Cytocentrifugation as an additional method to study echinoderm coelomocytes: a comparative approach combining living cells, stained preparations, and energy-dispersive x-ray spectroscopy

Introduction: Echinoderm coelomocytes have traditionally been investigated through a morphological approach using light microscopy, which relies on the idea of constant cell shape as a stable character. However, this can be affected by biotic or abiotic conditions. Objective: To analyze if the consistency in cell morphology offered by the cytocentrifugation method, might be used as a convenient tool to study echinoderm coelomocytes. Methods: Cells of Echinaster (Othilia) brasiliensis (Asteroidea), Holothuria (Holothuria) tubulosa (Holothuroidea), Eucidaris tribuloides, Arbacia lixula, Lytechinus variegatus, and Echinometra lucunter (Echinoidea) were spread on microscope slides by cytocentrifugation, stained, and analyzed through light microscopy. Additionally, fluorescence microscopy, scanning electron microscopy, and energy-dispersive x-ray spectroscopy were applied to cytospin preparations, to complement the analysis of granular and colorless spherulocytes of Eucidaris tribuloides. Results: Altogether, 11 cell types, including phagocytes, spherulocytes, vibratile cells, and progenitor cells were identified in the samples analyzed. The granular spherulocyte, a newly-described cell type, was observed in all Echinoidea and was very similar to the acidophilic spherulocytes of Holothuria (Holothuria) tubulosa. Conclusions: Cytocentrifugation proved to be versatile, either as the main method of investigation in stained preparations, or as a framework on which other procedures may be performed. Its ability to maintain a constant morphology allowed accurate correspondence between live and fixed/stained cells, differentiation among similar spherulocytes as well as comparisons between similar cells of Holothuroidea and Echinoidea.

Introducción: Los celomocitos de equinodermos se han investigado tradicionalmente a través de un enfoque morfológico utilizando microscopía óptica, que se basa en la idea de la forma celular constante como un carácter estable. Sin embargo, esto puede verse afectado por condiciones bióticas o abióticas. Objetivo: Analizar si la consistencia en la morfología celular que ofrece el método de citocentrifugación podría utilizarse como una herramienta conveniente para estudiar los celomocitos de equinodermos. Métodos: Células de Echinaster (Othilia) brasiliensis (Asteroidea), Holothuria (Holothuria) tubulosa (Holothuroidea), Eucidaris tribuloides, Arbacia lixula, Lytechinus variegatus y Echinometra lucunter (Echinoidea) se esparcieron en portaobjetos de microscopio por citocentrifugación, se tiñeron y analizaron mediante microscopía óptica. Adicionalmente, se aplicó microscopía de fluorescencia, microscopía electrónica de barrido y espectroscopía de rayos X con dispersión de energía a las preparaciones de citoespina, para complementar el análisis de los esferulocitos granulares e incoloros de Eucidaris tribuloides. Resultados: En total, se identificaron en las muestras analizadas 11 tipos de células, incluidos fagocitos, esferulocitos, células vibrátiles y células progenitoras. El esferulocito granular, un tipo de célula recién descrito, se observó en todos los Echinoidea y fue muy similar a los esferulocitos acidófilos de Holothuria (Holothuria) tubulosa. Conclusiones: La citocentrifugación demostró ser un método bastante versátil, ya sea como el método principal de investigación en preparaciones teñidas o como un marco en el que se pueden realizar otros procedimientos. Su capacidad para mantener una morfología constante permitió una correspondencia precisa entre las células vivas y las células fijas/teñidas, la diferenciación entre esferulocitos similares, así como comparaciones entre células similares de Holothuroidea y Echinoidea.

Isolation and cultivation of fungal strains from in vitro cell cultures of two marine sponges (Porifera: Halichondrida and Haplosclerida)

Despite the large number of reports describing sponge-microbe associations, limited knowledge is available about associated fungi and their relationships with the hosts. In this work, specific fungal strains were obtained directly from in vitro sponge cell cultures (primmorphs) and single sponge cells (cytospins) and compared with those obtained from whole tissue preparations. A total of 27 fungal strains were isolated from the marine sponges Hymeniacidon heliophila and Haliclona melana. Fifteen strains, nine from H. heliophila and six from H. melana, were obtained from whole tissue and were considered as possible mesohyl associated or transient fungi. Twelve strains were isolated from in vitro sponge cell cultures (primmorphs) and were, therefore, considered as cell associated. From these, five different strains were obtained from H. heliophila isolated cells, while five were identified from cytospins and two from primmorphs of H. melana. The fungal strains obtained from cell cultures from both sponge species were different, and none of them were detected in the whole tissue preparations of the same species. Nine H. heliophila and seven H. melana strains shows low similarity with the sequences available in public databases and belong to potentially new species. This is the first report of fungi isolated directly from sponge cells, which allowed the observation and selection of specific strains that probably would not be obtained by usual culture dependent techniques.

Neurotoxins in the polar fraction of the seaweed Galaxaura marginata (Rhodophyta, Nemaliales) / Neurotoxinas na fração polar da alga marinha Galaxaura marginata (Rhodophyta, Nemaliales)

A atividade neurotóxica das substâncias polares extraídas da alga vermelha Galaxaura marginata foi estudada por meio de ensaios farmacológicos, utilizando-se algas coletadas no canal de São Sebastião (45º25' W; 26º49' S), litoral norte do estado de São Paulo, Brasil. Altas concentrações da fração polar (PF) e da PF filtrada em membrana de 1000 Da, injetadas intraperitonealmente em camundongos, foram letais. Induzindo os animais à ataxia, desorientação e severa atonia muscular prévias à morte. Sintomas similares foram descritos para camundongos e ratos testados com aminoácidos neuroexcitatórios (NEA), sugerindo que alguns dos componentes da PF atravessam a barreira Hematoencefálica, da mesma forma que os NEA. No entanto, a análise da PF no HPLC e no espectro de massa não mostraram a presença dos aminoácidos neuroexcitatórios conhecidos, ácido Caínico, acido Domóico e NMDA. Assim concluímos que G. marginata possui substâncias com atividade letal e neurotóxica, diferentes às antes, descritas.

Neurotoxic activity of polar substances extracted from the red seaweed Galaxaura marginata was investigated in pharmacological assays, using seaweed collected at São Sebastião channel (45º25' W; 26º49' S) in the northern coast of the State of São Paulo, Brazil. High concentration of the polar fraction (PF) and PF filtered through a membrane (1000 Da) intraperitoneally (IP) injected in mice were lethal, inducing ataxia, disorientation, and severe muscle atonia prior to death. The same symptoms preceding death were described in mice and rat tested with neuroexcitatory amino acids (NEA), suggesting that some PF components cross the blood-brain barrier, the same as NEA. However, the HPLC and mass spectrometric analysis did not show the presence of the known neuroexcitatory amino acids, kainic acid, domoic acid and NMDA. We conclude that G. marginata possess substances with neurotoxic and lethal activities.

Intracellular increase of glutamate in neuroblatoma cells induced by polar substances of Galaxaura marginata (Rhodophyta, Nemaliales) / Aumento do glutamato intracelular induzido por substâncias polares de Galaxaura marginata (Rhodophyta, Nemaliales) em células de neuroblastoma

Four substances purified by HPLC from red seaweed Galaxaura marginata (Ellis & Solander) Lamouroux showed neuroprotective and mitochondria regulatory properties in neuroblastoma NEURO-2A cells line. Under oxidative basal conditions, each substance is capable of modifying the glutamate, glutamine and alanine intracellular concentrations, without to alter the oxidative equilibrium of these cells. These substances, also, produced a biphasic activity in the mitochondrial metabolism, inhibiting, in lower concentrations and stimulating, in higher concentrations the mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, that indicates the mitochondrial activity. These data suggest that the intracellular increase of glutamate can be related to the glutamate/glutamine cycle alterations induced by the blockade of intracellular glutamate transport. Moreover, we suggested that there are substances, exhibiting neuroprotective activity in neuroblastoma cells, protecting them from oxidative stress produced by the inhibition of the glutamate transporters.

Quatro substâncias purificadas da alga vermelha Galaxaura marginata (Ellis & Solander) Lamouroux, mediante HPLC, apresentaram propriedades neuroprotetoras e reguladoras da atividade mitocondrial quando testadas em células de neuroblastomas da cepa NEURO-2. Sob condições oxidativas basais, cada substância é capaz de modificar as concentrações intracelulares de glutamato, glutamina e alanina, sem alterar o equilíbrio oxidativo das células. Essas substâncias também produziram uma atividade bifásica no metabolismo mitocondrial, inibindo em baixas concentrações e estimulando em altas concentrações o mecanismo celular de redução do 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazol brometo (MTT), que indica atividade mitocondrial. Esses dados sugerem que o aumento do glutamato pode estar relacionado com a alteração do ciclo glutamato/glutamina induzido pelo bloqueio do transporte intracelular de glutamato. Além disso, nós sugerimos que essas substâncias exibem atividade neuroprotetora nas células de neuroblastoma, as protegendo do estresse oxidativo, produzido pela inibição dos transportadores de glutamato.