RESUMO
Selenium (Se) is a natural trace element, which shifts its action in a relatively narrow concentration range from nutritional role to toxicity. Although it has been well established that in plants chloroplasts are among the primary targets, the mechanism of toxicity on photosynthesis is not well understood. Here, we compared selenate and red-allotrope elemental selenium nanoparticles (red nanoSe) in in vitro tobacco cultures to investigate their effects on the structure and functions of the photosynthetic machinery. Selenate at 10 mg/L concentration retarded plant growth; it also led to a decreased chlorophyll content, accompanied with an increase in the carotenoid-to-chlorophyll ratio. Structural examinations of the photosynthetic machinery, using electron microscopy, small-angle neutron scattering and circular dichroism spectroscopy, revealed significant perturbation in the macro-organization of the pigment-protein complexes and sizeable shrinkage in the repeat distance of granum thylakoid membranes. As shown by chlorophyll a fluorescence transient measurements, these changes in the ultrastructure were associated with a significantly diminished photosystem II activity and a reduced performance of the photosynthetic electron transport, and an enhanced capability of non-photochemical quenching. These changes in the structure and function of the photosynthetic apparatus explain, at least in part, the retarded growth of plantlets in the presence of 10 mg/L selenate. In contrast, red nanoSe, even at 100 mg/L and selenate at 1 mg/L, exerted no negative effect on the growth of plantlets and affected only marginally the thylakoid membrane ultrastructure and the photosynthetic functions.
Assuntos
Nanopartículas/química , Nicotiana/metabolismo , Fotossíntese/fisiologia , Ácido Selênico/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Dicroísmo Circular , Tilacoides/metabolismoRESUMO
C-150 a Mannich-type curcumin derivative, exhibited pronounced cytotoxic effects against eight glioma cell lines at micromolar concentrations. Inhibition of cell proliferation by C-150 was mediated by affecting multiple targets as confirmed at transcription and protein level. C-150 effectively reduced the transcription activation of NFkB, inhibited PKC-alpha which are constitutively over-expressed in glioblastoma. The effects of C-150 on the Akt/ Notch signaling were also demonstrated in a Drosophila tumorigenesis model. C-150 reduced the number of tumors in Drosophila with similar efficacy to mitoxantrone. In an in vivo orthotopic glioma model, C-150 significantly increased the median survival of treated nude rats compared to control animals. The multi-target action of C-150, and its preliminary in vivo efficacy would render this curcumin analogue as a potent clinical candidate against glioblastoma.
Assuntos
Acrilamidas/química , Neoplasias Encefálicas/tratamento farmacológico , Curcumina/análogos & derivados , Curcumina/química , Glioblastoma/tratamento farmacológico , NF-kappa B/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Receptores Notch/antagonistas & inibidores , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Antineoplásicos/química , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Drosophila melanogaster , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Humanos , Concentração Inibidora 50 , Melanoma Experimental , Camundongos , Transplante de Neoplasias , Ratos , Ratos Nus , Receptores Notch/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
We identified and characterized a so far unrecognized cell type, dubbed the multinucleated giant hemocyte (MGH), in the ananassae subgroup of Drosophilidae. Here, we describe the functional and ultrastructural characteristics of this novel blood cell type as well as its characterization with a set of discriminative immunological markers. MGHs are encapsulating cells that isolate and kill the parasite without melanization. They share some properties with but differ considerably from lamellocytes, the encapsulating cells of Drosophila melanogaster, the broadly used model organism in studies of innate immunity. MGHs are nonproliferative effector cells that are derived from phagocytic cells of the sessile tissue and the circulation, but do not exhibit phagocytic activity. In contrast to lamellocytes, MGHs are gigantic cells with filamentous projections and contain many nuclei, which are the result of the fusion of several cells. Although the structure of lamellocytes and MGHs differ remarkably, their function in the elimination of parasites is similar, which is potentially the result of the convergent evolution of interactions between hosts and parasites in different geographic regions. MGHs are highly motile and share several features with mammalian multinucleated giant cells, a syncytium of macrophages formed during granulomatous inflammation.
Assuntos
Movimento Celular/imunologia , Células Gigantes/imunologia , Imunidade Celular , Fagocitose , Animais , Drosophila , Células Gigantes/citologia , HemócitosRESUMO
The expression pattern of aromatase (ARO), the enzyme converting androgens to estrogens, was analyzed in the olfactory bulb of adult male rats and was compared with the distribution of estrogen receptor beta (ERbeta), the main estrogen receptor isoform expressed in this brain region. A strong ARO immunolabeling obtained with a specificity tested antibody was observed in juxtaglomerular neurons of the glomerular layer and a weaker immunoreaction was detected in the mitral cell layer of the main olfactory bulb, while the granule cell layer of the main olfactory bulb as well as all layers in the accessory olfactory bulb showed faint immunolabeling. Fluorescence double labeling experiments revealed that ARO detected in juxtaglomerular neurons of the main olfactory bulb colocalized with tyrosine hydroxylase (TH) and glutamic acid decarboxylase 67 (GAD67), while no colocalization between ARO and the calcium binding proteins calretinin (CR) and calbindin (CB) was observed. Furthermore, the TH immunoreactive neurons expressed metabotropic glutamate receptor 1 (mGluR1) too. ERß immunoreactivity, in contrast to ARO, was detected in all layers of both the main and accessory olfactory bulb. In the glomerular layer of the main olfactory bulb it was expressed in TH and GAD67 containing juxtaglomerular neurons, and it colocalized with CR, CB and even with glial fibrillary acidic protein too. Our morphological findings suggest that ARO expression is a novel feature of dopaminergic/GABAergic juxtaglomerular neurons in the adult rat main olfactory bulb, and raise the possibility that ARO activity may change in function of olfactory input via mGluR1. In situ estrogen production in the olfactory bulb in turn may modulate interglomerular circuits through ERbeta.
Assuntos
Aromatase/metabolismo , Receptor beta de Estrogênio/metabolismo , Bulbo Olfatório/metabolismo , Animais , Calbindina 2/metabolismo , Calbindinas/metabolismo , Glutamato Descarboxilase/metabolismo , Masculino , Neurônios/metabolismo , Bulbo Olfatório/anatomia & histologia , Condutos Olfatórios/metabolismo , Ratos , Ratos Wistar , Receptores de Glutamato Metabotrópico/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Influence of the modification of the cyanobacterial light-harvesting complex [i.e. phycobilisomes (PBS)] on the surface electric properties and the functions of photosynthetic membranes was investigated. We used four PBS mutant strains of Synechocystis sp. PCC6803 as follows: PAL (PBS-less), CK (phycocyanin-less), BE (PSII-PBS-less) and PSI-less/apcE(-) (PSI-less with detached PBS). Modifications of the PBS content lead to changes in the cell morphology and surface electric properties of the thylakoid membranes as well as in their functions, such as photosynthetic oxygen-evolving activity, P700 kinetics and energy transfer between the pigment-protein complexes. Data reveal that the complete elimination of PBS in the PAL mutant causes a slight decrease in the electric dipole moments of the thylakoid membranes, whereas significant perturbations of the surface charges were registered in the membranes without assembled PBS-PSII macrocomplex (BE mutant) or PSI complex (PSI-less mutant). These observations correlate with the detected alterations in the membrane structural organization. Using a polarographic oxygen rate electrode, we showed that the ratio of the fast to the slow oxygen-evolving PSII centers depends on the partial or complete elimination of light-harvesting complexes, as the slow operating PSII centers dominate in the PBS-less mutant and in the mutant with detached PBS.
Assuntos
Fotossíntese , Complexo de Proteína do Fotossistema I/fisiologia , Complexo de Proteína do Fotossistema II/fisiologia , Synechocystis/genética , Tilacoides/fisiologia , Fenômenos Eletrofisiológicos , Deleção de Genes , Microscopia Eletrônica , Oxirredução , Oxigênio/análise , Complexo de Proteína do Fotossistema I/genética , Complexo de Proteína do Fotossistema II/genética , Synechocystis/fisiologia , Synechocystis/ultraestruturaRESUMO
Sixteen lytic bacteriophages that infect Pseudomonas tolaasii LMG 2342(T) were isolated from smashed sporocarps of oyster mushroom (Pleurotus ostreatus) showing necrotic symptoms. On the basis of the host range investigation of the phages, they have wide infection abilities against the genus Pseudomonas, mainly in the case of phages Bf3, Bf7, Bf10, and Bf15. Molecular investigations have revealed that they all have dsDNA genomes about 40 kbp in size. Identical restriction patterns resulting from restriction enzyme analysis suggest that the isolates probably belong to the same phage species. However, there was a difference between these phage isolates in their infecting abilities. Phage isolate Bf7 was investigated and characterized more deeply. Morphological characterization of Bf7 by transmission electron microscopy (TEM) has shown that it has a short, noncontractile tail, an icosahedral phage head, and the size is about 60 nm in diameter, suggesting that it belongs to the Podoviridae family. Complete genome sequence analysis of the Bf7 phage isolate revealed a 40 058 bp genome, 58.4% G+C content, 46 open reading frames encoding different proteins showing homology to proteins of the bacteriophage Caulobacter crescentus φCd1 from the Podoviridae family. On the basis of these results and comparative genomic studies, we classified the Bf7 phage to the subfamily of Autographivirinae, φKMV-like phages.
Assuntos
Genoma Viral , Fagos de Pseudomonas/genética , Fagos de Pseudomonas/isolamento & purificação , Pseudomonas/virologia , DNA Viral/química , DNA Viral/genética , Especificidade de Hospedeiro , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Fases de Leitura Aberta , Pleurotus/virologia , Podoviridae/genética , Podoviridae/isolamento & purificação , Podoviridae/fisiologia , Podoviridae/ultraestrutura , Polimorfismo de Fragmento de Restrição , Fagos de Pseudomonas/fisiologia , Fagos de Pseudomonas/ultraestrutura , Análise de Sequência de DNA , Homologia de Sequência , Vírion/ultraestruturaRESUMO
The naturally occurring steroid dehydroepiandrosterone (DHEA) is reported to reduce glial fibrillary acidic protein (GFAP) overexpression in a model of reactive gliosis due to its conversion to estradiol by the enzyme aromatase. In the present study we examined the biological effect of a new epimerized derivative of DHEA, 16alpha-iodomethyl-13alpha-dehydroepiandrosterone derivative (16alpha-iodomethyl-13alpha-DHEAd, 16alpha-iodomethyl-13alpha-androst-5-en-3beta,17beta-diol), using the same model system, and compared the 3D structure of this molecule with that of DHEA and two steroidal type aromatase inhibitors, formestane and exemestane. The synthetic compound, in contrast to the reported effect of DHEA, was able to reduce GFAP overexpression only if the enzyme aromatase was inhibited. Data obtained from computational calculations fortified by X-ray crystallography revealed that contrary to the nearly planar sterane framework of DHEA, the synthetic derivative 16alpha-iodomethyl-13alpha-DHEAd has a bent sterane skeleton, resulting in a 3D structure that is similar to that of formestane or exemestane. Moreover, 16alpha-iodomethyl-13alpha-DHEAd resulted to be metabolically more stable than DHEA. The results suggest that epimerization of the sterane skeleton of DHEA inclines the plane of the D ring, leading to a significantly altered biological activity. The synthetic molecule has a DHEA-like effect on GFAP overexpression when the enzyme aromatase is inhibited and the naturally occurring DHEA is ineffective in this respect. On the other hand, based on their structural similarity it can be hypothesized that 16alpha-iodomethyl-13alpha-DHEAd applied alone might have a biological effect similar to that of formestane or exemestane.
Assuntos
Desidroepiandrosterona/análogos & derivados , Desidroepiandrosterona/uso terapêutico , Gliose/tratamento farmacológico , Androstadienos/química , Androstadienos/metabolismo , Androstenodiona/análogos & derivados , Androstenodiona/química , Androstenodiona/metabolismo , Animais , Aromatase/química , Aromatase/metabolismo , Inibidores da Aromatase/química , Inibidores da Aromatase/metabolismo , Cristalografia por Raios X , Desidroepiandrosterona/química , Desidroepiandrosterona/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/patologia , Masculino , Dados de Sequência Molecular , Estrutura Molecular , Ratos , Ratos WistarRESUMO
BACKGROUND: Despite the fact that women are twice as likely to develop depression as men, our understanding of depression neurobiology in female subjects is limited. We have recently reported in male rats that development of helpless behavior is associated with a severe loss of hippocampal spine synapses, which is reversed by treatment with the antidepressant desipramine. Considering that estradiol has a hippocampal synaptogenic effect similar to those of antidepressants, the presence of estradiol during the female reproductive life might influence behavioral and synaptic responses to stress and depression. METHODS: With electron microscopic stereology, we analyzed hippocampal spine synapses in association with helpless behavior in ovariectomized female rats (n = 70), under different conditions of estradiol exposure. RESULTS: Stress induced an acute and persistent loss of hippocampal spine synapses, whereas subchronic treatment with desipramine reversed the stress-induced synaptic loss. Estradiol supplementation given either before stress or before escape testing of nonstressed animals increased the number of hippocampal spine synapses. Correlation analysis demonstrated a statistically significant negative correlation between the severity of helpless behavior and hippocampal spine synapse numbers. CONCLUSIONS: These findings suggest that hippocampal spine synapse remodeling might be a critical factor underlying learned helplessness and, possibly, the neurobiology of depression.
Assuntos
Região CA1 Hipocampal/citologia , Espinhas Dendríticas/efeitos dos fármacos , Estradiol/farmacologia , Estrogênios/farmacologia , Desamparo Aprendido , Neurônios/citologia , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Espinhas Dendríticas/ultraestrutura , Modelos Animais de Doenças , Eletrochoque/efeitos adversos , Reação de Fuga/efeitos dos fármacos , Feminino , Microscopia Eletrônica de Transmissão/métodos , Neurônios/efeitos dos fármacos , Ovariectomia/métodos , Ratos , Ratos Sprague-Dawley , Análise de Regressão , Técnicas Estereotáxicas , Estresse Psicológico/complicações , Estresse Psicológico/etiologia , Estresse Psicológico/patologia , Sinapses/efeitos dos fármacos , Sinapses/ultraestrutura , Fatores de TempoRESUMO
(1) Intranasal administration is a non-invasive and effective way for the delivery of drugs to brain that circumvents the blood-brain barrier. The aims of the study were to test a nasal delivery system for human beta-amyloid (A beta) peptides, to measure the delivery of the peptides to brain regions, and to test their biological activity in rats. (2) A beta(1-42), in the form of a mixture of oligomers, protofibrils, and fibrils was dissolved in a nasal formulation containing hydrophobic, hydrophylic, and mucoadhesive components. The peptide solution was administered intranasally to rats as a single dose or in repeated doses. (3) Nasally injected A beta labeled with the blue fluorescent dye amino-methyl coumarinyl acetic acid (AMCA) could be detected by fluorescent microscopy in the olfactory bulb and frontal cortex. The concentration of the peptide was quantified by fluorescent spectroscopy, and a significant amount of AMCA-A beta peptide could be detected in the olfactory bulb. Unlabeled A beta also reached the olfactory bulb and frontal cortex of rats as evidenced by intense immunostaining. (4) In behavioral experiments, nasal A beta treatment did not affect anxiety levels (open-field test) and short-term memory (Y-maze test), but significantly impaired long-term spatial memory in the Morris water maze. The treatments did not result in A beta immunization. (5) The tested intranasal delivery system could successfully target a bioactive peptide into the central nervous system and provides a basis for developing a non-invasive and cost effective, new model to study amyloid-induced dysfunctions in the brain.
Assuntos
Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/administração & dosagem , Encéfalo/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/fisiopatologia , Fragmentos de Peptídeos/administração & dosagem , Administração Intranasal , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Animais , Transporte Axonal/efeitos dos fármacos , Transporte Axonal/fisiologia , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Corantes Fluorescentes/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/metabolismo , Bulbo Olfatório/efeitos dos fármacos , Bulbo Olfatório/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Ratos , Ratos Wistar , Coloração e Rotulagem/métodosRESUMO
Recent evidence showed that neurotransmitters are synthesised in glial cells, such as the Schwann cells, which form myelin sheaths in the PNS. While the presence of GABA type A (GABA-A) receptors has been previously demonstrated in these cells, the evidence of GABA synthesis remained still elusive. In an attempt to demonstrate the presence of GABA in rat Schwann cells, we adopted a strategy, using several integrated neurochemical, molecular as well as immunocytochemical approaches. We first demonstrated the presence of glutamic acid decarboxylase of 67 kDa (GAD67) in Schwann cells, a crucial enzyme of the GABA synthesis mechanism. Second, we demonstrated that GABA is synthesized and localized in Schwann cells. As the third step we showed that allopregnanolone (10 nM), a potent allosteric modulator of GABA-A receptors, stimulates GABA synthesis through increased levels of GAD67 in Schwann cells. Analysis of intracellular signalling mechanisms revealed that the protein kinase A pathway, through enhanced cAMP levels and cAMP response element binding protein phosphorylation, modulates the allosteric action of allopregnanolone at the GABA-A receptor in Schwann cells. Our findings are the first to demonstrate that this GABA mechanism is active in Schwann cells thus establishing new potential therapeutic targets to control Schwann cell biology, which may prove useful in the treatment of several neurodegenerative disorders.
Assuntos
Anestésicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Pregnanolona/farmacologia , Células de Schwann/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Proteína de Ligação a CREB/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Colforsina/farmacologia , Biologia Computacional/métodos , AMP Cíclico/metabolismo , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Células de Schwann/metabolismo , Nervo Isquiático/citologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Regulação para CimaRESUMO
BACKGROUND: Although it has been postulated for many years that depression is associated with loss of synapses, primarily in the hippocampus, and that antidepressants facilitate synapse growth, we still lack ultrastructural evidence that changes in depressive behavior are indeed correlated with structural synaptic modifications. METHODS: We analyzed hippocampal spine synapses of male rats (n=127) with electron microscopic stereology in association with performance in the learned helplessness paradigm. RESULTS: Inescapable footshock (IES) caused an acute and persistent loss of spine synapses in each of CA1, CA3, and dentate gyrus, which was associated with a severe escape deficit in learned helplessness. On the other hand, IES elicited no significant synaptic alterations in motor cortex. A single injection of corticosterone reproduced both the hippocampal synaptic changes and the behavioral responses induced by IES. Treatment of IES-exposed animals for 6 days with desipramine reversed both the hippocampal spine synapse loss and the escape deficit in learned helplessness. We noted, however, that desipramine failed to restore the number of CA1 spine synapses to nonstressed levels, which was associated with a minor escape deficit compared with nonstressed control rats. Shorter, 1-day or 3-day desipramine treatments, however, had neither synaptic nor behavioral effects. CONCLUSIONS: These results indicate that changes in depressive behavior are associated with remarkable remodeling of hippocampal spine synapses at the ultrastructural level. Because spine synapse loss contributes to hippocampal dysfunction, this cellular mechanism may be an important component in the neurobiology of stress-related disorders such as depression.
Assuntos
Depressão/patologia , Reação de Fuga/efeitos dos fármacos , Desamparo Aprendido , Hipocampo/ultraestrutura , Sinapses/ultraestrutura , Animais , Anti-Inflamatórios/farmacologia , Antidepressivos Tricíclicos/administração & dosagem , Biomarcadores Farmacológicos/análise , Corticosterona/sangue , Corticosterona/farmacologia , Depressão/sangue , Depressão/tratamento farmacológico , Desipramina/administração & dosagem , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Masculino , Córtex Motor/efeitos dos fármacos , Córtex Motor/ultraestrutura , Ratos , Ratos Sprague-Dawley , Estresse Fisiológico , Sinapses/efeitos dos fármacosRESUMO
The numerous situations which can result in cerebral hypoxic damage occur in newborn infants and in the elderly. In research aimed at more effective therapeutic intervention in ischaemic disorders of the brain, the animal model used and the principles of the causal therapy should be better outlined. The effects of the non-peptide AVPR (V2) antagonist 5-dimethylamino-1-[4-(2-methylbenzoylamino) benzoyl]-2,3,4,5-tetrahydro-1H-benzazepine hydrochloride (OPC-31260) on the cerebral oedema induced by general cerebral hypoxia were studied in rats. The general cerebral hypoxia was produced by bilateral common carotid ligation in Sprague-Dawley rats of the CFY strain. By 6h after the ligation, half of the rats had died, but the survival rate was significantly higher following OPC-31260 administration. Electron microscopic examinations revealed typical ischaemic changes after the carotid ligation, and OPC-31260 treatment did not significantly reduce the hypoxic signs in the brain cortex; only a certain decrease in the pericapillary oedema was observed. The carotid ligation increased the brain contents of water and Na(+) and enhanced the plasma AVP level. The increased brain water and Na(+) accumulation was prevented by OPC-31260 administration, but the plasma AVP level was further enhanced by OPC-31260. These results demonstrate the important role of AVP in the development of the disturbances in brain water and electrolyte balance in response to general cerebral hypoxia. The carotid ligation-induced cerebral oedema was significantly reduced following oral OPC-31260 administration. The protective mechanism exerted by OPC-31260 stems from its influence on the renal AVPR (V2). These observations might suggest an effective approach to the treatment of global hypoxia-induced cerebral oedema in humans.
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos , Benzazepinas/uso terapêutico , Edema Encefálico/prevenção & controle , Hipóxia Encefálica/prevenção & controle , Animais , Arginina Vasopressina/sangue , Pressão Sanguínea/efeitos dos fármacos , Água Corporal/efeitos dos fármacos , Água Corporal/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/ultraestrutura , Artérias Carótidas/fisiologia , Modelos Animais de Doenças , Eletrólitos/metabolismo , Humanos , Hipóxia Encefálica/etiologia , Hipóxia Encefálica/mortalidade , Hipóxia Encefálica/patologia , Ratos , Ratos Sprague-Dawley , Receptores de Vasopressinas/efeitos dos fármacos , Taxa de SobrevidaRESUMO
To analyze the role of phosphatidylglycerol (PG) in photosynthetic membranes of cyanobacteria we used two mutants of Synechocystis sp. PCC6803: the PAL mutant which has no phycobilisomes and shows a high PSII/PSI ratio, and a mutant derived from it by inactivating its cdsA gene encoding cytidine 5'-diphosphate diacylglycerol synthase, a key enzyme in PG synthesis. In a medium supplemented with PG the PAL/DeltacdsA mutant cells grew photoautotrophically. Depletion of PG in the medium resulted (a) in an arrest of cell growth and division, (b) in a slowdown of electron transfer from the acceptor Q(A) to Q(B) in PSII and (c) in a modification of chlorophyll fluorescence curve. The depletion of PG affected neither the redox levels of Q(A) nor the S(2) state of the oxygen-evolving manganese complex, as indicated by thermoluminescence studies. Two-dimensional PAGE showed that in the absence of PG (a) the PSII dimer was decomposed into monomers, and (b) the CP43 protein was detached from a major part of the PSII core complex. [(35)S]-methionine labeling confirmed that PG depletion did not block de novo synthesis of the PSII proteins. We conclude that PG is required for the binding of CP43 within the PSII core complex.
Assuntos
Mutação/genética , Nucleotidiltransferases/metabolismo , Fosfatidilgliceróis/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Ficobilissomas/metabolismo , Synechocystis/enzimologia , Tamanho Celular , Clorofila/metabolismo , Clorofila A , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Ácidos Graxos/análise , Medições Luminescentes , Modelos Biológicos , Oxigênio/metabolismo , Fotossíntese , Pigmentos Biológicos/metabolismo , Subunidades Proteicas/biossíntese , Espectrometria de Fluorescência , Synechocystis/citologia , Synechocystis/crescimento & desenvolvimento , Synechocystis/ultraestruturaRESUMO
Gonadal steroids induce synaptic plasticity in several areas of the adult nervous system. In the arcuate nucleus of adult female rats, 17beta-estradiol triggers synaptic remodeling, resulting in a decrease in the number of inhibitory synaptic inputs, an increase in the number of excitatory synapses, and an enhancement of the frequency of neuronal firing. In the present paper, we studied the specificity of hormonal effects by determining the changes in synaptic connectivity of tyrosine hydroxylase (TH) immunoreactive (IR) neurons in the arcuate nucleus. We combined pre-embedding TH and post-embedding gamma-aminobutyric acid (GABA) immunostaining, and performed unbiased stereological measurements in gonadectomized and 17beta-estradiol-treated rats. We conclude that the synaptic connectivity of the TH-IR neurons is different from the other, nonlabeled population, and the response to estradiol is not uniform. TH-IR (dopaminergic) arcuate neurons of both male and female rats have more GABAergic (inhibitory) axosomatic inputs than the nondopaminergic population. Our study shows that the effect of 17beta-estradiol is sex and cell specific in the sense that not all arcuate neurons are affected by the structural synaptic remodeling. In ovariectomized females hormone treatment decreased the numerical density of GABAergic axosomatic synapses on TH-IR, but not on nondopaminergic, neurons, whereas in orchidectomized males, 17beta-estradiol treatment increased inhibitory synapses onto nondopaminergic neurons but did not affect the number of inhibitory terminals onto TH-IR neurons. The hormone-induced plastic changes in synaptic connectivity of TH-IR neurons may serve as the morphological basis for the cyclical regulation of the anterior pituitary.
Assuntos
Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Estradiol/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Castração , Contagem de Células , Feminino , Imuno-Histoquímica , Masculino , Neurônios/citologia , Neurônios/metabolismo , Ratos , Caracteres Sexuais , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/imunologia , Ácido gama-Aminobutírico/metabolismoRESUMO
Proper gonadal function requires coordinated (feedback) interactions between the gonads, adenohypophysis, and brain: the gonads elaborate sex steroids (progestins, androgens, and estrogens) and proteins (inhibin-activin family) during gamete development. In both sexes, the brain-pituitary gonadotrophin-regulating interaction is coordinated by estradiol through its opposing actions on pituitary gonadotrophs (sensitization of the response to gonadotrophin-releasing hormone [GnRH]) versus hypothalamic neurons (inhibition of GnRH secretion). This dynamic tension between the gonadotrophs and the GnRH cells in the brain regulates the circulating gonadotrophins and is termed reciprocal/negative feedback. In females, reciprocal/negative feedback dominates approximately 90% of the ovarian cycle. In a spectacular exception, the dynamic tension is broken during the surge of circulating estrogen that marks follicle and oocyte(s) maturation. The cause is an estradiol-induced disinhibition of the GnRH neurons that releases GnRH secretion to the highly sensitized pituitary gonadotrophs that in turn release the gonadotrophin surge (the estrogen-induced gonadotrophin surge [EIGS], also known as positive feedback). Studies during the past 4 decades have shown this disinhibition to result from estrogen-induced synaptic plasticity (EISP), including a reversible approximately 50% loss in arcuate nucleus synapses. The disinhibited GnRH secretion occurs during maximal gonadotroph sensitization and results in the EIGS. Specific immunoneutralization of estradiol blocks the EISP and EIGS. The EISP is accompanied by increases in insulinlike growth factor 1, polysialylated neural cell adhesion molecule, and ezrin, 3 proteins that the authors believe are the links between estrogen-induced astroglial extension and the EISP that releases GnRH secretion at the moment of maximal sensitization of the pituitary gonadotrophs. The result is the paradoxical surge of gonadotrophins at the peak of ovarian estrogen secretion and the triggering of ovulation. This enhanced understanding of the mechanics of gonadotrophin control clarifies elements of the involved feedback loops and opens the way to a better understanding of the neurobiology of reproduction.
Assuntos
Estrogênios/fisiologia , Gonadotropinas/fisiologia , Hipotálamo/fisiologia , Ciclo Menstrual/fisiologia , Plasticidade Neuronal/fisiologia , Hipófise/metabolismo , Animais , Estrogênios/metabolismo , Feminino , Hormônio Liberador de Gonadotropina/metabolismo , Hormônio Liberador de Gonadotropina/fisiologia , Gônadas/fisiologia , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/fisiologia , Neurônios/fisiologia , Ratos , Sinapses/fisiologiaRESUMO
Understanding the reaction of living cells in response to different extracellular stimuli, such as hyperosmotic stress, is of primordial importance. Mannitol, a cell-impermeable non-toxic alcohol, has been used successfully for reversible opening of the blood-brain barrier in hyperosmotic concentrations. In this study we analyzed the effect of hyperosmotic mannitol on the shape and surface structure of living cerebral endothelial cells by atomic force microscope imaging technique. Addition of clinically relevant concentrations of mannitol to the culture medium of the confluent cells induced a decrease of about 40% in the observed height of the cells. This change was consistent both at the nuclear and peripheral region of the cells. After mannitol treatment even a close examination of the contact surface between the cells did not reveal gap between them. We could observe the appearance of surface protrusions of about 100 nm. By force measurements the elasticity of the cells were estimated. While the Young's modulus of the control cells appeared to be 8.04 +/- 0.12 kPa, for the mannitol-treated cells it decreased to an estimated value of 0.93 +/- 0.04 kPa which points to large structural changes inside the cell.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Manitol/administração & dosagem , Fluidez de Membrana/efeitos dos fármacos , Microscopia de Força Atômica/métodos , Animais , Encéfalo/citologia , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Elasticidade , Células Endoteliais/citologia , Fluidez de Membrana/fisiologia , Pressão Osmótica/efeitos dos fármacos , Ratos , Estresse MecânicoRESUMO
Dehydroepiandrosterone and its sulfate (DHEAS) are sex hormone precursors that exert marked neurotrophic and/or neuroprotective activity in the central nervous system. The present study evaluated the effects of DHEAS and 17beta-estradiol (E2) in a focal cortical cold lesion model, in which DHEAS (50 mg/kg, sc) and E2 (35 mg/kg, sc) were administered either as pretreatment (two subsequent injections 1 d and 1 h before lesion induction) or posttreatment (immediately after lesion induction). The focal cortical cold lesion was induced in the primary motor cortex by means of a cooled copper cylinder placed directly onto the cortical surface. One hour later, the animals were killed, the brains cut into 0.4-mm-thick slices, and the sections stained with 1% triphenyltetrazolium chloride. The volume of the hemispheric lesion was calculated for each animal. The results demonstrated that the lesion area was significantly attenuated in both the DHEAS- and E2- pre- and posttreated groups and that in the presence of letrozole, a nonsteroidal aromatase inhibitor, no neuroprotection was observed, suggesting that the beneficial effect of DHEAS on the cold injury might depend on the conversion of DHEAS to E2 within the brain. It is concluded that even a single posttraumatic administration of DHEAS may be of substantial therapeutic benefit in the treatment of focal brain injury with vasogenic edema.
Assuntos
Lesões Encefálicas/prevenção & controle , Temperatura Baixa , Desidroepiandrosterona/administração & dosagem , Estradiol/fisiologia , Córtex Motor/lesões , Fármacos Neuroprotetores/administração & dosagem , Animais , Lesões Encefálicas/etiologia , Desidroepiandrosterona/metabolismo , Esquema de Medicação , Estradiol/administração & dosagem , Masculino , Modelos Animais , Córtex Motor/efeitos dos fármacos , Córtex Motor/metabolismo , Fármacos Neuroprotetores/metabolismo , Ratos , Ratos WistarRESUMO
Young adult male Wistar rats (24/group) were treated for 5 weeks with methyl mercury(II)chloride (corresponding to 0.5 and 2.0mgHg°/kg b.w., control: distilled water) by gavage, followed by a 19 weeks post-treatment period. Spontaneous motility, psychomotor performance and sensorimotor gating was repeatedly tested, electrophysiological recordings done, in the rats throughout the whole experiment. Decreased horizontal open field activity, reduced number of "noise positive" startle responses, as well as increase of startle response onset latency and peak time, and decrease of peak amplitude, was seen in the treated animals. Most changes disappeared in the post-treatment period. In the spontaneous cortical and hippocampal activity, altered distribution of the frequency bands was seen after 5 weeks of treatment but not at the end of the post-treatment period. Hippocampal population spikes in the treated animals were depressed and showed less potentiation, which effect was still present 19 weeks after finishing the treatment. The duration of the sensory cortical evoked potentials was shorter than in the controls. In the treated rats, tyrosine hydroxylase-immunoreactive boutons in the substantia nigra pars reticulata were shrunk; blood and brain Hg levels were significantly higher and decreased only slowly. Considering the continuous presence of low levels of mercurials in the human environment, effects of this kind may be supposed as the background of some human neurobehavioral abnormalities.
RESUMO
Effects of dehydroepiandrosterone (DHEA) on glial reactions of the peripherally denervated olfactory bulb were studied in adult male rats. Denervation was achieved by destroying the olfactory mucosa with ZnSO(4) (0.17 M) irrigation of the nasal cavities. In one series of experiments, chronic DHEA treatment was applied (daily injections for 7 days, i.p., 10 mg/kg b.w. and 25 mg/kg b.w.); in the other series of experiments, animals received a single injection of DHEA (i.p., 10 mg/kg b.w., 25 mg/kg b.w. and 50 mg/kg b.w.) 2 h following ZnSO(4) treatment. To determine whether DHEA conversion to estradiol was involved in the mechanism of DHEA action on glia, a third series of experiments was carried out in which the aromatase inhibitor fadrozole (4.16 mg/ml) was administered using subcutaneously implanted osmotic minipumps. Rats were killed on day 7 after chemical denervation, and the reaction of glial cells was monitored within the olfactory bulb, using GFAP and vimentin immunohistochemistry. Qualitative changes in GFAP expression were analyzed by Western blot. Chronic DHEA treatment with both doses (10 mg/kg b.w. and 25 mg/kg b.w.) and acute DHEA treatment with the highest dose applied (50 mg/kg b.w.), inhibited the increase in GFAP expression induced by the denervation of the olfactory bulb. Furthermore, GFAP and vimentin immunostaining in the glomerular layer of the olfactory bulb were diminished in the denervated and DHEA treated groups. However, when DHEA treatment was combined with fadrozole administration, such a decrease in GFAP expression could not be detected in the chemically denervated olfactory bulb. These findings indicate that DHEA, depending on the dose applied and the mode of administration, attenuates glial reaction to denervation and may regulate glial plasticity in the olfactory glomeruli. These effects are likely to be mediated at least in part by the conversion of DHEA to estradiol.