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Biomedical subjects

S Agrawal

Publications and source records attributed to S Agrawal.

At least 217 records · Page 12Linked to original sources
PubMed1995

Sequence identity of the n-1 product of a synthetic oligonucleotide.

After synthesis and purification of an oligonucleotide, the final product usually contains a low level of n-1 congeneric species. We have sequenced the n-1 population of a 25mer phosphodiester oligonucleotide. The n-1 band was cut from the gel and eluted. Oligonucleotides were tailed with dA and annealed to a dT-tailed plasmid. The recombinant plasmid was ligated and used to transform competent bacteria. Our results show that the n-1 population was heterogeneous. The frequency of truncated nucleotides at the 3'-end was much higher than at the 5'-end of the oligomer. No truncated nucleotides were found in the last four nucleotides at the 5'-end. Our results also show that the chain of oligonucleotides can grow on unreacted sites of a controlled-pore glass support.

Base Sequence↗
PubMed1995

Pharmacokinetics and tissue distribution in rats of an oligodeoxynucleotide phosphorothioate (GEM 91) developed as a therapeutic agent for human immunodeficiency virus type-1.

An antisense oligodeoxynucleotide phosphorothioate, namely gene expression modulator 91 (GEM 91), has been demonstrated to have significant anti-human immunodeficiency virus activity in various tissue culture models. The present study was undertaken to determine the pharmacokinetics and tissue distribution of GEM 91 in rats following i.v. bolus administration of 35S-radiolabeled GEM 91. Plasma disappearance curves for GEM 91-derived radioactivity could be described by the sum of two exponentials, with half-lives (mean +/- SEM) of 0.95 (+/- 0.07) and 47.57 (+/- 14.48) hr. Urinary excretion represented the major pathway of elimination of GEM 91, with 26.67 +/- 6.46% (mean +/- SD) of the administered dose excreted within 24 hr and 58.12 +/- 4.36% over 240 hr after GEM 91 administration. Fecal excretion was a minor pathway of elimination of GEM 91 with 1.4 +/- 0.62% (mean +/- SD) of the administered dose excreted over 24 hr and 8.54 +/- 0.64% over 240 hr. A wide tissue distribution of GEM 91 was observed. During the initial 30 min, the highest levels of tissue radioactivity were found in the kidney, liver, spleen, lungs, and heart. Radioactivity was retained over longer time periods in the kidneys, liver, heart, and intestine. Analyses of the extracted radioactivities from plasma, kidney, and liver by gel electrophoresis showed the presence of both intact GEM 91 and degradative products with smaller molecular weights. Radioactivity in urine was found to be degradative metabolites of GEM 91. Based on the experimental data, pharmacokinetic parameters for GEM 91 in each tissue and biological fluids were calculated using computer-based two-compartmental i.v. bolus or absorption models. This study is important not only in providing the basis for future studies of GEM 91 in humans, but also in understanding the pharmacology and toxicology of antisense oligodeoxynucleotide phosphorothioates, in general.

Animals↗
PubMed1995

Single strand targeted triplex-formation. Destabilization of guanine quadruplex structures by foldback triplex-forming oligonucleotides.

Oligonucleotides that can hybridize to single-stranded complementary polypurine nucleic acid targets by Watson-Crick base pairing as well as by Hoogsteen base pairing, referred to here as foldback triplex-forming oligonucleotides (FTFOs), have been designed. These oligonucleotides hybridize with target nucleic acid sequences with greater affinity than antisense oligonucleotides, which hybridize to the target sequence only by Watson-Crick hydrogen bonding [Kandimalla, E. R. and Agrawal, S. Gene(1994) 149, 115-121 and references cited therein]. FTFOs have been studied for their ability to destabilize quadruplexes formation by RNA or DNA target sequences. The influence of various DNA/RNA compositions of FTFOs on their ability to destabilize RNA and DNA quadruplexes has been examined. The ability of the FTFOs to destabilize quadruplex structures is related to the structurally and thermodynamically stable foldback triplex formed between the FTFO and its target sequence. Antisense oligonucleotides (DNA or RNA) that can form only a Watson-Crick double helix with the target sequence are unable to destabilize quadruplex structures of RNA and DNA target sequences and are therefore limited in their repertoire of target sequences. The quadruplex destabilization ability of FTFOs is dependent on the nature of the cation present in solution. The RNA quadruplex destabilization ability of FTFOs is -20% higher in the presence of sodium ion than potassium ion. The use of FTFOs, which can destabilize quadruplex structure, opens up new areas for development of oligonucleotide-based therapeutics, specifically, targeting guanine-rich sequences that exist at the ends of pro- and eukaryotic chromosomes and dimerization regions of retroviral RNA.

Base Sequence↗
PubMed1995

Diagnostic accuracy and role of fine needle aspiration cytology in management of thyroid nodules.

The diagnostic accuracy of fine needle aspiration cytology (FNAC) was evaluated in thyroid nodules in 100 consecutive cases, who subsequently underwent thyroidectomy between the years 1989-1991. FNAC as a diagnostic test for thyroid nodules demonstrated an accuracy of 90.9%, a sensitivity of 76.5%, a specificity of 95.9% with a false positivity of 2%, false negativity of 4%, positive and negative predictive values of 86.7% and 92.2%, respectively. A correct classification of the carcinoma type on the basis of FNAC was possible only in 69% patients. As a result, FNAC is the first line of investigation in most nontoxic nodular goiters and often the only procedure necessary to obtain an accurate diagnosis. However, it is recommended only as an adjunct to clinical judgment and is not intended to replace it.

Adolescent↗
PubMed1995

Stratification of the risk of thrombosis after intracoronary stenting for threatened or acute closure complicating coronary balloon angioplasty: a Cook registry study.

This study was carried out to stratify the risk of stent thrombosis by using three predictors: stent size, poststenting residual dissection, and residual filling defect. In the multicenter clinical trial, 1318 patients had successful deployment of Gianturco-Roubin coronary stent for threatened and acute closure. The 714 (54.2%) patients having none of these risk factors were designated a low-risk group; 484 (36.6%) had one factor and were designated an intermediate-risk group; 120 (9.1%) had two or all three factors and were designated a high-risk group. The incidence of stent thrombosis was 5.6%, 9.4%, and 16.7% in the low-, intermediate-, and high-risk groups; the difference among the three groups was highly significant (p < 0.0001). With these three predictors, the risk of stent thrombosis can be stratified. Avoiding the use of small stents (< 3.0 mm) and achieving optimal angiographic results after stenting for acute or threatened closure are useful strategies in reducing stent thrombosis.

Acute Disease↗
PubMed1995

Novel enzymatic and immunological responses to oligonucleotides.

Oligonucleotide phosphorothioates (PS-oligos) are being studied as antisense agents for viral infection and cancer. In preclinical studies, PS-oligos produced dose-dependent changes in heart rate and blood pressure and significantly reduced serum hemolytic complement, which could be avoided by slowing infusion rates. Here, in vitro PS-oligo treatment of either human, rhesus monkey or guinea pig serum reduced hemolytic complement and further inhibited in vitro coagulation when added to whole blood or citrated plasma. These effects were dependent upon both oligonucleotide dose and structure. Oligonucleotides having identical sequences but containing methylphosphonates (Chimeric), 2'-O-methyl ribonucleosides (Hybrid) or 3' hairpin loop (Self-stabilized) had altered effects on complement and coagulation in vitro.

Animals↗
PubMed1995

Modified oligonucleotides as therapeutic and diagnostic agents.

Modified oligonucleotides and their analogs represent an exciting new class of agents with potential therapeutic and diagnostic applications. Among the most extensively investigated analogs are oligonucleoside phosphorothioates, some of which incorporate 'hairpin' structures at the 3' end and 'chimeras' that bear two modified oligonucleotides, all potential therapeutic candidates. To further enhance their potential, bio-reversible analogs of oligonucleotides have been synthesized. Several isosteric analogs of phosphoric diester oligonucleotides with neutral achiral backbones, and other novel molecules, such as peptide nucleic acids and circular oligonucleotides, are also reported to form stable duplexes with complementary RNA/DNA and are currently under investigation. Additionally, oligonucleotides carrying a variety of fluorescent tags have been used as probes with potential for diagnostic applications.

Animals↗
PubMed1995

Single strand targeted triplex formation: strand displacement of duplex DNA by foldback triplex-forming oligonucleotides.

Linear oligonucleotides that bind to single-stranded nucleic acid targets by formation of both Watson-Crick (duplex) and Hoogsteen (triplex) hydrogen bonds simultaneously (foldback triplex-forming oligonucleotides; FTFOs) were studied for their ability to disrupt duplex DNA. Recently, we reported that FTFOs interfere with quadruplex forming ability of guanine rich RNA and DNA sequences and indicated that they might also disrupt duplex structures binding to the purine target strand by foldback triplex formation (Kandimalla and Agrawal, Nucleic Acids Res. (1995) 23, 1068-1074). We now obtained evidence for strand displacement of duplex DNA by FTFOs using nuclease assays and thermal melting studies. UV melting studies revealed that complementary strands of 16 to 31 bases long were completely displaced. Results of DNase I assays showed that the FTFOs bound to purine site by strand displacement probably by preassociating with the duplex DNA in the major groove via Hoogsteen hydrogen bonding and subsequently displacing the complementary strand. Experiments with S1 nuclease, an enzyme specific for single-stranded nucleic acids, confirmed the strand displacement ability of the FTFOs.

Base Sequence↗
PubMed1995

Antisense phosphorothioate oligodeoxynucleotides alter HIV type 1 replication in cultured human macrophages and peripheral blood mononuclear cells.

The use of antisense oligodeoxynucleotides as antiviral drugs to combat HIV-1 infection may offer an alternative to traditional pharmacological therapies. We compared the effects of two 28-mer antisense phosphorothioate oligodeoxynucleotides [PS-oligo(dN)] with non-sequence-specific controls on HIV-1 replication in long-term human monocyte/macrophage and PBMC cultures. The anti-rev PS-oligo(dN) was complementary to the messenger RNA (mRNA) sequences derived from the overlapping region of the HIV-1 regulatory genes tat and rev, while anti-gag targeted the translational initiation site of the gag mRNA. In vitro cytotoxicity of the PS-oligo(dN) was evaluated at concentrations ranging from 0.1 to 10.0 microM for a period of 20 days. Cell survival was 100% at 0.1 microM, but decreased to 5% at 10.0 microM in relation to the untreated control cultures. Our data demonstrate that replication of both the T cell-tropic and macrophage-tropic HIV-1 strains in primary cells can be inhibited by PS-oligo(dN) in a sequence-specific and dose-dependent manner at concentrations achievable in vivo. However, the sequence-dependent antiviral activity of the utilized PS-oligo(dN) was limited to a window of specificity at concentrations between 0.25 and 1.0 microM.

Antiviral Agents↗
PubMed1995

Uptake, intracellular distribution, and stability of oligodeoxynucleotide phosphorothioate by Schistosoma mansoni.

The in vitro uptake, cellular distribution, efflux, stability, and toxicity levels of an oligodeoxynucleotide phosphorothioate (PS-oligonucleotide) have been studied in mature Schistosoma mansoni worms. The intracellular accumulation of 35S-labeled PS-oligonucleotide occurred roughly in proportion to the worm body mass over a wide concentration range, whether the worms were exposed singly or in mating pairs. Cellular uptake was dependent on the extracellular concentration. A minor fraction (13%) of the PS-oligonucleotide taken up by the worm accumulated in the surface tegumental coat. Most of the PS-oligonucleotide taken up localized in the cytosol (54%) and the nuclei-enriched (33%) fractions. In a time course study on adult worms in culture, oligonucleotide uptake was observed within the first 2 h and peaked at about 36 h. A decrease in the intracellular concentration of the PS-oligonucleotide was observed by 42 h. Analysis of the extracted oligonucleotides showed that PS-oligonucleotide was digested slowly. Efflux of the oligonucleotide was time and temperature dependent. Significant toxicity to the cultured worms did not occur until the PS-oligonucleotide concentration was over 8 mg/ml (1 mM).

Animals↗
PubMed1995

Use of cyclodextrin and its derivatives as carriers for oligonucleotide delivery.

The use of antisense phosphorothioate oligodeoxynucleotides as tools for modulating gene expression represents a novel strategy for designing drugs to treat a variety of diseases. Several factors, including cellular uptake and internalization of the phosphorothioate oligodeoxynucleotide, are important parameters in determining the effectiveness of antisense agents as therapies. We have used cyclodextrin and its analogs as carriers to increase cellular uptake of phosphorothioate oligodeoxynucleotides. The studies were carried out using 35S-labeled and fluorescent-labeled phosphorothioate oligodeoxynucleotide in human T cell leukemia H9 cell line. Cellular uptake of phosphorothioate oligodeoxynucleotide in the presence of cyclodextrin was found to be concentration and time dependent. Using various cyclodextrin analogs, e.g., 2-hydroxypropyl beta-cyclodextrin (HPCD), hydroxyethyl beta-cyclodextrin (HECD), and a mixture of various hydroxypropyl beta-cyclodextrins (Encapsin), we observed increases in phosphorothioate oligodeoxynucleotide uptake, up to twofold to threefold in 48 hours. Confocal microscopy studies confirmed that oligonucleotide was present intracellularly. Cyclodextrin itself was not toxic at the concentration used. Cyclodextrins did not seem to affect the efflux of phosphorothioate oligodeoxynucleotide from cells. Stability of phosphorothioate oligodeoxynucleotide against endogenous cellular nucleases remained unchanged in the presence of cyclodextrins. These studies suggest that cyclodextrin and its analogs might be used successfully as carriers for oligonucleotide and analogs.

Amino Acid Sequence↗
PubMed1995

Outcome of pregnancy in women with recurrent spontaneous abortion following immunotherapy with allogeneic lymphocytes.

The efficacy of immunotherapy in the prevention of habitual abortion remains controversial. It has been suggested that the benefits are predominantly due to psychological factors. We have evaluated the success of pregnancy outcome following immunotherapy with allogeneic lymphocytes, in relation to the subsequent development of anti-paternal cytotoxic antibodies (APCA). It was observed that in women who developed an APCA titre of > or = 1:16, live births occurred in 16 out of 21 cases (76%), while only two out of seven (28%) women who failed to achieve an APCA titre of > or = 1:16 had successful pregnancies (P < 0.05). In eight women who had an APCA titre of 1:16 on initial screening, and were, therefore, excluded from the trial, successful pregnancy outcome was noted in 62.5% of cases. Although these results are based on a small sample and on an open, non-randomized trial, they show that the efficacy of immunotherapy is related to immune response to allogeneic lymphocytes, and is not simply a placebo effect. Measurement of APCA titre could serve as a marker for immunopotentiation.

Abortion, Habitual↗
PubMed1995

Compound heterozygosity of HLA-DR4 and DR1 antigens in Asian Indians increases the risk of extra-articular features in rheumatoid arthritis.

The relationship of HLA-DR antigens in rheumatoid arthritis (RA) patients with the presence of rheumatoid factor (RF), extra-articular features, joint erosions, functional status and response to disease modifying anti-rheumatic drugs (DMARDs) was studied. Seventy-four patients with RA were serologically typed for class II antigens. Clinical features were recorded before starting DMARDs and during follow-up to assess response and side-effects. HLA-DR1 and DR4 antigens were present more frequently in patients with RA than in the general population. The presence of DR4 correlated with RF seropositivity (P < 0.001) while DR1 was associated with seronegativity (P < 0.02). Ten of the 11 patients with extra-articular features had DR1 and/or DR4. Patients with both DR4 and DR1 had a significantly higher frequency of extra-articular features compared with those with only one of these (P < 0.01). HLA-DR4 and DR1 had no relation with the presence of subcutaneous nodules, erosive changes, deformities, response to drugs and side-effects of DMARDs.

Adult↗
PubMed1995

Recall lung pneumonitis due to carmustine after radiotherapy.

A patient who developed pneumonitis immediately after the administration of carmustine (BCNU), within exactly the same field as previous irradiation, is presented. The patient responded partially to corticosteroids. This case suggests that irradiation causes subclinical sensitisation of the lung and can therefore have an additive effect in precipitating lung damage when another pulmonary toxin is encountered at a later date.

Antineoplastic Agents, Alkylating↗
PubMed1995

Pharmacokinetics of antisense oligonucleotides.

Antisense oligonucleotides are promising therapeutic agents for the treatment of life-threatening diseases. Intravenous injection of phosphodiester oligonucleotide analogue (P-oligonucleotide) in monkeys shows that the oligonucleotide is degraded rapidly in the plasma with a half-life of about 5 minutes. Administration of a single dose of the phosphorothioate (S-oligonucleotide) in animals by the intravenous route reveals biphasic plasma elimination. An initial short half-life (0.53 to 0.83 hours) represents distribution out of the plasma compartment and a second long half-life (35 to 50 hours) represents elimination from the body. This elimination half-life was similar when the oligonucleotide was administered subcutaneously. In contrast, methylphosphonate oligonucleotides have an elimination half-life of 17 minutes in mice. S-Oligonucleotide was distributed into most of organs of rats and mice. Liver and kidney were the 2 organs with highest uptake of the oligonucleotide. The S-oligonucleotide was primarily excreted in urine. Up to 30% was excreted in the first 24 hours. Repeated daily intravenous injections of a 25-mer S-oligonucleotide into rats showed that the concentrations in the plasma are at steady-state during the 8 days' administration. The data represented here support the potential utility of phosphorothioate and methylphosphonate oligonucleotides as therapeutic agents in vivo.

Animals↗
PubMed1995

In vivo stability and disposition of a self-stabilized oligodeoxynucleotide phosphorothioate in rats.

The use of antisense oligonucleotides represents a novel, genetically based therapy. The biostability and pharmacokinetics of a 33-mer self-stabilized oligodeoxynucleotide with significant anti-HIV activity was determined in rats after intravenous administration of [35S]oligodeoxynucleotide. Plasma disappearance of the labeled oligodeoxynucleotide could be described by a two-compartment model, with half-lives of 0.54 and 41.44 h. The oligodeoxynucleotide in plasma remained mainly intact. Urinary excretion represented the major elimination pathway, with approximately 27% of the administered dose excreted within 24 h and 57% over 240 h. The majority of radioactivity in urine was attached to degradative products. Fecal excretion was a minor elimination pathway. A wide tissue distribution of the oligonucleotide was observed, with the majority of radioactivity in most tissues being intact. Compared with other linear oligonucleotide phosphorothioates, the self-stabilized oligonucleotide was more stable in vivo, which may be important in development of antisense oligonucleotides as therapeutic agents.

Animals↗