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Biomedical subjects

A Dasgupta

Publications and source records attributed to A Dasgupta.

At least 217 records · Page 12Linked to original sources
PubMed1989

Multiple isoelectric forms of poliovirus RNA-dependent RNA polymerase: evidence for phosphorylation.

Poliovirus-specific RNA-dependent RNA polymerase (3Dpol) was purified to apparent homogeneity. A single polypeptide of an apparent molecular weight of 63,000 catalyzes the synthesis of dimeric and monomeric RNA products in response to the poliovirion RNA template. Analysis of purified 3Dpol by two-dimensional electrophoresis showed multiple forms of 3Dpol, suggesting posttranslational modification of the protein in virus-infected cells. The two major forms of 3Dpol appear to have approximate pI values of 7.1 and 7.4. Incubation of purified 3Dpol with calf intestinal phosphatase resulted in almost complete disappearance of the pI 7.1 form and a concomitant increase in the intensity of the pI 7.4 form of 3Dpol. Addition of 32P-labeled Pi during infection of HeLa cells with poliovirus resulted in specific labeling of 3Dpol and 3CD, a viral protein which contains the entire 3Dpol sequence. Both 3Dpol and 3CD appear to be phosphorylated at serine residues. Ribosomal salt washes prepared from both mock- and poliovirus-infected cells contain phosphatases capable of dephosphorylating quantitatively the phosphorylated form (pI 7.1) of 3Dpol.

Amino Acids↗
PubMed1989

Multiple transcriptional regulatory domains in the human immunodeficiency virus type 1 long terminal repeat are involved in basal and E1A/E1B-induced promoter activity.

The human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR) is the site of activation of the HIV tat protein. However, additional transactivators, such as the adenovirus E1A and herpesvirus ICPO proteins, have also been shown to be capable of activating the HIV LTR. Analysis of adenovirus mutants indicated that complete transactivation of the HIV LTR was dependent on both the E1A and E1B proteins. To determine which regions of the HIV LTR were important for complete E1A/E1B activation, a variety of oligonucleotide-directed mutations in HIV transcriptional regulatory domains were assayed both in vivo and in vitro. S1 nuclease analysis of RNA prepared after transfection of these HIV constructs into HeLa cells infected with wild-type adenovirus indicated that the enhancer, SP1, TATA, and a portion of the transactivation-responsive element were each required for complete E1A/E1B-mediated activation of the HIV LTR. These same promoter elements were required for both basal and E1A/E1B-induced levels of transcription in in vitro transcription reactions performed with cellular extracts prepared from cells infected with dl434, an E1A/E1B deletion mutant, or wild-type adenovirus. No mutations were found that reduced only E1A/E1B-induced expression without proportionally reducing basal levels of transcription, suggesting that E1A/E1B-mediated induction of the HIV LTR requires multiple promoter elements which are also required for basal transcriptional levels. Unlike activation by the tat protein, there was not a rigid dependence on maintenance of the transactivation-responsive stem base pairing for E1A/E1B-mediated activation either in vivo or in vitro, indicating that activation occurs by a mechanism distinct from that of tat induction.

Adenovirus Early Proteins↗
PubMed1989

Inhibition of rRNA synthesis by poliovirus: specific inactivation of transcription factors.

Synthesis of rRNA by RNA polymerase I is almost completely inhibited soon after infection of human cells with poliovirus. We show that extracts prepared from poliovirus-infected HeLa cells are severely inhibited in their ability to transcribe from a human rDNA promoter compared with extracts from mock-infected cells. Two lines of evidence presented here suggest that a specific transcriptional activity required for rDNA transcription in vitro is impaired in virus-infected cells. First, fractionation of individual transcriptional components by phosphocellulose chromatography and subsequent reconstitution experiments showed that the specific transcriptional activity of fraction C (0.8 M KCl eluate) from virus-infected cells was reduced three- to fourfold relative to that isolated from mock-infected cells. The activities of other transcription factors needed for in vitro transcription from the rDNA promoter were unaffected. Second, fraction C derived from mock-infected cells specifically restored transcription in extracts prepared from virus-infected cells. Fraction C contained both a nonspecific RNA polymerase I elongation activity and a specific factor activity which was needed for accurate transcription initiation. It is the specific transcriptional activity and not the nonspecific chain elongation activity of fraction C that is affected in cells infected with poliovirus.

Cell Transformation, Viral↗
PubMed1989

Human transcription factor TFIIIC2 specifically interacts with a unique sequence in the Xenopus laevis 5S rRNA gene.

Transcription factor TFIIIC2 derived from human cells is required for tRNA-type gene transcription and binds with high affinity to the essential B-box promoter element of tRNA-type genes. Although 5S rRNA genes contain no homology with the tRNA-type gene B box, we show that TFIIIC2 is also required for Xenopus laevis 5S rRNA gene transcription. TFIIIC2 protected an approximately 30-base-pair (-10 to +18) region of a Xenopus 5S rRNA gene from DNase I digestion. This region, which spanned the transcription start site, included sequences that are highly conserved among eucaryotic 5S rRNA genes and have no homology with the B-box sequence of tRNA genes. Mutation of the TFIIIC2-binding site reduced transcription of the 5S rRNA gene by a factor of 10 in HeLa cell extracts. Methylation of C residues within the TFIIIC2-binding site interfered with binding of TFIIIC2. These results suggest a role of the TFIIIC2-binding sequence in 5S rRNA gene transcription. In addition, the 5S rRNA gene binding site and the tRNA-type gene B-box sequence did not compete with each other for binding to TFIIIC2 any better than did an unrelated DNA sequence, indicating that TFIIIC2 interacts with 5S rRNA genes and tRNA-type genes through separate DNA-binding domains or polypeptides.

Animals↗
PubMed1989

Fatty acid abnormalities in hemodialysis patients: effect of L-carnitine administration.

Serum fatty acid profiles were determined using gas chromatography/mass spectrometry in three groups of subjects. Compared to normal controls, eight patients on maintenance hemodialysis had significantly lower proportions of linoleic (28.15 +/- 1.3 vs. 26.5 +/- 1.4%, P less than 0.003), linolenic (0.49 +/- 0.2 vs. 0.21 +/- 0.2%, P less than 0.04) and arachidonic acids (8.42 +/- 0.9 vs. 6.0 +/- 1.5%, P less than 0.0001). Dialysis patients also had higher proportions of oleic (8.9 +/- 1.8 vs. 15.3 +/- 3.5%, P less than 0.0001) and n-9 eicosatrienoic acids (0.26 +/- 0.2 vs. 0.54 +/- 0.3%, P less than 0.04). Ratios between oleic and arachidonic acid and between eicosatrienoic and arachidonic acids were also significantly higher in the dialysis group. In a separate group of either chronic dialysis patients, L-carnitine was given intravenously for a period of 6 to 18 months. In this group linoleic, linolenic and eicosatrienoic acids were not significantly different from normal controls. The ratio between eicosatrienoic and arachidonic acids was also similar to normal controls. However, arachidonic acid remained significantly lower and oleic acid significantly elevated in the carnitine group. The ratio of saturated to unsaturated fatty acid was higher in the untreated dialysis patients compared to L-carnitine treated patients (0.75 +/- 0.07 vs. 0.65 +/- 0.06, P less than 0.01). Significant abnormalities in distribution of fatty acids was seen in hemodialyzed patients. Thus preliminary data suggest significant depletion of essential fatty acids in dialysis patients which is partially corrected by treatment of patients with L-carnitine.

Adult↗
PubMed1988

Spatial dependence of stress distribution for rod-shaped bacteria.

The stress distribution in the cylindrical portion of the cell envelope of a rod-shaped bacterial cell was compared with that at its polar ends. Using a symmetry argument it is shown that the critical internal pressure for the initiation of yielding of the envelope material has a non-uniform distribution and is significantly higher for the polar regions.

Animals↗
PubMed1988

Binding of sulfobromophthalein to rat and human ligandins: characterization of a binding-site peptide.

Photoaffinity techniques were employed to affect the covalent binding of [35S]sulfobromophthalein to proteins of rat and human liver cytosol. In rat liver cytosol at low concentrations, sulfobromophthalein bound to the 22 kDa subunit of ligandin. In human liver cytosol, binding to a 23.5 kDa subunit was observed. At higher concentrations, sulfobromophthalein also bound to 12, 23.5, 37, and 42 kDa peptides. When the peptides resulting from CNBr cleavage of [35S]sulfobromophthalein-ligandin complex were resolved by high-performance liquid chromatography, radioactivity was associated with two peptides. The peptide containing 80% of the radioactivity was isolated and characterized. Its molecular weight is 3.4 kDa, it contains the single tryptophan residue of ligandin and has a glutamate (glutamine) as the N-terminal amino acid.

Amino Acids↗
PubMed1988

Mass spectrometry studies of a novel digoxin-like substance (DLIS-2) isolated from human plasma ultrafiltrate.

Two unique low molecular weight (531) compounds with both digoxin-like immunoreactivity and Na, K-ATPase inhibitory properties have been isolated from human plasma. One of these, digoxin-like substance 2, (DLIS-2), was studied by fast atom bombardment mass spectrometry and collisionally activated dissociation mass spectrometry/mass spectrometry. The fragment patterns were interpreted as being derived from a lysophosphatidyl serine containing a novel 19:4 fatty acid side chain. The molecular formula C25H42O9NP is consistent with these observations.

Blood Proteins↗
PubMed1988

Characterisation of blast cells during blastic phase of chronic myeloid leukaemia by immunophenotyping--experience in 60 patients.

The blast cell population of 60 patients with chronic myeloid leukaemia in blast crisis (CML-BC) were analyzed with a panel of monoclonal antibodies to determine the cell surface antigen phenotypes. In addition, cytochemical stains periodic acid Schiff (PAS), myeloperoxidase (MP), Sudan black B (SBB) and terminal deoxynucleotidyl transferase (TdT) were also utilized for subtyping. Nineteen cases (31.6%) expressed lymphoid phenotypes characteristic of common ALL cells and one case with extramedullary lymph node crisis expressed T-cell surface phenotypes. Thirty cases (50%) expressed solely myelomonocytic surface antigens with significant TdT activity in three. Cytochemical stains contributed to recognize only 57% of these myeloid blasts. Seven cases (11.7%) were with a mixture of heterogenous group of cells expressing phenotypic characteristics for various haemopoietic cells of different lineage--five of them from the cells of non-lymphoid series (myelomono-erythromegakaryocytic series) and the other two with cells from both lymphoid and myeloid series. Additionally, in two cases (3.3%), the precursor cells reacted only with the erythroid monoclonals. Finally, in one case, the blast cells remained unclassified due to nonreactivity with any of the monoclonals used but expressed significant TdT positivity. The response to uniform vincristine and prednisolone (V + P) therapy has shown that lymphoid blast crisis cases were highly responsive in contrast to the cases with non-lymphoid blast crisis (complete remission rate 86 vs 21.4%). The results confirm the evidence of multilineage blast crisis involving either single or mixed haemopoietic differentiation pathway and the utility of having phenotypic characterisation for designing protocols for chemotherapy in the CML patients at the time of blast crisis.

Adolescent↗
PubMed1988

A heat-sensitive inhibitor in poliovirus-infected cells which selectively blocks phosphorylation of the alpha subunit of eucaryotic initiation factor 2 by the double-stranded RNA-activated protein kinase.

We partially purified an inhibitor from poliovirus-infected HeLa cells which specifically blocked phosphorylation of the alpha subunit of eucaryotic initiation factor 2 by the double-stranded RNA-activated protein kinase. The inhibitory activity eluted from a sizing column with an approximate molecular weight of 80,000 to 100,000 and was sensitive to heat, suggesting a protein nature for the inhibitor. No specific virus-encoded protein purified with the inhibitor. The inhibition of phosphorylation of the alpha subunit of eucaryotic initiation factor 2 was not due to a protein phosphatase associated with the inhibitor. The inhibitor did not seem to prevent phosphorylation of the double-stranded RNA-activated protein kinase but inhibited the phosphorylation of the alpha subunit of eucaryotic initiation factor 2 by the activated kinase. Double-stranded RNA-induced inhibition of in vitro protein synthesis in reticulocyte lysates could be prevented by the addition of the partially purified inhibitor during preincubation of lysate with double-stranded RNA.

Autoradiography↗
PubMed1988

An RNA polymerase II transcription factor inactivated in poliovirus-infected cells copurifies with transcription factor TFIID.

Inhibition of host cell RNA polymerase II-mediated transcription by poliovirus infection was studied in vitro. Whole-cell extracts prepared from poliovirus-infected HeLa cells at 3 h postinfection were shown to be deficient in a factor required for specific transcription from the adenovirus major late promoter. Three lines of evidence suggest that transcription factor TFIID is deficient in poliovirus-infected cells. First, the activity required to specifically restore transcription in poliovirus-infected cell extracts was shown to copurify with TFIID through three chromatographic steps. Second, transcription reactions reconstituted with phosphocellulose-derived chromatographic fractions revealed a fourfold decrease in the specific activity of the TFIID-containing fraction prepared from poliovirus-infected cells compared with that of the same fraction prepared from mock-infected cells. Finally, TFIID and the activity required to specifically restore transcription in virus-infected cell extracts were shown to have the same kinetics of heat inactivation. Together, these results suggest that inactivation of TFIID is an early event in the inhibition of host cell RNA polymerase II transcription by poliovirus.

Cell Transformation, Viral↗
PubMed1987

Two novel endogenous digoxin-like immunoreactive substances isolated from human plasma ultrafiltrate.

Two digoxin-like immunoreactive substances (DLIS 1 and DLIS-2) were isolated from human plasma by ultrafiltration, alkaline solvent extraction, and HPLC. Both inhibit canine Na, K-ATPase and cross-react with anti-digoxin antibodies. Their UV spectra are similar, with absorption peaks at 190 and 220 nm. FAB mass spectral analysis of both compounds yield a single dominant (M+H)+ component at m/z 532 otherwise, the fragmentation patterns are slightly different. The spectral data are consistent with the postulate that these compounds are related. Their identity as the natriuretic hormone is being investigated.

Chromatography, High Pressure Liquid↗
PubMed1987

High performance liquid chromatography and fast atom bombardment mass spectrometry of unusual branched and unsaturated phospholipid molecular species.

The unusual symmetrical molecular species 1,2-di-3,7,11,15-tetramethylhexadecanoyl-sn-glycero-3-phosphoglyce rol, 1,2-di-5,8,11,14-docosatetraenoyl-sn-glycero-3-phosphocholine, 1,2-di-5,9,19-octacosatrienoyl-sn-glycero-3-phosphoethanolamine, and 1,2-di-5,9,23-triacontatrienoyl-sn-glycero-3-phosphoethanolamine were isolated from the marine sponges Axinella verrucosa, Higginsia tethyoides, Tethya aurantia and Aplysina fistularis by HPLC and studied by fast atom bombardment (FAB) mass spectrometry. In addition to molecular weights, branching and double bonds were located in the fatty acyl chains of the intact phospholipid molecules, using FAB either in a positive or negative mode. Some mass spectral results were obtained on enriched phospholipid fractions rather than pure molecular species using MS/MS.

Animals↗
PubMed1987

Primer-dependent synthesis of covalently linked dimeric RNA molecules by poliovirus replicase.

Poliovirus-specific RNA-dependent RNA polymerase (replicase, 3Dpol) was purified from HeLa cells infected with poliovirus. The purified enzyme preparation contained two proteins of apparent molecular weights 63,000 and 35,000. The 63,000-Mr polypeptide was virus-specific RNA-dependent RNA polymerase, and the 35,000-Mr polypeptide was of host origin. Both polypeptides copurified through five column chromatographic steps. The purified enzyme preparation catalyzed synthesis of covalently linked dimeric RNA products from a poliovirion RNA template. This reaction was absolutely dependent on added oligo(U) primer, and the dimeric product appeared to be made of both plus- and minus-strand RNA molecules. Experiments with 5' [32P]oligo(U) primer and all four unlabeled nucleotides suggest that the viral replicase elongates the primer, copying the poliovirion RNA template (plus strand), and the newly synthesized minus strand snaps back on itself to generate a template-primer structure which is elongated by the replicase to form covalently linked dimeric RNA molecules. Kinetic studies showed that a partially purified preparation of poliovirus replicase contains a nuclease which can cleave the covalently linked dimeric RNA molecules, generating template-length RNA products.

Electrophoresis, Agar Gel↗
PubMed1987

Activation of double-stranded RNA-activated protein kinase in HeLa cells after poliovirus infection does not result in increased phosphorylation of eucaryotic initiation factor-2.

Protein kinase activity in general is stimulated at least 5- to 10-fold in ribosomal salt wash preparations from poliovirus-infected HeLa cells compared with those from mock-infected cells. The stimulation of kinase activity is manifested by increased phosphorylation of ribosome-associated polypeptides having approximate molecular weights of 135,000, 120,000, 85,000, 68,000, 65,000, 40,000, 28,000, 25,000, and 21,000. The Mr 68,000 phosphoprotein is structurally identical to the interferon-induced, double-stranded RNA-activated protein kinase (P1) which phosphorylates the alpha subunit of eucaryotic initiation factor-2 (eIF-2). A similar protein of Mr 68,000 is more phosphorylated in poliovirus-infected cells than in mock-infected cells. Increased phosphorylation of P1 protein in poliovirus-infected cells, however, does not result in an increased phosphorylation of the alpha subunit of endogenous or exogenously added eIF-2, both in vitro and in vivo. These results suggest that a mechanism must exist in poliovirus-infected HeLa cells which prevents further phosphorylation of eIF-2 by the activated kinase.

Enzyme Activation↗
PubMed1987

Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: inactivation of specific transcription factors.

The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoter, however, was not altered by infection of cells with the virus. We conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.

Cell Line↗