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S L Gupta

Publications and source records attributed to S L Gupta.

At least 19 recordsLinked to original sources
PubMed1996

Cooperative role of interferon regulatory factor 1 and p91 (STAT1) response elements in interferon-gamma-inducible expression of human indoleamine 2,3-dioxygenase gene.

Interferon (IFN)-gamma induces the expression of the indoleamine 2, 3-dioxygenase (INDO) gene in human cells, which plays a role in the inhibitory effect of IFN-gamma on intracellular pathogens and on cell proliferation. Earlier studies established that the IFN-gamma-inducible expression of the INDO gene was dependent on two upstream elements: (i) a 14-base pair sequence homologous to an interferon-stimulated response element (ISRE) sequence found in IFN-alpha-inducible genes and (ii) a 9-base pair palindromic sequence (palindromic element (PE) II) homologous to an interferon-gamma-activated site (GAS) element found in IFN-gamma-inducible genes. A second GAS element (PE I), between ISRE and PE II, was ineffective in supporting a response to IFN-gamma. Studies were carried out to determine the distinction between the two GAS elements and the relative role of the two elements (ISRE and PE II) required for a response to IFN-gamma. The PE I element was able to form a complex with IFN-gamma-activated p91 (STAT1) factor but with lower efficiency than the complex formed with PE II sequence. However, switching the positions of PE I and II sequences in reporter plasmid constructs (containing chloramphenicol acetyltransferase gene) showed that both PE I and PE II were able to support a response to IFN-gamma if located at the position of PE II but not at the position of PE I. Increasing the distance between the ISRE and PE II also affected the level of response, suggesting that the relative position of the two elements is important for optimal stimulus. To explore whether an interaction between the IFN-gamma-regulated factors (IRF-1 and p91) binding to the ISRE and PE II might be important, we tested whether the ISRE sequence could be replaced by another response element, NF-kappaB. The plasmid construct with NF-kappaB element in place of the ISRE was responsive to IFN-gamma, indicating that an interaction between the IRF-1 and p91 factors was not required. The results indicate that the response of INDO gene to IFN-gamma depends on a cooperative role of IFN-gamma-responsive factors binding to the ISRE and GAS elements.

Base Sequence↗
PubMed1995

Compatibility and activity of aldesleukin (recombinant interleukin-2) in presence of selected drugs during simulated Y-site administration: evaluation of three methods.

The compatibility and biological activity of aldesleukin (a form of recombinant interleukin-2) in the presence of selected i.v. drugs during simulated Y-site administration was studied. Five milliliters of aldesleukin 33,800 IU/mL in 5% dextrose injection was mixed in glass test tubes with 5 mL of each of 19 i.v. drugs prepared at concentrations used in routine clinical practice. The compatibility of the combinations was assessed by visual examination and spectrophotometry at 0, 0.5, 1, and 2 hours after preparation, and bioassays were conducted to determine the activity of aldesleukin in the combinations. Lorazepam was the only drug visually incompatible with aldesleukin. All the secondary drugs were spectrophotometrically compatible with aldesleukin. However, the bioassays showed that the following drugs reduced the activity of aldesleukin: ganciclovir sodium, lorazepam, pentamidine isethionate, prochlorperazine edisylate, and promethazine hydrochloride. Thus, aldesleukin became less biologically active when combined with four drugs for which visual examination suggested compatibility and when combined with five drugs for which spectrophotometry indicated compatibility. Aldesleukin 33,800 IU/mL in 5% dextrose injection lost significant biological activity in the presence of prochlorperazine edisylate, promethazine hydrochloride, lorazepam, ganciclovir sodium, and pentamidine isethionate during simulated Y-site administration. Visual assessment and spectrophotometry may not be valid methods for assessing possible changes in the biological activity of aldesleukin when combined with other agents.

Anti-Infective Agents↗
PubMed1995

Involvement of two regulatory elements in interferon-gamma-regulated expression of human indoleamine 2,3-dioxygenase gene.

Interferon (IFN)-gamma-induced expression of indoleamine 2,3-dioxygenase (IDO) gene is implicated in the antimicrobial and antiproliferative effects of IFN-gamma in cell cultures. Earlier studies identified a 96 base pair (bp) regulatory region upstream of the IDO gene that conferred IFN-gamma response to the chloroamphenicol acetyltransferase (CAT) gene linked to herpesvirus thymidine kinase promoter. The IFN-gamma-responsive region was further narrowed to a 67 bp fragment by 3' deletion. This 67 bp fragment contains several sequence elements of potential interest, including a 14 bp sequence homologous to the ISRE sequence found in IFN-alpha-inducible genes and two palindromic sequences (PE I and PE II) homologous to the GAS sequence identified in IFN-gamma-inducible genes. Site-directed mutagenesis studies showed that IFN-gamma-induced expression of IDO-CAT constructs involved cooperation between two elements: the ISRE homolog and the PE II (but not PE I). Either element alone with its flanking sequence was inadequate in conferring an IFN-gamma response to CAT reporter gene. Two IFN-gamma-regulated protein factors interacting with these two elements were identified. The factor binding to the ISRE region was induced with a slower kinetics, required new protein synthesis, and reacted with antibodies to IRF-1. The factor interacting with the PE II region appeared rapidly after treatment with IFN-gamma independently of new protein synthesis, and its binding to DNA probe was blocked by antibodies to p91 factor, reported to bind to GAS element.(ABSTRACT TRUNCATED AT 250 WORDS)

Antisense Elements (Genetics)↗
PubMed1994

Pulmonary lung surfactant synthetic peptide concentration-dependent modulation of DPPC and POPG acyl chain order in a DPPC:POPG:palmitic acid lipid mixture.

Lung surfactant-associated protein interaction with lipid matrices and the effects on lipid thermotropic phase behavior are areas of active research. Many studies limit the lipids to a single or two-component system. The current investigation utilizes a three-lipid component matrix (DPPC:POPG:palmitic acid) to investigate the impact of a synthetic surfactant protein B fragment (SP-B 53-78 DiACM) on the dynamic surface activity of the lipid admixture as measured by a Wilhelmy surface balance. Also, the modulation of the individual lipid acyl chain order by the peptide within the lipid matrix is studied through the use of thermal perturbation FTIR spectroscopy. The data clearly demonstrate a concentration-dependent effect of the peptide on the surface activity with an improvement in the dynamic surface tension diagram characteristics (decreased surface tension and increased collapse plateau) especially at low, 0.36 M%, peptide concentrations. These effects are diminished upon further addition of the peptide. FTIR spectral data demonstrate that the peptide addition results in a significant increase in the acyl chain order of the DPPC and POPG components as measured by the position of the methylene stretching vibrational bands. DPPC is most sensitive to the peptide presence, while the palmitic acid is least affected. The transition temperatures of the individual lipids are also increased with the addition of the peptide. The presence of POPG in the matrix achieves the surface activity similarly seen with natural lung surfactant relative to a DPPC/palmitic acid lipid matrix alone. Its presence increases the sensitivity of the DPPC acyl chains to the presence of the peptide. These effects on the chain order are most probably related to the increased acyl chain fluidity which POPG imparts to the lipid matrix because of the presence of the cis double bond. The phosphatidylglycerol headgroup also adds a negative charge to the lipid matrix which enhances the peptide-lipid interaction. Although the palmitic acid is minimally affected by the peptide, its presence, as suggested by surface balance measurements, results in the establishment of a stable lipid film with DPPC, capable of achieving low surface tension values.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Effect of a bovine lung surfactant protein isolate (SP-B/C) on egg phosphatidylglycerol acyl chain order in a lipid mixture with dipalmitoylphosphatidylcholine and palmitic acid.

Dynamic surface tension measurements of films of a d62 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine:L-alpha-phosphatidyl-DL - glycerol:d31 palmitic acid (d62-DPPC:EggPG:d31-PA) lipid matrix in the presence of a bovine pulmonary surfactant protein isolate (SP-B/C) demonstrate the improved surface activity over that of the lipids alone. Thus, significant interaction of the proteins with the lipid matrix is demonstrated. The effect of SP-B/C on the acyl chain order of the negatively charged EggPG within a d62-DPPC:EggPG:d31-PA lipid matrix in D2O saline was investigated in thermal perturbation Fourier transform IR spectroscopic studies. The EggPG thermotropic phase behavior was determined independently of the other lipid components with perdeuterated lipids and D2O. The data demonstrate the high degree of EggPG acyl chain disorder in the absence of the protein isolate. A broad transition occurs between 30 and 40 degrees C. The addition of the protein isolate did not alter the acyl chain order at 0.281 and 1.46 mg/mL of protein. However, alterations in the lipid carbonyl vibrational mode were observed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Antiparasitic and antiproliferative effects of indoleamine 2,3-dioxygenase enzyme expression in human fibroblasts.

Studies were carried out to evaluate the proposed role of indoleamine 2,3-dioxygenase (INDO) induction in the antimicrobial and antiproliferative effects of gamma interferon (IFN-gamma) in human fibroblasts. The INDO cDNA coding region was cloned in the pMEP4 expression vector, containing the metallothionein (MTII) promoter in the sense (+ve) or the antisense (-ve) orientation. Human fibroblasts (GM637) stably transfected with the sense construct expressed INDO activity after treatment with CdCl2 or ZnSO4, but cells transfected with the antisense construct did not. The growth of Chlamydia psittaci was strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+ or Zn2+. The inhibition correlated with the level of INDO activity induced and could be reversed by the addition of excess tryptophan to the medium. The growth of Toxoplasma gondii was also strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+. Expression of Cd(2+)-induced INDO activity also inhibited thymidine incorporation and led to cytotoxicity in INDO +ve cells but not in INDO -ve cells. Thus, the induction of INDO activity by IFN-gamma may be an important factor in the antimicrobial and antiproliferative effects of IFN-gamma in human fibroblasts.

Animals↗
PubMed1994

Molecular cloning of a novel receptor tyrosine kinase, tif, highly expressed in human ovary and testis.

Using a combination of polymerase chain reaction and conventional cDNA library screening approaches, we have cloned and characterized a putative receptor tyrosine kinase termed tif. The extracellular domain of tif has an immunoglobulin-like loop and a fibronectin type III structure. The intracellular domain contains a tyrosine kinase domain. Compared with ryk, a ubiquitously expressed receptor tyrosine kinase, tif expression is tissue-specific with human ovary and testis containing the highest amount of tif mRNA. Many other tested human tissues such as heart, liver, pancreas and thymus do not contain detectable levels of tif mRNA. The molecular cloning and characterization of tif cDNA will facilitate the identification of a potential ligand(s) for the putative receptor and the study of its biological role.

Amino Acid Sequence↗
PubMed1993

Enhanced gel mobility shift assay for DNA-binding factors.

Gel mobility shift assays are commonly used to study DNA-binding factors involved in the regulation of constitutive, tissue-specific, and inducible genes. We found that addition of 3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonate (Chaps, a zwitterionic detergent) at relatively high concentration (2.5% or more) to DNA-binding reactions for four different factors (AP-1, SP1, GATA-1, and interferon alpha-regulated factor ISGF3) assayed in cell extracts greatly enhanced the signal for DNA-protein complexes (up to about 20-fold). The amplified signal for DNA-protein complexes so obtained was (at least in part) due to increased binding efficiency, as revealed by greatly reduced amounts of the free probes in the gels. The binding specificity, however, was not compromised. The fact that DNA-protein complex formation with four different factors was stimulated by Chaps suggests that the enhancing effect of Chaps may be more general and not limited to certain types of DNA-binding factors. The results provide the basis for a highly sensitive assay for DNA-binding factors, which may be useful in several types of studies on such factors. Among other detergents tested, Chapso (another zwitterionic detergent), NP-40, and octylglucopyranoside (nonionic detergents) were found also to enhance the complex formation as tested for AP-1 binding, whereas sodium cholate and deoxycholate showed strong inhibition.

Base Sequence↗
PubMed1993

Differential regulation of human indoleamine 2,3-dioxygenase gene expression by interferons-gamma and -alpha. Analysis of the regulatory region of the gene and identification of an interferon-gamma-inducible DNA-binding factor.

The induction of indoleamine 2,3-dioxygenase (IDO) activity has been implicated in the antiproliferative action of interferon (IFN)-gamma on tumor cells and the inhibition of intracellular pathogens. Earlier studies have demonstrated that the expression of the IDO gene is induced strongly by IFN-gamma, but very poorly by IFN-alpha despite the presence of a sequence highly homologous to the IFN-alpha-responsive sequence element (interferon-stimulated response element (ISRE)) in its IFN-gamma-responsive control region. In addition, a sequence with a partial homology to the IFN-gamma-responsive sequence (GAS) identified by Lew et al. (Lew, D.J., Decker, T., Strehlow, I., and Darnell, J.E., Jr. (1991) Mol. Cell. Biol. 11, 182-191) in a human gene for a guanylate-binding protein and to the X box sequence found in all major histocompatibility complex class II genes was found. Deletion experiments have indicated that the ISRE homolog (but not the GAS-related or the X box-related sequence) was essential for an inducibility by IFN-gamma. To investigate the lack of inducibility by IFN-alpha despite the presence of an ISRE homolog, the binding of this ISRE homolog to the IFN-alpha-stimulated gene factor 3 (ISGF3) was examined. Gel mobility shift experiments and competition experiments indicated that this ISRE homolog did not form a stable complex with ISGF3. This may account for a poor inducibility by IFN-alpha. This inability to bind ISGF3 appears to be (at least in part) due to minor differences between the nucleotide sequence of the ISRE homolog present in the IDO gene promoter and the ISRE consensus sequence found in IFN-alpha-inducible genes. An IFN-gamma-inducible DNA-binding factor was identified with characteristics different from ISGF3: (i) the IFN-gamma-inducible factor was detected in the nuclear extracts, but not in the cytoplasmic extracts; and (ii) the appearance of this DNA-binding factor required new protein synthesis, which could explain the dependence on new protein synthesis for the induction of IDO by IFN-gamma.

Amino Acid Sequence↗
PubMed1991

Analysis of interferon-gamma resistant mutants that are possibly defective in their signaling mechanism.

Our previous observations indicated that mutants partially resistant to IFN-gamma cytotoxicity were defective in the induction of indoleamine 2,3-dioxygenase, (IDO). Two mutants highly resistant to IFN-gamma were isolated following a second round of mutagenesis. The resistance to IFN-gamma was inversely correlated with the inducibility of IDO in these mutants. Moreover, several other IFN-gamma responsive genes, including those encoding 2-5A synthetase, GTP cyclohydrolase and HLA-DR alpha, were also differentially altered in their expression upon INF-gamma treatment. IFN-gamma receptor gene expression was not changed nor was the binding of the receptor to IFN-gamma. Southern blot analysis failed to reveal any significant abnormality in the IDO gene structure in these mutants. We therefore suggest that these mutants are defective in the IFN-gamma signaling pathway and will be useful in further analysis of the biochemical mechanism of IFN-gamma activated gene expression in target cells.

Blotting, Northern↗
PubMed1990

Regulation of indoleamine 2,3-dioxygenase gene expression in human fibroblasts by interferon-gamma. Upstream control region discriminates between interferon-gamma and interferon-alpha.

The interferon (IFN)-gamma-mediated induction of indoleamine 2,3-dioxygenase (IDO) enzyme, which converts tryptophan into N-formylkynurenine, has been implicated in the inhibition of intracellular pathogens, e.g. Toxoplasma gondii and Chlamydia psittaci, and in the antiproliferative effect of IFN-gamma on tumor cells. The IDO activity is induced strongly in many cell types by IFN-gamma but rather poorly by IFN-alpha or -beta. A genomic DNA clone containing part of the transcribed region of the IDO gene and approximately 13 kilobases (kb) of the 5'-upstream DNA sequence was isolated and analyzed. An approximately 1.4-kb fragment of this clone, containing 329 nucleotides of the transcribed sequence and approximately 1.1 kb of the 5'-upstream sequence, when ligated to chloramphenicol acetyltransferase (CAT) structural gene made its expression inducible by IFN-gamma, but this construct responded poorly, if at all, to IFN-alpha 2. Deletion constructs derived from this plasmid narrowed down the IFN-gamma-responsive region to a 151-nucleotide segment (-495/-344) which also contained a 14-nucleotide sequence (GGTTTCAGTTTTCC) highly homologous to the IFN(alpha)-stimulated response element (ISRE) that has been found so far in all cellular genes inducible with IFN-alpha or -beta. Expression of CAT activity was stimulated by IFN-gamma more effectively than by IFN-alpha 2 when a 155-nucleotide fragment (-495/-340) containing the 151-nucleotide segment required for IFN-gamma response was inserted before herpes simplex virus thymidine kinase promoter linked to CAT structural gene. The results indicate that despite the presence of an ISRE, the control region of the IDO gene can distinguish between IFN-gamma and IFN-alpha. This may account for the differential activation of IDO gene expression by IFN-gamma as against IFN-alpha or -beta in intact cells, and suggests that the response of ISRE to IFN-alpha or -beta may be governed by other features in the upstream control region of this gene.

Base Sequence↗
PubMed1990

Molecular cloning, sequencing and expression of human interferon-gamma-inducible indoleamine 2,3-dioxygenase cDNA.

The antiproliferative action of human interferon (HuIFN)-gamma on human cells and the inhibition of intracellular pathogens, e.g. Toxoplasma gondii and Chlamydia psittaci, is at least in part due to an induction of indoleamine 2,3-dioxygenase (IDO) enzyme which degrades tryptophan, an essential amino acid. A cDNA clone (called C42) was isolated from a cDNA library made from poly(A)+ RNA obtained from HuIFN-gamma-treated human fibroblasts. Its nucleotide sequence revealed an open reading frame coding for a polypeptide of 403 amino acids, but no homology with any known gene in GenBank database was found. Evidence was obtained indicating that this cDNA codes for IDO: (i) Hybrid selected C42 specific poly(A)+ RNA from IFN-gamma-treated human cells coded for a polypeptide in vitro of approximately 42 kD (reported size of IDO, approximately 40 kD) which was immunoprecipitated by monoclonal anti-IDO antibody but not by a control antibody; and (ii) transfection of human fibroblasts with an expression plasmid containing C42 cDNA transcribed from chicken beta-actin promoter led to constitutive expression of C42 specific RNA as well as IDO activity. This cDNA clone will be useful in studying the role of IDO in the biological effects of IFN-gamma, and the regulation of IDO gene by IFN-gamma.

Amino Acid Sequence↗
PubMed1990

Regulation of cellular gene expression by interferon-gamma: involvement of multiple pathways.

Interferons (IFNs) classified as type I (IFN-alpha and -beta) and type II (IFN-gamma) interact with different receptors and regulate the expression of a number of genes in common, whereas the expression of certain other genes is regulated differentially by the type I or type II IFNs. Regulation of cellular gene expression by IFN-gamma was studied with the help of two cDNA clones (called C5-4 and C13) isolated in our laboratory and cDNA clones for human leukocyte antigen (HLA) class I (HLA-B) and class II (HLA-DR alpha, -DR beta) genes. The results indicate that IFN-gamma induced the expression of the cognate genes, but in different manners. IFN-gamma induced the transcription of all four genes as determined by nuclear run-on transcription analyses, but the induction of C5-4 genes transcription was inhibited by cycloheximide and anisomycin, indicating that some newly synthesized protein, presumably induced by IFN-gamma, was required for the transcriptional activation of the C5-4 gene. On the contrary, IFN-gamma-induced transcription of HLA class I, class II and C13 genes was unaffected by cycloheximide or anisomycin. However, these inhibitors completely blocked the accumulation of the HLA class II gene transcripts, but not HLA class I or C13 gene transcripts. Results suggest that some newly synthesized protein factor was required for IFN-gamma-induced accumulation of HLA class II gene transcripts and played a role at a step subsequent to the transcriptional activation by IFN-gamma. Evidence was obtained which suggests that the putative protein factor(s) required was induced by IFN-gamma. These studies indicate that IFN-gamma regulates the expression of cellular genes through multiple pathways.

Cloning, Molecular↗
PubMed1989

Involvement of an inducible factor in interferon-gamma-mediated accumulation of HLA gene transcripts.

Earlier studies with a cDNA clone (C5-4) complementary to an interferon (IFN)-gamma-inducible mRNA showed that in human fibroblasts (FS-4), IFN-gamma induced the transcription of the cognate gene, but it required new protein synthesis (Caplen and Gupta, J. Biol. Chem. 263, 332-339, 1988). To determine whether such a strategy is used for the regulation of other cellular genes by IFN-gamma, the regulation of the HLA class I and class II genes and another cellular gene for which a cDNA clone was isolated (C13) was studied. The results indicate that: (i) HLA-B (class I) and C13 gene expression was transcriptionally activated by IFN-gamma and IFN-alpha 2, and it did not require new protein synthesis. (ii) In contrast, the transcription of the HLA-DR alpha was activated by IFN-gamma (and not by IFN-alpha 2), but the accumulation of -DR alpha gene transcripts was strongly inhibited by cycloheximide or anisomycin, which indicated that there was a requirement for some newly synthesized protein factor(s) in this process, apparently at a step subsequent to transcriptional activation. We obtained evidence indicating that the putative protein factor(s) required is actually induced by IFN-gamma. (iii) IFN-gamma-induced transcription of the HLA-B gene was not inhibited by anisomycin or cycloheximide, but the accumulation of HLA-B transcripts plateaued sooner. This latter effect was not due to any toxicity of these inhibitors because it was observed if cycloheximide was added together with IFN-gamma, but not if it was added a few hours later. Furthermore, if cycloheximide was added 24 h after IFN-gamma, it actually caused a superinduction of HLA-B transcripts. The results suggest that some newly synthesized protein factor(s) may be required also for maximal accumulation of HLA-B gene transcripts following treatment with IFN-gamma. The results indicate a dual regulation of HLA class I and class II genes by IFN-gamma, and involvement of multiple mechanisms in the regulation of cellular gene expression by IFN-gamma.

Genes, MHC Class I↗
PubMed1988

Differential regulation of a cellular gene by human interferon-gamma and interferon-alpha.

Two cDNA clones complementary to interferon (IFN)-gamma inducible mRNAs in human cells were isolated, and one of these, C5-4, was used for studying the regulation of its cognate mRNA by IFNs. C5-4 cDNA hybridized to a 2.2-kilobase mRNA that coded for a 42,000 Mr polypeptide in vitro. This mRNA was induced by HuIFN-gamma as well as by HuIFN-alpha 2 but in different manners: (i) IFN-gamma induced this mRNA greater than 100-fold and this induction was sustained for at least 48 h, whereas IFN-alpha 2 induced it approximately 10-fold but only transiently. (ii) Cells that were pretreated with IFN-alpha 2 (18h) became refractory to induction by IFN-alpha 2 but responded normally to IFN-gamma. (iii) Cycloheximide (50 micrograms/ml) and anisomycin (10 micron or higher) inhibited the induction by IFN-gamma but not by IFN-alpha 2, suggesting the requirement for some newly synthesized, presumably IFN-gamma-induced, protein factor(s) in IFN-gamma-mediated but not in IFN-alpha 2-mediated induction. Interestingly, this inhibition by cycloheximide of the IFN-gamma-mediated induction was overcome by 24 h, whereas a sustained inhibition was obtained with anisomycin. Our results suggest that this release from cycloheximide-mediated inhibition was apparently due to a leakiness in its inhibitory action on protein biosynthesis. As analyzed by nuclear runoff transcription, IFN-gamma induced the transcription of C5-4-specific RNA within 2 h and it was inhibited by cycloheximide, indicating that the newly synthesized protein mediator(s) required plays a role in the transcriptional activation of the C5-4 gene by IFN-gamma. Southern blot analysis indicated that the C5-4 gene is present in a single copy in the genome. The results demonstrate that IFN-gamma and IFN-alpha induced the expression of a common gene through different mechanisms and suggest the involvement of an intermediary mechanism in IFN-gamma-mediated transcriptional activation of certain cellular genes.

Anisomycin↗