The piscine SAF-1 cell line: genetic stability and labeling

Fish cell lines are increasingly important research tools. The SAF-1 cell line, fibroblast-like culture derived from the marine fish gilthead seabream (Sparus aurata), has proved useful in many applications, especially in viral research. For cell lines intended as in vitro models, characterization of their properties and authentication are essential for deeper understanding of their performance and thus more precise experimental design and applicability. In this study we characterized the SAF-1 cell line in terms of genetic stability through time and genetic labeling. Methods for determining stability include telomerase activity, karyotyping, mapping of ribosomal RNA regions, and DNA content. For genetic labeling 12 microsatellite loci were used. The results indicate that telomerase has been activated in the course of SAF-1 development, and the highest levels of telomerase activity correlate with an increase in cell proliferation, thus supporting a permanent cell line. This stability is in agreement with the normal situation presented by the cytogenetic traits and DNA content values, and the genotypic profile allows SAF-1 authentication at the single individual level. This study increases the value of SAF-1 as an in vitro system, which is now one of the few well-characterized cell lines from a marine fish.     

Identification of a new cartilage-specific S100-like protein up-regulated during endo/perichondral mineralization in gilthead seabream

Calcium ions and calcium-binding proteins play a major role in many cellular processes, in particular skeletogenesis and bone formation. We report here the discovery of a novel S100 protein in fish and the analysis of its gene expression patterns. A 648-bp full-length cDNA encoding an 86-amino acid S100-like calcium-binding protein was identified through the subtractive hybridization of a gilthead seabream (Sparus aurata) cDNA library constructed to identify genes associated with in vitro mineralization. Deduced protein lacks an identifiable signal peptide and exhibits two EF-hand motifs characteristic of S100 proteins. Phylogenetic and bioinformatic analyses of S100 sequences suggested that gilthead seabream protein represents a novel and fish-specific member of the S100 protein family. Expression of S100-like gene was up-regulated during the in vitro mineralization of bone-derived cell lines and during seabream development, from larvae throughout adulthood, reflecting skeletogenesis. Restriction of S100-like gene expression to chondrocytes of cartilaginous tissues undergoing endo/perichondral mineralization in juvenile fish further confirmed the mineralogenic role of the protein in fish and emphasized the potential of S100-like as a marker of mineralizing cartilage in developing fish.     

Some properties of an established fish cell line fromXiphophorus helleri (red swordtail)

Summary A cell line (SWT) was established from embryonic tissue of the red swordtailXiphophorus helleri. The SWT cells grew optimally, at 26°C to 30°C in Eagle's basal medium plus 10% fetal calf serum but failed to grow at 16°C and 37°C. After 50 subcultivations, the cells remained contact-inhibited and were pseudodiploid with a chromosomal modal number of 46. Virological studies demonstrated that SWT cells supported replication of the following viruses at the indicated temperatures: IPN virus (22°C), FV-3 (30°C), and VSV (33°C). The following mammalian, viruses failed to replicate at 33°C: vaccinia, poliovirus 2, herpes simplex, and reovirus 2. Although not replicating, reovirus induced interferon in there cells. This work was supported in part by a grant from the University Research Council. A part of these results was presented at the 22nd Annual Meeting of the Tissue Culture Association, Lake Placid, N. Y. 1971.     

Development of two bone-derived cell lines from the marine teleost<Emphasis Type="Italic"> Sparus aurata</Emphasis>; evidence for extracellular matrix mineralization and cell-type-specific expression of matrix Gla protein and osteocalcin

A growing interest in the understanding of the ontogeny and mineralization of fish skeleton has emerged from the recent implementation of fish as a vertebrate model, particularly for skeletal development. Whereas several in vivo studies dealing with the regulation of bone formation in fish have been published, in vitro studies have been hampered because of a complete lack of fish-bone-derived cell systems. We describe here the development and the characterization of two new cell lines, designated VSa13 and VSa16, derived from the vertebra of the gilthead sea bream. Both cell types exhibit a spindle-like phenotype and slow growth when cultured in Leibovitzs L-15 medium and a polygonal phenotype and rapid growth in Dulbeccos modified Eagle medium (D-MEM). Scanning electron microscopy and von Kossa staining have revealed that the VSa13 and VSa16 cells can only mineralize their extracellular matrix when cultured in D-MEM under mineralizing conditions, forming calcium-phosphate crystals similar to hydroxyapatite. We have also demonstrated the involvement of alkaline phosphatase, a marker of bone formation in vivo, and Gla proteins (osteocalcin and matrix Gla protein, MGP) in the process of mineralization. Finally, we have shown that VSa13 and VSa16 cell lines express osteocalcin and MGP in a mutually exclusive manner. Thus, both cell lines are capable of mineralizing in vitro and of expressing genes found in chondrocyte and osteoblast cell lineages, emphasizing the suitability of these new cell lines as valuable tools for analyzing the expression and regulation of cartilage- and bone-specific genes.A.R. Pombinho and V. Laizé contributed equally to this workThis work was partially funded with grants from the Portuguese Science and Technology Foundation PRAXIS/BIA/11159/98, POCTI/34668/Fis/2000 and POCTI/BCI/48748/2002. V.L., S.M.P.M and A.P. were the recipients of a postdoctoral fellowship (BPD/1607/2000 and BPD/9403/2002) and a CCMAR/University of Algarve fellowship, respectively     

Establishment of primary cell cultures from fish calcified tissues

Fishes have been recently recognized as a suitable model organism to study vertebrate physiological processes, in particular skeletal development and tissue mineralization. However, there is a lack of well characterized in vitro cell systems derived from fish calcified tissues. We describe here a protocol that was successfully used to develop the first calcified tissue-derived cell cultures of fish origin. Vertebra and branchial arches collected from young gilthead seabreams were fragmented then submitted to the combined action of collagenase and trypsin to efficiently release cells embedded in the collagenous extracellular matrix. Primary cultures were maintained under standard conditions and spontaneously transformed to form continuous cell lines suitable for studying mechanisms of tissue mineralization in seabream. This simple and inexpensive protocol is also applicable to other calcified tissues and species by adjusting parameters to each particular case.     

Increase in activity of acetylcholinesterase by 20-OH-ecdysone in a Chironomus tentans cell line

Summary An epithelial cell line from Chironomus tentans exhibits acetylcholinesterase activity (specific activity 0.05–0.2 nkat/mg protein), which rises 30– to 40-fold after addition of 10^–6 M 20-OH-ecdysone. The first visible increase occurs after 4 days of incubation with hormone. The enzyme has an apparent K _m of 2.3±0.2×10^–4 M for acetylthiocholine iodide as substrate and is inhibited by eserine and BW284 C51 (50% inhibition at 5×10^–7 M for both inhibitiors) as well as by high concentrations of substrate, but not by tetraisopropylpyrophosphamide. The sensitivity against inhibitors is the same in extracts from hormone-treated cells and from controls. The cholinesterase activity correlates with morphological changes (shape and cell arrangement) and is indepenent of neuronal differentiation. We therefore propose a function for this activity during morphogenesis.     

Injection of xenogeneic cells into teleost fish elicits systemic and local cellular innate immune responses

The early innate immune response of the teleost gilthead seabream (Sparus aurata L.) against xenogeneic cells was studied. Fish received a single intraperitoneal injection of xenogeneic cells (tumour cell line), following which leucocyte mobilization, degranulation, peroxidase content, respiratory burst and phagocytic and cytotoxic activities were determined in both peritoneal exudate leucocytes (PELs) and head-kidney leucocytes (HKLs). The total number of PELs increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis of PEL and HKL suspensions revealed variations in the proportion of cell types. The percentage of HK acidophilic granulocytes significantly increased after 72 h, whereas PE acidophils increased after 4 h. Moreover, numbers of PE lymphocytes and monocyte-macrophages significantly increased during the experiment. The peroxidase content of the leucocytes was unaffected, although PEL degranulation was largely enhanced. This liberation of peroxidases correlated well with the enhancement of the oxidative respiratory burst activity in PELs, reflecting leucocyte activation. However, phagocytosis only increased in PELs 4 h after intraperitoneal injection, whereas the cytotoxic activity of HKLs increased 1 and 2 days post-injection but, in general, decreased in the PELs. Our data thus demonstrate that the appearance of xenogeneic cells involves leucocyte mobilization and innate immune-response activation at the site of invasion and in the head-kidney. Involvement of the various leucocyte types and potential modes of activation are discussed.This work was partially funded by the European Commission (QLRT-2001-00722). A. Cuesta and I. Salinas are fellows of Fundación CajaMurcia and Fundación Séneca, respectively.     

Induction of vesicle formation in a cell line derived from imaginal discs

Summary The ability of insect hemolymph to induce vesicles in a high passage insect cell line, IAL-TND1, is described. The factor responsible, designated VPA for ‘vesicle-promoting activity’, was determined to be heat sensitive, nondialyzable, and protease Type XIV sensitive but insensitive to trypsin digestion. In efforts to determine the source of VPA, hemolymph was collected from different developmental stages ofTrichoplusia ni, and certain tissues fromT. ni were cocultured with IAL-TND1 cells. Hemolymph from every developmental stage tested exhibited VPA although the effect was somewhat reduced in spinning-stage larvae. Additionally, several tissue, including fat body, tess, and imaginal discs, released VPA into the culture medium. Neural tissues and endocrine glands did not induce vesicle formation.     

Initiation and characterization of a diploid cell line from larval tissues ofAedes dorsalis (Meigen)

Summary Mosquito cell cultures were initiated from the minced tissues of newly hatchedAedes dorsalis (Meigen) larvae. Continuous cell division occurred only after an adaptive period of approximately 6 months. Optimal growth of the cells required a relatively low pH of 6.5. Karyological studies showed that the cells have remained diploid (2n=6) for 60 serial passages and that the cultures are free of contaminating cells. The cultures also were shown to be free of bacteria (includingMycoplasma), fungi and virions. Subpopulations (strains) of the original parental cultures have been selected and characterized on the basis of morphology, karyology, growth rate and monolayer formation. These studies were supported in part by funds from the Office of Naval Research, by Research Grant AI03028 from the National Institute of Allergy and Infectious Diseases, and by General Research Support Grant I-SO1-FR-05441 from the National Institutes of Health, U.S. Department of Health, Education and Welfare.     

Establishment and characterization of a cell line from the american opossum (Didelphys virginiana)

Summary A permanent tissue-cultured cell line (designated OK) has been established from kidney tissue of an adult American opossum. The OK line has been characterized with respect to morphology, chromosome constitution, tissue-culture requirements, and attainable mitotic arrest. The cells are epithelial-like with a stable nondiploid chromosomal modal number of 23. Cells grown in Eagle's minimal essential medium with 10% fetal bovine serum have a mean doubling time of 18 hr. The cell line OK is potentially useful for the isolation and purification of the mammalian X chromosome because of the size differential between the smaller X's and the larger autosomes. This work was supported by NIH grant HD-04420.     

20-OH-Ecdysone-induced morphogenetic movements in a Chironomus cell line are accompanied by expression of an embryonic muscarinic system

The permanent epithelial insect cell line used was derived from Chironomus tentans embryos. Cells are maintained in suspension culture, where they grow as single-layered vesicles. On treatment with the moulting hormone 20-OH-ecdysone cell division ceases. Patches of cuboidal epithelium appear in the vesicles which finally become multilayered and form bud-like protrusions at the outside. In the present study, we localized cholinesterase activity in the cell protrusions by histochemistry and demonstrated coexpression of cholinergic muscarinic receptors by immunofluorescence. Muscarinic receptors were visualized with the monoclonal antibody M35. Six hours after treatment with 20-OH-ecdysone, muscarinic receptors appeared in a few individual cells of the epithelial vesicles before morphological changes became visible. After 24 h, immunofluorescence was concentrated in multilayered patches which now also showed cholinesterase activity. After 3 days, muscarinic receptors and cholinesterase activity were localized in the epithelium protrusions. The results are discussed in the context of an embryonic cholinergic muscarinic system the expression of which has been described in vertebrate and non-vertebrate embryos and is correlated with phases of morphogenesis.     

Seasonal changes of zinc, copper, and iron in gilthead sea bream (Sparus aurata) fed fortified diets

Four groups of gilthead sea bream (Sparus aurata) were fed diets with additional metal contents: a basal diet (diet A) contained Zn at 60.9 ± 1.9 mg/kg diet, Cu at 3.9 ± 0.9 mg/kg diet, and Fe at 138.3 ± 6.8 mg/kg diet; the other diets were supplemented with copper (20 mg/kg, diet B), iron (100 mg/kg, diet C), or zinc (300 mg/kg, diet D). Two consecutive year-classes (0⁺ and 1⁺ age fish) from the same parent stock were examined. Several fish tissues were analyzed for metal contents in five different periods of each year in order to determine (1) the sensitivity of certain tissues as indicators of trace element metabolism and (2) possible seasonal variations. Growth data were similar for gilthead sea bream fed the basal diet and the metal-fortified diets. Mineral concentrations in tissues were found to be little affected by the dietary supplementation of trace elements, suggesting an efficient homeostatic control of these three metal concentrations. Tissues involved in metal metabolism (e.g., liver, kidney, gills) presented greater variations between minimum and maximum values with respect to other tissues (e.g., brain, muscle, eye). Seasonal variations were observed during the 2 yr of this study and were especially evident for zinc and copper concentrations in the liver. The overall pattern of metal variations showed a decreasing trend during the 2 yr. Results from this study indicate that (1) trace element concentrations in fish tissues vary with age and life cycle and (2) trace element requirements may vary in function of age and life cycle.     

Effect of age on the growth and response of a Drosophila cell line to moulting hormone

Insect cell lines in culture are used for a variety of studies. In this laboratory imaginal disc cell lines have been established from primary cultures from third instar larvae, and used for a number of experiments. The effect of ageing on the morphology and physiology of Drosophila cell lines has received very little attention, although problems of genotypic or phenotypic changes in cell lines with age are recognized in other areas of animal cell culture. We tested our cell line CI8+ for any difference in growth, morphology and response to 20-hydroxyecdysone (20HE) at different ages (passage numbers). The cells were found to multiply faster, adhere less firmly to the substrate and to lose the tendency to aggregate at higher passages. The response to 20HE in terms of cell numbers and induction of β-galactosidase was similar at all passage numbers but morphological changes in hormone-treated cells were less obvious in the higher passages. Cell lines are likely to vary in the extent of ageing effects but workers are advised to be aware of the possibilities. We suggest the effects of age on cell lines should be established, and passage numbers noted in experimental reports.     

Phenylobacterium zucineum sp. nov., a facultative intracellular bacterium isolated from a human erythroleukemia cell line K562

A bacterial strain HLK1(T) was isolated from the human erythroleukemia cell line K562. This bacterium is a Gram-negative rod, motile with a polar flagellum. It is strictly aerobic, nonfermentative, and oxidase and catalase positive. Its optimal growth occurs at 37 degrees C at pH between 6.5 and 7.5. Phylogenetically, although it shares 98% similarity with the 16S rRNA of Phenylobacterium lituiforme, the DNA-DNA hybridization value between the two species is only 43%. HLK1(T) has a DNA G+C content of 71.2+/-0.2 mol%. It is a facultative intracellular organism and may have pathogenic relevance with humans and mammals. On the basis of the phylogenetic and phenotypic characterization, strain HLK1(T) is proposed to be classified in the genus Phenylobacterium, as P. zucineum sp. nov. The type strain is HLK1(T) (=CGMCC 1.3786(T), DSM=18354).     

Changes in developmental profiles of brain gangliosides during ontogeny of a teleost fish (Sarotherodon mossambicus,Cichlidae)

Summary Ontogenetic changes of brain ganglioside concentration and composition have been followed in the teleost fishSarotherodon mossambicus Cichlidae) from the 1st day post hatching to the adult stage, in order to correlate these with findings in higher vertebrates. During the developmental period from hatching to the transition to free swimming, which comes along with maximum rate of synaptogenesis, a sharp rise in the brain ganglioside content occurs, which is mainly due to the trisialoganglioside GT1b. In the following phase of myelination (characterized in birds and mammals by an increase in GM1, GM1 and GM4) accretion of the novel and so far unidetified fraction (GM2) occurs, which is highly enriched in the brain stem. The results obtained are discussed with respect to gangliosides as useful biochemical markers for brain development and maturation in all vertebrates.     

Digestive enzymes in marine species. II. Amylase activities in gut from seabream (Sparus aurata), turbot (Scophthalmus maximus) and redfish (Sebastes mentella)

The amylase activity of the digestive tract of three carnivorous fish species (Sparus aurata, Scophthalmus maximus and Sebastes mentella) has been studied. The activity of seabream and turbot showed its maximum at neutral pH (7.0–7.5); meanwhile, the activity of redfish had an optimum pH at 4.5-5.0. The t° function ranged between 35 and 45°C for the three species. The Arrhenius plots of the intestinal activities of seabream and turbot showed breakpoints at temperatures close to those of their physiological activities. High saline concentrations inhibited the activity of seabream and turbot and activated the activity of redfish. Seabream activity was absolutely dependent on calcium ions. On the contrary, redfish activity was only detected in the absence of this metal. Studies carried out by using several effectors suggested that the activities found in these three species are different. Considering our results from a point of view of the environmental conditions of these species, it might be concluded that enzymatic digestion of dietary carbohydrates proceeds at very low rate. Physiological implications are discussed.     

Early innate immune response and redistribution of inflammatory cells in the bony fish gilthead seabream experimentally infected with <Emphasis Type="Italic">Vibrio anguillarum</Emphasis>

An obvious difference between the immune system of fish and mammals is that fish lack both bone marrow and lymph nodes; in their place, the head-kidney acts as a major haematopoietic and lymphoid organ in adult fish, whereas the thymus, spleen and mucosa-associated lymphoid tissues are common to both fish and mammals. This suggests that differences exist in antigen presentation and naïve lymphocyte stimulation, a prerequisite for the initiation of adaptive immune responses. Intraperitoneal injection of the bony fish gilthead seabream (Sparus aurata L.) with intact Vibrio anguillarum, as a particulate bacterial antigen, results in the mobilisation of head-kidney leucocytes to the peritoneal cavity and priming of their respiratory burst activity. We have also observed the rapid infiltration of acidophilic granulocytes, which are leucocytes functionally equivalent to mammalian neutrophils, into the spleen. These cells may be involved in antigen capture and transport to the spleen, since an apparent association between mobilised acidophilic granulocytes, bacterial antigens and proliferating lymphocytes has been seen in this organ. Collectively, these results suggest that, in addition to being actively involved in bacterial clearance, fish phagocytic granulocytes play a role in the initiation and support of the adaptive immune response.This work was supported by the Spanish Ministry of Science and Technology (grants BIO2001-2324-C02-02, AGL2002-03529 and AGL2002-04306-C03-01 to V.M. and J.M. and Programa Ramón y Cajals contract to P.M.), Spanish Ministry of Education, Culture and Sport (fellowship to E.C.-P.) and the Fundación Séneca (grant PI-51/00782/FS/01 to J.M. and fellowship to P.P.).