Mechanistic Evaluation of a Nucleoside Tetraphosphate with a Thymidylyltransferase

Pyrimidine polyphosphates were first detected in cells 5 decades ago; however, their biological significance remains only partially resolved. Such nucleoside polyphosphates are believed to be produced nonspecifically by promiscuous enzymes. Herein, synthetically prepared deoxythymidine 5′-tetraphosphate (p₄dT) was evaluated with a thymidylyltransferase, Cps2L. We have identified p₄dT as a substrate for Cps2L and evaluated the reaction pathway by analysis of products using high-performance liquid chromatography, liquid chromatography and tandem mass spectrometry, and ³¹P nuclear magnetic resonance spectroscopy. Product analysis confirmed production of dTDP-Glc and triphosphate (P₃) and showed no trace of dTTP-Glc and PP_i, which could arise from alternative pathways for the reaction mechanism

Synthesis of α‑Deoxymono and Difluorohexopyranosyl 1‑Phosphates and Kinetic Evaluation with Thymidylyl- and Guanidylyltransferases

Eight fluorinated isosteric α-d-glucopyranosyl 1-phosphate (<b>Glc 1P</b>) analogues have been synthesized. A promiscuity investigation of the thymidylyltransferase Cps2L and the guanidylyltansferase GDP-ManPP with these analogues showed that all were accepted by either enzyme, with the exception of 1,6-diphosphate <b>6</b>. Kinetic parameters were determined for these analogues using a continuous coupled assay. These data demonstrated the broad substrate promiscuity of Cps2L, with <i>k</i>_cat/<i>K</i>_m changes for monofluoro substitution at C-2, C-4, and C-6 and difluoro substitution at C-2 within two orders of magnitude. In contrast, the kinetic analysis of GDP-ManPP was only possible with three out of eight analogues. The p<i>K</i>a₂ values of analogues (<b>1</b>–<b>3</b>) were determined by proton decoupled ³¹P and ¹⁹F NMR titration experiments. Counterintuitively, the axial fluoro substituent in <b>3</b> did not change chemical shift upon titration, and there was no significant increase in acidity for the difluoro analogue over the monofluoro analogues. No strong Brønsted linear free-energy correlations were observed among all five substrates (<b>1</b>–<b>3</b>, <b>Glc 1P</b>, and <b>Man 1P</b>) for either enzyme-catalyzed reactions. However, Brønsted correlations were observed among selected substrates, indicating that the acidity of the nucleophilic phosphate and the configuration of the hexose each plays a significant role in determining the substrate specificity

Characterization of l‑Digitoxosyl-phenanthroviridin from <i>Streptomyces venezuelae</i> ISP5230

The jadomycin-derived compound l-digitoxosyl-phenanthroviridin was isolated from fermentations of <i>Streptomyces venezuelae</i> ISP5230 grown in nutrient-deficient media with l-lysine as the sole nitrogen source. Structural elucidation was accomplished using a combination of high-resolution MS, LC-MS/MS, and 1D- and 2D-NMR. The compound was evaluated against the National Cancer Institute (NCI) 60 human tumor cell line screen in both the one-dose and five-dose screens, and cytotoxicity was compared to a small library of jadomycin analogues to probe the structure–activity relationship

Synthesis and Evaluation of l‑Rhamnose 1C-Phosphonates as Nucleotidylyltransferase Inhibitors

We report the synthesis of a series of phosphonates and ketosephosphonates possessing an l-rhamnose scaffold with varying degrees of fluorination. These compounds were evaluated as potential inhibitors of α-d-glucose 1-phosphate thymidylyltransferase (Cps2L), the first enzyme in Streptococcus pneumoniae l-rhamnose biosynthesis, and a novel antibiotic target. Enzyme–substrate and enzyme–inhibitor binding experiments were performed using water-ligand observed binding via gradient spectroscopy (WaterLOGSY) NMR for known sugar nucleotide substrates and selected phosphonate analogues. IC₅₀ values were measured and <i>K</i>_i values were calculated for inhibitors. New insights were gained into the binding promiscuity of enzymes within the prokaryotic l-rhamnose biosynthetic pathway (Cps2L, RmlB–D) and into the mechanism of inhibition for the most potent inhibitor in the series, l-rhamnose 1C-phosphonate

Peripheral intravenous infusion in newborns infants: reasons for infusion discontinuance and involveed factors

A terapia intravenosa periferica, parte da assistencia de enfermagem ao recem-nascido, fica sob cuidados e responsabilidade da enfermagem. A partir da prescricao medica, e a enfermeira que determinara o local, tipo e calibre do dispositivo, modo de fixacao e de instalacao desta terapia intravenosa, incluindo aspectos relacionados a diluicao e incompatibilidade medicamentosa. Com o objetivo de identificar os motivos que acarretam a interrupcao da infusao intravenosa, analisar o tempo de permanencia de cada dispositivo intravascular e solucoes utilizadas, realizou-se um estudo descritivo correlacional prospectivo em 650 insercoes intravenosas perifericas, tendo sido 331(50,9 por cento) realizadas com dispositivo agulha com asas e 319(4 9,1 por cento) com dispositivo cateter platico. Foram estudados 100 recem-nascidos pre-termo, subdivididos em tres grupos de acordo com a idade gestacional. Os dados deste estudo foram coletados no periodo de outubro de 1996 a outubro de 1997. Os resultados revelam que os principais motivos de interrupcao intravenosa foram, em ordem decrescente, a infiltracao , a flebite e a necrose. A agulha com asas e o catetor plastico nao apresentaram diferencas significante em relacao ao local de insercao(cabeca, membros superiores e membros inferiores) e tempo de permanencia. A presenca de flebite foi mais frequente (19,1 por cento) no dispositivo cateter plastico do que no dispositivo agulha com asas (8,5 por cento). Oteste de Fisher mostrou associacao significante entre o uso do gluconato de calcio 10 por cento, do cloreto de potassio 19,1 por cento e a presenca de necrose e entre o uso associado de cloreto de sodio 3 por cento, cloreto de potassio 19,1 por cento e glicose 50 por cento e a presenca de flebite. A escolha inadequada do dispositivo intravascular pode pesar na qualidade de assistencia de enfermagem, de forma a ocasionar sofrimento adicional e, sobretudo, desnecessario ao recem-nascido. Medidas para evitar a infiltracao, flebite e necrose continuam sendo imprescindiveis, pois estas ocorrencias implicam necessariamente, na retirada da insercao e consequente reinsercao, quando incontrolavelmente repetida resulta na inacessibilidade de veias perifericas e, por conseguinte, na inevitabilidade do acesso por via centralBV UNIFESP: Teses e dissertaçõe

CASA-F: Uma Ferramenta para Obtenção de Pontos de Controle por Casamento de Feições

Um problema freqüentemente encontrado em registro de imagens é a obtenção de pontos de controle. Uma quantidade razoável de pontos de controle é necessária para que um processo de registro de imagens seja realizado com sucesso. Em imagens que cobrem uma vasta região, nem sempre estes pontos de controle são encontrados com facilidade. Este artigo apresenta uma ferramenta, que está em fase em desenvolvimento, para a obtenção de pontos de controle a partir da extração e casamento de feições

Jadomycins Derived from the Assimilation and Incorporation of Norvaline and Norleucine

<i>Streptomyces venezuelae</i> ISP5230 is recognized for the production of chloramphenicol and the jadomycin family of natural products. The jadomycins are angucycline natural products containing a unique oxazolone ring incorporating an amino acid present in the minimal culture media. Substitution of different amino acids results in products of varying biological activity. Analysis of cultures of <i>S. venezuelae</i> ISP5230 incubated with l- and d-norvaline and l- and d-norleucine indicated that only the d-configured amino acids were incorporated into the natural products. Subsequently, jadomycin DNV and jadomycin DNL were isolated and characterized (titers 4 and 9 mg L^–1, respectively). The compounds were evaluated in the National Cancer Institute cell line cancer growth inhibition and cytotoxicity screens, for antimicrobial activity against selected Gram-positive and Gram-negative bacteria, and as DNA-cleavage agents <i>in vitro</i>

Eight-Membered Ring-Containing Jadomycins: Implications for Non-enzymatic Natural Products Biosynthesis

Jadomycin Oct (<b>1</b>) was isolated from Streptomyces venezuelae ISP5230 and characterized as a structurally unique eight-membered l-ornithine ring-containing jadomycin. The structure was elucidated through the semisynthetic derivatization of starting material via chemoselective acylation of the l-ornithine α-amino group using activated succinimidyl esters. Incorporation of 5-aminovaleric acid led to jadomycin AVA, a second eight-membered ring-containing jadomycin. These natural products illustrate the structural diversity permissible from a non-enzymatic step within a biosynthetic pathway and exemplifies the potential for discovery of novel scaffolds

Post Polyketide Synthase Carbon–Carbon Bond Formation in Type-II PKS-Derived Natural Products from <i>Streptomyces venezuelae</i>

Polyketide synthase (PKS) derived natural products are biosynthesized by head-to-tail addition of acetate and malonate extender units resulting in linear extended-polyketide chains. Despite the well-documented structural diversity associated with PKS-derived natural products, C–C chain branching deviating from the usual linear pattern is relatively rare. Herein, type-II PKS angucyclic natural products containing a hemiaminal functionality were identified and proposed as the parent of a series of C–C-branched analogues. These C–C linked acetate or pyruvate branching units were located at the α-positions on the extended polyketide chains of jadomycins incorporating 3- and 4-aminomethylbenzoic acids. Labeling studies utilizing [1-¹³C]-d-glucose provided mechanistic evidence that the C–C bond formation occurred as a result of a previously unidentified post-PKS processing, additional to the enzymes encoded within the biosynthetic gene cluster. Selected compounds were evaluated in cytotoxic or antimicrobial assays