Synthesis, inhibition and binding of simple non-nitrogen inhibitors of monoamine transporters

A series of simple truncated analogues of phenyl tropanes, 2-arylcycloalk-1-enyl carboxylic acid methylesters, were prepared and investigated for their activity towards the dopamine, serotonin and norepinephrine transporters. The compounds were prepared from cyclic ketoesters, which were converted to enolic triflates and reacted with arylboronates using the Suzuki coupling. For comparison the corresponding piperidines were also made and investigated. The new compounds inhibit monoamine-transporters with Ki values ranging from 0.1 to 1000 microM.     

A stromal cell line from rainbow trout spleen, RTS34st, that supports the growth of rainbow trout macrophages and produces conditioned medium with mitogenic effects of leukocytes

Summary A rainbow trout spleen cell line, RTS34, was developed from a long-term hemopoietic culture. This cell line consisted of a mixed stromal cell layer with an associated cell population of macrophage-like cells that formed proliferative foci and released nonadherent progeny cells into the culture medium. A stromal cell line, RTS34st, was isolated from the RTS34 cell line. RTS34st cultures contained cells with fibroblast-like and epithelial-like morphologies and showed enhanced [³H]thymidine incorporation in response to either FBS or rainbow trout serum. The combination of FBS and trout serum was synergistic. Conditioned medium from RTS34st stimulated thymidine incorporation by peripheral blood and head kidney leukocytes, but not by leukocytes from the spleen. In addition, RTS34st provided a hemopoietic inductive microenvironment for immature precursor cells, selectively supporting the growth of macrophage-like cells. Therefore, RTS34st appears useful for studying the different roles of the stroma in regulating hemopoiesis in fish.     

Identification of bovine serum albumins that support salmonid cell proliferation in the absence of serum

This investigation was supported by a Natural Sciences and Engineering Research Council of Canada grant to N. C. B. and a scholarship to A. B. from the Canada-Indonesia Graduation Program through the Indonesian Ministry of Education and Culture.     

The impact of simplified boundary conditions and aortic arch inclusion on CFD simulations in the mouse aorta: a comparison with mouse-specific reference data

Computational fluid dynamics (CFD) simulations allow for calculation of a detailed flow field in the mouse aorta and can thus be used to investigate a potential link between local hemodynamics and disease development. To perform these simulations in a murine setting, one often needs to make assumptions (e.g. when mouse-specific boundary conditions are not available), but many of these assumptions have not been validated due to a lack of reference data. In this study, we present such a reference data set by combining high-frequency ultrasound and contrast-enhanced micro-CT to measure (in vivo) the time-dependent volumetric flow waveforms in the complete aorta (including seven major side branches) of 10 male ApoE -/- deficient mice on a C57Bl/6 background. In order to assess the influence of some assumptions that are commonly applied in literature, four different CFD simulations were set up for each animal: (i) imposing the measured volumetric flow waveforms, (ii) imposing the average flow fractions over all 10 animals, presented as a reference data set, (iii) imposing flow fractions calculated by Murray's law, and (iv) restricting the geometrical model to the abdominal aorta (imposing measured flows). We found that - even if there is sometimes significant variation in the flow fractions going to a particular branch - the influence of using average flow fractions on the CFD simulations is limited and often restricted to the side branches. On the other hand, Murray's law underestimates the fraction going to the brachiocephalic trunk and strongly overestimates the fraction going to the distal aorta, influencing the outcome of the CFD results significantly. Changing the exponential factor in Murray's law equation from 3 to 2 (as suggested by several authors in literature) yields results that correspond much better to those obtained imposing the average flow fractions. Restricting the geometrical model to the abdominal aorta did not influence the outcome of the CFD simulations. In conclusion, the presented reference dataset can be used to impose boundary conditions in the mouse aorta in future studies, keeping in mind that they represent a subsample of the total population, i.e., relatively old, non-diseased, male C57Bl/6 ApoE -/- mice.     

In vitro growth of microsporidia Anncaliia algerae in cell lines from warm water fish

Anncaliia algerae is an aquatic microsporidium that most commonly infects mosquitoes but can be grown on the rabbit kidney cell line, RK-13. Spores were purified from RK-13 cultures and added to cell lines from warm water fish and from an insect. The cell lines were GFSK-S1 and GFB3C-W1 from goldfish skin and brain respectively, ZEB2J from zebrafish embryos, FHMT-W1 from fathead minnow testis, and Sf9 from ovaries of a fall armyworm moth. All cultures were maintained at 27°C. Infection was judged to have taken place by the appearance of sporonts and/or spores in cells and occurred in all cell lines. Spores were also isolated from ZEB2J cultures and used to successfully infect new cultures of ZEB2J, RK-13 and Sf9. These results suggest that cells of a wide range of vertebrates support A. algerae growth in vitro and fish cells can produce spores infectious to cells of mammals, fish, and insects.     

Survival and Growth of Tetrahymena thermophila in Media that are Conventionally Used for Piscine and Mammalian Cells

The transfer of Tetrahymena thermophila from normosmotic solutions (~20–80 mOsm/kg H₂O) to hyperosmotic solutions (> 290 mOsm/kg H₂O) was investigated. During the first 24 h of transfer from proteose peptone yeast extract (PPYE) to either 10 mM HEPES or PPYE with added NaCl to give ~300 mOsm/kg H₂O, most ciliates died in HEPES but survived in PPYE. Supplementing hyperosmotic HEPES or PPYE with fetal bovine serum (FBS) enhanced survival. When ciliates were transferred from PPYE to a basal medium for vertebrate cells, L‐15 (~320 mOsm/kg H₂O), only a few survived the first 24 h but many survived when the starting cell density at transfer was high (100,000 cells/ml) or FBS was present. These results suggest that nutrients and/or osmolytes in either PPYE or FBS helped ciliates survive the switch to hyperosmotic solutions. FBS also stimulated T. thermophila growth in normosmotic HEPES and PPYE and in hyperosmotic L‐15. In L‐15 with 10% FBS, the ciliates proliferated for several months and could undergo phagocytosis and bacterivory. These cell culture systems and results can be used to explore how some Tetrahymena species function in hyperosmotic hosts and act as opportunistic pathogens of vertebrates.     

Applications and potential uses of fish gill cell lines: examples with RTgill-W1

Gills are unique structures involved in respiration and osmoregulation in piscinids as well as in many aquatic invertebrates. The availability of the trout-derived gill cell line, RTgill-W1, is beginning to make impacts in fish health and toxicology. These cells are available from the American Type Culture Collection as ATCC CRL 2523. The cells have an epithelioid morphology and form tight monolayer sheets that can be used for testing epithelial resistance. The cells can be grown in regular tissue culture surfaces or in transwell membranes in direct contact with water on their apical surfaces. The ability of RTgill-W1 to withstand hypo- and hyper-osmotic conditions and their optimal growth capacity at room temperature, make these cells ideal sentinel models for in vitro aquatic toxicology as well as model systems to study fish gill function and gill diseases. RTgill-W1 support growth of paramyxoviruses and orthomyxoviruses like salmon anemia virus. RTgill-W1 also support growth of Neoparamoeba pemaquidensis, the causative agent of amoebic gill disease. The cells have been used to understand mechanisms of toxicity, ranking the potencies of toxicants, and evaluating the toxicity of environmental samples. These cells are also valuable for high throughput toxicogenomic and toxicoproteomic studies which are easier to achieve with cell lines than with whole organisms. RTgill-W1 cell line could become a valuable complement to whole animal studies and in some cases as gill replacements in aquatic toxicology.     

Development of a zebrafish spleen cell line, ZSSJ, and its growth arrest by gamma irradiation and capacity to act as feeder cells

A zebrafish spleen cell line, ZSSJ, was developed and its growth arrest by gamma radiation determined and its capacity to stimulate the proliferation of the zebrafish blastula cell line, ZEB2J, measured. ZSSJ was initiated by explant outgrowth, grew adherent with mainly an epithelial-like morphology, and stained strongly for alkaline phosphatase. ZSSJ was not only grown in L-15 with 15% fetal bovine serum at 26°C to 28°°C but also grew at room temperature. Cultures of ZSSJ have undergone approximately 40 population doublings, had few cells staining for b-galactosidase activity, which is commonly present in senescent cultures, and many cells with an aneuploid karyotype, which is frequently associated with immortalization. ZSSJ growth was arrested by 30 to 50 Gy of g-irradiation, whereas after 20 Gy, some slight growth was observed. By contrast, growth of the rainbow trout spleen stromal cell line, RTS34st, which has been used as a feeder for zebrafish ES cell cultures, was arrested completely by 20 Gy. In cocultures, nongrowth-arrested ZSSJ stimulated ZEB2J proliferation better than growth-arrested ZSSJ and better than RTS34st. ZSSJ should be useful as a feeder cell line for zebrafish ES cell cultures.