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Biomedical subjects

F A Gonzalez

Publications and source records attributed to F A Gonzalez.

At least 19 recordsLinked to original sources
PubMed2003

Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents.

Upregulation and activation of phospholipases A2 (PLA2) and cyclooxygenases (COX) leading to prostaglandin E2(PGE2) production have been implicated in a number of neurodegenerative diseases. In this study, we investigated PGE2 production in primary rat astrocytes in response to agents that activate PLA2 including pro-inflammatory cytokines (IL-1beta, TNFalpha and IFNgamma), the P2 nucleotide receptor agonist ATP, and oxidants (H2O2 and menadione). Exposure of astrocytes to cytokines resulted in a time-dependent increase in PGE2 production that was marked by increased expression of secretory sPLA2 and COX-2, but not COX-1 and cytosolic cPLA2. Although astrocytes responded to ATP or phorbol ester (PMA) with increased cPLA2 phosphorylation and arachidonic acid release, ATP or PMA only caused a small increase in levels of PGE2. However, when astrocytes were first treated with cytokines, further exposure to ATP or PMA, but not H2O2 or menadione, markedly increased PGE2 production. These results suggest that ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines on PGE2 production and may contribute to chronic inflammation seen in Alzheimer's disease.

Adenosine Triphosphate↗
PubMed1998

Structural basis of agonist-induced desensitization and sequestration of the P2Y2 nucleotide receptor. Consequences of truncation of the C terminus.

Molecular determinants of P2Y2 receptor desensitization and sequestration have been investigated. Wild-type P2Y2 receptors and a series of five C-terminal truncation mutants of the receptor were epitope-tagged and stably expressed in 1321N1 cells. These constructs were used to assess the importance of the intracellular C terminus on 1) UTP-stimulated increases in intracellular calcium concentration, 2) homologous desensitization of the receptor, and 3) agonist-induced decreases in cell-surface density (receptor sequestration) of epitope-tagged receptors using fluorescence-activated cell sorting. The potency and efficacy of UTP were similar for the wild-type and all mutant P2Y2 receptors. Truncation of 18 or more amino acids from the C terminus increased by approximately 30-fold the concentration of UTP necessary to desensitize the receptor. Both the rate and magnitude of UTP-induced receptor sequestration were decreased with progressively larger truncations of the C terminus. Furthermore, the recovery from sequestration was slower for the most extensively truncated receptor. Complete desensitization was obtained with >50% of the original receptor complement remaining on the cell surface. Protein kinase C activation, which desensitizes the P2Y2 receptor, had no effect on sequestration, consistent with the ideas that desensitization and sequestration are discrete events and that agonist occupancy is required for receptor sequestration.

Amino Acid Sequence↗
PubMed1997

DNA binding-independent anti-proliferative action of benzazolo[3,2-alpha]quinolinium DNA intercalators.

The proposed mechanism of action of the antineoplastic drug 3-nitrobenzothiazolo[3,2-alpha]quinolinium chloride (NBQ-2) involves its interaction with DNA by intercalation and inhibition of topoisomerase II activity by arresting the enzyme in a covalent cleavage complex. In an attempt to identify some structural determinants for activity and develop a molecular structure/cytotoxicity correlation, four new structural analogs of the antitumor NBQ-2 were prepared and their cytotoxic activity and DNA binding properties were investigated. The cytotoxic activity was evaluated against six different human tumor cell lines: U937, K-562, HL-60, HT-29, HeLa, and A431. The results showed that these new drugs elicit pronounced cytotoxic effects against U937, K-562, HL-60 and A431 while HeLa and HT-29 were less sensitive to the new drugs. This apparent selectivity was different to that of m-AMSA, a drug currently used for cancer treatment. Since the interaction of NBQ-2 to DNA by intercalation has been proposed as the initial step leading to its antineoplastic activity, DNA binding and changes in DNA contour length induced by the new NBQ-2 structural analogs were also investigated using calf thymus and human DNA. The drug, 7-(1-propenyl)-3-nitrobenzimidazolo[3,2-alpha]quinolinium chloride (NBQ-59) was the most cytotoxic agent of the analog series (IC50 = 16 microM for HL-60 cells), however, it demonstrated the weakest binding to DNA (Kint = 0.9 x 10[5] M-1 for calf thymus DNA). NBQ-59 was also found to be a poor intercalator into the DNA double helix. Therefore, our results suggest that DNA binding is not the primary mechanism of drug action for this family of compounds. In addition structural determinants important for cytotoxicity of the benzazolo quinolinium chlorides were suggested by our results. In particular, the nitro group in the 3 position does not seem to be necessary for bioactivity, while substitutions in the benzazolo moiety have striking effects on the biological activity of the drugs.

Antineoplastic Agents↗
PubMed1994

Alpha-thalassemia-1 (--CAL mutation) in a Spanish family.

We have detected a second family (five members affected) with a large (32 kb) deletion involving the alpha 1, alpha 2, psi alpha 1, psi alpha 2, psi zeta 1, and zeta-globin genes. This mutation has been previously described in Calabria, Italy, in a child and his mother and has been named alpha-thalassemia--)AL.

Adult↗
PubMed1994

Genomic loci of human mitogen-activated protein kinases.

Mitogen-activated protein (MAP) kinases [also known as Erks] have been established to function as important mediators of signal transduction by growth factor receptors. Several components of the MAP kinase signal transduction pathway have been demonstrated to be oncogenically activated in malignant tumors. These include growth factor receptors, the GTP-binding protein Ras, and the protein kinase Raf. The genes that encode MAP kinases therefore represent potential targets of carcinogenic insults. Here, we report the genomic loci of three MAP kinase genes are widely distributed within the human genome: p41mapk (Erk2) at 22q11.2; p44mapk (Erk1) at 16p11.2; and p63mapk (Erk3-related) at 18q12-21.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1993

Serum-induced translocation of mitogen-activated protein kinase to the cell surface ruffling membrane and the nucleus.

The mitogen-activated protein (MAP) kinase signal transduction pathway represents an important mechanism by which growth factors regulate cell function. Targets of the MAP kinase pathway are located within several cellular compartments. Signal transduction therefore requires the localization of MAP kinase in each sub-cellular compartment that contains physiologically relevant substrates. Here, we show that serum treatment causes the translocation of two human MAP kinase isoforms, p40mapk and p41mapk, from the cytosol into the nucleus. In addition, we report that p41mapk (but not p40mapk) is localized at the cell surface ruffling membrane in serum-treated cells. To investigate whether the protein kinase activity of MAP kinase is required for serum-induced redistribution within the cell, we constructed mutated kinase-negative forms of p40mapk and p41mapk. The kinase-negative MAP kinases were not observed to localize to the cell surface ruffling membrane. In contrast, the kinase-negative MAP kinases were observed to be translocated to the nucleus. Intrinsic MAP kinase activity is therefore required only for localization at the cell surface and is not required for transport into the nucleus. Together, these data demonstrate that the pattern of serum-induced redistribution of p40mapk is different from p41mapk. Thus, in addition to common targets of signal transduction, it is possible that these MAP kinase isoforms may differentially regulate targets located in distinct sub-cellular compartments.

Amino Acid Sequence↗
PubMed1992

Signal transduction within the nucleus by mitogen-activated protein kinase.

The nucleus is an important target of signal transduction by growth factor receptors that stimulate mitogen-activated protein (MAP) kinases. We tested the hypothesis that MAP kinases have a signaling role within the nucleus by examining the effect of the expression of a human MAP kinase isoform (p41mapk) in tissue culture cells. The expressed p41mapk was found to be localized in both the cytoplasmic and nuclear compartments of the cells. Significantly, the expression of p41mapk caused an increase in the phosphorylation of a nuclear substrate: Ser62 of c-Myc. Phosphorylation at Ser62 stimulated the activity of the NH2-terminal transactivation domain of c-Myc. Thus, p41mapk causes the phosphorylation and regulation of a physiologically significant nuclear target of signal transduction. These data establish that at least one MAP kinase isoform has a nuclear role during signal transduction.

Amino Acid Sequence↗
PubMed1992

Heterogeneous expression of four MAP kinase isoforms in human tissues.

Mitogen-activated protein kinases (MAP kinases) are a group of closely related enzymes implicated in signal transduction pathways. We report the molecular cloning of four human proteins (p40mapk, p41mapk, p44mapk and p63mapk) with high homology to members of the MAP kinase family. Sequence analysis demonstrated that p44mapk and p63mapk were the products of distinct genes. However, the p40mapk and p41mapk were found to be related, and are likely to result from alternative processing of transcripts from a single gene. The heterogeneous expression of these human MAP kinase isoforms in different tissues may reflect the diversity of signal transduction pathways in differentiated cells.

Amino Acid Sequence↗
PubMed1991

Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases.

Two epidermal growth factor-stimulated protein kinases that correspond to ERK1 and ERK2 have been purified from human epidermoid carcinoma cells (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). A consensus primary sequence for substrates of ERK1 has been identified as -Pro-Leu-Ser/Thr-Pro- (Alvarez, E., Northwood, I. C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C., Curran, T., and Davis, R. J. (1991) J. Biol. Chem. 266, 15277-15285). However, the structural determinants for substrate recognition are not understood. We performed a systematic analysis of the effect of point mutations in the primary sequence of peptide substrates on the rate of phosphorylation by ERK1 and ERK2. The results of this investigation demonstrate that the substrate specificities of the ERK1 and ERK2 protein kinases are very similar. We propose that the primary sequence of substrates for ERK1 and ERK2 protein kinases can be generalized as -Pro-Xaan-Ser/Thr-Pro- (where Xaa is a neutral or basic amino acid and n = 1 or 2).

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of two growth factor-stimulated protein kinases that phosphorylate the epidermal growth factor receptor at threonine 669.

A growth factor-stimulated protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669 has been described (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Anion-exchange chromatography demonstrated that this protein kinase activity was accounted for by two enzymes. The first peak of activity eluted from the column corresponded to the microtubule-associated protein 2 (MAP2) kinase. However, the second peak of activity was found to be a distinct enzyme. We present here the purification of this enzyme from human tumor KB cells by sequential ion-exchange chromatography. The isolated protein kinase was identified as a 46-kDa protein by polyacrylamide gel electrophoresis and silver staining. Gel filtration chromatography demonstrated that the enzyme was functional in a monomeric state. A kinetic analysis of the purified enzyme was performed at 22 degrees C using a synthetic peptide substrate based on the primary sequence of the EGF receptor (KREL VEPLT669PSGEAPNQALLR). The Km(app) for ATP was 40 +/- 5 microM (mean +/- S.D., n = 3). GTP was not found to be a substrate for the purified enzyme. The Km(app) for the synthetic peptide substrate was 260 +/- 40 microM (mean +/- S.D., n = 3). The Vmax(app) for the isolated protein kinase was determined to be 400-900 nmol/mg/min. The purified enzyme was designated EGF receptor Thr669 (ERT) kinase. It is likely that the MAP2 and ERT kinases account for the phosphorylation of the EGF receptor at Thr669 observed in cultured cells. The marked stimulation of protein kinase activity caused by growth factors indicates that these enzymes may have an important function during signal transduction.

Amino Acid Sequence↗
PubMed1991

Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase.

A growth factor-stimulated (MAP2-related) protein kinase, ERT, that phosphorylates the epidermal growth factor receptor at Thr669 has been purified from KB human tumor cells by Northwood and co-workers (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). The ERT protein kinase has a restricted substrate specificity, and the structural determinants employed for substrate recognition by this enzyme have not been defined. As an approach toward understanding the specificity of substrate phosphorylation, we have used an in vitro assay to identify additional substrates for the ERT protein kinase. In this report we describe two novel substrates: (a) the human c-myc protein at Ser62 and (b) the rat c-jun protein at Ser246. Alignment of the primary sequences surrounding the phosphorylation sites located within the epidermal growth factor receptor (Thr669), Myc (Ser62), and Jun (Ser246) demonstrated a marked similarity. The observed consensus sequence was Pro-Leu-Ser/Thr-Pro. We propose that this sequence forms part of a substrate structure that is recognized by the ERT protein kinase.

Amino Acid Sequence↗
PubMed1991

Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells: I. Involvement of protein kinase C-dependent and -independent pathways.

We recently reported that extracellular ATP was mitogenic for Swiss 3T3, 3T6, and A431 cells (Huang et al.: Proc. Natl. Acad. Sci. USA, 86:7904-7908, 1989). Here we examined the possible involvement of activation of the protein kinase C (PKC) signal transduction pathway in the mechanism of action of extracellular ATP. A potent synergistic stimulation of DNA synthesis in quiescent cultures of 3T3 and 3T6 cells was observed when ATP was presented in combination with growth factors that activate PKC, such as bombesin, vasopressin, or tumor-promoting phorbol esters. This finding suggests that ATP and these mitogens do not act through a common mechanism. In contrast, ATP was unable to show synergism with phorbol esters in A431 cells. We discovered striking differences when we examined the kinetics of formation of diacylglycerol (DAG) stimulated by ATP among these cell lines. Thus, ATP stimulated a sustained biphasic increase of DAG in A431 cells, but only a rapid transient increase of DAG formation was observed in 3T3 and 3T6 cells. The breakdown of phosphatidylcholine was stimulated by ATP in A431 cells; however, a significantly reduced effect was displayed in 3T6 cells. Furthermore, we found that the diacylglycerol-kinase inhibitor, 1-monooleoylglycerol, greatly potentiated ATP-stimulated DNA synthesis in A431 cells. Finally, down-regulation of PKC by long-term exposure to phorbol dibutyrate (PDBu) prevented stimulation of DNA synthesis induced by bombesin, vasopressin, or phorbol esters in 3T3 or 3T6 cells, while it had no such effect on ATP-stimulated mitogenesis in the presence of insulin or epidermal growth factor. On the other hand, PDBu-mediated down-regulation of PKC partially inhibited [3H [thymidine incorporation stimulated by ATP in A431 cells. Taken together, we conclude that a protein kinase C-dependent pathway is partially involved in ATP-stimulated DNA synthesis in A431 cells, but a protein kinase C-independent pathway exists in 3T3 and 3T6 cells. Pertussis toxin (PTX) inhibited the sustained phase of DAG formation and the breakdown of phosphatidylcholine stimulated by ATP in A431 cells. This suggests involvement of a PTX-sensitive G protein.

Adenosine Triphosphate↗
PubMed1991

Extracellular ATP and some of its analogs induce transient rises in cytosolic free calcium in individual canine keratinocytes.

Changes in intracellular free calcium ([Ca++]i) play an important role in a variety of biochemical reactions that lead to cellular responses such as proliferation and differentiation. The response of [Ca++]i to extracellular nucleotides (ATP, UTP, ITP, and AMP-PNP) was determined in individual canine keratinocytes using the fluorescent probe fura-2 and digital video fluorescence imaging microscopy. In the presence of 1.8 mM extracellular Ca++, 100 and 500 microM ATP caused a rapid (less than 9 sec) three- to twelvefold rise in [Ca++]i above resting levels of 50-150 nM followed by occasional fluctuations. Small responses were elicited with doses as low as 0.1 microM ATP. The response of cells stimulated with 500 microM ATP in Ca(++)-free medium was characterized by 1.5 to 3 times rapid initial peak followed by a decrease of [Ca++]i below resting levels. Loss of response occurred in the majority of keratinocytes preincubated for 30 min in Ca(++)-free medium. UTP was as effective as ATP in stimulating rises in [Ca++]i in keratinocytes. Smaller elevations in [Ca++]i up to four- to fivefold resting levels were noted with 100 microM AMP-PNP or 500 microM ITP. Desensitization of cells was demonstrated when a second stimulation followed the primary ATP or UTP treatment. These results are suggestive of the presence of purinergic receptors in the cytoplasmic membrane of canine keratinocytes. Experiments using the calcium channel blocker lanthanum suggest that ATP-induced initial rises and sustained levels of [Ca++]i are dependent on the release of Ca++ from intracellular stores. These intracellular Ca++ stores appear to be rapidly depleted after removal of extracellular calcium ([Ca++]e), thereby abolishing ATP-induced [Ca++]i increases.

Adenosine Triphosphate↗