RESUMO
Neuroendocrine differentiation (NED) has been implicated in prostate cancer progression and hormone-therapy failure. Neuroendocrine cells are non-proliferating and escape apoptotic cell death, although their origin and the causes of their apoptotic resistance have as yet been poorly elucidated. This study demonstrates a new mechanism involved in controlling NED. We report that epidermal growth factor (5-50 ng/ml) promotes neuroendocrine-like differentiation of androgen-independent DU145 prostate cancer cells. This differentiation is associated with an increase in the expression of Neuron Specific Enolase (NSE) and a reduction in cell proliferation and is blocked by inhibiting tyrosine kinase activity with genistein and with compound 56 (C56). An increase in the cAMP level, using dibutryl cAMP (db-cAMP) (1 mM) and isobutylmethylxanthine (100 microM), does not promote NED by itself, but does increase the effect of EGF on NED. In addition, EGF-induced NED protects cells from apoptosis induced with thapsigargin (1 microM) by reducing the thapsigargin-induced cytosolic calcium overload. In order to describe how EGF-induced NED protects cells against thapigargin-induced calcium overload we investigated the spatiotemporal calcium signalling linked to apoptosis. By using thapsigargin in various conditions on DU145 cells and using micro-fluorimetric calcium measurements, we show that depletion of intracellular calcium store induces apoptosis and that the amplitude and duration of the capacitive calcium entry are two apoptosis-modulating parameters. We show that protection against thapsigargin-induced apoptosis conferred by NED is achieved by reducing the amount and the speed of calcium that can be released from calcium pools, as well as modulating the amplitude of the subsequent calcium entry.
Assuntos
Androgênios/metabolismo , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Neoplasias Hormônio-Dependentes/patologia , Sistemas Neurossecretores/efeitos dos fármacos , Neoplasias da Próstata/patologia , Antineoplásicos/farmacologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Humanos , Masculino , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias Hormônio-Dependentes/metabolismo , Fosfopiruvato Hidratase/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Tapsigargina/farmacologiaRESUMO
TRPM8 (melastatine-related transient receptor potential member 8), a member of the transient receptor potential (TRP) superfamily of cation channels, has been shown to be a calcium-channel protein. TRPM8 mRNA has also been shown to be overexpressed in prostate cancer and is considered to play an important role in prostate physiology. This study was designed to determine the androgen-regulation mechanisms for TRPM8 mRNA expression and to identify the phenotype of TRPM8-expressing cells in the human prostate. Our findings show that trpm8 gene expression requires a functional androgen receptor. Furthermore, this article argues strongly in favour of the fact that the trpm8 gene is a primary androgen-responsive gene. Single-cell reverse transcriptase PCR and immunohistochemical experiments also showed that the trpm8 gene was mainly expressed in the apical secretory epithelial cells of the human prostate and trpm8 down-regulation occurred during the loss of the apical differentiated phenotype of the primary cultured human prostate epithelial cells. The androgen-regulated trpm8 expression mechanisms are important in understanding the progression of prostate cancer to androgen-independence. These findings may contribute to design a strategy to predict prostate cancer status from the TRPM8 mRNA level. Furthermore, as the TRPM8 channel is localized in human prostate cells, it will be interesting to understand its physiological function in the normal prostate and its potential role in prostate cancer development.
Assuntos
Regulação Neoplásica da Expressão Gênica , Canais Iônicos/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Receptores Androgênicos/fisiologia , 5-alfa-Di-Hidroprogesterona/metabolismo , 5-alfa-Di-Hidroprogesterona/farmacologia , Androgênios/metabolismo , Células Epiteliais/química , Células Epiteliais/metabolismo , Humanos , Canais Iônicos/metabolismo , Masculino , Miócitos de Músculo Liso/química , Miócitos de Músculo Liso/metabolismo , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas/genética , Próstata/citologia , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores Androgênicos/genética , Elementos de Resposta , Canais de Cátion TRPM , Células Tumorais CultivadasRESUMO
In the present study, we have assessed whether a putative calcium channel α2δ2 auxiliary subunit (CACNA2D2 gene) could be involved in prostate cancer (PCA) progression. We therefore carried out experiments to determine whether this protein is expressed in PCA LNCaP cells and in PCA tissues, and whether its expression may be altered during cancer development. In addition, we evaluated the influence on cell proliferation of overexpressing or downregulating this subunit. In vitro experiments show that α2δ2 subunit overexpression is associated with increased cell proliferation, alterations of calcium homeostasis and the recruitment of a nuclear factor of activated T-cells pathway. Furthermore, we carried out in vivo experiments on immuno-deficient nude mice in order to evaluate the tumorigenic potency of the α2δ2 subunit. We show that α2δ2-overexpressing PCA LNCaP cells are more tumorigenic than control LNCaP cells when injected into nude mice. In addition, gabapentin, a ligand of α2δ2, reduces tumor development in LNCaP xenografts. Finally, we show that the action of α2δ2 on tumor development occurs not only through a stimulation of proliferation, but also through a stimulation of angiogenesis, via an increased secretion of vascular endothelial growth factor in cells overexpressing α2δ2.
Assuntos
Canais de Cálcio/fisiologia , Proliferação de Células , Transformação Celular Neoplásica , Neovascularização Patológica/etiologia , Neoplasias da Próstata/etiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Linhagem Celular Tumoral , Homeostase , Humanos , Masculino , Camundongos , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
This study investigates the calcium mechanisms involved in growth arrest induced by extracellular ATP in DU-145 androgen-independent human prostate cancer cells. Exposure of DU-145 cells to 100 microM ATP produced an increase in cytoplasmic calcium concentration ([Ca(2+)](i)), due to a mobilization of calcium from the endoplasmic reticulum stores and to subsequent capacitative calcium entry (CCE). We have shown that this [Ca(2+)](i) increase occurs after stimulation by ATP of the phospholipase C (PLC) pathway. For the first time, we have identified the inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms expressed in this cell line and have demonstrated a participation of protein kinase C in CCE. Using fluorescence imaging, we have shown that a long-term treatment with ATP leads to a decrease in the intraluminal endoplasmic reticulum calcium concentration as well as in the amount of releasable Ca(2+). Modulating extracellular free calcium concentrations indicated that variations in [Ca(2+)](i) did not affect the ATP-induced growth arrest of DU-145 cells. However, treating cells with 1 nM thapsigargin (TG) to deplete intracellular calcium pools prevented the growth arrest induced by ATP. Altogether, these results indicate that growth arrest induced in DU-145 cells by extracellular ATP is not correlated with an increase in [Ca(2+)](i) but rather with a decrease in intracellular calcium pool content.
Assuntos
Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Carcinoma/metabolismo , Neoplasias da Próstata/metabolismo , Trifosfato de Adenosina/farmacologia , Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carcinoma/fisiopatologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Masculino , Neoplasias da Próstata/fisiopatologia , Proteína Quinase C/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Tapsigargina/farmacologia , Fosfolipases Tipo C/efeitos dos fármacos , Fosfolipases Tipo C/metabolismoRESUMO
A series of compounds including 4-amino (1), 3-amino (2), 4-nitro (3), 2-methyl-3-amino (4), 2-methyl-3-nitro (5), 2-methyl-4-amino (6), 2-methyl-4-nitro (7), 2-methyl-5-amino (8), 2-methyl-5-nitro (9), 2-methyl-6-amino (10), 2-methyl-6-nitro (11), 2,6-dimethyl (12), 2-methyl-3-carboxy (13), 2-methoxycarbonyl (14), 2-methyl-4-methoxy (15), 2,4-dimethoxy (16), 2-chloro-4-amino (17), and 2-chloro-4-nitro (18) N-phenyl substituents of phthalimide were evaluated along with N-[3-methyl-(2-pyridinyl)]phthalimide (19), N-(3-amino-2-methylphenyl)succinimide (20), and phenytoin for anticonvulsant and neurotoxic properties. Initial screening in the intraperitoneal (ip) maximal electroshock-induced seizure (MES) test and the subcutaneous pentylenetetrazol-induced seizure (scPtz) test in mice led to the selection of 1, 2, 4, 10, 12, 17, and 19 for oral MES evaluation in rats. The resultant ED(50) values for 4, 10, 17, and phenytoin were 8.0, 28.3, 5.7 and 29.8 mg/kg, respectively. In the batrachotoxin affinity assay, IC(50) values for 17 and phenytoin were 0.15 and 0.93 microM, respectively, and in the recently validated magnesium deficiency-dependent audiogenic seizure test, ED(50) values of 5.2 and 23 mg/kg were obtained for 17 and phenytoin, respectively. Electrophysiology studies on compound 17 point out its ability to (i) potentiate GABA-evoked current responses with a failure to directly activate the GABAA receptor and (ii) to affect, at 100 microM excitatory non NMDA, but not NMDA, receptors with a 25% block of kainate-evoked response. Electrophysiology measurements on voltage-gated sodium channels in N1E-115 neuroblastoma cells confirm voltage-dependent block of these channels by compound 17. In view of its interaction with multiple ion channels, one would predict that compound 17 might be active in a wide range of seizure models.
Assuntos
Anticonvulsivantes/farmacologia , Ftalimidas/farmacologia , Administração Oral , Animais , Anticonvulsivantes/síntese química , Anticonvulsivantes/toxicidade , Batraquiotoxinas/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiologia , Convulsivantes , Eletrochoque , Agonistas de Aminoácidos Excitatórios/farmacologia , Técnicas In Vitro , Ativação do Canal Iônico , Ácido Caínico/farmacologia , Deficiência de Magnésio/complicações , Camundongos , N-Metilaspartato/farmacologia , Técnicas de Patch-Clamp , Pentilenotetrazol , Fenitoína/farmacologia , Ftalimidas/síntese química , Ftalimidas/toxicidade , Ratos , Convulsões/tratamento farmacológico , Convulsões/etiologia , Bloqueadores dos Canais de Sódio , Relação Estrutura-Atividade , Sinaptossomos/metabolismo , Células Tumorais Cultivadas , Ácido gama-Aminobutírico/farmacologiaRESUMO
Severe magnesium deficiency protects mice against infections by Plasmodium Spp. and Babesia hylomysci which invade mature erythrocytes. By contrast severe magnesium deficiency does not protect against parasite infections by P. berghei which invades reticulocytes, Toxoplasma gondii which invades macrophages, and Trypanosoma brucei which lives free in blood. The results indicate that the infectious response depends on the severity of magnesium deficiency and on the parasite species. The decrease in red blood cell magnesium and increased oxidant stress are possible explanations for the protective effect of magnesium deficiency.
Assuntos
Deficiência de Magnésio/parasitologia , Infecções por Protozoários/metabolismo , Animais , Babesia/crescimento & desenvolvimento , Babesia/ultraestrutura , Babesiose/metabolismo , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Imunidade Inata , Magnésio/metabolismo , Deficiência de Magnésio/sangue , Malária/metabolismo , Camundongos , Plasmodium/genética , Plasmodium/crescimento & desenvolvimento , Plasmodium/ultraestrutura , Infecções por Protozoários/parasitologia , Reticulócitos/parasitologia , Fatores de Tempo , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose Animal/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/ultraestrutura , Tripanossomíase/metabolismoRESUMO
Accumulating data point to K(+) channels as relevant players in controlling cell cycle progression and proliferation of human cancer cells, including prostate cancer (PCa) cells. However, the mechanism(s) by which K(+) channels control PCa cell proliferation remain illusive. In this study, using the techniques of molecular biology, biochemistry, electrophysiology and calcium imaging, we studied the expression and functionality of intermediate-conductance calcium-activated potassium channels (IK(Ca1)) in human PCa as well as their involvement in cell proliferation. We showed that IK(Ca1) mRNA and protein were preferentially expressed in human PCa tissues, and inhibition of the IK(Ca1) potassium channel suppressed PCa cell proliferation. The activation of IK(Ca1) hyperpolarizes membrane potential and, by promoting the driving force for calcium, induces calcium entry through TRPV6, a cation channel of the TRP (Transient Receptor Potential) family. Thus, the overexpression of the IK(Ca1) channel is likely to promote carcinogenesis in human prostate tissue.
Assuntos
Cálcio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/fisiologia , Neoplasias da Próstata/patologia , Benzimidazóis/farmacologia , Canais de Cálcio/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/análise , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27 , Fase G1 , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/análise , Peptídeos e Proteínas de Sinalização Intracelular/análise , Masculino , Potenciais da Membrana , Neoplasias da Próstata/metabolismo , RNA Mensageiro/análise , Proteínas S100/análise , Canais de Cátion TRPV/fisiologia , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Mice were fed diets containing 960 mg (control), 100 mg (moderately Mg deficient) and 30 mg (severely Mg deficient) of Mg/kg. After 20 days, mice were inoculated with Babesia hylomysci (from Dr. Wery, Anvers, Belgium). Significant increases in RBC Mg levels were observed following infection. All the control and moderately deficient mice died from infection, whereas the severely Mg-deficient diet protected mice against infection, as shown by a decrease in parasitaemia and mortality. The decrease in RBC Mg, modifications in membrane properties and increased oxidant stress are possible explanations for the protective effect of severe Mg deficiency. When mice were maintained for 2 months after inoculation on a severely Mg-deficient diet and were then switched to a control diet, all survived and had low parasitaemias. After 1 month, these mice were rechallenged with B. hylomysci and 89% survived.
Assuntos
Babesiose/imunologia , Deficiência de Magnésio/imunologia , Magnésio/administração & dosagem , Animais , Babesiose/sangue , Babesiose/complicações , Eritrócitos/química , Eritrócitos/parasitologia , Feminino , Magnésio/sangue , Deficiência de Magnésio/complicações , CamundongosRESUMO
The role of endogenous and internalized catalase in the protection of Plasmodium against oxidant stress was studied. Catalase activities were measured in isolated Plasmodium falciparum at different stages of intererythrocytic development. Activities measured at late schizont stages were compared to parasite markers (glutamate dehydrogenase, SOD) and to red blood cell markers (haemoglobin, Cu/Zn-SOD). The fate of the host cell catalase in the parasite digestive system was studied by immunoelectron microscopy using monoclonal antibodies. The internalized catalase appeared to be dissociated in the digestive system of the parasite and inactivated. To examine the protective role of the endogenous and internalized catalase in the parasite protection against oxidant stress, parasites were cultivated at two oxygen concentrations (5% and 20%) in inhibited catalase red blood cells. These experiments suggested that the catalases present both in red blood cell and parasite are not essential when parasites are cultivated under 5% oxygen, but are necessary to protect the parasite under 20% oxygen. Catalase may not be the main protective enzyme involved in the protection of P. falciparum in standard in vitro culture conditions, but may become critical under the higher oxygen tensions conditions encountered in vivo.
Assuntos
Catalase/metabolismo , Eritrócitos/enzimologia , Oxigênio/fisiologia , Plasmodium falciparum/enzimologia , Animais , Eritrócitos/parasitologia , Humanos , Imuno-HistoquímicaRESUMO
The authors describe a case of so-called "intra-atrial reentry," associated with sinus node dysfunction. The spontaneous initiation of the tachycardia was always preceded by a sinus pause, and external atrial pacing prevented the appearance of tachycardia. Thus an atrial pacemaker was implanted, and after several months, the atrial dysrhythmias disappeared completely, despite interruption of the antiarrhythmic drugs. When a pacemaker dysfunction occurred, the sinus node dysfunction and the tachycardia reappeared. This is analogous with the "incessant" tachycardias seen in the WPW syndrome, and must be differentiated from the usual forms of the sick sinus syndrome which require both a pacemaker and antiarrhythmic drugs.
Assuntos
Marca-Passo Artificial , Taquicardia/prevenção & controle , Adulto , Diagnóstico Diferencial , Eletrodos Implantados , Eletrofisiologia , Feminino , Átrios do Coração/fisiopatologia , Sistema de Condução Cardíaco/fisiologia , Humanos , Recidiva , Síndrome do Nó Sinusal/fisiopatologia , Nó Sinoatrial/fisiopatologia , Taquicardia/etiologiaRESUMO
MCF-7 cells express voltage-activated K+ channels. In the present study, we used the patch-clamp and RT-PCR techniques to investigate the involvement of these channels during the cell cycle progression. The outward rectifier current (IK) recorded during depolarization was almost completely suppressed by the classical K+ channel blocker tetraethylammonium (TEA) in MCF-7 cells. TEA also inhibited cell proliferation, as measured with 3H-thymidine incorporation. Moreover, profound changes were observed in both the resting membrane potential (RMP) and IK during the release from the G0/G1 phase of the cell cycle. MCF-7 cells arrested in G0/G1 were depolarized (-26.3 +/- 10 mV, n = 30) and IK-density was small (9.4 +/- 5.6 pA/pF, n = 60) compared to cells progressing in the G1 phase (RMP = -60 +/- 7.9 mV; n = 35 and IK-density = 30.2 +/- 8.5 pA/pF; n = 76). IK was highly sensitive to Mg2+, astemizole and TEA (10 mM). Extracellular perfusion of 5 mM Mg2+ dramatically slowed the activation and perfusion of 2 microM astemizole inhibited both IK (20 +/- 3%) and cell proliferation (23%). Moreover, the h-EAG mRNA expression was modulated during the cell cycle. Thus, these data suggested that h-EAG K+ channels play a role in controlling the proliferation and/or cell cycle.
Assuntos
Neoplasias da Mama/metabolismo , Canais de Potássio/metabolismo , Potássio/metabolismo , Astemizol/farmacologia , Transporte Biológico , Ciclo Celular/efeitos dos fármacos , Condutividade Elétrica , Canais de Potássio Éter-A-Go-Go , Feminino , Fase G1/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Humanos , Potenciais da Membrana , Bloqueadores dos Canais de Potássio/farmacologia , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Tetraetilamônio/farmacologia , Células Tumorais CultivadasRESUMO
Previous studies have shown that ferrochloroquine (FQ) exhibited an antimalarial activity against Plasmodium spp. The present work confirmed this activity, described the curative effect on P. vinckei and investigated the FQ toxicity in vitro and in vivo. The in vitro and in vivo growth inhibition of P. falciparum and P. berghei N, respectively, showed that FQ antimalarial activity was 1.5-10 times more potent than chloroquine. FQ completely inhibited the in vivo development of both chloroquine-susceptible and resistant P. vinckei strains and protected mice from lethal infection at a dose of 8.4 mg kg(-1) day(-1) given for 4 days subcutaneously or orally. This curative effect was 5-20 times more potent than chloroquine, according to the strains' resistance to chloroquine. At this curative dose, no clinical changes were observed in mice up to 14 days after the last administration. Nevertheless, the acute toxicity and lethality of ferrochloroquine seemed to be dependent on gastric surfeit. The FQ security index determined in vitro confirmed that it might be a promising compound.
Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Malária Falciparum/veterinária , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Doenças dos Roedores/prevenção & controle , Administração Oral , Animais , Células Cultivadas , Cloroquina/análogos & derivados , Resistência a Medicamentos , Feminino , Compostos Ferrosos , Injeções Subcutâneas , Linfoma , Malária Falciparum/prevenção & controle , Camundongos , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
Electrophysiological, immunocytochemical, and RT-PCR methods were used to identify a K(+) conductance not yet described in MCF-7 human breast cancer cells. A voltage-dependent and TEA-sensitive K(+) current was the most commonly observed in these cells. The noninactivating K(+) current (I(K)) was insensitive to iberiotoxin (100 nM) and charybdotoxin (100 nM) but reduced by alpha-dendrotoxin (alpha-DTX). Perfusion of alpha-DTX reduced a fraction of I(K) amplitude in a dose-dependent manner (IC(50) = 0.6 +/- 0.3 nM). This DTX sensitive I(K) exhibited a voltage threshold at -20 mV and was not inactivated. The time constant of activation was 5.3 +/- 2.2 ms measured at +60 mV. The averaged half-activation potential and slope factor values were 14 +/- 1.6 mV and 10 +/- 1.4, respectively. Immunocytochemical analysis demonstrated that plasma membrane was labeled by anti-Kv1.1 but not by anti-Kv1.2 nor anti-Kv1.3 antibodies. Furthermore, only Kv1.1 mRNA was detected in MCF-7 cells. Incubation in 1 and 10 nM alpha-DTX reduced cell proliferation by 20 and 30%, respectively. These data provide the first evidence of Kv1.1 K(+) channels expression in MCF-7 cells and indicate that these channels are implicated in cell proliferation.
Assuntos
Neoplasias da Mama/metabolismo , Divisão Celular , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Neoplasias da Mama/patologia , Venenos Elapídicos/farmacologia , Humanos , Imuno-Histoquímica , Canal de Potássio Kv1.1 , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
A calcium dependent-chloride current (I(ni)) was recorded in Xenopus oocytes injected with total RNA from chicory leaf tissues, following depolarization from -35 to +60 mV. However, the signal transduction mechanism mediating I(ni) is unknown. The development of this current was mimicked by intracellular injection of the second messenger InsP(3) in control (non-injected) oocytes. Moreover, InsP(3) injection after I(ni) rundown did not reinitiate the current. The same phenomenon was observed following a second injection into control oocytes. Measurement of InsP(3) production in injected oocytes showed a net increase in the InsP(3) level on depolarization. Moreover, extracellular application of caffeine (5 mM) significantly reduced the number of oocytes displaying I(ni). Also, extracellular application of U-73122, a potent PLC inhibitor, clearly reduced the occurrence of I(ni). These data provide the first evidence that the calcium homeostasis mechanism induced by heterologous expression of total RNA from chicory leaves involves the InsP(3) signaling pathway.
Assuntos
Cálcio/metabolismo , Cichorium intybus , Inositol 1,4,5-Trifosfato/fisiologia , Folhas de Planta/genética , RNA de Plantas/fisiologia , Sistemas do Segundo Mensageiro/fisiologia , Animais , Canais de Cloreto , Eletrofisiologia , Microinjeções , Oócitos , RNA de Plantas/biossíntese , RNA de Plantas/isolamento & purificação , Fosfolipases Tipo C/metabolismo , Xenopus laevisRESUMO
BACKGROUND: Human natural antibodies have been found that owe their candidacidal action to the mimicry of a yeast killer toxin produced by the yeast Pichia anomala (PaKT). Candidacidal human natural antibodies (KTAb) are elicited by and bind to a KT receptor (PaKTR) present on the cell surface of infectious PaKT-sensitive microorganisms. Because of the recognized susceptibility of Pneumocystis carinii organisms to PaKT upon the occurrence of specific PaKTR, we examined whether human natural KTAb could also bind to and inhibit P. carinii. MATERIALS AND METHODS: Immunoaffinity-purified KTAb from the vaginal fluid of patients affected by candidiasis were tested and compared with PaKT for their ability to inhibit rat-derived P. carinii attachment to epithelial lung cells as well as infectivity to nude rats. Immunofluorescence studies were also performed by biotinylated PaKT in competition with human KTAb to establish their specific binding to PaKTR on the surface of rat-derived and human P. carinii organisms. RESULTS: Human natural candidacidal KTAb exerted a strong, specific inhibitory activity against rat-derived P. carinii organisms that are susceptible to PaKT itself. The antimicrobial activity of human KTAb was abolished by adsorption with a specific PaKT-neutralizing mAb KT4. Immunofluorescence studies of competition with PaKT showed that human KTAb efficiently bind to the specific PaKTR on the surface of rat-derived and human P. carinii organisms. CONCLUSIONS: The results strongly suggest that human KTAb, elicited by a common transphyletic receptor of different pathogenic microorganisms during infection, may play a role in antibody-mediated cross-immunity and, if properly engineered, as functionally equivalent recombinant antibodies they could exert a therapeutic activity against pneumocystosis in vivo.