RESUMEN

A manganese porphyrin complex, Mn-TMPyP, associated with KHSO(5) is a chemical nuclease able to selectively recognize the minor groove of three consecutive AT base pairs of DNA and to mediate very precise cleavage chemistry at that particular site. This specific recognition and cleavage were used to probe the accessibility of the minor groove of DNA duplexes composed of one phosphodiester strand and one phosphorothioate strand. The cleavage of 5'-GCAAAAGC/5'-GCTTTTGC duplexes by Mn-TMPyP/KHSO(5) was monitored by HPLC coupled to electrospray mass analysis. Each single strand was synthesized with all-phosphate, all- Rp-phosphorothioate and all- Sp-phosphorothioate internucleotide bonds. We found that the manganese porphyrin was able to recognize its favorite (AT)(3)-box binding site within the heteroduplexes, as in the case of natural DNA. Molecular modeling studies on the interactions of the reactive porphyrin manganese-oxo species with both types of duplexes confirmed the experimental data.

Asunto(s)

Desoxirribonucleasas/química , Ácidos Nucleicos Heterodúplex/química , Compuestos de Fósforo/química , Tionucleótidos/química , Sitios de Unión , Hidrólisis , Sondas Moleculares/química

RESUMEN

Isis 3521 and G3139 are 20- and 18-mer phosphorothioate oligonucleotides, respectively, targeted to the protein kinase C (PKC)-alpha and bcl-2 mRNAs. Treatment of T24 bladder and PC3 prostate carcinoma cells with full-length and 3'-truncation mutants of Isis 3521 causes down-regulation of PKC-alpha protein and mRNA. However, at the level of a 15-mer and shorter, down-regulation of mRNA expression is no longer observed. Further, no diminution in cellular viability, as measured by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide assay, in response to increasing concentrations of paclitaxel, can be observed for these shorter oligomers. These observations not only indicate that PKC-alpha protein expression can be down-regulated by both RNase H-dependent and -independent mechanisms but also that down-regulation of PKC-alpha is insufficient by itself to "chemosensitize" cells. G3139, which down-regulates bcl-2 protein and mRNA expression, also down-regulates PKC-alpha protein and mRNA expression but not that of PKC-betaI, -epsilon, or -zeta. However, the down-regulation of PKC-alpha and bcl-2 are not linked. When the carrier Eufectin 5 is employed, only bcl-2 is down-regulated in both T24 and PC3 cells at 50 nM oligonucleotide concentration. At 100 nM, both bcl-2 and PKC-alpha expression are down-regulated, and only at this concentration can "chemosensitization" to paclitaxel and carboplatin be observed. In contrast, the down-regulation of bcl-2 seems to be linked with that of RelA (p65). However, this too is also not sufficient for chemosensitization, even though it leads to the loss of expression of genes under the putative control of nuclear factor-kappaB and to detachment of the cells from plastic surfaces. These results underscore the complexity of the intracellular requirements for the initiation of chemosensitization to anti-neoplastic agents.

Asunto(s)

Apoptosis , Isoenzimas/antagonistas & inhibidores , Oligodesoxirribonucleótidos Antisentido/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Tionucleótidos/farmacología , Antineoplásicos Fitogénicos/farmacología , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Eliminación de Gen , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , FN-kappa B/metabolismo , FN-kappa B/fisiología , Paclitaxel/farmacología , Neoplasias de la Próstata , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteína Quinasa C-alfa , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , Ribonucleasa H/metabolismo , Factor de Transcripción ReIA , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria

RESUMEN

Endonuclease from Serratia marcescens hydrolyzes internucleotide phosphorothioate linkages of R(P) configuration with inversion of configuration at P-atom. This observation supports a reported architecture of the active site, with 3'-bridging and pro-S(P) non-bridging oxygen atoms of the scissile phosphate group involved in direct contact with hydrated magnesium cation, while His-89 activates a water molecule which attacks the phosphorus atom according to a one-step in-line mechanism. The presence of a phosphorothioate bond of S(P) configuration downstream to that one being cleaved reduces the rate of hydrolysis. This suggests participation of the pro-S(P) oxygen atom of that phosphate bond in the mechanism of action of the enzyme, which was not detected in published crystallographic analyses.

Asunto(s)

Endonucleasas/metabolismo , Serratia marcescens/enzimología , Tionucleótidos/metabolismo , Catálisis , Dominio Catalítico , Endonucleasas/química , Hidrólisis , Cinética , Magnesio/química , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estereoisomerismo , Tionucleótidos/química

RESUMEN

Diastereomerically pure, partially modified (in selected positions) or fully modified phosphorothioate oligomers of the [PS]-d(CG)(4) and [PS]-d(GC)(4) series were investigated with respect to their ability to adopt the left-handed conformation at high sodium chloride concentration. NaCl induces the B-Z transition of [All-S(P)R(P)-PS]-d(CG)(4) with a midpoint of transition at ca. 2 M, which is approximately 1 M less than for unmodified d(CG)(4). Also, [All-R(P)S(P)-PS]-d(GC)(4) at 5 M NaCl converts to the Z form to the extent of ca. 55%, while the unmodified d(GC)(4) counterpart does not convert at all. This enhanced ability of stereodefined phosphorothioate oligomers to adopt the Z conformation is discussed in terms of already known structural factors (hydrogen bonding and water bridges) facilitating the B-Z transition, identified for unmodified d(CG)(n) oligonucleotides. By CD spectroscopy, the [All-S(P)-PS]-d(CG)(4) oligomer at a NaCl concentration higher than 0.01 M adopts a unique conformation as assessed from the presence of an additional negative band centered at 282 nm.

Asunto(s)

ADN/química , Fosfatos de Dinucleósidos/química , Oligodesoxirribonucleótidos/química , Tionucleótidos/química , Composición de Base , Secuencia de Bases , Quimera , Dicroismo Circular , Desoxiguanosina/química , Estructura Molecular , Conformación de Ácido Nucleico , Cloruro de Sodio/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estereoisomerismo , Termodinámica

RESUMEN

The stability of stereoregular oligo(nucleoside phosphorothioate)s (PS-oligos) in human plasma has been studied. 3'-Exonuclease present in human plasma appeared to be RP specific, that is, it cleaves internucleotide phosphorothioate linkages of [RP]-configuration and not those of [SP]-configuration. Therefore, PS-oligos containing all phosphorothioate internucleotide linkages of [RP]-configuration [RP-PS-oligos]) are more effectively degraded by the enzyme than PS-oligos prepared via nonstereo-controlled methods (so-called random mixture of diastereomers [Mix-PS-oligos]), whereas oligo(nucleoside phosphorothioate)s of [S(P)]-configuration remain intact. The enzyme activity depends on the sequence of nucleobases. The presence of deoxycytidine units (three or more residues) at the 3'-end of PS-oligo substrate significantly inhibits the enzyme activity.

Asunto(s)

Exodesoxirribonucleasas/sangre , Tionucleótidos/sangre , Estabilidad de Medicamentos , Electroforesis en Gel de Poliacrilamida , Exodesoxirribonucleasas/antagonistas & inhibidores , Exodesoxirribonucleasas/metabolismo , Humanos , Conformación de Ácido Nucleico , Estereoisomerismo , Tionucleótidos/síntesis química , Tionucleótidos/química

RESUMEN

Antisense oligodeoxynucleotides can selectively inhibit the expression of individual genes and thus have potential applications in anticancer and antiviral therapy. A critical prerequisite to their use as therapeutic agents is the understanding of their non-specific interactions with biological structures, e.g. proteins. In this study we examined the interactions of P-chiral phosphorothioate oligodeoxynucleotides with several proteins. The Rp- and Sp- diastereomers, and racemic machine-made mixtures, or M-oligodeoxynucleotides were used independently as competitors of the binding of a probe, phosphodiester oligodeoxynucleotide bearing a 5' alkylating moiety, to rsCD4, bFGF and laminin. These oligodeoxynucleotides were also used as competitors of the binding of a non-alkylating probe M-phosphorothioate oligodeoxynucleotide, 5'-32P-SdT18 to fibronectin. The average values of and quantitative estimates for the IC50 of competition and the constant of competition (Kc) of Rp-, Sp- and M-stereoisomers of several homo- and heteropolymer oligodeoxynucleotides were determined and compared. Surprisingly, in the proteins we studied, the values of IC50 and Kc for the Rp-, Sp- and M-oligodeoxynucleotides were essentially identical. Thus, the ability of the phosphorothioate oligodeoxynucleotides we employed, to bind to the proteins studied in this work, is virtually independent of P-chirality. Our results also imply that the role of the purine and pyrimidine bases in oligodeoxynucleotide-protein interactions, as well as the nature of the contact points (sulfur versus oxygen) between the oligomer and the protein, may be relatively unimportant.

Asunto(s)

Oligodesoxirribonucleótidos/metabolismo , Proteínas/metabolismo , Tionucleótidos/metabolismo , Secuencia de Bases , Unión Competitiva , Antígenos CD4/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fibronectinas/metabolismo , Isomerismo , Laminina/metabolismo , Sondas Moleculares , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Unión Proteica , Relación Estructura-Actividad , Tionucleótidos/química

RESUMEN

Granulocyte factor (GF) is a substance secreted selectively from the specific granules of granulocytes in the first minutes of adherence or phagocytosis. GF possesses many immunoregulatory functions. We applied flow cytometric analysis to evaluate the target cells responsible for GF action. Resting lymphocytes did not display binding sites for GF, but 93 +/- 4.6% of the neutrophils isolated from peripheral blood bound GF (mean fluorescence = 122 +/- 11). GF was bound by 26 +/- 7.6% of the lymphocytes from nonstimulated and 42.6 +/- 12% of the lymphocytes from PHA-stimulated cultures, and the mean fluorescence intensity was 14.5 +/- 1 and 54 +/- 10.6, respectively. PHA-stimulated CD8+ cells presented higher expression of binding sites for GF than CD4+ cells. Binding sites for GF were found on 66 +/- 5.1% of activated cells expressing the receptor molecule to IL-2 (CD25) and 50.3 +/- 7.4% of cultured lymphocytes expressing the early activation marker CD69. These values were significantly higher in comparison to the percentage of CD25- and CD69- cells that bound FITC-labeled GF (10.3 +/- 3.3% and 11.3 +/- 3.5%, respectively). The binding was specific, and preincubation of the cells with GF almost completely abolished FITC-labeled GF binding. It has also been shown that GF binds to lymphocytes via molecules other than CD16. High-performance liquid chromatography (HPLC) with fractionated GF showed three separate fractions and two of them had GF-like activity, as demonstrated in the mixed lymphocyte reaction (MLR).

Asunto(s)

Factores Biológicos/inmunología , Granulocitos/metabolismo , Antígenos CD/biosíntesis , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos de Diferenciación de Linfocitos T/metabolismo , Factores Biológicos/metabolismo , Células Cultivadas , Citometría de Flujo , Humanos , Inmunoglobulina G/inmunología , Lectinas Tipo C , Linfocitos/inmunología , Receptores de Interleucina-2/biosíntesis , Receptores de Interleucina-2/metabolismo

RESUMEN

The pleiotropic cytokine TNF has been implicated in the regulation of many immune and inflammatory responses in vivo, and in addition exerts a wide range of effects on target cells in vitro. However, although two cell surface receptors for TNF have been identified, and their cDNAs cloned, the amino acid residues necessary for the biological activity of TNF have not been characterized. We have therefore constructed derivatives of TNF termed 'muteins', in which the first 3 to 7 amino acids of native TNF-alpha have been replaced, using synthetic cDNA expressed in E. coli. In the present study we compare the effects of native TNF-alpha and muteins III, IV, V and VI in several different in vitro systems and in one in vivo model. We observed binding to the p75 TNF receptor on Jijoye Burkitt lymphoma cells with native TNF-alpha and mutein III alone, whereas the p55 TNF receptor on the human epithelioid carcinoma cell line HeLa bound TNF-alpha, mutein III and mutein V. Muteins IV and VI failed to recognize either TNF receptor. WEHI 164 fibrosarcoma cells were killed by muteins III, V and VI. Human umbilical vein endothelial cells responded to native TNF-alpha and to muteins III, IV and V, but not to mutein VI, by increasing the surface expression of ICAM-1 antigen and secretion of the cytokines GM-CSF and IL-6. All four compounds were pro-inflammatory in a mouse in vivo model. The results presented in this report confirm that N-terminal amino acids are critical for both receptor binding and biological activity of TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Endotelio Vascular/efectos de los fármacos , Receptores de Superficie Celular/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión/efectos de los fármacos , Moléculas de Adhesión Celular/biosíntesis , Células Cultivadas/efectos de los fármacos , Endotelio Vascular/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Inflamación , Molécula 1 de Adhesión Intercelular , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Necrosis Tumoral , Células Tumorales Cultivadas/metabolismo , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Tumour necrosis factor-alpha (TNF-alpha) has been implicated as an important inflammatory mediator. In vitro, TNF-alpha is reported to activate human polymorphonuclear neutrophils (PMN), inducing responses such as phagocytic activity, degranulation and oxidative metabolism. Biological responses to TNF-alpha are initiated by its binding to specific cell surface receptors, and various studies have shown that the major TNF receptor species on PMN is the 75 kDa receptor. To verify the suggestion that the receptor binding domain includes the region close to the N-terminus of the TNF-alpha molecule, four TNF-alpha derivatives termed muteins were constructed, using a synthetic cDNA fragment substituting the N-terminal 3-7 selected hydrophilic or hydrophobic amino acids in the original TNF-alpha genomic DNA. Binding of muteins to PMN was assessed using monoclonal antibodies recognizing either the 55 kDa (p55) or the 75 kDa (p75) TNF receptor subtypes. Blocking by muteins of anti-p75 antibody binding to PMN was as expected from their N-terminal amino acid composition and hydrophilic properties. Hydrophilic muteins competed well with anti-TNF receptor antibodies for binding to the p75 receptor. In contrast, hydrophobic muteins were unable to block anti-p75 binding. Similarly, degranulation, chemiluminescence or enhancement of the PMN response to specific stimuli by the muteins correlated with the hydrophilic properties of the muteins. The significance of these observations in relation to the molecular structure of TNF-alpha is discussed.

RESUMEN

A gene coding for human tumor necrosis factor (h alpha TNF) has been assembled by ligating short oligodeoxyribonucleotides and cloning into plasmid vectors. These oligonucleotides were prepared by the modified phosphoramidite methodology using isopropoxyacetyl (IPA) as a protecting group for exoamino- functions of nucleosides. Gene was expressed in E. coli and the protein product was purified to homogeneity by ion-exchange chromatography.

Asunto(s)

Factor de Necrosis Tumoral alfa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/genética , Escherichia coli/genética , Expresión Génica , Variación Genética , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Factor de Necrosis Tumoral alfa/aislamiento & purificación

RESUMEN

Diastereomers of the title compound were obtained and absolute configuration was assigned by means of stereochemical correlation. Their reaction with 3'-O-methoxyacetylthymidine in the presence of triisopropylbenzenesulphonyl (4-nitro) triazole is neither chemo- nor stereo-selective and leads to diastereomeric pairs of dithymidyl (3',5')methanephosphonate and -methanephosphonothioate. Obtained results are discussed in terms of mechanism of activation of phosphodiesters under conditions known as "phosphotriester approach to oligonucleotide synthesis".

Asunto(s)

Compuestos Organotiofosforados/síntesis química , Estereoisomerismo , Timidina/análogos & derivados , Timidina/síntesis química