Gary K. Ostrander

Capillary electrophoresis of carboxylated carbohydrates. I. Selective precolumn derivatization of gangliosides with UV absorbing and fluorescent tags.

We demonstrate that the precolumn derivatization reaction, recently introduced by our laboratory for the selective labeling of carboxylated monosaccharides, can be readily transposed to other glycoconjugates containing carboxylated sugar residues, namely sialogangliosides. The selective derivatization reaction described here involved the attachment of sulfanilic acid (a UV-absorbing tag) or 7-aminonaphthalene-1,3-disulfonic acid (a UV-absorbing and also fluorescing tag) to the sialic acid moiety of the gangliosides via the carboxylic group in the presence of water-soluble carbodiimide. This labeling of the sialic acid moiety of the gangliosides with a chromophore and/or fluorophore leads to the formation of an amide bond between the carboxylic group of the sugar residue and the amino group of the derivatizing agent, thus replacing the weak carboxylic acid group of the carbohydrate species by the stronger sulfonic acid group which is ionized over the entire pH range. Furthermore, novel electrolyte systems were introduced and evaluated for the separation of the derivatized and underivatized gangliosides. The addition of acetonitrile or alpha-cyclodextrin (alpha-CD) to the running electrolyte was necessary to break-up the aggregation of amphiphilic gangliosides and allowed for their efficient separation as monomers in aqueous media using capillary electrophoresis. Several operating parameters were investigated with these electrolyte systems including the additive concentration as well as the ionic strength, pH and nature of the running electrolyte. Acetonitrile at 50% (v/v) in 5 mM sodium phosphate at high and low pH or 15 mM alpha-CD in 100 mM sodium borate, pH 10.0, proved ideal, in terms of resolution and separation efficiency, for the group separation of mono-, di- and trisialogangliosides. On the other hand, the complete resolution of disialoganglioside isomers (e.g., GD1a and GD1b) necessitated the superimposition of a chromatographic component on the electrophoretic process. This was achieved by adding either a hydrophobic (e.g., decanoyl-N-methylglucamide-borate surfactant complex) or hydrophilic [e.g., poly(vinyl alcohol) or hydroxypropyl cellulose] selectors to the running electrolyte.

Coral bleaching: a potential biomarker of environmental stress.

Coral bleaching refers to the loss of symbiotic algae by host corals, or to the loss of pigmentation by the algae themselves, causing corals to appear white or bleached. Some corals may regain algae or pigmentation and survive, but when bleaching is severe the host coral dies. Coral bleaching events have increased dramatically in the last two decades, and coral reefs throughout the world have been extensively degraded as a result. This article reviews coral bleaching for investigators working in the field of toxicology and environmental health, a group of scientists not normally exposed to this issue. Several environmental stressors have been correlated with bleaching, including fluctuations in sea surface temperatures and salinity, increased sedimentation, increased solar radiation, and contaminants such as oil and herbicides. Molecular mechanisms of bleaching are only beginning to be investigated and are thus far poorly understood. Toxicologists have the potential to make significant contributions toward understanding anthropogenic aspects of coral bleaching and elucidating molecular mechanisms of this important environmental problem.

LONG-TERM PRIMARY CULTURE OF EPITHELIAL CELLS FROM RAINBOW TROUT (ONCORHYNCHUS MYKISS) LIVER

SummaryLong-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (>97%) populations of hepatocytes were placed into primary culture and remained viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures for at least 30 d. Finally, a third type of epithelial cell, which we have termed a “spindle cell,” consistently appeared and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and passaged.The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis, and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation or pluripotent cells, were observed with increased time in primary culture.

A ceramide analogue (PDMP) inhibits glycolipid synthesis in fish embryos

Glycolipids were depleted from medaka embryos using 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glucosylceramide synthetase. Embryos cultured in the presence of 20 microM PDMP exhibited a dramatic decline in glycolipid synthesis and cell surface expression. Metabolic labeling of glucosylceramide declined by 87% on Days 3-6 of development and 72% on Days 7-10 (hatching occurred on Day 10). In parallel, PDMP-treated embryos exhibited a striking loss of several tissue-specific glycolipid antigens, including 9-O-acetyl GD3 from brain and retina, GT3/GQ1C from brain, neural tube, and retina, and sulfated glycolipid from skin and gut. Despite these changes in glycolipid expression, PDMP-treated embryos were fully viable with no evidence of developmental abnormality. PDMP appears to provide a useful tool for identifying glycolipid antigens in embryos and investigating their role in development.

Relationships among petroleum refining, water and sediment contamination, and fish health

Water, sediment, and fish were sampled from three streams that were receiving or had received effluents from oil refineries. Water and sediment samples were analyzed by gas chromatography/mass spectrometry. Each stream contained aromatic carbons including substituted benzenes and naphthalenes, which are related to oil refinery operations. Fish were identified, counted, and examined for external lesions. Lengths and weights were recorded for older bullhead catfish, and their livers were examined histologically. Differences were seen in the diversity and abundance of fish among the upstream, impacted (effluent-receiving), and downstream stations. In one stream, differences in liver pathology were observed between reference bullhead, collected from an upstream station, and those collected at impacted stations with more than 50% of the bullheads taken from impacted stations having some sort of pathological change, including one with a liver clear-cell focus, which is considered a preneoplastic lesion in rodents. These data suggest a correlation between contamination of water and sediments with aromatic hydrocarbons, presumably from refinery effluents, and compromised fish health.

Isolation and characterization of biliary epithelial cells from rainbow trout liver

SummaryLectin binding and density gradient centrifugation were explored for isolating epithelial cells from trout liver. Hepatocytes exhibited preferential attachment to coverslips coated withPhaseolus vulgaris erythroagglutinin. Biliary epithelial cells attached with glycine max agglutinin; however, significant attachment of cellular debris limited the use of glycine max agglutinin. Percoll-density gradient centrifugation separated liver cells into two distinct populations with biliary cells and hepatocytes banding at densities of 1.04 and 1.09, respectively. A discontinuous gradient composed of 13% Ficoll (wt/wt) separated biliary cells from hepatocytes. The recovery of highly enriched biliary epithelial cells from trout liver using Ficoll gradients yielded approximately 8 million cells (0.1 ml packed cells) from 10 g liver. Western blot analysis demonstrated that the cytokeratin profile for extracts from biliary epithelial cell-enriched populations differ significantly from those seen with whole liver extracts or with extracts from hepatocyte-enriched populations. Ficoll-gradient purified biliary cells and hepatocytes attached to culture plates coated with trout skin extract and carried out linear incorporation of leucine into protein and thymidine into DNA for 24 h. A mixture of growth hormones (insulin, epidermal growth factor, and dexamethasone) stimulated thymidine incorporation into DNA; however, long-term culture of dividing biliary epithelial cells was not achieved. Chemical analysis of neutral and acidic glycolipids indicated that hepatocytes and biliary cells have similar glycolipid profiles with an exception in the region of GM3 mobility, which is attributable to differences in the ceramide moiety. These studies provide a starting point for further characterization of unique cell types of the trout liver that may be important in their response to toxic and carcinogenic agents.

Response of rainbow trout liver to partial hepatectomy

Abstract Existing procedures for partial hepatectomy in rainbow trout (Oncorhynchus mykiss) liver were modified and a greater than 95% success rate achieved. The initial response of the rainbow trout liver to partial hepatectomy was evaluated. DNA synthesis, determined by tritiated thymidine incorporation into DNA in liver slices, increased significantly in the first 24–48 h following partial hepatectomy and remained elevated for at least 10–14 days. Protein content based on wet weight of the liver, however, appeared to remain constant in the two weeks following partial hepatectomy. The procedure was equally effective on both large (600 g) and small (6–80 g) fish and should be useful for either promotion in complete carcinogenesis regimens or other toxicological studies requiring liver cell proliferation.

Isolation and characterization of the major glycosphingolipids from the liver of the rainbow trout (Oncorhynchus mykiss): Identification of an abundant source of 9-O-acetyl GD3

The carbohydrate structures of the major glycosphingolipids from the liver of the rainbow trout Oncorhynchus mykiss have been examined. We have isolated and identified four major neutral (glucosylceramide, galactosylceramide, lactosylceramide, and globoside) and five acidic (sulfatide, GM3, GM2, GD1a, and 9-O-Acetyl GD3) glycosphingolipids from trout liver. They have been characterized by 1H nuclear magnetic resonance spectroscopy, methylation analysis, fast atom bombardment mass spectrometry, and specific monoclonal antibodies. Significantly, the relatively scarce ganglioside 9-O-acetyl GD3 was found to comprise approximately 23% of the total ganglioside content of normal rainbow trout liver. 9-O-Acetyl GD3 is, however, abundant in human melanoma and as such, trout liver may be a suitable source of this antigen.

Poorly Differentiated Mesenchymal Neoplasm in the Dermis of a White Bass

Abstract A poorly differentiated subcutaneous neoplasm occurred in a white bass Morone chrysops collected from a freshwater lake in Oklahoma. The neoplasm was a solitary soft round mass that bulged from the anal fin. Histologically, the neoplasm was lobular and consisted of islands of spindle-to- stellate cells separated by clefts. The islands frequently contained capillary profiles and deposits of an amorphous eosinophilic material. The lesion was mesenchymal and suggestive of a poorly differentiated hemangiopericytoma, though it might have derived from nerve sheath, pigment cells, fibroblasts, or smooth muscle. An epizootic of another type of dermal neoplasm, a poorly differentiated spindle cell tumor, has been reported in gizzard shad Dorosoma cepedianum residing in the same lake. However, etiology of the neoplasms from both the white bass and the shad remains uncertain.

Characterization of a CMPNeuAc : lactosylceramide α2→3sialyltransferase from rainbow trout hepatoma (RTH-149) cells

1. The rainbow trout (Oncorhynchus mykiss) CMPNeuAc:lactosylceramide alpha 2----3sialytransferase enzyme from RTH-149 cells has been characterized. 2. Transfer of sialic acid to lactosylceramide was optimal at a pH of 5.9, temperature of 25 degrees C, and in the pressure of 0.3% CF-54, 10 mM Mn2+, 0.1 M sodium cacodylate, and 2 mM ATP. 3. Golgi-rich membrane fractions of RTH-149 cells were found to be enriched in sialidase activity and as such the addition of 40 microM 2,3-dehydro-2-deoxy-N-acetylneuraminic acid was necessary to assay alpha 2----3sialyltransferase activity optimally. 4. Apparent Km for donor (CMPNeuAc) and acceptor (lactosylceramide) were found to be 243 microM and 34 microM, respectively. 5. The alpha 2----3sialyltransferase characterized was found to be primarily specific for lactosylceramide though minor activity with other glycolipid acceptors was observed. 6. The presence of another sialyltransferase with differing substrate specificity was noted. 7. Properties of this enzyme, compared to analogous mammalian enzymes, are discussed.