Marine Biotechnology

A tunicate (Botryllus schlosseri) cDNA reveals similarity to vertebrate antigen receptors

Characterization of cells with different mitochondrial membrane potential during apoptosis.

Background Until now, the simultaneous analysis of several parameters during apoptosis, including DNA content and mitochondrial membrane potential (ΔΨ), has not been possible because of the spectral characteristics of the commonly used dyes. Using polychromatic flow cytometry based upon multiple laser and UV lamp excitation, we have characterized cells with different ΔΨ during apoptosis. Methods U937 cells were treated with the flavonoid quercetin (Qu) and stained with JC-1 to detect ΔΨ, propidium iodide (PI) for cell viability, Hoechst 33342 for DNA content, Annexin V conjugated with Alexa Fluor-647 for detection of phosphatidilserine (PS) exposure, marker of early apoptosis, or Mitotracke…

Immunological Activity of Ascidian Hemocytes

In ascidians, various hemocyte types and their differentiation stages may be responsible for several immune functions. A central role in the immune effector mechanisms can be assigned to PO-containing hemocytes and prophenoloxidase system. Morula cell in Styela plicata and univacuolar refringent granulocyte in Ciona intestinalis, a probable intermediate stages in the differentiation pathway of morula cell, may be cytotoxic cells. They are involved in immune reactions of solitary and colonial ascidians. We have shown that they are provided with a PO-linked spontaneous cytotoxic activity and recognize mammalian erythrocytes or tumor cells markers. The toxic molecules can be radical oxygen int…

Comparative immunologic models can enhance analyses of environmental immunotoxicity

Abstract To treat immune systems and how environments affect them is a unique challenge especially when the environment is considered in its broadest perspective: internal and external. Internal focuses on relationships between immune, nervous and endocrine systems (neuroendocrine) and how they interact to maintain homeostasis. External considers physical and chemical influences that act to change the internal. Using animal models is based upon phylogeny which focuses on invertebrates, fish, amphibians, and reptiles, including mammalian results and relationships to humans. Emphasizing primitive animals is due to a growing interest in using them as models, sentinels, surrogates—predictors of…

A urochordate putative homolog of human EB1, the protein which binds APC1

Abstract The human EB1 protein has been cloned by virtue of its interaction with the C-terminus of the APC (adenomatous polyposis coli) protein, whose C-terminal truncated forms have been shown to accompany sporadic and familial forms of colorectal cancer. We have cloned a putative EB1 homolog from Botryllus schlosseri (Urochordata, Ascidiacea). The deduced protein is 287 amino acids long, and is identical with 48% of the residues in human EB1 and 24–25% in two yeast hypothetical proteins. We propose that such a high degree of conservation among EB1 homologs is indicative of an essential regulatory mechanism in eukaryotic cells.

Confinement stress in sea bass (Dicentrarchus labrax) depresses peritoneal leukocyte cytotoxicity

Fish respond to stressful conditions via neuroendocrine responses (primary response) which result in increased levels of plasma cortisol which is considered immunosuppressive. Sea bass were confined at low (10 kg/m3) and high (60 kg/m3) density for 3-48 h. Plasma cortisol and glucose were evaluated and two principal cellular immune responses were assayed. A significant increase in plasma cortisol and glucose levels, as well as osmolarity, was found following stress. In addition, phagocytic activity, as shown by reactive oxygen species (ROS) production by challenged head kidney phagocytes and cytotoxic activity of eosinophilic granule cells from peritoneal cavity against K562 tumour cell lin…

Immunodefense in Tunicates: Cells and Molecules

There are several reasons for analyzing tunicate immune systems. First, they can be established as primitive models for understanding fundamental immunological mechanisms by analyzing their individual cells and molecular products either in vivo or in vitro. Discovered mechanisms could provide alternatives to traditional (vertebrate) mammalian (mouse, rat) and emerging models (fish, amphibian reptile) in answering basic questions concerning immunity and disease in protochordates, the ancestors of vertebrates. Second in vitro, biochemical, immunochemical, and serological analyses coupled with molecular approaches are useful as we search for common molecules (e.g. markers of lymphocyte-like ce…

Tributyltin affects phagocytic activity of Ciona intestinalis hemocytes

Organotin compounds have been used in marine anti-fouling paints as biocides. Because tunicates are vulnerable to these compounds in their natural habitats, we used Ciona intestinalis to establish an assay for phagocytosis in vitro of yeast by hemocytes after exposure to different concentrations (0.0015, 0.015, 0.15 and 1.5 microM) of four organotin compounds: tributyltin (TBT), triphenyltin (TPT), dibutyltin (DBT) and diphenyltin (DPT). To evaluate the phagocytic activity, we used a method based on fluorescence excitation of yeast pre-treated with eosin-Y. The percentage of phagocytosis decreased from 45.1 +/- 3.49 to 22.4 +/- 5.14 at 1.5 microM of TBT (P0.001); it was significantly reduce…