RESUMO
In recent years, several studies aimed to investigate the metabolic effects of non-functioning or absent cyclophilin D (CypD), a crucial regulatory component of mitochondrial permeability transition pores. It has been reported that the lack of CypD affects glucose and lipid metabolism. However, the findings are controversial regarding the metabolic pathways involved, and most reports describe the effect of a high-fat diet on metabolism. We performed a lipidomic analysis of plasma and liver samples of CypD-/- and wild-type (WT) mice to reveal the lipid-specific alterations resulting from the absence of CypD. In the CypD-/- mice compared to the WT animals, we found a significant change in 52% and 47% of the measured 225 and 201 lipid species in liver and plasma samples, respectively. The higher total lipid content detected in these tissues was not accompanied by abdominal fat accumulation assessed by nuclear magnetic resonance imaging. We also documented characteristic changes in the lipid composition of the liver and plasma as a result of CypD ablation with the relative increase in polyunsaturated membrane lipid species. In addition, we did not observe remarkable differences in the lipid distribution of hepatocytes using histochemistry, but we found characteristic changes in the hepatocyte ultrastructure in CypD-/- animals using electron microscopy. Our results highlight the possible long-term effects of CypD inhibition as a novel therapeutic consideration for various diseases.
Assuntos
Lipidômica , Proteínas de Transporte da Membrana Mitocondrial , Animais , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Ciclofilinas/metabolismo , Glucose , Fígado/metabolismo , Lipídeos de Membrana , Camundongos , Camundongos Knockout , Proteínas de Transporte da Membrana Mitocondrial/metabolismoRESUMO
Sepsis caused by LPS is characterized by an intense systemic inflammatory response affecting the lungs, causing acute lung injury (ALI). Dysfunction of mitochondria and the role of reactive oxygen (ROS) and nitrogen species produced by mitochondria have already been proposed in the pathogenesis of sepsis; however, the exact molecular mechanism is poorly understood. Oxidative stress induces cyclophilin D (CypD)-dependent mitochondrial permeability transition (mPT), leading to organ failure in sepsis. In previous studies mPT was inhibited by cyclosporine A which, beside CypD, inhibits cyclophilin A, B, C and calcineurin, regulating cell death and inflammatory pathways. The immunomodulatory side effects of cyclosporine A make it unfavorable in inflammatory model systems. To avoid these uncertainties in the molecular mechanism, we studied endotoxemia-induced ALI in CypD(-/-) mice providing unambiguous data for the pathological role of CypD-dependent mPT in ALI. Our key finding is that the loss of this essential protein improves survival rate and it can intensely ameliorate endotoxin-induced lung injury through attenuated proinflammatory cytokine release, down-regulation of redox sensitive cellular pathways such as MAPKs, Akt, and NF-κB and reducing the production of ROS. Functional inhibition of NF-κB was confirmed by decreased expression of NF-κB-mediated proinflammatory genes. We demonstrated that impaired mPT due to the lack of CypD reduces the severity of endotoxemia-induced lung injury suggesting that CypD specific inhibitors might have a great therapeutic potential in sepsis-induced organ failure. Our data highlight a previously unknown regulatory function of mitochondria during inflammatory response.
RESUMO
By means of whole mount NADPH-diaphorase histochemistry the distribution pattern of primary sensory cells (PSC) and the pathway of their central processes in the ventral nerve cord (VNC) ganglia were investigated in the lumbricid earthworms, Eisenia fetida and Lumbricus terrestris. The distribution pattern of the stained structures seemed to be the same in both species investigated. Strong labelling occurred in sensory fibre branches of segmental nerves and in each of the sensory longitudinal axon bundles of VNC ganglia. Based on their anatomical location some NADPH-d positive central sensory cells were identified from among which the putative tactile receptors were characterized by constant, strong staining.
Assuntos
Sistema Nervoso Central/metabolismo , Gânglios Sensitivos/metabolismo , NADPH Desidrogenase/metabolismo , Oligoquetos/metabolismo , Nervos Periféricos/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Corantes , Gânglios/metabolismo , HistocitoquímicaRESUMO
Little is known about the potential threats of silver nanoparticles (AgNPs) to ecosystem health, with no detailed report existing on the stress and immune responses of soil invertebrates. Here we use earthworm primary cells, cross-referencing to human cell cultures with a particular emphasis on the conserved biological processes, and provide the first in vitro analysis of molecular and cellular toxicity mechanisms in the earthworm Eisenia fetida exposed to AgNPs (83 ± 22 nm). While we observed a clear difference in cytotoxicity of dissolved silver salt on earthworm coelomocytes and human cells (THP-1 cells, differentiated THP-1 cells and peripheral blood mononuclear cells), the coelomocytes and differentiated (macrophage-like) THP-1 cells showed a similar response to AgNPs. Intracellular accumulation of AgNPs in the coelomocytes, predominantly in a phagocytic population, was evident by several methods including transmission electron microscopy. Molecular signatures of oxidative stress and selected biomarker genes probed in a time-resolved manner suggest early regulation of oxidative stress genes and subsequent alteration of immune signaling processes following the onset of AgNP exposure in the coelomocytes and THP-1 cells. Our findings provide mechanistic clues on cellular innate immunity toward AgNPs that is likely to be evolutionarily conserved across the animal kingdom.
Assuntos
Evolução Biológica , Imunidade/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Oligoquetos/efeitos dos fármacos , Oligoquetos/imunologia , Prata/toxicidade , Estresse Fisiológico/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunidade/genética , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Nanopartículas Metálicas/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/genética , Fatores de TempoRESUMO
The cytochemical and functional characteristics of chloragocytes of both 'control' and cold-stressed Eisenia fetida were examined. Flow cytometry revealed the heterogeneity of chloragocytes: the first group was characterized by low, the second one by high acid phosphatase (AcP) content. In 'control' animals the former, in cold-stressed ones the latter type were the dominant form. The elevated AcP-activity correlated with the accumulation of autophagic vacuoles (AVs) in chloragocytes. Both AVs and all small chloragosomes showed high AcP activity, while most of the large chloragosomes did not display any. Most 'control' granules (0.75-1.25 µm) contained high amounts of Ca and P, with less and variable quantities of S, Cl, K, Fe and Zn. Small chloragosomes with low Ca and P concentrations were seldom found. In cold-stressed animals the number of small granules (0.25-0.75 µm) increased up to 40% of total population. Their Ca and P contents were significantly lower; S and Fe concentrations were higher than those of large chloragosomes (1.0-1.5 µm). Our results prove that the formation and elemental composition of chloragosomes can be influenced by environmental stressors and suggest that the mature chloragosomes are tertiary lysosomes and their formation is coupled to autophagocytosis.
Assuntos
Vasos Sanguíneos/citologia , Cálcio/metabolismo , Grânulos Citoplasmáticos/metabolismo , Oligoquetos/metabolismo , Fósforo/metabolismo , Estresse Fisiológico , Fosfatase Ácida/metabolismo , Animais , Temperatura Baixa , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/ultraestrutura , Sistema Digestório/irrigação sanguínea , Oligoquetos/citologia , Oligoquetos/fisiologia , Forma das OrganelasRESUMO
The fungal mycotoxin patulin is produced by several molds, especially by Aspergillus and Penicillium. The aim of this study was to clarify whether patulin causes alterations in plasma membrane permeability of Schizosaccharomyces pombe lead to cellular shrinkage charateristic to apoptosis or increases cell size indicating necrosis in cells. Transmission and scanning electronmicroscopy revealed that lower concentrations of patulin induced cellular shrinkage and blebbing, higher concentration caused expansion without cellular disruption. Large-scale morphological changes of individual cells were followed by time lapse video microscopy. Patulin caused the elongation and stickiness of cells or rounded up their shapes. To visualize chromatin structures of S. pombe nuclei upon patulin treatment, protoplasts were isolated from S. pombe and subjected to fluorescent microscopy. Chromatin changes in the presence of 50 µM patulin concentration were characterized by elongated nuclei containing sticky fibrillary chromatin and enlarged round shaped nuclei trapped at the fibrillary stage of chromatin condensation. Short (60 min) incubation of S. pombe cells in the presence of high (500 µM) patulin concentration generated patches of condensed chromatin bodies inside the nucleus and caused nuclear expansion, with the rest of chromatin remaining in fibrillary form. Longer (90 min, 500 µM) incubation resulted in fewer highly condensed chromatin patches and in nuclear fragmentation. Although, high patulin concentration increased the size of S. pombe size, it did not lead to necrotic explosion of cells, neither did the fragmented nuclei resemble apoptotic bodies that would have indicated programmed cell death. All these morphological changes and the high rate of cell survival point to rapid adaptation and mixed type of fungistatic effects.
Assuntos
Cromatina/efeitos dos fármacos , Micotoxinas/farmacologia , Patulina/farmacologia , Schizosaccharomyces/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Cromatina/genética , Necrose/genética , Necrose/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
This study investigated the effect of CAPE on planktonic growth, biofilm-forming abilities, mature biofilms, and cell death of C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis strains. Our results showed a strain- and dose-dependent effect of CAPE on Candida, and the MIC values were between 12.5 and 100 µg/mL. Similarly, the MBIC values of CAPE ranging between 50 and 100 µg/mL highlighted the inhibition of the biofilm-forming abilities in a dose-dependent manner, as well. However, CAPE showed a weak to moderate biofilm eradication ability (19-49%) on different Candida strains mature biofilms. Both caspase-dependent and caspase-independent apoptosis after CAPE treatment were observed in certain tested Candida strains. Our study has displayed typical apoptotic hallmarks of CAPE-induced chromatin margination, nuclear blebs, nuclear condensation, plasma membrane detachment, enlarged lysosomes, cytoplasm fragmentation, cell wall distortion, whole-cell shrinkage, and necrosis. In conclusion, CAPE has a concentration and strain-dependent inhibitory activity on viability, biofilm formation ability, and cell death response in the different Candida species.
RESUMO
Terahertz (THz) irradiation of excised Eisenia andrei earthworms is shown to cause overriding of the genetically determined, endogenously mediated segment renewing capacity of the model animal. Single-cycle THz pulses of 5 µJ energy, 0.30 THz mean frequency, 293 kV/cm peak electric field, and 1 kHz repetition rate stimulated the cell proliferation (indicated by the high number of mitotic cells) and both histogenesis and organogenesis, producing a significantly higher number of regenerated segments. The most conspicuous alteration in THz-treated animals was the more intense development of the new central nervous system and blood vessels. These results clearly demonstrate that THz pulses are capable to efficiently trigger biological processes and suggest potential applications in medicine.
RESUMO
Pituitary adenylate cyclase activating polypeptide (PACAP)-like molecules have been shown to be present in cocoon albumin and in Eisenia fetida embryos at an early developmental stage (E1) by immunocytochemistry and radioimmunoassay. Here, we focus on detecting the stage at which PAC1 receptor (PAC1R)-like immunoreactivity first appears in germinal layers and structures, e.g., various parts of the central nervous system (CNS), in developing earthworm embryos. PAC1R-like immunoreactivity was revealed by Western blot and Far Western blot as early as the E2 developmental stage, occurring in the ectoderm and later in specific neurons of the developing CNS. Labeled CNS neurons were first seen in the supraesophageal ganglion (brain) and subsequently in the subesophageal and ventral nerve cord ganglia. Ultrastructurally, PAC1Rs were located mainly on plasma membranes and intracellular membranes, especially on cisternae of the endoplasmic reticulum. Therefore, PACAP-like compounds probably influence the differentiation of germinal layers (at least the ectoderm) and of some neurons and might act as signaling molecules during earthworm embryonic development.
Assuntos
Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Oligoquetos/embriologia , Oligoquetos/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Gânglios/citologia , Gânglios/embriologia , Gânglios/metabolismo , Gânglios/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Hipófise/metabolismo , Extratos de Tecidos/metabolismoRESUMO
The regeneration of the ventral nerve cord ganglion and peripheral tissues was investigated by radioimmunoassay and immunohistochemistry in the model animal, Eisenia fetida (Annelida, Oligochaeta). It is now well-established that pituitary adenylate cyclase-activating polypeptide (PACAP) is a neurotrophic factor, playing important roles in the development of the nervous system in vertebrate animals. Based on the apparent evolutionary conservation of PACAP and on the several common mechanisms of vertebrate and invertebrate nervous regeneration, the question was raised whether PACAP has any role in the regeneration of the earthworm nervous system. As a first step, we studied the distribution, concentration, and time-course of PACAP-like immunoreactivity during caudal regeneration of both lost segments and the ventral nerve cord ganglia in E. fetida. A strong upregulation of PACAP-like immunoreactivity was observed in most tissues following injury as determined by radioimmunoassay and immunohistochemistry. Significant increases in the concentration of PACAP-like compounds were found in the body wall, alimentary canal, and in coelomocytes. The most characteristic morphological feature was the accumulation of immunolabeled neoblasts in the injured tissues, especially in the ventral nerve cord ganglion that initiates and mediates regeneration processes. Our present results show that PACAP/PACAP-like peptides accumulate in the regenerating tissues of the earthworm, suggesting trophic functions of these compounds in earthworm tissues similarly to vertebrate species.
Assuntos
Oligoquetos/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Regeneração/fisiologia , Animais , Imuno-Histoquímica , Oligoquetos/anatomia & histologia , RadioimunoensaioRESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) is expressed at very early stages in the vertebrate nervous system, and its functions in the embryonic development have been shown by various studies. PACAP is an extremely conserved molecule in phylogeny; however, little is known about its presence and functions in invertebrates. Our previous studies have shown the occurrence of PACAP-like immunoreactivity in the invertebrate nervous system. The aim of this study was to investigate the presence and localization of PACAP-like compounds during the embryonic development of earthworms from cocoon deposition to hatching using immunological methods (radioimmunoassay, dot blot, immunohistochemistry). PACAP-like immunoreactive compounds were detected at very early stages of the embryonic development of the earthworm Eisenia fetida. No significant changes were observed during the early stages in the developing embryo, but a marked increase occurred before hatching. In contrast, during the embryonic development, the level of PACAP-like compounds gradually decreased in cocoon fluids. Immunohistochemistry revealed the presence of PACAP-like immunoreactive cell bodies and processes in the developing body wall, prostomium, pharyngeal wall, and central nervous system. Cells located in the body wall correspond to putative progenitor cells of primary sensory cells. In the present study, we also showed that the clitellum (reproductive organ) of sexually mature worms contained significantly higher levels of PACAP-like immunoreactivity than other regions of the same animals or the clitellar region of a non-reproducing animal. In summary, these observations provide a morphological basis and suggest a role of PACAP(-like peptides) in the reproductive and developmental functions of invertebrates.
Assuntos
Oligoquetos/embriologia , Oligoquetos/crescimento & desenvolvimento , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Animais , Imuno-Histoquímica , Oligoquetos/anatomia & histologia , Oligoquetos/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genéticaRESUMO
FXPRLamides are insect neuropeptides that mediate such diverse functions as pheromone biosynthesis, visceral muscle contraction, and induction of diapause. Although multiple forms occur in every insect studied so far, little is known about a possible functional differentiation and/or differences in the cellular expression pattern of these messenger molecules. In this study, we performed a mass spectrometric survey of all FXPRLamide-expressing neurosecretory neurons in the CNS of Periplaneta americana. That species combines a very well characterized peptidergic system with relatively easy accessible neurosecretory cells suitable for dissection. In addition to the extensive mass spectrometric analyses of single cells, the projection of the FXPRLamide-expressing neurons was studied with three antisera specifically recognizing different FXPRLamides. The following conclusions can be drawn from this first comprehensive peptidomic approach on insect neurons. 1) A high degree of differentiation in the expression of FXPRLamides exists; not fewer then four cell types containing different sets of FXPRLamides were observed. 2) A low level of colocalization with other neuropeptides was found in these neurons. 3) A comparison with FXPRLamide-expressing neurons of other insects shows a high degree of conservation in the localization and projection of these neurons, which is not corroborated by a similar conservation of the corresponding peptide sequences. 4) Although the methods for cell identification, dissection, and sample preparation for mass spectrometry were kept as simple as possible, it was unambiguously shown that this approach is generally suitable for routine analysis of single identified neurons of insects.
Assuntos
Sistema Nervoso Central/metabolismo , Neurônios/metabolismo , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Sistemas Neurossecretores/metabolismo , Periplaneta/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Sistema Nervoso Central/ultraestrutura , Gânglios dos Invertebrados/metabolismo , Gânglios dos Invertebrados/ultraestrutura , Imuno-Histoquímica , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Neurônios/ultraestrutura , Neuropeptídeos/análise , Sistemas Neurossecretores/ultraestrutura , Periplaneta/ultraestrutura , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Proteômica/métodos , Especificidade da EspécieRESUMO
By means of a whole mount immunocytochemical approach, the distribution patterns of pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 were identified in the ventral nerve cord (VNC) ganglia of the earthworms Eisenia fetida and Lumbricus terrestris. Each PACAP form appears to occur in a distinct neuron population. Positions of these populations, as well as numbers and sizes of the constituting neurons do not essentially differ between the two species. The data suggest that in Lumbricid Oligochaetes, PACAP-27 and PACAP-38 neuron populations may mediate distinct physiological processes.
Assuntos
Gânglios dos Invertebrados/imunologia , Gânglios dos Invertebrados/metabolismo , Oligoquetos/imunologia , Oligoquetos/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/imunologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Animais , Gânglios dos Invertebrados/anatomia & histologia , Oligoquetos/anatomia & histologiaRESUMO
Flow cytometry is a common approach to study invertebrate immune cells including earthworm coelomocytes. However, the link between light-scatter- and microscopy-based phenotyping remains obscured. Here we show, by means of light scatter-based cell sorting, both subpopulations (amoebocytes and eleocytes) can be physically isolated with good sort efficiency and purity confirmed by downstream morphological and cytochemical applications. Immunocytochemical analysis using anti-EFCC monoclonal antibodies combined with phalloidin staining has revealed antigenically distinct, sorted subsets. Screening of lectin binding capacity indicated wheat germ agglutinin (WGA) as the strongest reactor to amoebocytes. This is further evidenced by WGA inhibition assays that suggest high abundance of N-acetyl-d-glucosamine in amoebocytes. Post-sort phagocytosis assays confirmed the functional differences between amoebocytes and eleocytes, with the former being in favor of bacterial engulfment. This study has proved successful in linking flow cytometry and microscopy analysis and provides further experimental evidence of phenotypic and functional heterogeneity in earthworm coelomocyte subsets.
Assuntos
Anticorpos Monoclonais/metabolismo , Citometria de Fluxo/métodos , Microscopia/métodos , Oligoquetos/imunologia , Fagócitos/imunologia , Animais , Separação Celular , Difusão Dinâmica da Luz , Glucosamina/metabolismo , Imuno-Histoquímica , Imunofenotipagem , Lectinas/metabolismoRESUMO
A unique costorage of neuropeptides was recently found in the abdominal perisympathetic organs (PSOs) of the American cockroach, Periplaneta americana. Having specific antisera directed against all peptides belonging to this neurosecretory system, we examined the sorting of PSO-peptides in the soma of the median neurosecretory cells of abdominal ganglia by using immunoelectron microscopic double stainings. The data indicate that all six abundant neuropeptides of this neurohormonal system, which includes three capa-gene related products, are primarily incorporated into separate vesicles. These vesicles fuse with each other in the cytoplasm and become translucent on their way to the axon hillock. By means of light microscopy and MALDI-TOF mass spectrometry, an identical population of neuropeptides was found in interneurons of the brain. As revealed by subsequent immunoelectron microscopic analysis, the peptides of these cells are separately packed into dense core vesicles but do not fuse with each other. Thus, hitherto unknown cell-type-specific sorting mechanisms occur in neurosecretory cells and interneurons, respectively.
Assuntos
Proteínas de Insetos/metabolismo , Interneurônios/metabolismo , Neuropeptídeos/metabolismo , Sistemas Neurossecretores/metabolismo , Periplaneta/metabolismo , Abdome/inervação , Animais , Feminino , Imunofluorescência , Gânglios dos Invertebrados/metabolismo , Gânglios dos Invertebrados/ultraestrutura , Gânglios Parassimpáticos/metabolismo , Gânglios Parassimpáticos/ultraestrutura , Interneurônios/ultraestrutura , Masculino , Sistemas Neurossecretores/ultraestrutura , Periplaneta/ultraestrutura , Transporte Proteico/fisiologia , Vesículas Secretórias/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Earthworm decerebration causes temporary inhibition of reproduction which is mediated by certain brain-derived neurohormones; thus, cocoon production is an apposite supravital marker of neurosecretory center functional recovery during brain regeneration. The core aim of the present study was to investigate aspects of the interactions of nervous and immune systems during brain regeneration in adult Dendrobaena veneta (Annelida; Oligochaeta). Surgical brain extirpation was combined, either with (i) maintenance of immune-competent coelomic cells (coelomocytes) achieved by surgery on prilocaine-anesthetized worms or (ii) prior extrusion of fluid-suspended coelomocytes by electrostimulation. Both brain renewal and cocoon output recovery were significantly faster in earthworms with relatively undisturbed coelomocyte counts compared with individuals where coelomocyte counts had been experimentally depleted. These observations provide empirical evidence that coelomocytes and/or coelomocyte-derived factors (e.g. riboflavin) participate in brain regeneration and, by implication, that there is close functional synergy between earthworm neural and immune systems.
Assuntos
Encéfalo/fisiologia , Regeneração Nervosa/imunologia , Oligoquetos/fisiologia , Animais , Encéfalo/anatomia & histologia , Oligoquetos/anatomia & histologia , ReproduçãoRESUMO
Earthworm's innate immunity is maintained by cellular and humoral components. Our objective was to characterize the cytotoxicity leading to target cell death caused by earthworm coelomocytes. Coelomocyte lysates induced strong cytotoxicity in tumor cell lines. Transmission electron microscopy revealed cell membrane and intracellular damage in cells treated with coelomocyte lysates. Using TUNEL-assay, within 5 min of incubation we detected DNA fragmentation. Moreover, we found phosphatidylserine translocation in target cell-membranes. Furthermore, we detected dose-dependent Ca(2+) influx and decrease of mitochondrial membrane potential in coelomocyte lysate-treated cells. Interestingly, caspase 3/8 activation was undetectable in exposed tumor cells. One such cytotoxic molecule, lysenin identified in earthworms binds to sphingomyelin and causes target cell lysis in vertebrates. Pretreatment with our anti-lysenin monoclonal antibody rescued the majority but not all target cells from coelomocyte induced death. These data suggest that, not only lysenin but also other factors participate in the caspase-independent apoptosis induced by coelomocytes.
Assuntos
Apoptose/efeitos dos fármacos , Extratos Celulares/farmacologia , Oligoquetos/citologia , Animais , Anexina A5/metabolismo , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Caspases/metabolismo , Adesão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Dano ao DNA , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Humanos , Marcação In Situ das Extremidades Cortadas , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Células Jurkat , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Fosfatidilserinas/metabolismo , Toxinas Biológicas/farmacologiaRESUMO
Although the number of identified avian-borne picornaviruses (family Picornaviridae) is continuously increasing there remains several species-rich avian host groups, such as the order Falconiformes (with 290 bird species) from which picornaviruses have not been identified. This study reports the first complete genome of a novel, highly divergent picornavirus, named as Falcovirus A1 (KP230449), from the carnivorous bird, the common kestrel (Falco tinnunculus, order Falconiformes). Falcovirus A1 has the longest 3D(RdRp) genome region and distant phylogenetic relationship to the Hepatitis A virus 1 (Hepatovirus) and Avian encephalomyelitis virus 1 (Tremovirus). It has a type-I (enterovirus-like) IRES in the 5'UTR - identified for the first time among avian-borne picornaviruses suggesting that type-I IRES is not restricted only to enteroviruses and providing further evidence of mosaicism of this region among different picornavirus genera.
Assuntos
Falconiformes/virologia , Genoma Viral , Picornaviridae/genética , Proteínas Virais/genética , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , Picornaviridae/classificação , Picornaviridae/isolamento & purificação , RNA Viral/genética , Análise de Sequência de RNARESUMO
The median neurosecretory cells in abdominal ganglia of insects synthesize a number of putative hormones, which are abundant in the abdominal perisympathetic organs (PSOs). The peptide inventory of these prominent neurohemal release sites is best investigated in the American cockroach and strongly differs from that of head/thoracic neurohemal organs. In this study, we found a complete colocalization of all abundant neuropeptides in this hormonal system, including periviscerokinin-1 and -2, pyrokinin-5, YLSamide, VEAacid, and SKNacid. The first immunoreactive cells were detected on day 18 of embryonic development and already contained the complete set of peptides. By using antisera against the above-mentioned peptides, the development of this neurohormonal system could be studied and is described in detail. Subsequent electron microscopic immunogold stainings in PSO preparations revealed the costorage of PSO peptides in a single vesicle species. Surprisingly, all these peptides were found in axons containing clear vesicles, whereas all axons with dense core vesicles were totally devoid of immunoreactivity. Unlike the axons with dense core vesicles, immunostained axons ramify in the center of the PSO but exhibit only rare morphological signs of exocytosis. Instead, putative release sites of the clear vesicle-containing axons were detected peripherally to the PSOs, namely, on the hyperneural muscle.
Assuntos
Hormônios de Inseto/metabolismo , Neuropeptídeos/metabolismo , Sistemas Neurossecretores/metabolismo , Sistemas Neurossecretores/ultraestrutura , Periplaneta/metabolismo , Animais , Vesículas Citoplasmáticas/metabolismo , Gânglios dos Invertebrados/embriologia , Gânglios dos Invertebrados/metabolismo , Gânglios dos Invertebrados/ultraestrutura , Larva , Microscopia Eletrônica , Microscopia de Fluorescência , Sistemas Neurossecretores/embriologia , Periplaneta/embriologia , Periplaneta/ultraestruturaRESUMO
Peptidergic neurons are not easily integrated into current connectomics concepts, since their peptide messages can be distributed via non-synaptic paracrine signaling or volume transmission. Moreover, the polarity of peptidergic interneurons in terms of in- and out-put sites can be hard to predict and is very little explored. We describe in detail the morphology and the subcellular distribution of fluorescent vesicle/dendrite markers in CCAP neurons (NCCAP), a well defined set of peptidergic neurons in the Drosophila larva. NCCAP can be divided into five morphologically distinct subsets. In contrast to other subsets, serial homologous interneurons in the ventral ganglion show a mixed localization of in- and output markers along ventral neurites that defy a classification as dendritic or axonal compartments. Ultrastructurally, these neurites contain both pre- and postsynaptic sites preferably at varicosities. A significant portion of the synaptic events are due to reciprocal synapses. Peptides are mostly non-synaptically or parasynaptically released, and dense-core vesicles and synaptic vesicle pools are typically well separated. The responsiveness of the NCCAP to ecdysis-triggering hormone may be at least partly dependent on a tonic synaptic inhibition, and is independent of ecdysteroids. Our results reveal a remarkable variety and complexity of local synaptic circuitry within a chemically defined set of peptidergic neurons. Synaptic transmitter signaling as well as peptidergic paracrine signaling and volume transmission from varicosities can be main signaling modes of peptidergic interneurons depending on the subcellular region. The possibility of region-specific variable signaling modes should be taken into account in connectomic studies that aim to dissect the circuitry underlying insect behavior and physiology, in which peptidergic neurons act as important regulators.