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Biomedical subjects

S Agrawal

Publications and source records attributed to S Agrawal.

At least 235 records · Page 13Linked to original sources
PubMed1995

Expression of porcine transforming growth factor-beta.

Altered porcine transforming growth factor-beta (TGF-B)-cDNA was ligated to the expression vector. The resultant plasmid, pSA10041 was cotransfected with pSV2neo plasmid in NIH3T3 cells and clones were selected using G418. The clones were checked for integration of TGF-B-cDNA by PCR. The western blotting results confirm the expression of TGF-B in NIH3T3 cells. This construct can be used to develop transgenic mice to study its effect in vivo.

3T3 Cells↗
PubMed1994

Single-strand-targeted triplex formation: stability, specificity and RNase H activation properties.

Single-stranded (ss) oligodeoxyribonucleotides (oligos) containing both Watson-Crick and Hoogsteen hydrogen bonding domains joined by either a 5-nucleotide loop or a flexible hexaethylene-glycol linker, called foldback triplex-forming oligos (FTFOs), are designed and studied for their binding affinity and specificity to their ss DNA/RNA targets. Thermal denaturation studies revealed an increased affinity of FTFOs, due to addition of a Hoogsteen hydrogen bonding domain at the binding site, as the Watson-Crick domain forms a double helix with the target, when compared to conventional antisense and antigene oligos. DNase I hydrolysis and electrophoretic mobility shift analysis confirmed the formation of foldback triplexes relative to conventional double- and triple-stranded structures. The FTFOs showed increased sequence specificity mainly arising from their ability to recognize the target sequence twice, first by Watson-Crick base pairing and a second time by Hoogsteen base pairing. An FTFO with DNA components in both duplex- and triplex-forming domains showed preference for a DNA homopurine target strand.

Base Sequence↗
PubMed1994

Antisense oligodeoxynucleotide phosphorothioate complementary to Gag mRNA blocks replication of human immunodeficiency virus type 1 in human peripheral blood cells.

Gene-expression modulator 91 (GEM91) is a 25-nt antisense oligodeoxynucleotide phosphorothioate complementary to the Gag mRNA of human immunodeficiency virus type 1 (HIV-1). Cellular uptake and intracellular distribution of GEM91 within cells suggest that this oligomer is readily available for antisense activity. GEM91 inhibited HIV-1 replication in a dose-dependent and sequence-specific manner. In a comparative study, 2 microM GEM91 was as effective as 5 microM 3'-azido-3'-deoxythymidine in blocking virus replication during the 28-day treatment of an HIV-1-infected T-cell line. GEM91 also completely inhibited (> 99%) the growth of three different HIV-1 isolates in primary lymphocytes and prevented the cytopathic effect of the virus in primary CD4+ T cells. Similarly, treatment with GEM91 for 3 weeks of HIV-1/BaL-infected primary macrophages blocked virus replication. Based on GEM91 anti-HIV-activity, safety, and pharmacokinetic profile in animals, a clinical trial was started using this compound as an antisense oligonucleotide drug for the treatment of the acquired immunodeficiency syndrome.

Antigens, CD↗
PubMed1994

Modulation of the PC12 cell response to nerve growth factor by antisense oligonucleotide to amyloid precursor protein.

1. The amyloid precursor protein (APP) is widely distributed among eukaryotic cells, however, its precise role in cellular functioning is not fully clarified. APP is glycoprotein membrane constituent and it may facilitate membrane associated functions. 2. The aim of the present study was to examine the possibility that APP may play a role in mediating cellular trophic responses. The methods made use of an antisense oligonucleotide that was prepared to the 5' terminus of APP and shown specifically to reduce the level of APP isoforms. 3. In sequential mixing experiments it was observed that the APP antisense oligonucleotide did not significantly modify the trophic response of PC12 cells pretreated with nerve growth factor (NGF). However, pretreatment of cells with the antisense oligonucleotide diminished NGF-induced increases in cellular size and neurite length. 4. These observations suggest that APP may play a role in modulating the trophic response. The combined use of APP antisense oligonucleotides and neurotrophic agents may find clinical utility in the treatment of Alzheimer-type dementia since it is known that NGF normally causes increases in APP levels.

Alzheimer Disease↗
PubMed1994

Persistent polyclonal B-cell lymphocytosis.

We document a case of persistent lymphocytosis in which the characteristic binucleated circulating lymphocytes were shown immunologically to be polyclonal B-cells. Cytogenetic and molecular studies failed to show a clonal population. A review of the literature on this rare condition, persistent polyclonal B-cell lymphocytosis, highlights an association with the female sex, HLA-DR7 antigen, smoking and raised levels of IgM, all of which were present in our patient.

B-Lymphocytes↗
PubMed1994

Large-scale synthesis, purification, and analysis of oligodeoxynucleotide phosphorothioates.

Synthesis of oligonucleotides has been carried out on 1-, 2-, and 5-mmol scales using an appropriately modified automated, commercially available DNA synthesizer. The reaction cycles were optimized to obtain efficient coupling (> or = 97%). The synthesized oligonucleotide was purified by preparative reversed-phase liquid chromatography, followed by detritylation and desalting to obtain the oligonucleotides in the Na+ form. The purified oligonucleotides were characterized by 31P NMR, mass spectrometry, capillary gel electrophoresis, and ion-exchange high-performance liquid chromatography. By using these protocols, a 25-mer oligodeoxyribonucleotide can be synthesized on a 1-, 2-, or 5-mmole scale to obtain approximately 2.4, 4.8, or 12 g of purified product.

Base Sequence↗
PubMed1994

Complement activation and hemodynamic changes following intravenous administration of phosphorothioate oligonucleotides in the monkey.

Rapid intravenous infusion of GEM 91, a 25-mer phosphorothioate oligonucleotide complementary to the gag site of HIV, in the monkey produces transient decreases in peripheral total WBC and neutrophil counts, hemoconcentration, and a brief increase followed by a prolonged decrease in arterial blood pressure. These changes are preceded by and are likely mediated by activation of C5 complement. These effects are dose and infusion rate dependent and can be avoided by administering GEM 91 by slow intravenous infusion. Similar hemodynamic effects are produced with rapid intravenous infusion of other phosphorothioate oligonucleotides varying in length from 20- to 33-mer, and are, therefore, not sequence specific but a property of this chemical structure.

Animals↗
PubMed1994

Inhibition of in vitro transcription by oligodeoxynucleotides.

Single-stranded short phosphodiester oligodeoxynucleotides have been used to inhibit in vitro T7 transcription system. These oligodeoxynucleotides were complementary to either the 3'-5' or 5'-3' strand of the transcription initiation site of a plasmid containing the gag region of HIV. Our results show that incubation of this plasmid DNA with the oligodeoxynucleotide complementary to the template DNA strand (3'-5', sense oligo) showed efficient inhibition of transcription. Incubation of this plasmid with the oligodeoxynucleotide complementary to the 5'-3' strand (antisense oligo) or a random oligodeoxynucleotide failed to do so. The inhibition of gag transcription was specific since the sense oligo failed to prevent transcription of a plasmid containing U2 RNA sequences. The inhibition of transcription was not limited to T7 RNA polymerase but was also observed with SP6 RNA polymerase.

Bacteriophage T7↗
PubMed1994

Cellular uptake of oligodeoxynucleotide phosphorothioates and their analogs.

The uptake and intracellular distribution of oligonucleotide phosphorothioates (S-oligonucleotides) and their analogs by various cells lines were studied using internally 35S-labeled oligonucleotides. Intracellular accumulation starts in the first 2 hr and reaches a maximum at about 16 hr. A decrease of intracellular concentration of the S-oligonucleotide was observed after 16 hr, probably due to a significant efflux transport. Cellular uptake was dependent on the extracellular concentration. The intracellular concentration was significantly higher than that in the medium. The uptake and the intracellular distribution were different in the various cell lines studied. Comparative studies of the uptake of the S-oligonucleotide and various 3' end-modified S-oligonucleotides show that end modified S-oligonucleotides with a hydrophobic group significantly increases the uptake. The introduction of methylphosphonothioate linkages at the 3' end of the S-oligonucleotide also resulted in an increase in uptake.

3T3 Cells↗
PubMed1994

First report of fludarabine in gamma-heavy chain disease.

We describe a case of gamma-heavy chain disease presenting with lymphadenopathy, splenomegaly, lytic lesions and bone marrow infiltration. Chemotherapy with epirubicin, chlorambucil and prednisolone produced a partial remission which lasted 5 months. The patient was then treated with fludarabine. After six courses of fludarabine a good partial remission was achieved which converted to complete remission with disappearance of the monoclonal IgG3 in the ensuing 12 months without further therapy. This response has, so far, been sustained for 2 1/2 years after stopping treatment.

Aged↗
PubMed1994

Cimetidine-induced myelosuppression after bone marrow transplantation.

A strong temporal correlation was observed between cessation of cimetidine and a sustained increase in blood counts in two marrow transplant recipients. Both were receiving cimetidine from the day of transplantation for prophylaxis of stress ulceration and gastritis. The blood counts of both patients were not increasing satisfactorily 5-6 weeks after marrow transplantation without any obvious cause of marrow suppression. A similar observation has been made with the use of ranitidine after marrow transplantation suggesting that histamine H2-receptor antagonists should be used very cautiously, if at all, in the setting of bone marrow transplantation.

Adolescent↗