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S Gopalakrishnan

Publications and source records attributed to S Gopalakrishnan.

At least 37 records · Page 2Linked to original sources
PubMed1996

Induction of a complex between rasGAP and a novel 110 kD protein is required for immortalization of primary epithelial cells by the E1A 12S oncoprotein of adenovirus.

Although established cell lines can be transformed with oncogenic ras, primary epithelial cells cannot, but require the coexpression of an immortalizing oncogene, such as the E1A region of adenovirus. We have previously shown that immortalization of primary epithelial cells by E1A 12S requires the expression of five regions encoded by both the first and second exons of the gene. However, only three of these regions, located in the first exon, are required for cotransformation of primary cells with an activated ras oncogene. Thus, the expression of oncogenic ras is able to abrogate the need for the E1A function(s) encoded by the second exon that are required for immortalization. This suggested the possibility that the functions encoded by the second exon of E1A may involve or interact with the normal ras signal transduction pathway. The results described herein demonstrate that immortalization-competent 12S gene products induce the expression of a novel 110 kD protein, p110, that forms a stable complex with rasGAP. Failure to induce the p110-rasGAP complex results in the concomitant loss of ability of 12S to immortalize primary epithelial cells. The appearance of this complex parallels the expression of the 12S protein and is sensitive to the levels of E1A 12S. p110 induction is independent of the ability of 12S to activate the cell cycle and of the presence of adenovirus E1B and is not observed in the presence of the large T antigen of SV40. Thus, it is not a general response to proliferation or tumorigenic transformation, but rather seems to be specific to the immortalization function(s) of E1A 12S.

Adenovirus E1A Proteins↗
PubMed1996

Magnetohydrodynamic Aggregation of Cholesterol and Polystyrene Latex Suspensions

The aggregation state of flowing (0.49 m s-1 linear velocity) colloidal dispersions of polystyrene latex microspheres (certified particle diameter of 156 ± 6 nm; measured diameter, 156 ± 3 nm at pH 5.3 in 50 mM NaCl) in NaCl solution and cholesterol (measured diameter, 533 ± 9 nm at pH 5.3), stabilized in NaCl solution by sodium taurodeoxycholate, was studied using photon correlation spectroscopy. For cholesterol suspensions having electrolyte concentrations close to the critical coagulation concentration (50 mM NaCl), pronounced aggregation was observed after 15 to 30 min of recirculation in the presence of an orthogonally applied magnetic field (2.0, 1.0 and 0.15 T). In all experiments with cholesterol, aggregation was followed by a period of deaggregation, after which aggregation again occurred. Comparable effects were not observed when cholesterol suspensions were recirculated in the absence of the magnetic field or when the suspensions were exposed to an equivalent magnetic field in the absence of flow. For cholesterol suspensions, the increase in particle size was most pronounced at 0.15 and 1.0 T rather than at 2.0 T. Aggregation effects were also observed when suspensions of polystyrene latex in 200 mM NaCl were made to flow through a 1.0-T field. In both systems, the magnetic aggregation does not appear to involve direct interaction between the field and the solid phase, but is interpreted in terms of orthokinetic effects involving magnetohydrodynamic changes in the flow profile resulting from the presence of the transverse field.

Journal Article↗
PubMed1995

Chemical and chemotaxonomical studies on Dicranopteris species.

Clerodane glycosides and flavonoids in Dicranopteris pedata and three varieties of D. linearis were investigated. All the ferns contained a new glycoside, (6S,13S)-6-[6-O-acetyl-beta-D-glucopyranosyl-(1-->4)-alpha-L-rhamnopy - ranosyloxy]-13-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-fucopyra nosyloxy]- cleroda-3,14-diene, as a chemical marker of this group. Flavonoids were limited to flavonol 3-O-glycosides. The ferns and isolated flavonoids are as follows; D. pedata: afzelin, quercitrin. D. linearis var. brevis: afzelin, quercitrin. D. linearis var. tenuis: quercitrin, isoquercitrin. D. linearis var. sebastiana: astragarin, isoquercitrin, rutin, kaempferol 3-O-(4-O-p-coumaroyl-3-O-alpha-L-rhamnopyranosyl)-alpha-L-rhamn opy ranosyl- (1-->6)-beta-D-glucopyranoside.

Carbohydrate Sequence↗
PubMed1995

Modulation of E-cadherin localization in cells expressing wild-type E1A 12S or hypertransforming mutants.

The adenovirus E1A 12S gene can immortalize primary epithelial cells such that they retain expression of epithelial cell characteristics. E1A 12S can also cooperate with an activated ras oncogene to cause tumorigenic transformation of primary cells. Specific substitution and deletion mutants of E1A 12S cooperate more efficiently with ras to produce foci with a hypertransformed phenotype, wherein the cells are less differentiated and exhibit invasive properties. Loss of epithelial differentiation in carcinomas is often a consequence of reduced intercellular adhesion involving loss of a functional cell-cell adhesion molecule, E-cadherin. Therefore, expression of E-cadherin was analyzed in mock-infected primary epithelial cells, cells expressing E1A 12S protein, and in hypertransformed cells. Primary epithelial cells express E-cadherin and correctly localize it to the cell-cell junctions, as detected by immunofluorescence. The level of E-cadherin expression decreases with time in culture. Primary cells expressing the E1A 12S protein maintain the expression and correct localization of E-cadherin. This effect of E1A 12S on E-cadherin expression is not at the transcriptional level. Immortalized cells expressing the 12S protein and primary epithelial cells transformed with the E1A 12S gene and ras continue to express E-cadherin at the cell-cell junctions. In contrast to the 12S-immortalized and wild-type transformed cells, hypertransformed cells are defective for localization of E-cadherin and exhibit altered subcellular distribution of E-cadherin, as detected by immunoprecipitation with an anti-E-cadherin antibody. Furthermore, the aberrant localization exhibited by the hypertransformed cells can be overcome by superinfection of the hypertransformed cells with a virus expressing the E1A 12S cDNA or treatment with retinoic acid. Thus, the E1A 12S protein appears to act as a differentiation factor to maintain the differentiated characteristics of epithelial cells.

Adenovirus E1A Proteins↗
PubMed1994

Alpha-A crystallin: quantitation of C-terminal modification during lens aging.

Previous studies have demonstrated that the C-terminal region of alpha-A crystallin is susceptible to age-dependent, posttranslational modification. To quantitate the amount of modification, alpha-A crystallin was purified from total proteins of the aging bovine lens, then digested with lys-C endoproteinase. Reverse phase, high pressure liquid chromatography was used to resolve and quantitate the resulting peptides, to determine the amount of C-terminal peptide relative to peptides from other regions of the protein that have not been reported to undergo modification. The results indicate that relative to alpha-A crystallin from newborn lens, posttranslational modification has occurred in approximately 45-55% of the C-terminal region from mature lens. These results demonstrate extensive modification of the C-terminal region of alpha-A crystallin from the mature lens, indicating that during the aging process, posttranslational modifications in this region may make significant contributions to the aggregated state and/or molecular chaperone properties of the molecule.

Aging↗
PubMed1994

Preferential interaction of alpha crystallin with denatured forms of gamma crystallin.

PURPOSE: To characterize the possible interaction of alpha crystallin with partially denatured forms of gamma crystallin. METHODS: Gamma crystallin was denatured in the presence of guanidine hydrochloride, then dialyzed in the presence or absence of alpha crystallin. The high-molecular-weight complex formed in the presence of alpha was characterized by gel filtration chromatography, electron microscopy, and quantitative Western blot analysis. RESULTS: Relative to native alpha or reconstituted aggregates of purified alpha, the higher molecular weight complex possessed a greater mean diameter and contained increased amounts of gamma crystallin. CONCLUSIONS: Alpha crystallin preferentially interacts with partially denatured forms of a lens protein, consistent with its putative role as a functional molecular chaperone in the intact lens.

Animals↗
PubMed1993

Localization of the chaperone binding site.

The hypothesis derived from models of the multi-oligomeric chaperone complex suggests that partially denatured proteins bind in a central cavity in the aggregate. To test this hypothesis, the molecular chaperone, alpha crystallin, was bound to partially denatured forms of gamma crystallin, and the binding site was visualized by immunogold localization. In an alternative approach, gold particles were directly complexed with gamma crystallin, followed by binding to the alpha crystallin aggregate. In both cases, binding was localized to the central region of the aggregate, confirming for the first time that partially denatured proteins do indeed bind to a central region of the molecular chaperone aggregate.

Animals↗
PubMed1993

Association of actin with alpha crystallins.

The alpha crystallins are cytosolic proteins that co-localize and co-purify with actin-containing microfilaments. Affinity column chromatography employing both covalently-coupled actin or alpha crystallin was used to demonstrate specific and saturable binding of actin with alpha crystallin. This conclusion was confirmed by direct visualization of alpha aggregates bound to actin polymerized in vitro. The significance of this interaction in relation to the functional properties of these two polypeptides will be discussed.

Actin Cytoskeleton↗
PubMed1992

Solution structure of the covalent sterigmatocystin-DNA adduct.

Sterigmatocystin and aflatoxin are potent mutagens that contaminate foodstuffs stored under conditions that permit fungal growth. These food mycotoxins can be metabolically activated to their epoxides, which subsequently form covalent adducts with DNA and can eventually induce tumor development. We have generated the sterigmatocystin-d(A1-A2-T3-G4-C5-A6-T7-T8) covalent adduct (two sterigmatocystins per duplex) by reacting sterigmatocystin-1,2-epoxide with the self-complementary d(A-A-T-G-C-A-T-T) duplex and determined its solution structure by the combined application of two-dimensional NMR experiments and molecular dynamics calculations. The self-complementary duplex retains its 2-fold symmetry following covalent adduct formation of sterigmatocystin at the N7 position of G4 residues on each strand of the duplex. The H8 proton of [ST]G4 exchanges rapidly with water and resonates at 9.58 ppm due to the presence of the positive charge on the guanine ring following adduct formation. We have assigned the exchangeable and nonexchangeable proton resonances of sterigmatocystin and the duplex in the covalent adduct and identified the intermolecular proton-proton NOEs that define the orientation and mode of binding of the mutagen to duplex DNA. The analysis was aided by intermolecular NOEs between the sterigmatocystin protons with both the major groove and minor groove protons of the DNA. The molecular dynamics calculations were aided by 180 intramolecular nucleic acid constraints, 16 intramolecular sterigmatocystin constraints, and 56 intermolecular distance constraints between sterigmatocystin and the nucleic acid protons in the adduct. The sterigmatocystin chromophore intercalates between the [ST]G4.C5 and T3.A6 base pairs and stacks predominantly over the modified guanine ring in the adduct duplex. The overall conformation of the DNA remains right-handed on adduct formation with unwinding of the helix, as well as widening of the minor groove. Parallel NMR studies on the sterigmatocystin-d(A1-A2-A3-G4-C5-T6-T7-T8) covalent adduct (two sterigmatocystins per duplex) provide supportive evidence that the mutagen covalently adducts the N7 position of G4 and its chromophore intercalates to the 5' side of the guanine and stacks over it. The present NMR-molecular dynamics studies that define a detailed structure for the sterigmatocystin-DNA adduct support key structural conclusions proposed previously on the basis of a qualitative analysis of NMR parameters for the adduct formed by the related food mutagen aflatoxin B1 and DNA [Gopalakrishnan, S., Harris, T. M., & Stone, M. P. (1990) Biochemistry 29, 10438-10448].

Base Composition↗
PubMed1992

Binding of actin to lens alpha crystallins.

Actin has been coupled to a cyanogen bromide-activated Sepharose 4B column, then tested for binding to alpha, beta, and gamma crystallin preparations from the bovine lens. Alpha, but not beta or gamma, crystallins bound to the actin affinity column in a time dependent and saturable manner. Subfractionation of the alpha crystallin preparation into the alpha-A and alpha-B species, followed by incubation with the affinity column, demonstrated that both species bound approximately the same. Together, these studies demonstrate a specific and saturable binding of lens alpha-A and alpha-B with actin.

Actins↗
PubMed1992

An assay for intermolecular exchange of alpha crystallin.

An affinity column of alpha crystallin linked to cyanogen bromide-activated Sepharose was developed to study the exchange of alpha subunits. Alpha crystallin bound to the Sepharose-alpha complex was dissociated with 8 mol/l urea, followed by quantitation using high-performance reverse-phase liquid chromatography. The time course of binding at 37 degrees C showed a hyperbolic binding pattern reaching equilibrium between 6-18 hr. Under these conditions, binding of beta and gamma crystallins to the same matrix was less than 10% of the alpha values, as was binding of alpha to glycine-coupled Sepharose. This assay was used to demonstrate changes in the subunit exchange of alpha crystallins present in high molecular weight versus lower molecular weight aggregates of the human lens. These results show that this binding procedure was a specific reproducible assay that might be used to study intermolecular interactions of the alpha crystallins.

Animals↗
PubMed1991

Modulation of transformation of primary epithelial cells by the second exon of the Ad5 E1A12S gene.

Expression of the Ad5 E1A first exon is necessary and sufficient to cooperate with an activated RAS oncogene to transform primary epithelial cells. The second exon, although necessary for immortalization and induction of an epithelial cell growth factor, is not essential for co-transformation with T24 RAS. To determine whether the second exon has a role in the cooperation of E1A with an activated RAS gene, we have performed an extensive mutational analysis of this region. All of the deletion and point mutants that we have generated and analyzed retained the ability to enable the transformants to grow in serum-free media and in soft agar. A region in the C-terminus of the E1A polypeptide encoded by nucleotides 1437-1488 appears to modulate the level of transformation. Co-transfections of T24 RAS and E1A genes with mutations that bring about specific amino acid substitutions or deletions in the C-terminus result in enhanced transformation. There is an increase in the number of transformed foci and they appear earlier. A single amino acid change can bring about this phenotype, which is dominant over wild type. Thus, it seems that expression of the wild-type second exon retards or suppresses transformation. The hypertransforming phenotype does not correlate with any differences in the expression of the mutated E1A or the co-cotransfected RAS gene. The C-terminus encodes a nuclear localization signal for E1A, however the subcellular localization of the mutant polypeptides does not affect their co-transforming ability.

Adenoviridae↗
PubMed1990

Intercalation of aflatoxin B1 in two oligodeoxynucleotide adducts: comparative 1H NMR analysis of d(ATCAFBGAT).d(ATCGAT) and d(ATAFBGCAT)2.

8,9-Dihydro-8-(N7-guanyl-[d(ATCGAT)])-9-hydroxyaflatoxin B1.d(ATCGAT) and 8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1.8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1 were prepared by direct addition of afltoxin B1 8,9-epoxide to d(ATCGAT)2 and d(ATGCAT)2, respectively. In contrast to reaction of aflatoxin B1 8,9-epoxide with d(ATCGAT)2 which exhibits a limiting stoichiometry of 1:1 aflatoxin B1:d(ATCGAT)2 [Gopalakrishnan, S., Stone, M. P., & Harris, T. M. (1989) J. Am. Chem. Soc. 111, 7232-7239], reaction of aflatoxin B1 8,9-epoxide with d(ATGCAT)2 exhibits a limiting stoichiometry of 2:1 aflatoxin B1:d(ATGCAT)2. 1H NOE experiments, nonselective 1H T1 relaxation measurements, and 1H chemical shift perturbations demonstrate that in both modified oligodeoxynucleotides the aflatoxin moiety is intercalated above the 5'-face of the modified guanine. The oligodeoxynucleotides remain right-handed, and perturbation of the B-DNA structure is localized adjacent to the adducted guanine. Aflatoxin-oligodeoxynucleotide 1H NOEs are observed between aflatoxin and the 5'-neighbor base pair and include both the major groove and the minor groove. The aflatoxin methoxy and cyclopentenone ring protons face into the minor groove; the furofuran ring protons face into the major groove. No NOE is observed between the imino proton of the modified base pair and the imino proton of the 5'-neighbor base pair; sequential NOEs between nucleotide base and deoxyribose protons are interrupted in both oligodeoxynucleotide strands on the 5'-side of the modified guanine. The protons at C8 and C9 of the aflatoxin terminal furan ring exhibit slower spin-lattice relaxation as compared to other oligodeoxynucleotide protons, which supports the conclusion that they face into the major groove. Increased shielding is observed for aflatoxin protons; chemical shift perturbations of the oligodeoxynucleotide protons are confined to the immediate vicinity of the adducted base pair. The imidazole proton of the modified guanine exchanges with water and is observed at 9.75 ppm. The difference in reaction stoichiometry is consistent with an intercalated transition-state complex between aflatoxin B1 8,9-epoxide and B-DNA. Insertion of aflatoxin B1-8,9 epoxide above the 5'-face of guanine in d(ATCGAT)2 would prevent the binding of a second molecule of aflatoxin B1 8,9-epoxide. In contrast, two intercalation sites would be available with d(ATGCAT)2.(ABSTRACT TRUNCATED AT 400 WORDS)

Aflatoxin B1↗
PubMed1989

Role of protein X in the function of the mammalian pyruvate dehydrogenase complex.

Two lipoyl-bearing subunits--the dihydrolipoyl transacetylase and protein X--form the core of the mammalian pyruvate dehydrogenase complex. Selective removal of the lipoyl domain of protein X results in loss in the activity of the complex with a relationship suggesting the involvement of the lipoyl domain of protein X in a key but not rate limiting step. The dihydrolipoyl dehydrogenase component markedly reduces both the cleavage of protein X and the loss in activity. Using a microplate binding assay, we demonstrate that the lipoyl domain of protein X and the transacetylase component contribute to the binding of the dihydrolipoyl dehydrogenase component. These roles of protein X in the catalytic function and organization of the complex require new reactions and afford an explanation for the unusual stoichiometry of dihydrolipoyl dehydrogenase dimers in the complex.

Animals↗
PubMed1989

Subunit associations in the mammalian pyruvate dehydrogenase complex. Structure and role of protein X and the pyruvate dehydrogenase component binding domain of the dihydrolipoyl transacetylase component.

We have further distinguished the structures and roles of the two lipoyl-bearing components of the pyruvate dehydrogenase complex, the dihydrolipoyl transacetylase (E2) component and the component designated as protein X. The amino acid sequences of the NH2-terminal regions of the lipoyl-bearing domain of the E2 component and protein X are different but related. The dihydrolipoyl dehydrogenase (E3) component but not the pyruvate dehydrogenase (E1) component protected protein X against proteolytic degradation by trypsin and protease Arg C. Protein X-specific polyclonal antibodies inhibit reconstitution of the overall reaction catalyzed by the complex (E2-X subcomplex recombined with the E1 and E3 components). The rate of development of this inhibition was reduced by pretreatment of E2-X subcomplex with the E3 component. These data strongly suggest the E3 component associates with protein X. The E1 component (an alpha 2 beta 2 tetramer), but not the E3 component, reduced trypsin cleavage of E2 subunits at 4 degrees C and altered the patterns of cleavage at 22 degrees C. At 22 degrees C a large (Mr congruent to 49,000) outer domain (E2LB) of the E2 component was produced. E2LB had the same NH2-terminal amino acid sequence as the smaller (Mr congruent to 38,000) lipoyl-bearing domain (E2L). E2LB, in contrast to E2L, interacted with both the E1 component and the beta subunit of the E1 component. Thus the E1 component is bound through an E1-binding domain that is located in E2 subunits between the inner domain and the outer, lipoyl-bearing domain.

Acetyltransferases↗
PubMed1989

Carcinogen-nucleic acid interactions: equilibrium binding studies of aflatoxin B1 with the oligodeoxynucleotide d(ATGCAT)2 and with plasmid pBR322 support intercalative association with the B-DNA helix.

Equilibrium binding of aflatoxin B1 (AFB1) to the oligodeoxynucleotide d(ATGCAT)2 was examined by using 1H NMR. AFB1 binds to double-stranded d(ATGCAT)2 with an apparent binding constant of 3.7 x 10(3) M-1. The equilibrium is rapid on the NMR time scale; the observed 1H NMR spectrum represents the population-weighted average of the chemical shifts arising from the free and bound states of the oligodeoxynucleotide and the AFB1. The spectrum of d(ATGCAT)2 exhibits exchange broadening in the presence of AFB1, manifested as decreases in apparent T2 relaxation times for the d(ATGCAT)2 base protons. Upon binding to d(ATGCAT)2, the AFB1 signals are shifted upfield, indicative of increased shielding. The adenine H2 protons are also shifted upfield in the presence of the carcinogen. Small changes in chemical shift are observed for other d(ATGCAT)2 protons. A substantial decrease in the nonselective T1 relaxation time is observed for the adenine H2 protons in the presence of AFB1. Competition binding experiments in which the competing ligands actinomycin D, ethidium bromide, and spermidine were individually added to an AFB1-d(ATGCAT)2 equilibrium mixture showed that addition of 1 equiv of actinomycin D or 4 equiv of ethidium bromide was sufficient to displace bound AFB1 from d(ATGCAT)2. In contrast, the addition of spermidine did not result in the displacement of bound AFB1 molecules and may have slightly enhanced binding, presumably due to stabilization of the DNA duplex. 1H NOESY experiments confirmed that the overall conformation for the d(ATGCAT)2 duplex was right-handed both in the absence and in the presence of AFB1. Equilibrium binding of AFB1 to d(ATGCAT)2 is greatly diminished at higher temperatures at which the oligodeoxynucleotide is single-stranded.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxin B1↗
PubMed1989

Domain structures of the dihydrolipoyl transacetylase and the protein X components of mammalian pyruvate dehydrogenase complex. Selective cleavage by protease Arg C.

Treatment of the dihydrolipoyl transacetylase-protein X-kinase subcomplex (E2-X-KcKb) with protease Arg C selectively converted protein X into an inner domain fragment (Mr approximately equal to 35,000) and an outer (lipoyl-bearing) domain fragment (Mr approximately equal to 15,500). These fragments were larger and much smaller, respectively, than the inner domain and outer domain fragments derived from the E2 component, supporting the conclusion that protein X is distinct from the E2 component. Protease Arg C cleaved the Kb subunit more slowly than protein X. An increase in kinase activity correlated with this cleavage of the Kb subunits. An even slower cleavage of E2 subunits generated an inner domain fragment (Mr approximately equal to 31,500) and a lipoyl-bearing domain fragment (Mr approximately equal to 49,000) which had Mr values at least 3,000 and 10,000 larger, respectively, than the corresponding E2 fragments generated by trypsin treatment of the subcomplex. Following various extents of cleavage with protease Arg C or trypsin, residual oligomeric subcomplexes were isolated and characterized. We found that selective removal of the lipoyl-bearing domain of protein X did not alter lipoyl-mediated regulation of the kinase indicating that the lipoyl residues bound to E2 subunits are effective, that the inner domain of protein X remained associated with the inner domain of E2 subunits following the complete removal of the outer domains of both E2 and protein X, that, with only 10% of the E2 subunits intact, nearly half of the catalytic (Kc) subunits of the kinase were bound by the residual subcomplex, and that removal of the remaining outer domains from E2 subunits released the Kc subunits. Thus, protein X is unique among the subunits of the complex in binding tightly to the oligomeric inner domain of the transacetylase, and the outer domain of the transacetylase serves to bind to and facilitate the regulation of the catalytic subunit of the kinase.

Acyltransferases↗