Jean-Louis Vigne, and Synthia H. Mellon. "Novel Role for the Nuclear Phosphoprotein SET in Transcriptional Activation of P450c17 and Initiation of Neurosteroidogenesis." Molecular Endocrinology 14, no. 6 (June 1, 2000): 875–88. http://dx.doi.org/10.1210/mend.14.6.0469.
Full textAbstract:
Abstract Neurosteroids are important endogenous regulators of γ-aminobutryic acid (GABAA) and N-methyl-d-aspartate (NMDA) receptors and also influence neuronal morphology and function. Neurosteroids are produced in the brain using many of the same enzymes found in the adrenal and gonad. The crucial enzyme for the synthesis of DHEA (dehydroepiandrosterone) in the brain is cytochrome P450c17. The transcriptional strategy for the expression of P450c17 is clearly different in the brain from that in the adrenal or gonad. We previously characterized a novel transcriptional regulator from Leydig MA-10 cells, termed StF-IT-1, that binds at bases −447/−399 of the rat P450c17 promoter, along with the known transcription factors COUP-TF (chicken ovalbumin upstream promoter transcription factor), NGF-IB (nerve growth factor inducible protein B), and SF-1 (steroidogenic factor-1). We have now purified and sequenced this protein from immature porcine testes, identifying it as the nuclear phosphoprotein SET; a role for SET in transcription was not established previously. Binding of bacterially expressed human and rat SET to the DNA site at −418/−399 of the rat P450c17 gene transactivates P450c17 in neuronal and in testicular Leydig cells. We also found SET expressed in human NT2 neuronal precursor cells, implicating a role in neurosteroidogenesis. Immunocytochemistry and in situ hybridization in the mouse fetus show that the ontogeny and distribution of SET in the developing nervous system are consistent with SET being crucial for initiating P450c17 transcription. SET’s developmental pattern of expression suggests it may participate in the early ontogenesis of the nervous, as well as the skeletal and hematopoietic, systems. These studies delineate an important new factor in the transcriptional regulation of P450c17 and consequently, in the production of DHEA and sex steroids.
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Zhou, MX, HW Findley, LH Ma, SR Zaki, T. Hill, M. Hamid, WC Hooper, and AH Ragab. "Effect of tumor necrosis factor-alpha on the proliferation of leukemic cells from children with B-cell precursor-acute lymphoblastic leukemia (BCP-ALL): studies of primary leukemic cells and BCP-ALL cell lines." Blood 77, no. 9 (May 1, 1991): 2002–7. http://dx.doi.org/10.1182/blood.v77.9.2002.2002.
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Abstract The effect of recombinant tumor necrosis factor-alpha (rTNF-alpha) on the primary leukemic blasts and leukemic cell lines derived from children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) was studied. The proliferation of leukemic cells from the bone marrow of 11 of 13 patients (seven at diagnosis, four in relapse) and from the 697 (BCP-ALL) cell line was significantly inhibited by rTNF-alpha at the lowest dose tested (0.1 ng/mL), as measured by 3H-TdR uptake. The degree of inhibition was variable, ranging from 17% to 78%. Furthermore, a dose-dependent inhibitory effect was observed, with approximately 70% mean inhibition of DNA synthesis detected when cells from 12 of 13 patients were incubated with 100 ng/mL of rTNF-alpha for 3 days. In contrast, rTNF-alpha did not inhibit another BCP-ALL cell line (EU-1/ALL) established recently in our laboratory. Studies indicated that the TNF-alpha gene was expressed by the primary leukemic blasts of one TNF-resistant case in his third relapse and by EU-1 cells. Also, TNF-alpha protein was detected by Western blot analysis and enzyme-linked immunoabsorbent assay in the supernatant of EU-1 cells; this is the first report of TNF production by a BCP-ALL cell lines. The production of TNF-alpha mRNA and protein was not detected in the 697 cell line and in the primary leukemic blasts from six patients (four at diagnosis, two in relapse) whose leukemic cells were inhibited by TNF. The partially purified TNF-alpha obtained from the EU-1 cell line also suppressed the proliferation of TNF-sensitive primary leukemic cells, and this inhibitory activity was abolished by an anti- TNF-alpha specific antibody. Our results demonstrate that TNF-alpha is an inhibitor of in vitro proliferation of BCP-ALL cells from most patients. The TNF-resistant leukemic cells from a few patients and the EU-1 cell line express TNF mRNA, suggesting that the induction of TNF gene expression is associated with the development of TNF resistance.
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Zhou, MX, HW Findley, LH Ma, SR Zaki, T. Hill, M. Hamid, WC Hooper, and AH Ragab. "Effect of tumor necrosis factor-alpha on the proliferation of leukemic cells from children with B-cell precursor-acute lymphoblastic leukemia (BCP-ALL): studies of primary leukemic cells and BCP-ALL cell lines." Blood 77, no. 9 (May 1, 1991): 2002–7. http://dx.doi.org/10.1182/blood.v77.9.2002.bloodjournal7792002.
Full textAbstract:
The effect of recombinant tumor necrosis factor-alpha (rTNF-alpha) on the primary leukemic blasts and leukemic cell lines derived from children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) was studied. The proliferation of leukemic cells from the bone marrow of 11 of 13 patients (seven at diagnosis, four in relapse) and from the 697 (BCP-ALL) cell line was significantly inhibited by rTNF-alpha at the lowest dose tested (0.1 ng/mL), as measured by 3H-TdR uptake. The degree of inhibition was variable, ranging from 17% to 78%. Furthermore, a dose-dependent inhibitory effect was observed, with approximately 70% mean inhibition of DNA synthesis detected when cells from 12 of 13 patients were incubated with 100 ng/mL of rTNF-alpha for 3 days. In contrast, rTNF-alpha did not inhibit another BCP-ALL cell line (EU-1/ALL) established recently in our laboratory. Studies indicated that the TNF-alpha gene was expressed by the primary leukemic blasts of one TNF-resistant case in his third relapse and by EU-1 cells. Also, TNF-alpha protein was detected by Western blot analysis and enzyme-linked immunoabsorbent assay in the supernatant of EU-1 cells; this is the first report of TNF production by a BCP-ALL cell lines. The production of TNF-alpha mRNA and protein was not detected in the 697 cell line and in the primary leukemic blasts from six patients (four at diagnosis, two in relapse) whose leukemic cells were inhibited by TNF. The partially purified TNF-alpha obtained from the EU-1 cell line also suppressed the proliferation of TNF-sensitive primary leukemic cells, and this inhibitory activity was abolished by an anti- TNF-alpha specific antibody. Our results demonstrate that TNF-alpha is an inhibitor of in vitro proliferation of BCP-ALL cells from most patients. The TNF-resistant leukemic cells from a few patients and the EU-1 cell line express TNF mRNA, suggesting that the induction of TNF gene expression is associated with the development of TNF resistance.
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Newton, CJ, G. Ran, YX Xie, D. Bilko, CH Burgoyne, I. Adams, A. Abidia, PT McCollum, and SL Atkin. "Statin-induced apoptosis of vascular endothelial cells is blocked by dexamethasone." Journal of Endocrinology 174, no. 1 (July 1, 2002): 7–16. http://dx.doi.org/10.1677/joe.0.1740007.
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Statins block de novo synthesis of cholesterol by inhibiting the enzyme, HMG CoA reductase. The product of this reaction, mevalonic acid, is also a precursor of isoprenoids, molecules required for the activation of signalling G-proteins, such as Ras. Signal transduction pathways involving Ras are important for cell survival and this may be why statins induce apoptotic death of several cell types. Given that statins are used to treat vascular disease, it is surprising that no studies have been conducted on vascular endothelial cells. For this reason, we have tested the effect of fluvastatin (FS) on the endothelial cell line EA.hy 926. Here we show that FS, at concentrations from 1 to 2 microM, blocks growth and induces apoptosis of the endothelial cell line, EA.hy 926. As considerable redundancy exists in cell signalling pathways for cell survival, toxicity of FS under more physiological conditions might be prevented by pathways that do not require Ras, such as those activated by adrenal or sex steroids. To test this hypothesis, first RT-PCR analysis was performed for nuclear receptor mRNA expression. This revealed the presence of mRNA for the androgen receptor (AR) and glucocorticoid receptor (GR). The effect of the AR agonist, dihydrotestosterone (DHT), and the GR agonist, dexamethasone (Dex), was then tested. Whilst DHT (100 nM) had no effect on FS-induced cell death, Dex (1 microM) blocked FS-induced apoptosis. Cell cycle analysis revealed that 24 h exposure to FS prevented cells from leaving G(1) and 24-48 h later a marked sub-G(1) peak was observed. Dex was able to reduce the sub-G(1) peak, but it failed to reduce accumulation of cells in G(1). Further studies revealed that, in addition to blocking FS-induced apoptosis, Dex was able to block apoptosis of EA.hy 926 cells induced by serum deprivation, tumour necrosis factor-alpha, oxidants, DNA damage and mitochondrial disruption. This study strongly suggests that glucocorticoids have a role to play in preventing vascular injury and they may provide a reason why statins are apparently not toxic to vascular endothelial cells in vivo.
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Leclerc, Guy J., and Julio C. Barredo. "Histone Deacetylase Inhibitor Induces FPGS mRNA Expression in Childhood B-Precursor and T Acute Lymphoblastic Leukemia: Implication of Combination Therapy with Methotrexate To Enhance Cytotoxicity." Blood 110, no. 11 (November 16, 2007): 864. http://dx.doi.org/10.1182/blood.v110.11.864.864.
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Abstract Methotrexate (MTX) is an antifolate widely used to treat childhood acute lymphoblastic leukemia (ALL). MTX is retained within cells as long-chain polyglutamates (MTX-PGs), after metabolism by the enzyme folylpoly-γ-glutamate synthetase (FPGS). Intracellular retention of MTX-PGs results in enhanced cytotoxicity due to prolonged inhibition of dihydrofolate reductase (DHFR), and the additional inhibition of thymidylate synthetase (TS). The FPGS gene was shown to be regulated by the transcription factors Sp1 and NFY. We performed DNaseI hypersensitive assays and identified a hypersensitive site mapping closely upstream of exon 1 suggesting that chromatin remodeling may contribute to FPGS gene regulation. Using co-immunoprecipitation and Western blotting we investigated the role of histone modifications and chromatin remodeling on the expression of FPGS and uncovered interactions between NFY, Sp1 and HDAC1. Our results demonstrate that HDAC1 complexes with NFY and Sp1 transcription factors in both B- and T-ALL cells. DNA affinity precipitation assay (DAPA) revealed that the HDAC1-NFY and HDAC1-Sp1 complex binds to the NFY and Sp1 binding sites in the FPGS promoter. These findings suggest that transcription of the FPGS gene may be regulated by acetylation of NFY and Sp1 factors and interaction with HDAC1, and/or chromatin remodeling. We then examined the effect of the histone deacetylase inhibitor (HDACi) sodium butyrate (NaBu) on the expression of FPGS and other folate-related genes. The level of FPGS, ATP-binding cassette subfamily C (ABCC1), ATP-binding cassette subfamily G (ABCG2), DHFR, γ-glutamyl hydrolase (GGH), solute carrier family 19/folate transporter (SLC19A1), and TS mRNA gene expression was determined by qRT-PCR in NALM6 (Bp-ALL), REH (Bp-ALL, t(12,21)/TEL-AML1), SupB15 (Bp-ALL, t(9,22)/BCR-ABL), and CCRF-CEM (T-ALL) cells treated with NaBu [2mM-5mM]. In all cell lines examined, treatment with NaBu induced 2- to 5-fold the level of FPGS and ABCC1 mRNA expression whereas the level of DHFR, SLC19A1, and TS mRNA expression was decreased. Expression of GGH and ABCG2 mRNAs was increased 2-fold in CCRF-CEM but remained unaltered in Bp-ALL NaBu treated cells. Promoters of butyrate-responsive genes have been shown to contain genetic elements such as Sp1/Sp3 binding sites which interact with HDAC1 to mediate the action of NaBu. On this basis, we hypothesized that pre-treatment of ALL cells with NaBu should lead to induction of FPGS expression and subsequently, higher synthesis of MTX-PG and enhanced MTX cytotoxicity in ALL cells. To test this hypothesis, CCRF-CEM, NALM6, REH, and SupB15 cells were treated sequentially with NaBu (24h) and MTX (4h), and assessed for cell viability. Treatment of NaBu and MTX increased cell death by ∼40% in NALM6, REH, and SupB15 Bp-cells, and ∼60% in CCRF-CCEM cells when compared to treatment with each drug alone. These data suggest that combination of HDACi and MTX may represent a novel therapeutic strategy for treatment of ALL. This strategy may be particularly useful to overcome MTX resistance in patients diagnosed with phenotypes that accumulate low levels of MTX-PGs and for patients after relapse.
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Dashnamoorthy, Ravi, Afshin Beheshti, Xiaoyang Su, Ying Chen, Maisarah Mokhtar, Gregory Dolnikowski, Frederick Lansigan, William B. Kinlaw, Sandeep Dave, and Andrew M. Evens. "Identification of FASN-Dependent Onco-Metabolic Regulation of the Pentose Phosphate Pathway (PPP) and Nucleotide Metabolism in Non-Hodgkin Lymphoma (NHL)." Blood 134, Supplement_1 (November 13, 2019): 1573. http://dx.doi.org/10.1182/blood-2019-126382.
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Introduction: FASN catalyzes de novo fatty acid (FA) biosynthesis, which is an oncogenic function observed in many cancers, including NHL. HIF1a inducible FASN activity is responsible for overcoming negative hypoxic influence and upregulation of glucose metabolism as observed during premalignant transformation. FASN catalytic activity is also dependent on precursors derived from glucose metabolism. However, implications of FASN targeted therapies on these interdependent metabolic interactions and the overall impact on cancer cell proliferation remains unknown. Methods: FASN small molecule inhibitors cerulenin, orlistat, TVB3657 and TVB3166 were evaluated using a diverse panel of B cell NHL lines and primary NHL cells for the impact on FASN inhibition signaling & induction of cell death (MTT, caspases & AnnexinV/PI). Global transcriptomics were done with Affymetrix Human 2.0 ST Genechip with Gene Set Enrichment and Ingenuity Pathway Analysis (GSEA & IPA). Metabolomic profiling was performed using mass spectrometry. TXNRD1, GSR, NQO1 expression and activity were evaluated by western blot and using enzyme assay kits. Global DNA & RNA synthesis were evaluated using ClickIt Edu and EU assay kits. RNAseq data available from 775 NHL patients were utilized for metabolic transcriptome mapping. Results: Treatment with all FASN inhibitors resulted in dose- and time-dependent reduction (>90%) in cell viability and cell death in all NHL cell lines. GSEA and IPA identified cell cycle & RNA metabolism as downregulated biological processes with carbohydrate and oxidative stress as upregulated biological processes observed. "Key gene" analysis predicted TNF signaling as a prominent response to FASN inhibition, validated as increased TNF secretion by ELISA with cell fractionation and western blot analysis revealing activation of TNF-PI3K signaling. Activation of PI3K with FASN inhibition also corresponded with increased expression of PI3K-dependent metabolic genes associated with glucose and lipid metabolism. Furthermore, metabolomic profiling in SUDHL10 cells revealed accumulation of the FASN precursor acetyl-CoA with FASN inhibition that was accompanied by increased ketogenic glycolytic and citric acid cycle activity. In addition, NADPH accumulation (FASN substrate) occurred with FASN inhibition, which was accompanied with reduction in ribose-phosphate and nucleotide pools as these processes are relevant to NADPH-generating PPP function. G6PD, TXNRD1, GSR & NQO1 were identified as "key genes" responsive to FASN inhibition. Interestingly, this cluster of "key genes" represented the entire enzymatic activity related to the first rate-limiting step in PPP. Subsequently, we determined that increased NADP(H) pools were responsible for impaired NADPH regenerating functions of TXNRD1 and GSR antioxidant enzymes, with PI3K-dependent activation of NQO1 and uptake of glucose facilitating de novo glutathione synthesis, which substituted for the loss of antioxidant functions in SUDHL10, SUDHL4 and Raji cells. All responses were sensitive to inhibition by PI3K inhibition (e.g., BKM120) with co-targeting via FASN & PI3K inhibition resulting in markedly increased oxidative stress, loss of mitochondrial membrane potential & synergistic cell death in NHL cell lines and primary NHL cells. Finally, FASN inhibition was associated with reduction in ribo/deoxy-ribonucleotide pools that decreased global transcriptional activity (de novo RNA synthesis, by EU labeling) and replication (by EdU incorporation in DNA) (Fig 1). Analysis of RNAseq data and mapping of metabolic transcriptome from 772 NHL patients showed consistently elevated expression of genes related to glycolysis, citric acid cycle, fatty acid and nucleotide metabolism in patients with mutations in p53, MYC, BCL2, mTOR, MYD88, PIM2 & CREBP genes, suggesting that these onco-metabolic interactions may be important for lymphomagenesis. Conclusions: Taken together, FASN oncogenic activity appears to extend beyond de novo fatty acid biosynthesis, serving as a central onco-metabolic regulator of malignant cell proliferation vis-à-vis integrating glucose, nucleotides, and antioxidant metabolic functions in NHL. In addition, co-targeting FASN and PI3K induced synergistic cell death. Altogether, blocking FASN and the dependent onco-metabolic functions represent highly novel targets for therapeutic strategies in NHL. Disclosures Chen: Oncomics, LLC: Consultancy, Patents & Royalties. Dave:Data Driven Bioscience: Equity Ownership. Evens:Seattle Genetics: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Tesaro: Research Funding; Verastem: Consultancy, Honoraria.
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Piya, Sujan, Marla Weetall, Josephine Sheedy, Balmiki Ray, Huaxian Ma, Kensuke Kojima, Vivian Ruvolo, et al. "The Novel Dihydroorotate Dehydrogenase (DHODH) Inhibitor PTC299 Inhibit De Novo Pyrimidine Synthesis with Broad Anti-Leukemic Activity Against Acute Myeloid Leukemia." Blood 136, Supplement 1 (November 5, 2020): 8–9. http://dx.doi.org/10.1182/blood-2020-139940.
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Introduction: Acute myeloid leukemia (AML) is characterized by both aberrant proliferation and differentiation arrest at hematopoietic progenitor stages 1,2. AML relies upon de novo nucleotide synthesis to meet a dynamic metabolic landscape and to provide a sufficient supply of nucleotides and other macromolecules 3,4. Hence, we hypothesized that inhibition of de novo nucleotide synthesis would lead to depletion of the nucleotide pool and pyrimidine starvation in leukemic cells compared to their non-malignant counterparts and impact proliferative and differentiation inhibition pathways. PTC299 is an inhibitor of dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme for de novo pyrimidine nucleotide synthesis that is currently in a clinical trial for the treatment of AML. Aim: We investigated the pre-clinical activity of PTC299 against AML in primary AML blasts and cytarabine-resistant cell lines. To confirm that PTC299 effects are due to inhibition of de novo pyrimidine nucleotide synthesis for leukemic growth, we specifically tested the impact of uridine and orotate rescue. In addition, a comprehensive analysis of alteration of metabolic signaling in PI3K/AKT pathways, apoptotic signatures and DNA damage responses were analyzed by Mass cytometry based proteomic analysis (CyTOF) and immunoblotting. The potential clinical relevance of DHODH inhibition was confirmed in an AML-PDX model. Results: The IC50s for all tested cell lines (at 3 day) and primary blasts (at 5-7 day) were in a very low nanomolar range: OCI-AML3 -4.43 nM, HL60 -59.7 nM and primary samples -18-90 nM. Treatment of AML in cytarabine-resistant cells demonstrated that PTC299 induced apoptosis, differentiation, and reduced proliferation with corresponding increase in Annexin V and CD14 positive cells (Fig.1). PTC299-induced apoptosis and inhibition of proliferation was rescued by uridine and orotate. To gain more mechanistic insights, we used an immunoblotting and mass cytometry (CyTOF) based approach to analyze changes in apoptotic and cell signaling proteins in OCI-AML3 cells. Apoptotic pathways were induced (cleaved PARP, cleaved Caspase-3) and DNA damage responses (TP53, γH2AX) and the PI3/AKT pathway were downregulated in response to PTC299. In isogenic cell lines, p53-wildtype cells were sustained and an increased DNA damage response with corresponding increase in apoptosis in comparison to p53-deficient cells was shown. (Fig.2) In a PDX mouse model of human AML, PTC299 treatment improved survival compared to mice treated with vehicle (median survival 40 days vs. 30 days, P=0.0002) (Fig.3). This corresponded with a reduction in the bone marrow burden of leukemia with increased expression of differentiation markers in mice treated with PTC299 (Fig.3). Conclusion: PTC299 is a novel dihydroorotate dehydrogenase (DHODH) inhibitor that triggers differentiation, apoptosis and/or inhibition of proliferation in AML and is being tested in a clinical trials for the treatment of acute myeloid malignancies. Reference: 1. Thomas D, Majeti R. Biology and relevance of human acute myeloid leukemia stem cells. Blood 2017; 129(12): 1577-1585. e-pub ahead of print 2017/02/06; doi: 10.1182/blood-2016-10-696054 2. Quek L, Otto GW, Garnett C, Lhermitte L, Karamitros D, Stoilova B et al. Genetically distinct leukemic stem cells in human CD34- acute myeloid leukemia are arrested at a hemopoietic precursor-like stage. The Journal of experimental medicine 2016; 213(8): 1513-1535. e-pub ahead of print 2016/07/06; doi: 10.1084/jem.20151775 3. Villa E, Ali ES, Sahu U, Ben-Sahra I. Cancer Cells Tune the Signaling Pathways to Empower de Novo Synthesis of Nucleotides. Cancers (Basel) 2019; 11(5). e-pub ahead of print 2019/05/22; doi: 10.3390/cancers11050688 4. DeBerardinis RJ, Chandel NS. Fundamentals of cancer metabolism. Sci Adv 2016; 2(5): e1600200. e-pub ahead of print 2016/07/08; doi: 10.1126/sciadv.1600200 Disclosures Weetall: PTC Therapeutic: Current Employment. Sheedy:PTC therapeutics: Current Employment. Ray:PTC Therapeutics Inc.: Current Employment. Konopleva:Genentech: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Ablynx: Research Funding; Ascentage: Research Funding; Agios: Research Funding; Kisoji: Consultancy; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; AbbVie: Consultancy, Research Funding; Calithera: Research Funding; Cellectis: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Sanofi: Research Funding. Andreeff:Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees. Borthakur:BioLine Rx: Consultancy; BioTherix: Consultancy; Nkarta Therapeutics: Consultancy; Treadwell Therapeutics: Consultancy; Xbiotech USA: Research Funding; Polaris: Research Funding; AstraZeneca: Research Funding; BMS: Research Funding; BioLine Rx: Research Funding; Cyclacel: Research Funding; GSK: Research Funding; Jannsen: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Incyte: Research Funding; PTC Therapeutics: Research Funding; FTC Therapeutics: Consultancy; Curio Science LLC: Consultancy; PTC Therapeutics: Consultancy; Argenx: Consultancy; Oncoceutics: Research Funding.
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Bornemann, Claus, and Hartmut Follmann. "Deoxyribonucleotide Synthesis in Phycovirus-Infected Green Algae. A New Virus-Induced Ribonucleotide Reductase." Zeitschrift für Naturforschung C 48, no. 1-2 (February 1, 1993): 113–18. http://dx.doi.org/10.1515/znc-1993-1-222.
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Infection of Chlorella-like green algae with freshwater phycoviruses is associated with a large and rapid demand for DNA precursors which cannot be met by the algal deoxyribonucleotide-synthesizing enzymes. We have demonstrated in these cells an up to ten-fold increase of the key enzyme, ribonucleotide reductase, 1-2 h post infection. The enzyme activity has been partially enriched from cell extracts. In vitro, it differs from that of uninfected algae in three characteristic parameters, viz. eight-fold higher resistance to millimolar hydroxyurea concentrations, much higher optimum concentration of an allosteric effector nucleotide, thymidine triphosphate, and an unusually low temperature optimum at 20 °C. We conclude that the large DNA phycoviruses, like Herpes and pox viruses, code for their own specific ribonucleotide reductase.
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Chen, Qing, Nancy Fassinger, Ronald Thomas, Ana C. Xavier, Yubin Ge, Larry H. Matherly, and Jeffrey W. Taub. "Altered Folate Metabolism Maybeo the Develop Linked tment of Acute Lymphoblastic Leukemia in African American Children." Blood 114, no. 22 (November 20, 2009): 4118. http://dx.doi.org/10.1182/blood.v114.22.4118.4118.
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Abstract Abstract 4118 Background Acute lymphoblastic leukaemia (ALL) is the most common form of childhood cancer in the United States. The incidence of ALL is approximately 2-3-fold higher in Caucasian compared to African American (AA) children, suggesting potential differences in genetic susceptibility and/or exogenous exposures. Multiple epidemiologic studies have examined both genetic and environmental factors linked to the development of childhood ALL, primarily in Caucasian populations. Hence, identifying factors associated with racial differences in incidence of leukemia may provide new insights into the role of endogenous versus exogenous factors in the development of leukemia. A number of studies have reported relationships between folate metabolism and the risk of developing ALL including: i)maternal folate supplementation during pregnancy (reduced risk of ALL in offspring); and ii)polymorphisms of genes encoding enzymes involved in folate metabolism, including 5,10-methylenetetrahydrofolate reductase (MTHFR) (increased and decreased risks). To date, no studies have been performed specifically examining the role of folate metabolism in AA children. The objective of this study was to identify factors associated with folate metabolism which may be linked to the development of ALL in AA children compared to healthy controls. Patients and Methods AA children with B-precursor (BP) ALL were enrolled from the Hematology/Oncology Division of Children's Hospital of Michigan, while healthy AA children were enrolled as controls. Patients' racial backgrounds were based on parental reporting. The frequencies of polymorphisms in the MTHFR [677C>T, 1298A>C], thymidylate synthase [TS 2R3R], cystathionine-β-synthase [CBS 844ins(68)], and reduced folate carrier [RFC 80G>A] genes were determined by genotyping between AA childhood BP-ALL [n=26; 14 males] and healthy AA children [n=87; 47 males]. The distributions of genotypes between cases and controls were compared using Fisher's exact test. Results The genotype distributions of the polymorphisms of the folate pathway genes are summarized in Table 1. The frequencies of the MTHFR gene variants 677 CT/TT were 2-fold higher in the ALL cohort than that in the healthy control cohort. MTHFR 677 CT/TT was significantly associated with a risk of developing ALL in the AA patients. There were no significant differences in the distributions of the TS, CBS, or RFC polymorphisms between the groups. High birth weight has been associated with an increased risk of developing ALL, though we found no significant difference in birth weights between ALL and control groups. Conclusion Our study is the first to demonstrate that there is a higher frequency of the variant MTHFR C677T polymorphism (associated with reduced enzyme activity and altered distribution of folate forms) in AA children with ALL compared to healthy controls. Low MTHFR enzyme activity leads to imbalances in the thymidylate and de novo purine biosynthetic pathways, ultimately affecting DNA synthesis and repair and likely increasing the risk of leukemia. Thus, the role of altered folate metabolism may contribute to the development of ALL in AA children similar to Caucasian children, although additional studies are still required to identify factors linked to the higher incidence of ALL in Caucasian children and/or low incidence in AA children. Disclosures: No relevant conflicts of interest to declare.
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Murialdo, Helios, and Wendy L. Fife. "Synthesis of a trans-Acting Inhibitor of DNA Maturation by Prohead Mutants of Phage Λ." Genetics 115, no. 1 (January 1, 1987): 3–10. http://dx.doi.org/10.1093/genetics/115.1.3.
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ABSTRACT Bacteriophage λ with mutations in genes that control prohead assembly and other head precursors cannot mature their DNA. In this paper we present evidence that the failure of these phage mutants to mature DNA is a reflection of a mechanism that modulates terminase nicking activity during normal phage development. We have constructed plasmids that contain the λ-cohesive end site (cos) and the genes that code for DNA terminase, the enzyme that matures DNA by cutting at cos. The DNA terminase genes are under control of a thermosensitive cI repressor. These plasmids lack most of the genes involved in prohead morphogenesis and other head precursors. However, when repression is lifted by destruction of the thermosensitive repressor, the terminase synthesized is able to cut almost 100% of the plasmids. Therefore, these plasmids can mature in the absence of proheads and other head gene products. The plasmids are also able to complement mutants of λ deficient in terminase and DNA maturation. However, in these complementation experiments, if the phage carry mutations in prohead genes E or B, not only is phage DNA maturation blocked, but the plasmid also fails to mature. These experiments show that, in the absence of proheads, phage λ produces a trans-acting inhibitor of maturation. The genetic determinant of this inhibitor maps in a region extending from the middle of gene B to the end of gene C. A model is proposed in which the nicking activity of DNA-bound terminase is inhibited by the trans-acting inhibitor. Prohead (and other factors) binding to this complex would release the block to allow DNA cleavage and packaging.
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Wang, Wenqing, Andrew Devilbiss, Thomas Mathews, Martin Arreola, Misty Martin, Zhiyu Zhao, Avni Awani, et al. "Reticular Dysgenesis-Associated Adenylate Kinase 2 Deficiency Impairs Hematopoietic Stem and Progenitor Cell Function through Reductive Stress." Blood 136, Supplement 1 (November 5, 2020): 33. http://dx.doi.org/10.1182/blood-2020-138560.
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Energy deficiency and redox stress are hallmarks of mitochondrial pathology. Reductive stress is marked by an accumulation of reducing species and can arise from defects in the electron transport chain (ETC) that prevent NAD+ regeneration from NADH. Reticular Dysgenesis (RD) is a particularly grave form of severe combined immunodeficiency (SCID), characterized by maturation arrest of both myeloid and lymphoid lineages. Unlike other forms of SCID, RD is a mitochondriopathy caused by biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2). AK2 catalyzes the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP), and maintains ADP availability for ATP synthase. We hypothesize that AK2 deficiency leads to decreased ETC activities and defective NAD+ regeneration. Emerging evidence suggests that the development of hematopoietic stem and progenitor cells (HSPCs) is intricately intertwined with various aspects of mitochondrial function. Investigating the cellular and molecular consequences of AK2 deficiency during myelopoiesis provides fundamental insight into the pathology of many mitochondrial disorders. Methods: To recapitulate RD myeloid maturation defects, we developed an AK2 biallelic knock out model in human HSPCs using CRISPR gene editing. HSPCs were edited at the AK2 locus, and cells with biallelic AK2 knock out were enriched using homologous recombination-mediated dual reporters. HSPCs edited at the safe harbor locus AAVS1 were used as a control. When differentiated along the myeloid lineage in vitro, AK2-/- HSPCs showed significantly decreased proliferation, lower commitment to the granulocytic lineage, and maturation arrest at the promyelocyte stage, mimicking the presentation of RD patients. To dissect differentiation stage specific changes in metabolism, metabolomics analysis (LC-MS/MS), metabolic flux analysis (Seahorse assays) and RNA-seq were performed on FACS sorted populations of promyelocytes (PMs), metamyelocytes (MCs) and neutrophils (NPs). Additionally, mitochondrial membrane potential and ribosomal RNA (rRNA) content were quantified using TMRM and pyronin Y staining. Results: AK2-/- MCs and NPs showed higher AMP levels, and increased AMP/ADP and AMP/ATP ratios, in line with AK2's function to regenerate ADP from AMP. Mitochondrial oxygen consumption rate decreased, and mitochondrial membrane potential increased in AK2-/- MCs and NPs, indicating defective ETC function and ATP synthesis. Consistent with these results, TCA cycle metabolites were downregulated while pathways that fuel the TCA cycle, i.e. glycolysis and fatty acid oxidation, were upregulated. Interestingly, we observed a significant decrease in NAD+ levels, and an increase in NADH/NAD+ and GSH/GSSG ratios in AK2-/- MCs and NPs, indicative of reductive stress. These results suggest that AK2 deficiency compromises mitochondrial respiration, leading to NAD+ depletion and reductive stress in later stages of myeloid development. Defective mitochondrial respiration has been shown to impair NAD+-dependent aspartate and purine biosynthesis. In AK2-/- MCs and NPs, we observed a profound aspartate depletion and build-up of the purine precursor inosine monophosphate (IMP). As a building block for DNA and RNA, purine deficiency is known to block cell proliferation. Genes in cell cycle and ribosomal biogenesis pathways were down regulated in AK2-/- MCs and NPs. In addition, rRNA content was significantly decreased. These data raise the possibility that purine deficiency in AK2-/- HSPCs compromises nucleotide/protein synthesis along with cell cycle progression. Conclusions: Using an AK2 biallelic knock out HSPC model for RD, we have shown that defective mitochondrial respiration in AK2-/- HSPCs leads to reductive stress, NAD+ and purine depletion resulting in compromised nucleotide/protein synthesis and impaired cell cycle progression. Notably, these defects worsen as myeloid maturation progresses, possibly reflecting the increasing mitochondrial metabolic demand. We are currently exploring whether correcting the NADH/NAD+ ratio in AK2-/- HSPCs improves purine synthesis and restores myelopoiesis. Understanding how redox metabolism governs HSPC differentiation will not only allow us to delineate metabolic changes during development, but enable us to develop novel therapies for RD and other mitochondrial disorders. Disclosures Dever: Integral Medicines: Current Employment.
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Naffouje, Grover, Yu, Sendilnathan, Wolfe, Majd, Smith, et al. "Anti-Tumor Potential of IMP Dehydrogenase Inhibitors: A Century-Long Story." Cancers 11, no. 9 (September 11, 2019): 1346. http://dx.doi.org/10.3390/cancers11091346.
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The purine nucleotides ATP and GTP are essential precursors to DNA and RNA synthesis and fundamental for energy metabolism. Although de novo purine nucleotide biosynthesis is increased in highly proliferating cells, such as malignant tumors, it is not clear if this is merely a secondary manifestation of increased cell proliferation. Suggestive of a direct causative effect includes evidence that, in some cancer types, the rate-limiting enzyme in de novo GTP biosynthesis, inosine monophosphate dehydrogenase (IMPDH), is upregulated and that the IMPDH inhibitor, mycophenolic acid (MPA), possesses anti-tumor activity. However, historically, enthusiasm for employing IMPDH inhibitors in cancer treatment has been mitigated by their adverse effects at high treatment doses and variable response. Recent advances in our understanding of the mechanistic role of IMPDH in tumorigenesis and cancer progression, as well as the development of IMPDH inhibitors with selective actions on GTP synthesis, have prompted a reappraisal of targeting this enzyme for anti-cancer treatment. In this review, we summarize the history of IMPDH inhibitors, the development of new inhibitors as anti-cancer drugs, and future directions and strategies to overcome existing challenges.
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MD, Matthew Garrett. "103 Metabolic Characterization of IDH1mutant and IDH Wildtype Gliomaspheres Uncovers Cell-type Specific Vulnerabilities." Neurosurgery 64, CN_suppl_1 (August 24, 2017): 220. http://dx.doi.org/10.1093/neuros/nyx417.103.
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Abstract INTRODUCTION Large scale sequencing of tumor banks has revealed a subset of tumors that have a mutation in the isocitrate dehydrogenase (IDH1) enzyme, which bestows a novel function of reducing alpha-ketoglutarate into 2-hydroxyglutarate (2-HG). There has been considerable interest in defining metabolic differences of IDH1mutant tumors to exploit in therapy. However, most studies are limited by over-expressing the mutant IDH1 gene on an IDH1WT background. In this study we attempt to define metabolic differences between a cohort of patient-derived IDH1mutant and IDH1WT gliomaspheres to design patient-specific therapy. METHODS We propagated 59 patient-derived gliomasphere lines (7 bearing IDH1mutations) and performed microarray expression and KEGG analysis to define the pathways that were differentially enriched in IDH1mutant and IDH1WT cells. We used mass spectroscopy with labeled glucose and glutamine to determine differences in metabolite uptake and utilization. We then used inhibitors of de novo synthesis and Xray radiation treatment to test the predictions made by our expression analysis. RESULTS >Expression analysis showed IDH1WT cells to be enriched for pathways involving de novo DNA synthesis while IDH1mutant cells were enriched for pathways involving DNA repair after radiation. Using LC-MS we were able to define labeling patterns between IDH1WT and IDH1mutant cells particularly in regards to glucose utilization in nucleotide precursors. IDH1WT cells utilize more of the de novo pathway to synthesize nucleotides and consequently to be more sensitive to inhibitors of de novo synthesis. IDH1mutant cells were found to show less DNA damage after radiation and to more quickly repair that damage. They also showed better growth after radiation. CONCLUSION In this study we have identified key metabolic differences between IDH1mutant and IDH1wildtype tumors that suggest these two groups require different treatment modalities.
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Summers, E. F., V. A. Letts, P. McGraw, and S. A. Henry. "Saccharomyces cerevisiae cho2 mutants are deficient in phospholipid methylation and cross-pathway regulation of inositol synthesis." Genetics 120, no. 4 (December 1, 1988): 909–22. http://dx.doi.org/10.1093/genetics/120.4.909.
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Abstract Five allelic Saccharomyces cerevisiae mutants deficient in the methylation of phosphatidylethanolamine (PE) have been isolated, using two different screening techniques. Biochemical analysis suggested that these mutants define a locus, designated CHO2, that may encode a methyltransferase. Membranes of cho2 mutant cells grown in defined medium contain approximately 10% phosphatidylcholine (PC) and 40-50% PE as compared to wild-type levels of 40-45% PC and 15-20% PE. In spite of this greatly altered phospholipid composition, cho2 mutant cells are viable in defined medium and are not auxotrophic for choline or other phospholipid precursors such as monomethylethanolamine (MME). However, analysis of yeast strains carrying more than one mutation affecting phospholipid biosynthesis indicated that some level of methylated phospholipid is essential for viability. The cho2 locus was shown by tetrad analysis to be unlinked to other loci affecting phospholipid synthesis. Interestingly, cho2 mutants and other mutant strains that produce reduced levels of methylated phospholipids are unable to properly repress synthesis of the cytoplasmic enzyme inositol-1-phosphate synthase. This enzyme was previously shown to be regulated at the level of mRNA abundance in response to inositol and choline in the growth medium. We cloned the CHO2 gene on a 3.6-kb genomic DNA fragment and created a null allele of cho2 by disrupting the CHO2 gene in vivo. The cho2 disruptant, like all other cho2 mutants, is viable, exhibits altered regulation of inositol biosynthesis and is not auxotrophic for choline or MME.
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Romine, Margaret F., Dmitry A. Rodionov, Yukari Maezato, Lindsey N. Anderson, Premchendar Nandhikonda, Irina A. Rodionova, Alexandre Carre, et al. "Elucidation of roles for vitamin B12 in regulation of folate, ubiquinone, and methionine metabolism." Proceedings of the National Academy of Sciences 114, no. 7 (January 30, 2017): E1205—E1214. http://dx.doi.org/10.1073/pnas.1612360114.
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Only a small fraction of vitamin B12-requiring organisms are able to synthesize B12 de novo, making it a common commodity in microbial communities. Initially recognized as an enzyme cofactor of a few enzymes, recent studies have revealed additional B12-binding enzymes and regulatory roles for B12. Here we report the development and use of a B12-based chemical probe to identify B12-binding proteins in a nonphototrophic B12-producing bacterium. Two unexpected discoveries resulted from this study. First, we identified a light-sensing B12-binding transcriptional regulator and demonstrated that it controls folate and ubiquinone biosynthesis. Second, our probe captured proteins involved in folate, methionine, and ubiquinone metabolism, suggesting that it may play a role as an allosteric effector of these processes. These metabolic processes produce precursors for synthesis of DNA, RNA, and protein. Thereby, B12 likely modulates growth, and by limiting its availability to auxotrophs, B12-producing organisms may facilitate coordination of community metabolism.
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Sato, T., and A. Theologis. "Cloning the mRNA encoding 1-aminocyclopropane-1-carboxylate synthase, the key enzyme for ethylene biosynthesis in plants." Proceedings of the National Academy of Sciences 86, no. 17 (September 1989): 6621–25. http://dx.doi.org/10.1073/pnas.86.17.6621.
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Ethylene is the plant hormone that controls several features of plant growth and development. The rate-limiting step in its synthesis is the formation of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) from S-adenosylmethionine (AdoMet), catalyzed by ACC synthase. We have isolated a complementary DNA sequence encoding ACC synthase from zucchini (Cucurbita) fruits. The biological activity of the clone was confirmed by the ability of the cloned sequence to direct ACC synthase activity in Escherichia coli and yeast. In vivo studies using the ACC cDNA as probe showed that the ACC synthase gene is induced by a diverse group of inducers, including wounding, Li+ ions, and the plant hormone auxin.
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Skinner, M. K., P. S. Tung, and I. B. Fritz. "Cooperativity between Sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components." Journal of Cell Biology 100, no. 6 (June 1, 1985): 1941–47. http://dx.doi.org/10.1083/jcb.100.6.1941.
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We examined the synthesis and deposition of extracellular matrix (ECM) components in cultures of Sertoli cells and testicular peritubular cells maintained alone or in contact with each other. Levels of soluble ECM components produced by populations of isolated Sertoli cells and testicular peritubular cells were determined quantitatively by competitive enzyme-linked immunoabsorbent assays, using antibodies shown to react specifically with Type I collagen, Type IV collagen, laminin, or fibronectin. Peritubular cells in monoculture released into the medium fibronectin (432 to 560 ng/microgram cell DNA per 48 h), Type I collagen (223 to 276 ng/microgram cell DNA per 48 h), and Type IV collagen (350 to 436 ng/microgram cell DNA per 48 h) during the initial six days of culture in serum-free medium. In contrast, Sertoli cells in monoculture released into the medium Type IV collagen (322 to 419 ng/microgram cell DNA per 48 h) but did not form detectable amounts of Type I collagen or fibronectin during the initial six days of culture. Neither cell type produced detectable quantities of soluble laminin. Immunocytochemical localization investigations demonstrated that peritubular cells in monoculture were positive for fibronectin, Type I collagen, and Type IV collagen but negative for laminin. In all monocultures most of the ECM components were intracellular, with scant deposition as extracellular fibrils. Sertoli cells were positive immunocytochemically for Type IV collagen and laminin but negative for fibronectin and Type I collagen. Co-cultures of peritubular cells and Sertoli cells resulted in interactions that quantitatively altered levels of soluble ECM components present in the medium. This was correlated with an increased deposition of ECM components in extracellular fibrils. The data correlated with an increased deposition of ECM components in extracellular fibrils. The data presented here we interpret to indicate that the two cell types in co-culture act cooperatively in the formation and deposition of ECM components. Results are discussed with respect to the nature of interactions between mesenchymal peritubular cell precursors and adjacent epithelial Sertoli cell precursors in the formation of the basal lamina of the seminiferous tubule.