Inhibition of DNA polymerase reaction by pyrimidine nucleotide analogues lacking the 2-keto group

To investigate the influence of the pyrimidine 2-keto group on selection of nucleotides for incorporation into DNA by polymerases, we have prepared two C nucleoside triphosphates that are analogues of dCTP and dTTP, namely 2-amino-5-(2'-deoxy-beta-d-ribofuranosyl)pyridine-5'-triphosphate (d*CTP) and 5-(2'-deoxy- beta-d-ribofuranosyl)-3-methyl-2-pyridone-5'-triphosphate (d*TTP) respectively. Both proved strongly inhibitory to PCR catalysed by Taq polymerase; d*TTP rather more so than d*CTP. In primer extension experiments conducted with either Taq polymerase or the Klenow fragment of Escherichia coli DNA polymerase I, both nucleotides failed to substitute for their natural pyrimidine counterparts. Neither derivative was incorporated as a chain terminator. Their capacity to inhibit DNA polymerase activity may well result from incompatibility with the correctly folded form of the polymerase enzyme needed to stabilize the transition state and catalyse phosphodiester bond formation.

Nucleic-Acid Analogs with Restricted Conformational Flexibility in the Sugar-Phosphate Backbone (‘Bicyclo-DNA’). Part 5. Synthesis, Characterization, and Pairing Properties of Oligo-αlpha-D-(bicyclodeoxynucleotides) of the Bases Adenine and Thymine (αlpha-Bicyclo-DNA)

10.1002/hlca.19950780816.abs A conformational analysis of the (3′S,5′R)-2′-deoxy-3′,5′-ethano-α-D-ribonucleosides (a-D-bicyclodeoxynucleosides) based on the X-ray analysis of N4-benzoyl-α-D-(bicyclodeoxycytidine) 6 and on 1H-NMR analysis of the α-D-bicyclodeoxynucleoside derivatives 1-7 reveals a rigid sugar structure with the furanose units in the l′-exo/2′-endo conformation and the secondary OH groups on the carbocyclic ring in the pseudoequatorial orientation. Oligonucleotides consisting of α-D-bicyclothymidine and α-D-bicyclodeoxyadenosine were successfully synthesized from the corresponding nucleosides by phosphoramidite methodology on a DNA synthesizer. An evaluation of their pairing properties with complementary natural RNA and DNA by means of UV/melting curves and CD spectroscopy show the following characteristics: i) α-bcd(A10) and α-bcd(T10) (α = short form of α-D)efficiently form complexes with complementary natural DNA and RNA. The stability of these hybrids is comparable or slightly lower as those with natural β-d(A10) or β-d(T10)( β = short form ofβ-D). ii) The strand orientation in α-bicyclo-DNA/β-DNA duplexes is parallel as was deduced from UV/melting curves of decamers with nonsymmetric base sequences. iii) CD Spectroscopy shows significant structural differences between α-bicyclo-DNA/β-DNA duplexes compared to α-DNA/β-DNA duplexes. Furthermore, α-bicyclo-DNA is ca. 100-fold more resistant to the enzyme snake-venom phosphodiesterase with respect to β-DNA and about equally resistant as α-DNA.

A Method for Preparing Oligodeoxynucleotides Containing an Apurinic Site

10.1002/hlca.19900730309.abs In three steps, 2-deoxy-D-ribose has been converted into a phosphoramidite building block bearing a (t-Bu)Me2Si protecting group at the OH function of the anomeric centre of the furanose ring. This building block was subsequently incorporated into DNA oligomers of various base sequences using the standard phosphoramidite protocol for automated DNA synthesis. The resulting silyl-oligomers have been purified by HPLC and selectively desilylated to the corresponding free apurinic DNA sequences. The hexamer d (A-A-A-A-X-A) (X representing the apurinic site) which was prepared in this way was characterized by 1H- and 31P-NMR spectroscopy. The other sequences as well as their fragments, which formed upon treatment with alkali base, were analyzed by polyacrylamide gel electrophoresis.

Lipid- and mechanosensitivities of sodium/hydrogen exchangers analyzed by electrical methods.

Sodium/hydrogen exchangers (NHEs) are ubiquitous ion transporters that serve multiple cell functions. We have studied two mammalian isoforms, NHE1 (ubiquitous) and NHE3 (epithelial-specific), by measuring extracellular proton (H+) gradients during whole-cell patch clamp with perfusion of the cell interior. Maximal Na(+)-dependent H+ fluxes (JH+) are equivalent to currents >20 pA for NHE1 in Chinese hamster ovary fibroblasts, >200 pA for NHE1 in guinea pig ventricular myocytes, and 5-10 pA for NHE3 in opossum kidney cells. The fluxes are blocked by an NHE inhibitor, ethylisopropylamiloride, and are absent in NHE-deficient AP-1 cells. NHE1 activity is stable with perfusion of nonhydrolyzable ATP [adenosine 5'-(beta,gamma-imido)triphosphate], is abolished by ATP depletion (2 deoxy-D-glucose with oligomycin or perfusion of apyrase), can be restored with phosphatidylinositol 4,5-bisphosphate, and is unaffected by actin cytoskeleton disruption (latrunculin or pipette perfusion of gelsolin). NHE3 (but not NHE1) is reversibly activated by phosphatidylinositol 3,4,5-trisphosphate. Both NHE1 and NHE3 activities are disrupted in giant patches during gigaohm seal formation. NHE1 (but not NHE3) is reversibly activated by cell shrinkage, even at neutral cytoplasmic pH without ATP, and inhibited by cell swelling. NHE1 in Chinese hamster ovary fibroblasts (but not NHE3 in opossum kidney cells) is inhibited by agents that thin the membrane (L-alpha-lysophosphatidylcholine and octyl-beta-D-glucopyranoside) and activated by cholesterol enrichment, which thickens membranes. Expressed in AP-1 cells, however, NHE1 is insensitive to these agents but remains sensitive to volume changes. Thus, changes of hydrophobic mismatch can modulate NHE1 but do not underlie its volume sensitivity.

Total Synthesis of Prostaglandin 15d-PGJ(2) and Investigation of its Effect on the Secretion of IL-6 and IL-12.

An efficient synthesis of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2, 1) is reported. The route described allows for diversification of the parent structure to prepare seven analogues of 1 in which the positioning of electrophilic sites is varied. These analogues were tested in SAR studies for their ability to reduce the secretion of proinflammatory cytokines. It was shown that the endocyclic enone is crucial for the bioactivity investigated and that the conjugated ω-side chain serves in a reinforcing manner.

Membrane Lipid Integrity Relies on a Threshold of ATP Production Rate in Potato Cell Cultures Submitted to Anoxia

In this paper we report on our study of the changes in biomass, lipid composition, and fermentation end products, as well as in the ATP level and synthesis rate in cultivated potato (Solanum tuberosum) cells submitted to anoxia stress. During the first phase of about 12 h, cells coped with the reduced energy supply brought about by fermentation and their membrane lipids remained intact. The second phase (12–24 h), during which the energy supply dropped down to 1% to 2% of its maximal theoretical normoxic value, was characterized by an extensive hydrolysis of membrane lipids to free fatty acids. This autolytic process was ascribed to the activation of a lipolytic acyl hydrolase. Cells were also treated under normoxia with inhibitors known to interfere with energy metabolism. Carbonyl-cyanide-4-trifluoromethoxyphenylhydrazone did not induce lipid hydrolysis, which was also the case when sodium azide or salicylhydroxamic acid were fed separately. However, the simultaneous use of sodium azide plus salicylhydroxamic acid or 2-deoxy-D-glucose plus iodoacetate with normoxic cells promoted a lipid hydrolysis pattern similar to that seen in anoxic cells. Therefore, a threshold exists in the rate of ATP synthesis (approximately 10 μmol g−1 fresh weight h−1), below which the integrity of the membranes in anoxic potato cells cannot be preserved.

HETEROGENEITY IN VIRUS EXPRESSION, TUMORIGENICITY, AND IN VITRO GROWTH PROPERTIES OF DRUG-RESISTANT CLONES OF AN RIII MOUSE MAMMARY TUMOR CELL LINE

Four 8-azaguanine (AG)-resistant and 5-bromodeoxyuridine (BUdR)-resistant clones of a mouse mammary adenocarcinoma cell line, RIII 7387, were developed and analyzed for their tumorigenic properties, in vitro characteristics, and virus expression. These characteristics were analyzed for relationships of any of the cellular parameters and the ability of these lines to produce tumors in syngeneic animals.^ The results of this study demonstrated that the parental line consists of a heterogeneous population of cells. Doubling times, saturation densities, and 2-deoxy-D-glucose uptake varied between sublines. In addition, while all sublines were found to express both B-type and C-type viral antigenic markers, levels of the major B-type and C-type viral proteins varied in the subclones. The sublines also differed markedly in their response to the presence of dexamethasone, glutathione, and insulin in the tissue culture medium.^ Variations in retrovirus expression were convirmed by electron microscopy. Budding and extracellular virus particles were seen in the majority of the cell lines. Virus particles in one of the BUdR-resistant lines, BUD9, were found however, only in inclusions and vacuoles. The AG-resistant subline AGE11 was observed to be rich in intracytoplasmic A particles. The examination of these cell lines for the presence of retroviral RNA-dependent DNA polymerase (RT) activity revealed that some B-type RT activity could be found in the culture fluid of most of the cell lines but that little C-type RT activity could be found suggesting that the C-type virus particles expressed by these RIII clones contain a defective RT.^ Tumor clones also varied in their ability to form tumors in syngeneic RIII mice. Tumor incidence ranged from 50% to 100%. The majority of the tumors regressed within 30 days post infection.^ Statistical analysis indicated that while these clones varied in their characteristics, there was no correlation between the ability of these cell lines to form tumors in syngeneic mice and any of the other characteristics examined.^ These studies have confirmed and extended the growing evidence that tumors, regardless of their natural origin, consist of heterogeneous subpopulations of cells which may vary widely in their in vitro growth behavior, their antigenic expression, and their malignant properties. ^

ASSESSMENT OF SKELETAL MUSCLE BLOOD FLOW AND GLUCOSE METABOLISM WITH POSITRON EMITTING RADIONUCLIDES

In order to evaluate factors regulating substrate metabolism in vivo positron emitting radionuclides were used for the assessment of skeletal muscle blood flow and glucose utilization. The potassium analog, Rb-82 was used to measure skeletal muscle blood flow and the glucose analog, 18-F-2-deoxy-2-fluoro-D-glucose (FDG) was used to examine the kinetics of skeletal muscle transport and phosphorylation.^ New Zealand white rabbits' blood flow ranged from 1.0-70 ml/min/100g with the lowest flows occurring under baseline conditions and the highest flows were measured immediately after exercise. Elevated plasma glucose had no effect on increasing blood flow, whereas high physiologic to pharmacologic levels of insulin doubled flow as measured by the radiolabeled microspheres, but a proportionate increase was not detected by Rb-82. The data suggest that skeletal muscle blood flow can be measured using the positron emitting K+ analog Rb-82 under low flow and high flow conditions but not when insulin levels in the plasma are elevated. This may be due to the fact that insulin induces an increase in the Na+/K+-ATPase activity of the cell indirectly through a direct increase in the Na+/H+pump activity. This suggests that the increased cation pump activity counteracts the normal decrease in extraction seen at higher flows resulting in an underestimation of flow as measured by rubidium-82.^ Glucose uptake as measured by FDG employed a three compartment mathematical model describing the rates of transport, countertransport and phosphorylation of hexose. The absolute values for the metabolic rate of FDG were found to be an order of magnitude higher than those reported by other investigators. Changes noted in the rate constant for transport (k1) were found to disagree with the a priori information on the effects of insulin on skeletal muscle hexose transport. Glucose metabolism was however, found to increase above control levels with administration of insulin and electrical stimulation. The data indicate that valid measurements of skeletal muscle glucose transport and phosphorylation using the positron emitting glucose analog FDG requires further model application and biochemical validation. (Abstract shortened with permission of author.) ^

Metabolism and actions of 2 -chloro-2$\sp\prime$-fluoro-arabinosyladenine

2-Chloro-9-(2-deoxy-2-fluoro-$\beta $-D-arabinofuranosyl)adenine(Cl-F-ara-A) is a new deoxyadenosine analogue which is resistant to phosphorolytic cleavage and deamination, and exhibits therapeutic activity for both leukemia and solid tumors in experimental systems. To characterize its mechanism of cytotoxicity, the present study investigated the cellular pharmacology and the biochemical and molecular mechanisms of action of Cl-F-ara-A, from entrance of the drug into the cell, chemical changes to active metabolites, targeting on different cellular enzymes, to final programmed cell death response to the drug treatment.^ Cl-F-ara-A exhibited potent inhibitory action on DNA synthesis in a concentration-dependent and irreversible manner. The mono-, di-, and triphosphates of Cl-F-ara-A accumulated in cells, and their elimination was non-linear with a prolonged terminal phase, which resulted in prolonged dNTP depression. Ribonucleotide reductase activity was inversely correlated with the cellular Cl-F-ara-ATP level, and the inhibition of the reductase was saturated at higher cellular Cl-F-ara-ATP concentrations. The sustained inhibition of ribonucleotide reductase and the consequent depletion of deoxynucleotide triphosphate pools result in a cellular Cl-F-ara-ATP to dATP ratio which favors analogue incorporation into DNA.^ Incubation of CCRF-CEM cells with Cl-F-ara-A resulted in the incorporation of Cl-F-ara-AMP into DNA. A much lesser amount was associated with RNA, suggesting that Cl-F-ara-A is a more DNA-directed compound. The site of Cl-F-ara-AMP in DNA was related to the ratio of the cellular concentrations of the analogue triphosphate and the natural substrate dATP. Clonogenicity assays showed a strong inverse correlation between cell survival and Cl-F-ara-AMP incorporation into DNA, suggesting that the incorporation of Cl-F-ara-A monophosphate into DNA is critical for the cytotoxicity of Cl-F-ara-A.^ Cl-F-ara-ATP competed with dATP for incorporation into the A-site of the extending DNA strand catalyzed by both DNA polymerase $\alpha$ and $\varepsilon$. The incorporation of Cl-F-ara-AMP into DNA resulted in termination of DNA strand elongation, with the most pronounced effect being observed at Cl-F-ara-ATP:dATP ratio $>$1. The presence of Cl-F-ara-AMP at the 3$\sp\prime$-terminus of DNA also resulted in an increased incidence of nucleotide misincorporation in the following nucleotide position. The DNA termination and the nucleotide misincorporation induced by the incorporation of Cl-F-ara-AMP into DNA may contribute to the cytotoxicity of Cl-F-ara-A. ^

FDG-PET Intra-tratamiento como valor pronóstico en el tratamiento del linfoma Hodgkin

Introducción: La utilización de regímenes de tratamiento más individualizados requiere de mejores sistemas de estratificación temprana en Linfoma Hodgkin (LH). El estudio Tomografía por Emisión de Positrones utilizando 2-[18F] fluoro-2-deoxi-Dglucosa (FDG-PET) intra-tratamiento podría jugar un rol muy importante en esta evaluación. Objetivo: Determinar el valor pronóstico del FDG-PET intra-tratamiento en pacientes con LH para predecir sobrevida libre de progresión y sobrevida global. Material y método: El estudio fue llevado a cabo en el Servicio de Hematología del Hospital Central de Mendoza incluyendo pacientes con diagnóstico de LH confirmados por histología. De acuerdo al estadio y sitio de presentación, los pacientes recibieron quimioterapia sola o la combinación de radioterapia y quimioterapia, con el uso del esquema ABVD (adriamicina, bleomicina, vinblastina y dacarbazina) como protocolo estándar. Los estudios FDG-PET fueron practicados como parte de la evaluación intra-tratamiento y a la finalización. Resultados: En total fueron evaluados 8 pacientes, Sexo: F/M: 4/4, Edad: 18-58 años (Mediana: 29 años), Estadios: IIB:1, IIIA:2, IIIB:1, IVA:1, IVB:3, regiones nodales: 2-10 (Mediana:4), compromiso extranodal: 4/8, síntomas B: 5/8, enfermedad bulky 2/8 . Subtipos: Escleronodular: 6/8, Celularidad mixta: 1/8, Depleción linfocítica: 1/8. IPS: 1: 3/8 2: 3/8 3: 1/8 4: 0/8 ≥ 5: 1/8. Tratamientos: ABVD x 6: 6/8, ABVD x 6 + Radioterapia: 2/8. PET intermedio: 8/8 negativos (6/8 PET 3, 2/8 PET 2). PET final: 7/8 PET negativo, 1/8 PET positivo. Recaída: 1/8 (10° mes). Seguimiento: 11-37 meses (mediana de 24 meses). Discusión y Conclusiones: Al momento actual el FDG-PET intra-tratamiento demostró tener un importante valor predictivo negativo dado que todos los pacientes, menos uno, se encuentran en remisión completa sin progresión de enfermedad. Resta aún determinar el rol que esta herramienta pueda tener en el futuro en la terapia adaptada al riesgo de pacientes con LH.

Ordered water molecules as key allosteric mediators in a cooperative dimeric hemoglobin

One of the most remarkable structural aspects of Scapharca dimeric hemoglobin is the disruption of a very well-ordered water cluster at the subunit interface upon ligand binding. We have explored the role of these crystallographically observed water molecules by site-directed mutagenesis and osmotic stress techniques. The isosteric mutation of Thr-72 → Val in the interface increases oxygen affinity more than 40-fold with a surprising enhancement of cooperativity. The only significant structural effect of this mutation is to destabilize two ordered water molecules in the deoxy interface. Wild-type Scapharca hemoglobin is strongly sensitive to osmotic conditions. Upon addition of glycerol, striking changes in Raman spectrum of the deoxy form are observed that indicate a transition toward the liganded form. Increased osmotic pressure, which lowers the oxygen affinity in human hemoglobin, raises the oxygen affinity of Scapharca hemoglobin regardless of whether the solute is glycerol, glucose, or sucrose. Analysis of these results provides an estimate of six water molecules lost upon oxygen binding to the dimer, in good agreement with eight predicted from crystal structures. These experiments suggest that the observed cluster of interfacial water molecules plays a crucial role in communication between subunits.

Insulin-like growth factor 1 regulates developing brain glucose metabolism

The brain has enormous anabolic needs during early postnatal development. This study presents multiple lines of evidence showing that endogenous brain insulin-like growth factor 1 (Igf1) serves an essential, insulin-like role in promoting neuronal glucose utilization and growth during this period. Brain 2-deoxy-d- [1-14C]glucose uptake parallels Igf1 expression in wild-type mice and is profoundly reduced in Igf1−/− mice, particularly in those structures where Igf1 is normally most highly expressed. 2-Deoxy-d- [1-14C]glucose is significantly reduced in synaptosomes prepared from Igf1−/− brains, and the deficit is corrected by inclusion of Igf1 in the incubation medium. The serine/threonine kinase Akt/PKB is a major target of insulin-signaling in the regulation of glucose transport via the facilitative glucose transporter (GLUT4) and glycogen synthesis in peripheral tissues. Phosphorylation of Akt and GLUT4 expression are reduced in Igf1−/− neurons. Phosphorylation of glycogen synthase kinase 3β and glycogen accumulation also are reduced in Igf1−/− neurons. These data support the hypothesis that endogenous brain Igf1 serves an anabolic, insulin-like role in developing brain metabolism.

An important 2′-OH group for an RNA–protein interaction

We have investigated the role of 2′-OH groups in the specific interaction between the acceptor stem of Escherichia coli tRNACys and cysteine-tRNA synthetase. This interaction provides for the high aminoacylation specificity observed for cysteine-tRNA synthetase. A synthetic RNA microhelix that recapitulates the sequence of the acceptor stem was used as a substrate and variants containing systematic replacement of the 2′-OH by 2′-deoxy or 2′-O-methyl groups were tested. Except for position U73, all substitutions had little effect on aminoacylation. Interestingly, the deoxy substitution at position U73 had no effect on aminoacylation, but the 2′-O-methyl substitution decreased aminoacylation by 10-fold and addition of the even bulkier 2′-O-propyl group decreased aminoacylation by another 2-fold. The lack of an effect by the deoxy substitution suggests that the hydrogen bonding potential of the 2′-OH at position U73 is unimportant for aminoacylation. The decrease in activity upon alkyl substitution suggests that the 2′-OH group instead provides a monitor of the steric environment during the RNA–synthetase interaction. The steric role was confirmed in the context of a reconstituted tRNA and is consistent with the observation that the U73 base is the single most important determinant for aminoacylation and therefore is a site that is likely to be in close contact with cysteine-tRNA synthetase. A steric role is supported by an NMR-based structural model of the acceptor stem, together with biochemical studies of a closely related microhelix. This role suggests that the U73 binding site for cysteine-tRNA synthetase is sterically optimized to accommodate a 2′-OH group in the backbone, but that the hydroxyl group itself is not involved in specific hydrogen bonding interactions.

The human mitochondrial deoxynucleotide carrier and its role in the toxicity of nucleoside antivirals

The synthesis of DNA in mitochondria requires the uptake of deoxynucleotides into the matrix of the organelle. We have characterized a human cDNA encoding a member of the family of mitochondrial carriers. The protein has been overexpressed in bacteria and reconstituted into phospholipid vesicles where it catalyzed the transport of all four deoxy (d) NDPs, and, less efficiently, the corresponding dNTPs, in exchange for dNDPs, ADP, or ATP. It did not transport dNMPs, NMPs, deoxynucleosides, nucleosides, purines, or pyrimidines. The physiological role of this deoxynucleotide carrier is probably to supply deoxynucleotides to the mitochondrial matrix for conversion to triphosphates and incorporation into mitochondrial DNA. The protein is expressed in all human tissues that were examined except for placenta, in accord with such a central role. The deoxynucleotide carrier also transports dideoxynucleotides efficiently. It is likely to be medically important by providing the means of uptake into mitochondria of nucleoside analogs, leading to the mitochondrial impairment that underlies the toxic side effects of such drugs in the treatment of viral illnesses, including AIDS, and in cancer therapy.

Comparison of the Ability of Partially N-Acetylated Chitosans and Chitooligosaccharides to Elicit Resistance Reactions in Wheat Leaves1

Chitin, a linear polysaccharide composed of (1→4)-linked 2-acetamido-2-deoxy-β-d-glucopyranose (GlcNAc) residues, and chitosan, the fully or partially N-acetylated, water-soluble derivative of chitin composed of (1→4)-linked GlcNAc and 2-amino-2-deoxy-β-d-glucopyranose (GlcN), have been proposed as elicitors of defense reactions in higher plants. We tested and compared the ability of purified (1→4)-linked oligomers of GlcNAc (tetramer to decamer) and of GlcN (pentamer and heptamer) and partially N-acetylated chitosans with degrees of acetylation (DA) of 1%, 15%, 35%, 49%, and 60% and average degrees of polymerization between 540 and 1100 to elicit phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, lignin deposition, and microscopically and macroscopically visible necroses when injected into the intercellular spaces of healthy, nonwounded wheat (Triticum aestivum L.) leaves. Purified oligomers of (1→4)-linked GlcN were not active as elicitors, whereas purified oligomers of (1→4)-linked GlcNAc with a degree of polymerization ≥ 7 strongly elicited POD activities but not PAL activities. Partially N-acetylated, polymeric chitosans elicited both PAL and POD activities, and maximum elicitation was observed with chitosans of intermediate DAs. All chitosans but not the chitin oligomers induced the deposition of lignin, the appearance of necrotic cells exhibiting yellow autofluorescence under ultraviolet light, and macroscopically visible necroses; those with intermediate DAs were most active. These results suggest that different mechanisms are involved in the elicitation of POD activities by GlcNAc oligomers, and of PAL and POD activities by partially N-acetylated chitosan polymers and that both enzymes have to be activated for lignin biosynthesis and ensuing necrosis to occur.