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Biomedical subjects

G Zon

Publications and source records attributed to G Zon.

At least 55 records · Page 3Linked to original sources
PubMed1993

Immune stimulation by an antisense oligomer complementary to the rev gene of HIV-1.

Mice developed massive splenomegaly and polyclonal hypergammaglobulinemia within 2 days after intravenous injection of a phosphorothioate oligomer that is antisense to a portion of the rev region of the HIV-1 genome. Histologic examination of spleens from injected animals showed marked expansion of a uniform-appearing population of small lymphocytes and many mitoses. Spleen mononuclear cells (SMNCs) from injected animals showed approximately a 10-fold-increased uptake of [3H]thymidine and production of IgM and IgG. Flow cytometry analysis indicated that the responding cells were predominantly B-lymphocytes. The anti-rev oligomer also was mitogenic in vitro and stimulated immunoglobulin production by normal mouse SMNCs and human peripheral blood mononuclear cells. Similar immunologic effects were observed with an anti-rev 21-mer phosphorothioate, truncated at the 3' end, but not with a 20-mer human p53 antisense phosphorothioate or a 28-mer anti-rev phosphodiester. These observations are consistent with the possibility that DNA sequences homologous to the rev gene participate in the regulation of mammalian lymphocyte activation, proliferation and maturation.

Animals↗
PubMed1993

Single-stranded phosphodiester and phosphorothioate oligonucleotides bind actinomycin D and interfere with tumor necrosis factor-induced lysis in the L929 cytotoxicity assay.

Antisense oligonucleotides may prove useful tools to elucidate the biological functions of cytokines and to address regulatory control of cytokine expression. The functional activity of cytokines generally is determined by biological assays, and a standard biological assay for TNF activity measures the cytotoxic effect of TNF on actinomycin D-sensitized L929 cells (Matthews and Neale, 1987). We observed that phosphorothioate and phosphodiester oligonucleotides, at concentrations > 100 nM in supernatants tested in this bioassay, prevented TNF-induced lysis of L929 cells. This "protective" effect was due to an interaction of the single-stranded oligonucleotides with actinomycin D as demonstrated by UV spectra of an actinomycin D-oligonucleotide solution. Substitution of cycloheximide for actinomycin D in the L929 assay eliminated the protective effect of the oligonucleotide. Our results reinforce the importance of controlling for nonspecific effects of oligonucleotides, particularly when a functional assay for protein activity is used to screen for antisense-mediated reductions in target protein expression.

Animals↗
PubMed1993

Inhibition of HIV-1 replication in cultured cells with antisense oligonucleotides encapsulated in immunoliposomes.

Antisense oligonucleotides inhibit HIV replication in vitro, but their activity is limited by their sensitivity to nucleases and low cellular uptake. To see whether these problems could be circumvented, we compared effects of HIV-1 rev and tat gene-specific antisense phosphodiester or phosphorothioate oligonucleotides, either free in solution or encapsulated in antibody-targeted liposomes (immunoliposomes), on acutely or chronically infected cells. Phosphodiester antisense oligonucleotides were inactive in their free form in acutely and chronically infected cells (up to a concentration of 50 microM). When encapsulated in immunoliposomes directed to HLA class I molecules expressed by targeted cells, they inhibited viral replication (at a concentration of 0.5 microM) in acutely infected cells in a sequence-specific manner. The same phosphodiester antisense oligonucleotides in liposomes had no antiviral activity in chronically infected cells. In acutely infected cells, phosphorothioate oligonucleotides free in solution inhibited the replication of HIV without sequence specificity and had slightly greater activity, also nonspecific, when encapsulated in liposomes. Phosphorothioate antisense (anti-rev) oligonucleotides specifically blocked HIV replication in chronically infected cells. When encapsulated in targeted liposomes the efficiency of inhibition for these cells was increased by at least 60-fold relative to the same oligonucleotide free in solution.

Animals↗
PubMed1993

Systemic administration of a phosphorothioate oligonucleotide with a sequence complementary to p53 for acute myelogenous leukemia and myelodysplastic syndrome: initial results of a phase I trial.

A synthetic phosphorothioate oligonucleotide was administered systemically to five patients with either relapsed or refractory acute myelogenous leukemia (AML), or myelodysplastic syndrome (MDS). Patients received a 10-day continuous intravenous infusion of this compound, which is complementary to p53 mRNA. No major toxicity attributable to a dose of 0.05 mg/kg/hr was observed. A range of approximately 9 to 18% of the administered dose was recovered in the urine as intact oligonucleotide. Evaluation of malignant cells recovered from bone marrow and peripheral blood at intervals before, during, and after treatment reveals no enhanced growth potential following oligonucleotide administration. Hence, a phosphorothioate oligonucleotide complementary to p53 mRNA can be administered at this dose level to humans without major toxicity. Higher doses need to be evaluated for toxicity and potential clinical efficacy.

Adolescent↗
PubMed1993

Inhibition of the protease of human immunodeficiency virus blocks replication and infectivity of the virus in chronically infected macrophages.

Because of the importance of monocytes/macrophages (M/M) as an in vivo reservoir of human immunodeficiency virus (HIV), a study was done to investigate whether viral replication in chronically infected macrophages (HIV M/M) could be inhibited by various drugs, including U-75875, an inhibitor of HIV protease. HIV replication in M/M and in chronically infected T cells was dramatically decreased by U-75875, while other drugs, including zidovudine, interferon-alpha, and an antisense oligodeoxynucleotide against the rev gene, were effective antiviral agents only in de novo-infected cells. Virus titer in HIV M/M was reduced approximately 10(5)-fold by nontoxic concentrations of U-75875, while no effect on HIV DNA or virus antigen expression on cell membrane was achieved in M/M infected either chronically or de novo. Thus, U-75875 essentially worked against late stages of viral replication. These data support the use of protease inhibitors, alone or in combination, in the therapy of HIV-infected patients.

Antiviral Agents↗
PubMed1993

In vitro evaluation of phosphorothioate oligonucleotides targeted to the E2 mRNA of papillomavirus: potential treatment for genital warts.

Papillomaviruses induce benign proliferative lesions, such as genital warts, in humans. The E2 gene product is thought to play a major role in the regulation of viral transcription and DNA replication and may represent a rational target for an antisense oligonucleotide drug action. Phosphorothioate oligonucleotides complementary to E2 mRNAs were synthesized and tested in a series of in vitro bovine papillomavirus (BPV) and human papillomavirus (HPV) models for the ability to inhibit E2 transactivation and virus-induced focus formation. The most active BPV-specific compounds were complementary to the mRNA cap region (ISIS 1751), the translation initiation region for the full-length E2 transactivator (ISIS 1753), and the translation initiation region for the E2 transrepressor mRNA (ISIS 1755). ISIS 1751 and ISIS 1753 were found to reduce E2-dependent transactivation and viral focus formation in a sequence-specific and concentration-dependent manner. ISIS 1755 increased E2 transactivation in a dose-dependent manner but had no effect on focus formation. Oligonucleotides with a chain length of 20 residues had optimal activity in the E2 transactivation assay. On the basis of the above observations, ISIS 2105, a 20-residue phosphorothioate oligonucleotide targeted to the translation initiation of both HPV type 6 (HPV-6) and HPV-11 E2 mRNA, was designed and shown to inhibit E2-dependent transactivation by HPV-11 E2 expressed from a surrogate promoter. These observations support the rationale of E2 as a target for antiviral therapy against papillomavirus infections and specifically identify ISIS 2105 as a candidate antisense oligonucleotide for the treatment of genital warts induced by HPV-6 and HPV-11.

Animals↗
PubMed1993

Inhibition of Rev activity and human immunodeficiency virus type 1 replication by antisense oligodeoxynucleotide phosphorothioate analogs directed against the Rev-responsive element.

The interaction between the Rev protein of human immunodeficiency virus type 1 and its highly structured and conserved RNA target, the Rev-responsive element, is required for virus replication. We demonstrate that antisense oligodeoxynucleotide phosphorothioate analogs directed against the Rev-responsive element effectively inhibit Rev activity, as well as human immunodeficiency virus type 1 replication, and are candidates for antiviral therapy.

Base Sequence↗
PubMed1993

Stabilities of duplexes and triplexes of dA19 + dT19 with alternating methylphosphonate and phosphodiester linkages.

Results from UV thermal melting, polyacrylamide gel electrophoresis and circular dichroism (CD) experimetns are used to compare stabilities of complexes formed from 1:1 and 1:2 molar ratio mixtures of dA19 or dA*P19 (alternating phosphodiester and methylphosphonate linkages on the A chain) with dT19 or dT*P19 (alternating phosphodiester and methylphosphonate linkages on the T chain). Complexes formed from 1:1 and 1:2 molar ratio mixtures of dA*P19 with dT19 are more stable than the respective unmodified 'parent' complexes, but less stable than complexes of dA*19 (17 methylphosphonates on the A chain) + dT19. At 0.1 M Na+ complexes containing dT*P19 are less stable than those with dT19, yet more stable than complexes containing the highly modified dT*19 (17 methylphosphonates on the T chain). A value of 2.6 kcal/mol base triplets is calculated for delta H of dA19.2dT19-->dA19.dT19+dT19.

Circular Dichroism↗
PubMed1992

Crystal and molecular structure of d(GTGCGCAC): investigation of the effects of base sequence on the conformation of octamer duplexes.

The structure of the self-complementary deoxyoctanucleotide d(GTGCGCAC), which crystallized as an A-type helix in the space group P4(3)2(1)2, with one strand in the crystallographic asymmetric unit has been determined and refined to a final R-value of 0.154 using 1.64-A diffraction data collected on an area detector. In contrast to the closely related sequence d(GTGTACAC)tet, there was no evidence for an ordered spermine molecule in the major groove of this octamer. Ordered water is found associated with almost all the exposed hydrogen bonding groups of the octamer. A pentagonal ring of water molecules is hydrogen bonded to O6 and N7 of G3 and the N4 and O6 of the C4.G13 base pair. A detailed comparison of the local helical parameters of d(GTGCGCAC) and d(GTGTACAC)tet is presented. The base sequence change at the center of the octamers affects several of the local helical parameters, via both intra- and interduplex interactions within the crystal.

Base Composition↗
PubMed1992

Crystal and molecular structure of the alternating dodecamer d(GCGTACGTACGC) in the A-DNA form: comparison with the isomorphous non-alternating dodecamer d(CCGTACGTACGG).

The crystal structure of the alternating dodecamer d(GCGTACGTACGC) (5'-GC) has been determined to a resolution of 2.55A using oscillation film data. The crystals belong to space group P6(1) 22, a = b = 46.2A, c = 71.5A with one strand in the asymmetric unit, and are isomorphous with a previously described non-alternating dodecamer, d(CCGTACGTACGG) (5'-CC). Refinement by X-PLOR/NUCLSQ gave a final R factor of 14.2% for 1089 observations. The molecule adopts the A-DNA form. The interchange of the terminal base pairs in the two dodecamers results in differences in the intermolecular contacts and may account for the differences in the bending. This dodecamer shows an axial deflection of 30 degrees, in the direction of the major groove compared to 20 degrees in 5'-CC and may be a consequence of additional contacts generated in 5'-GC by the interchange of end base pairs. The high helical axis deflection appreciably influences the local helical parameters. The molecule exhibits relatively high inclination angles, and has a narrow major groove. The helical parameters when described relative to the dyad-related hexamer halves of the molecule give more reasonable values. The crystal packing, local helical parameters, torsion angles, and hydration are described and also compared with the non-alternating 5'-CC dodecamer.

Amides↗
PubMed1992

In vivo treatment of human leukemia in a scid mouse model with c-myb antisense oligodeoxynucleotides.

The c-myb protooncogene encodes proteins that are critical for hematopoietic cell proliferation and development. Disrupting c-myb function might, therefore, prove an effective therapeutic strategy for controlling leukemic cell growth. Antisense oligodeoxynucleotides have been utilized for this purpose in vitro, but their in vivo efficacy has not been reported. We therefore established human leukemia-scid mouse chimeras with K562 cells and treated diseased animals with phosphorothioate-modified antisense oligodeoxynucleotides. K562 cells express the c-myb protooncogene, which served as the target for the antisense DNA. They also express the tumor-specific bcr-abl oncogene that was utilized to track the human cells in the mouse host. Once circulating leukemic blast cells had been detected, the survival of untreated control mice was 6 +/- 3 days (mean +/- SD). The survival of animals treated for 7 or 14 days with either sense or scrambled-sequence c-myb oligodeoxynucleotides was not statistically different from the control animals. In distinct contrast, animals treated for similar lengths of time with antisense c-myb oligodeoxynucleotides survived at least 3.5 times longer than the various control animals. In addition, animals receiving antisense c-myb DNA had significantly less disease at the two sites most frequently manifesting leukemic cell infiltration, the central nervous system and the ovary. These results suggest that phosphorothioate-modified antisense DNA may be efficacious for the treatment of human leukemia in vivo, and by analogy, for the treatment of other human neoplasias.

Animals↗
PubMed1992

Sequencing of peptides and proteins from the carboxy terminus.

A new chemical method for carboxy-terminal (C-terminal) protein sequencing has been developed. This approach has been successfully used to sequence 5 residues of standard proteins and 5 to 10 residues of synthetic peptides at low nanomole levels. The sequencing procedure consists of converting the C-terminal amino acid into a thiohydantoin (TH) derivative, followed by transformation of the TH into a good leaving group by alkylation. Next, the alkylated TH is cleaved mildly and efficiently with (N = C V S)- anion, which simultaneously forms a TH on the newly truncated protein or peptide. Thus, after the initial TH derivatization, there is no return to a free carboxyl group at the C-terminus. An additional benefit of this method is that the alkylating moiety can be chosen with a variety of properties allowing for variation in the detection method. This chemistry has been adapted to automated protein sequencers with a cycle time of about 1 h.

Amino Acid Sequence↗
PubMed1992

Crystal and molecular structure of the A-DNA dodecamer d(CCGTACGTACGG). Choice of fragment helical axis.

The crystal structure of the dodecamer d(CCGTACGTACGG) has been determined at 2.5 A resolution. The crystals grow in the hexagonal space group P6(1)22, a = b = 46.2 A, c = 71.5 A with one strand as the asymmetric unit. Diffraction data were collected by the oscillation film method yielding 1664 unique reflections with an Rmerge of 0.04. The structure was solved by real-space rotational translational searches with idealized helical models of A, B and Z-DNA. The best agreement was given by an A-DNA model with its dyad axis along the diagonal crystallographic dyad axis, with an R-factor 0.43 and correlation coefficient of 0.59 for data between 10 and 5 A. Iterative map fitting and restrained least-squares refinement and addition of 40 solvent molecules brought the R-factor to 0.15 and the correlation coefficient to 0.97 for all data between 8.0 and 2.5 A. The stereochemistry of the atomic model is good, with a root-mean-square deviation in bond distances of 0.006 A. This is the first example of an A-DNA containing a full helical turn. The dodecamer displays a novel packing motif. In addition to the characteristic contacts between the terminal base-pairs and the minor grooves of symmetry-related molecules, there are also minor groove to minor groove interactions not previously observed. The packing leaves an approximately 25 A diameter solvent channel around the origin, along the c-axis. The presence of a prominent 3.4 A meridional reflection and other diffuse features in the diffraction pattern provided evidence for the presence of disordered B-DNA along the c-axis, which can be accommodated in these solvent channels. The molecular conformation of the dodecamer also displays novel features. The dyad-related halves of the molecule are bent at an angle of 20 degrees, and the helical parameters are affected by this bend. Unlike the shorter A-DNA octamers, the dimensions of the major groove can be directly measured. Novel correlations between local helical parameters and global conformational features are presented. Most of the solvent molecules are associated with the major groove and the sugar-phosphate backbone.

Base Sequence↗