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Biomedical subjects

S Agrawal

Publications and source records attributed to S Agrawal.

At least 199 records · Page 11Linked to original sources
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

Enzymatic labeling of nucleic acids.

Enzymatic labeling of nucleic acids is a fundamental tool in molecular biology with virtually every aspect of nucleic acid hybridization technique involving the use of labeled probes. Different methods for enzymatic labeling of DNA, RNA and oligonucleotide probes are available today. In this review, we will describe both radioactive and nonradioactive labeling methods, yet the choice of system for labeling the probe depends on the application under study.

DNA↗
PubMed1996

Antisense oligonucleotides: towards clinical trials.

Antisense oligonucleotides have the ability to selectively block disease-causing genes, thereby inhibiting production of disease-associated proteins. The specificity and application of antisense oligonucleotides have been strongly validated in animal models for various disease targets. Based on the pharmacological, pharmacodynamic and pharmacokinetic profiles, the first generation of antisense oligonucleotides--phosphorothioates--have reached the stage of human clinical trials for various diseases. While ongoing human clinical trials are being carried out to further establishing the safety and efficacy of these oligonucleotides, the experience gained is providing a basis for designing a second generation of antisense oligonucleotides.

Administration, Oral↗
PubMed1996

Hybrid oligonucleotides: synthesis, biophysical properties, stability studies, and biological activity.

We have designed and synthesized hybrid oligonucleotides 2-5, as analogues of oligodeoxynucleoside phosphorothioates, in an effort to have agents with improved 'antisense activity' with reduced phosphorothioate content. The hybrid oligonucleotides contain segments of 2'-O-methyl ribonucleoside phosphoric diesters and oligodeoxynucleoside phosphorothioates. Thus, compared with the 'all' phosphorothioate analogues 1 and 6, the analogues 2-5 showed significantly reduced effect on complement activation. In addition, thermal denaturation studies with complementary RNA revealed that the analogues 2-5 had higher Tm compared with that with oligodeoxynucleoside phosphorothioates. Additionally, the RNA component of the oligo/ RNA duplex is efficiently cleaved by RNase H, the site of endonucleolytic cleavage being dictated by the length of the oligodeoxynucleoside phosphorothioate segment.

Anti-HIV Agents↗
PubMed1996

Single stand targeted triplex formation: physicochemical and biochemical properties of foldback triplexes.

Oligodeoxyribonucleotides containing both Watson-Crick and Hoogsteen hydrogen bonding domains joined by a nucleotide loop (FTFOs) are studied for their binding affinity and specificity to the DNA and RNA single-stranded targets. Thermal denaturation studies reveal that FTFOs have high binding affinity for their targets than do antisense (duplex forming) and antigene (triplex forming) oligonucleotides, because of involvement of both the Watson-Crick and Hoogsteen domains in the interaction. Studies with FTFOs containing different sizes and sequences of loops show that 4-6 bases long loops are optimum for binding; loop sequence does not have a dramatic effect on binding. The FTFOs have greater sequence specificity than do antisense and antigene oligonucleotides because they read the target sequence twice. SI-, PI- and mung bean nuclease protection assays show that the DNA FTFO forms a stable triplex with the DNA target strand, but a weak or no triplex with the RNA target strand. Gel mobility shift assay is used to determine binding of FTFOs to DNA and RNA targets. The circular dichroism (CD) spectrum of the foldback triplex formed with the DNA target strand resembles the B-DNA spectrum, suggesting that the triplex has a B-type of conformation.

Binding Sites↗
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↗
PubMed1996

Are patients who undergo open-access endoscopy more anxious about their procedures than patients referred from the GI clinic?

OBJECTIVES: Our academic hospital center operates an open access endoscopy service that allows referring physicians to directly schedule patients for outpatient esophagogastroduodenoscopy and colonoscopy without having them first seen in the GI clinic. Although patients from the GI clinic have an opportunity to meet the endoscopist and discuss their procedures directly beforehand, patients who are scheduled to undergo endoscopy through our open access service receive only a brief telephone call from a GI fellow or nurse that is made to screen for contraindications to the procedure and to give instructions. Our objective was to find out whether patients who used our open access service were more anxious about their procedures than those referred from the GI clinic. METHODS: On arrival to our endoscopy center, consecutive patients from the open access service (n = 142) and the GI clinic (n = 85) completed a questionnaire that included three measures of anxiety. After completion of the endoscopic procedure, the attending endoscopist rated patient cooperation. Patients also completed a phone questionnaire on the day after their procedures assessing their experience with endoscopy. RESULTS: We found no difference between patients from the open access service and those from the GI clinic in any of the measures of preprocedure anxiety, in scores measuring cooperation during procedures, or in patient ratings of their experience with endoscopy as assessed on the day after procedures. CONCLUSIONS: Patients who used our open access service were no more anxious about their procedures or less cooperative during them than patients first seen in the GI clinic. In both groups, procedure-related anxiety was decreased in patients who had previously undergone endoscopy.

Adolescent↗
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↗