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USGS2003

Effects of brevetoxins on murine myeloma SP2/O cells: Aberrant cellular division

Massive deaths of manatees (Trichechus manatus latirostris) during the red tide seasons have been attributed to brevetoxins produced by the dinoflagellate Karenia brevis (formerly Ptychodiscus breve and Gymnodinium breve). Although these toxins have been found in macrophages and lymphocytes in the lung, liver, and secondary lymphoid tissues of these animals, the molecular mechanisms of brevetoxicosis have not yet been identified. To investigate the effects of brevetoxins on immune cells, a murine myeloma cell line (SP2/O) was used as a model for in vitro studies. By adding brevetoxins to cultures of the SP2/O cells at concentrations ranging from 20 to 600 ng/ml, an apparent increase in proliferation was observed at around 2 hours post challenge as compared to the unchallenged cell cultures. This was followed by a drop in cell number at around 3 hours, suggesting an aberrant effect of brevetoxins on cellular division, the cells generated at 2 hours being apparently short-lived. In situ immunochemical staining of the SP2/O cells at 1 and 2 hour post challenge showed an accumulation of the toxins in the nucleus. A 21-kDa protein was subsequently isolated from the SP2/O cells as having brevetoxin-binding properties, and immunologically identified as p21, a nuclear factor known to down-regulate cellular proliferation through inhibition of cyclin-dependent kinases. These data are the first on a possible effect of brevetoxins on the cell cycle via binding to p21, a phenomenon that needs to be further investigated and validated in normal immune cells.

International Journal of Toxicology↗

USGS2011

Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity

Previously, we demonstrated that a representative M genogroup type strain of infectious haematopoietic necrosis virus (IHNV) from rainbow trout grows well in rainbow trout‐derived RTG‐2 cells, but a U genogroup type strain from sockeye salmon has restricted growth, associated with reduced genome replication and mRNA transcription. Here, we analysed further the mechanisms for this growth restriction of U‐type IHNV in RTG‐2 cells, using strategies that assessed differences in viral genes, host immune regulation and phosphorylation. To determine whether the viral glycoprotein (G) or non‐virion (NV) protein was responsible for the growth restriction, four recombinant IHNV viruses were generated in which the G gene of an infectious IHNV clone was replaced by the G gene of U‐ or M‐type IHNV and the NV gene was replaced by NV of U‐ or M‐type IHNV. There was no significant difference in the growth of these recombinants in RTG‐2 cells, indicating that G and NV proteins are not major factors responsible for the differential growth of the U‐ and M‐type strains. Poly I:C pretreatment of RTG‐2 cells suppressed the growth of both U‐ and M‐type IHNV, although the M virus continued to replicate at a reduced level. Both viruses induced type 1 interferon (IFN1) and the IFN1 stimulated gene Mx1, but the expression levels in M‐infected cells were significantly higher than in U‐infected cells and an inhibitor of the IFN1‐inducible protein kinase PKR, 2‐aminopurine (2‐AP), did not affect the growth of U‐ or M‐type IHNV in RTG‐2 cells. These data did not indicate a role for the IFN1 system in the restricted growth of U‐type IHNV in RTG‐2 cells. Prediction of kinase‐specific phosphorylation sites in the viral phosphoprotein (P) using the NetPhosK program revealed differences between U‐ and M‐type P genes at five phosphorylation sites. Pretreatment of RTG‐2 cells with a PKC inhibitor or a p38MAPK inhibitor did not affect the growth of the U‐ and M‐type viruses. However, 100 μ m of the casein kinase II (CKII) inhibitor, 5,6‐dichloro‐1‐β‐ d ‐ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3‐fold at 24 h post‐infection. In contrast, 100 μ m of the CKII inhibitor reduced the titre of the M type only 1.3‐fold at 48 h post‐infection. Our data suggest that the different growth of U‐ and M‐type IHNV in RTG‐2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.

Journal of Fish Diseases↗

USGS1999

Sperm-cell ultrastructure of North American sturgeons II: The shortnose sturgeon (Acipenser brevirostrum, Lesueur, 1818)

The fine structure of the sperm cell of the shortnose sturgeon ( Acipenser brevirostrum ) was examined using transmission electron microscopy and selected metrics. The cell possesses a distinct acrosome, a defined head region, a midpiece, and a single flagellum. The mean length of the sperm cell body (acrosome + nucleus + midpiece) is approximately 9.71 µm, and the length of the flagellum is about 37 µm, resulting in a total cell length of about 46 µm. The sperm cell of the shortnose sturgeon is much longer and slightly wider than that of the Atlantic sturgeon. The nuclei of shortnose, white, and stellate sturgeon sperm cells are elongate trapezoids with the anterior (acrosome) end narrowest, the opposite of that of the Atlantic sturgeon. Although slightly smaller in total length and width than the sperm cells of the stellate and white sturgeons, that of the shortnose sturgeon is most similar to them in overall ultrastructure, as all three cells have three endonuclear canals. A structural connection of unknown function between the nuclear fossa and the proximal centriole, which is similar to the fibrous body in other species, is present in the shortnose sturgeon sperm cell. Our results suggest a more recent evolutionary link between the shortnose, white, and stellate sturgeons than between any of these and the Atlantic sturgeon. This is the first description of sperm cell ultrastructure in the shortnose sturgeon, an endangered species.

Canadian Journal of Zoology↗

USGS2009

Distinct freshwater and seawater isoforms of Na+/K+-ATPase in gill chloride cells of Atlantic salmon

Gill Na(+)/K(+)-ATPase (NKA) in teleost fishes is involved in ion regulation in both freshwater and seawater. We have developed and validated rabbit polyclonal antibodies specific to the NKA alpha1a and alpha1b protein isoforms of Atlantic salmon (Salmo salar Linnaeus), and used western blots and immunohistochemistry to characterize their size, abundance and localization. The relative molecular mass of NKA alpha1a is slightly less than that for NKA beta1b. The abundance of gill NKA alpha1a was high in freshwater and became nearly undetectable after seawater acclimation. NKA alpha1b was present in small amounts in freshwater and increased 13-fold after seawater acclimation. Both NKA isoforms were detected only in chloride cells. NKA alpha1a was located in both filamental and lamellar chloride cells in freshwater, whereas in seawater it was present only as a faint background in filamental chloride cells. In freshwater, NKA alpha1b was found in a small number of filamental chloride cells, and after seawater acclimation it was found in all chloride cells on the filament and lamellae. Double simultaneous immunofluorescence indicated that NKA alpha1a and alpha1b are located in different chloride cells in freshwater. In many chloride cells in seawater, NKA alpha1b was present in greater amounts in the subapical region than elsewhere in the cell. The combined patterns in abundance and immunolocalization of these two isoforms can explain the salinity-related changes in total NKA and chloride cell abundance. The results indicate that there is a freshwater and a seawater isoform of NKA alpha-subunit in the gills of Atlantic salmon and that they are present in distinct chloride cells.

Journal of Experimental Biology↗

USGS2010

Establishment and partial characterization of a cell line from burbot Lota lota maculosa: susceptibility to IHNV, IPNV and VHSV.

This study describes the development and partial characterization of a continuous fibroblastic-like cell line (BEF-1) developed from late stage embryos of North American burbot Lota lota maculosa. This cell line has been maintained for over 5 yr and 100 passages in vitro. Cells were cultured using Eagle’s minimum essential medium with Earle’s salts (MEM) supplemented with GlutaMAX™, and 10% fetal bovine serum (FBS), pH 7.4. The addition of penicillin-streptomycin-neomycin (PSN) antibiotic mixture (0.05, 0.05, 0.1 mg ml–1, respectively) did not negatively influence cell replication; however, the antimycotic Fungizone™ (2.5 µg ml–1, amphotericin B) caused cell rounding and resulted in a severe decrease in cell proliferation. Optimal incubation temperature has been observed between 15 and 23°C, and at these temperatures cultures are routinely passed using standard trypsinization methods every 5 to 7 d at a split ratio of 1:3 or 1:4. The cell line was susceptible to isolates of the M and U North American genotypes of infectious hematopoietic necrosis virus (IHNV), and to isolates of genotypes I, IVa, and IVb of viral hemorrhagic septicemia virus (VHSV). In contrast, the cell line was refractory to infection by 2 North American isolates of infectious pancreatic necrosis virus (IPNV) from serotypes A1 and A9. This cell line provides a new laboratory tool, will allow further investigation into viral diseases of burbot and possibly other species, and is the first immortalized cell line reported from a species in the Gadidae (cod) family.

Diseases of Aquatic Organisms↗

USGS2022

Histochemical evidence for nitrogen‐transfer Endosymbiosis in non‐photosynthetic cells of leaves and inflorescence bracts of angiosperms

We used light and confocal microscopy to visualize bacteria in leaf and bract cells of more than 30 species in 18 families of seed plants. Through histochemical analysis, we detected hormones (including ethylene and nitric oxide), superoxide, and nitrogenous chemicals (including nitric oxide and nitrate) around bacteria within plant cells. Bacteria were observed in epidermal cells, various filamentous and glandular trichomes, and other non-photosynthetic cells. Most notably, bacteria showing nitrate formation based on histochemical staining were present in glandular trichomes of some dicots (e.g., Humulus lupulus and Cannabis sativa ). Glandular trichome chemistry is hypothesized to function to scavenge oxygen around bacteria and reduce oxidative damage to intracellular bacterial cells. Experiments to assess the differential absorption of isotopic nitrogen into plants suggest the assimilation of nitrogen into actively growing tissues of plants, where bacteria are most active and carbohydrates are more available. The leaf and bract cell endosymbiosis types outlined in this paper have not been previously reported and may be important in facilitating plant growth, development, oxidative stress resistance, and nutrient absorption into plants. It is unknown whether leaf and bract cell endosymbioses are significant in increasing the nitrogen content of plants. From the experiments that we conducted, it is impossible to know whether plant trichomes evolved specifically as organs for nitrogen fixation or if, instead, trichomes are structures in which bacteria easily colonize and where some casual nitrogen transfer may occur between bacteria and plant cells. It is likely that the endosymbioses seen in leaves and bracts are less efficient than those of root nodules of legumes in similar plants. However, the presence of endosymbioses that yield nitrate in plants could confer a reduced need for soil nitrogen and constitute increased nitrogen-use efficiency, even if the actual amount of nitrogen transferred to plant cells is small. More research is needed to evaluate the importance of nitrogen transfer within leaf and bract cells of plants

Biology↗

USGS1991

Effects of prolacton chloride cells opercular membrane of seawater-adapted tilapia

Effects of prolactin on morphology and numbers of chloride cells in the opercular membrane of seawater-adapted tilapia ( Oreochromis mossambicus ) have been examined. Following five daily injections of ovine prolactin at a dose of 10 μg · g body wt −1 , blood samples were taken and opercular membranes were removed and stained with a fluorescent mitochondrial dye (dimethylaminostyrylethylpyridiniumiodine), a fluorescent derivative of ouabain (anthroylouabain), and a histological stain specific for the extensive tubular system of chloride cells (zinc-osmium-iodine). Mean plasma osmolarity and sodium increased 23–24% following prolactin injection. An increase in the relative frequency of chloride cells between 20 and 180 μm 2 in cross-sectional area and a decrease in the relative frequency of chloride cells greater than 180 μm 2 were observed following prolactin injections. Average cell size decreased 46–70% and cell height decreased 26–38% following prolactin injections. There was no significant change in cell density. Anthroylouabain staining was observed in both prolactin- and saline-injected fish, and no significant effect on Na + ,K + -adenosinetri-phosphatase activity was seen in either opercular membrane or gill tissue. The results demonstrate an effect of prolactin on chloride cell size and provide a morphological correlate for decreased secretory activity of chloride cells following prolactin injections.

General and Comparative Endocrinology↗

USGS2018

Development of a dual luciferase activity and fluorescamine protein assay adapted to a 384 micro-well plate format: Reducing variability in human luciferase transactivation cell lines aimed at endocrine active substances

There is a need to adapt cell bioassays to 384-well and 1536-well formats instead of the traditional 96-well format as high-throughput screening (HTS) demands increase. However, the sensitivity and performance of the bioassay must be re-verified in these higher micro-well plates, and verification of cell health must also be HT (high-throughput). We have adapted two commonly used human breast luciferase transactivation cell bioassays, the recently re-named estrogen agonist/antagonist screening VM7Luc4E2 cell bioassay (previously designated BG1Luc4E2) and the androgen/glucocorticoid screening MDA-kb2 cell bioassay, to 384-well formats for HTS of endocrine-active substances (EASs). This cost-saving adaptation includes a fast, accurate, and easy measurement of protein amount in each well via the fluorescamine assay with which to normalize luciferase activity of cell lysates without requiring any transfer of the cell lysates. Here we demonstrate that by accounting for protein amount in the cell lysates, antagonistic agents can easily be distinguished from cytotoxic agents in the MDA-kb2 and VM7Luc4E2 cell bioassays. Additionally, we demonstrate via the fluorescamine assay improved interpretation of luciferase activity in wells along the edge of the plate (the so-called “edge effect”), thereby increasing usable wells to the entire plate, not just interior wells.

Toxicology in Vitro↗

USGS1996

Uruguay Roselli 1938 and Rosellichnus, N. Ichnogenus: Two ichnogenera for clusters of fossil bee cells

The systematics of the fossil bee nest ichnogenus Uruguay from the Uruguayan Cretaceous or lower Tertiary is reviewed and two new ichnospecies of a new ichnogenus, Rosellichnus, are proposed for fossil bee nests from the Miocene of the United Arab Emirates and Patagonian Argentina. Uruguay contains U. auroranormae (the ich‐notype) and U. rivasi, which differs from the type ichnospecies in having clustered cells arranged in three rows, with one central and two marginal rows of subparallel cells. No bee nest architecture known to us unequivocally fits with any ichnospecies described herein. Uruguay was constructed in the subsoil and may be of halictid origin; however, its large cell size, thick cell walls, and the presence of a vestibular cell do not fit the architecture of nests of extant halictids. Rosellichnus arabicus lacks the complete cavity surrounding the cell cluster that is typical in nests of Old World halictids, but it may, nonetheless, still be of halictid origin. R. pa‐tagonicus has an architecture more similar to nests of anthophorine bees than to those of Neotropical Halictidae. Therefore, the ichnogenus Rosellichnus may include both halictid and anthophorine constructions. Although the smooth inner cell walls, spiral cell caps, and clustering of cells indicate that both Uruguay and Rosellichnus are trace fossils of bee origin, it is likely that both represent nest architectures that are no longer employed by extant bees, or those that are as yet unknown. The paleoenvironments of both the Argentine and Emirates species of Rosellichnus indicate that the trace‐makers of this ichnogenus constructed their nests in open ground sandy soils.

Ichnos: An International Journal for Plant and Ani↗

USGS2003

Nuclear transfer of synchronized African wild cat somatic cells into enucleated domestic cat oocytes

The African wild cat is one of the smallest wild cats and its future is threatened by hybridization with domestic cats. Nuclear transfer, a valuable tool for retaining genetic variability, offers the possibility of species continuation rather than extinction. The aim of this study was to investigate the ability of somatic cell nuclei of the African wild cat (AWC) to dedifferentiate within domestic cat (DSH) cytoplasts and to support early development after nuclear transplantation. In experiment 1, distributions of AWC and DSH fibroblasts in each cell-cycle phase were assessed by flow cytometry using cells cultured to confluency and disaggregated with pronase, trypsin, or mechanical separation. Trypsin (89.0%) and pronase (93.0%) yielded higher proportions of AWC nuclei in the G0/G1 phase than mechanical separation (82.0%). In contrast, mechanical separation yielded higher percentages of DSH nuclei in the G0/G1 phase (86.6%) than pronase (79.7%) or trypsin (74.2%) treatments. In both species, pronase induced less DNA damage than trypsin. In experiment 2, the effects of serum starvation, culture to confluency, and exposure to roscovitine on the distribution of AWC and DSH fibroblasts in various phases of the cell cycle were determined. Flow cytometry analyses revealed that the dynamics of the cell cycle varied as culture conditions were modified. Specifically, a higher percentage of AWC and DSH nuclei were in the G0/G1 phase after cells were serum starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition (61% vs. 88%), or after roscovitine (56% vs. 84%) treatment, respectively. In experiment 3, we evaluated the effects of cell synchronization and oocyte maturation (in vivo vs. in vitro) on the reconstruction and development of AWC-DSH- and DSH-DSH-cloned embryos. The method of cell synchronization did not affect the fusion and cleavage rate because only a slightly higher percentage of fused couplets cleaved when donor nuclei were synchronized by serum starvation (83.0%) than after roscovitine (80.0%) or contact-inhibition (80.0%). The fusion efficiency of in vivo and in vitro matured oocytes used as recipient cytoplasts of AWC donor nuclei (86.6% vs. 85.2%) was similar to the rates obtained with DSH donor nuclei, 83.7% vs. 73.0%, respectively. The only significant effect of source of donor nucleus (AWC vs. DSH) was on the rate of blastocyst formation in vitro. A higher percentage of the embryos derived from AWC nuclei developed to the blastocyst stage than did embryos produced from DSH nuclei, 24.2% vs. 3.3%, respectively (P < 0.05). In experiment 4, the effect of calcium in the fusion medium on induction of oocyte activation and development of AWC-DSH-cloned embryos was determined. The presence of calcium in the fusion medium induced a high incidence of cleavage of DSH oocytes (54.3%), while oocyte cleavage frequency was much lower in the absence of calcium (16.6%). The presence or absence of calcium in the fusion medium did not affect the fusion, cleavage, and blastocyst development of AWC-DSH-cloned embryos. In experiment 5, AWC-DSH-cloned embryos were transferred to the uteri of 11 synchronized domestic cat recipients on Day 6 or 7 after oocyte aspiration. Recipients were assessed by ultrasonography on Day 21 postovulation, but no pregnancies were observed. In the present study, after NT, AWC donor nuclei were able to dedifferentiate in DSH cytoplasts and support high rates of blastocyst development in vitro. Incomplete reprogramming of the differentiated nucleus may be a major constraint to the in vivo developmental potential of the embryos.

Biology of Reproduction↗

USGS2018

Development and characterization of two cell lines from gills of Atlantic salmon

Gill disease in Atlantic salmon, Salmo salar L., causes big losses in the salmon farming industry. Until now, tools to cultivate microorganisms causing gill disease and models to study the gill responses have been lacking. Here we describe the establishment and characterization of two cell lines from the gills of Atlantic salmon. Atlantic salmon gill cell ASG-10 consisted of cells staining for cytokeratin and e-cadherin and with desmosomes as seen by transmission electron microscopy suggesting the cells to be of epithelial origin. These structures were not seen in ASG-13. The cell lines have been maintained for almost 30 passages and both cell lines are fully susceptible to infection by infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), infectious pancreatic necrosis virus (IPNV), Atlantic salmon reovirus TS (TSRV) and Pacific salmon paramyxovirus (PSPV). While infectious salmon anemia virus (ISAV) did not cause visible CPE, immunofluorescent staining revealed a sub-fraction of cells in both the ASG-10 and ASG-13 lines may be permissive to infection. ASG-10 is able to proliferate and migrate to close scratches in the monolayer within seven days in vitro contrary to ASG-13, which does not appear to do have the same proliferative and migratory ability. These cell lines will be useful in studies of gill diseases in Atlantic salmon and may represent an important contribution for alternatives to experimental animals and studies of epithelial–mesenchymal cell biology.

PLoS ONE↗

USGS1999

Establishment and characterization of 13 cell lines from a green turtle (Chelonia mydas) with fibropapillomas

Thirteen cell lines were established and characterized from brain, kidney, lung, spleen, heart, liver, gall bladder, urinary bladder, pancreas, testis, skin, and periorbital and tumor tissues of an immature male green turtle (Chelonia mydas) with fibropapillomas. Cell lines were optimally maintained at 30A? C in RPMI 1640 medium supplemented with 10% fetal bovine serum. Propagation of the turtle cell lines was serum dependent, and plating efficiencies ranged from 13 to 37%. The cell lines, which have been subcultivated more than 20 times, had a doubling time of approximately 30 to 36 h. When tested for their sensitivity to several fish viruses, most of the cell lines were susceptible to a rhabdovirus, spring viremia carp virus, but refractory to channel catfish virus (a herpesvirus), infectious pancreatic necrosis virus (a birnavirus), and two other fish rhabdoviruses, infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus. During in vitro subcultivation, tumor-like cell aggregates appeared in cell lines derived from lungs, testis, and periorbital and tumor tissues, and small, naked intranuclear virus particles were detected by thin-section electron microscopy. These cell lines are currently being used in attempts to isolate the putative etiologic virus of green turtle fibropapilloma.

In Vitro Cellular and Developmental Biology - Anim↗

USGS2009

Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (20 C)

Usually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 °C/min in a low-temperature (−80 °C) freezer (LTF) before storage in liquid nitrogen (LN 2 ); however, a LTF is not always available. The purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen in a LTF or conventional freezer (CF; −20 °C) and their subsequent ability for development to blastocyst stage after fusion with enucleated bovine oocytes. Percentages of live cells frozen in LTF (49.5%) and CF (50.6%) were similar, but significantly less than non-frozen control (88%). In both CF and LTF, percentages of live apoptotic cells exposed to LN 2 after freezing were lower (4% and 5%, respectively) as compared with unexposed cells (10% and 18%, respectively). Cells frozen in a CF had fewer cell doublings/24 h (0.45) and required more days (9.1) to reach 100% confluence at the first passage (P) after thawing and plating as compared with cells frozen in a LTF (0.96 and 4.0 days, respectively). Hypoploidy at P12 was higher than at P4 in cells frozen in either a CF (37.5% vs. 19.2%) or in a LTF (30.0% vs. 15.4%). A second-generation cryo-solution reduced the incidence of necrosis (29.4%) at 0 h after thawing as compared with that of a first generation cryo-solution (DMEM + DMSO, 60.2%). The percentage of apoptosis in live cells was affected by cooling rate (CF = 1.9% vs. LFT = 0.7%). Development of bovine cloned embryos to the blastocyst stage was not affected by cooling rate or freezer type.

Zygote↗

USGS2006

The proinflammatory cytokine interleukin-18 alters multiple signaling pathways to inhibit natural killer cell death

The proinflammatory cytokine, interleukin-18 (IL-18), is a natural killer (NK) cell activator that induces NK cell cytotoxicity and interferon-?? (IFN-??) expression. In this report, we define a novel role for IL-18 as an NK cell protective agent. Specifically, IL-18 prevents NK cell death initiated by different and distinct stress mechanisms. IL-18 reduces NK cell self-destruction during NK-targeted cell killing, and in the presence of staurosporin, a potent apoptotic inducer, IL-18 reduces caspase-3 activity. The critical regulatory step in this process is downstream of the mitochondrion and involves reduced cleavage and activation of caspase-9 and caspase-3. The ability of IL-18 to regulate cell survival is not limited to a caspase death pathway in that IL-18 augments tumor necrosis factor (TNF) signaling, resulting in increased and prolonged mRNA expression of c-apoptosis inhibitor 2 (cIAP2), a prosurvival factor and caspase-3 inhibitor, and TNF receptor-associated factor 1 (TRAF1), a prosurvival protein. The cumulative effects of IL-18 define a novel role for this cytokine as a molecular survival switch that functions to both decrease cell death through inhibition of the mitochondrial apoptotic pathway and enhance TNF induction of prosurvival factors. ?? Mary Ann Liebert, Inc.

Journal of Interferon and Cytokine Research↗

USGS1976

Differential inhibition of host protein synthesis in L cells infected with RNA - temperature-sensitive mutants of vesicular stomatitis virus

The response of mouse L cells to infection with wild-type (wt) and temperature-sensitive (ts) mutants of vesicular stomatitis virus was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to delineate the synthesis of host cell and viral proteins. Experiments utilized transcriptase mutants of complementation group I (ts114 and ts13), a group IV mutant (ts44) that is restricted in total RNA synthesis (RNA-1) but not in primary transcription, and a group II mutant (ts52) variably restricted in RNA synthesis (RNA +/-). L cells infected with ts mutants at permissive temperature exhibited the wt response of progressive inhibition of host cell protein synthesis accompanied by accumulation of all five viral proteins. Mutant ts44 (IV) also switched off cell protein synthesis at restrictive temperature and accumulated all five viral proteins, but with disproportionate ratios of N and G proteins. At restrictive temperature, cells infected with group I ts mutants failed to accumulate any viral protein and did not exhibit significant reduction in host cell protein synthesis. These data suggest that vesicular stomatitis virus inhibits cell protein synthesis at a stage of viral infection after transcription and possibly translation but preceding replication of progeny viral RNA.

Journal of Virology↗

USGS1989

Enhanced detection of infectious hematopoietic necrosis virus by pretreatment of cell monolayers with polyethylene glycol

To improve quantification of very low levels of infectious hematopoietic necrosis virus (IHNV) in samples of tissue, ovarian fluid, or natural water supplies, we tested the ability of polyethylene glycol (PEG) to enhance the sensitivity and speed of the plaque assay system. We compared 4, 7, and 10% solutions of PEG of molecular weight 6,000, 8,000, or 20,000 applied at selected volumes and for various durations. When cell monolayers of epithelioma papulosum cyprini (EPC), fathead minnow (FHM), chinook salmon embryo (CHSE-214), and bluegill fry (BF2) were pretreated with 7% PEG-20,000, they produced 4-17-fold increases in plaque assay titers of IHNV. The plaque assay titers of viral hemorrhagic septicemia virus, chum salmon reovirus, and chinook salmon paramyxovirus were also enhanced by exposure of CHSE-214 cells to PEG, but the titers of infectious pancreatic necrosis virus and Oncorhynchus masou virus were not substantially changed. Plaques formed by IHNV on PEG-treated EPC cells incubated at 15°C had a larger mean diameter at 6 d than those on control cells at 8 d; this suggests the assay could be shortened by use of PEG. Pretreatment of EPC cell monolayers with PEG enabled detection of IHNV in some samples that appeared negative with untreated cells. For example, when ovarian fluid samples from chinook salmon Oncorhynchus tshawytscha were inoculated onto untreated monolayers of EPC cells, IHNV was detected in only 11 of 51 samples; 17 of the samples were positive when PEG-treated EPC cells were used. PDF

Journal of Aquatic Animal Health↗

USGS1971

Blood cell lineage in the sea lamprey, Petromyzon marinus (Pisces: Petromyzontidae)

Blood cell types of the sea lamprey, Petromyzon marinus , are described and identified and the lineage of mature circulating cells in peripheral blood is traced to blast cells in the hematopoietic fat body. The fat body appears to be the phylogenetic precursor of bone marrow in higher forms, since blood cells originate and begin maturation in this tissue. Experimental animals were injected first with a hematopoietic stimulant and then (at an experimentally determined time) with pertussis vaccine to release proliferated blood cells into peripheral blood. Peripheral blood for smears was collected by cardiac exsanguination; hematopoietic tissue was extirpated for imprints; and leucocyte preparations were made by a special technique. Blood cells of the sea lamprey are apparently products of at least four distinct blast cells, each of which has a 'one end' maturation process. Results of this investigation support the polyphyletic theory of blood cell formation.

Copeia↗

USGS2003

Alternative Sources of Energy - An Introduction to Fuel Cells

Fuel cells are important future sources of electrical power and could contribute to a reduction in the amount of petroleum imported by the United States. They are electrochemical devices similar to a battery and consist of a container, an anode, a cathode, catalysts, an intervening electrolyte, and an attached electrical circuit. In most fuel cell systems, hydrogen is supplied to the anode and oxygen to the cathode which results in the production of electricity, water, and heat. Fuel cells are comparatively efficient and reliable, have no moving parts, operate without combustion, and are modular and scale-able. Their size and shape are flexible and adaptable. In operation, they are nearly silent, are relatively safe, and generally do not pollute the environment. During recent years, scientists and engineers have developed and refined technologies relevant to a variety of fuel cells. Types of fuel cells are commonly identified by the composition of their electrolyte, which could be either phosphoric acid, an alkaline solution, a molten carbonate, a solid metal oxide, or a solid polymer membrane. The electrolyte in stationary power plants could be phosphoric acid, molten carbonates, or solid metal oxides. For vehicles and smaller devices, the electrolyte could be an alkaline solution or a solid polymer membrane. For most fuel cell systems, the fuel is hydrogen, which can be extracted by several procedures from many hydrogen-bearing substances, including alcohols, natural gas (mainly methane), gasoline, and water. There are important and perhaps unresolved technical problems associated with using fuel cells to power vehicles. The catalysts required in several systems are expensive metals of the platinum group. Moreover, fuel cells can freeze and not work in cold weather and can be damaged by impacts. Storage tanks for the fuels, particularly hydrogen, must be safe, inexpensive, of a reasonable size, and contain a supply sufficient for a trip of several hundred miles. Additional major problems will be the extensive and costly changes in the national infrastructure to obtain, store, and distribute large amounts of the fuels, and in related manufacturing

Bulletin↗