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The Mode of Action of 5Fluorouracil and Its Derivatives

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... It has been prescribed to treat different types of cancers, including skin, colon, breast, ovarian, and lung cancers [5,6]. The anticancer drug, 5-FU, acts by inhibition of essential biosynthetic processes, or by interfering with macromolecules, like DNA and RNA, and interrupting their normal function [7]. Based on the above, the main objective of this work was the development of a thermosensitive hydrogel from the physical mixture of CS and Pluronic F 127/β-GP for the controlled release of 5-FU. ...
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The treatment of breast cancer requires long chemotherapy management, which is accompanied by severe side effects. Localized delivery of anticancer drugs helps to increase the drug concentration at the site of action and overcome such a problem. In the present study, chitosan hydrogel was prepared for local delivery of 5-Fluorouracil. The in vitro release behavior was investigated and the anticancer activity was evaluated against MCF-7 cells using MTT assay. The in vivo studies were investigated via intra-tumoral injection of a 5-FU loaded hydrogel into breast cancer of female rats. The results indicated that the modified hydrogel has excellent physicochemical properties with a sustained in vitro release profile matching a zero-order kinetic for one month. In addition, the hydrogel showed superior inhibition of cell viability compared with the untreated control group. Moreover, the in vivo studies resulted in antitumor activity with minor side effects. The tumor volume and level of tumor markers in blood were inhibited significantly by applying the hydrogel compared with the untreated control group. In conclusion, the designed injectable hydrogels are potential drug delivery systems for the treatment of breast cancer with a controlled drug release profile, which could be suitable for decreasing the side effects of chemotherapy agents.
... 1,2 Interest in the fluorination of drug leads started in the 1950s after the success of 9-α-fluorohydrocortisone, 3 and this was followed by 5-fluorouracil, which is still used in chemotherapy. 4,5 The introduction of even a single fluorine often enhances and/or alters pharmacological properties, 1,6 such as those associated with potency, pK a , membrane permeability, metabolic stability, and pharmacokinetics. 7−9 Fluorinated compounds increased from 2% of marketed drugs in the 1970s to approximately 20−30% today. ...
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Precursor-directed biosynthesis was used to generate a series of fluorinated verticillins. The biosynthesis of these epipolythiodioxopiperazine alkaloids was monitored in situ via the droplet liquid microjunction surface sampling probe (droplet probe), and a suite of NMR and mass spectrometry data were used for their characterization. All analogues demonstrated nanomolar IC 50 values vs a panel of cancer cell lines. This approach yielded new compounds that would be difficult to generate via synthesis.
... 9,10 Substitution into native substrate dUMP with fluorine is a successful inhibitor (F-dUMP) of thymidylate synthase. 11 F-dUMP is a metabolite form of the anti-cancerous drug 5-fluorouracil (5-FU) and is known to inhibit both ThyX and ThyA. 12,13 Thus, the application of F-dUMP as a drug for M. tuberculosis is limited by poor selectivity. ...
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Thymidylate synthase is an enzyme that catalyzes deoxythymidine monophosphate (dTMP) synthesis from substrate deoxyuridine monophosphate (dUMP). Thymidylate synthase of Mycobacterium tuberculosis (MtbThyX) is structurally distinct from its human analogue human thymidylate synthase (hThyA), thus drawing attention as an attractive drug target for combating tuberculosis. Fluorodeoxyuridylate (F-dUMP) is a successful inhibitor of both MtbThyX and hThyA, thus limited by poor selectivity. Understanding the dynamics and energetics associated with substrate/inhibitor binding to thymidylate synthase in atomic details remains a fundamental unsolved problem, which is necessary for a new selective inhibitor design. Structural studies of MtbThyX and hThyA bound substrate/inhibitor complexes not only revealed the extensive specific interaction network between protein and ligands but also opened up the possibility of directly computing the energetics of the substrate versus inhibitor recognition. Using experimentally determined structures as a template, we report extensive computer simulations (∼4.5 μs) that allow us to quantitatively estimate ligand selectivity (dUMP vs F-dUMP) by MtbThyX and hThyA. We show that MtbThyX prefers deprotonated dUMP (enolate form) as the substrate, whereas hThyA binds to the keto form of dUMP. Computed energetics clearly show that MtbThyX is less selective between dUMP and F-dUMP, favoring the latter, relative to hThyA. The simulations reveal the role of tyrosine at position 135 (Y135) of hThyA in amplifying the selectivity. The protonation state of the pyrimidine base of the ligand (i.e., keto or enolate) seems to have no role in MtbThyX ligand selectivity. A molecular gate (consists of Y108, K165, H203, and a water molecule) restricts water accessibility and offers a desolvated dry ligand-binding pocket for MtbThyX. The ligand-binding pocket of hThyA is relatively wet and exposed to bulk water.
... Compounds were chosen based on our initial findings that the EMT subtype increased glutathione biosynthesis and TCA cycle metabolism, while the papillary subtype increased de novo nucleotide biosynthesis. The three selected compounds were: (1) Buthionine sulfoximine (BSO), an inhibitor of glutamate-cysteine ligase (GCL) in glutathione biosynthesis [31]; (2) CPI-613, which targets pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (AKGDH) in the TCA cycle [32,33]; and (3) 5-Fluorouracil (5FU), which is an inhibitor of thymidylate synthase (TYMS) in de novo nucleotide biosynthesis (Supplementary Fig. 4) [34,35]. ...
Article
PurposeBreast cancer is a heterogeneous disease with several subtypes that currently do not have targeted therapeutic options. Metabolomics has the potential to uncover novel targeted treatment strategies by identifying metabolic pathways required for cancer cells to survive and proliferate.Methods The metabolic profiles of two histologically distinct breast cancer subtypes from a MMTV-Myc mouse model, epithelial-mesenchymal-transition (EMT) and papillary, were investigated using mass spectrometry-based metabolomics methods. Based on metabolic profiles, drugs most likely to be effective against each subtype were selected and tested.ResultsWe found that the EMT and papillary subtypes display different metabolic preferences. Compared to the papillary subtype, the EMT subtype exhibited increased glutathione and TCA cycle metabolism, while the papillary subtype exhibited increased nucleotide biosynthesis compared to the EMT subtype. Targeting these distinct metabolic pathways effectively inhibited cancer cell proliferation in a subtype-specific manner.Conclusions Our results demonstrate the feasibility of metabolic profiling to develop novel personalized therapy strategies for different subtypes of breast cancer.Graphical abstract Schematic overview of the experimental design for drug selection based on breast cancer subtype-specific metabolism. The epithelial mesenchymal transition (EMT) and papillary tumors are histologically distinct mouse mammary tumor subtypes from the MMTV-Myc mouse model. Cell lines derived from tumors can be used to determine metabolic pathways that can be used to select drug candidates for each subtype.
... These antimetabolites are also potent antimicrobial agents since their toxicity originates from a key metabolic pathway found in all cell-types -nucleotide synthesis. In bacteria, intermediate compounds metabolized from these fluoropyrimidines interfere with DNA and RNA synthesis, inhibit the synthesis of deoxythymidine monophosphate (Cohen et al., 1958), and lead to harmful accumulation of cell wall precursors (Tomasz and Borek, 1962;Tomasz and Borek, 1960). Recent studies on these drugs revealed that mutations in the bacterial gene network for nucleotide synthesis can both increase and decrease drug efficacy in the C. elegans host feeding on mutated bacteria ( Figure 1A). ...
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Metabolism of host-targeted drugs by the microbiome can substantially impact host treatment success. However, since many host-targeted drugs inadvertently hamper microbiome growth, repeated drug administration can lead to microbiome evolutionary adaptation. We tested if evolved bacterial resistance against host-targeted drugs alters their drug metabolism and impacts host treatment success. We used a model system of Caenorhabditis elegans , its bacterial diet, and two fluoropyrimidine chemotherapies. Genetic screens revealed that most of loss-of-function resistance mutations in Escherichia coli also reduced drug toxicity in the host. We found that resistance rapidly emerged in E. coli under natural selection and converged to a handful of resistance mechanisms. Surprisingly, we discovered that nutrient availability during bacterial evolution dictated the dietary effect on the host – only bacteria evolving in nutrient-poor media reduced host drug toxicity. Our work suggests that bacteria can rapidly adapt to host-targeted drugs and by doing so may also impact the host.
... It has been prescribed to treat different types of cancers, including skin, colon, breast, ovarian, and lung cancers [5,6]. The anticancer drug, 5-FU, acts by inhibition of essential biosynthetic processes, or by interfering with macromolecules, like DNA and RNA, and interrupting their normal function [7]. Based on the above, the main objective of this work was the development of a thermosensitive hydrogel from the physical mixture of CS and Pluronic F 127/β-GP for the controlled release of 5-FU. ...
Article
Citation: Abdellatif, A.A.H.; Mohammed, A.M.; Saleem, I.; Alsharidah, M.; Al Rugaie, O.; Ahmed, F.; Osman, S.K. Smart Injectable Chitosan Hydrogels Loaded with 5-Fluorouracil for the Treatment of Breast Cancer.
Chapter
As a result of clinical research over the last ten years, the simultaneous use of multiple pharmacologic agents in the therapy of malignant disease has become the accepted form of treatment for acute leukemia (Holland, 1968), advanced Hodgkin’s disease (Devita et al., 1970), lymphosarcoma (Bagley et al., 1972), and other tumors (Cooper, 1969; James et al., 1965). These therapeutic advances were largely the outgrowth of earlier experiences with single agents, which despite obvious antitumor activity, elicited incomplete responses or complete remissions of only brief duration. The limitations of single drug therapy were two-fold: (1) the toxicity of the drug limited the amount and duration of drug exposure tolerated by the host, and thus restricted cell kill, and (2) adaptive mechanisms allowed survival and proliferation of a fraction of resistant neoplastic cells despite a metabolic block lethal to the bulk of the tumor. Noting the effectiveness of combined chemotherapy in infectious diseases such as malaria (Blount, 1967), tuberculosis (Barry, 1964), and enterococcal bacterial endocarditis (Lerner and Weinstein, 1966), oncologists reasoned that the use of several active agents in combination might produce greater cell kill, while delaying the appearance of resistance to the individual drugs. The success of such combinations has established a permanent and expanding role for chemotherapy in the treatment of human malignancy.
Chapter
Les composés organiques du fluor ont fait leur apparition en chimie il y a exactement 125 ans, avec la description faite par les chimistes français Dumas et Peligot, en 1835, d’une méthode de préparation du fluorure de méthyle par réaction du sulfate de méthyle sur le fluorure de potassium. La chimie organique du fluor a devancé ainsi d’environ un demi-siècle la découverte de l’élément fluor lui-même (Moissan, 1886), et n’a jamais cessé d’être assidûment étudiée, autant sur le plan théorique que sur celui des applications pratiques possibles. Mais en ce qui concerne les dérivés organiques fluorés d’intérêt phar-macologique, leur importance ne s’est dégagée que très lentement, et depuis seulement ces vingt dernières années. Les étapes essentielles de ce développement nous semble être, successivement: 1) la découverte de l’activité antagoniste de certains dérivés organiques fluorés vis-à-vis de la fonction thyroïdienne, 2) la découverte de la toxicité remarquablement élevée des esters de l’acide fluorophosphonique d’une part, et de l’acide fluoroacétique et de ses dérivés d’autre part, et, enfin, beaucoup plus récemment, 3) la découverte de la potentialisation des propriétés biologiques des hormones Stéroïdes par l’introduction du fluor dans leurs molécules.
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The ability of chemotherapeutic agents to affect the growth of common bacterial pathogens and the relationship between the effects of chemotherapeutics and antimicrobials is largely unknown. The purpose of this study was to describe the susceptibility of canine bacterial isolates to chemotherapeutic agents and to compare these results to their antimicrobial susceptibility. The effects of bleomycin, doxorubicin, cytarabine, cyclophosphamide, methotrexate, 5‐fluorouracil and gemcitabine on the growth of 33 S. pseudintermedius isolates and 32 E. coli isolates from dogs was determined by agar dilution. In addition to MICs, the lowest drug concentration associated with a decreased colony size was recorded. Results were compared to the MICs of a panel of antimicrobial agents. Bleomycin consistently inhibited bacterial growth of S. pseudintermedius and E. coli. Doxorubicin inhibited S. pseudintermedius but not E. coli while the opposite was seen for gemcitabine. Reduction in colony size on exposure to 5‐fluorouracil for both organisms, and methotrexate for S. pseudintermedius was seen. No observable effect of cyclophosphamide or cytarabine was observed. Associations between elevated MICs to chemotherapeutic drugs and antimicrobial resistance were not found. These results indicate that chemotherapeutic agents affect the growth of bacteria, but do not support a role in the selection of antimicrobial resistance.
Article
Kinetoplastid and apicomplexan parasites comprise a group of protozoans responsible for human diseases, with a serious impact in human health and the socioeconomic growth of developing countries. Chemotherapy is the main option to control these pathogenic organisms and nucleotide metabolism is considered a promising area for the provision of antimicrobial therapeutic targets. Impairment of thymidylate (dTMP) biosynthesis severely diminishes the viability of parasitic protozoa and the absence of enzymatic activities specifically involved in the formation of dTMP (e.g. dUTPase, thymidylate synthase, dihydrofolate reductase or thymidine kinase) results in decreased deoxythymidine triphosphate (dTTP) levels and the so-called thymineless death. In this process the ratio of deoxyuridine triphosphate (dUTP) versus dTTP in the cellular nucleotide pool has a crucial role. A high dUTP/dTTP ratio leads to uracil misincorporation into DNA, the activation of DNA repair pathways, DNA fragmentation and eventually cell death. The essential character of dTMP synthesis has stimulated the interest in the identification and development of drugs that specifically block the biochemical steps involved in thymine nucleotide formation. Here, we review the available literature in relation to drug discovery studies targeting thymidylate biosynthesis in kinetoplastid (genera Trypanosoma and Leishmania) and apicomplexan (Plasmodium spp and Toxoplasma gondii) protozoans. The most relevant findings concerning novel inhibitory molecules with antiparasitic activity against these human pathogens are presented herein.
Chapter
Chemical and physical agents lead to direct DNA damage and chromosome rearrangements, which are detected using microscope techniques. The changes in chromosomal morphology are usually identified using classical cytogenetic techniques, which can fail in the analysis of small changes. More information is required in order to understand the biological consequences of DNA damage and the molecular action of mutagens in cells. Recently introduced modern molecular cytogenetic methods allow a detailed analysis of mutagenic effects, both at the chromosomal and DNA levels. One of the techniques used to improve the assessment of the effectiveness of mutagenic effects is fluorescence in situ hybridisation (FISH). It allows the detection and a more detailed analysis of the involvement of specific chromosomes or chromosome fragments in chromosome aberration formations. Repetitive DNA sequences that recognise specific chromosome regions, such as the centromere and telomere, as well as rDNA are the most extensively used probes for FISH for plant chromosomes. Interphase cytogenetics using FISH with specific DNA probes provides a sensitive tool with which to detect small chromosome rearrangements in nondividing cells. DNA fragmentation can be estimated using the TUNEL test and the single cell gel electrophoresis (comet assay). Additionally, the effectivity of repair processes can be estimated using these approaches. The relatively new technique, comet-FISH, permits the localisation of a specific chromosome, regions of chromosomes or specific genes within the comets. It is suitable for a detailed quantification of any DNA damage and repair within specific DNA sequences in plant mutagenesis. Chemical and physical agents lead to direct DNA damage and chromosome rearrangements, which are detected using microscope techniques. The changes in chromosomal morphology are usually identified using classical cytogenetic techniques, which can fail in the analysis of small changes. More information is required in order to understand the biological consequences of DNA damage and the molecular action of mutagens in cells. Recently introduced modern molecular cytogenetic methods allow a detailed analysis of mutagenic effects, both at the chromosomal and DNA levels. One of the techniques used to improve the assessment of the effectiveness of mutagenic effects is fluorescence in situ hybridisation (FISH). It allows the detection and a more detailed analysis of the involvement of specific chromosomes or chromosome fragments in chromosome aberration formations. Repetitive DNA sequences that recognise specific chromosome regions, such as the centromere and telomere, as well as rDNA are the most extensively used probes for FISH for plant chromosomes. Interphase cytogenetics using FISH with specific DNA probes provides a sensitive tool with which to detect small chromosome rearrangements in nondividing cells. DNA fragmentation can be estimated using the TUNEL test and the single cell gel electrophoresis (comet assay). Additionally, the effectivity of repair processes can be estimated using these approaches. The relatively new technique, comet-FISH, permits the localisation of a specific chromosome, regions of chromosomes or specific genes within the comets. It is suitable for a detailed quantification of any DNA damage and repair within specific DNA sequences in plant mutagenesis.
Chapter
The classic biochemical studies of Peters and co-workers, demonstrating the biosynthesis of the extremely toxic fluorocitrate from fluoroacetate, provided the initial example of the profound biological consequences of lethal synthesis (Chapter 1). The syntheses of fluorinated steroids by Fried and co-workers, based in part on the precedent provided by the altered biological activity of fluorocitrate, was a second development of historical proportions (Chapter 3). Of comparable scientific impact was the synthesis of 5-fluorouracil (fl5ura), 5-fluoroorotic acid (fl5oro), and 5-fluorocytosine (fl5cyt) (Fig. 5-1) by Heidelberger et al. (1957).
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Magnetotactic bacteria (MTB) are a group of organisms that form intracellular nanometer-scale magnetic crystals though a complex process involving lipid and protein scaffolds. These magnetic crystals and their lipid membranes, termed magnetosomes, are model systems for studying bacterial cell biology and biomineralization and are potential platforms for biotechnological applications. Due to a lack of genetic tools and unculturable representatives, the mechanisms of magnetosome formation in phylogenetically deeply branching MTB remain unknown. These MTB contain elongated bullet-/tooth-shaped magnetite and greigite crystals that likely form in a manner distinct from that of the cubooctahedral-shaped magnetite crystals of the genetically tractable MTB within the Alphaproteobacteria . Here, we present a method for genome editing in Desulfovibrio magneticus RS-1, a cultured representative of the deeply branching MTB of the class Deltaproteobacteria . This marks a crucial step in developing D. magneticus as a model for studying diverse mechanisms of magnetic particle formation by MTB.
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C 10 H 7 FN 2 O 5 , monoclinic, P 2 1 / n (no. 14), a = 8.5781(17) Å, b = 5.3121(11) Å, c = 23.578(5) Å, β = 96.68(3)°, V = 1067.1(4) Å ³ , Z = 4, Rgt ( F ) = 0.0375, wRref ( F² ) = 0.1138, T = 293(2) K.
Article
The purine analogs, 8-azaadenine, 8-azaguanine, 8-azaxanthine and 8-azahypoxanthine, and the pyrimidine analogs, 2-thiocytosine, 5-fluorouracil, 2-thiouracil and 6-azauracil, inhibited the induction of 2-dimensional growth in the gametophytes of the fern Asplenium nidus L. In contrast, thymine analogs such as 5-fluorodeoxyuridine, 2-thiothymine, 6-azathymine and 5-bromouracil caused non-specific growth inhibitions without suppressing 2-dimensional growth. Subinhibitory concentrations of 8-azaxanthine, 8-azahypoxanthine, and 2-thiouracil promoted both 1-dimensional and 2-dimensional phases of growth of the gametophytes. Inhibitory effects of the analogs were observed on treatment of the spores or of gametophytes of different ages. Gametophytes growing in the analogs for different periods of time recovered from inhibition on transfer to the basal medium.
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A large and rapidly increasing body of knowledge is available concerning the effects of ionizing radiations on the metabolism of normal tissues and malignant tumours (see Bibliography, especially reviews and books). Nevertheless it is clear that a great deal of essential evidence is still lacking. There is inadequate detailed knowledge of the fundamental biology of living cells, including molecular biology in a wide sense. Moreover, there is insufficient relevant biochemical information concerning differences between the effects of radiations on proliferating normal tissues and malignant tumours, particularly for malignant tumours which show cellular differentiation and for irradiation at therapeutic levels of dose.
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Fluoropyrimidines—5-fluorouracil (5-FU) and capecitabine—have been implicated as cardiotoxic chemotherapy agents. This rare, albeit potentially serious toxicity has been described in nearly four decades of case reports, case series, and in vitro modeling; however, there is a paucity in clinical trials and prospective analyses focused on cardioprotective strategies and cardiotoxic surveillance of these agents. While much attention has focused on the well-known cardiac toxicity of anthracyclines and monoclonal antibody agents such as trastuzumab, fluoropyrimidines remain one of the most common causes of chemotherapy-associated cardiotoxicity. The introduction of capecitabine, an oral prodrug of 5-FU, has made the treatment of solid tumors more convenient along with a subsequent rise in documented cardiotoxic cases. This review discusses the symptomatology, clinical manifestations, and proposed molecular mechanisms that attempt to describe the heterogeneous spectrum of fluoropyrimidine-induced cardiotoxicity. Four case examples showcasing the varied manifestations of cardiotoxicity are presented. Finally, several proposed management strategies for cardiotoxicity and post-hospital course precautions are discussed.
Chapter
Die Bezeichnung »Pochen« ist ein klinischer Sammelbegriff für eine Reihe von Krankheiten des Menschen und der Tiere, deren wesentliches gemeinsames Merkmal ein bläschen-bis papelförmiger Ausschlag der Haut und Schleimhäute ist.
Chapter
Unna (1883) wies erstmalig auf die engen Beziehungen zwischen Herpes febrilis, Herpes genitalis und Zoster hin und unterschied diese Krankheitsgruppe als »Herpes-Gruppe« von der Pocken-Gruppe (s. auch Schönfeld, 1947). Heute faßt man das Herpes hominis-Virus (HV), das Herpes simiae (B)-Virus (BV), das Herpes suis = Pseudowut-Virus (PWV) und das Varicellae-Zoster-Virus (VZV) zu einer Virusgruppe zusammen. Es handelt sich bei ihnen meist um hochinfektiöse Erreger einer Gruppe neurotroper Ektodermosen, die sehr oft in den schweren Formen mit neurologischen Syndromen einhergehen. Sie führen aber auch zu latenten Infektionen, die lebenslänglich bestehen bleiben, was auf eine nur teilweise, durch verschiedene endogene und exogene Einwirkungen oder Reize unterbrochene Immunität zurückzuführen ist. Alle 4 Viren führen zu bläschenförmigen Eruptionen der Haut und Schleimhäute, die in ihrer histologischen Struktur weitgehend übereinstimmen. Alle bilden anfangs basophile Feulgen-positive Kerneinschlüsse (»karyotrope Viren«), die virushaltig sind, später aber eosinophil werden und kein Virus mehr enthalten müssen. Die Einschlüsse scheinen vielmehr durch Viruseinwirkung auf den Kern zu entstehen (s. auch Haagen, 1931a, 1934). Nach Andrewes (1962, 1965) handelt es sich bei ihnen nur um Fixierungs-Kunstprodukte, die in frischen Präparaten nicht sichtbar sind (s. auch Reissig und Melnick, 1955; Barski und Robineaux, 1959). Die Einschlüsse gehören zum Typ A von Cowdry.
Chapter
The concept of inhibition of growth of cells by an organic compound having specific structural features which would cause it to bind to a cell surface or protein molecule was introduced by Ehrlich in the late nineteenth century. However the concept of the antimetabolite, i e inhibition of a biochemical reaction by a compound similar in structure to a metabolite because of competition for a site on an enzyme molecule, has developed during the past twenty years. It is noteworthy that this development closely parallels the development of use of isotopes in the study of biochemical problems. That the two areas have not grown in different directions is evident from a survey of articles in any recent biochemical journal. Many investigators have used a combination of radioactive isotopes and anti-metabolites to gain information about biochemical mechanisms. It is, perhaps, in this area that the use of isotopes in the study of antimetabolites has achieved its greatest importance. It is the purpose of this chapter to point out how these concepts have been brought to bear in gaining information about a number of specific metabolic reactions. The choice of reactions and examples is dictated by personal interest, but the principles involved would be the same for an entirely different group of examples.
Chapter
Angiosperm morphogenesis is regulated largely by the processes of cell division, cell elongation, cell differentiation, and cell senescence. During shoot development the regions of primary cell division, elongation, and differentiation are in the shoot tip. The meristem proper is the site of active cell division, and is associated with an adjacent zone of cell elongation and differentiation. Thus, an understanding of the mechanisms that control shoot meristem activity is essential if we are to learn how basic cellular processes regulate plant development.
Chapter
During the last two decades it became clear that a significant fraction of the biological cellular damage caused by high-energy radiation is actually due to reactions induced by low-energy electrons (<20 eV). In this energy regime electrons can efficiently decompose molecules such as DNA or DNA building blocks by dissociative electron attachment (DEA) . Experiments on single DNA building blocks have been performed in the gas phase revealing that DEA can proceed with remarkable site selectivity. Low-energy electron-induced DNA strand breakage is typically investigated using plasmid DNA in the condensed phase. Very recently, a pronounced dependence of electron induced DNA strand breakage on the nucleotide sequence was found using different experimental approaches suggesting that at least part of the observed strand breaks are due to initial electron attachment to the nucleobases . Currently, a strong research focus is on the fundamental understanding of DEA to therapeutically administered radiosensitizers. In the near future DEA to novel potential radiosensitizers will be explored, and the electron induced damage of biomolecules within complex environments has to be investigated. Considerable attention has been paid to the theoretical research of the DEA in the context of the DNA damage. With respect to this, the theoretical part of the chapter reviews all the computational approaches that have been used to study DEA to biomolecules over the last decade. These approaches are divided into two classes. The first class consists of electronic structure methods studying the transient negative ions formed by electrons captured by the neutral building blocks of the DNA. Approaches dealing with the complicated nuclear dynamics of the DEA to biomolecules form the second class explored in this chapter.
Chapter
1. Bei der Chemotherapie von Tumoren wurde 5-Fluoruridin (FUrd) klinisch bisher relativ selten angewandt [6], obwohl es bei einigen experimentellen Tumoren [6,9] stärker wachstumshemmend wirkt als das häufig angewandte 5-Fluoruracil (FUra). Unsere Untersuchungen an Rattenhepatomzellen und Mammatumorzellen der Maus mit einer Kombination von FUrd und anderen Antipyrimidinen, insbesondere mit Aminozuckern und Inhibitoren der De novo-Pyrimdinnukleotidsynthese dienen dem Ziel, eine wirksamere Hemmung des Tumorzellwachstums und eine gesteigerte Selektivität der Chemotherapie zu erreichen [14,15]. Die Selektivität für das hepatozelluläre Karzinom wird gesteigert durch die Ausnutzung spezifischer Tumoreigenschaften: Die in diesem Gewebe nachgewiesene Galaktosamin (GalN)-Wirkung [11, 12, 17] und die höhere Uridinkinaseaktivität in den Tumorzellen [25] machen diese empfindlieher für FUrd und andere Uridin- und Cytidinanaloge; die Bildung und intrazelluläre Anreicherung von Fluoruridindiphosphat(FUDP)aminozuckern [10] können zu einer Verlängerung der zytostatischen Wirkung von FUrd beitragen (Abb. 1).
Chapter
Sister chromatid exchanges (SCEs) are usually scored in cells which have incorporated 5-bromodeoxyuridine (BrdUrd) for two cell cycles or, alternatively, for just one cell cycle followed by another in the absence of the halogenated nucleoside. By using this approach, however, the yield of SCEs observed at second mitosis is the sum of exchanges which occur in either of the two cell cycles. Therefore, it is impossible to distinguish which SCEs took place during the first or the second cell cycle.
Chapter
The development of the fluoropyrimidines as anticancer drugs began in 1957 with the publication by Heidelberger of a method for the synthesis of 5-fluorouracil (FU) (1). This compound was soon found to possess significant antitumor activity in both mice (2) and men (3) and today FU and some of its derivatives, notably the deoxyribonucleoside 5-fluoro-2′-deoxyuridine (FdUrd), are extensively used in the treatment of metastatic cancers, singly or in combination with other drugs. In addition to anticancer drugs, the fluoropyrimidines have been used as biochemical tools to study a wide variety of problems in biochemistry and cell biology. As might be expected, a vast literature on fluoropyrimidines has been generated describing studies on their metabolism, mechanism of action, biochemical pharmacology, synthesis of new derivatives, and clinical antitumor activity.
Chapter
Physiologically active and structurally central substances, such as DNA, RNA, proteins, polysaccharides and lipids are usually after fixation insoluble in a wide range of solvents. The presence, the localization and the quantity of radioactive isotopes incorporated into these compounds, can be demonstrated by autoradiography in organelles, cells or tissues, and often simultaneously in one preparation. Accordingly, autoradiography may be used to solve problems involving the site, initiation, duration and rate of synthesis of substances. Often it is even possible to investigate the type of synthesis, e.g. DNA replication or repair synthesis. The redistribution of substances from parent to daughter cells can also be analysed. Autoradiography is especially useful for demonstrating and measuring small amounts of radioactivity, and is therefore an independent though complementary method to liquid scintillation and gas flow counting techniques. The sensitivity is sufficient to detect a few decays per week.
Article
Grula, E. A. (Oklahoma State University, Stillwater) and Mary M. Grula. Cell division in a species of Eriwinia. V. Effect of metabolic inhibitors on terminal division and composition of a “division” medium. J. Bacteriol. 84 492–499. 1962.—Terminal division in Erwinia spp. involves a triggering action and subsequent septum synthesis. It is a metabolic process requiring organic nitrogen and carbon and energy. The process, when triggered by pantoic acid or pantoyl lactone, is inhibited strongly by 2,4-dinitrophenol, hydroxylamine, mitomycin C, and Hg ion and to a lesser degree by cyanide, azide, 5-fluorouracil, and diisopropylfluorophosphate. Ethylenediaminetetraacetic acid completely inhibits division only when calcium ion is the triggering agent. Heating of the cells at 43 C for 10 to 20 min also completely inhibits division. Hydroxylamine, iodoacetate, and mitomycin C cause extensive lysis of growing cells. No evidence has been obtained to demonstrate the need for protein or normal ribonucleic acid synthesis in terminal division. Requirement for intact deoxyribonucleic acid (DNA) or DNA synthesis is questionable; —SH groups are not directly involved. Once triggering has occurred, septum formation appears to require synthesis of at least cell-wall mucopeptide. A “division” medium is reported and discussed.
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Background: Rhodobacter sphaeroides is a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-β-hydroxybutyrate (PHB), has been an important metabolic engineering target for R. sphaeroides. However, the lack of efficient markerless genome editing tools for R. sphaeroides is a bottleneck for fundamental studies and biotechnological exploitation. The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not for R. sphaeroides. Results: Here we describe the development of a highly efficient SpCas9-based genomic DNA targeting system for R. sphaeroides, which we combine with plasmid-borne homologous recombination (HR) templates developing a Cas9-based markerless and time-effective genome editing tool. We further employ the tool for knocking-out the uracil phosphoribosyltransferase (upp) gene from the genome of R. sphaeroides, as well as knocking it back in while altering its start codon. These proof-of-principle processes resulted in editing efficiencies of up to 100% for the knock-out yet less than 15% for the knock-in. We subsequently employed the developed genome editing tool for the consecutive deletion of the two predicted acetoacetyl-CoA reductase genes phaB and phbB in the genome of R. sphaeroides. The culturing of the constructed knock-out strains under PHB producing conditions showed that PHB biosynthesis is supported only by PhaB, while the growth of the R. sphaeroides ΔphbB strains under the same conditions is only slightly affected. Conclusions: In this study, we combine the SpCas9 targeting activity with the native homologous recombination (HR) mechanism of R. sphaeroides for the development of a genome editing tool. We further employ the developed tool for the elucidation of the PHB production pathway of R. sphaeroides. We anticipate that the presented work will accelerate molecular research with R. sphaeroides.
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Approximately twenty drugs have been shown to be effective for the treatment of colorectal cancer (CRC). These drugs are from several classes of agents and include cytotoxic drugs, therapeutics that target cell signaling pathways at the extracellular and/or intracellular levels, and combination therapies that contain multiple targeted agents and/or cytotoxic compounds. Targeted therapeutics can include monoclonal antibodies, fusion proteins, and small molecule drugs. 5‐Fluorouracil (5‐FU) was first introduced into clinical use in the late 1950s and remains the foundation for most CRC treatments in both adjuvant therapy and in advanced (metastatic) treatment regimens. As with other cancers, the consideration of biomarkers has the potential to improve CRC therapy through patient stratification. The biomarkers can include germline genetic markers, tumor‐specific genetic markers, immune markers, and other biomarkers that can predict antitumor efficacy or the likelihood of toxicity prior to administration of a specific drug. Consistent with the benefit of considering biomarkers in treatment, many newer targeted therapies are developed and approved simultaneously with a companion diagnostic test to determine efficacy. This review will focus on biomarkers that have demonstrated clinical utility in CRC treatment; however, it is noted that many additional biomarkers have been theorized to contribute to drug response and/or toxicity based on known biological pathways but thus far have not attained widespread use in the clinic. The importance of pre‐treatment biomarker testing is expected to increase as future drug development will likely continue to focus on the concurrent development of companion diagnostics.
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A new protocol for inducing mutations in mammalian cells in culture by exposure to the thymidine analog 5-bromodeoxyuridine (BrdUrd) was established. This protocol, called "DNA-dependent" mutagenesis, involved the incorporation of BrdUrd into DNA under nonmutagenic conditions and the subsequent replication of the 5-bromouracil (BrUra)-containing DNA under mutagenic conditions but with no BrdUrd present in the culture medium. The mutagenic conditions were induced by allowing BrUra-containing DNA to replicate in the presence of high concentrations of thymidine. This generated high intracellular levels of dTTP and dGTP, causing nucleotide pool imbalance. The mutagenesis induced by this protocol was found to correlate with the level of BrUra substituted for thymine in DNA.
Article
Two protocols have been developed, both of which utilize the thymidine analog 5-bromodeoxyuridine (BrdUrd) to induce mutations in mammalian cells in culture (E. R. Kaufman and R. L. Davidson, Proc. Natl. Acad. Sci. USA 75:4982-4986, 1978; E. R. Kaufman, Mol. Cell. Biol. 4:2449-2454, 1984). The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP. The second protocol, termed replicational (REP) mutagenesis, entails the incorporation of BrdUrd into DNA under nonmutagenic conditions, the removal of all BrdUrd from the culture medium, and the subsequent replication of the bromouracil-containing DNA in the presence of high intracellular levels of dTTP and dGTP. Genetic studies using reversion analysis at the hypoxanthine-guanine phosphoribosyltransferase locus were used to determine whether the mechanisms of these two BrdUrd mutagenesis protocols had enough specificity to be distinguishable by their ability to revert various mutants. The results of these studies indicated that (i) mutants induced by INC mutagenesis were induced to revert only by REP mutagenesis and not by INC mutagenesis, (ii) mutants induced by REP mutagenesis were more efficiently reverted by INC mutagenesis than by REP mutagenesis, and (iii) both spontaneous mutants and mutants induced by the chemical mutagen ethyl methanesulfonate showed a high degree of specificity when tested for reversion by the BrdUrd mutagenesis protocols.
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A big challenge in natural product research of today is rapid dereplication of already known substances, to free capacities for the exploration of new agents. Prompt information on bioactivities and mode of action (MOA) speeds up the lead discovery process and is required for rational compound optimization. Here, we present a bioreporter approach as a versatile strategy for combined bioactivity- and MOA-informed primary screening for antimicrobials. The approach is suitable for directly probing producer strains grown on agar, without need for initial compound enrichment or purification, and works along the entire purification pipeline with culture supernatants, extracts, fractions, and pure substances. The technology allows for MOA-informed purification to selectively prioritize activities of interest. In combination with high-resolution mass spectrometry, the biosensor panel is an efficient and sensitive tool for compound deconvolution. Concomitant information on the affected metabolic pathway enables the selection of appropriate follow-up assays to elucidate the molecular target.
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Infection of Escherichia coli with T-even bacteriophage or T5 leads to the production of large amounts of dihydrofolate reductase. As shown previously, this represents the synthesis of a new, virus-specific protein. Since appreciable levels of this enzyme exist in the cell before infection, an attempt was made to demonstrate a specific metabolic requirement for the viral enzyme. This included (a) a study of the structure of tetrahydrofolate produced enzymatically by both the bacterial and the viral reductase and (b) measurements of levels of reduced pyridine nucleotides in the cell before and after infection. Both the cellular and the phage-specific reductase catalyze the formation of the l, l diastereoisomer of tetrahydrofolate, as does the same enzyme isolated from chicken liver. Experiments on pyridine nucleotide concentrations reveal a 2- to 3-fold increase in the reduced forms of both di- and triphosphopyridine nucleotide occurring within about 10 min after infection, followed by a return to preinfection values. These observations are discussed with reference to the reductive biosynthetic steps in the production of deoxyribonucleic acid.
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Goss, William A. (Sterling-Winthrop Research Institute, Rensselaer, N.Y.), William H. Dietz, and Thomas M. Cook. Mechanism of action of nalidixic acid on Escherichia coli. J. Bacteriol. 88 1112–1118. 1964.—Nalidixic acid was lethal for proliferating cultures of Escherichia coli. Associated with this lethal effect was the formation of elongated, serpentine forms. Cultures treated with nalidixic acid were osmotically stable; lethality was observed in the presence of stabilizers. Although it was possible to demonstrate leakage of intracellular components from treated cells, this effect occurred only after 99% of the cells were nonviable. Nalidixic acid had little or no effect on respiration with glucose as substrate. If cellular growth was restricted by suboptimal temperature or nutritional deficiencies, the drug was not lethal. Chemical analysis of cellular constituents revealed that lipid, protein, and ribonucleic acid levels were of the same order of magnitude in control and drug-treated cells. Only deoxyribonucleic acid (DNA) levels were markedly lowered in drug-treated cells. These facts are consistent with the view that nalidixic acid interferes with the synthesis of E. coli DNA.
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Abstract CircRNAs are a novel class of RNA molecules with a unique closed continuous loop structure. CircRNAs are abundant in eukaryotic cells, have unique stability and tissue specificity, and can play a biological regulatory role at various levels, such as transcriptional and posttranscriptional levels. Numerous studies have indicated that circRNAs serve a crucial purpose in cancer biology. CircRNAs regulate tumor behavioral phenotypes such as proliferation and migration through various molecular mechanisms, such as miRNA sponging, transcriptional regulation, and protein interaction. Recently, several reports have demonstrated that they are also deeply involved in resistance to anticancer drugs, from traditional chemotherapeutic drugs to targeted and immunotherapeutic drugs. This review is the first to summarize the latest research on circRNAs in anticancer drug resistance based on drug classification and to discuss their potential clinical applications.
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Antibiotics halt the growth of bacteria by targeting core, essential physiology that is required for life on standard microbiological media. Many more biochemical and virulence processes, however, are required for bacteria to cause infection in a host. Indeed, chemical inhibitors of the latter processes are overlooked using conventional antibiotic drug discovery approaches. Here, we use human blood serum as an alternative growth medium to explore new targets and compounds. High-throughput screening of genetic and chemical libraries identified compounds targeting biological activities required by Klebsiella pneumoniae to grow in serum, such as nucleobase biosynthesis and iron acquisition, and showed that serum can chemically transform compounds to reveal cryptic antibacterial activity. One of these compounds, ruthenium red, was effective in a rat bloodstream infection model. Our data demonstrate that human serum is an effective tool to find new chemical matter to address the current antibiotic resistance crisis.
Chapter
The discovery and clinical utilization of antibiotics is one of the most important breakthroughs that has revolutionized medical science and significantly contributed to saving millions of lives. However, due to irrational, rampant misuse of antibiotics along with poor patient compliance has led to current state of almost entering postantibiotic era because of a phenomenon called antimicrobial resistance (AMR). Furthermore, the problem has become compounded due to the dearth of novel and effective antibacterials being discovered and developed owing to a multitude of factors, including ever increasing costs of discovery and development as well as complex and costly clinical trials for regulatory approval. As a result of this, recent reports suggest an estimated death toll of 700,000 deaths each year attributable to AMR along with future projected deaths reaching ∼10 million by 2050. Thus new policies to invigorate antibiotic discovery and development are required to avoid postantibiotic era.
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The aim of the current study is to present a strategy to improve the efficiency of 5-fluorouracil (5-FU), which is widely used as antineoplastic agent against solid tumors-based on the use of gold nanocarriers to overcome the resistance of colorectal cancer cells. 5-FU was loaded on gold nanoparticles (AuNP) coated with anti-EGFR antibodies in order to target them towards colorectal cancer cells that overexpress epidermal growth factor receptors (EGFR). Physicochemical characterization has shown that AuNP size was approximately 20 nm and that AuNP functionalization led to spherical nanoparticles. Flow cytometry allowed observing that some compounds synthesized by our research group have induced apoptosis/necrosis and impaired the proliferation of colon cancer cell lines ‘HCT-116′ and ‘HT-29′. The antibody/drug combination in AuNP (AuNP 5FU EGFR) has improved the apoptosis rate and impaired cell proliferation in both cell lines, regardless of the exposure time. Overall, these results have shown that AuNP functionalization with monoclonal antibodies focused on delivering 5-FU to tumor cells is an exciting strategy against colorectal cancer.
Chapter
Substrate analogs may be defined as compounds which bear a close chemical and/or physical resemblance to a constituent of normal tissue and which may replace the latter in one or more of its normal reactions, usually leading to a metabolic block. The substrate analogs, or antimetabolites, usually compete with the natural substrate at the same site on an enzyme. Actually, only a portion of the antimetabolite molecule must be analogous for competition.
Article
Studies have been described with a thymine-requiring strain of E. coli. This organism dies in the absence of its requirement. It has been shown that nuclear and cytoplasmic syntheses are no longer balanced under conditions of thymine deficiency and continuing unbalanced synthesis results in the loss of the power to multiply. Thymine deficiency and death of other bacterial strains may be provoked by sulfanilamide in the presence of certain metabolites which then support unbalanced growth. The killing action of low doses of ultraviolet irradiation also appears to depend on growth and may be prevented by inhibiting protein synthesis. It was shown that many bactericidal treatments are similar in that they appear to affect nucleic acid metabolism and to require continuing growth for their lethal actions to be manifest. A selective inhibition of DNA synthesis appears to be capable of inducing death by unbalanced growth.
  • S S Cohen
  • H D Barner
S. S. Cohen and H. D. Barner, these PROCEEDINGS, 40, 885, 1954.
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S. Cohen, Texas Repts. Biol. and Med., 15, 154, 1957.
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J. Rutman, A. Cantarow, and K. E. Paschkis, Cancer Research, 14, 119, 1954. VOL. 44) 1958 BIOCHEMISTRY: COHEN ET AL.
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Heidelberger, N. K. Chaudhuri, P. Danneberg, D. Mooren, L. Griesbach, R. Duschinsky, R. J. Schnitzer, E. Pleven, and J. Scheiner, Nature, 179, 663, 1957. 10 L. Bosch, E. Harbers, and C. Heidelberger, Cancer Research, 18, 335, 1958. wa J. M. Scheiner, E. Kostelak, and R. Duschinsky, Fed. Proc., 16, 242, 1957.
this strain with T2 bacteriophage induced the organism , in some manner, to synthesize both thymine and HMC It has now been shown in this laboratory that the syntheses of these pyrimidines occur at the nucleotide levels by means of two different enzymes
  • D Howeverh
  • S S Barner
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  • Bact
However, it was observed that infection of this strain with T2 bacteriophage induced the organism, in some manner, to synthesize both thymine and HMC (H. D. Barner and S. S. Cohen, J. Bact., 68, 80, 1954). It has now been shown in this laboratory that the syntheses of these pyrimidines occur at the nucleotide levels by means of two different enzymes. Thymidylate synthetase: UDRP + HCHO + 2H THFA TI)RP. Deoxycytidylate hydroxymethylase: CDRP + HCHO THFA HMCDRP.
15 A sample of enzymatically synthesized FURP was kindly given to us by Dr
  • S Cohen
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S. Cohen, J. Lichtenstein, H. D. Barner, and M. Green, J. Biol. Chem., 228, 611, 1957. 14 R. L. Sinsheimer and J. F. Koerner, Science, 114, 42, 1951. 15 A sample of enzymatically synthesized FURP was kindly given to us by Dr. Charles Heidelberger, of the University of Wisconsin.
70 vols. ethanol :30 vols. M ammonium acetate (pH 9) saturated with sodium tetraborate
  • Plesner
Plesner, Acta Chem. Scand., 9, 197, 1955. 70 vols. ethanol :30 vols. M ammonium acetate (pH 9) saturated with sodium tetraborate.
  • W G Farkas
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16a W. G. Farkas, N. C. Iacono, and R. Duschinsky, Abstracts Fourth Inter. Congress Biochemistry, Vienna, Sept. 1958.
Purification from the bacterial extract involved streptomycin precipitation of nucleic acid, heat denaturation at 55-60°, and fractional ethanol precipitation of the enzyme
  • Arthur Kornberg Of Washington
  • University
  • St
  • Louis
Arthur Kornberg of Washington University, St. Louis. Purification from the bacterial extract involved streptomycin precipitation of nucleic acid, heat denaturation at 55-60°, and fractional ethanol precipitation of the enzyme.
we have synthesized the deoxyriboside in gram quantities by exchanging fluorouracil with thymidine in a ratio of 3 moles of FU to 1 mole of TDR in the presence of a bacterial nucleoside phosphorylase The reaction could readily be followed by paper electrophoresis of the incubation mixture for 2
  • N J Nutley
Nutley, N. J. In addition, with the participation of Mr. A. Balsam and Mr. E. Kean, we have synthesized the deoxyriboside in gram quantities by exchanging fluorouracil with thymidine in a ratio of 3 moles of FU to 1 mole of TDR in the presence of a bacterial nucleoside phosphorylase (M. Green and S. S. Cohen, J. Biol. Chem., 225, 397, 1957). The reaction could readily be followed by paper electrophoresis of the incubation mixture for 2. 5 hours in 0.2 M borate buffer at pH 9.2
  • S S Cohen
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S. S. Cohen and H. D. Barner, J. Bact., 71, 588, 1956.
  • H D Barner
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H. D. Barner and S. S. Cohen, J. Bact., 74, 350, 1957. 4H. D. Barner and S. S. Cohen, Biochem. Biophys. Acta (in press).
  • L Bosch
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L. Bosch, E. Harbers, and C. Heidelberger, Cancer Research, 18, 335, 1958. wa J. M. Scheiner, E. Kostelak, and R. Duschinsky, Fed. Proc., 16, 242, 1957.
  • R L Sinsheimer
  • J F Koerner
R. L. Sinsheimer and J. F. Koerner, Science, 114, 42, 1951.
  • W G Farkas
  • N C Iacono
  • R Duschinsky
W. G. Farkas, N. C. Iacono, and R. Duschinsky, Abstracts Fourth Inter. Congress Biochemistry, Vienna, Sept. 1958.
  • J G Flaks
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J. G. Flaks and S. S. Cohen, Biochem. Biophys. Acta., 25, 667, 1957; Fed. Proc., 17, 220, 1958.