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1.
J Chem Phys ; 137(6): 064507, 2012 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-22897293

RESUMEN

Raman spectroscopy and synchrotron x-ray diffraction measurements of ammonia (NH(3)) in laser-heated diamond anvil cells, at pressures up to 60 GPa and temperatures up to 2500 K, reveal that the melting line exhibits a maximum near 37 GPa and intermolecular proton fluctuations substantially increase in the fluid with pressure. We find that NH(3) is chemically unstable at high pressures, partially dissociating into N(2) and H(2). Ab initio calculations performed in this work show that this process is thermodynamically driven. The chemical reactivity dramatically increases at high temperature (in the fluid phase at T > 1700 K) almost independent of pressure. Quenched from these high temperature conditions, NH(3) exhibits structural differences from known solid phases. We argue that chemical reactivity of NH(3) competes with the theoretically predicted dynamic dissociation and ionization.


Asunto(s)
Amoníaco/química , Congelación , Termodinámica , Difusión , Calor , Presión , Espectrometría Raman , Temperatura , Difracción de Rayos X
2.
Genes Immun ; 12(4): 270-9, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21270825

RESUMEN

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disorder with a complex pathogenesis in which genetic, hormonal and environmental factors have a role. Rare mutations in the TREX1 gene, the major mammalian 3'-5' exonuclease, have been reported in sporadic SLE cases. Some of these mutations have also been identified in a rare pediatric neurological condition featuring an inflammatory encephalopathy known as Aicardi-Goutières syndrome (AGS). We sought to investigate the frequency of these mutations in a large multi-ancestral cohort of SLE cases and controls. A total of 40 single-nucleotide polymorphisms (SNPs), including both common and rare variants, across the TREX1 gene, were evaluated in ∼8370 patients with SLE and ∼7490 control subjects. Stringent quality control procedures were applied, and principal components and admixture proportions were calculated to identify outliers for removal from analysis. Population-based case-control association analyses were performed. P-values, false-discovery rate q values, and odds ratios (OR) with 95% confidence intervals (CI) were calculated. The estimated frequency of TREX1 mutations in our lupus cohort was 0.5%. Five heterozygous mutations were detected at the Y305C polymorphism in European lupus cases but none were observed in European controls. Five African cases incurred heterozygous mutations at the E266G polymorphism and, again, none were observed in the African controls. A rare homozygous R114H mutation was identified in one Asian SLE patient, whereas all genotypes at this mutation in previous reports for SLE were heterozygous. Analysis of common TREX1 SNPs (minor allele frequency (MAF)>10%) revealed a relatively common risk haplotype in European SLE patients with neurological manifestations, especially seizures, with a frequency of 58% in lupus cases compared with 45% in normal controls (P=0.0008, OR=1.73, 95% CI=1.25-2.39). Finally, the presence or absence of specific autoantibodies in certain populations produced significant genetic associations. For example, a strong association with anti-nRNP was observed in the European cohort at a coding synonymous variant rs56203834 (P=2.99E-13, OR=5.2, 95% CI=3.18-8.56). Our data confirm and expand previous reports and provide additional support for the involvement of TREX1 in lupus pathogenesis.


Asunto(s)
Exodesoxirribonucleasas/genética , Lupus Eritematoso Sistémico/genética , Fosfoproteínas/genética , Estudios de Cohortes , Femenino , Haplotipos , Humanos , Lupus Eritematoso Sistémico/epidemiología , Masculino , Mutación , Fenotipo , Polimorfismo de Nucleótido Simple
3.
J Chem Phys ; 132(8): 084509, 2010 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-20192309

RESUMEN

We have used reactive force field (ReaxFF) to investigate the mechanism of interaction of alanes on Al(111) surface. Our simulations show that, on the Al(111) surface, alanes oligomerize into larger alanes. In addition, from our simulations, adsorption of atomic hydrogen on Al(111) surface leads to the formation of alanes via H-induced etching of aluminum atoms from the surface. The alanes then agglomerate at the step edges forming stringlike conformations. The identification of these stringlike intermediates as a precursor to the bulk hydride phase allows us to explain the loss of resolution in surface IR experiments with increasing hydrogen coverage on single crystal Al(111) surface. This is in excellent agreement with the experimental works of Go et al. [E. Go, K. Thuermer, and J. E. Reutt-Robey, Surf. Sci. 437, 377 (1999)]. The mobility of alanes molecules has been studied using molecular dynamics and it is found that the migration energy barrier of Al(2)H(6) is 2.99 kcal/mol while the prefactor is D(0)=2.82 x 10(-3) cm(2)/s. We further investigated the interaction between an alane and an aluminum vacancy using classical molecular dynamics simulations. We found that a vacancy acts as a trap for alane, and eventually fractionates/annihilates it. These results show that ReaxFF can be used, in conjunction with ab initio methods, to study complex reactions on surfaces at both ambient and elevated temperature conditions.

4.
J Chem Phys ; 131(4): 044501, 2009 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-19655888

RESUMEN

A reactive force field, REAXFF, for aluminum hydride has been developed based on density functional theory (DFT) derived data. REAXFF(AlH(3)) is used to study the dynamics governing hydrogen desorption in AlH(3). During the abstraction process of surface molecular hydrogen charge transfer is found to be well described by REAXFF(AlH(3)). Results on heat of desorption versus cluster size show that there is a strong dependence of the heat of desorption on the particle size, which implies that nanostructuring enhances desorption process. In the gas phase, it was observed that small alane clusters agglomerated into a bigger cluster. After agglomeration molecular hydrogen was desorbed from the structure. This thermodynamically driven spontaneous agglomeration followed by desorption of molecular hydrogen provides a mechanism on how mobile alane clusters can facilitate the mass transport of aluminum atoms during the thermal decomposition of NaAlH(4).

5.
Genes Immun ; 10(5): 446-56, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19440200

RESUMEN

In our earlier study, we utilized a Bayesian design to probe the association of approximately 1000 genes (approximately 10,000 single-nucleotide polymorphisms (SNPs)) with systemic lupus erythematosus (SLE) on a moderate number of trios of parents and children with SLE. Two genes associated with SLE, with a multitest-corrected false discovery rate (FDR) of <0.05, were identified, and a number of noteworthy genes with FDR of <0.8 were also found, pointing out a future direction for the study. In this report, using a large population of controls and adult- or childhood-onset SLE cases, we have extended the earlier investigation to explore the SLE association of 10 of these noteworthy genes (109 SNPs). We have found that seven of these genes exhibit a significant (FDR<0.05) association with SLE, both confirming some genes that have earlier been found to be associated with SLE (PTPN22 and IRF5) and presenting novel findings of genes (KLRG1, interleukin-16, protein tyrosine phosphatase receptor type T, toll-like receptor (TLR)8 and CASP10), which have not been reported earlier. The results signify that the two-step candidate pathway design is an efficient way to study the genetic foundations of complex diseases. Furthermore, the novel genes identified in this study point to new directions in both the diagnosis and the eventual treatment of this debilitating disease.


Asunto(s)
Predisposición Genética a la Enfermedad , Lupus Eritematoso Sistémico/genética , Polimorfismo de Nucleótido Simple , Edad de Inicio , Teorema de Bayes , Estudios de Casos y Controles , Estudio de Asociación del Genoma Completo , Humanos , Lupus Eritematoso Sistémico/epidemiología
6.
J Chem Phys ; 128(16): 164714, 2008 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-18447486

RESUMEN

We have parametrized a reactive force field for NaH, ReaxFF(NaH), against a training set of ab initio derived data. To ascertain that ReaxFF(NaH) is properly parametrized, a comparison between ab initio heats of formation of small representative NaH clusters with ReaxFF(NaH) was done. The results and trend of ReaxFF(NaH) are found to be consistent with ab initio values. Further validation includes comparing the equations of state of condensed phases of Na and NaH as calculated from ab initio and ReaxFF(NaH). There is a good match between the two results, showing that ReaxFF(NaH) is correctly parametrized by the ab initio training set. ReaxFF(NaH) has been used to study the dynamics of hydrogen desorption in NaH particles. We find that ReaxFF(NaH) properly describes the surface molecular hydrogen charge transfer during the abstraction process. Results on heat of desorption versus cluster size shows that there is a strong dependence on the heat of desorption on the particle size, which implies that nanostructuring enhances desorption process. To gain more insight into the structural transformations of NaH during thermal decomposition, we performed a heating run in a molecular dynamics simulation. These runs exhibit a series of drops in potential energy, associated with cluster fragmentation and desorption of molecular hydrogen. This is consistent with experimental evidence that NaH dissociates at its melting point into smaller fragments.

7.
J Chem Phys ; 129(24): 244506, 2008 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-19123516

RESUMEN

A parametrized reactive force field model for aluminum ReaxFF(Al) has been developed based on density functional theory (DFT) data. A comparison has been made between DFT and ReaxFF(Al) outputs to ascertain whether ReaxFF(Al) is properly parametrized and to check if the output of the latter has correlation with DFT results. Further checks include comparing the equations of state of condensed phases of Al as calculated from DFT and ReaxFF(Al). There is a good match between the two results, again showing that ReaxFF(Al) is correctly parametrized as per the DFT input. Simulated annealing has been performed on aluminum clusters Al(n) using ReaxFF(Al) to find the stable isomers of the clusters. A plot of stability function versus cluster size shows the existence of highly stable clusters (magic clusters). Quantum mechanically these magic clusters arise due to the complete filling of the orbital shells. However, since force fields do not care about electrons but work on the assumption of validity of Born-Oppenheimer approximation, the magic clusters are therefore correlated with high structural symmetry. There is a rapid decline in surface energy contribution due to the triangulated nature of the surface atoms leading to higher coordination number. The bulk binding energy is computed to be 76.8 kcal/mol. This gives confidence in the suitability of ReaxFF for studying and understanding the underlying dynamics in aluminum clusters. In the quantification of the growth of cluster it is seen that as the size of the clusters increase there is preference for the coexistence of fcc/hcp orders at the expense of simple icosahedral ordering, although there is some contribution from distorted icosahedral ordering. It is found that even for aluminum clusters with 512 atoms distorted icosahedral ordering exists. For clusters with N>/=256 atoms fcc ordering dominates, which implies that at this point we are already on the threshold of bulklike bonding.

8.
J Urol ; 166(2): 461-9, 2001 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-11458048

RESUMEN

PURPOSE: We investigated antisense inhibition of anti-apoptotic bcl-xL and bcl-2 proteins to increase chemosensitization in the T24 and 5637 bladder carcinoma cell lines. MATERIALS AND METHODS: A T24 bladder carcinoma cell line stably over expressing bcl-xL protein was constructed. Apoptosis by cytotoxic agents was estimated by cell cycle analysis and Annexin V binding. To eliminate bcl-xL expression T24 and 5637 cells were treated with C5-propynylated and 2'-O-methylribo-oligonucleotides. Levels of protein and messenger RNA were measured by Western and Northern blot analysis. Cell viability after combined treatment with oligonucleotides and various cytotoxic agents was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and evaluated statistically by Student's 2-sample t test. RESULTS: Forced over expression of bcl-xL protein desensitized the T24 bladder carcinoma cell line to cytotoxic agents. C5-propynylated and 2'-O-methylribo-oligonucleotides down-regulated bcl-xL protein expression in the T24 and 5637 cell lines, and increased their sensitivity to cytotoxic agents. The efficiency of antisense down-regulation of bcl-xL protein expression depended on the type of delivery agent. CONCLUSIONS: Antisense down-regulation of bcl-xL protein sensitizes bladder carcinoma cells to cytotoxic agents. However, it is possible that cellular chemosensitization results from a combination of effects, including nonsequence specificity, irrelevant cleavage and effects of the carriers combined with the specific antisense effects.


Asunto(s)
Carcinoma , Resistencia a Antineoplásicos/fisiología , Oligonucleótidos Antisentido/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Neoplasias de la Vejiga Urinaria , Apoptosis , Carcinoma/tratamiento farmacológico , Línea Celular , Regulación hacia Abajo/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Proteína bcl-X
9.
Cancer Res ; 60(21): 6052-60, 2000 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-11085527

RESUMEN

Both Bcl-xL and Bcl-2, antiapoptotic members of the Bcl family, are found in prostate cancer cell lines. Although these proteins may have similar antiapoptotic functions, it is not clear to what extent each serves as an antiapoptotic effector in prostate cancer cells. We engineered LNCaP and PC-3 cells to overexpress Bcl-xL protein and demonstrated that this desensitized them to the effects of cytotoxic chemotherapy. We then used two "antisense" strategies to down-regulate Bcl-xL protein expression in the parental lines. The first strategy used CS-propynylated phosphorothioate-phosphodiester oligonucleotides and co-down-regulated both Bcl-xL and Bcl-2; the second strategy used isosequential "gap-mer" phosphorothioate oligonucleotides containing 2'-O-methyl oligoribonucleotides at the 3' and 5' termini. In this case, only Bcl-xL protein expression was affected. The most active oligonucleotides of both types decreased the level of Bcl-xL protein expression to 5-30% of the control level. Multiple controls were inactive. Experiments combining oligonucleotide treatment with cytotoxic chemotherapeutic agents (paclitaxel, docetaxel, etoposide, vinblastine, carboplatin, and mitoxantrone) demonstrated a marked increase in the sensitivity of these prostate cancer cells. However, the increase in chemosensitivity in PC-3 cells was statistically identical (except mitoxantrone) for both "antisense" strategies, indicating that basal expression of Bcl-2, in contrast to that of Bcl-xL, may play little cytoprotective role in these cells.


Asunto(s)
Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Fenotipo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína X Asociada a bcl-2 , Proteína bcl-X
10.
Methods ; 18(3): 244-51, 1999 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-10454982

RESUMEN

Antisense oligodeoxynucleotides (ODNs) are being explored as therapeutic agents for the treatment of many disorders including viral infections, cancers, and inflammatory disorders. In addition, antisense technology can be of great benefit to those attempting to assign function to the multitude of new genes being uncovered in the genomics initiative. However, the demonstration that the gene-regulating effects produced by antisense-designed ODNs are attributable to an antisense mechanism of action requires carefully designed experimentation. Critical to the assignment of an antisense mechanism of action is the availability of nuclease-stable ODNs, inside cells, that have a high binding affinity with the target mRNA and modulate gene functions in a sequence-dependent manner. To help us achieve a goal of sequence-specific antisense activity we designed antisense ODNs containing C(5)-propyne-modified 2'-deoxyuracil and N(7)-propyne-modified 7-deaza-2'-deoxyguanosine bases and partially modified (phosphorothioate) internucleoside linkages. These modified ODNs were found to have enhanced binding affinity to their target mRNA sequences as well as reduced sequence-independent side effects. We used these ODNs to specifically inhibit p55 tumor necrosis factor receptor type 1 expression and tumor necrosis factor alpha-mediated functions in culture assays.


Asunto(s)
Antígenos CD/genética , Desoxiguanosina/química , Oligodesoxirribonucleótidos Antisentido/farmacología , Receptores del Factor de Necrosis Tumoral/genética , Antígenos CD/química , Células Cultivadas , Sondas de ADN , Desoxiguanosina/análogos & derivados , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-6/análisis , Interleucina-8/análisis , Estructura Molecular , Hibridación de Ácido Nucleico , Oligorribonucleótidos/química , ARN Mensajero/genética , Receptores del Factor de Necrosis Tumoral/química , Receptores Tipo I de Factores de Necrosis Tumoral , Termodinámica , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/genética
11.
Nucleosides Nucleotides ; 17(1-3): 379-96, 1998.
Artículo en Inglés | MEDLINE | ID: mdl-9708354

RESUMEN

Two convenient, practical routes to the synthesis of non-nucleotide bridged cyclic oligonucleotides have been developed. The first procedure included circularization of oligonucleotides by template-directed ligation on solid phase, while the second procedure involved preparation of a circular oligomer by non-template chemical ligation of a linear precursor in solution. Using these approaches, a series of single- and double-stranded cyclic oligonucleotides with non-nucleotide bridges has been synthesized.


Asunto(s)
ADN Circular/síntesis química , Oligonucleótidos/síntesis química , Fármacos Anti-VIH , Línea Celular , Humanos , Inhibidores de Integrasa/síntesis química , Conformación Molecular , Estructura Molecular , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Hibridación de Ácido Nucleico , Glicoles de Propileno/síntesis química , Transcripción Genética/efectos de los fármacos , Transfección/genética
12.
Antimicrob Agents Chemother ; 42(7): 1646-53, 1998 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-9660998

RESUMEN

The objective of the proposed study was to determine the distribution in plasma lipoprotein of free all-trans retinoic acid (ATRA) and liposomal ATRA (Atragen; composed of dimyristoyl phosphatidylcholine and soybean oil) following incubation in human, rat, and dog plasma. When ATRA and Atragen at concentrations of 1, 5, 10, and 25 micrograms/ml were incubated in human and rat plasma for 5, 60, and 180 min, the majority of the tretinoin was recovered in the lipoprotein-deficient plasma fraction. However, when ATRA and Afragen were incubated in dog plasma, the majority of the tretinoin (> 40%) was recovered in the high-density lipoprotein (HDL) fraction. No differences in the plasma distribution between ATRA and Atragen were found. These data suggest that a significant percentage of tretinoin associates with plasma lipoproteins (primarily the HDL fraction) upon incubation in human, dog, and rat plasma. Differences between the lipoprotein lipid and protein profiles in human plasma and in dog and rat plasma influenced the plasma distribution of ATRA and Atragen. Differences in lipoprotein distribution between ATRA and Atragen were not observed, suggesting that the drug's distribution in plasma in not influenced by its incorporation into these liposomes.


Asunto(s)
Lipoproteínas/sangre , Tretinoina/farmacocinética , Animales , Proteínas Sanguíneas/metabolismo , Perros , Humanos , Lípidos/sangre , Liposomas/metabolismo , Ratas , Tretinoina/sangre
13.
Antimicrob Agents Chemother ; 42(6): 1412-6, 1998 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-9624486

RESUMEN

The in vitro activity of a multilamellar liposomal formulation of nystatin (Nyotran) was compared with those of free nystatin and four pharmaceutical preparations of amphotericin B. MICs for 200 isolates of two Aspergillus spp., seven Candida spp., and Cryptococcus neoformans were determined by a broth microdilution adaptation of the method recommended by the National Committee for Clinical Laboratory Standards. Minimum lethal concentrations (MLCs) of the six antifungal preparations were also determined. Both nystatin formulations possessed fungistatic and fungicidal activities against the 10 species tested. Liposomal nystatin appeared to be as active as free nystatin, with MICs and MLCs that were similar to, or lower than, those of the latter. Neither formulation of nystatin was as active as amphotericin B deoxycholate (Fungizone) or amphotericin B lipid complex (Abelcet), but both were more effective than liposomal amphotericin B (AmBisome). Our results suggest that further evaluation of liposomal nystatin is justified.


Asunto(s)
Anfotericina B/farmacología , Antifúngicos/farmacología , Nistatina/farmacología , Antifúngicos/química , Aspergillus/efectos de los fármacos , Candida/efectos de los fármacos , Cryptococcus neoformans/efectos de los fármacos , Portadores de Fármacos , Liposomas , Pruebas de Sensibilidad Microbiana , Nistatina/química
14.
Antivir Chem Chemother ; 9(1): 53-63, 1998 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-9875377

RESUMEN

The synthesis and in vitro antiviral activity of certain hydroxyalkoxymethyl, hydroxyalkyl, hydroxyalkenyl and phosphonoalkenyl derivatives of the guanine congener 5-aminothiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione are reported. The compounds of this study were selected for their structural similarity to acyclonucleosides with known anti-herpesvirus activity. 5-Amino-3-[(Z)-4-hydroxy-2-buten-1-yl]thiazolo[4,5-d]pyrimidine-2, 7(3H,6H)- dione was the only member of the series to display significant in vitro activity against human cytomegalovirus (HCMV); however, this compound did not inhibit other herpesviruses, human immunodeficiency virus type 1 or murine cytomegalovirus. It was found to have a cytotoxicity profile similar to that of ganciclovir (DHPG). The antiviral effect was found to be sensitive to the initial viral input and the time of addition during the virus replication cycle. Significantly, the compound was found to have equal anti-HCMV activity, against standard virus strains, to DHPG, but also showed potent activity against DHPG-resistant virus strains, except for a strain mutated in the UL97 (phosphotransferase) gene.


Asunto(s)
Antivirales/química , Citomegalovirus/efectos de los fármacos , Nucleósidos/síntesis química , Nucleótidos/síntesis química , Pirimidinas/síntesis química , Pirimidinas/farmacología , Pirimidinonas/química , Tiazoles/química , Línea Celular , Citomegalovirus/crecimiento & desarrollo , Citomegalovirus/fisiología , Humanos , Espectroscopía de Resonancia Magnética , Nucleósidos/farmacología , Nucleótidos/farmacología , Espectrofotometría Ultravioleta , Ensayo de Placa Viral , Replicación Viral/efectos de los fármacos
15.
Mol Pharmacol ; 52(5): 771-80, 1997 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-9351967

RESUMEN

Oligonucleotides that can form a highly stable intramolecular four-stranded DNA structure containing two stacked guanosine-quartets (G-quartets) have been reported to inhibit the replication of the human immunodeficiency virus type 1 (HIV-1) in cell culture. Two possible mechanisms for the observed antiviral activity have been proposed: interference with virus adsorption to the cell and/or inhibition of HIV-1 integrase. We investigated the molecular interaction of G-quartet-containing oligonucleotides with HIV-1 integrase in comparison with random oligonucleotides and dextran sulfate. The prototypical G-quartet-containing oligonucleotide, T30177 (Zintevir), inhibited the overall integration reaction with an IC50 value of 80 nM. A random oligonucleotide was 10-fold less potent, but dextran sulfate was more potent, with an IC50 value of 7 nM. We developed novel kinetic assays to dissect the overall integration reaction in three steps: the formation of the initial stable complex (ISC), the 3'-processing reaction, and the DNA strand-transfer step. We then analyzed the kinetics of the ISC formation and 3'-processing. The rate constant determined for the conversion of ISC into the cleaved product was 0.08 +/- 0.01 min-1. T30177 did not inhibit 3'-processing or DNA strand transfer, whereas dextran sulfate inhibited DNA strand transfer to some extent. Binding studies using surface plasmon resonance technology revealed that both T30177 and dextran sulfate were capable of preventing the binding of integrase to specific DNA. We propose a model in which the interaction of HIV-1 integrase with G-quartets results in the inhibition of the formation of the ISC between integrase and substrate DNA. Finally, we selected for an HIV-1 strain that was resistant to T30177 in cell culture. DNA sequence analysis revealed mutations in the envelope glycoprotein gp120 but not in the integrase gene. Although gp120 seems to be the main target for the antiviral activity in cell culture of G-quartets, the study of their specific inhibition of HIV-1 integrase may lead to the development of effective integrase inhibitors.


Asunto(s)
ADN Viral/efectos de los fármacos , Guanosina , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/efectos de los fármacos , VIH-1/enzimología , Oligonucleótidos/farmacología , Integración Viral/efectos de los fármacos , ADN Viral/análisis , Integrasa de VIH/genética , Integrasa de VIH/metabolismo , VIH-1/genética , Cinética , Proteínas Virales/metabolismo
16.
Antisense Nucleic Acid Drug Dev ; 7(5): 447-59, 1997 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-9361904

RESUMEN

Tumor necrosis factor-alpha (TNF-alpha) is a highly pleiotropic cytokine produced mainly by activated macrophages. This cytokine has been found to mediate the growth of certain tumors, the replication of HIV-1, septic shock, cachexia, graft-versus-host disease, and autoimmune diseases. The binding of TNF-alpha to the p55 tumor necrosis factor receptor type I (TNFRI) is considered one of the initial steps responsible for the multiple physiologic effects mediated by TNF-alpha. The role of TNF-alpha as an inflammatory mediator through TNFRI makes both of these genes attractive targets for intervention in both acute and chronic inflammatory diseases. We have designed antisense oligodeoxynucleotides (ODNs) containing chemically modified purine and pyrimidine bases that specifically inhibit TNFRI expression and functions. These ODNs were designed to hybridize to the 3'-polyadenylation signal region of the TNFRI gene. In cell-based assays, gene-specific antisense inhibition occurred in a dose-dependent fashion at submicromolar concentrations in the presence of cellular uptake enhancing agents. Within ODN sets with a common pattern of stabilizing backbone substitution, the inhibition of the gene expression is found to be correlated with the affinity of the ODNs for their cognate mRNA target sites, providing direct evidence for an antisense mechanism of action. In addition, events triggered by the binding of TNF-alpha to TNFRI, such as the production of IL-6 and IL-8, were significantly reduced by treatment of cells with the anti-TNFRI ODN. Therefore, antisense ODNs can be used to control biologic processes mediated by TNF-alpha and may be useful as therapeutic agents to treat conditions resulting from overproduction of TNF-alpha.


Asunto(s)
Antígenos CD/genética , Desoxiguanosina/análisis , Expresión Génica/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Receptores del Factor de Necrosis Tumoral/genética , Línea Celular , Fluoresceína , Humanos , Oligonucleótidos Antisentido/química , ARN Mensajero/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Relación Estructura-Actividad , Termodinámica
17.
Biochemistry ; 36(20): 6033-45, 1997 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-9166774

RESUMEN

Tumor necrosis factor alpha (TNF alpha), a polypeptide produced by activated macrophages, is a highly pleiotropic cytokine which elicits inflammatory and immunological reactions. The binding of TNF alpha to tumor necrosis factor receptor type I (TNFRI) is considered the initial step responsible for some of the multiple biological functions mediated by TNF alpha. The role of TNF alpha as an inflammatory mediator through human TNFRI makes TNFRI an attractive target for intervention in both acute and chronic inflammatory diseases. In this study, we have identified partial phosphorothioate oligodeoxyribonucleotides (ODNs) containing C-5 propynyl or hexynyl derivatives of 2'-deoxyuridine which specifically inhibited TNFRI and subsequently inhibited the functions of TNF alpha mediated through TNFRI. The most active ODNs were directed against the 3'-poly adenylation signal site on the TNFRI mRNA, and in a cellular assay, gene-specific antisense inhibition occurred in a dose-dependent fashion at submicromolar concentrations, in the presence of Cellfectin. The inhibition of gene expression correlated with the binding affinity of the ODN for the target mRNA. The ODNs lowered TNFRI protein levels and TNF alpha-mediated functions by specifically reducing levels of TNFRI mRNA. These anti-TNFRI ODNs offer a novel approach for controlling biological functions of TNF alpha and may be useful as human therapeutic agents for treating diseases in which TNF alpha has been implicated.


Asunto(s)
Antígenos CD/genética , Oligonucleótidos Antisentido/farmacología , Receptores del Factor de Necrosis Tumoral/genética , Tionucleótidos/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Antígenos CD/biosíntesis , Células Cultivadas , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Fibroblastos , Expresión Génica/efectos de los fármacos , Humanos , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral , Pruebas de Toxicidad
18.
Biochemistry ; 35(43): 13762-71, 1996 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-8901518

RESUMEN

An oligonucleotide (T30177) composed entirely of deoxyguanosine and thymidine has previously been shown to fold upon itself in the presence of potassium into a highly stable four-stranded DNA structure containing two stacked deoxyguanosine quartets (G4s). T30177 also protects host cells from the cytopathic effects of human immunodeficiency virus type 1 (HIV-1). We report that this G4 oligonucleotide is the most potent inhibitor of HIV-1 integrase identified to date, with IC50 values in the nanomolar range. Both the number of quartets formed and the sequence of the loops between the quartets are important for optimal activity. T30177 binds to HIV-1 integrase without being processed and blocks the binding of the normal viral DNA substrate to the enzyme. The normal DNA substrate was not able to compete off T30177 binding to HIV-1 integrase, indicating a tight binding of G4s to the enzyme. Experiments with truncated HIV-1 integrases indicate that the N-terminal region containing a putative zinc finger is required for inhibition by T30177 and that T30177 binds better to full-length or deletion mutant integrases containing the zinc finger region than to a deletion mutant consisting of only the central catalytic domain. The N-terminal region of integrase alone is able to bind efficiently to T30177, but not the linear viral DNA substrate, in the presence of zinc. Hence, G4s represent the first class of compounds that inhibit HIV-1 integrase by interacting with the enzyme N-terminal domain. The greater inhibitory potency of T30177 in buffer containing magnesium versus manganese suggests that divalent metal ion coordination along the phosphodiester backbone may play a role in the inhibitory activity. T30177 inhibited HIV-2 integrase with similar potency as HIV-1 but inhibited feline and simian immunodeficiency virus integrases at higher concentrations, suggesting selectivity can be achieved. We propose that novel AIDS therapies could be based upon guanosine quarters as inhibitors of HIV-1 integrase.


Asunto(s)
Desoxiguanosina/análogos & derivados , Inhibidores de Integrasa VIH/farmacología , VIH-1/enzimología , Oligodesoxirribonucleótidos/metabolismo , Oligodesoxirribonucleótidos/farmacología , Unión Competitiva , Reactivos de Enlaces Cruzados/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN , Desoxiadenosinas/metabolismo , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores de Integrasa VIH/química , VIH-2/enzimología , Virus de la Inmunodeficiencia Felina/enzimología , Magnesio/farmacología , Manganeso/farmacología , Mutación/genética , Oligodesoxirribonucleótidos/química , Oligonucleótidos/farmacología , Eliminación de Secuencia/genética , Rayos Ultravioleta , Dedos de Zinc/genética
19.
J Biol Chem ; 271(10): 5698-703, 1996 Mar 08.
Artículo en Inglés | MEDLINE | ID: mdl-8621435

RESUMEN

We have identified a potentially therapeutic anti-human immunodeficiency virus (HIV)-1 oligonucleotide composed entirely of deoxyguanosines and thymidines (T30177, also known as AR177: 5'-g.tggtgggtgggtggg.t-3', where asterisk indicates phosphorothioate linkage). In acute assay systems using human T-cells, T30177 and its total phosphodiester homologue T30175 inhibited HIV-1-induced syncytium production by 50% at 0.15 and 0.3 microM, respectively. Under physiological conditions, the sequence and composition of the 17-mer favors the formation of a compact, intramolecularly folded structure dominated by two stacked guanine quartet motifs that are connected by three loops of TGs. The molecule is stabilized by the coordination of a potassium ion between the two stacked quartets. We now show that these guanine quartet-containing oligonucleotides are highly resistant to serum nucleases, with t1/2 of 5 h and >4 days for T30175 and T30177, respectively. Both oligonucleotides were internalized efficiently by cells, with intracellular concentrations reaching 5-10-fold above the extracellular levels after 24 h of incubation. In contrast, single-base mutated variants or random sequence control oligonucleotides that could not form the compactly folded structure had markedly reduced half-lives (t1/2 from approximately 3 to 7 min), low cellular uptake, and no sequence-specific anti-HIV-1 activity. These data suggest that the tertiary structure of an oligonucleotide is a key determinant of its nuclease resistance, cellular uptake kinetics, and biological efficacy.


Asunto(s)
Antivirales/química , Antivirales/farmacología , VIH-1/efectos de los fármacos , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/farmacología , Antivirales/metabolismo , Composición de Base , Secuencia de Bases , Transporte Biológico , Gráficos por Computador , Farmacorresistencia Microbiana , Células Gigantes/efectos de los fármacos , VIH-1/fisiología , Células HeLa , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/metabolismo , Relación Estructura-Actividad , Tionucleótidos
20.
Antimicrob Agents Chemother ; 39(11): 2426-35, 1995 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-8585721

RESUMEN

T30177, an oligonucleotide composed of only deoxyguanosine and thymidine, is 17 nucleotides in length and contains single phosphorothioate internucleoside linkages at its 5' and 3' ends for stability. This oligonucleotide does not share significant primary sequence homology with or possess any complementary (antisense) sequence motifs to the human immunodeficiency virus type 1 (HIV-1) genome. T30177 inhibited replication of multiple laboratory strains of HIV-1 in human T-cell lines, peripheral blood lymphocytes, and macrophages. T30177 was also found to be capable of inhibiting multiple clinical isolates of HIV-1 and preventing the cytopathic effect of HIV-1 in primary CD4+ T lymphocytes. In assays with human peripheral blood lymphocytes there was no observable toxicity associated with T30177 at the highest concentration tested (100 microM), while the median inhibitory concentration was determined to be in the range of 0.1 to 1.0 microM for the clinical isolates tested, resulting in a high therapeutic index for this drug. In temporal studies, the kinetics of addition of T30177 to infected cell cultures indicated that, like the known viral adsorption blocking agents dextran sulfate and Chicago sky blue, T30177 needed to be added to cells during or very soon after viral infection. However, analysis of nucleic acids extracted at 12 h postinfection from cells treated with T30177 at the time of virus infection established the presence of unintegrated viral cDNA, including circular proviral DNA, in the treated cells. In vitro analysis of viral enzymes revealed that T30177 was a potent inhibitor of HIV-1 integrase, reducing enzymatic activity by 50% at concentrations in the range of 0.050 to 0.09 microM. T30177 was also able to inhibit viral reverse transcriptase activity; however, the 50% inhibitory value obtained was in the range of 1 to 10 microM, depending on the template used in the enzymatic assay. No observable inhibition of viral protease was detected at the highest concentration of T30177 used (10 microM). In experiments in which T30177 was removed from infected cell cultures at 4 days post-HIV-1 infection, total suppression of virus production was observed for more than 27 days. PCR analysis of DNA extracted from cells treated in this fashion was unable to detect the presence of viral DNA 11 days after removal of the drug from the infected cell cultures. The ability of T30177 to inhibit both laboratory and clinical isolates of HIV-1 and the experimental data which suggest that T30177 represents a novel class of integrase inhibitors indicate that this compound is a viable candidate for evaluation as a therapeutic agent against HIV-1 in humans.


Asunto(s)
Antivirales/farmacología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Oligonucleótidos/farmacología , Secuencia de Bases , Fusión Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , ADN Nucleotidiltransferasas/antagonistas & inhibidores , ADN Viral/biosíntesis , Citometría de Flujo , VIH-1/enzimología , VIH-1/fisiología , Humanos , Integrasas , Linfocitos/efectos de los fármacos , Linfocitos/virología , Macrófagos/virología , Datos de Secuencia Molecular , Inhibidores de la Transcriptasa Inversa/farmacología , Subgrupos de Linfocitos T/virología , Replicación Viral/efectos de los fármacos
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