RESUMO
Paraphaeoketones A-C (1-3) were isolated from the culture broth of Paraphaeosphaeria sp. KT4192. Their structures and relative configurations were determined using spectroscopic analysis and verified through density functional theory (DFT)-based chemical shift calculations. The absolute configurations of these compounds were determined by comparing the experimental electronic circular dichroism (ECD) spectra with those based on DFT calculations. We also propose a plausible biosynthetic route to 1-3. While our prior studies on the isolation and structural elucidation of paraphaeolactones (e.g., 4) led us to suggest a Favorskii rearrangement for their biosynthesis, the isolation of 2 prompted the proposal of an alternative biosynthesis for 4, featuring a benzilic acid rearrangement of 2. Moreover, an in vitro conversion of 2 into 4 was achieved successfully, suggesting that a biosynthetic pathway for paraphaeolactones involving a benzilic acid rearrangement is more plausible than the previously presumed Favorskii rearrangement pathway. Arguments based on DFT calculations for these pathways are also described.
Assuntos
Ascomicetos , Cetonas , Ascomicetos/química , Ascomicetos/metabolismo , Lactonas/química , Lactonas/metabolismo , Estrutura Molecular , Cetonas/química , Cetonas/metabolismoRESUMO
Paraphaeolactones A1, A2, B1, and B2 (1-4, respectively), known arthropsadiol D (5), massariphenone (6) and its positional isomer 7, and massarilactones E (8) and G (9) were isolated from the culture broth of Paraphaeosphaeria sp. KT4192. Although the structural resemblance between 1 and 2 implies that these comprised a diastereomeric pair at the C-2 stereogenic center, electronic circular dichroism (ECD) spectral analyses revealed that they were pseudo-enantiomers possessing the common (2R)-configuration. Paraphaeolactones B1 and B2 (3 and 4) were the derivatives of 2, which equipped the 3-(1-hydroxy-2-oxopropyl)-4-methylcatechol moiety via an acetal bond at C-10. The relative configurations of their acetal carbons were elucidated by NOE experiments, and those of C-8' were deduced independently by ECD spectral analysis. The present study disclosed that 1-5, 8, and 9 contain a methylcyclohexene substructure with the same absolute configuration. This prompted us to reinvestigate the absolute configurations of known structurally related fungal metabolites, allowing us to conclude that the methylcyclohexene moieties of these natural products have the same absolute configuration despite the variety of configurations of other stereogenic centers. The plausible biosynthetic routes for 1-9 are discussed on the basis of the above conclusion. We propose a Favorskii rearrangement as the key transformation for biosyntheses of 1-4.
Assuntos
Acetais , Ascomicetos , Lactonas , Dicroísmo Circular , Conformação Molecular , Estrutura Molecular , Estereoisomerismo , Lactonas/químicaRESUMO
Zebrafish is a useful model to study vertebrate hematopoiesis, but lack of antibodies to zebrafish proteins has limited purification of hematopoietic cells. Here, we purified neutrophils from larval and adult zebrafish using the lectin Phaseolus vulgaris erythroagglutinin (PHA-E) and DRAQ5, a DNA-staining fluorescent dye. In adult kidney marrow, we purified neutrophil-like PHA-E4low DRAQ5low cells, which neutrophil-type granules. Specifically, at 96-hr post-fertilization, we sorted large-sized cells from larvae using forward scatter and found that they consisted of PHA-Elow DRAQ5low populations. These cells had myeloperoxidase activity, were Sudan Black B-positive and expressed high levels of neutrophil-specific (csf3r and mpx) mRNAs, all neutrophil characteristics. Using this method, we conducted functional analysis suggesting that zyxin (Zyx) plays a role in neutrophil generation in zebrafish larvae. Overall, PHA-E and DRAQ5-based flow cytometry serves as a tool to purify zebrafish neutrophils.
Assuntos
Citometria de Fluxo/métodos , Hematopoese , Neutrófilos/citologia , Cultura Primária de Células/métodos , Animais , Células Cultivadas , Lectinas/metabolismo , Neutrófilos/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
A series of cyclohumulanoids, i.e., tricocerapicanols A-C (1a-1c), tricoprotoilludenes A (2a) and B (3), tricosterpurol (4), and tricoilludins A-C (5-7) were isolated along with known violascensol (2b) and omphadiol (8) from the culture broth of Daedaleopsis tricolor, an inedible but not toxic mushroom. The structures were fully elucidated on the basis of NMR spectroscopic analysis, and the suggested relative structures were confirmed via density functional theory (DFT)-based chemical shift calculations involving a DP4 probability analysis. In the present study, the 1H chemical shifts were more informative than the 13C chemical shifts to distinguish the diastereomers at C-11. The absolute configurations of 1-5 were determined by comparing the experimental and calculated electronic circular dichroism (ECD) spectra. For 6 and 7, the same chirality was assigned according to their biosynthetic similarities with the other compounds. The successful assignment of some Cotton effects was achieved by utilizing DFT calculations using simple model compounds. The plausible biosynthesis of 1-7 was also discussed on the basis of the structural commonality and general cyclohumulanoid biosynthesis. Compounds 2a and 5 were found to simultaneously induce hyphal swelling and branching at 5.0 µg/mL against a test fungus Cochliobolus miyabeanus.
Assuntos
Meios de Cultura/química , Polyporaceae/crescimento & desenvolvimento , Sesquiterpenos , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologiaRESUMO
Hematopoietic stem and progenitor cell (HSPC) transplantation is a curative treatment of hematological disorders that has been utilized for several decades. Although umbilical cord blood (UCB) is a promising source of HSPCs, the low dose of HSPCs in these preparations limits their use, prompting need for ex vivo HSPC expansion. To establish a more efficient method to expand UCB HSPCs, we developed the bioactive peptide named SL-13R and cultured UCB HSPCs (CD34+ cells) with SL-13R in animal component-free medium containing a cytokine cocktail. Following 9 days of culture with SL-13R, the numbers of total cells, CD34+, CD38- cells, and hematopoietic stem cell (HSC)-enriched cells were significantly increased relative to control. Transplantation of cells cultured with SL-13R into immunodeficient NOD/Shi-scid/IL-2Rγ knockout mice confirmed that they possess long-term reconstitution and self-renewal ability. AHNAK, ANXA2, and PLEC all interact with SL-13R. Knockdown of these genes in UCB CD34+ cells resulted in reduced numbers of hematopoietic colonies relative to SL-13R-treated and non-knockdown controls. In summary, we have identified a novel bioactive peptide SL-13R promoting expansion of UCB CD34+ cells with long-term reconstitution and self-renewal ability, suggesting its clinical use in the future.
Assuntos
Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Antígenos CD34/metabolismo , Biomarcadores , Proteínas de Transporte , Técnicas de Cultura de Células , Diferenciação Celular , Autorrenovação Celular , Células Cultivadas , Imunofluorescência , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunofenotipagem , Camundongos , Ligação ProteicaRESUMO
Fetal liver (FL) is the major embryonic hematopoietic organ and a site where circulating hematopoietic stem/progenitor cells (HSPCs) reside. However, HSPC migration/retention mechanisms in FL remain unclear. A chemokine screen revealed that the CCR4 ligands CCL17 and CCL22 are highly expressed in mouse embryonic day (E) 12.5 FL. Flow cytometric analysis confirmed CCR4 expression in FL HSPCs. To identify sources of CCL17 and CCL22, we fractionated FL into various cell types and found that Ccl17 and Ccl22 were predominantly expressed in HPCs/matured HCs. In vitro cell migration analysis confirmed enhanced HSPC migration in the presence of HPCs/matured HCs. Furthermore, exo-utero injection of anti-CCR4 neutralizing antibody into pregnant mice significantly reduced the number of FL HSPCs in embryos. These data demonstrate a paracrine mechanism by which HSPC migration/retention is regulated by CCL17 and CCL22 secreted from HPCs or matured HCs in FL.
Assuntos
Quimiocina CCL17/metabolismo , Quimiocina CCL22/metabolismo , Células-Tronco Hematopoéticas/citologia , Fígado/embriologia , Transdução de Sinais , Animais , Movimento Celular , Células Cultivadas , Células-Tronco Hematopoéticas/metabolismo , Fígado/citologia , Camundongos , Camundongos Endogâmicos C57BL , Comunicação ParácrinaRESUMO
Ants (Hymenoptera, Apocrita, Aculeata, Formicoidea) comprise a well-succeeded group of animals. Like bees and wasps, ants are mostly venomous, having a sting system to deliver a mixture of bioactive organic compounds and peptides. The predatory giant ant Dinoponera quadriceps belongs to the subfamily Ponerinae that includes one of the largest known ant species in the world. In the present study, low molecular weight compounds and peptides were identified by online peptide mass fingerprint. These include neuroactive biogenic amines (histamine, tyramine, and dopamine), monoamine alkaloid (phenethylamine), free amino acids (e.g. glutamic acid and proline), free thymidine, and cytosine. To the best of our knowledge, most of these components are described for the first time in an ant venom. Multifunctional dinoponeratoxin peptide variants (pilosulin- and ponericin-like peptides) were characterized that possess antimicrobial, hemolytic, and histamine-releasing properties. These venom components, particularly peptides, might synergistically contribute to the overall venom activity and toxicity, for immobilizing live prey, and for defending D. quadriceps against aggressors, predators, and potential microbial infection.
Assuntos
Venenos de Formiga/química , Peptídeos/química , Animais , Formigas , Peso MolecularRESUMO
Ants (Hymenoptera, Apocrita, Aculeata, Formicoidea) comprise a well-succeeded group of animals. Like bees and wasps, ants are mostly venomous, having a sting system to deliver a mixture of bioactive organic compounds and peptides. The predatory giant ant Dinoponera quadriceps belongs to the subfamily Ponerinae that include one of the largest known ant species in the world. In the present study, low molecular weight compounds and peptides were identified by on-line peptide mass fingerprint. These include neuroactive biogenic amines (histamine, tyramine, and dopamine), monoamine alkaloid (phenethylamine), free amino acids (e.g., glutamic acid and proline), free thymidine and cytosine. To the best of our knowledge most of these components are described for the first time in an ant venom. Multifunctional dinoponeratoxin peptides variants (pilosulin- and ponericin-like peptides) were characterized that possess antimicrobial, hemolytic, and histamine-releasing properties. These venom components, particularly peptides, might synergistically contribute to the overall venom activity and toxicity, for immobilizing live prey, and defending D. quadriceps against aggressors, predators and potential microbial infection.
RESUMO
The novel trichothecene 12-deoxytrichodermin (3) was isolated from the fungus Trichoderma sp. 1212-03, and included with other known natural trichothecenes in a structure-activity relationship investigation against a human colon cancer cell line (COLO201) and filamentous fungus Cochliobolus miyabeanus. This revealed that the 12-epoxide functionality is critical for the cytotoxicity of simple trichothecenes trichodermin (4) and deoxynivalenol (2), while not critical for the cytotoxicity of roridin J (6) and epiisororidin E (8). In contrast, 12-epoxide is essential for the antifungal activity.
Assuntos
Antifúngicos/química , Ascomicetos/metabolismo , Compostos de Epóxi/química , Tricotecenos/química , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Fungos Mitospóricos/efeitos dos fármacos , Conformação Molecular , Relação Estrutura-Atividade , Tricotecenos/isolamento & purificação , Tricotecenos/farmacologiaRESUMO
Food poisoning caused by natural toxins, especially poisonous plants, is characterized by severe symptoms and a relatively high mortality rate. Therefore, rapid and accurate identification of the causative agent is extremely important. From plant toxin food poisoning data published by the Ministry of Health, Labour and Welfare of Japan from 1989 to 2015, we selected five plants (Veratrum spp., Datura spp., Aconitum spp., Narcissus spp. and Colchicum spp.) that are frequently involved in poisoning outbreaks, and developed a PCR-RFLP assay to discriminate them. Separation of the PCR-RFLP products by electrophoresis resulted in detection of two fragments from poisonous plants and one from edible plants. The PCR-RFLP method is rapid and straightforward and does not require expensive analytical devices. This assay was also confirmed to be applicable to cooked samples.
Assuntos
Doenças Transmitidas por Alimentos , Plantas Tóxicas , Humanos , Japão , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
NAD biosynthesis is of substantial interest because of its important roles in regulating various biological processes. Nicotinamide mononucleotide adenylyltransferase 3 (Nmnat3) is considered a mitochondria-localized NAD synthesis enzyme involved in de novo and salvage pathways. Although the biochemical properties of Nmnat3 are well documented, its physiological function in vivo remains unclear. In this study, we demonstrated that Nmnat3 was localized in the cytoplasm of mature erythrocytes and critically regulated their NAD pool. Deficiency of Nmnat3 in mice caused splenomegaly and hemolytic anemia, which was associated with the findings that Nmnat3-deficient erythrocytes had markedly lower ATP levels and shortened lifespans. However, the NAD level in other tissues were not apparently affected by the deficiency of Nmnat3. LC-MS/MS-based metabolomics revealed that the glycolysis pathway in Nmnat3-deficient erythrocytes was blocked at a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step because of the shortage of the coenzyme NAD. Stable isotope tracer analysis further demonstrated that deficiency of Nmnat3 resulted in glycolysis stall and a shift to the pentose phosphate pathway. Our findings indicate the critical roles of Nmnat3 in maintenance of the NAD pool in mature erythrocytes and the physiological impacts at its absence in mice.
Assuntos
Anemia Hemolítica/metabolismo , Eritrócitos/metabolismo , Glicólise , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Trifosfato de Adenosina/metabolismo , Anemia Hemolítica/genética , Animais , Western Blotting , Cromatografia Líquida , Citoplasma/enzimologia , Eritrócitos/ultraestrutura , Redes e Vias Metabólicas/genética , Metabolômica/métodos , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , NAD/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/deficiência , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Esplenomegalia/genética , Esplenomegalia/metabolismo , Análise de Sobrevida , Espectrometria de Massas em TandemRESUMO
Axonal regeneration might contribute to the restoration of damaged neuronal networks and improvement of memory deficits in a murine Alzheimer's disease (AD) model. A search for axonal regenerative drugs was performed to discover novel therapeutic options for AD. In this study, an aqueous extract of Drynaria fortunei rhizomes reversed Aß25-35-induced axonal atrophy in cultured cortical neurons of mice. Bioassay-guided fractionation of this extract led to the isolation and identification of compounds 1-5. Among them, (2S)-neoeriocitrin (2) and caffeic acid 4-O-glucoside (4) showed significant axonal elongation effects on Aß25-35-induced atrophy.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Fragmentos de Peptídeos/farmacologia , Polypodiaceae/química , Doença de Alzheimer/tratamento farmacológico , Animais , Atrofia/induzido quimicamente , Axônios/efeitos dos fármacos , Medicamentos de Ervas Chinesas/química , Camundongos , Estrutura Molecular , Neurônios/efeitos dos fármacos , Rizoma/químicaRESUMO
Polyamines (PAs) are polycationic biogenic amines ubiquitously present in all life forms and are involved in molecular signaling and interaction, determining cell fate (e.g., cell proliferation, dif-ferentiation, and apoptosis). The intricate balance in the PAs' levels in the tissues will determine whether beneficial or detrimental effects will affect homeostasis. It's crucial to note that endoge-nous polyamines, like spermine and spermidine, play a pivotal role in our understanding of neu-rological disorders as they interact with membrane receptors and ion channels, modulating neuro-transmission. In spiders and wasps, monoamines (histamine, dopamine, serotonin, tryptamine) and polyamines (spermine, spermidine, acyl polyamines) comprise, with peptides and other sub-stances, the low molecular weight fraction of the venom. Acylpolyamines are venom components exclusively from spiders and a species of solitary wasp, which cause inhibition chiefly of iono-tropic glutamate receptors (AMPA, NMDA, and KA iGluRs) and nicotinic acetylcholine receptors (nAChRs). The first venom acylpolyamines ever discovered (argiopines, Joro and Nephila toxins, and philanthotoxins) have provided templates for the design and synthesis of numerous analogs. Thus far, analogs with high potency exert their effect at nanomolar concentrations, with high se-lectivity toward their ionotropic and ligand receptors. These potent and selective acylpolyamine analogs can serve biomedical purposes and pest control management. The structural modification of acylpolyamine with photolabile and fluorescent groups converted these venom toxins into use-ful molecular probes to discriminate iGluRs and nAchRs in cell populations. In various cases, the linear polyamines, like spermine and spermidine, constituting venom acyl polyamine backbones, have served as cargoes to deliver active molecules via a polyamine uptake system on diseased cells for targeted therapy. In this review, we examined examples of biogenic amines that play an essential role in neural homeostasis and cell signaling, contributing to human health and disease outcomes, which can be present in the venom of arachnids and hymenopterans. With an empha-sis on the spider and wasp venom acylpolyamines, we focused on the origin, structure, derivatiza-tion, and biomedical and biotechnological application of these pharmacologically attractive, chemically modular venom components.
Assuntos
Inseticidas , Poliaminas , Venenos de Aranha , Vespas , Animais , Poliaminas/química , Venenos de Aranha/química , Venenos de Aranha/toxicidade , Inseticidas/farmacologia , Inseticidas/química , Inseticidas/toxicidade , Humanos , AranhasRESUMO
Particles containing alpha (α) nuclides were identified from sediment in stagnant water in the Unit 3 reactor building of the Fukushima Daiichi Nuclear Power Station (FDiNPS). We analyzed different concentrations of α-nuclide samples collected at two sampling sites, the torus room and the main steam isolation valve (MSIV) room. The solids in the stagnant water samples were classified, and the uranium (U) and total alpha concentrations of each fraction were measured by dissolution followed by inductively coupled plasma mass spectrometry and α-spectrometry. Most of the α-nuclides in the stagnant water samples from the torus and MSIV rooms were in particle fractions larger than 10 µm. We detected uranium-bearing particles ranging from sub-µm to 10 µm in size by scanning electron microscopy-energy-dispersive X-ray (SEM-EDX) observations. The chemical forms of U particles were determined in U-Zr oxides, oxidized UO2, and U3O8 with micro-Raman spectroscopy. Other short-lived α-nuclides (plutonium [Pu], americium [Am], and curium [Cm]) were detected by alpha track detection, and the particles with α-nuclides was characterized by SEM-EDX analysis. α-nuclide-containing particles with several tens to several 100 µm in size mainly comprised iron (Fe) oxyhydroxides. In addition, we detected adsorbed U onto Fe oxyhydroxide particles in the MSIV room sample, which indicated nuclear fuel dissolution and secondary U accumulation. This study clarifies the major characteristics of U and other α-nuclides in sediment in stagnant water in the FDiNPS Unit 3 reactor building, which significantly contribute to the consideration of removal methods for particles containing α-nuclides in the stagnant water.
RESUMO
Envenoming by the contact of human skin with Lonomia obliqua caterpillars promotes a hemorrhagic syndrome characterized by a consumptive coagulopathy. Losac (Lonomia obliqua Stuart factor activator) is a component of the bristle of L. obliqua that is probably partially responsible for the observed syndrome because it activates factor X and is recognized by an effective antilonomic serum. Here we unveil the proteolytic activity of Losac and demonstrate the feasibility of its recombinant production. On the other hand, Losac has no homology to known proteases, but it can be inhibited by PMSF, a serine protease inhibitor. Instead, it shows closer homology to members of the hemolin family of proteins, a group of cell adhesion molecules. The recombinant protein (rLosac) shortened the coagulation time of normal and deficient plasmas, whereas it was ineffective in factor X-deficient plasma unless reconstituted with this protein. rLosac was able to activate factor X in a dose- and time-dependent manner but not γ-carboxyglutamic acid domainless factor X. Moreover, phospholipids and calcium ions increased rLosac activity. Also, rLosac had no effect on fibrin or fibrinogen, indicating its specificity for blood coagulation activation. Linear double reciprocal plots indicate that rLosac follows a Michaelis-Menten kinetics. Cleavage of factor X by rLosac resulted in fragments that are compatible with those generated by RVV-X (a well known factor X activator). Together, our results validate Losac as the first protein from the hemolin family exhibiting procoagulant activity through selective proteolysis on coagulation factor X.
Assuntos
Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Mariposas/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Coagulação Sanguínea/efeitos dos fármacos , Simulação por Computador , Cisteína Endopeptidases/farmacologia , Fator X/metabolismo , Fator Xa/metabolismo , Imunoglobulinas/farmacologia , Proteínas de Insetos/farmacologia , Dados de Sequência Molecular , Mariposas/metabolismo , Proteínas de Neoplasias/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Crotalphine, a 14 amino acid peptide first isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, induces a peripheral long-lasting and opioid receptor-mediated antinociceptive effect in a rat model of neuropathic pain induced by chronic constriction of the sciatic nerve. In the present study, we further characterized the molecular mechanisms involved in this effect, determining the type of opioid receptor responsible for this effect and the involvement of the nitric oxide-cyclic GMP pathway and of K⺠channels. Crotalphine (0.2 or 5 µg/kg, orally; 0.0006 µg/paw), administered on day 14 after nerve constriction, inhibited mechanical hyperalgesia and low-threshold mechanical allodynia. The effect of the peptide was antagonized by intraplantar administration of naltrindole, an antagonist of δ-opioid receptors, and partially reversed by norbinaltorphimine, an antagonist of κ-opioid receptors. The effect of crotalphine was also blocked by 7-nitroindazole, an inhibitor of the neuronal nitric oxide synthase; by 1H-(1,2,4) oxadiazolo[4,3-a]quinoxaline-1-one, an inhibitor of guanylate cyclase activation; and by glibenclamide, an ATP-sensitive K⺠channel blocker. The results suggest that peripheral δ-opioid and κ-opioid receptors, the nitric oxide-cyclic GMP pathway, and ATP-sensitive K⺠channels are involved in the antinociceptive effect of crotalphine. The present data point to the therapeutic potential of this peptide for the treatment of chronic neuropathic pain.
Assuntos
Analgésicos/farmacologia , Arginina/fisiologia , GMP Cíclico/fisiologia , Canais KATP/fisiologia , Neuralgia/tratamento farmacológico , Óxido Nítrico/fisiologia , Peptídeos/farmacologia , Animais , Masculino , Ratos , Ratos Wistar , Receptores Opioides delta/fisiologia , Receptores Opioides kappa/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Plipastatin A1 and fengycin IX were experimentally proven to be identical compounds, while these had been considered as diastereomers due to the permutation of the enantiomeric pair of Tyr in most papers. The (1)H NMR spectrum changed to become quite similar to that of plipastatin A1, when the sample which provided resembled spectrum of fengycin IX was treated with KOAc followed by LH-20 gel filtration. Our structural investigations disclosed that the structures of these molecules should be settled into that of plipastatin A1 by Umezawa (L-Tyr4 and D-Tyr10).
Assuntos
Ácidos Graxos/química , Oligopeptídeos/química , Peptídeos Cíclicos/química , Bacillus subtilis/química , Bacillus subtilis/isolamento & purificação , Espectroscopia de Ressonância Magnética , Estrutura Molecular , EstereoisomerismoRESUMO
A toxic protein, dubbed molybdophyllysin, was isolated from the tropical toadstool Chlorophyllum molybdites by following its lethal effect in mice. Analysis of the protein using SDS-PAGE revealed a single 23-kDa band. Sequence analysis of molybdophyllysin tryptic fragments showed that this protein is highly homologous to metalloendopeptidases (MEPs) obtained from edible mushrooms, such as Grifola frondosa, Pleurotus ostreatus, and Armillaria mellea. These proteins include a HEXXH+D zinc-binding motif known as aspzincin. Accordingly, molybdophyllysin is a member of the deuterolysin family of zinc proteases. Molybdophyllysin retained its proteolytic activity at temperatures up to 60°C with an optimum pH of 7.0. The activity was inhibited by both 1,10-phenanthroline and N-bromosuccinimide, but molybdophyllysin exhibited strong resistance to SDS.
Assuntos
Agaricales/enzimologia , Metaloendopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Bromosuccinimida/química , Concentração de Íons de Hidrogênio , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/química , Metaloendopeptidases/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Fenantrolinas/química , Homologia de Sequência de Aminoácidos , Temperatura , Zinco/química , Zinco/metabolismoRESUMO
Arthropods comprise the largest group of living animals, including thousands of species that inhabit marine and terrestrial niches in the biosphere [...].
Assuntos
Artrópodes , Peçonhas , AnimaisRESUMO
The zebrafish is a useful model to identify genes functioning in hematopoiesis, owing to high conservation of hematopoiesis. Flow cytometry is widely used to isolate and quantitatively characterize human and mouse hematopoietic cells, often using fluorescently labeled antibodies. However, such analysis is limited in zebrafish due to lack of antibodies that recognize zebrafish hematopoietic cells. We here describe methods for isolation of hematopoietic cells by antibody-free flow cytometry in the zebrafish embryo. Hematopoietic stem cells (HSCs) are specified from a specific subset of vascular endothelial cells, the hemogenic endothelial cell (HEC), by a high level of Notch signaling. In combination with a Notch reporter line (Tp1:GFP) and a vascular specific line (fli1a:dsRed), HECs can be isolated as Tp1:GFPhigh fli1a:dsRed+ cells at 20-22 hours post-fertilization (hpf). Zebrafish erythrocytes can be purified using 1,5-bis{[2-(dimethylamino)ethyl]amino}-4, 8-dihydroxyanthracene-9,10-dione (DRAQ5), a DNA-staining fluorescent dye, and gata1:dsRed (erythroid marker line). DRAQ5high dsRed+ cells are morphologically erythrocyte-like, hemoglobin-stained positive, and express erythropoiesis-related genes. Zebrafish neutrophils can be also isolated using the lectin Phaseolus vulgaris erythroagglutinin (PHA-E) and DRAQ5. PHA-Elow DRAQ5low cells have myeloperoxidase activity, are Sudan Black B-positive, and express neutrophil-related genes. These methods will help to perform genetical and functional assays for various types of hematopoietic cells in zebrafish embryos.