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Biomedical subjects

S Agrawal

Publications and source records attributed to S Agrawal.

At least 19 recordsLinked to original sources
PubMed1996

Effect of different chemically modified oligodeoxynucleotides on immune stimulation.

Based on previous studies that certain oligonucleotides can stimulate cell proliferation and immunoglobulin production, this study was carried out to establish the relationship between the stimulatory effect and the chemical modification of the oligonucleotide. First, the effects of oligonucleotide and analogs on immune stimulation were studied in vitro using murine splenic lymphocytes. Our results show that cell proliferation and immunoglobulin production (IgG and IgM) depend on the sequence and the chemical modification of the oligonucleotide. Phosphorothioate oligodeoxynucleotides displayed a greater stimulatory effect than partially modified phosphorothioate oligonucleotides. Second, we studied the effects of these chemically modified oligonucleotides after injection in mice. Massive splenomegaly and stimulation of cell proliferation were observed with some phosphorothioate oligonucleotides. These effects were minimized markedly by chimeric and hybrid oligonucleotides. We also demonstrate that in vitro the effects of oligonucleotides on murine lymphocytes were unaffected by T cell depletion, suggesting that oligonucleotides exert their effects mainly on the B cells.

Adjuvants, Immunologic↗
PubMed1996

Differential effect of IFN-alpha and IFN-gamma on phosphorylation of p65 and p50 (rel) in the K562 cell line: implications for altered interaction with RXR beta.

Phosphorylation of nuclear transcription factors plays an important role in their ability to regulate genes in a differential manner. Recent studies indicate that the rel homology domain mediates interaction of rel proteins with other transcription factors. The rel domain is multiply phosphorylated. Thus, altered phosphorylation in this domain would affect synergy of rel proteins with members of other transcription factor families. This could affect differential expression of genes that are regulated by a combination of the two transcription factors. We have observed that in the K562 cell line IFN-alpha treatment leads to deficient phosphorylation of nuclear p65/p50 (rel). We propose that this prevents interaction between p65/p50 and RXR beta in IFN-alpha treated K562 cells. This could be a major reason for the lack of globin gene transcription by IFN-alpha.

Base Sequence↗
PubMed1996

Acute hepatitis associated with the use of a Chinese herbal product, ma-huang.

Herbal medicines are widely perceived by the public as being healthful and innocuous. A number of herbal medicines have now been linked with hepatotoxicity. We report a case of acute hepatitis associated with the use of ma-huang, a herbal product derived from plants of the Ephedra species, which is advertised as being useful for causing weight loss and enhancing energy levels. Given the lack of reports in the literature of hepatotoxicity with ma-huang and ephedrine, we speculate that the ma-huang product our patient took contained some other ingredient or contaminant or was misidentified. Our report and others in the literature, which we review, indicate that the clinician should consider herbal medicines as a possible cause of unexplained liver injury.

Acute Disease↗
PubMed1996

Pharmacokinetics and tissue disposition of a chimeric oligodeoxynucleoside phosphorothioate in rats after intravenous administration.

Antisense oligonucleotides represent a novel therapeutic principle for designing drugs against various diseases. Oligonucleotides can be chemically modified to improve their pharmacokinetics and in vivo stability, and it is important to understand the effect of these modifications. In the present study, the pharmacokinetics of a 25-mer phosphorothioate oligonucleotide containing four contiguous, internucleotide, methylphosphonate linkages at the 3'- and 5'-ends (chimeric oligonucleotide) were determined in rats after i.v. administration of the 35S-labeled oligonucleotide at a dose of 30 mg/kg. Plasma disappearance of the oligonucleotide could be described by a two-compartment model, with half-lives of 0.38 and 52.9 hr. The intact chimeric oligonucleotide was detected in plasma up to 6 hr after dosing. Urinary excretion represented the major elimination pathway, with approximately 21% of the administered dose being excreted within 24 hr and 35% being excreted over a 240-hr period after dosing. The majority of the radioactivity in urine was associated with the intact oligonucleotide within 6 hr after dosing and with increasing degradation products thereafter. Fecal excretion was a minor elimination pathway. The oligonucleotide was widely distributed in tissues, with the majority of the radioactivity in most tissues being intact up to 48 hr after dosing. Compared with oligodeoxynucleotide phosphorothioates, the chimeric oligonucleotide was significantly more stable in vivo. The presence of intact oligonucleotide in plasma and tissues even 12 hr after dosing is a significant advantage over an "all"-phosphorothioate analog. Thus, the chimeric oligonucleotide could provide a longer duration of action as an antisense agent after its administration.

Animals↗
PubMed1996

Comparative study of immunoglobulin G and immunoglobulin M among neonates in caesarean section and vaginal delivery.

The present study comprised 50 cases of normal vaginal delivery and 50 cases of caesarean section. The cord blood level of immunoglobulin G was significantly higher in normal vaginal delivery cases than in caesarean section cases (mean level is 1653.2 +/- 443.1 mg/dl and 898.3 +/- 415.6 mg/dl respectively). The immunoglobulin G was low in cord blood but had higher concentration in all the 7 cases in study group who had fever with rigor in antenatal period. Thus the babies delivered with caesarean section had lower immunoglobulin level than normal vaginal delivery.

Adult↗
PubMed1995

Single strand targeted triplex formation: targeting purine-pyrimidine mixed sequences using abasic linkers.

Foldback triplex-forming oligonucleotides (FTFOs) that contain an abasic linker, [2-(4-aminobutyr-1-yl)-1,3-propanediol] (APD linker), in the Hoogsteen domain against pyrimidine bases of a C:G and a T:A base pair were studied for their relative stability and sequence specificity of triplex formation. In general, the APD linker has less destabilizing effect against a C:G base pair than a T:A base pair. Incorporation of three APD linker moieties resulted in decreased binding to the target, which was comparable to results observed with three imperfectly matched natural base triplets. The APD linker incorporation did not result in the loss of sequence specificity of FTFOs, unlike in the case of normal triplex-forming oligonucleotides (TFOs). The introduction of a positively charged abasic linker, however, resulted in decreased stability of the triplex, because of loss of hydrogen bonding and stacking interactions in the major groove. The results of a molecular modeling study show that APD linker can be readily incorporated without any change in the conformation of the natural sugar-phosphate backbone conserving overall triple helix geometry. Further, the modeling study suggests a hydrogen bond formation between the amino group of linker and N4 of cytosine mediated by a solvent molecule (water) in the floor of the base triplet in addition to a contribution from the positive charge on the APD linker amino group. Either a direct or water-mediated hydrogen bond between the amino group of the APD linker and the O4 of thymine is unlikely when the linker is placed against a T:A base pair.

Base Composition↗
PubMed1995

Role of sodium in the pathophysiology of secondary spinal cord injury.

STUDY DESIGN: Experimental study using an in vitro model of compressive injury to isolated adult rat dorsal column axons. OBJECTIVES: To examine the role of extracellular Na+ (Na+e) in mediating secondary injury to spinal cord axons after compressive trauma. The mechanisms of intracellular sodium entry were examined using ion substitution techniques and pharmacologic blockers. SUMMARY OF BACKGROUND DATA: There is evidence that intracellular Na+ entry potentiates hypoxic-ischemic cell death by causing cytotoxic cell swelling, intracellular acidosis, and gating of Ca++ entry through reverse activation of the Na(+)-Ca++ exchanger. In the present study, we have examined the role of Na+e in the pathophysiology of spinal cord injury. METHODS: Dorsal column segments isolated from the thoracic cord of adult rats (n = 40) were pinned in a recording chamber and superfused with oxygenated Ringer's solution. Extracellular field potentials were recorded from glass microelectrodes (150 mmol KCl; 5-10 mol). Injury was accomplished in vitro by compression with a modified aneurysm clip (closing force, 2 g) for 15 seconds. The effect of zero Na+e (equimolar substitution with NMDG+), the Na(+)-H+ exchange blocker amiloride, the Na+ channel blocker procaine, and the Na(+)-Ca++ exchanger blocker benzamil on CAP recovery after compressive injury were assessed. RESULTS: Pretreatment with zero Na+, amiloride and procaine conferred significant neuroprotection (P < 0.05). In contrast, the NCE blocker benzamil was ineffective in attenuation secondary injury. CONCLUSIONS: Reduction of extracellular Na+, inhibition of the Na(+)-H+ exchanger or blockade of voltage gated Na+ channels is neuroprotective after spinal cord injury. The mechanism of Na(+)-associated cytotocity does not involve reverse gating of the Na(+)-Ca++ exchanger.

Action Potentials↗
PubMed1995

Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides.

Short oligonucleotides that can bind to adjacent sites on target mRNA sequences are designed and evaluated for their binding affinity and biological activity. Sequence-specific binding of short tandem oligonucleotides is compared with a full-length single oligonucleotide (21mer) that binds to the same target sequence. Two short oligonucleotides that bind without a base separation between their binding sites on the target bind cooperatively, while oligonucleotides that have a one or two base separation between the binding oligonucleotides do not. The binding affinity of the tandem oligonucleotides is improved by extending the ends of the two oligonucleotides with complementary sequences. These extended sequences form a duplex stem when both oligonucleotides bind to the target, resulting in a stable ternary complex. RNase H studies reveal that the cooperative oligonucleotides bind to the target RNA with sequence specificity. A short oligonucleotide (9mer) with one or two mismatches does not bind at the intended site, while longer oligonucleotides (21mers) with one or two mismatches still bind to the same site, as does a perfectly matched 21mer, and evoke RNase H activity. HIV-1 inhibition studies reveal an increase in activity of the cooperative oligonucleotide combinations as the length of the dimerization domain increases.

Antiviral Agents↗
PubMed1995

In vivo stability, disposition and metabolism of a "hybrid" oligonucleotide phosphorothioate in rats.

Oligodeoxynucleotide phosphorothioates containing segments of 2'-O-methyloligoribonucleotide phosphorothioates at both 3'- and 5'-ends (hybrid oligonucleotide) have been shown to be potent antisense agents. In the present study, in vivo biostability, disposition, and excretion of a 25-mer hybrid oligonucleotide were determined in rats after i.v. bolus administration of the 35S-labeled oligonucleotide at a dose of 30 mg/kg. The plasma disappearance curve for the hybrid oligonucleotide could be described by a two-compartmental model, with half-lives of 0.34 and 52.02 hr, respectively. The majority of the radioactivity in plasma was associated with the intact hybrid oligonucleotide. Urinary excretion represented the major pathway of elimination, with 21.98 +/- 3.21% (mean +/- SD) of the administered dose excreted within 24 hr and 38.13 +/- 2.99% over 240 hr post-dosing. The majority of the radioactivity in urine was associated with the degradative products with lower molecular weights, but the intact form was also detected by HPLC analysis. Fecal excretion was a minor pathway of elimination with 2.34 +/- 0.13% of the administered dose excreted over 24 hr and 6.74 +/- 0.40% over 240 hr post-dosing. A wide tissue distribution of hybrid oligonucleotide was observed based on radioactivity levels, and analysis by HPLC showed that the majority of the radioactivity in tissues was associated with the intact hybrid oligonucleotide. Further analyses of the experimental data provided a comprehensive pharmacokinetic analysis of hybrid oligonucleotide in each tissue. Compared with a previously examined oligodeoxynucleotide phosphorothioate (GEM 91) that has a similar nucleotide sequence, the hybrid oligonucleotide had a shorter distribution half-life and a longer elimination half-life, based on the quantitation of radioactivity in plasma. Although it had a similar tissue distribution pattern compared with other oligonucleotide phosphorothioates such as GEM 91, the hybrid oligonucleotide was more stable in vivo, which may be important in the development of antisense oligonucleotides as therapeutic agents.

Animals↗
PubMed1995

Absorption, tissue distribution and in vivo stability in rats of a hybrid antisense oligonucleotide following oral administration.

In vivo stability and oral bioavailability of an oligodeoxynucleotide phosphorothioate containing segments of 2'-O-methyloligoribonucleotide phosphorothioates at both the 3'- and 5'-ends (hybrid oligonucleotide) were studied. A 25-mer 35S-labeled hybrid oligonucleotide was administered to rats by gavage at a dose of 50 mg/kg body weight. HPLC analysis revealed that this hybrid oligonucleotide was stable in the gastrointestinal tract for up to 6 hr following oral administration. Radioactivity associated with the hybrid oligonucleotide was detectable in portal venous plasma, systemic plasma, various tissues, and urine. Intact hybrid oligonucleotide was detected, by HPLC analysis, in portal venous plasma, systemic plasma, and various tissues. The majority of the radioactivity in urine was associated with degradative products with lower molecular weights, but the intact form was also detected. In summary, the hybrid oligonucleotide was absorbed intact through the gastrointestinal tract, indicating the possibility of oral administration of oligonucleotides, a finding that may be important in the development of antisense oligonucleotides as therapeutic agents.

Administration, Oral↗
PubMed1995

Oligonucleotides containing G.A pairs: effect of flanking sequences on structure and stability.

Sixteen oligodeoxyribonucleotides, 5'd(GXGAYC)3', X and Y = G, A, C, or T, have been synthesized and studied by UV melting and 1H and 31P NMR methods. By varying X and Y, the sixteen resulting oligonucleotides can theoretically form 10 duplexes with all possible Watson-Crick base pairs flanking the center two G.A base pairs. Two-dimensional 1H NMR data on 5'd(GCGAGC)3' revealed that the center bases G and A pair through G amino hydrogen bonding and that the two consecutive G.A pairs form excellent purine-purine stacks. The concurrent appearance of one or more upfield-shifted imino proton peaks (approximately 10.5 ppm) and both upfield- and downfield- shifted 31P signals (approximately -2 and approximately -5.1 ppm) was a unique characteristic in imino 1H and 31P NMR spectra and was used as a conformational probe for this type of G.A pairs. Using this probe, seven out of 10 duplexes of 5'GXGAYC3' were found to adopt the G.A base pairing with G amino proton bonding and G to G and A to A base stacking. Three were in the group comprising 5'pyrimidine-GA-purine3', and four were in the group comprising 5'purine-GA-purine3'/5'pyrimidine-GA-pyrimidine3'. The G.A pairs in 5'purine-GA-pyrimidine3' adopted a totally different conformation. Thermodynamic analysis indicated that duplexes in the 5'pyrimidine-GA-purine3' group were more stable than the duplexes in the 5'purine-GA-pyrimidine3' group. Overall, G.C base pairs were preferred as neighbors to this type of G.A pairs.

Base Composition↗
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↗