RESUMO
Melatonin is synthesized in the pineal gland during nighttime in response to nocturnal increase in the activity of the enzyme aralkylamine N-acetyltransferase (AANAT), the transcription of which is modulated by several homeodomain transcription factors. Recent work suggests that the homeodomain transcription factor ISL LIM homeobox 2 (ISL2) is expressed in the pineal gland, but its role is currently unknown. With the purpose of identifying the mechanisms that control pineal expression of Isl2 and the possible function of Isl2 in circadian pineal biology, we report that Isl2 is specifically expressed in the pinealocytes of the rat pineal gland. Its expression exhibits a 24 h rhythm with high transcript and protein levels during the day and a trough in the second half of the night. This rhythm persists in darkness, and lesion studies reveal that it requires intact function of the suprachiasmatic nuclei, suggesting intrinsic circadian regulation. In vivo and in vitro experiments show that pineal Isl2 expression is repressed by adrenergic signaling acting via cyclic AMP; further, Isl2 is negatively regulated by the nocturnal transcription factor cone-rod homeobox. During development, pineal Isl2 expression is detectable from embryonic day 19, preceding Aanat by several days. In vitro knockdown of Isl2 is accompanied by an increase in Aanat transcript levels suggesting that ISL2 represses its daytime expression. Thus, rhythmic expression of ISL2 in pinealocytes is under the control of the suprachiasmatic nucleus acting via adrenergic signaling in the gland to repress nocturnal expression, while ISL2 itself negatively regulates daytime pineal expression of Aanat and thereby suggestively enhances the circadian rhythm in melatonin synthesis.
RESUMO
Charge separation and charge transfer across interfaces are key aspects in the design of efficient photocatalysts for solar energy conversion. In this study, we investigate the hydrogen generating capabilities and underlying photophysics of nanostructured photocatalysts based on CdSe nanowires (NWs). Systems studied include CdSe, CdSe/CdS core/shell nanowires and their Pt nanoparticle-decorated counterparts. Femtosecond transient differential absorption measurements reveal how semiconductor/semiconductor and metal/semiconductor heterojunctions affect the charge separation and hydrogen generation efficiencies of these hybrid photocatalysts. In turn, we unravel the role of surface passivation, charge separation at semiconductor interfaces and charge transfer to metal co-catalysts in determining photocatalytic H2 generation efficiencies. This allows us to rationalize why Pt nanoparticle decorated CdSe/CdS NWs, a double heterojunction system, performs best with H2 generation rates of â¼434.29 ± 27.40 µmol h(-1) g(-1) under UV/Visible irradiation. In particular, we conclude that the CdS shell of this double heterojunction system serves two purposes. The first is to passivate CdSe NW surface defects, leading to long-lived charges at the CdSe/CdS interface capable of carrying out reduction chemistries. Upon photoexcitation, we also find that CdS selectively injects charges into Pt NPs, enabling simultaneous reduction chemistries at the Pt NP/solvent interface. Pt nanoparticle decorated CdSe/CdS NWs thus enable reduction chemistries at not one, but rather two interfaces, taking advantage of each junction's optimal catalytic activities.
Assuntos
Hidrogênio/química , Nanopartículas Metálicas/química , Nanofios/química , Processos Fotoquímicos , Compostos de Cádmio/química , Oxirredução , Platina/química , Compostos de Selênio/química , Água/químicaRESUMO
Supercontinuum spatial modulation spectroscopy is a facile tool for conducting single molecule/particle extinction spectroscopy throughout the visible and near infrared (420-1100 nm). The technique's capabilities are benchmarked using individual Au nanoparticles (NPs) as a standard since they are well studied and display a prominent plasmon resonance in the visible. Extinction spectra of individual Au NPs with diameters (d) ranging from d ~ 8 to 40 nm are resolved with extinction cross sections (σ(ext)) of σ(ext) ~ 1 × 10(-13)-1 × 10(-11) cm(2). Corresponding signal-to-noise ratios range from ~30 to ~1400. The technique's limit of detection is determined to be 4.3 × 10(-14) cm(2) (4.3 nm(2)). To showcase supercontinuum spatial modulation spectroscopy's broader applicability, extinction spectra are acquired for other model systems, such as individual single-walled carbon nanotubes (SWCNTs) and CdSe nanowires. We show for the first time extinction spectra of individual (8,3) and (6,5) SWCNTs. For both chiralities, their E11 [(8,3) 1.30 eV (952 nm); (6,5) 1.26 eV (986 nm)] and E22 [(8,3) 1.86 eV (667 nm); (6,5) 2.19 eV (567 nm)] excitonic resonances are seen with corresponding cross sections of σ(ext) ~ 10(-13) cm(2) µm(-1).
RESUMO
The rumen has several important physiological functions: absorption, transport, metabolic activity, and protection. To clarify the molecular basis underlying the physiological function of the rumen, reticulum, omasum, and abomasum, we used mRNA differential display to isolate and identify differentially expressed genes in these tissues. We isolated 18 transcripts that coexpressed in the rumen, reticulum, and omasum. Five genes, ribosomal protein 19 (RPS19), basic helix-loop-helix domain containing class B2 (BHLHB2), NADH dehydrogenase flavoprotein 2 (NDUFV2), exosome component 9 (EXOSC9), and ribosomal protein 23 (RPS23), were highly expressed in the rumen of adult Holstein and Japanese Black cattle. Significant differences of expression were observed in the abomasum compared with the rumen, reticulum, and omasum. To investigate the expression pattern of these genes during the neonatal growth stage, the relative levels of gene expression were analyzed in the rumen and abomasum of 3-wk-, 13-wk-, and 18- to 20-mo-old Holstein cattle. The expression level of RPS19 did not change with age in the rumen and abomasum. The levels of BHLHB2, NDUFV2, and EXOSC9 mRNA in the abomasum decreased (P < 0.05) after weaning and declined (P < 0.05) further in adults; in contrast, expression in the rumen was not altered. Interestingly, the levels of RPS23 mRNA in the rumen increased (P < 0.05) after weaning and further increased in the adult; however, the level of expression of this gene decreased (P < 0.05) in the abomasum with weaning and age. This indicates that the 4 tissues, especially the rumen and abomasum, have different developmental pathways after birth and subsequent onset of rumination.
Assuntos
Abomaso/crescimento & desenvolvimento , Bovinos/crescimento & desenvolvimento , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Rúmen/crescimento & desenvolvimento , Abomaso/fisiologia , Animais , Bovinos/fisiologia , Primers do DNA/química , Perfilação da Expressão Gênica/métodos , Genes/fisiologia , Masculino , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Rúmen/fisiologia , Fatores de Tempo , DesmameRESUMO
We analyze photon statistics of blinking CdSe-ZnS nanocrystals interacting with a continuous wave laser field, showing that the process is described by a ballistic Lévy walk. In particular, we show that Mandel's Q parameter, describing the fluctuations of the photon counts, is increasing with time even in the limit of long time. This behavior is in agreement with the theory of Silbey and co-workers (Jung et al. Chem. Phys. 2002, 284, 181), and in contrast to all existing examples where Q approaches a constant, independent of time in the long time limit. We then analyze the distribution of the time averaged intensities, showing that they exhibit a nonergodic behavior, namely, the time averages remain random even in the limit of a long measurement time. In particular, the distribution of occupation times in the on-state compares favorably to a theory of weak ergodicity breaking of blinking nanocrystals. We show how our data analysis yields information on the amplitudes of power-law decaying on and off time distributions, information not available using standard data analysis of on and off time histograms. Photon statistics reveals fluctuations in the intensity of the bright state indicating that it is composed of several states. Photon statistics exhibits a Lévy walk behavior also when an ensemble of 100 dots is investigated, indicating that the strange kinetics can be observed already at the level of small ensembles.
RESUMO
An elevation in the extracellular Ca(2+) concentration ([Ca(2+)](o)) is a key signal for bone remodeling by inhibiting the resorbing activity of osteoclasts. The [Ca(2+)](o)-sensing responses include a variety of morphological and functional changes, but the underlying mechanisms are yet to be defined. This study was aimed at investigating the [Ca(2+)](o)-sensing mechanisms leading to the activation of the Cl(-) channel in murine osteoclasts. A rise in either Ca(2+) or Gd(3+) activated an outwardly rectifying Cl(-) (OR(cl)) channel reversibly and dose-dependently, which was characterized by rapid activation kinetics, little inactivation, and blockage by DIDS. The concentration required for a half-maximal response was estimated to be >20-30 mmol/L for Ca(2+). Intracellular dialysis with an ATP-free pipette solution or application of an actin destabilizer, cytochalasin D, decreased the [Ca(2+)](o)-activated OR(cl) current. Substitution of extracellular Na(+) by an impermeable cation, N-methyl-D-glucamine(+), inhibited the [Ca(2+)](o)-activated OR(cl) channel, suggesting that the activation depended on extracellular Na(+). A blocker for the Na(+)-Ca(2+) exchanger, 2'4'-dichlorobenzamil hydrochloride (DCB), inhibited the [Ca(2+)](o)-activated OR(cl) channel as well. Although 10 mmol/L Ca(2+) activated the OR(cl) current only slightly at a standard intracellular pH (7.3), decreasing pH by dialyzing cells with an acidic pipette solution (pH 6.6) enhanced the [Ca(2+)](o)-activated OR(cl) current. This potentiation by cell acidosis was eliminated by amiloride, a blocker for the Na(+)-H(+) exchanger. Zinc ion (0.1 mmol/L) and a polycation, neomycin (0.2 mmol/L), activated the OR(cl) current at intracellular pH 6.6, whereas the effects of those cations were negligible at intracellular pH 7.3. These results suggest that [Ca(2+)](o)-sensing mechanisms, leading to activation of the OR(cl) channel in murine osteoclasts, are regulated by ATP and actin cytoskeletal organization, and are sensitized greatly by cell acidosis. Contributions of Na(+)-dependent transporters in this activating process are examined in the context of a possible intermediate signal of cell swelling caused by Na(+) influx.
Assuntos
Cálcio/farmacologia , Canais de Cloreto/metabolismo , Espaço Extracelular/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Sódio/farmacologia , Potenciais de Ação , Animais , Cálcio/metabolismo , Células Cultivadas , Espaço Extracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Osteoclastos/metabolismo , Sódio/metabolismoRESUMO
We describe the preparation and characterization of bioinorganic conjugates in which luminescent semiconductor CdSe-ZnS core-shell nanocrystal quantum dots (QDs) were coupled to antibodies through the use of an avidin bridge adsorbed to the nanocrystal surface via electrostatic self-assembly. Avidin, a highly positively charged protein, was found to adsorb tightly to QDs modified with dihydrolipoic acid, which gives their surface a homogeneous negative charge. QD conjugation to biotinylated antibodies subsequently is readily achieved. Fluoroimmunoassays utilizing these antibody conjugated QDs were successful in the detection of protein toxins (staphylococcal enterotoxin B, cholera toxin). QD-antibody conjugates formed in such a facile manner permit their use as a common immuno reagent, and in the development of multianalyte detection.
Assuntos
Anticorpos/química , Avidina/química , Imunoconjugados/química , Antígenos de Bactérias/análise , Biotina/química , Cádmio/química , Proteínas de Transporte/química , Toxina da Cólera/análise , Enterotoxinas/análise , Fluorometria/métodos , Imunoensaio/métodos , Zíper de Leucina , Proteínas Ligantes de Maltose , Estrutura Terciária de Proteína , Semicondutores , Sulfetos/química , Compostos de Zinco/químicaRESUMO
T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has been found to have potent and selective inhibitory activity against influenza virus. In an in vitro plaque reduction assay, T-705 showed potent inhibitory activity against influenza A, B, and C viruses, with 50% inhibitory concentrations (IC(50)s) of 0.013 to 0.48 microg/ml, while it showed no cytotoxicity at concentrations up to 1,000 microg/ml in Madin-Darby canine kidney cells. The selectivity index for influenza virus was more than 2,000. It was also active against a neuraminidase inhibitor-resistant virus and some amantadine-resistant viruses. T-705 showed weak activity against non-influenza virus RNA viruses, with the IC(50)s being higher for non-influenza virus RNA viruses than for influenza virus, and it had no activity against DNA viruses. Orally administered T-705 at 100 mg/kg of body weight/day (four times a day) for 5 days significantly reduced the mean pulmonary virus yields and the rate of mortality in mice infected with influenza virus A/PR/8/34 (3 x 10(2) PFU). These results suggest that T-705 may be a compound that is useful and highly selective against influenza virus infections and that has a mode of action different from those of commercially available drugs, such as amantadine, rimantadine, and neuraminidase inhibitors.
Assuntos
Amidas/farmacologia , Amidas/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Influenza Humana/tratamento farmacológico , Orthomyxoviridae/efeitos dos fármacos , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Animais , Linhagem Celular , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Influenza Humana/virologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Vírus/efeitos dos fármacosRESUMO
p16, cyclin D1 and retinoblastoma protein (pRB) regulate G1 to S transition and are commonly targeted in various cancers. However, few studies have simultaneously examined all components of the p16/cyclin D1/pRB pathway (RB pathway) in hepatocellular carcinoma (HCC). To clarify the role of the disruption of the RB pathway in HCC, we analyzed p16, pRB and cyclin D1 in 47 HCCs. Inactivation of p16 was detected in 30 of 47 HCCs (64%) by Western blot analysis and significantly correlated with hypermethylation of the promoter of this gene. pRB expression was found to be absent in 13 of 47 HCCs (28%) by immunohistochemistry. We found that 38 of 47 HCCs (81%) contained at least one inactivation in either pRB or p16. Furthermore, there was a significant inverse correlation between p16 and pRB inactivation (p = 0.041). Overexpression of cyclin D1 was detected in 5 of 47 HCCs (11%) by immunohistochemistry. The cases with cyclin D1 overexpression exhibited an advanced clinicopathological appearance and also contained inactivation of pRB and/or p16. These findings suggest that inactivation of pRB and/or p16 is a major event in human hepatocarcinogenesis, while cyclin D1 overexpression may confer additional growth advantages to the tumor in addition to pRB and/or p16 inactivation in HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Ciclina D1/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Neoplasias Hepáticas/metabolismo , Proteína do Retinoblastoma/metabolismo , Adulto , Idoso , Carcinoma Hepatocelular/patologia , Núcleo Celular/patologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , DNA de Neoplasias/análise , Feminino , Deleção de Genes , Humanos , Técnicas Imunoenzimáticas , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Análise de Sequência de DNARESUMO
Basic electrical profiles of cultured neurons are modified by multiple factors, such as cell growth, differentiation and cell damage from the isolation procedure. In the present study, we assessed development of electrophysiological properties of rat spinal neurons over the late embryonic and early postnatal period in a neuron-enriched culture. After recovery from acute damage within 2 days after plating, the input conductance and amplitudes of voltage-gated Na(+) and K(+) currents increased parallel to the increase in the cell capacitance. Whether this depended on the period or the growth of the cell area was estimated by normalizing the parameters with the cell capacitance. The input conductance per unit area, the membrane time constant and the Na(+) current density remained constant for two weeks. However, densities of two types of outward K(+) currents, an A-current and a delayed rectifier, required 3--5 days to reach the maximum, although neither thresholds for activation nor sensitivities to blockers (TEA and 4AP) altered. The hyperpolarizing shift of the resting membrane potential became stabilized within 6--8 days, suggesting that the development of the K(+) currents underlay the shift. These results show that passive electrical properties and voltage-gated currents of rat spinal neurons in the neuron-rich culture differ in temporal patterns of development but stabilize at the latest within a week, corresponding to the day of birth.
Assuntos
Diferenciação Celular/fisiologia , Células Cultivadas/metabolismo , Potenciais da Membrana/fisiologia , Neurônios/metabolismo , Canais de Potássio/metabolismo , Canais de Sódio/metabolismo , Medula Espinal/embriologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Feto , Proteína Glial Fibrilar Ácida , Imuno-Histoquímica , Potenciais da Membrana/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Ratos , Canais de Sódio/efeitos dos fármacos , Medula Espinal/citologia , Medula Espinal/metabolismo , Tetrodotoxina/farmacologiaRESUMO
The zona pellucida, a transparent envelope surrounding the mammalian oocyte, comprises three glycoproteins, ZPA, ZPB and ZPC, and plays important roles in fertilization. We have previously reported that apparent relative molecular masses of bovine zona glycoproteins on SDS/PAGE under nonreducing conditions after removal of poly N-acetyllactosamine at the nonreducing portion of sugar chains with endo-beta-galactosidase are 72 000, 58 000 and 45 000 [Noguchi, S., Yonezawa, N., Katsumata, T., Hashizume, K.,Kuwayama, M., Hamano, S., Watanabe, S. & Nakano, M. (1994) Biochim. Biophys. Acta 1201, 7-14]. The N-terminal amino-acid sequences and crossreactivity to antibodies specific to each porcine zona component show that the bovine components correspond to porcine ZPA, ZPB and ZPC, respectively. In this study, we deduced amino-acid sequences of bovine ZPA and ZPB by cDNA cloning and sequencing. Identities in amino-acid sequences between bovine and porcine counterparts were 77% for ZPA and 75% for ZPB, whereas between bovine and murine counterparts identities were 57% for ZPA and 37% for ZPB. The positions of Cys were completely conserved in bovine ZPA and ZPB compared with counterparts of other mammalian species. Bovine ZPA was processed between Ala and Asp on fertilization, suggesting that the consensus motif for the processing is Ala-Asp-Asp/Glu. We purified bovine zona components and examined their sperm-binding activity with an in vitro competition assay and sperm-bead-binding assay. As a result, ZPB showed the strongest sperm-binding activity among the components. ZPC also showed sperm-binding activity and the activity per molecule was about one-sixth that of ZPB according to the result of the sperm-bead-binding assay. We could not determine if ZPA has significant sperm-binding activity, but the activity may be much lower than that of ZPB even if ZPA has significant activity. Thus, ZPB may play a major role in sperm binding in bovine zona.
Assuntos
Proteínas do Ovo/genética , Glicosídeo Hidrolases , Glicoproteínas de Membrana/genética , Receptores de Superfície Celular , Interações Espermatozoide-Óvulo , Espermatozoides/metabolismo , Zona Pelúcida/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Cromatografia Líquida de Alta Pressão , DNA Complementar , Proteínas do Ovo/química , Proteínas do Ovo/metabolismo , Masculino , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Glicoproteínas da Zona Pelúcida , beta-Galactosidase/metabolismoRESUMO
BACKGROUND: We tested for the presence of alpha-fetoprotein (AFP) mRNA by using nested RT-PCR in the peripheral blood of hepatocellular carcinoma (HCC) patients who had undergone curative surgery, and investigated the occurrence of intrahepatic and/or extrahepatic metastasis thereafter, to reveal the optimal timing of blood sampling for the prediction of metastatic recurrence. METHODS: Twenty-nine patients with HCC, who had been operated on were analyzed with RT-PCR at several points during the clinical course, and examined for metastatic recurrence for 3-28 months (mean = 18.7 months) after surgery. RESULTS: The presence of AFP mRNA before surgery was significantly correlated with the tumor size (P = 0.017). Metastatic recurrence was associated with the postoperative detection of AFP mRNA (P < 0.001), but not with the preoperative and/or perioperative detection. Furthermore, AFP mRNA was detected in some cases that showed low serum AFP levels at recurrence. The recurrence-free period after the detection of AFP mRNA varied from 1 to 12 months. CONCLUSIONS: The postoperative detection of AFP mRNA is useful for the prediction of metastatic recurrence, and long-term follow up with this method should be conducted.
Assuntos
Carcinoma Hepatocelular/secundário , Carcinoma Hepatocelular/cirurgia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/cirurgia , RNA Mensageiro/sangue , alfa-Fetoproteínas/genética , Adulto , Idoso , Carcinoma Hepatocelular/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , PrognósticoRESUMO
Voltage-gated proton (H(+)) channels are unique mechanisms to extrude a massive amount of H(+), and are proposed to regulate intracellular pH of microglia during respiratory bursts. Temporal variations of the H(+) current were studied in rat spinal microglia cultivated on the glial cell layer using the voltage-ramp protocol. Repetitive applications of the large and long-lasting depolarization decreased the amplitudes of the H(+) current transiently and reversibly. This decrease was accompanied by a shift of the reversal potential to a more positive direction, indicating that a drop in the transmembrane pH gradient (delta pH) by the H(+) efflux through the channel reduced the current. The decline of the H(+) current during depolarizations was also observed in a rat microglial cell line (GMI-R1). An increase in the extracellular buffer suppressed the reduction of the current, suggesting that H(+) secreted into the extracellular space contributed to the drop in delta pH. On the other hand, the amplitudes of the H(+) current often fluctuated greatly at intervals of 5-20 min without changes in delta pH. These results suggest that the H(+) current of microglia is tuned via both delta pH-dependent and -independent mechanisms, which may regulate both microglial behavior and the pH environments of the surrounding neural tissue.
Assuntos
Ativação do Canal Iônico , Canais Iônicos/fisiologia , Microglia/fisiologia , Prótons , Medula Espinal/fisiologia , Animais , Células Cultivadas , Condutividade Elétrica , Eletrofisiologia , Concentração de Íons de Hidrogênio , Oscilometria , Ratos , Ratos Wistar , Medula Espinal/citologia , Fatores de TempoRESUMO
alpha-Mannosidase and beta-galactosidase were released from boar sperm into the medium by treatment with calcium ionophore A23187 or by 0.2% Brij-35/2% acetic acid. About half as much alpha-mannosidase activity as that in the acid extract was recovered by digestion with phosphatidylinositol-specific phospholipase C (PI-PLC), whereas the liberation rate of beta-galactosidase treated with PI-PLC was low. These results suggest that some alpha-mannosidase is anchored in the plasma membrane of the acrosomal region by attachment to the lipid phosphatidylinositol and that beta-galactosidase is localized mainly in the acrosome or integrated in the plasma membrane by a spanning stretch of hydrophobic peptides. beta-Galactosidase, which is present as an oligomers in the acid extract of sperm, dissociated into monomers under weakly alkaline conditions; under acidic conditions, the monomers associated again. No pH-sensitive association-dissociation of alpha-mannosidase was observed.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Manosidases/metabolismo , Espermatozoides/enzimologia , Acrossomo/enzimologia , Acrossomo/metabolismo , Reação Acrossômica , Animais , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Estabilidade Enzimática , Glicosilfosfatidilinositóis/isolamento & purificação , Concentração de Íons de Hidrogênio , Masculino , Manosidases/isolamento & purificação , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Espermatozoides/metabolismo , Suínos , Fosfolipases Tipo C , alfa-Manosidase , beta-Galactosidase/isolamento & purificação , beta-Galactosidase/metabolismoRESUMO
Microglia are equipped with a strong proton (H(+)) extrusion pathway, a voltage-gated H(+) channel, probably to compensate for the large amount of H(+) generated during phagocytosis; however, little is known about how this channel is regulated in pathological states. Because neural damage is often associated with intracellular and extracellular acidosis, we examined the regulatory mechanisms of the H(+) current of rat spinal microglia in acidic environments. More than 90% of round/amoeboid microglia expressed the H(+) current, which was characterized by slow activation kinetics, dependencies on both intracellular and extracellular pH, and blockage by Zn(2+). Extracellular lactoacidosis, pH 6.8, induced intracellular acidification and cell swelling. Cell swelling was also induced by intracellular dialysis with acidic pipette solutions, pH 5.5-6.8, at normal extracellular pH 7.3 in the presence of Na(+). The H(+) currents were increased in association with cell swelling as shown by shifts of the half-activation voltage to more negative potentials and by acceleration of the activation kinetics. The acidosis-induced cell swelling and the accompanying potentiation of the H(+) current required nonhydrolytic actions of intracellular ATP and were inhibited by agents affecting actin filaments (phalloidin and cytochalasin D). The H(+) current was also potentiated by swelling caused by hypotonic stress. These findings suggest that the H(+) channel of microglia can be potentiated via cell swelling induced by intracellular acidification. This potentiation might operate as a negative feedback mechanism to protect microglia from cytotoxic acidification and hence acidosis-induced swelling in pathological states of the CNS.
Assuntos
Acidose/metabolismo , Microglia/metabolismo , Microglia/patologia , Bombas de Próton/metabolismo , Prótons , ATPases Vacuolares Próton-Translocadoras , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Ácidos/farmacologia , Trifosfato de Adenosina/metabolismo , Álcalis/farmacologia , Animais , Células Cultivadas , Canais de Cloreto/antagonistas & inibidores , Cloretos/farmacologia , Citocalasina D/farmacologia , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Soluções Hipotônicas/farmacologia , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Ácido Láctico/metabolismo , Ácido Láctico/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Microglia/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Técnicas de Patch-Clamp , Faloidina/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Ratos , Ratos Wistar , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Compostos de Zinco/farmacologiaRESUMO
In our continuing research for dual CCK-A and -B antagonists, according to our hypothesis that dual CCK-A and -B antagonists should be more efficacious than selective CCK-A antagonists for the treatment of pancreatitis, we have prepared various 5-alkyl-9-methyl-1,4-benzodiazepines. From the compounds prepared, 1-cyclohexyl-carbonylmethyl-5-ethyl-9-methyl-3- (m-tolylureido)-2-oxo-1,4-benzodiazepine, (40) was selected as a candidate for development due to its well-balanced high affinity for both receptors. The R-enantiomer of 40, (R)-40 (FR 208419), had 27-fold higher affinity for the CCK-A receptor and 8-fold more potent CCK-B receptor binding activity than (S)-40. The biological activity after p.o. administration of (R)-40, estimated from the ID50 value (0.23 mg/kg p.o.) obtained by preliminary evaluation by gastric emptying effects, is considered to be high enough for further development. This compound is now undergoing further biological evaluations with a view to clinical development.
Assuntos
Benzodiazepinonas/síntese química , Compostos de Fenilureia/síntese química , Receptores da Colecistocinina/antagonistas & inibidores , Animais , Benzodiazepinonas/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Esvaziamento Gástrico/efeitos dos fármacos , Cobaias , Técnicas In Vitro , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Compostos de Fenilureia/farmacologia , Ratos , Receptor de Colecistocinina A , Receptor de Colecistocinina B , Sincalida/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The rapidly (< 1 h) developing neuronal death induced by a 15-min-exposure to N-methyl-D-aspartate (NMDA) in rat hippocampal slice cultures is associated with cell swelling. We examined whether the swelling directly leads to neuronal death. The rapid neuronal death assayed by propidium iodide was Cl(-)-dependent, as reported for the cell swelling. However, the dose-dependence for NMDA-induced neuronal death differed from that for the cell swelling. Also, cell swelling alone induced by hypotonic insults led to neuronal death only when the cell size increased far more than the extent achieved by NMDA insults. Moreover, contrary to the previous notion, the rapid neuronal death was Ca2+-dependent. Thus, the primary cause of the rapid neuronal death induced by NMDA cannot be attributed to cell swelling.
Assuntos
Morte Celular/efeitos dos fármacos , Hipocampo/citologia , Neurotoxinas/farmacologia , Células Piramidais/citologia , Animais , Cálcio/farmacologia , Morte Celular/fisiologia , Tamanho Celular/efeitos dos fármacos , Cloretos/farmacologia , Corantes , Agonistas de Aminoácidos Excitatórios/farmacologia , Soluções Hipotônicas/farmacologia , N-Metilaspartato/farmacologia , Técnicas de Cultura de Órgãos , Propídio , RatosRESUMO
Excitotoxic neuronal death is mediated primarily by the N-methyl-D-aspartate receptor. N-methyl-D-aspartate induces two forms of excitotoxicity in CA1 pyramidal neurons of cultured rat hippocampal slices: the rapidly developing form that depends on external Na+ and Cl-, and the delayed form that requires external Ca2+ but not Cl-. Consistent with this notion, replacement of external Cl- with glucuronate, isethionate or methylsulfate attenuated or delayed selectively the rapid excitotoxicity. However, gluconate substituting for Cl- blocked both rapid and delayed forms of excitotoxicity. Gluconate also reduced N-methyl-D-aspartate-induced membrane currents recorded from CA1 neurons in a dose-dependent manner. This dose-dependence was remarkably similar to that observed for protection of N-methyl-D-aspartate-induced neuronal death by gluconate. Although gluconate chelated Ca2+ most strongly among the four Cl- substitutes examined, excitotoxic neuronal death could be protected by 7 mM gluconate without Ca2+ chelating action. The voltage-dependent Mg2+ block of N-methyl-D-aspartate receptors was not affected by gluconate. Gluconate suppressed the N-methyl-D-aspartate component of excitatory synaptic currents evoked in CA1 neurons. We conclude that protection of excitotoxic neuronal death by gluconate at low doses (<20 mM) is due to its antagonistic action on N-methyl-D-aspartate receptors. Gluconate is a widely used substitute for Cl-. Our unexpected findings give a warning that the results of any of the experiments concerning excitotoxicity or glutamate receptors obtained by gluconate substituting for Cl- must be interpreted with caution.
Assuntos
Morte Celular/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Gluconatos/farmacologia , Fármacos Neuroprotetores/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Cálcio/farmacologia , Células Cultivadas , Corantes , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Propídio , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/fisiologiaRESUMO
1. An outwardly rectifying Cl- (ORCl) current of murine osteoclasts was activated by hypotonic stimulation. The current was characterized by rapid activation, little inactivation, strong outward rectification, blockage by DIDS and permeability to organic acids (pyruvate and glutamate). 2. The hypotonically activated ORCl current was inhibited by intracellular dialysis with an ATP-free pipette solution, but not by replacement of ATP with a poorly hydrolysable ATP analogue adenosine 5'-O-(3-thiotriphosphate). The current amplitude was reduced when intracellular alkalinity increased over the pH range 6.6-8.0. 3. Intracellular application of cytochalasin D occasionally activated the ORCl current without hypotonic stress, but inhibited activation of the ORCl current by hypotonic stimulation. The hypotonically activated ORCl current was unaffected by a non-actin-depolymerizing cytochalasin, chaetoglobosin C, but partially inhibited by deoxyribonuclease I. 4. Removal of extracellular Ca2+ inhibited activation of the ORCl current by hypotonic shock, but did not reduce the current once activated. The hypotonically activated ORCl current was partially decreased by intracellular dialysis with 20 mM EGTA. 5. With 10 mM Ca2+ in the extracellular medium, the ORCl current was activated in response to more minor decreases in osmolarity than with 1 mM Ca2+. The increased sensitivity to hypotonicity was mimicked by increasing the intracellular Ca2+ level (pCa 6.5). 6. These results suggest that hypotonic stimulation and a rise in the extracellular Ca2+ level synergistically activate the ORCl channel of murine osteoclasts, and that the activating process is modified by multiple intracellular factors (pH, ATP and actin cytoskeletal organization).