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G Shaulsky

Publications and source records attributed to G Shaulsky.

32 records · Page 2Linked to original sources
PubMed1994

CRAC, a cytosolic protein containing a pleckstrin homology domain, is required for receptor and G protein-mediated activation of adenylyl cyclase in Dictyostelium.

Adenylyl cyclase in Dictyostelium, as in higher eukaryotes, is activated through G protein-coupled receptors. Insertional mutagenesis into a gene designated dagA resulted in cells that cannot activate adenylyl cyclase, but have otherwise normal responses to exogenous cAMP. Neither cAMP treatment of intact cells nor GTP gamma S treatment of lysates stimulates adenylyl cyclase activity in dagA mutants. A cytosolic protein that activates adenylyl cyclase, CRAC, has been previously identified. We trace the signaling defect in dagA- cells to the absence of CRAC, and we demonstrate that dagA is the structural gene for CRAC. The 3.2-kb dagA mRNA encodes a predicted 78.5-kD product containing a pleckstrin homology domain, in agreement with the postulated interaction of CRAC with activated G proteins. Although dagA expression is tightly developmentally regulated, the cDNA restores normal development when constitutively expressed in transformed mutant cells. In addition, the megabase region surrounding the dagA locus was mapped. We hypothesize that CRAC acts to connect free G protein beta gamma subunits to adenylyl cyclase activation. If so, it may be the first member of an important class of coupling proteins.

Adenylyl Cyclases↗
PubMed1993

Cell type regulation in response to expression of ricin A in Dictyostelium.

Expression of ricin A in either prespore or prestalk cells of Dictyostelium discoideum results in cell-autonomous lethality. Strains expressing the toxic gene under the control of a prestalk-specific regulatory region fail to culminate or form stalks, but form spores normally. Strains expressing ricin A under the control of a prespore-specific regulatory region form neither spores nor stalks. Regulation of the cell types results in conversion of prestalk cells to prespore cells when the prespore cells are poisoned. The newly converted cells then express ricin A and die. In contrast, we could not detect any significant conversion of prespore cells to prestalk cells when the prestalk cells are poisoned under our experimental conditions. This regulation of cell types suggests that the tendency of prestalk cells to regulate and become prespore cells is inhibited by the already established prespore cells. It appears that prespore cells control prestalk cell regulation by producing an inhibitor of prespore differentiation to which they themselves are insensitive.

Animals↗
PubMed1991

Involvement of wild-type p53 in pre-B-cell differentiation in vitro.

Wild-type p53 protein is a growth modulator whose inactivation has been found to be a key event in malignant transformation. Reconstitution of wild-type p53 in the p53-nonproducer, Abelson murine leukemia virus-transformed pre-B-cell line L12 gave rise to stably growing clones. Wild-type p53-producer derived cell lines exhibit an altered cell cycle, however. More cells with an extended G0/G1 phase were found than in the p53-nonproducer parental cell line. Furthermore, when injected into syngeneic mice, these cells induced a lower incidence of tumors and these tumors were less aggressive. Analysis of immunoglobulin expression revealed that wild-type p53 induced the expression of cytoplasmic immunoglobulin mu heavy chain. In addition, these derived cells lines exhibited increased levels of a B-cell-specific surface marker, B220. These results suggest that wild-type p53 may function as a cell differentiation factor that can induce development of pre-B cells into a more advanced stage in the pathway of B-cell maturation. In these pre-B cells, wild-type p53 may induce cell differentiation without terminal growth arrest of the cell population.

Abelson murine leukemia virus↗
PubMed1991

Alterations in tumor development in vivo mediated by expression of wild type or mutant p53 proteins.

To study the mechanism of p53 involvement in malignant transformation, we compared the tumor development patterns induced by a parental p53 nonproducer pre-B cell line with those by cell lines generated from this parental cell line following transfection of either wild type or mutant p53. It was found that whereas mutant p53 facilitated tumor development, expression of wild type p53 restrained tumor development. Cell lines expressing the wild type p53 induced the development of faster regressing tumors than the parental cell line. The parental p53 nonproducer and the wild type p53 producer regressor tumors underwent in vivo cell differentiation, manifested as IgG production. Mutant p53, producer cell lines, on the other hand, failed to show any immunoglobulin synthesis and gave rise to highly proliferative lethal tumors. Our results support the conclusion that these pre-B cells develop regressor tumors because they have undergone differentiation. Whereas the wild type p53 facilitates this differentiation, mutant p53 cells block it. We suggest that, in addition to inactivating the growth-suppressive activity of wild type p53, the expression of mutant p53 facilitates malignant transformation.

Animals↗
PubMed1991

Nuclear localization is essential for the activity of p53 protein.

p53 appears to be a growth regulator, the perturbation of which induces changes in normal cell proliferation. Wild-type p53 protein is thought to function as a growth arrest gene, whereas mutant p53, which accumulates in transformed cells, has been shown to enhance malignant transformation. Both wild-type and mutant p53 migrate into the cell nucleus by means of identical nuclear localization signals (NLS) inherent in their primary sequences. Results presented here show that the suppressive activity of wild-type p53 measured as the reduction of transformation of primary rat fibroblasts induced by co-transfection with ras and either E1A or mutant p53, as well as the transformation enhancement of mutant p53 estimated by cooperation with ras in transformation of primary rat fibroblasts, is dependent upon nuclear localization signals in p53 protein. While transfection of unmodified wild-type p53 significantly reduces the number of rat embryonic fibroblast-transformed foci induced by E1A and ras or mutant p53 and ras, the wild-type p53 protein without NLS has completely lost this suppressive activity. Partially defective NLS wild-type p53, with a reduced nuclear accumulation ability, still exhibits some suppressive activity. In addition, we found that plasmids coding for intact mutant p53 protein efficiently cooperate with the ras oncogene, whereas the corresponding plasmids without NLS are totally inert. On this basis we conclude that nuclear localization of both wild-type and mutant p53 is a fundamental feature for manifesting the activities of these proteins. Both the suppressor activity mediated by the wild-type p53 and enhancement of transformation mediated by the mutant p53 require nuclear localization of the proteins to function.

Amino Acid Sequence↗
PubMed1991

Involvement of wild-type p53 protein in the cell cycle requires nuclear localization.

Transfection of wild-type p53 into a pre-B, p53 nonproducer cell line yielded the generation of stable clones. Although constitutively expressing the growth-suppressor wild-type p53 protein, these cells proliferate continuously in vitro. However, expression of wild-type p53 in these cells altered their cell cycle pattern and reduced their growth in vivo. When the same parental cells were transfected with a plasmid coding for a wild-type p53 lacking nuclear localization signals, a wild-type cytoplasmic p53 protein was expressed. Expression of this cytoplasmic p53 product did not exert any changes in the growth of the parental cells, suggesting that wild-type p53 affects the cell cycle only when localized in the nuclear cell compartment.

Animals↗
PubMed1990

Properties of aflatoxin-DNA adducts formed by photoactivation and characterization of the major photoadduct as aflatoxin-N7-guanine.

Aflatoxin-DNA adducts were formed by microsomal and photoactivation, using nick-translated DNA labelled with 14C in each of the DNA bases [3H]AFB1 and [3H]AFB2. DNA adducts were analysed by HPLC of DNA hydrolysates, and were characterized as double labelled peaks with specific retention times. The only AF-DNA adducts which were detected in significant amounts were guanine adducts, irrespective of the type of aflatoxin used or the mode of its activation. No stable adduct with adenine, cytosine or thymine was detected. UV spectra, proton NMR spectroscopy and mass spectrometry are consistent with the notion that the major AFB1-DNA photoadduct is the N7-guanine adduct. This report provides direct evidence for the existence of aflatoxin photoadducts formed on DNA.

Aflatoxin B1↗
PubMed1990

Involvement of singlet oxygen in photoactivation of aflatoxins B1 and B2 to DNA-binding forms in vitro.

UVA-activated aflatoxin B1 (AFB1) and AFB2 bound covalently to DNA. The rate of binding increased in D2O versus H2O and decreased by diazabicyclo-octane, azide, aniline, 2-naphthylamine and by efficient stripping of oxygen. Similarly to DNA-binding, singlet oxygen (1O2) formation increased in D2O and was inhibited by singlet scavengers, as judged by the formation of malonaldehyde from linolenic acid. The above argue for the involvement of 1O2 in aflatoxin photoactivation to DNA-binding species. A model for the mechanism is proposed.

2-Naphthylamine↗
PubMed1990

Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis.

The basic carboxy terminus of p53 plays an important role in directing the protein into the nuclear compartment. The C terminus of the p53 molecule contains a cluster of several nuclear localization signals (NLSs) that mediate the migration of the protein into the cell nucleus. NLSI, the most active domain, is highly conserved in genetically diverged species and shares perfect homology with consensus NLS sequences found in other nuclear proteins. The other two NLSs, II and III, appear to be less effective and less conserved. Although nuclear localization is dictated primarily by the NLSs inherent in the primary amino acid sequence, the actual nuclear homing can be modified by interactions with other proteins expressed in the cell. Comparison between wild-type p53 and naturally occurring mutant p53 showed that both protein categories could migrate into the nucleus of rat primary embryonic fibroblasts by essentially similar mechanisms. Nuclear localization of both proteins was totally dependent on the existence of functional NLS domains. In COS cells, however, we found that NLS-deprived wild-type p53 molecules could migrate into the nucleus by complexing with another nuclear protein, simian virus 40 large-T antigen. Wild-type and mutant p53 proteins differentially complexed with viral or cellular proteins, which may significantly affect the ultimate compartmentalization of p53 in the cell; this finding suggests that the actual subcellular compartmentalization of proteins may differ in various cell type milieux and may largely be affected by the ability of these proteins to complex with other proteins expressed in the cell. Experiments designed to test the physiological significance of p53 subcellular localization indicated that nuclear localization of mutant p53 is essential for this protein to enhance the process of malignant transformation of partially transformed cells, suggesting that p53 functions within the cell nucleus.

Amino Acid Sequence↗
PubMed1990

Subcellular distribution of the p53 protein during the cell cycle of Balb/c 3T3 cells.

The expression of p53, a transformation associated protein, has been found to be regulated during the cell cycle. We show here that the subcellular localization of p53 varies throughout the cell cycle. In growth stimulated Balb/c 3T3 cells, p53 is produced at elevated levels and the newly synthesized protein accumulates in the cytoplasm during the G1 phase. Around the beginning of the S phase, p53 accumulates in the cell nucleus, where it stays for about 3 h. Following this initial step of DNA synthesis, p53 is no longer found in the nuclear compartment, but rather accumulates in the cytoplasm. This modulation in the subcellular localization of p53 suggests that the protein is spatially regulated during cell cycle.

Animals↗
PubMed1989

Specific L-arginine taste receptor sites in the catfish, Ictalurus punctatus: biochemical and neurophysiological characterization.

We report here the characterization of the arginine binding site(s) and corroborative neurophysiological studies. Binding of L-[3H]arginine to Fraction P2 from taste epithelium was measured by a modification of the method of Krueger and Cagan. Parameters for measuring maximal binding activity were established for both duration of incubation and pH of medium. At pH 7.8, the apparent single rate constant for association (kobs) at 4 degrees C was 4.72 x 10(+5).M-1.min-1. Dissociation was more complex, yielding two rate constants of 1.77.min-1 and 8.34 x 10(-3).min-1. These data suggest the presence of two affinity states for L-arginine. The KD values as calculated from the ratio k-1/k+1 were 1.3 x 10(-6) M and 1.8 x 10(-8) M. Homologous inhibition studies of L-arginine binding were not fit by a simple mass action relationship (Hill Coefficient 0.79), but were best fit by a two-site model with IC50 values of 1.6 x 10(-6) M for the high affinity state and 9 x 10(-4) M for the low affinity state. Multiunit neural recordings examined the stimulatory effectiveness of a number of guanidinium-containing compounds. Compared with L-arginine, only L-arginine methyl ester and L-alpha-amino-beta-guanidino propionic acid (L-AGPA) were effective stimuli. Cross-adaptation experiments demonstrated that at 10(-4) M L-arginine methyl ester, L-AGPA and, to a lesser extent, D-arginine were effective cross-adapting stimuli to 10(-6) M L-arginine. In competition binding studies L-arginine methyl ester, L-AGPA and D-arginine also inhibited binding of L-[3H]arginine (10(-6) M), but each recognized only one affinity state. Inhibition by the poorly cross-adapting stimuli L-glutamate, glycine and L-alanine occurred only above 10(-3) M, indicating that the binding sites for L-arginine are selective. These studies suggest that there are at least two affinity states of L-arginine binding, that the binding sites are specific, and that effective agonists of L-arginine receptors must contain a guanidinium group and an unblocked L-alpha-amino group.

Animals↗
PubMed1988

Meth A fibrosarcoma cells express two transforming mutant p53 species.

Expression plasmids directing the synthesis of various forms of the p53 cellular tumor antigen were compared with respect to their biological activities. All plasmids encoding wild type p53, derived from two different cDNA libraries, had absolutely no detectable activity when assayed for transformation of primary rat embryo fibroblasts in collaboration with Ha-ras. In contrast, p53 variants carrying point mutations in the protein coding region exhibited at least some transforming activity. Most notably, this was true for both types of mutant p53 cDNA clones isolated from Meth A cells. The data indicate that these cells, derived from a chemically-induced tumor, carry two independently mutated p53 alleles, each encoding a transformationally activated protein. This may imply that the mutations in the p53 gene played a role in the development of the Meth A tumor. Finally, cells overexpressing a transfected mutant p53 exhibit a physical complex between this exogenous p53 and its endogenous counterpart, possibly resulting in the stabilization of the latter.

Animals↗
PubMedDate not supplied

Toward the functional analysis of the Dictyostelium discoideum genome.

Dictyostelium discoideum is a useful model for molecular studies of cell biology and development. The 34-megabase Dictyostelium genome is currently being sequenced through the efforts of an international consortium. The genome is expected to encode 8-10,000 genes, including all those required for a free-living eukaryote capable of multicellular development. A complete description of the Dictyostelium genome will open the way toward the application of genome-based experimental approaches to studies of cell biology and development in this organism, and allow detailed physiological and evolutionary comparisons to other species.

Animals↗