Glycosidase activities in Chinese hamster ovary cell lysate and cell culture supernatant

To probe the potential for extracellular degradation of glycoprotein oligosaccharides in conjunction with Chinese hamster ovary (CHO) cell culture, an initial characterization of several CHO cell glycosidases was performed using 4-methylumbelliferyl substrates. CHO cell lysates contained sialidase, beta-galactosidase, beta-hexosaminidase, and fucosidase activities with pH optimums near 5.5, 4, 6, and 6.5, respectively. These glycosidase activities were also present in cell-free supernatant samples from commercial CHO cell cultures. The sialidase activity was further characterized. In contrast to previous reports concerning mammalian sialidases, the sialidase activity in CHO cell lysate retained considerable activity at pH 7 and was very stable, with a half-life of 57 h at 37 degrees C. Both the Km and Vmax of CHO lysate sialidase for 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (4MU-NeuAc) varied with pH, and this activity was competitively inhibited by 2,3-dehydro-2-deoxy-N-acetylneuraminic acid and by free N-acetylneuraminic acid. The kinetic characteristics and pH-activity profiles of the CHO cell lysate and cell culture supernatant sialidase activities were essentially identical, and both released sialic acid from the glycoprotein fetuin at pH 7.5. These results suggest that the oligosaccharides of glycoproteins secreted by CHO cells can potentially be modified extracellularly by sialidase under culture conditions which promote the release and extracellular accumulation of this enzyme.     

Lectin histochemistry of rainbow trout (Oncorhynchus mykiss) gill and skin

In order to characterize the glycoconjugate residues in skin and gills of the adult rainbow trout, the binding pattern of five biotinylated lectins with different carbohydrate specificities was examined. In the skin, mucous cells revealed binding sites for PNA and SBA; filament-containing cells were additionally labelled with Con A. However, the basal cell layer showed no reaction. In the gill, subpopulations of mucous cells reacted with Con A, PNA, SBA and UEA-I. This broader spectrum of glycoconjugates in gill mucous cells compared with the epidermal mucous cells could point to the additional function of gill mucus in ion and osmoregulation. Lectin binding sites were less common in the respiratory epithelial cells of the secondary lamellae than in those of the primary lamellae. Chloride cells revealed mannose, galactose and fucose residues. Immature chloride cells, as indicated by a comparison with Na+/K+ ATPase immunolabelling, reacted with Con A; subpopulations of them reacted with PNA, SBA and UEA-I. The results form the basis for further investigations in which these cell populations can be analysed under different environmental conditions.     

Reproductive success of female rainbow trout (Oncorhynchus mykiss) in response to graded dietary ascorbyl monophosphate levels

Ascorbic acid is an essential nutrient in rainbow trout diets and has been shown to play an important role in fish reproduction. Recommended dietary levels are based on immature fish, and the specific requirements for brood stock are unknown. To establish the optimum dietary level for mature rainbow trout, six graded levels of ascorbyl-2-monophosphate were fed to groups of female fish over a period of 10 mo until spawning. Increasing dietary levels of ascorbyl monophosphate resulted in significantly increased ascorbic acid concentrations in liver, kidney, ovaries, and ovulated eggs. Liver and egg concentrations were saturable at 109.3 and 266.6 micrograms ascorbic acid/g tissue, respectively. Tissue saturation levels of 83.7% and 91.2%, respectively, were reached at the highest dietary level (870 mg ascorbyl monophosphate/kg diet) tested. Both fecundity and embryo survival increased significantly with dietary ascorbyl monophosphate levels. The results indicated that the present National Research Council recommended dietary level of 50 mg ascorbic acid/kg diet for rainbow trout is inadequate for brood stock fish. An amount 8 times higher is necessary to optimize tissue ascorbic acid levels and achieve maximum reproductive success.     

Cloning of the rainbow trout (Oncorhynchus mykiss) Mx2 and Mx3 cDNAs and characterization of trout Mx protein expression in salmon cells

Two rainbow trout (Oncorhynchus mykiss) Mx cDNAs were cloned by using RACE (rapid amplification of cDNA ends) PCR and were designated RBTMx2 and RBTMx3. The deduced RBTMx2 and RBTMx3 proteins were 636 and 623 amino acids in length with molecular masses of 72 and 70.8 kDa, respectively. These proteins, along with the previously described RBTMx1 protein (G. D. Trobridge and J. A. Leong, J. Interferon Cytokine Res. 15:691-702, 1995), have between 88.7 and 96.6% identity at the amino acid level. All three proteins contain the tripartite GTP binding domain and leucine zipper motif common to Mx proteins. A monospecific polyclonal antiserum to an Escherichia coli-expressed fragment of RBTMx3 was generated, and that reagent was found to react with all three rainbow trout Mx proteins. Subsequently, endogenous Mx production in RTG-2 cells induced with poly(IC) double-stranded RNA was detected by immunoblot analysis. The cellular localization of the rainbow trout proteins was determined by transient expression of the RBTMx cDNAs in CHSE-214 (chinook salmon embryo) cells. A single-cell transient-transfection assay was used to examine the ability of each Mx cDNA clone to inhibit replication of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV). No significant inhibition in the accumulation of the IHNV nucleoprotein was observed in cells expressing either trout Mx1, Mx2, or Mx3 in transiently transfected cells.     

Three-dimensional visualization of physiologically based kinetic model outputs

Outputs from a physiologically based toxicokinetic (PB-TK) model for fish were visualized by mapping time-series data for specific tissues onto a three-dimensional representation of a rainbow trout. The trout representation was generated in stepwise fashion: 1) cross-section images were obtained from an anesthetized fish using a magnetic resonance imaging system, 2) images were processed to classify tissue types and eliminate unnecessary detail. 3) processed images were imported to a visualization software package (Application Visualization System) to create a three-dimensional representation of the fish, encapsulating five volumes corresponding to the liver, kidney, muscle, gastrointestinal tract, and fat, Kinetic data for the disposition of pentachloroethane in trout were generated using a PB-TK model. Model outputs were mapped onto corresponding tissues volumes, representing chemical concentration as color intensity. The workstation software was then used to animate the images, illustration the accumulation of pentachloroethane in each tissue during a continuous branchial (gill) exposure.     

Immune response to synthetic peptides representing antigenic sites on the glycoprotein of infectious hematopoietic necrosis virus

Monoclonal antibodies against infectious hematopoietic necrosis virus have been used to react with recombinant expression products in immunoblots and to select neutralization-resistant mutants for sequence analysis. These strategies identified neutralizing and non-neutralizing antigenic sites on the viral glycoprotein. Synthetic peptides based upon the amino acid sequences of these antigenic sites were synthesized and were injected together with an adjuvant into rainbow trout. The constructs generally failed to stimulate neutralizing antibodies in the fish. These results indicate that we need to understand more about the ability of peptide antigens to stimulate fish immune systems.     

cDNA cloning of the beta subunit of teleost thyrotropin

cDNA clones encoding the beta subunit of thyrotropin (thyroid-stimulating hormone; TSH) were isolated from a cDNA library made from the pituitaries of immature rainbow trout and sequenced. The precursor of rainbow trout TSH beta consists of 147 aa, which can be cleaved into a signal peptide (20 aa) and a mature protein (127 aa) containing one potential N-glycosylation site and 12 cysteine residues. The protein showed highest homology with human TSH beta (51%) and lesser homology with human follitropin (42%), human lutropin (32%), and salmon gonadotropin (31-33%) beta subunits. The identification of TSH in addition to two gonadotropins (gonadotropins I and II) in the teleost fish suggests that the divergence of three kinds of glycoprotein hormones from an ancestral molecule took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods. Northern blot analysis showed that the expression of the rainbow trout TSH beta gene is specific to the pituitary gland and is significantly higher in immature fish than in mature fish, suggesting that TSH plays some role in the biological processes of immature fish.     

Magnetosensitivity of the epiphysis

Small intestine goblet cell numbers and the composition of their mucus were compared in guinea pigs with genetically determined differences in responsiveness to Trichostrongylus colubriformis infection. Prior to infection, no differences between high responder and low responder animals were detected. However, following primary infection with T. colubriformis, pronounced goblet cell hyperplasia developed and the proportion of sulphomucin in these cells increased. Both changes developed significantly earlier in high responder animals.     

Intestinal mucin distribution in the germ-free rat and in the heteroxenic rat harbouring a human bacterial flora: effect of inulin in the diet

A colorimetric method was used on water-soluble mucin extracted from mucosal scrapings and contents of the caecum and the colon of five germ-free (GF) rats and five heteroxenic (HE) rats harbouring a human flora (GF rats associated with a human flora). These rats were fed on a diet containing either 100 g sucrose/kg or 100 g inulin/kg. Histological stains, periodic acid-Schiff, alcian blue pH 2.5 and alcian blue pH 0.5 were used to discriminate between neutral, acidic and acidic sulphated mucins respectively. Spectrocolorimetric assays led to a calculated absorbance value for 1 mg of the initial mucin extract. Each mucin type was compared between treatments. The caecal contents of GF rats contained more acidic mucin than sulphomucin, which was present in the same proportion as neutral mucin. Their colonic contents contained more acidic mucins than sulphomucin, which in turn was more abundant than neutral mucin. Their caecal mucosa mucin distribution differed from that of the contents: very little acidic mucin was present and neutral and sulphomucin proportions were of the same order of magnitude. Inulin increased the amount of neutral mucin in the caecal contents and of sulphated mucins in the colonic contents and increased the amounts of neutral and acidic mucins in the caecal mucosa. Mucin distribution in the HE rats was very different from that in the GF rats: the caecal contents contained a high proportion of acidic mucins and very little sulphomucin. The same distribution of mucins was observed in the colonic contents. The caecal mucosa contained less acidic mucin and more sulphomucin than the caecal contents. Inulin decreased acidic mucins and increased sulphated mucins in the caecal contents and increased neutral and sulphated mucins in the colonic contents. Inulin increased sulphomucin in the caecal mucosa and decreased acidic mucin in the caecal and colonic mucosas. The very low amount of mucin that was recovered in the colonic mucosa suggests that, in the presence of the bacterial flora and associated with inulin in the diet, mucin was extensively released from the mucosa to the colonic lumen. This might be related to the bacterial metabolites produced.     

Ability of 16 priority PAHs to be photocytotoxic to a cell line from the rainbow trout gill

Sixteen polycyclic aromatic hydrocarbons (PAHs) were screened for their ability to be photocytotoxic to a cell line from the rainbow trout gill, RTgill-W1. PAHs could be divided into one of three groups: incapable of being photocytotoxic, able to be both photocytotoxic and directly cytotoxic, or capable of being only photocytotoxic. Photocytotoxicity was distinct from direct cytotoxicity in that EC50 values were lower with the neutral red assay immediately after the PAH/UV treatment than with alamar Blue or CFDA-AM, indicating a more specific action on lysosomes. As well, in photocytotoxicity but not in direct cytotoxicity, the three assays showed increased impairment 24 h after treatment. Most PAHs were found to be strictly photocytotoxic; however, only six compounds were photocytotoxic at concentrations theoretically achievable in water. When photocytotoxic PAHs were ranked relative to fluoranthene to establish fluoranthene equivalent factors (FEFs), benzo[a]pyrene and benzo[g,h,i]perylene were found to be most potent. However, when the water solubility of each compound was taken into account in order to calculate the potential environmental photocytotoxic potency (PEPP), fluoranthene and pyrene appeared to have the most potential to impact fish through photocytotoxicity.     

Immunolocalization of a novel annexin-like protein encoded by a stress and abscisic acid responsive gene in alfalfa

Rainbow trout (Oncorhynchus mykiss, 1-5 g) were exposed to approximately 7.5 microM Co in soft water for 2-3 hr at pH approximately 6.5. The water contained either complexing ligands such as nitrilotriacetic acid and natural dissolved organic matter (DOM) or competing cations such as Ca, Na, or H. After exposure, gills were excised and analyzed for total bound Co using a graphite furnace atomic absorption spectrophotometer. A Langmuir isotherm was used to calculate the conditional equilibrium binding constant (K) for Co binding to trout gills plus the concentrations of gill Co binding sites. The calculated binding constant for Co to trout gills was log KCo-gillCo = 5.1, with 88 nmol Co binding sites per g of wet gill tissue. Conditional equilibrium binding constants were also calculated for Ca, Na, and H binding to the gill Co sites and for Co binding to DOM. The experimentally determined binding constants were entered into an aquatic equilibrium chemistry program, MINEQL+, to predict Co binding by fish gills. Predicted and observed results indicate that Co would not accumulate on or in gills of trout held in a series of natural and 1:1 diluted natural waters supplemented with approximately 8.7 microM Co. Model analysis of the reasons for Co being kept off gills of trout held in natural waters indicated that Ca competition and DOM complexation were the most important factors in preventing Co binding by trout gills.     

Variation of influenza viruses and their recognition of the receptor sialo-sugar chains

Influenza A, B viruses contain 2 viral specific, membrane associated glycoprotein antigens, hemagglutinin and sialidase. Hemagglutinin is essential for the initial binding of the virus to the cell membrane receptors that contain sialic acid such as gangliosides and sialo-glycoproteins. Hemagglutinin is also important for the intracellular viral uncoating by the low pH fusion processes. The evolution of the influenza viruses and host range variation come from the mutation of hemagglutinin and sialidase genes and change of their sialo-sugar chain recognition together with alteration in the antigenic epitopes. In this report, the molecular mechanism of the relationship between the evolutional change of the viral glycoproteins, especially hemagglutinin molecules and the change of the receptor binding specificity is reported, and also the strategy for the development of a new universal vaccine which generates the antibody whose supervariable region mimics the common receptor sialo-sugar chains for all the subtypes of influenza viruses is also described.     

Biological and toxicological effects of solvent extraction chemicals: Range finding acute toxicity in the rainbow trout (Salmo gairdnerii Rich.) and in the rat (Rattus norwegicus L.)

The acute toxicity of twelve solvent extraction chemicals was studied in the albino rat after intraperitoneal injection of the chemicals, and in the rainbow trout (Salmo gairdnerii Rich.) after exposure to the chemicals in the aquaria water. The tested chemicals included the following technical products: one primary aliphatic amine (Primene JM-T), two secondary aliphatic amines (Amberlite LA-1 and Adogen 283), two tertiary aliphatic amines (Adogen 383 and Alamine 336), one quaternary amine (Aliquat 336), two organophosphorus compounds (TBP, tributyl phosphate and HDEHP, di-(2-ethyl)-hexylphosphoric acid), one synthetic carboxylic acid (Versatic 10), one naphthalene-sulphonic acid (NA-SUL AS-50), one substituted oxime (LIX 64N) and one aliphatic alcohol (2-ethylhexanol). The intraperitoneal 96 hours LD 50 in the rat for the tested chemicals varied between less than 50 mg/kg b.w. to more than 5000 mg/kg b.w. Ninety six hours LC 50 in the rainbow trout was not possible to determine for three of the twelve tested chemicals due to their low water solubility. When determined, the LC 50 at 15°C was in the range 0.11–56 mg/l based on the amounts added.The present study discusses the acute toxicity of the chemicals mentioned above, gross pathological changes in the rat and behavioural changes in the fish.     

Aah VI, a novel, N-glycosylated anti-insect toxin from Androctonus australis hector scorpion venom: isolation, characterisation, and glycan structure determination

A full-length cDNA for retinoblastoma (RB1) has been cloned from a cDNA library prepared from 3-week-old rainbow trout (Oncorhynchus mykiss) eyed embryos. The trout RB1 cDNA encodes a predicted protein of 910 amino acids and is the most divergent cloned retinoblastoma gene sequence to date. RT-PCR studies reveal high levels of RB1 expression by the second week of embryogenesis, which remains uniformly expressed until hatching. Expression studies of adult fish tissues show the RB1 gene to be expressed in all tissues examined, including the oesophagus, eye, liver, intestine, posterior and anterior kidney, skin, stomach, muscle, spleen, gill, swim bladder, gonads and brain. The RB1 gene appears to be a single copy gene based on Southern analysis, and maps to linkage group XVI in the trout genome map. Polymorphisms in the RB1 gene and in closely linked markers should facilitate LOH analysis of RB1.     

Evaluation of the critical body burden concept based on inorganic and organic mercury toxicity to rainbow trout (Oncorhynchus mykiss)

Subadult rainbow trout (Oncorhynchus mykiss) were exposed to four waterborne concentrations each of 64-426 microg/L mercuric chloride (HgCl2) and 4-34 microg/L methylmercury chloride (CH3HgCl) until death to evaluate the critical body burden concept. Mean days to death for fish exposed to the highest and lowest concentrations of HgCl2 were 1 and 58 d, and 2 and > 100 d for fish exposed to CH3HgCl. Time to death was an important factor that influenced Hg tissue concentration, and was most evident among fish that died within a few days of exposure. Critical body burdens for Hg could be difficult to establish at the tissue level because no threshold concentrations were clearly indicated among the liver, kidney, spleen, brain, muscle, and gill that were monitored in this study. A critical burden for Hg was derived on a whole body basis for Hg in its organic form. An evaluation of this and other studies suggests whole body concentrations of 10-20 mg/kg Hg could be lethal to fish. Extrapolation from other studies indicate whole body concentrations of 1-5 mg/kg Hg could have chronic effects on fish and possibly other aquatic organisms. This concept could be used to assess the toxicological significance of chemical concentrations that are monitored in feral aquatic organisms. The tissue-based approach appears to have some advantages over current assessment protocols that focus on waterborne concentrations.     

Purification and characterization of urotensin II from the brain of a teleost (trout, Oncorhynchus mykiss) and an elasmobranch (skate, Raja rhina)

Peptides related to urotensin II have been isolated in pure form from an extract of whole brain of a teleost, the rainbow trout (Oncorhynchus mykiss) and of an elasmobranch, the longnose skate (Raja rhina). The primary structure of the trout peptide [Gly-Gly-Asn-Ser-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val] is similar to that of urotensin II peptides isolated from the urophyses of other teleost fish. For example, trout urotensin II contains only one amino acid substitution (Thr4-->Ser) compared with urotensin II beta 1 isolated from the urophysis of the carp. The primary structure of the skate peptide [Asn-Asn-Phe-Ser-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val] is the same as urotensin II isolated from the caudal spinal cord region of the dogfish Scyliorhinus canicula. The data provide chemical evidence to support the conclusion of earlier morphological studies [Yulis, C. R., and Lederis, K. (1988) Gen. Comp. Endocrinol. 70, 301-311) that certain species of fish possess an extensive extraurophyseal distribution of urotensin II-immunoreactive neurons.     

Evidence for an endogenous clock in the retina of rainbow trout: II. Circadian rhythmicity of serotonin metabolism

The purpose of the present study was to investigate patterns of circadian rhythmicity in the retina of a salmonid fish, the rainbow trout (Oncorhynchus mykiss). Our data present the first demonstration of intraretinal variations of serotonergic substances under light/dark-conditions (LD) and during continuous darkness (DD). All substances examined (serotonin, N-acetyl serotonin, 5-hydroxytryptophol, 5-hydroxyindole acetic acid) were rhythmic in LD. Serotonin, N-acetyl-serotonin, and 5-hydroxyindole acetic acid showed a preservation of specific features of rhythmicity in DD indicating the involvement of an endogenous pacemaker in the regulation of serotonin metabolism in the rainbow trout eye.     

Purification and characterization of metallothionein and its activation of pyridoxal phosphokinase in trout (Salmo gairdneri) brain

1. Brain metallothionein was isolated and purified for the first time from rainbow trout. 2. Brain metallothionein exhibited an elution volume (Ve/Vo) of 2.0, had a molecular weight of 6762 Daltons, and contained a zinc content of 9 micrograms/mg protein. 3. Brain pyridoxal phosphokinase was isolated and assayed for the first time in rainbow trout. 4. Zinc (0.20 microM) or zinc metallothionein (6-30 microM) stimulated the activity of brain pyridoxal kinase in a linear fashion. 5. The results of these studies are interpreted to suggest that in trout brain zinc metallothionein may participate in metabolism of vitamin B6 and formation of pyridoxal phosphate, the active coenzyme.     

T-cell receptors in channel catfish: structure and expression of TCR alpha and beta genes

Herein are reported full length cDNA sequences for TCR alpha- and beta-chains of the channel catfish. Included are sequences belonging to four Valpha and six Vbeta families which share hallmarks in common with the Valpha and Vbeta genes of other species. Similar to the situation in other vertebrates, the catfish Calpha and Cbeta sequences exhibit distinct immunoglobulin, connecting peptide, transmembrane and cytoplasmic domains. However, the catfish TCR Calpha and Cbeta regions are shorter than those of mammals and the catfish Cbeta chain lacks a cysteine in its connecting peptide region. Two different catfish Cbeta cDNA sequences were identified, suggesting the existence of either two Cbeta loci or allotypes. Based on Southern blot analyses, each of the catfish TCR gene loci appear to be arranged in a translocon (as opposed to multicluster) organization with multiple V elements and a single or few copies of C region DNA. At the deduced amino acid level, the catfish Cbeta sequence exhibits 42% identity with the Cbeta of Atlantic salmon, 41% identity with the Cbeta of rainbow trout and 26% identity with Cbeta of the horned shark. The catfish Calpha amino acid sequence exhibits 44 and 29% identity with Calpha of the rainbow trout and southern pufferfish, respectively. TCRalpha and beta messages are selectively expressed and rearranged in a catfish clonal cell line that appears to be of the T lineage. This TCR alpha/beta expressing clonal lymphocyte line, designated 28S.1, has T-cell like function in that it constitutively produces a supernatant factor(s) with growth promoting activity. These findings should facilitate functional studies of fish TCRs and T cells in ways not previously possible with other 'lower' vertebrate models.