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At least 19 recordsLinked to original sources
USGS2006

Cell proliferation detected with flow cytometric cell cycle analysis and immunohistochemical detection of Proliferating Cell Nuclear Antigen (PCNA) from somatic tissues of Eastern Oysters, Crassostrea virginica

Two novel biomarker of response assays were developed and compared for use with bivalves. Bivalve mollusks are often used as bioindicators to monitor contaminant body burdens and are employed globally in pollution monitoring and as sentinels of environmental quality. The prevalence of proliferating cells in tissues of the eastern oyster, Crassostrea virginica , (n = 10) was investigated by using immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and by flow cytometric analysis of DNA in cell cycle phases. The PCNA protein is highly conserved and central to DNA synthesis in dividing cells. This protein was detected by using a commercial antibody in the histological staining of gills, mantle, digestive gland, stomach, and labial palps. Cells obtained from these same organs and from the hearts of the oysters were used for flow cytometry (FCM). The proportions of nuclei in the S plus G2/M and G2/M fractions were compared with percentages of PCNA-positive cells scored through microscopy. Proliferation responses generated by microscopy and FCM (S plus G2/M) were equal for digestive gland, gills, stomach, and mantle. The proliferation response in labial palps was significantly higher by PCNA microscopy than by DNA FCM (S plus G2/M). For PCNA microscopy results, a significantly higher proliferation response was noted both for labial palps as compared to gills and mantle and for stomach and digestive gland as compared to gills (P < 0.005). For DNA FCM results, the percentages of proliferating cells in G2M fractions were significantly higher for labial palps and heart when compared to gills, mantle, and digestive gland. By FCM (S plus G2M), the percentages of proliferating cells followed a similar trend, although no significant differences were found between organs. The choice of a relatively highly proliferative tissue, such as labial palps as determined by this study, has utility for developing a cell line, for examining proliferation as a biomarker due to stressor impacts in feral oysters, and for detecting impacts of compounds on proliferative mechanisms.

Environmental Bioindicators↗
USGS2020

Cytology reveals diverse cell morphotypes and cellin-cell interactions in normal collector sea urchins Tripneustes gratilla

Echinoderms such as sea urchins are important in marine ecosystems, particularly as grazers, and unhealthy sea urchins can have important ecological implications. For instance, unexplained mortalities of Diadema antillarum in the Caribbean were followed by algal overgrowth and subsequent collapse of coral reef ecosystems. Unfortunately, few tools exist to evaluate echinoderm health, making management of mortalities or other health issues problematic. Hematology is often used to assess health in many animal groups, including invertebrates, but is seldom applied to echinoderms. We used a standard gravitometric technique to concentrate fixed coelomocytes from the collector sea urchin Tripneustes gratilla onto microscope slides, permitting staining and enumeration. Using Romanowsky stain and electron microscopy to visualize cell details, we found that urchin cells could be partitioned into different morphotypes. Specifically, we enumerated phagocytes, phagocytes with perinuclear cytoplasmic dots, vibratile cells, colorless spherule cells, red spherule cells, and red spherule cells with pink granules. We also saw cell-in-cell interactions characterized by phagocytes apparently phagocytizing mainly the motile cells including red spherule cells, colorless spherule cells, and vibratile cells disproportionate to underlying populations of circulating cells. Cell-in-cell interactions were seen in 71% of sea urchins, but comprised <1% of circulating cells. Finally, about 40% of sea urchins had circulating phagocytes that were apparently phagocytizing spicules. The coelomic fluid collection and slide preparation methods described here are simple, field portable, and might be a useful complementary tool for assessing health of other marine invertebrates, revealing heretofore unknown physiological phenomena in this animal group.

Hawaii↗
USGS2010

Derivation of cat embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells

The domestic cat is a focal mammalian species that is used as a model for developing assisted reproductive technologies for preserving endangered cats and for studying human diseases. The generation of stable characterized cat embryonic stem cells (ESC) lines to use as donor nuclei may help to improve the efficiency of interspecies somatic cell nuclear transfer for preserving endangered cats and allow the creation of knockout cell lines to generate knockout cats for studying function of specific genes related to human diseases. It will also enable the possibility of producing gametes in vitro from ESC of endangered cats. In the present study, we report the generation of cat embryonic stem-like (cESL) cells from blastocysts derived entirely in vitro. We generated 32 cESL cell lines from 331 in vitro derived blastocysts from which inner cell masses were isolated by immunosurgery or by a mechanical method. Inhibition of cat dermal fibroblast (CDF) proliferation after exposure to mitomycin-C was both dose and time dependent, where doses of 30 to 40 microg/mL for 5 h were most efficient. These dosages were higher than that required to inhibit cell proliferation of mouse fetal fibroblasts (MFF; 10 microg/mL for 2.5 h). Mitomycin-C did not significantly increase necrosis of cells from either species, and had an anti-proliferative effect at concentrations below cytotoxicity. A clear species-specific relationship between feeder layers and derivation of cESL cell lines was observed, where higher numbers of cESL cell lines were generated on homologous cat feeder layers (n = 26) than from those derived on heterologous mouse feeder layers (n = 6). Three cESL cell lines generated from immunosurgery and cultured on CDF maintained self-renewal and were morphologically undifferentiated for nine and twelve passages (69-102 days). These lines showed a tightly packed dome shaped morphology, exhibited alkaline phosphatase activity and immuno-expression of the pluripotent marker OCT-4 and surface marker SSEA-1. Primary colonies at P0 to P3 and cat blastocysts expressed transcription factors OCT-4, NANOG and SOX-2 and the proto-oncogene C-MYC. However, expression was at levels significantly lower than in vitro produced blastocysts. During culture, cESL colonies spontaneously differentiated into fibroblasts, cardiomyocytes, and embryoid bodies. Development of techniques to prevent differentiation of cESL cells will be essential for maintaining defined cell lines

Theriogenology↗
USGS2001

Antibody-producting cells correlated with body weight in juvenile Chinook salmon Oncorhynchus tshawytscha acclimated to optimal and elevated temperatures

The immune response of juvenile chinook salmon ( Oncorhynchus tshawytscha ) ranging in weight from approximately 10 to 55 g was compared when the fish were acclimated to either 13 or 21° C. A haemolytic plaque assay was conducted to determine differences in the number of antibody-producing cells (APC) among fish of a similar age but different body weights. Regression analyses revealed significant increases in the number of APC with increasing body weight when fish were acclimated to either water temperature. These results emphasise the importance of standardising fish weight in immunological studies of salmonids before exploring the possible effects of acclimation temperatures.

Fish & Shellfish Immunology↗
USGS2002

IL-2 and IL-12 alter NK cell responsiveness to IFN-γ-inducible protein 10 by down-regulating CXCR3 expression

Cytokine treatment of NK cells results in alterations in multiple cellular responses that include cytotoxicity, cytokine production, proliferation, and chemotaxis. To understand the molecular mechanisms underlying these responses, microarray analysis was performed and the resulting gene expression patterns were compared between unstimulated, IL-2, IL-2 plus IL-12, and IL-2 plus IL-18-stimulated NK92 cells. RNase protection assays and RT-PCR confirmed microarray predictions for changes in mRNA expression for nine genes involved in cell cycle progression, signal transduction, transcriptional activation, and chemotaxis. Multiprobe RNase protection assay also detected changes in the expression of CCR2 mRNA, a gene that was not imprinted on the microarray. We subsequently expanded our search for other chemokine receptor genes absent from the microarray and found an IL-2- and IL-12-dependent decrease in CXCR3 receptor mRNA expression in NK92 cells. A detailed analysis of CXCR3 expression in primary NK cells revealed that an IL-2 and an IL-12 together significantly decreased the CXCR3 receptor mRNA and receptor surface expression by 6 and 24 h of treatment, respectively. This decrease in receptor expression was associated with a significant reduction in chemotaxis in the presence of IFN-γ-inducible protein-10. The decline in CXCR3 mRNA was due to transcriptional and posttranscriptional mechanisms as the addition of actinomycin D to IL-2- and IL-12-treated NK92 slightly altered the half-life of the CXCR3 mRNA. Collectively, these data suggest that IL-2 and IL-12 directly affect NK cell migratory ability by rapid and direct down-regulation of chemokine receptor mRNA expression. Natural killer (NK) cells are large granular lymphocytes that play an important role in the defense against virally infected or malignant cells (1). Their activity can be characterized as nonadaptive and independent of MHC restriction (1, 2). A variety of NK cell functions including cytotoxicity, proliferation, chemotaxis, and cytokine production are modulated by regulatory cytokines including IFN-αβ, IL-2, IL-12, IL-18, IL-10, and TNF (reviewed in Refs. 3 and 4). Because cytokines induce such a broad range of effects in NK cells, the potential for alterations in gene expression in stimulated cells is very great. To determine which genes are regulated in response to cytokine stimulation, our laboratory has used cDNA microarray technology to examine gene expression in NK cells. Microarray technology is very useful because it allows for large-scale examination of gene expression. Additionally, this technology has proved useful in identifying physiologically relevant gene expression patterns in eukaryotic systems such as yeast (5) and fibroblasts (6) as well as predicting patterns of gene expression in tumor cells (7, 8). To examine gene expression in response to cytokine stimulation, a human NK cell line, NK92, was stimulated with IL-2 alone or in combination with IL-12 or IL-18. These cytokines were chosen because of their ability to induce NK cell responses; however, little is known about the repertoire of genes that are activated by these cytokines. Microarray analysis of gene expression in NK92 cells identified a variety of genes whose mRNA expression patterns change in response to cytokine stimulation. The genes encoding the mRNAs are not specific to any one pathway; however, changes in cytokine, chemokine, and chemokine receptor gene mRNAs were prevalent. Our mRNA studies on chemokine receptor gene expression were extended to cell surface analysis of receptor densities in cytokine-treated primary NK cells. Using FACS analysis, we observed a significant decrease in CXCR3 receptor expression in NK cells treated for 24 h with IL-2 and IL-12 alone or in combination. Recently, alterations in chemokine receptor expression were reported in IL-2-stimulated NK cells (9); however, the cells were cultured in IL-2 for 8–10 days. In contrast, our data demonstrate that cytokines can modify chemokine receptor function within hours, thus supporting a model whereby cytokines, in particular IL-2 and IL-12, regulate chemokine receptor expression in a direct, rapid, and novel manner.

Journal of Immunology↗
USGS1998

Immunocyctochemical and ultrastructural identification of pituitary cell types in the protogynous Thalassoma duperrey during adult sexual ontogeny

Protogynous wrasses ( Thalassoma duperrey ): females (F), primary males (PM) along with a few terminal-phase males (TM) and sex-changed males (SM), were used to characterize the topographical organization of the pituitary. In general, immunocytochemical and ultrastructural features of the adenohypo-physeal cell types of the saddleback wrasse pituitary resemble those of other teleosts. In the rostral pars distalis (RPD), corticotropic cells were found bordering the neurohypophysis (NH) and surrounding the centroventrally located prolactin cells. Thyrotropic cells formed a small group in the anteriodorsal part of the rostral and proximal pars distalis (PPD). The somatotropic cells were distributed in large clusters, mostly organized in cell cords around the interdigitations of the NH of the dorsal PPD. Cells containing gonadotropin Ib subunit were localized in the dorsal parts of the PPD, in close association with somatotropic cells and gonadotropin IIb subunit containing cells were seen in the centroventral parts of the PPD and along the periphery of the pars intermedia (PI). The pars intermedia was composed of melanotropic cells and somatolactin cells that lined the neurohypohysis. Distinct ultrastructural differences in corticotropic and somatotropic cells were not observed between the four groups. In all groups, prolactin cells in the ventral-most RPD could be immature cells or actively secreting prolactin. Gonadotropic II cells of PM and F had relatively higher incidence of “nuclear budding” and cell organelles compared to TM and SM. Besides gonadotropic, the active melanotropic and somatolactin cells might be associated with some aspect(s) of reproduction.

Zoological Science↗
USGS1998

Immunocytochemical and ultrastructural identification of pituitary cell types in the protogynous Thalassoma duperrey during adult sexual ontogeny

Protogynous wrasses (Thalassoma duperrey): females (F), primary males (PM) along with a few terminal-phase males (TM) and sex-changed males (SM), were used to characterize the topographical organization of the pituitary. In general, immunocytochemical and ultrastructural features of the adenohypophyseal cell types of the saddleback wrasse pituitary resemble those of other teleosts. In the rostral pars distalis (RPD), corticotropic cells were found bordering the neurohypophysis (NH) and surrounding the centroventrally located prolactin cells. Thyrotropic cells formed a small group in the anteriodorsal part of the rostral and proximal pars distalis (PPD). The somatotropic cells were distributed in large clusters, mostly organized in cell cords around the interdigitations of the NH of the dorsal PPD. Cells containing gonadotropin I?? subunit were localized in the dorsal parts of the PPD, in close association with somatotropic cells and gonadotropin II?? subunit containing cells were seen in the centroventral parts of the PPD and along the periphery of the pars intermedia (PI). The pars intermedia was composed of melanotropic cells and somatolactin cells that lined the neurohypohysis. Distinct ultrastructural differences in corticotropic and somatotropic cells were not observed between the four groups. In all groups, prolactin cells in the ventral-most RPD could be immature cells or actively secreting prolactin. Gonadotropic II cells of PM and F had relatively higher incidence of "nuclear budding" and cell organelles compared to TM and SM. Besides gonadotropic, the active melanotropic and somatolactin cells might be associated with some aspect(s) of reproduction.

Zoological Science↗
USGS1996

Hydrogeology of the area near the J4 test cell, Arnold Air Force Base, Tennessee

The U.S. Air Force operates a major aerospace systems testing facility at Arnold Engineering Development Center (AEDC) in Coffee County, Tennessee. Dewatering operations at one of the test facilities, the J4 test cell, has affected the local ground-water hydrology. The J4 test cell is approximately 100 feet in diameter, extends approximately 250 feet below land surface, and penetrates several aquifers. Ground water is pumped continuously from around the test cell to keep the cell structurally intact. Because of the test cell's depth, dewatering has depressed water levels in the aquifers surrounding the site. The depressions that have developed exhibit anisotropy that is controlled by zones of high permeability in the aquifers. Additionally, contaminants - predominately volatile organic compounds - are present in the ground-water discharge from the test cell and in ground water at several other Installation Restoration Program (IRP) sites within the AEDC facility. The dewatering activities at J4 are drawing these contaminants from the nearby sites. The effects of dewatering at the J4 test cell were investigated by studying the lithologic and hydraulic characteristics of the aquifers, investigating the anisotropy and zones of secondary permeability using geophysical techniques, mapping the potentiometric surfaces of the underlying aquifers, and developing a conceptual model of the ground-water-flow system local to the test cell. Contour maps of the potentiometric surfaces in the shallow, Manchester, and Fort Payne aquifers (collectively, part of the Highland Rim aquifer system) show anisotropic water-level depressions centered on the J4 test cell. This anisotropy is the result of features of high permeability such as chert-gravel zones in the regolith and fractures, joints, and bedding planes in the bedrock. The presence of these features of high permeability in the Manchester aquifer results in complex flow patterns in the Highland Rim aquifers near the J4 test cell. The occurrence, distribution, and orientation of these features has a great effect on ground-water flow to the J4 test cell. The depression caused by dewatering extends out horizontally through the aquifers along the most permeable pathways. Since the aquifers above the Chattanooga Shale are not separated by distinct confining units, areas in adjacent aquifers above and below these zones of high permeability in the Manchester aquifer are also dewatered. Conditions in all Highland Rim aquifers approximate steady-state equilibrium because ground-water withdrawal at the test cell has been continuous since the late 1960's. The average ground-water discharge from the dewatering system at the J4 test cell was 105 gallons per minute, for 1992-95. The ground-water capture areas in each aquifer extend into all or parts of landfill #2 and leaching pit #2 (IRP site 1), the main testing area (IRP site 7), and the old fire training area (IRP site 10). IRP sites 8 and 12 are outside the ground-water capture areas. Of the 35 sampled wells in the J4 area, 10 produced water samples containing chlorinated organic compounds such as 1,2-dichloroethane (1,2-DCA), 1,1-dichloroethylene (1,1-DCE), and trichloroethylene (TCE) in concentrations which exceeded the Tennessee Department of Environment and Conservation Maximum Contaminant Levels (MCL's) for public water-supply systems. The highest concentrations were detected in samples from well AEDC-274 with 45 micrograms per liter (mg/L) 1,2-DCA, 320 mg/L 1,1-DCE, and 1,200 mg/L TCE. These compounds are synthetic and do not occur naturally in the environment. A sample of the ground-water discharge from the J4 test cell also contained concentrations of these compounds that exceed MCL's. Chlorinated organic compounds, including 1,2-DCA; 1,1-DCE; and TCE also have been detected at IRP sites 1, 7, 8, nd 10. The six dewatering wells surrounding the J4 test cell penetrate the Chattanooga Shale and are open to the Highland Rim aquifer system, there

Water-Resources Investigations Report↗
USGS2005

Functional classification of mitochondrion-rich cells in euryhaline Mozambique tilapia (Oreochromis mossambicus) embryos, by means of triple immunofluorescence staining for Na+/K+-ATPase, Na +/K+/2Cl- cotransporter and CFTR anion channel

Mozambique tilapia Oreochromis mossambicus embryos were transferred from freshwater to seawater and vice versa, and short-term changes in the localization of three major ion transport proteins, Na+/K +-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR) were examined within mitochondrion-rich cells (MRCs) in the embryonic yolk-sac membrane. Triple-color immunofluorescence staining allowed us to classify MRCs into four types: type I, showing only basolateral Na+/K +-ATPase staining; type II, basolateral Na+/K +-ATPase and apical NKCC; type III, basolateral Na+/K +-ATPase and basolateral NKCC; type IV, basolateral Na +/K+-ATPase, basolateral NKCC and apical CFTR. In freshwater, type-I, type-II and type-III cells were observed. Following transfer from freshwater to seawater, type-IV cells appeared at 12 h and showed a remarkable increase in number between 24 h and 48 h, whereas type-III cells disappeared. When transferred from seawater back to freshwater, type-IV cells decreased and disappeared at 48 h, type-III cells increased, and type-II cells, which were not found in seawater, appeared at 12 h and increased in number thereafter. Type-I cells existed consistently irrespective of salinity changes. These results suggest that type I is an immature MRC, type II is a freshwater-type ion absorptive cell, type III is a dormant type-IV cell and/or an ion absorptive cell (with a different mechanism from type II), and type IV is a seawater-type ion secretory cell. The intracellular localization of the three ion transport proteins in type-IV cells is completely consistent with a widely accepted model for ion secretion by MRCs. A new model for ion absorption is proposed based on type-II cells possessing apical NKCC.

Journal of Experimental Biology↗
USGS2008

An assessment of the effects of cell size on AGNPS modeling of watershed runoff

This study investigates the changes in simulated watershed runoff from the Agricultural NonPoint Source (AGNPS) pollution model as a function of model input cell size resolution for eight different cell sizes (30 m, 60 m, 120 m, 210 m, 240 m, 480 m, 960 m, and 1920 m) for the Little River Watershed (Georgia, USA). Overland cell runoff (area-weighted cell runoff), total runoff volume, clustering statistics, and hot spot patterns were examined for the different cell sizes and trends identified. Total runoff volumes decreased with increasing cell size. Using data sets of 210-m cell size or smaller in conjunction with a representative watershed boundary allows one to model the runoff volumes within 0.2 percent accuracy. The runoff clustering statistics decrease with increasing cell size; a cell size of 960 m or smaller is necessary to indicate significant high-runoff clustering. Runoff hot spot areas have a decreasing trend with increasing cell size; a cell size of 240 m or smaller is required to detect important hot spots. Conclusions regarding cell size effects on runoff estimation cannot be applied to local watershed areas due to the inconsistent changes of runoff volume with cell size; but, optimal cells sizes for clustering and hot spot analyses are applicable to local watershed areas due to the consistent trends.

Cartography and Geographic Information Science↗
USGS1990

Establishment of a cell line with reticulo-endothelial characteristics from a rainbow trout spleen explant

A cell line from rainbow trout ( Oncorhynchus mykiss ) with phagocytic-like characteristics was established. First we found that it was necessary to inject the fish with antibiotics (penicillinstreptomycin) in order to clear tissue of bacterial contamination. Two days later, the spleen was excised and minced with scissors in MEM10. The large number of erythrocytes in the suspension were removed by low centrifugation, then the tissue fragments were incubated on tissue culture flasks at 18°C for 30 days with complete changes of media every other day. After 30 days in culture, primary explants from spleen were composed of three types of cells. The majority were large mononucleate, plecmorphic cells with thinly spread cytoplasm. These cells were trypsinized and subcultured. Another cell types was simple, round cells, somewhat similar to mature lymphocytes, while the third type appeared to be similar to fibroblastic cells. After 9 months with the culture in 17 to 20 passages, the predominate cell type was showing similarities to phagocytic cell. These cells had a marked reticular cytoplasm and grew individually without parallel orientations. By enzyme-specific stains, the cells were observed to have abundant acid phosphatase and β-glucuronidase activities of the cytoplasm. They were negative for peroxidase. The cells were also found to have highly adherent and phagocytic properties when incubated with latex particles. The morphological, enzymatic, and functional findings suggested the possibility that these cells in culture have some properties similar to the cells which are composed of reticulo-endothelial system.

Fish Pathology↗
USGS2011

Fish T cells: recent advances through genomics

This brief review is intended to provide a concise overview of the current literature concerning T cells, advances in identifying distinct T cell functional subsets, and in distinguishing effector cells from memory cells. We compare and contrast a wealth of recent progress made in T cell immunology of teleost, elasmobranch, and agnathan fish, to knowledge derived from mammalian T cell studies. From genome studies, fish clearly have most components associated with T cell function and we can speculate on the presence of putative T cell subsets, and the ability to detect their differentiation to form memory cells. Some recombinant proteins for T cell associated cytokines and antibodies for T cell surface receptors have been generated that will facilitate studying the functional roles of teleost T cells during immune responses. Although there is still a long way to go, major advances have occurred in recent years for investigating T cell responses, thus phenotypic and functional characterization is on the near horizon.

Developmental and Comparative Immunology↗
USGS2006

Temperature comparisons for antibody production in vitro by plaque-forming cells from trout Salmo gairdneri (Richardson), and mice

Anterior kidney and splenic cells were taken from rainbow trout and splenic cells from BALB/c mice immunized with a T-dependent (sheep red blood cells) or T-independent (DNP-Ficoll) antigen. The cells were incubated at different temperatures in Jerne plaque assays (direct or passive haemolytic plaque assays). The optimum numbers of in vitro plaque-forming cells (PFC) after incubation with homologous complement were directly correlated with normal body temperatures of the respective species. The optimum incubation temperature was 37°C for mouse cells and 10°C for fish cells. Incubation of mouse cells at lower temperatures of 30, 20, 10, 4 or 0°C appeared to yield a direct line reduction in numbers of PFC. Trout cells developed significantly fewer PFC at 4 and 20°C and none at 30°C or above; however, significant numbers still appeared at 0°C. More PFC per million white blood cells were obtained from the anterior kidney; however, related to temperatures, no differences in development of numbers of PFC could be seen between the spleen and anterior kidney cells of trout. When the incubation time was lengthened for both trout and mouse cells held at low temperatures, the numbers of PFC approached those of the cells incubated at the optimum temperatures for 10 h.

Journal of Fish Biology↗