Search PubMed⌕ Search

Biomedical subjects

R Bravo

Publications and source records attributed to R Bravo.

At least 235 records · Page 13Linked to original sources
PubMed1988

Two mouse genes encoding potential transcription factors with identical DNA-binding domains are activated by growth factors in cultured cells.

We previously reported the identification of a mouse gene, Krox-20, encoding a protein with three "zinc fingers" (DNA-binding domains with coordinated zinc ions) whose expression is regulated during G0/G1 transition (cell-cycle reentry). We now have isolated cDNAs corresponding to a related gene, Krox-24. Krox-24 encodes a protein with zinc fingers nearly identical to those encoded by Krox-20 and similar to those of transcription factor Sp1. Similarity between Krox-20 and Krox-24 proteins also extends to several blocks of amino acid sequence located upstream of the finger region. Like Krox-20, Krox-24 is transiently activated in quiescent cells after treatment with fetal bovine serum or purified growth factors. The kinetics of activation are similar to those of the protooncogene c-fos. The induction does not require de novo protein synthesis, and cycloheximide treatment of the cells leads to superinduction due, at least in part, to mRNA stabilization. In the mouse, the two genes are expressed in a tissue-specific manner, with slightly different patterns. These properties suggest that Krox-20 and Krox-24 may encode transcription factors with identical DNA target sequences and that these factors may be involved in the modulation of cell proliferation and differentiation.

Amino Acid Sequence↗
PubMed1988

Changes in cyclin/proliferating cell nuclear antigen distribution during DNA repair synthesis.

UV irradiation of quiescent human fibroblasts immediately triggers the appearance of the nuclear protein cyclin/proliferating cell nuclear antigen (PCNA) as detected by indirect immunofluorescent staining after methanol fixation. This was found to be independent of new synthesis of cyclin/PCNA by two-dimensional gel analysis and cycloheximide treatment. The intensity of the immunofluorescent staining of cyclin/PCNA observed in UV-irradiated cells corresponded with the UV dose used and with the DNA repair synthesis detected by autoradiography. The nuclear staining remains as long as DNA repair activity is detected in the cells. By extracting the UV-irradiated quiescent cells with Triton X-100 and fixing with formaldehyde, it was possible to demonstrate by indirect immunofluorescence rapid changes in the cyclin/PCNA population after irradiation, a small proportion (5-10%) of which is tightly associated to the nucleus as determined by high salt extraction. By incubating at low temperature and depleting the ATP pools of the cells before UV irradiation, we have demonstrated that the changes in cyclin/PCNA distribution observed involve at least two different nuclear associations.

Aphidicolin↗
PubMed1988

Characterization of a mouse multigene family that encodes zinc finger structures.

The Drosophila segmentation gene Krüppel encodes multiple tandemly repeated units predicted to form DNA-binding zinc fingers. We have isolated 23 bacteriophages, containing nonoverlapping inserts from a mouse genomic DNA library, on the basis of cross-hybridization under nonstringent conditions to a probe corresponding to the Krüppel finger region. Nucleotide sequence analysis of six phage DNAs indicated that they all contained regions with similarity to Krüppel and potentially encoded zinc finger domains. Within these regions, the level of similarity to Krüppel was particularly high between successive fingers. Northern (RNA) blotting analysis suggested that the mouse sequences belonged to different genes, the expression of some of which was modulated during cell differentiation and development. Hybridization experiments suggested that the similarity between some of the genes extended outside of the finger regions. In conclusion, our data suggest that the mouse genome contains a large family of evolutionarily related genes encoding possible trans-acting factors. These genes are likely to play a regulatory role at the transcriptional level.

Amino Acid Sequence↗
PubMed1988

Complexity of the early genetic response to growth factors in mouse fibroblasts.

Genes whose expression is growth factor regulated are likely to be important components in the mechanisms controlling cell proliferation and differentiation. With the aim of identifying some of those genes, a lambda cDNA library was prepared with poly(A)+ RNA from quiescent NIH 3T3 cells stimulated with serum for 4 h in the presence of cycloheximide. Differential screening of approximately 200,000 recombinant phage plaques revealed 2,540 clones that cross hybridized preferentially with [32P]cDNA derived from RNA of stimulated cells rather than with cDNA derived from nonstimulated cells. Cross hybridization of these clones identified 82 independent sequences, including c-fos and c-myc. Seventy-one clones were further studied. Analysis of the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction vary dramatically between the different genes. Cycloheximide in all cases superinduced the mRNA levels by two mechanisms, inhibiting the shutoff of transcription and prolonging the half-lives of the mRNAs. Our results showed that induction of proliferation is accompanied by the onset of a complex genetic program.

Animals↗
PubMed1987

Involvement of common and cell type-specific pathways in c-fos gene control: stable induction of cAMP in macrophages.

The c-fos proto-oncogene is rapidly and transiently induced by PDGF in fibroblast and by CSF-1 in macrophages. In both cells, the breakdown of phospholipids with the ensuing activation of protein kinase C (PKC) and intracellular release of Ca2+ seems to play a role in the induction of c-fos. The transient induction of c-fos mRNA and protein by PDGF is both increased and prolonged by inhibitors of calmodulin, apparently by inhibiting the degradation of c-fos mRNA. While no response to cyclic nucleotides is observed in fibroblasts, cAMP is a strong inducer of c-fos in macrophages. In contrast to the transient induction by PKC/Ca2+, cAMP induces stable transcription of the c-fos gene for many hours, suggesting the existence of different mechanisms regulating c-fos transcription in the same cell.

Animals↗
PubMed1987

Phosphorylation changes associated with the early cell cycle in Xenopus eggs.

Enucleated and nondividing amphibian eggs undergo cyclic changes in cell morphology and in the level of maturation promoting factor (MPF) with a period similar to the early cleavage cycle. We show here that there is a corresponding phosphorylation and dephosphorylation of specific proteins associated with this fundamental cell cycle. M-phase is associated with a general increase in phosphatase activity and specific phosphorylation of a small set of M-phase proteins, reflected in an increased stochiometry of phosphate and increased turnover. At the end of metaphase and correlated with a drop in MPF the phosphoproteins are rapidly lost. By microinjecting M-phase phosphoproteins into arrested interphase and metaphase eggs we could show that the specific M-phase phosphorylation was not due to specificity in phosphatase action. The ability to segregate synthesis from phosphorylation demonstrates that regulation is not on the level of synthesis of the M-phase proteins. Taken together these data suggest that regulation of kinase activity in M-phase in the face of general rapid phosphate turnover in the egg plays an important role in the regulation of the fundamental mitotic cycle.

Adenosine Triphosphate↗
PubMed1987

Immediate induction of a 45 K secreted glycoprotein by serum and growth factors in quiescent mouse 3T3 cells. Two-dimensional gel analysis.

Stimulation of quiescent 3T3 cells by serum dramatically induces the synthesis of a group of secreted polypeptides with a molecular weight (MW) of 45 K (p45 A, B, C, D). The synthesis of these polypeptides increases 10-fold during the first 2 h. Cycloheximide superinduces the 45 K polypeptides and actinomycin D (actD) blocks completely their induction by serum. Peptide mapping analysis and pulse-chase experiments revealed that p45 A is a precursor of polypeptides p45 B, C, D. Tunicamycin treatment inhibits the synthesis of all four polypeptides but a new related protein appears, p-p45, the unglycosylated precursor. In the presence of tunicamycin, p-p45 is also found in the medium, demonstrating that glycosylation is not essential for the secretion. In vitro translation experiments show that the levels of p45 mRNA present in stimulated cells are severalfold higher than that of non-stimulated cells. Purified growth factors, such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) induce the synthesis of p45 in quiescent cells.

Animals↗
PubMed1987

Bombesin induces c-fos and c-myc expression in quiescent Swiss 3T3 cells. Comparative study with other mitogens.

We have studied the effect of the potent mitogen bombesin on the expression of c-fos and c-myc genes in quiescent mouse fibroblasts. We have demonstrated that bombesin rapidly induces a transient expression of c-fos mRNA followed by a more protracted elevation in c-myc mRNA levels. The intensity of the induction of expression of both proto-oncogenes depended on the dose of bombesin used. Prolonged treatment of the cells with TPA, which causes a selective decrease in protein kinase C activity, partially inhibited the induction of c-fos and c-myc gene expression by bombesin, similar to what has been observed with PDGF. However, a dramatic inhibition of the mitogenic response to bombesin--but not to PDGF--was found in TPA-treated cells. In contrast, TPA-treated cells showed an increased response to EGF with regard to proto-oncogene expression. The role of protein kinase C and Ca2+-dependent pathways in proto-oncogene induction by bombesin is discussed.

Animals↗
PubMed1987

Specific antibody against a protein (p27) present in nonestablished fibroblasts. A putative microfilament-associated protein.

A specific polyclonal antibody has been raised against a basic cytoplasmic protein (p27) which is induced by serum in growth-arrested NIH 3T3 cells but is constitutively expressed in nonestablished fibroblasts. Immunoblotting analysis and [35S]methionine labeling show that p27 is absent in tissues and established cell lines of different types. However, it is present in fibroblasts from human, rat, mouse, and chicken origin and is highly conserved as determined by two-dimensional gel electrophoresis. Double immunofluorescence shows that p27 colocalizes with actin filaments. These observations would suggest that p27 is an actin-associated protein expressed in nonestablished fibroblasts.

Aging↗
PubMed1987

Cloning and sequence of the human nuclear protein cyclin: homology with DNA-binding proteins.

A full-length cDNA clone for the human nuclear protein cyclin has been isolated by using polyclonal antibodies and sequenced. The sequence predicts a protein of 261 amino acids (Mr 29,261) with a high content of acidic (41, aspartic and glutamic acids) versus basic (24, lysine and arginine) amino acids. The identity of the cDNA clone was confirmed by in vitro hybrid-arrested translation of cyclin mRNA. Blot-hybridization analysis of mouse 3T3 and human MOLT-4 cell RNA revealed a mRNA species of approximately the same size as the cDNA insert. Expression of cyclin mRNA was undetectable or very low in quiescent cells, increasing after 8-10 hr of serum stimulation. Inhibition of DNA synthesis by hydroxyurea in serum-stimulated cells did not affect the increase in cyclin mRNA but inhibited 90% the expression of H3 mRNA. These results suggest that expression of cyclin and histone mRNAs are controlled by different mechanisms. A region of the cyclin sequence shows a significant homology with the putative DNA binding site of several proteins, specially with the transcriptional-regulator cAMP-binding protein of Escherichia coli, suggesting that cyclin could play a similar role in eukaryotic cells.

Amino Acid Sequence↗
PubMed1987

Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites.

Pulse-chase experiments have revealed that cyclin, the auxiliary protein of DNA polymerase-delta, is stable during the transition from growth to quiescence in 3T3 cells. Immunoblotting together with immunofluorescence analysis has shown that the amount of cyclin after 24 h of quiescence is 30-40% of that of growing cells and that it presents a nucleoplasmic staining. Immunofluorescence studies show the existence of two populations of cyclin during the S phase, one that is nucleoplasmic as in quiescent cells and is easily extracted by detergent, and another that is associated to specific nuclear structures. By using antibromodeoxyuridine immunofluorescence to detect the sites of DNA synthesis, it was shown that the staining patterns of the replicon clusters and their order of appearance throughout the S phase are identical to those observed for cyclin. Two-dimensional gel analysis of Triton-extracted cells show that 20-30% of cyclin remains associated with the replicon clusters. This population of cyclin could not be released from the nucleus using high-salt extractions. This demonstrates that cyclin is tightly associated to the sites of DNA replication and that it must have a fundamental role in DNA synthesis in eukaryotic cells.

Animals↗
PubMed1987

Different types of modification in c-fos and its associated protein p39: modulation of DNA binding by phosphorylation.

We have studied the biosynthesis and biochemical properties of the c-fos gene product and its associated protein (p39) in growth factor-stimulated fibroblasts. c-fos is a markedly acidic protein that is extensively post-translationally modified by phosphorylation and another type of modification not changing its relative molecular mass (Mr). More than 10 different forms of c-fos protein can be identified by two-dimensional gel analysis. In c-fos-transformed cells, however, most of the highly modified forms are missing. The affinity for DNA of less phosphorylated c-fos-protein complexes is higher than that of the highly modified ones. The transforming potential of c-fos protein and its affinity for DNA thus seems to be inversely correlated with the extent of its phosphorylation. In contrast to c-fos, p39 is a basic protein that is rendered even more basic by post-translational modification. Two other forms of p39 differing in specific domains of the protein (p41 and p43) were also found to be complexed with c-fos.

Animals↗
PubMed1987

Anchorage-independent growth of v-myc-transformed Balb/c 3T3 cells is promoted by platelet-derived growth factor or co-transformation by other oncogenes.

Murine fibroblasts transformed by the myc oncogene have a reduced growth factor dependence for both anchorage-dependent and anchorage-independent proliferation. Here we show that v-myc-transformed Balb/c 3T3 cells require, in addition to insulin, only platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) for anchorage-dependent growth in serum-free media. PDGF, however, cannot efficiently be substituted by EGF for anchorage-independent growth. The results suggest that constitutive v-myc expression reduces cellular growth factor requirements by non-autocrine mechanisms for proliferation in monolayer cultures. In contrast, v-myc-transformed cells require plasma components and growth factors of the 'competence' type for anchorage-independent growth. We also demonstrate that the requirement for PDGF by the myc-transformed cells can be abrogated by v-K-ras, v-src and v-fos but not the v-raf oncogene. The results demonstrate that oncogenes can cooperate in the expression of the transformed phenotype by also drastically reducing cellular growth factor requirements.

Animals↗
PubMed1986

Synthesis, processing, and secretion of rat immunoglobulin E made in Xenopus oocytes.

Rat immunoglobulin E (IgE) synthesized in Xenopus laevis oocytes, injected with rat plasmacytoma mRNA, was analysed by specific immunoprecipitation and SDS-polyacrylamide gel electrophoresis under reducing as well as non-reducing conditions. The results indicate that the oocytes will translate and correctly process the rat IgE heavy and light chains, resulting in secretion of a correctly assembled, normal immunoglobulin molecule. The normal, extensive glycosylation of the IgE heavy chain (e-chain) is faithfully carried out by the oocytes; therefore, this posttranslational modification is apparently of an unspecific nature, and does not depend upon a mechanism specific for plasma cells.

Animals↗
PubMed1986

Effect of pH on the induction of competence and progression to the S-phase in mouse fibroblasts.

The pH dependence of competence induction and progression to the S-phase in quiescent stimulated cells has been studied. The results show that: (i) induction of competence by fibroblast growth factor in these cells is relatively independent of the external pH between pH 5.6-7.6; (ii) progression of cells to the S-phase is highly sensitive to pH and shows a dramatic increase between pH 6.8-7.2. These observations suggest that the intracellular alkalinization triggered by growth factors is fundamental for progression but not for competence induction.

Animals↗
PubMed1986

Identification of proliferation-sensitive human proteins amongst components of the 40 S hnRNP particles. Identity of hnRNP core proteins in the HeLa protein catalogue.

The core proteins of HeLa 40 S hnRNP monoparticles have been identified in the HeLa protein catalogue. Human proteins previously identified as proliferation-sensitive [NEPHGE 21 and 17; Bravo, R. and Celis, J.E. (1982) Clin. Chem. 28, 766], as well as two proteins characterized in this study (NEPHGE 16 W and 16 W1), are shown to be components of these particles. These basic nuclear polypeptides correspond to core proteins A1, B1a, B2 and C4, respectively. The significance of these results in terms of composition and function of hnRNP particles is discussed.

Amino Acids↗
PubMed1986

Evidence that expression of c-fos protein in amnion cells is regulated by external signals.

While c-fos expression is normally very low in certain cell types but transiently inducible by growth factors (e.g. in fibroblasts), other cells (e.g. amnion cells) exhibit an apparently constitutive expression in vivo. Here we show that in primary amnion cells c-fos protein expression rapidly drops to undetectable levels following plating in normal growth medium. However, c-fos expression is inducible by, and maintained at high levels in the presence of, dialyzed placenta- or embryo-conditioned medium. These observations suggest that c-fos expression in primary amnion cells is regulated by placenta- and embryo-derived factor(s), providing further evidence for the hypothesis that transcription of the c-fos gene may generally be controlled by external signals. We also show that proliferation of primary amnion cells is not dependent on a high c-fos expression, suggesting that the function of c-fos is more likely to be associated with other cellular functions in the differentiated amnion cell.

Amnion↗