Disparate actions of hydroxyurea in potentiation of purine and pyrimidine 2',3'-dideoxynucleoside activities against replication of human immunodeficiency virus.

We and other groups have recently reported the potentiation by ribonucleotide reductase inhibitors such as hydroxyurea of the anti-human immunodeficiency virus type 1 (HIV-1) activity of purine and pyrimidine 2',3'-dideoxynucleosides in both resting and phytohemagglutinin-stimulated peripheral blood mononuclear cells. Little agreement prevails, however, as to the mechanism of the synergistic effects described. We report here that in phytohemagglutinin-stimulated peripheral blood mononuclear cells, two mechanisms exist for the potentiation of the anti-HIV-1 activity by low-dose hydroxyurea of the purine-based dideoxynucleoside 2',3'-dideoxyinosine and the pyrimidine-based dideoxynucleosides 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine. For 2',3'-dideoxyinosine, the enhancement arises from a specific depletion of dATP by hydroxyurea, resulting in a favorable shift of the 2',3'-dideoxyadenosine 5'-triphosphate/dATP ratio. For the pyrimidine dideoxynucleosides 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine, the more modest anti-HIV enhancement results from hydroxyurea-induced increases of pyrimidine kinase activities in the salvage pathway and, hence, increased 5'-phosphorylation of these drugs, while depletion of the corresponding deoxynucleoside 5'-triphosphates (dTTP and dCTP) plays no significant role.

Close association of the alpha subunits of Gq and G11 G proteins with actin filaments in WRK1 cells: relation to G protein-mediated phospholipase C activation.

A selective polyclonal antibody directed toward the C-terminal decapeptide common to the alpha subunits of Gq and G11 G proteins (G alpha q/G alpha 11) was prepared and used to investigate the subcellular distribution fo these proteins in WRK1 cells, a rat mammary tumor cell line. In immunoblots, the antibody recognized purified G alpha q and G alpha 11 proteins and labeled only two bands corresponding to these alpha subunits. Functional studies indicated that this antibody inhibited vasopressin- and guanosine 5'-[alpha-thio]triphosphate-sensitive phospholipase C activities. Immunofluorescence experiments done with this antibody revealed a filamentous labeling corresponding to intracytoplasmic and perimembranous actin-like filament structures. Colocalization of G alpha q/G alpha 11 and F-actin filaments (F-actin) was demonstrated by double-labeling experiments with anti-G alpha q/G alpha 11 and anti-actin antibodies. Immunoblot analysis of membrane, cytoskeletal, and F-actin-rich fractions confirmed the close association of G alpha q/G alpha 11 with actin. Large amounts of G alpha q/G alpha 11 were recovered in the desmin- and tubulin-free F-actin-rich fraction obtained by a double depolymerization-repolymerization cycle. Disorganization of F-actin filaments with cytochalasin D preserved G alpha q/G alpha 11 and F-actin colocalization but partially inhibited vasopressin- and fluoroaluminate-sensitive phospholipase C activity, suggesting that actin-associated G alpha q/G alpha 11 proteins play a role in signal transduction.

Frequency encoding in excitable systems with applications to calcium oscillations.

A number of excitable cell types respond to a constant hormonal stimulus with a periodic oscillation in intracellular calcium. The frequency of oscillation is often proportional to the hormonal stimulus, and one says that the stimulus is frequency encoded. Here we develop a theory of frequency encoding in excitable systems and apply it to intracellular calcium oscillations that results from increases in the intracellular level of inositol 1,4,5-triphosphate.

Depletion-activated calcium current is inhibited by protein kinase in RBL-2H3 cells.

Whole-cell patch-clamp recordings and single-cell Ca2+ measurements were used to study the control of Ca2+ entry through the Ca2+ release-activated Ca2+ influx pathway (ICRAC) in rat basophilic leukemia cells. When intracellular inositol 1,4,5-trisphosphate (InsP3)-sensitive stores were depleted by dialyzing cells with high concentrations of InsP3, ICRAC inactivated only slightly in the absence of ATP. Inclusion of ATP accelerated inactivation 2-fold. The inactivation was increased further by the ATP analogue adenosine 5'-[gamma-thio]triphosphate, which is readily used by protein kinases, but not by 5'-adenylyl imidodiphosphate, another ATP analogue that is not used by kinases. Neither cyclic nucleotides nor inhibition of calmodulin or tyrosine kinase prevented the inactivation. Staurosporine and bisindolylmaleimide, protein kinase C inhibitors, reduced inactivation of ICRAC, whereas phorbol ester accelerated inactivation of the current. These results demonstrate that a protein kinase-mediated phosphorylation, probably through protein kinase C, inactivates ICRAC. Activation of the adenosine receptor (A3 type) in RBL cells did not evoke much Ca2+ influx or systematic activation of ICRAC. After protein kinase C was blocked, however, large ICRAC was observed in all cells and this was accompanied by large Ca2+ influx. The ability of a receptor to evoke Ca2+ entry is determined, at least in part, by protein kinase C. Antigen stimulation, which triggers secretion through a process that requires Ca2+ influx, activated ICRAC. The regulation of ICRAC by protein kinase will therefore have important consequences on cell functioning.

Cloning and expression of two brain-specific inwardly rectifying potassium channels.

We have cloned two inwardly rectifying K+ channels that occur selectively in neurons in the brain and are designated BIRK (brain inwardly rectifying K+) channels. BIRK1 mRNA is extremely abundant and is enriched in specific brainstem nuclei, BIRK1 displays a consensus phosphate-binding loop, and expression in Xenopus oocytes has shown that its conductance is inhibited by ATP and adenosine 5'-[gamma-thio]triphosphate. BIRK2 is far less abundant and is selectively localized in telencephalic neurons. BIRK2 has a consensus sequence for cAMP-dependent phosphorylation.

Evidence that the 50-kDa substrate of brefeldin A-dependent ADP-ribosylation binds GTP and is modulated by the G-protein beta gamma subunit complex.

Brefeldin A, a fungal metabolite that inhibits membrane transport, induces the mono(ADP-ribosyl)ation of two cytosolic proteins of 38 and 50 kDa as judged by SDS/PAGE. The 38-kDa substrate has been previously identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We report that the 50-kDa BFA-induced ADP-ribosylated substrate (BARS-50) has native forms of 170 and 130 kDa, as determined by gel filtration of rat brain cytosol, indicating that BARS-50 might exist as a multimeric complex. BARS-50 can bind GTP, as indicated by blot-overlay studies with [alpha-32P]GTP and by photoaffinity labeling with guanosine 5'-[gamma-32P] [beta,gamma-(4-azidoanilido)]triphosphate. Moreover, ADP-ribosylation of BARS-50 was completely inhibited by the beta gamma subunit complex of G proteins, while the ADP-ribosylation of GAPDH was unmodified, indicating that this effect was due to an interaction of the beta gamma complex with BARS-50, rather than with the ADP-ribosylating enzyme. Two-dimensional gel electrophoresis and immunoblot analysis shows that BARS-50 is a group of closely related proteins that appear to be different from all the known GTP-binding proteins.

Function of the p86 subunit of eukaryotic initiation factor (iso)4F as a microtubule-associated protein in plant cells.

The isozyme form of eukaryotic initiation factor 4F [eIF-(iso)4F] from wheat germ is composed of a p28 subunit that binds the 7-methylguanine cap of mRNA and a p86 subunit having unknown function. The p86 subunit was found to have limited sequence similarity to a kinesin-like protein encoded by the katA gene of Arabidopsis thaliana. Native wheat germ eIF-(iso)4F and bacterially expressed p86 subunit and p86-p28 complex bound to taxol-stabilized maize microtubules (MTs) in vitro. Binding saturation occurred at 1 mol of p86 per 5-6 mol of polymerized tubulin dimer, demonstrating a substoichiometric interaction of p86 with MTs. No evidence was found for a direct interaction of the p28 subunit with MTs. Unlike kinesin, cosedimentation of eIF-(iso)4F with MTs was neither reduced by MgATP nor enhanced by adenosine 5'-[gamma-imido]triphosphate. Both p86 subunit and p86-p28 complex induced the bundling of MTs in vitro. The p86 subunit was immunolocalized to the cytosol in root maize cells and existed in three forms: fine particles, coarse particles, and linear patches. Many coarse particles and linear patches were colocalized or closely associated with cortical MT bundles in interphase cells. The results indicate that the p86 subunit of eIF-(iso)4F is a MT-associated protein that may simultaneously link the translational machinery to the cytoskeleton and regulate MT disposition in plant cells.

Phospholipase D is present on Golgi-enriched membranes and its activation by ADP ribosylation factor is sensitive to brefeldin A.

ADP ribosylation factor (ARF) is a small guanosine triphosphate (GTP)-binding protein that regulates the binding of coat proteins to membranes and is required for several stages of vesicular transport. ARF also stimulates phospholipase D (PLD) activity, which can alter the lipid content of membranes by conversion of phospholipids into phosphatidic acid. Abundant PLD activity was found in Golgi-enriched membranes from several cell lines. Golgi PLD activity was greatly stimulated by ARF and GTP analogs and this stimulation could be inhibited by brefeldin A (BFA), a drug that blocks binding of ARF to Golgi membranes. Furthermore, in Golgi membranes from BFA-resistant PtK1 cells, basal PLD activity was high and not stimulated by exogenous ARF or GTP analogs. Thus, ARF activates PLD on the Golgi complex, suggesting a possible link between transport events and the underlying architecture of the lipid bilayer.

Evidence for a role of pituitary ATP receptors in the regulation of pituitary function.

Despite a rapidly increasing acceptance for a role of ATP as an extracellular mediator in several biological systems, the present report shows that ATP may mediate physiological responses in pituitary cells. We have now been able to demonstrate a specific action of ATP receptors to mediate the release of luteinizing hormone from gonadotropes and have coupled them with further studies that clearly show that ATP can be exocytotically released from cultured rat pituitary cells. Both ATP and UTP (100 microM) caused a > 14-fold increase in the rate of luteinizing hormone release from superfused cells. Adenosine 5'-[alpha, beta-methylene]triphosphate and 5'-[beta,gamma-methylene triphosphate were ineffective, and 2-methylthio-ATP had only a modest stimulatory effect. Homologous and heterologous desensitization occurred with UTP and ATP, and these did not have additive effects. Thus, nucleotides can be effective stimulators of luteinizing hormone release through a single class of ATP receptor (P2U subtype). The calcium ionophore A23187 provoked release of a substantial amount of ATP from pituitary cells in a concentration- and Ca(2+)-dependent manner, which was desensitized by pretreatment with A23187. This implies a possible paracrine and/or autocrine mechanism by which nucleotides may exert their effects on pituitary cells. In conclusion, we have provided strong evidence for a novel role of extracellular nucleotides as mediators in pituitary--in particular, in gonadotrope--function.

DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions.

DNA-strand exchange promoted by Escherichia coli RecA protein normally requires the presence of ATP and is accompanied by ATP hydrolysis, thereby implying a need for ATP hydrolysis. Previously, ATP hydrolysis was shown not to be required; here we demonstrate furthermore that a nucleoside triphosphate cofactor is not required for DNA-strand exchange. A gratuitous allosteric effector consisting of the noncovalent complex of ADP and aluminum fluoride, ADP.AIF4-, can both induce the high-affinity DNA-binding state of RecA protein and support the homologous pairing and exchange of up to 800-900 bp of DNA. These results demonstrate that induction of the functionally active, high-affinity DNA-binding state of RecA protein is needed for RecA protein-promoted DNA-strand exchange and that there is no requirement for a high-energy nucleotide cofactor for the exchange of DNA strands. Consequently, the free energy needed to activate the DNA substrates for DNA-strand exchange is not derived from ATP hydrolysis. Instead, the needed free energy is derived from ligand binding and is transduced to the DNA via the associated ligand-induced structural transitions of the RecA protein-DNA complex; ATP hydrolysis simply destroys the effector ligand. This concept has general applicability to the mechanism of energy transduction by proteins.

Efeito da estimulação purinérgica sobre a produção de melatonina em macrófagos da linhagem RAW 264.7

A melatonina é um hormônio produzido de forma rítmica e no período de escuro pela glândula pineal bem como de forma não rítmica por diversos tecidos e células imunocompetentes. É sintetizada pela acetilação e metilação da serotonina pela ação das enzimas arilalquilamina N-acetiltransferase (AA-NAT) e acetilserotonina -O-metiltransferase (ASMT) que levam à formação de N-acetilserotonina (NAS) e melatonina (MEL), respectivamente. Nos últimos anos temos demonstrado que síntese de melatonina pela pineal pode ser negativamente modulada por mediadores inflamatórios e pelo ATP que atua como co-transmissor juntamente com a noradrenalina liberada no terminal nervoso simpático que a inerva. Perifericamente, contudo, estes mediadores inflamatórios apresentam um efeito contrário induzindo a produção de melatonina em células imunocompetentes. Estas observações levaram à criação da hipótese de um eixo imune-pineal. Esse trabalho teve como objetivo verificar o efeito do ATP sobre produção de melatonina em macrófagos da linhagem RAW 264.7 Os dados desse trabalho mostram que o ATP é capaz de induzir de maneira dose dependente a produção de melatonina em macrófagos através da modulação das enzimas AA-NAT e ASMT. Foi demostrado também que esse efeito é mediado pelo receptor P2X7 e que a melatonina produzida age autocrina e paracrinamente aumentando a fagocitose de particulas de zimosan. Com isso, podemos concluir que o ATP é um ativador endógeno do eixo imune-pineal

Investigating the role of glycine in astrocyte signalling using calcium imaging of rat primary cultures of cortical astrocytes

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Control of neuronal signalling pathways by CYP46A1, an enzime involved in brain cholesterol homeostasis

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2016

(Table 1) Porosity, particle diameter, ATP content and carbon and nitrogen composition of ODP Site 164-994 sediments

Sediment samples were obtained for detailed Adenosine 5'-Triphosphate (ATP) analysis down to 57.8 m below the seafloor (mbsf). The samples were also analyzed for particle-size distribution, calcium carbonate (CaCO3), organic carbon, and total nitrogen. The concentrations of ATP ranged between 360 and 7050 pg/g (dry weight sediment), which agree well with a limited number of direct bacteria counts. Principal component analyses show that 63% of the total variance can be accounted for by the first two principal components. The concentration of ATP (bacterial numbers by inference) is virtually independent of the concentration of sedimentary organic carbon, but correlates with CaCO3 and coarse particles.