RESUMO
Caffeine increases the amplitude of the Cl- currents evoked by capacitative Ca2+ entry (CCE) on thapsigargin-treated Xenopus oocytes. The caffeine-induced potentiation of the CCE process appears to rest on two distinct and additive components. The first component involves the cAMP second messenger system since it can be mimicked by either IBMX perfusion or cAMP microinjection into the oocyte and inhibited by the PKA inhibitory peptide i-PKA. The second component, although activatory, is dynamically related to the caffeine-evoked inhibition of InsP3-mediated Ca+ release and may arise from an interaction between caffeine and the InsP3 receptor in the context of a conformational coupling between the InsP, receptor and the channels responsible for CCE.
Assuntos
Cafeína/farmacologia , Cálcio/metabolismo , Estimulantes do Sistema Nervoso Central/farmacologia , Oócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Canais de Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Transporte de Íons/efeitos dos fármacos , XenopusRESUMO
The effects of the carcinogen Cd(2+) on Xenopus oocyte were evaluated by Inositol (1,4,5)-trisphosphate (InsP(3)) assays and electrophysiological experiments. The stimulation of the Ca(2+)-dependent Cl(-) current by Cd(2+) is clearly linked to InsP(3) formation since the effects of the metal are antagonized by neomycin, heparin and caffeine. A similar inhibition of the Cd(2+) effects is observed when the oocytes are pretreated with thapsigargin. Moreover, the use of sulfhydryl groups reductors such as 2-mercaptoethanol or N-ethylmaleimide strongly suggests that the Cd(2+) response is mediated by an extracellular receptor. Finally, measurements of InsP(3) production demonstrate that Cd(2+) superfusion actually leads to a PIP(2) breakdown. We conclude that extracellular Cd(2+) evokes an increase in [Ca(2+)](i) by stimulating the emptying of the InsP(3)-sensitive Ca(2+) stores, and that it may do so by interacting with a specific cell-surface ion receptor. This putative ion receptor may be important in allowing oocytes to respond to heavy metals.
Assuntos
Cádmio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Transporte/efeitos dos fármacos , Inositol 1,4,5-Trifosfato/fisiologia , Proteínas de Membrana/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Animais , Antracenos/farmacologia , Cafeína/farmacologia , Quelantes/farmacologia , Ácido Egtázico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Feminino , Heparina/farmacologia , Transporte de Íons/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Mercaptoetanol/farmacologia , Microinjeções , Neomicina/farmacologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol Diacilglicerol-Liase , Tapsigargina/farmacologia , Fosfolipases Tipo C/metabolismo , Xenopus laevisRESUMO
Rat cerebellar RNA injected into Xenopus oocytes leads to the expression of putative P-type voltage-dependent Ca2+ channels (VDCCs). The monitoring of intracellular Ca2+ variations by recording the Ca2+ dependent chloride current in voltage clamped oocytes indicates that activation of these Ca2+ channels by depolarization gives rise to two distinct components of cytosolic Ca2+ elevation. If the early component (T1) can be directly attributed to the Ca2+ entry through VDCCs, the second delayed one (T2) is related to a Ca2+ release from InsP3 sensitive stores activated following Ca2+ entry. Modifications of cytosolic Ca2+ by direct injection of Ca2+ into oocytes or by increasing the Ca2+ influx through VDCCs suggest that the Ca2+ release from intracellular InsP3 sensitive stores can be modulated in a differential manner. Namely, discrete elevations of cytosolic Ca2+ switch on the Ca2+ release whereas higher Ca2+ concentrations dampen the release. These results suggest a functional coupling between P-type VDCCs and InsP3 receptors.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Fosfatos de Inositol/fisiologia , Animais , Canais de Cálcio/genética , Cerebelo/química , Expressão Gênica , Técnicas In Vitro , Transporte de Íons , Oócitos/química , RNA Mensageiro/genética , Ratos , Ratos Wistar , Xenopus laevisRESUMO
The effect of Ca2+ on inositol (1,4,5)-trisphosphate 3-kinase (3-kinase) activity was measured on Xenopus oocyte cytosolic extracts. The Ca2+-evoked elevation in 3-kinase activity appeared to be mediated by calmodulin (CaM) and the calmodulin-dependent protein kinase II (CaMKII). The results observed in vitro were totally retrieved in intact oocytes and tend to demonstrate the involvement of a CaMKII-mediated phosphorylation in the regulation of 3-kinase activity. Finally, electrophysiological recordings of InsP3-elicited chloride current transients in the presence of CaM/CaMKII inhibitors allowed to postulate an involvement of 3-kinase activity in the regulation of InsP3-mediated Ca2+ release.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Ativação Enzimática , Oócitos , Xenopus laevisRESUMO
Xenopus oocytes injected with rat cerebellar mRNA expressed functional voltage-dependent Ca channels detected as an inward Ba current (IBa). The pharmacological resistance to dihydropyridines and omega-conotoxin together with the blockade obtained with Agelenopsis aperta venom suggest that these channels could be somehow assimilated to P-type Ca channels. The precise nature of the transplanted Ca channels was assessed by hybrid-arrest experiments using a specific oligonucleotide antisense-derivated from the recently cloned alpha 1-subunit of P channels (BI-1 clone). In addition, we demonstrate that exogenous Ca channel activity was enhanced by two different PKC activators (a phorbol ester and a structural analog to diacylglycerol). The general electrophysiological and pharmacological properties of the stimulated Ca channels remain unchanged. This potentiation induced by PKC activators is antagonized by a PKC inhibitor (staurosporine) and by a monoclonal antibody directed against PKC. It is concluded that P-type Ca channels are potentially regulated by PKC phosphorylation and the functional relevance of this intracellular pathway is discussed.
Assuntos
Canais de Cálcio/metabolismo , Cerebelo/metabolismo , Proteína Quinase C/metabolismo , RNA Mensageiro/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Clonagem Molecular , Eletrofisiologia , Ativação Enzimática , Oócitos , Proteína Quinase C/antagonistas & inibidores , Ratos , XenopusRESUMO
The slow inward Na current observed during sustained depolarization of the Xenopus oocyte membrane is due to a complex mechanism described as the induction of the channels. The present work investigates the role of protein phosphorylation in Na channel function. Injection of alkaline phosphatase in the oocytes decreased inward current. Therefore, the possible involvement of protein kinase in Na channel induction was explored. Treatment of oocytes with two activators of protein kinase C (PKC) resulted in enhanced Na current amplitude, whereas the treatment of oocytes with two potent PKC inhibitors decreased the inward current. These results imply that PKC phosphorylation is a fundamental step of Na channel induction. The possibility that the depolarization of the oocyte membrane may be the factor involved in PKC activation is discussed.
Assuntos
Oócitos/fisiologia , Proteína Quinase C/metabolismo , Canais de Sódio/fisiologia , Alcaloides/farmacologia , Animais , Diglicerídeos/farmacologia , Ativação Enzimática , Feminino , Potenciais da Membrana/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Ésteres de Forbol/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/efeitos dos fármacos , Canais de Sódio/efeitos dos fármacos , Estaurosporina , Tamoxifeno/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Tetrodotoxina/farmacologia , Xenopus laevisRESUMO
We have conducted a phase II trial of cisplatinumdiamminodichloride (CPDD) which not only demonstrated its remarkable activity in embryonic carcinoma of the testes, but also in ovarian carcinoma, in melanoma, and in epidermoid carcinoma, especially of the head and of the uterus cervix. Its toxicity, manifested mainly in the digestive and renal tracts, confines its administration to hospitalized patients only. This compound is now indicated in combination therapy for the above-mentioned tumors.
Assuntos
Cisplatino/uso terapêutico , Neoplasias/tratamento farmacológico , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Cisplatino/efeitos adversos , Avaliação de Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
An improved procedure is described involving gel permeation and anion-exchange chromatography for the purification of four major hen egg white proteins. The procedure involves a first-step purification of ovomucin and lysozyme by gel permeation on a Superose 6 Prep Grade column. In the second step, anion-exchange chromatography on Q Sepharose Fast Flow led to the isolation of ovotransferrin and ovalbumin from a gel permeation chromatographic peak. The purities were estimated as ca. 80, 100, 80 and 100% for ovomucin, lysozyme, ovotransferrin and ovalbumin, respectively. The purification yield was over 60% for each protein. Further characterization of purified lysozyme revealed that it was fully active and homogeneous in relation to the electrospray ionization mass spectrum. The electrospray ionization mass spectrum showed different ovotransferrin species. The amino acid composition of purified ovomucin was compared to those published previously.
Assuntos
Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Conalbumina/isolamento & purificação , Muramidase/isolamento & purificação , Ovalbumina/isolamento & purificação , Ovomucina/isolamento & purificação , Aminoácidos/análise , Animais , Galinhas , Clara de Ovo/análise , Feminino , Espectrometria de MassasRESUMO
A simple rapid procedure for preparation of large quantities of highly purified homogeneous ovalbumin from egg white by using an anion exchanger is described. It is based on the principle of frontal chromatography. The volume of "mucin-free" egg white loaded onto the column was determined in order to exceed resin capacity. Thus, competition between proteins for resin sites was created. Owing to its high negative charge density, ovalbumin drives other egg white proteins from the column progressively. Two hundred and fifty milliliters of Q-Sepharose FF gel eluted isocratically with 0. 5 M NaCl extracted 9.55 g of ovalbumin with a purity rate of 83%. A 6.75 g amount of ovalbumin, with a purity rate of 94%, was recovered with an isocratic elution program using 0.14 M NaCl. Purified ovalbumins were compared by electrophoresis and analytical chromatography with other ovalbumin preparations.
Assuntos
Clara de Ovo/análise , Ovalbumina/isolamento & purificação , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Mucinas/químicaRESUMO
The aim of this study was to evaluate the consequences of the egg white-drying process on egg white ability to limit Salmonella Enteritidis growth in addition to the elucidation of the factors involved. We observed rapid growth of Salmonella Enteritidis inoculated in egg white reconstituted from industrial powder in comparison with that observed in liquid egg white collected in the laboratory: Salmonella cell counts rose from 10(3) to 10(8) cells/ml of egg white from powder during 24 h incubation at 30 degrees C. This rapid growth was observed in powder from all egg-breaking factories investigated, and it was comparable to that observed in optimum medium (tryptone soy broth). In view of the mechanism of egg white resistance and the major role played by iron availability and by ovotransferrin, we investigated several hypotheses to explain this rapid growth: iron provided during the drying process and/or denaturation of protein (especially ovotransferrin). The rapid growth observed in egg white reconstituted from powder was in relation to egg white protein denaturation and especially ovotransferrin denaturation during powder pasteurization that enhanced the availability of iron necessary for Salmonella growth. The major role played by ovotransferrin and iron deficiency on Salmonella growth in egg white was illustrated in this study.
Assuntos
Clara de Ovo/microbiologia , Salmonella enteritidis/crescimento & desenvolvimento , Animais , Galinhas , Contagem de Colônia Microbiana , Conalbumina/química , Meios de Cultura , Dessecação , Gema de Ovo/microbiologia , Conservação de Alimentos , Indústria de Processamento de Alimentos , Ferro/análise , Desnaturação ProteicaRESUMO
High-intensity electric fields have been successfully applied to the destruction of Salmonella Enteritidis in diaultrafiltered egg white. The effects of electric field strength (from 20 to 35 kV x cm(-1)), pulse frequency (from 100 to 900 Hz), pulse number (from 2 to 8), temperature (from 4 to 30 degrees C), pH (from 7 to 9), and inoculum size (from 10(3) to 10(7) CFU x ml(-1)) were tested through a multifactorial experimental design. Experimental results indicate that, for Salmonella inactivation, the electric field intensity is the dominant factor with a strongly positive effect, strengthened by its positive interaction with pulse number. Pulse number, temperature, and pH have also significant positive effects but to a lesser extent. In the most efficient conditions, the pulsed electric field (PEF) treatment is capable of 3.5 log10 reduction in viable salmonellae. Simultaneously, the measure of surface hydrophobicity does not indicate any increase after PEF treatment. These results suggest that no protein denaturation occurs, unlike what is observed after comparable heat treatment in terms of Salmonella inactivation (55 degrees C for 15 min).
Assuntos
Proteínas do Ovo/química , Clara de Ovo/microbiologia , Eletricidade , Microbiologia de Alimentos , Desnaturação Proteica , Salmonella enteritidis/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Conservação de Alimentos/métodos , Indústria de Processamento de Alimentos , Temperatura AltaRESUMO
Rates of calcium uptake by and calcium release from sarcoplasmic reticulum vesicles isolated from skeletal muscle of the crab seem to depend on membrane potential generated by potassium (K) and chloride (Cl) gradients. This does not appear to be due to an effect of the ions themselves since media of different ionic compositions leading to the same membrane potential, also lead to the same ATP hydrolysis and the same Ca uptake by SR vesicles. From a large positive intravesicular potential (conditions termed "normal" in this paper), membrane depolarization of passively Ca loaded vesicles, produced by changes in K and Cl concentrations in the media, resulted in: i) decrease in rate of calcium uptake; ii) decrease in calcium loading; iii) increase in rate of calcium release despite a decrease in the driving force for calcium ions. Moreover, the addition of caffeine (5 mmol/l) to the different polarization media resulted in a increase in calcium release.
Assuntos
Cálcio/metabolismo , Retículo Sarcoplasmático/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Braquiúros , Cafeína/farmacologia , ATPases Transportadoras de Cálcio/metabolismo , Cloretos/farmacologia , Cinética , Potenciais da Membrana/efeitos dos fármacos , Músculos/fisiologia , Potássio/farmacologia , Retículo Sarcoplasmático/efeitos dos fármacosRESUMO
Pain is a frequent and complex symptom in cancer patients. For successful treatment, it is necessary to recognize the exact origin. Pain is often the direct or indirect consequence of neoplastic spread to osseous or peripheral nervous tissues. But for one patient out of five, it is induced by previous treatments and rarely is unrelated to the cancer. Surgery, radiotherapy, hormonotherapy and chemotherapy often improve, temporarily at least, painful symptoms induced by tumor growth. For chronic persisting pain, the oncologist may use peripheral analgesics, anxiolytics, morphinics, local and neurosurgical treatment. Moreover the specific indications of each modality must be recognized and if necessary combined if the analgesic effect is inadequate or too brief.
Assuntos
Neoplasias/complicações , Dor/etiologia , HumanosRESUMO
A phase II trial was conducted in 57 patients with advanced metastatic breast cancer given 2-N-methyl 9-hydroxy-ellipticine (NMHE) as 100 mg/m2 weekly. Evaluation of response, after at least 4 injections, was possible in 46 patients. Two complete regressions (of 3 and 12 months) and 7 regressions of over 50 p. cent were observed, a total regression rate of 19 p. cent. Regression was mainly observed in cutaneous or subcutaneous metastases. No objective regression was noted for pulmonary or hepatic metastases. Bone metastases were not taken in account when assessing response to treatment. Absence of haematological changes must be emphasized. The most frequent side effects were anorexia, nausea +/- vomiting and dryness of mouth. Major toxicity was intravascular haemolysis, observed in 6 of 175 patients receiving NMHE in the Institut Gustave-Roussy, always controlled by symptomatic treatment. This product, of acceptable efficacy in breast cancer treatment, will probably occupy an original place in anti-cancer chemotherapy because of its lack of myelotoxicity.
Assuntos
Alcaloides/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias da Mama/secundário , Elipticinas/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Ensaios Clínicos como Assunto , Elipticinas/efeitos adversos , Feminino , Hemólise/efeitos dos fármacos , HumanosRESUMO
Duborimycin is a new antimitotic agent approaching danorubicin and adriamycin in activity which has been tried on 151 patients suffering from cancer of different types, is an advanced local/regional stage and/or metastatic disease. It was administered intravenously every fortnight in a mean unit dose of 400 mg, and the duration of the treatment ranged from 2 to 52 weeks. Objective improvement was registered in 56 patients of the 135 cases in whcih the results were assessed (around 41.4% of cases). In 4 cases the regression of tumour volume was greater than 50% (one of these cases was in melanoma, the other a sarcoma) and in 2 cases regression was complete (a squamous cell carcinoma and an embryonal testicular tumour). The subjective effects were appreciable in 53 of the 115 cases which could be studied (46%) and above all in the refractory pain of bony secondaries from breast cancer (a favourable response in 78% of cases). Manifestations of intolerance/toxicity were of a minor nature on the haematologic side, that cardiologic ones relatively frequent (18% of treated cases) and occasionally serious (2 cases of asystole). Great care is therefore necessary in supervision of the treatment. However, the first results obtained by this line of approach, notably in chemo-resistant forms of tumour such as melanoma and sarcomas, utilizing the very strict criteria in one analysis encourage further study of duborimycin in cases of this sort (preferably in association and in accordance with protocols of comparative trials) so that its place in cancer chemotherapy may be more precisely defined.
Assuntos
Daunorrubicina/análogos & derivados , Adenocarcinoma/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Carcinoma de Células Escamosas/tratamento farmacológico , Daunorrubicina/uso terapêutico , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Metástase Linfática , Masculino , Melanoma/tratamento farmacológico , Metástase Neoplásica , Neoplasias Cutâneas/tratamento farmacológicoRESUMO
The results of palliative chemotherapy applied to 123 patients with disseminated soft tissue sarcomas are reviewed with the recent literature data. With the exception of embryonal rhabdomyosarcomas of children, which are considered separately, the response of these tumors to chemotherapy is poor but not negligible. The association of several active drugs should permit substantial progress, especially in the initial stage of local treatment, when the number of disseminated cells is still reduced. A follow-up of controlled clinical studies must specify the modalities of multidisciplinary treatment in an effort to reduce regional recurrence or metastasis.
Assuntos
Antineoplásicos/uso terapêutico , Sarcoma/tratamento farmacológico , Neoplasias de Tecidos Moles/tratamento farmacológico , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Ciclofosfamida/efeitos adversos , Ciclofosfamida/uso terapêutico , Dactinomicina/efeitos adversos , Dactinomicina/uso terapêutico , Avaliação de Medicamentos , Feminino , Seguimentos , Humanos , Lactente , Masculino , Metotrexato/efeitos adversos , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Metástase Neoplásica , Recidiva Local de Neoplasia , Sarcoma/mortalidade , Neoplasias de Tecidos Moles/mortalidade , Vimblastina/efeitos adversos , Vimblastina/uso terapêutico , Vincristina/efeitos adversos , Vincristina/uso terapêuticoRESUMO
Two hundred and nine patients with breast cancer in an advanced stage were treated according to a chemotherapeutic regimen associating doxorubicin, vincristine and methotrexate, administered in courses of 5 days every three weeks. This analysis deals only with the short range results; they confirm those of a previous randomized study. A global objective response was obtained in 187 cases (89%) and a measurable regression of the lesions in 150 cases (71%); in these later cases 90 (43%) had a regression of more than 50 per cent. The most striking effects, often rapidly observed, involve sites which are not generally sensitive: liver (40%), pleura (24%) and bone (only 6%, but 8 times out of 10 a definite action on the pain syndrome). Side effects were, on the whole, acceptable (only one severe hematologic complication); however, the risk of myocardiac toxocity due to the accumulation of doxorubicin limits the utilization of this association. It thus needs to be relayed by other drug regimens which are included in a program of long term action, but has interesting characteristics as induction chemotherapy.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/efeitos adversos , Adulto , Idoso , Ensaios Clínicos como Assunto , Avaliação de Medicamentos , Quimioterapia Combinada , Feminino , Humanos , Metotrexato/efeitos adversos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Vincristina/efeitos adversosRESUMO
P-type calcium channels are expressed in Xenopus oocytes after injection of rat cerebellar mRNA. The FTX and omega-Aga-IVa toxins extracted from Agelenopsis aperta venom are known to inhibit the activity of this channel. The present results demonstrate that 8RN-DAGal is also a antagonist of P-type calcium channels. The inhibition of the current, obtained with Ba2+, as charge carrier, is voltage dependent.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Galactose/análogos & derivados , Oócitos/metabolismo , Animais , Canais de Cálcio/efeitos dos fármacos , Galactose/farmacologia , Oócitos/efeitos dos fármacos , RNA Mensageiro/biossíntese , Ratos , XenopusRESUMO
Endogenous calcium channels of Xenopus oocyte membrane do not fit with pharmacological classification of calcium channels. The present study demonstrates that the saccharidic derivate, OC8-MAGlu-MAGlu, has potent inhibitory effect on this channel activity.
Assuntos
Bloqueadores dos Canais de Cálcio/síntese química , Dissacarídeos/síntese química , Oócitos/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Dissacarídeos/farmacologia , Oócitos/efeitos dos fármacos , XenopusRESUMO
Ethers and thioethers of monosaccharides have been synthesised which show potent toxicity to mouse (LD50 > or = 4 g.kg-1 O.W. and 0.2 to 1.5 g.kg-1 I.P.W.). A study of calcium antagonist activity for the full series of compounds indicated that the activity was similar for both O- and S- ethers and maximum activities were observed for monoacetoneglucose ethers possessing carbon chain close to 8 carbons.