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The aim of this study was to investigate the seasonal changes in the epidermal structure and the innate immunity parameters of skin mucus in rainbow trout. The skin epidermis and mucus samples were collected over three consecutive seasons including winter, spring and late summer from three different weight groups i.e., 2-20 g (W1), 100-200 g (W2) and 400-600 g (W3) fish. The skin mucosal immunity analysis of rainbow trout showed that the haemagglutination activity increased significantly with increasing fish size from W1 to W3 in all three seasons, while no significant seasonal changes occurred in haemagglutination activity. Moreover, the bactericidal activity against fish pathogens increased significantly with increasing water bacterial load in late summer. The SDS-PAGE analysis of mucus showed a high amount of low molecular weight proteins (<35 kDa) in the late summer that was correlated with the increase in bactericidal activity. Histological analysis of the epidermis structure of rainbow trout skin showed that the density and size of goblet cells and consequently the mucus secretion significantly increased in W3 group in all seasons. In all three weight groups of fish, the density of goblet cells significantly increased from winter to spring and late summer along with increasing water temperature. Moreover, the goblet cell density showed a significant positive relationship with the soluble protein concentration and haemagglutination activity (p < 0.01). The results of this study demonstrated the more active immune role of the skin epidermal cells and mucus in rainbow trout during summer to protect fish against the pathogenic microorganisms. Given its potent bactericidal properties and the lack of haemolytic activity, the rainbow trout mucus might be used as a safe and inexpensive source for developing antimicrobial agents to prevent and treat some bacterial diseases in human and fish., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The traditional approach to extracting estrogens from water matrices, solid-phase extraction (SPE), presents a number of challenges when applied to complex wastewater matrices. Conversely, the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) clean-up method offers an alternative sample preparation approach that omits sample filtration and overcomes additional challenges associated with SPE. The objective of this study was to implement and validate a scaled QuEChERS method, using a standard addition approach, for extracting estrone (E1), 17β-estradiol (E2), and estriol (E3) from the estrogenic influent of a recirculating aquaculture system containing American eels (Anguilla rostrata). While traditional QuEChERS protocols do not facilitate considerable sample concentration, a 500-fold concentration factor was implemented for reliable quantitation of parts-per-trillion concentrations of estrogens from an initial sample volume of 20 mL to a final extract volume of 40 μL. Following analysis via high-performance liquid chromatography-mass spectrometry, excellent process efficiencies were observed at spiked concentrations of 10 and 50 ng L -1 for E2 and E1 (101 to 111%; %RSD ≤ 16), and moderate to acceptable process efficiencies were achieved for E3 (75 to 87%; %RSD ≤ 16). Validation of method parameters, including specificity, linearity, accuracy (recovery and process efficiencies), precision (intra-day precision, and inter-day precision), matrix effects, method detection limit, and limit of quantitation, led to reliable quantitation of unknown concentrations of E1, E2, and E3 in the aquaculture influent as low as 52, 20, and 33 ng L -1 , respectively. This study provides a validated analytical method for waste systems requiring quantitation of estrogens in their complex wastewater matrices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Rationale: 17β-Estradiol (E2), estrone (E1) and estriol (E3) are steroid hormones responsible for the regulation of the female reproductive system. Estradiol is planned to be used to feminize eels in aquaculture in order to improve their size and marketability. The residual levels of these hormones in fish tissue must be monitored to meet the requirements of food regulatory agencies. Few studies have studied these hormones in complex biological matrices such as fish tissue., Methods: We developed a method to analyze E1, E2 and E3 in fish tissue using liquid chromatography in combination with differential ion mobility spectrometry (DMS) and tandem mass spectrometry (MS/MS). The mass spectrometer was operated in negative polarity selected reaction monitoring (SRM) mode. To test the performance of this method, residual levels of E1, E2 and E3 were measured in the muscle tissue of juvenile eels subjected to feminization treatment with E2., Results: We report that following 17β-estradiol treatment, E2 is rapidly metabolized from the eel tissue, with a 50% depletion rate per day. Five days post-treatment, E2 returned to the level found in non-treated controls, similar to levels found in wild mature female eels., Conclusions: The method presented herein allows the quantitative analysis of E1, E2 and E3 in fish tissue samples. Under the experimental conditions, E2 in fish tissue samples returned to physiological levels post hormonal treatment. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

This research investigated how ploidy level (diploid versus triploid) affects the heat shock protein (HSP) response in erythrocytes under different thermal stress regimes, both in vivo and in vitro, in Atlantic salmon (Salmo salar) and brook charr (Salvelinus fontinalis) in order to address the question of why triploids typically have reduced thermal tolerance. A preliminary study confirmed that identical volumes of diploid and triploid erythrocytes (which equates to a smaller number of larger cells for triploids compared to diploids) did not differ in total protein synthesis rates. After chronic (100d) acclimation of fish to 5, 15 and 25°C, triploid erythrocytes had lower HSP70, HSP90, heat shock factor 1 (HSF1) and ubiquitin (free and total) levels than diploids in both species. Furthermore, Atlantic salmon erythrocytes showed significantly higher protein breakdown (based on conjugated ubiquitin levels) in triploids than diploids after acute heat stress in vitro, but no significant difference was detected between ploidies after acute cold stress. These results indicate that: 1) triploid erythrocytes synthesize more total protein per cell than diploids as a result of increased cell size; 2) triploids have sufficient total HSP levels for survival under low stress conditions; and 3) the lower basal titres of HSPs in triploids may be a handicap when combating acute stress. Taken together, this suggests that triploids are limited in their ability to withstand thermal stress because of a reduced ability to maintain proteostasis under stressful conditions., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis; a serious infectious disease in aquaculture raised salmonids. Iron acquisition has been shown to be critical for the survival of pathogenic bacteria during the course of infection. Previous work has demonstrated that A. salmonicida expresses iron-repressible IROMP proteins, suggesting the presence of iron acquisition systems that are under the control of a ferric uptake regulator (Fur). In this study, the A. salmonicida fur has been sequenced and a fur deletion strain generated. The A. salmonicida fur gene has an open reading frame of 428 bp, coding for a protein of 143 amino acids, and with high homology to previously described Fur proteins. The Fur protein product had a 94% sequence identity and 96% sequence similarity to the Aeromonas hydrophila Fur protein product. Transcription of the A. salmonicida fur gene was not regulated by the iron status of the bacterium and is not autoregulated, as in Escherichia coli. Proteomic analysis of the A. salmonicida fur mutant, fails to repress iron-regulated outer membrane proteins in the presence of iron. The A. salmonicida fur::KO mutant shows significantly reduced pathogenicity compared to the wild-type parental strain. In addition, the A. salmonicida fur mutant provides an important tool for further investigation of the iron acquisition mechanisms utilized by A. salmonicida., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Contaminants are ubiquitous in the environment and their impacts are of increasing concern due to human population expansion and the generation of deleterious effects in aquatic species. Oxidative stress can result from the presence of persistent organic pollutants, metals, pesticides, toxins, pharmaceuticals, and nanomaterials, as well as changes in temperature or oxygen in water, the examined species, with differences in age, sex, or reproductive cycle of an individual. The antioxidant role of glutathione (GSH), accompanied by the formation of its disulfide dimer, GSSG, and metabolites in response to chemical stress, are highlighted in this review along with, to some extent, that of glutathione S-transferase (GST). The available literature concerning the use and analysis of these markers will be discussed, focusing on studies of aquatic organisms. The inclusion of GST within the suite of biomarkers used to assess the effects of xenobiotics is recommended to complement that of lipid peroxidation and mixed function oxygenation. Combining the analysis of GSH, GSSG, and conjugates would be beneficial in pinpointing the role of contaminants within the plethora of causes that could lead to the toxic effects of reactive oxygen species.

This study examined changes in Atlantic salmon Salmo salar epidermal mucus proteins following short- and long-term handling stress. Short-term stress consisted of a single removal of fish from water for 15 s with long-term stress consisting of daily removal of fish from water for 15 s over 21 days. In the long-term handling stress study, there was a high level of individual variability with respect to mucus alkaline phosphatase, cathepsin B and lysozyme activities, with no correlation to treatment group. There was limited or no positive correlation between lysozyme, cathepsin B or alkaline phosphatase activities and plasma cortisol. There was a significant difference in lysozyme activity for both control and stressed fish at day 21 compared to other sampling days. In the short-term study, there was again high variability in mucus enzyme activities with no difference observed between groups. Immunoblotting also showed variability in mucus actin breakdown products in both short- and long-term handling stress studies. There appeared, however, to be a shift towards a more thorough breakdown of actin at day 14 in the stressed group. This shift suggested changes in mucus proteases in response to long-term handling stress. In summary, there were correlations of some mucus enzyme/protein profiles with stress or cortisol; however, the variability in S. salar mucus enzyme levels and actin fragmentation patterns suggested other triggers for inducing changes in mucus protein composition that need to be investigated further in order to better understand the role of mucus in the response of S. salar to external stressors., (© 2010 The Authors. Journal of Fish Biology © 2010 The Fisheries Society of the British Isles.)

Fish epidermal mucus contains innate immune components that provide a first line of defense against various infectious pathogens. This study reports the bioassay-guided fractionation and characterization of a novel antimicrobial peptide, myxinidin, from the acidic epidermal mucus extract of hagfish (Myxine glutinosa L.). Edman sequencing and mass spectrometry revealed that myxinidin consists of 12 amino acids and has a molecular mass of 1,327.68 Da. Myxinidin showed activity against a broad range of bacteria and yeast pathogens at minimum bactericidal concentration (MBC) ranging from 1.0 to 10.0 microg/mL. Screened pathogens, Salmonella enterica serovar Typhimurium C610, Escherichia coli D31, Aeromonas salmonicida A449, Yersinia ruckeri 96-4, and Listonella anguillarum 02-11 were found to be highly sensitive to myxinidin at the MBC of 1.0-2.5 microg/mL; Staphylococcus epidermis C621 and yeast (Candida albicans C627) had an MBC of 10.0 microg/mL. The antimicrobial activity of myxinidin was found to be two to 16 times more active than a potent fish-derived antimicrobial peptide, pleurocidin (NRC-17), against most of the screened pathogens. The microbicidal activity of myxinidin was retained in the presence of sodium chloride (NaCl) at concentrations up to 0.3 M and had no hemolytic activity against mammalian red blood cells. These results suggest that myxinidin may have potential applications in fish and human therapeutics.

The mucus protein profile of Atlantic salmon (Salmo salar) and changes due to infection with sea lice (Lepeophtheirus salmonis) were examined. Two-dimensional gel electrophoresis was performed on salmon skin mucus and comparisons between control and infected fish mucus were made. LC MS/MS identified intracellular proteins, calmodulin, actin, and hemopexin and plasma proteins, such as apolipoproteins, lectin, plasminogen and transferrin. Plasma proteins in the mucus may result from either direct expression by epidermal cells, leakage of plasma or via a secondary circulation system. Therefore, RT-PCR was used to measure mRNA of transferrin and lectin in Atlantic salmon skin. Transferrin expression was observed suggesting direct expression by the epidermis. Lectin expression was not detected suggesting another mechanism of entry into mucus, either leakage from plasma or secondary circulation. The lack of observable albumin on 2D gels, suggests that mucus lectin may arise from the secondary circulation route. Interestingly, ?-actin was a significant component of Atlantic salmon mucus. Cleaved actin and transferrin fragments were observed and positively correlated with sea lice infection suggestive of proteolytic activity. Increased levels of cleaved transferrin during sea lice infection may activate the nitrous oxide response of salmon macrophages, as part of the fish's immune response to sea lice infection.

Cellular defence against accumulation of toxic xenobiotics includes metabolism by phase I and II enzymes and export of toxicants and their metabolites via ATP-binding cassette (ABC) transporters. Liver gene expression of representatives of these three protein groups was examined in a population of multixenobiotic-resistant killifish (Fundulus heteroclitus) from the Sydney Tar Ponds, Nova Scotia, Canada. The Tar Ponds are heavily polluted with polycyclic aromatic hydrocarbons, polychlorinated biphenyls and heavy metals. The relationship among ABC transporters ABCB1, ABCB11, ABCC2, ABCG2, phase I enzyme cytochrome P4501A1 (CYP1A1) and phase II enzyme glutathione-S-transferase (GST-mu) was investigated by quantifying hepatic transcript abundance. In Tar Pond killifish, hepatic mRNA expression levels of ABCC2, ABCG2, CYP1A1 and GST-mu were elevated compared to reference sites, suggesting that hydrophobic contaminants undergo phase I and II metabolism and are then excreted into the bile of these fish. Hepatic ABCB1 and ABCB11 mRNA were not up-regulated in Tar Pond fish compared to two reference sites, indicating that these two proteins are not involved in conferring multixenobiotic resistance to Tar Pond killifish. The results suggest instead that liver up-regulation of phase I and II enzymes and complementary ABC transporters ABCC2 and ABCG2 may confer contaminant resistance to Tar Pond fish.

Hagfish (Myxine glutinosa) secrete normal epidermal mucus and extruded slime. The epidermal mucus is produced continuously to prevent pathogen adherence while the extruded slime is observed predominantly during feeding, provocation or stress. To date little is known about the involvement of extruded slime in the physiological functions of hagfish. In this preliminary study, innate immune enzymes and the protein composition of hagfish normal epidermal mucus and extruded slime were analysed and compared. The lysozyme specific activity of hagfish was observed approximately two-fold higher in extruded slime than that of epidermal mucus. The extruded slime had approximately 3.5-5.0 fold increased levels of alkaline phosphatase, cathepsin B and proteases in comparison to epidermal mucus. Protease characterization using specific inhibitors showed that the extruded slime had higher levels of serine trypsin-like proteases compared to metalloproteases whereas epidermal mucus showed equal proportion of both serine and metalloproteases. SDS-PAGE analysis showed high levels of three proteins with molecular masses in the range of 13-16kDa in the extruded slime. The LC/MS/MS analysis of protein bands 1, 2 and 3 showed closest matches to hemoglobulin-3, histone H3 and H2B proteins, respectively. The observation of elevated levels of innate immune parameters in the extruded slime suggested that the extruded slime has a significant role in innate immunity of hagfish against infectious pathogens.

The mucus layer on the surface of fish consists of several antimicrobial agents that provide a first line of defense against invading pathogens. To date, little is known about the antimicrobial properties of the mucus of Arctic char (Salvelinus alpinus), brook trout (S. fontinalis), koi carp (Cyprinus carpio sub sp. koi), striped bass (Morone saxatilis), haddock (Melanogrammus aeglefinus) and hagfish (Myxine glutinosa). The epidermal mucus samples from these fish were extracted with acidic, organic and aqueous solvents to identify potential antimicrobial agents including basic peptides, secondary metabolites, aqueous and acid soluble compounds. Initial screening of the mucus extracts against a susceptible strain of Salmonella enterica C610, showed a significant variation in antimicrobial activity among the fish species examined. The acidic mucus extracts of brook trout, haddock and hagfish exhibited bactericidal activity. The organic mucus extracts of brook trout, striped bass and koi carp showed bacteriostatic activity. There was no detectable activity in the aqueous mucus extracts. Further investigations of the activity of the acidic mucus extracts of brook trout, haddock and hagfish showed that these fish species had specific activity for fish and human pathogens, demonstrating the role of fish mucus in antimicrobial protection. In comparison to brook trout and haddock, the minimum bactericidal concentrations of hagfish acidic mucus extracts were found to be approximately 1.5 to 3.0 times lower against fish pathogens and approximately 1.6 to 6.6 folds lower for human pathogens. This preliminary information suggests that the mucus from these fish species may be a source of novel antimicrobial agents for fish and human health related applications.

Intense physical activity results in transient immunosuppression in a wide range of animals. We tested the hypothesis that competition between immune function and lipid transport for the protein apolipophorin III (apoLpIII) can cause transient immunosuppression in crickets. Both flying, an energetically demanding behavior, and an immune challenge reduced the amount of monomeric (free) apoLpIII in the hemolymph of crickets. Because both immune function and flying depleted free apoLpIII, these two phenomena could be in competition for this protein. We showed that immune function was sensitive to the amount of free apoLpIII in the hemolymph. Reducing the amount of free apoLpIII in the hemolymph using adipokinetic hormone produced immunosuppression. Increasing apoLpIII levels after flight by pre-loading animals with trehalose reduced immunosuppression. Increasing post-flight apoLpIII levels by injecting purified apoLpIII also reduced flight-induced immunosuppression. These results show that competition between lipid transport and immune function for the same protein can produce transient immunosuppression after flight-or-fight behavior. Intertwined physiological systems can produce unexpected trade-offs.

Fish epidermal mucus and its components provide the first line of defense against pathogens. Little is known about the role of epidermal mucus enzymes in the innate immune system of fish species such as Arctic char (Salvelinus alpinus), brook trout (S. fontinalis), koi carp(Cyprinus carpio), striped bass (Morone saxatilis), haddock, (Melanogrammus aeglefinus), Atlantic cod (Gadus morhua) and hagfish (Myxine glutinosa). The epidermal mucus samples from these fish were analysed for the specific activities of various hydrolytic enzymes including lysozyme, alkaline phosphatase, cathepsin B and proteases and the enzyme levels were compared among the fish species. Of all the species hagfish mucus showed a high activity for lysozyme and proteases and koi carp mucus had the highest levels of alkaline phosphatase and cathepsin B. A wide variation in enzyme activities was observed among the seven species and also between species of same family such as Arctic char and brook trout (salmonidae), haddock and cod (gadidae). Only lysozyme levels showed a marked variation with salinity where seawater fish showed approximately two times higher lysozyme activity than freshwater-reared fish species. Characterization of proteases with specific inhibitors showed Arctic char, brook trout, haddock and cod having higher levels of serine over metalloproteases whereas koi carp and striped bass had higher levels of metalloproteases over serine proteases. In contrast, hagfish had almost equal proportion of both serine and metalloproteases. This study demonstrates variation in the level of hydrolytic enzymes in the epidermal mucus of fish. These results provide preliminary information for a better understanding of the role of epidermal mucus and its components in the fish innate immune system.

We have previously shown that Lepeophtheirus salmonis produces trypsin and prostaglandin E(2) (PGE(2)) that are most likely responsible for the limited inflammatory response of Atlantic salmon to infection. After removal of the dopamine and PGE(2), the immunomodulatory activity of unfractionated and pools of the fractionated secretions was determined by examining the effects of the secretions on Atlantic salmon immune gene expression. Incubation of macrophage-enriched isolates of Atlantic salmon head kidney cells with the unfractionated secretion + PGE(2) revealed a significant inhibition of interleukin-1beta (IL-1beta) and major histocompatibility class I gene expression. Inhibition of lipopolysaccharide-induced IL-1beta expression in the Atlantic salmon head kidney cell line (SHK-1) was observed when three pools of the secretory/excretory products were tested. Further purification of products within these pools revealed that fraction 1-2 could account fully for the inhibition of IL-1beta expression in SHK-1 cells observed in pooled fraction 1. This study demonstrates that there are other immunomodulatory compounds produced by L. salmonis, in addition to PGE(2) and trypsin, that can inhibit the expression of Atlantic salmon immune-related genes in vitro.

This study was conducted to determine the effects of a high level of infection of the parasitic copepod L. salmonis on the stress response and immunological status of Atlantic salmon. An initial low-level initial infection was carried out 14d prior to a second infection in which twice as many parasites were introduced. Plasma cortisol and prostaglandin E(2) (PGE(2)) levels were monitored concurrent to the expression of six immune-related genes over five sample times (9, 21, 26, 33 and 40days post initial infection, dpii). The mean lice counts on the infected fish increased significantly from the first infection (16.3+/-1.89 at 9dpii) to the second (142.8+/-12.8 at 26dpii). Plasma cortisol levels increased significantly at 26, 33 and 40dpii in infected fish compared to controls. Plasma PGE(2) levels were significantly higher in infected fish at 9, 33 and 40dpii, when compared to controls. At 9dpii, expression of interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNFalpha)-like cytokine, major histocompatibility class II (MH II), transforming growth factor-beta (TGFbeta)-like cytokine and cyclooxygenase-2 genes were increased in infected fish compared to controls. The expression of most of these genes returned to control levels at 21dpii when the highest expression of the MH class I gene was observed in infected fish (significantly higher than controls). Major histocompatibility class I gene expression remained higher in infected fish at 26 and 33dpii compared to controls and this was observed for the TNFalpha-like gene. By 33dpii, MH class II and TGFbeta-like genes had higher expression in infected fish compared to controls. Interleukin-1beta and TNFalpha-like gene were the only genes that showed significantly higher expression in infected fish compared to controls at 40dpii, while MH class I gene expression was significantly depressed in infected fish at this time. The expression of nearly all immune-related genes studied here increased following initial infection with L. salmonis, however, immunological stimulation did not reduce parasite numbers or protect against re-infection.

A study was conducted to compare astaxanthin binding ability of solubilized muscle proteins of Atlantic salmon (Salmo salar L.), haddock (Melanogrammus aeglefinus L.) and Atlantic halibut (Hippoglossus hippoglossus L.). Muscle proteins of juvenile Atlantic salmon, haddock and halibut were solubilized by sequential extraction of muscle tissue using low ionic strength solutions. Electrophoretic protein profiles of the six solubilized fractions from these species were similar. Each solubilized fraction from the three species was examined for its relative astaxanthin binding capacity. The amount of bound astaxanthin was significantly different (P<0.05) among the six fractions of each species. Significant differences in astaxanthin binding were only found for fractions A and E among the species. The amount of bound astaxanthin in various fractions of each species showed a good correlation (R2=0.80-0.92) with the ANS (8-anilino-1-naphthalenesulfonate) fluorescence intensity of those fractions. The pattern and extent of astaxanthin binding to the muscle proteins of juvenile salmon, haddock and halibut is comparable to that reported previously for adult Atlantic salmon (Saha, M.R., Ross, N.W., Gill, T.A., Olsen, R.E., Lall, S.P., 2005. Development of a method to assess binding of astaxanthin to Atlantic salmon S. salar L. muscle proteins. Aquacult. Res. 36, 336-343.). These combined observations suggest that the carotenoid binding capacity of the muscle proteins of salmon is not the limiting factor in the deposition of carotenoid in their flesh.

The rubicund pigmentation in salmon and trout flesh is unique and is due to the deposition of dietary carotenoids, astaxanthin and canthaxanthin in the muscle. The present study was undertaken to determine which protein was responsible for pigment binding. Salmon muscle proteins were solubilized by sequential extractions with non-denaturing, low ionic strength aqueous solutions and segregated as such into six different fractions. Approximately 91% of the salmon myofibrillar proteins were solubilized under non-denaturing conditions using a protocol modified from a method described by Krishnamurthy et al. [Krishnamurthy, G., Chang, H.S., Hultin, H.O., Feng, Y., Srinivasan, S., Kelleher. S.D., 1996. Solubility of chicken breast muscle proteins in solutions of low ionic strength. J. Agric. Food Chem. 44: 408-415.] for the dissolution of avian muscle. To our knowledge, this is the first time this solubilization approach has been applied to the study of molecular interactions in myofibrillar proteins. Astaxanthin binding in each fraction was determined using an in vitro binding assay. In addition, SDS-PAGE and quantitative densitometry were used to separate and determine the relative amounts of each of the proteins in the six fractions. The results showed that alpha-actinin was the only myofibrillar protein correlating significantly (P<0.05) with astaxanthin binding. Alpha-actinin was positively identified using electrophoretic techniques and confirmed by tandem mass spectroscopy. Purified salmon alpha-actinin bound synthetic astaxanthin in a molar ratio of 1.11:1.00. The study was repeated using halibut alpha-actinin, which was found to have a molar binding ratio of astaxanthin to alpha-actinin of 0.893:1. These results suggest that the difference in pigmentation between white fish and Atlantic salmon is not due to binding capacity in the muscle, but rather differences in the metabolism or transport of pigment.

Aeromonas salmonicida subsp. salmonicida is the aetiological agent of furunculosis, a disease of farmed and wild salmonids. The type III secretion system (TTSS) is one of the primary virulence factors in A. salmonicida. Using a combination of differential proteomic analysis and reverse transcriptase (RT)-PCR, it is shown that A. salmonicida A449 induces the expression of TTSS proteins at 28 degrees C, but not at its more natural growth temperature of 17 degrees C. More modest increases in expression occur at 24 degrees C. This temperature-induced up-regulation of the TTSS in A. salmonicida A449 occurs within 30 min of a growth temperature increase from 16 to 28 degrees C. Growth conditions such as low-iron, low pH, low calcium, growth within the peritoneal cavity of salmon and growth to high cell densities do not induce the expression of the TTSS in A. salmonicida A449. The only other known growth condition that induces expression of the TTSS is growth of the bacterium at 16 degrees C in salt concentrations ranging from 0.19 to 0.38 M NaCl. It is also shown that growth at 28 degrees C followed by exposure to low calcium results in the secretion of one of the TTSS effector proteins. This study presents a simple in vitro model for the expression of TTSS proteins in A. salmonicida.

Following lipopolysaccharide (LPS)-stimulation of Atlantic salmon (Salmo salar) macrophage-like SHK-1 cells, prostaglandin E(2) (PGE(2)) exhibited dose-dependent inhibition of the antigen presenting molecules major histocompatability class I and II and the pro-inflammatory cytokine interleukin-1 beta gene expression. Prostaglandin E(2) was found to be stimulatory towards cyclooxygenase-2 (COX-2) expression at higher concentrations (1 x 10(-6) and 1 x 10(-8)M) and inhibitory at lower concentrations (1 x 10(-10) and 1 x 10(-12)M) after 4h exposure. After 24h exposure, however, LPS-induced COX-2 expression decreased and was completely inhibited by all PGE(2) concentrations (1 x 10(-6)-1 x 10(-10)M). Incubation of SHK-1 cells with LPS alone had no effect on tumour necrosis factor alpha (TNFalpha)-like gene or transforming growth factor beta-like gene expression after 4h, however, LPS and PGE(2) showed a synergistic effect on TNFalpha-like gene expression after 24h. This study provides evidence for the existence of a PGE(2)-mediated negative feedback mechanism in the control of PGs through down-regulation of COX-2, as well as for inflammatory responses by the down-regulation of both COX-2 and IL-1 beta. The differential regulation of immune-related genes under these conditions further demonstrates the usefulness of the SHK-1 cell line for studying aspects of salmonid immunology.

Lepeophtheirus salmonis is an ectoparasitic copepod that causes serious disease outbreaks in both wild and farmed salmonids. As the relationship between L. salmonis and its hosts is not well understood, the current investigation was undertaken to investigate whether any immunomodulatory compounds could be identified from secretions of L. salmonis. By incubating live L. salmonis adults with the neurotransmitter dopamine in seawater, we were able to obtain secretions from the parasite. These were analyzed by RP-HPLC column, as well as LC-MS. L. salmonis secretions contained a compound with the same retention time and mass of PGE(2). The identity of this compound as PGE(2) was confirmed by MS-in source dissociation. The concentrations of PGE(2) in L. salmonis secretions ranged from 0.2 to 12.3 ng/individual and varied with incubation temperature and time kept off the host. Prostaglandin E(2) is a potent vasodilator and thought to aid in parasite evasion from host immune responses. This is the first reported evidence of prostaglandin production in parasitic copepod secretions and its implications for the host-parasite relationship are discussed.

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis, a serious infectious disease of salmonids. Bacterial phenotypes are known to change in vivo compared to the in vitro state. Proteomic analysis of in vivo phenotypes is usually not possible due to insufficient biomass. Using an in vivo growth chamber model, the pathogenic fish bacterium A. salmonicida was cultured in pure culture in vivo in its host, the Atlantic salmon, to obtain sufficient biomass to allow proteomic analysis. Growth of A. salmonicida under in vitro iron-restricted conditions resulted in the expression of outer membrane proteins of 73, 76 and 85 kDa, which were not present when grown under in vitro iron-replete conditions. Mass spectrometry analysis identified the 73 kDa protein as a colicin receptor, the 76 kDa protein as an outer membrane heme receptor, and the 85 kDa protein as a ferric siderophore receptor. When cultured in vivo, A. salmonicida up-regulated the identical 73, 76 and 85 kDa proteins. The results of this study also suggest, at least with respect to the outer membrane proteins, that the in vitro iron-restricted growth model largely reproduces the results obtained from growth of A. salmonicida within the peritoneal cavity of salmon.

Aeromonas salmonicida subsp. salmonicida is a facultatively intracellular gram-negative bacterium that is the etiological agent of furunculosis, a bacterial septicemia of salmonids that causes significant economic loss to the salmon farming industry. The mechanisms by which A. salmonicida evades intracellular killing may be relevant in understanding virulence and the eventual design of appropriate treatment strategies for furunculosis. We have identified two open reading frames (ORFs) and related upstream sequences that code for two putative superoxide dismutases (SODs), sodA and sodB. The sodA gene encoded a protein of 204 amino acids with a molecular mass of approximately 23.0 kDa (SodA) that had high similarity to other prokaryotic Mn-SODs. The sodB gene encoded a protein of 194 amino acids with a molecular mass of approximately 22.3 kDa that had high similarity to other prokaryotic Fe-SODs. Two enzymes with activities consistent with both these ORFs were identified by inhibition of O(2)(-)-catalyzed tetrazolium salt reduction in both gels and microtiter plate assays. The two enzymes differed in their expression patterns in in vivo- and in vitro-cultured bacteria. The regulatory sequences upstream of putative sodA were consistent with these differences. We could not identify other SOD isozymes such as sodC either functionally or through data mining. Levels of SOD were significantly higher in virulent than in avirulent strains of A. salmonicida subsp. salmonicida strain A449 when cultured in vitro and in vivo.

Haddock (Melanogrammus aeglefinus) is a gadoid fish species that deposits dietary lipid mainly in the liver. The fatty acid (FA) beta-oxidation activity of various tissues was evaluated in juvenile haddock fed graded levels of lipid. The catabolism of a radiolabelled FA, [1-(14)C]palmitoyl-CoA, through peroxisomal and mitochondrial beta-oxidation was determined in the liver, red and white muscle of juvenile haddock fed 12, 18 and 24% lipid in the diet. There was no significant increase in the mitochondrial or peroxisomal beta-oxidation activity in the tissues tested as the dietary lipid level increased from 12 to 24%. Peroxisomes accounted for 100% of the beta-oxidation observed in the liver, whereas mitochondrial beta-oxidation dominated in the red (91%) and white muscle (97%) of juvenile haddock. Of the tissues tested, red muscle possessed the highest specific activity for beta-oxidation expressed on a per mg protein or per g wet weight basis. However, white muscle, which forms over 50% of the body mass in gadoid fish was the most important tissue in juvenile haddock for overall FA catabolism. The total lipid and FA composition of these tissues were also determined. This study confirmed that the liver was the major lipid storage organ in haddock. The hepatosomatic index (HSI; 10.0-15.2%) and lipid (73.8-79.3% wet wt.) in the liver increased significantly as dietary lipid was increased from 12 to 24% lipid. There was no significant increase in the lipid composition of the white muscle (0.8% wet wt.), red muscle (1.9% wet wt.) or heart (2.5% wet wt.).

Adult and mobile preadult sea lice Lepophtheirus salmonis were incubated with mucus samples from rainbow trout (Oncorhynchus mykiss), coho salmon (O. kisutch), Atlantic salmon (Salmo salar), and winter flounder (Pseudopleuronectes americanus) to determine the response of L. salmonis to fish skin mucus as assessed by the release of proteases and alkaline phosphatase. There was variation in the release of respective enzymes by sea lice in response to different fish. As well, sealice collected from British Columbia responded differently than New Brunswick sea lice to coho salmon mucus. Fish mucus and seawater samples were also analyzed using protease gel zymography to observe changes in the presence of low molecular weight (LMW) proteases after L. salmonis incubation. Significantly higher proportions of sea lice secreted multiple bands of L. salmonis-derived LMW proteases after incubation with rainbow trout or Atlantic salmon mucus in comparison with seawater, coho salmon, or winter flounder mucus. Susceptibility to L. salmonis infections may be related to the stimulation of LMW proteases from L. salmonis by fish mucus. The resistance of coho salmon to L. salmonis infection may be due to agents in their mucus that block the secretion of these LMW proteases or factors may exist in the mucus of susceptible species that stimulate their release.

The salmon louse, Lepeophtheirus salmonis, is a marine ectoparasitic copepod that infects salmonid fishes. We are studying the interactions between this parasite and its salmonid hosts, as it is a common cause of disease in both wild and farmed stocks of salmon. In this paper, we report on the cloning and sequencing of seven trypsin-like enzymes from a cDNA library prepared from whole body preadult female and male L. salmonis. The predicted trypsin activation peptides are 23 or 24 residues in length, considerably longer than previously reported activation peptides of other animals. Differences in the putative signal and activation peptide sequences of the trypsin isoforms suggest that these forms differ in their regulation and function. The calculated molecular weights of the trypsins range from 23.6 to 23.7 kDa. There are eight cysteine residues, which suggest the presence of four disulfide bridges. These trypsins are very similar (>or=46% aa identity) to other crustacean trypsins and insect hypodermins. Using in situ hybridization techniques trypsinogen expression could be identified in all three cell types of the midgut.

Silver stained proteins of a wide molecular weight (MW) range (20-116 kDa) were successfully recovered by both electroblot and electroelution. The recovery was demonstrated for nanogram loads of proteins separated by SDS-PAGE and visualized by silver staining methods compatible and incompatible with mass spectrometry (MS). It was shown that the alcohol/acid and glutaraldehyde fixation steps present in a number of staining procedures did not prevent recovery of intact proteins from gels. It was found that the recovery of intact proteins from silver stained gels was substantially increased upon pre-equilibration in a buffer containing the reducing agent, dithiothreitol (DTT). The effect of destaining on the recovery of silver stained proteins was also investigated. Comparable recovery of intact proteins within a wide MW range from silver stained gels with and without destaining step was demonstrated. Recovery of model proteins from gels visualized using silver staining method compatible with MS showed 52 to 76% yield of that from the unstained gel, depending upon method of the transfer. Comparison of the recovery of intact proteins from gels visualized using other staining procedures was also made. The above findings have implications as to the supposed irreversible nature of protein "fixation" inside polyacrylamide matrix, and confirm lack of binding of proteins in the gel to metal silver deposited on its surface. This method has the potential to be suitable for direct characterization of proteins by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) without additional purification steps.

Susceptibility to different diseases among related species, such as coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhyncus mykiss) and Atlantic salmon (Salmo salar), is variable. The prominence of these species in aquaculture warrants investigation into sources of this variability to assist future disease management. To develop a better understanding of the basis for species variability, several important non-specific humoral parameters were examined in juvenile fish of these three economically important species. Mucous protease, alkaline phosphatase and lysozyme, as well as plasma lysozyme activities and histological parameters (epidermal thickness and mucous cell density, and size) were characterized and compared for three salmonids: rainbow trout, Atlantic salmon and coho salmon. Rainbow trout had a thicker epidermis and significantly more mucous cells per cross-sectional area than the other two species. Rainbow trout also had significantly higher mucous protease activity than Atlantic salmon and significantly higher lysozyme (plasma and mucus) activities than coho and Atlantic salmon, in seawater. Atlantic salmon, on the other hand, had the lowest activities of mucous lysozyme and proteases, the thinnest epidermal layer and the sparsest distribution of mucous cells, compared with the two other salmonids in seawater. Only coho salmon had sacciform cells. Atlantic and coho salmon had higher mucous lysozyme activities in freshwater as compared to seawater. There was no significant difference between mucous lysozyme activities in any of the three species reared in freshwater; however, rainbow trout still had a significantly higher plasma lysozyme activity compared with the other two species. All three species exhibited significantly lower mucous alkaline phosphatase and protease activities in freshwater than in seawater. Our results demonstrate that there are significant histological and biochemical differences between the skin and mucus of these three salmonid species, which may change as a result of differing environments. Variation in these innate immune factors is likely to have differing influences on each species response to disease processes.

The effects of microwave irradiation on the staining of electrophoresed and electroblotted proteins have been assessed using currently available detection methods. Although the absorption of microwave radiation was found to be uneven, band intensity following microwave-assisted protein staining (MAPS) was comparable and in some cases exceeded the intensity of the bands visualised by the original staining methods. It was found that microwave treatment drastically reduced the duration of the staining protocols for visualisation of the proteins separated by both one- and two-dimensional electrophoresis. Application of MAPS methods did not affect peptide mass fingerprinting analysis by mass spectrometry and subsequent identification of the protein by database searching. The peptide mass maps corresponding to the proteins visualised using both the conventional and MAPS methods did not show significant difference in signal/noise ratio. Moreover, it appeared that microwave treatment of the gels resulted in the increased recovery of the peptides following in-gel trypsin digestion. Briefly, microwave-assisted protein staining methods were rapid, compatible with mass spectrometry and were equally effective on thin (0.75-mm) and thick (1.5-mm) gels (such as those used in 2D electrophoresis)., (Copyright 2002 Crown in the right of Canada.)

As part of an investigation of the biochemical interactions between the salmon louse Lepeophtheirus salmonis and Atlantic salmon Salmo salar, we characterized protease activity in the skin mucus of noninfected Atlantic salmon and Atlantic salmon infected with L. salmonis and in an L. salmonis whole-body homogenate. Zymography revealed that mucus from infected salmon contained a series of low-molecular-mass (17-22 kDa) serine proteases that were not present in the mucus of noninfected salmon. Based on molecular mass, inhibition studies, and affinity chromatography, the series of proteases was identified as being trypsin-like. Similar proteases were observed in the L. salmonis homogenate and in mucus from noninfected Atlantic salmon following a 1-hr incubation with live L. salmonis. An antibody raised against Atlantic salmon trypsin failed to recognize any proteases in the mucus of noninfected salmon or infected salmon or in the L. salmonis homogenate. Collectively, these findings suggest that the trypsin-like proteases present in the mucus of infected Atlantic salmon were produced by L. salmonis, possibly to aid in feeding and evasion of host immune responses.

Urastoma cyprinae occurs on the gills of various bivalves species, including the eastern oyster Crassostrea virginica. While the worm is known to cause severe gill disruption in mussels, no evidence of this nature has been described for oysters. Nonetheless, high levels of U. cyprinae have been reported in oysters, which may, in turn, reduce the oyster's overall condition. U. cyprinae is strongly attracted to oyster gill mucus, which is suggested to play an active role in the worm's feeding activities. Furthermore, host mucus contains many active components, including proteases, which have been suggested to play a defensive role against invading organisms. It follows, therefore, that some of the interactions between U. cyprinae and oysters take place in host gill mucus. Studies were undertaken to determine whether the presence of U. cyprinae altered the electrophoretic profiles of oyster gill mucus, using proteases as indicators. Findings reveal that oyster gill mucus contains three proteases, a putative acid protease at 96 kDa, a zinc metalloprotease at 64 kDa, and a serine protease at 33 kDa. Results based on experiments using mucus preparations extracted from infected and noninfected oysters, along with those using lyophilized mucus incubated with live U. cyprinae, confirm that the presence of U. cyprinae alters the protease composition of gill mucus. The present data demonstrate that both U. cyprinae and host gill mucus actively secrete proteases. While the precise roles of these enzymes still need to be defined, one of their functions may be associated with digestion-related activities induced by the worm.

We investigated the effects of a calcium-dependent mannose-binding lectin isolated from the serum of Atlantic salmon on Aeromonas salmonicida viability and the anti-A. salmonicida activity of Atlantic salmon macrophages. In the absence of other factors, binding of this lectin at concentrations of 0.8, 4.0 and 20.0 ng ml(-1) to virulent A. salmonicida failed to significantly reduce (P> 0.05) cell viability. However, binding of the lectin to A. salmonicida did result in significant (P < or = 0.05) dose-dependent increases in phagocytosis, and bactericidal activity. Significant increases (P < or = 0.05) were also observed in phagocyte respiratory burst activity within the lectin concentration range of 4.0-20.0 ng ml(-1) but the stimulation was not dose dependent at these lectin concentrations. At the lowest lectin concentration tested (0.32 ng ml(-1)), a significant decrease (P < or = 0.05) in respiratory burst was observed. The structure and activity of this lectin are similar to that of mammalian mannose-binding lectins, which are known to play a pivotal role in innate immunity. The presence of this lectin may be an important defense mechanism against Gram-negative bacteria such as A. salmonicida.

A mannose-binding lectin was isolated from the blood serum of Atlantic salmon (Salmo salar). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and non-reducing conditions revealed a multimeric structure composed of 17000 Mr subunits. Hexosamine analysis and glycosidase digestion showed that the lectin is not glycosylated and amino acid analysis revealed no unusual compositional features. Using ruthenium red staining, the lectin was shown to bind Ca2+ ions. N-terminal sequencing by Edman degradation gave: H2N-TGAKGAEEGVVPAETRNQXPTGWFQFGS. A database search revealed no similarity to protein sequences deposited to date. Binding experiments using biotinylated lectin revealed that it specifically recognizes and binds to mannose on the surfaces of two salmon pathogens, Vibrio anguillarum and Aeromonas salmonicida, implying an immunological role for this lectin in Atlantic salmon.

The cross-reactivity of the 6/50 monoclonal anti-okadaic acid antibody (mAb) to the recently discovered diarrhetic shellfish poisoning (DSP) metabolites dinophysistoxin-4 (DTX-4), dinophysistoxin-5 (DTX-5), and an okadaic acid (OA) diol ester was determined using a competitive indirect enzyme-linked immunosorbent assay (ELISA). The reactivity of the antibody to these molecules was compared to that with OA; the 6/50 mAb recognized all of these DSP compounds with equal sensitivity within the working range of the antibody (10-100 nM for OA). This confirms the ability of the antibody to detect all DSP compounds when used in analyses including ELISA and immunocytochemistry.

A fluorescence assay for streptogramin B lyase, an enzyme that confers resistance to streptogramin B antibiotics, has been developed. The antibiotic substrates are fluorescent and the linear peptide products formed in the lyase-catalyzed reaction are relatively nonfluorescent. The assay has potential for assessing bacterial resistance to streptogramin B antibiotics and will be utilized to direct the purification of streptogramin B lyase from bacterial extracts.

A simple, sensitive silver staining method for sodium dodecyl sulfate polyacrylamide gel electrophoresis has been developed which yields high resolution of proteins with a transparent background. The method includes the incorporation of a water wash after a short fixation step in ethanol/ acetic acid and prior to glutaraldehyde cross-linking, which appears to be necessary for the high resolution staining of protein bands and the low background staining.

A factor in colon carcinogenesis might be the partial defeat in colon epithelial cells of the protective enzymic barrier against xenobiotics, via bile acid inhibition of enzymes that detoxify mutagens. The applicability of aspects of this concept to glucuronosyltransferase, a phenol detoxification enzyme, was tested in a colon cancer cell line. Inhibition of glucuronidation of the test substrate, 4-methylumbelliferone, occurred at bile acid concentrations found in faecal water, and depended on pH for some bile acids. Lithocholate was the most inhibitory: the concentration causing 50% inhibition of the initial rate of glucuronidation (IC50) was about 3 microM at pH 7.4 and at pH 6.2. The inhibitory potency of deoxycholate and chenodeoxycholate increased when pH decreased, but still remained less than that of lithocholate: the IC50 for deoxycholate was 88.5 microM at pH 7.4, and 14.8 microM at pH 6.2, and for chenodeoxycholate the IC50 was 67.4 microM at pH 7.4, and 21.7 microM at pH 6.2. Cholate did not cause appreciable inhibition. The inhibitory effects were additive when lithocholate was present together with either deoxycholate or chenodeoxycholate. The results provide a mechanism for the comutagenicity of bile acids, a feature of which is the inter-relation of bile acid comutagenicity specifically with mutagens that are inactivated by a bile acid-inhibitable enzyme. The results are also in accord with the view that high concentrations of bile acids in solution in faecal water, especially lithocholate, are a risk factor for colon cancer.

Quantitative aspects of bile acid cytotoxicity to colon cancer cell lines were investigated because of the etiological role in colon carcinogenesis attributed to the toxic effects of bile acids on colon mucosal cells. The cytotoxicity of major colonic bile acids differed. Lithocholate was the most toxic, followed by chenodeoxycholate and deoxycholate, with cholate being non-toxic over the concentration range studied. Cytotoxicity increased with time of exposure. Values for IC50 for some of the acids were determined to be in the physiological range, as estimated from their concentrations in fecal water. The results suggest dietary factors that contribute to bile acid mucosal damage. They also identify factors of possible importance in the association of high concentrations of bile acids in fecal water with risk for colon cancer.

The composition of extracellular material produced by non-mucoid strains of Pseudomonas aeruginosa differed when grown on surfaces that either did or did not induce the formation of slime under conditions where the medium was identical. The nature of the changes in protein composition indicated that protein expression differed in the course of growth on the two surfaces, and hence that there were physiological consequences associated with growth under conditions which do or do not lead to slime formation. The compositional differences also included elevated levels in extracellular material from the slimy colonies of two virulence factors, protease and rhamnolipids.

Candida rugosa lipase was enzymatically active when coated on glass beads and exposed to mixtures of substrate and water vapor over a range of relative humidities up to 100%. Evidence was obtained for operation of the enzyme in a thin liquid film of concentrated buffer on the surface of the glass beads. Formation of the thin film was associated with hygroscopicity of the buffer used to suspend the enzyme in preparation of the enzyme-coated beads. At some buffer concentrations estimated to be on the bead surface, the enzyme was partially soluble and both soluble and insoluble forms were enzymatically active. The vapor mode of operation over a range of relative humidities had comparatively small effects on kinetic constants for hydrolysis of ethyl acetate, which were also similar to those in phosphate buffer. The extent of reaction occurred in the order hydrolysis greater than alcoholysis greater than ester interchange greater than esterification. Reaction preference between alcoholysis and hydrolysis changed as acyl chain length of substrate increased with C. rugosa lipase, as well as with Rhizopus arrhizus lipase and porcine liver esterase, with details depending on the enzyme. The vapor mode approach has the potential of being used with a wide variety of substrates, as shown by the ability to obtain hydrolysis at 30 degrees C with substrate vapor pressures as low as 0.08 mm Hg and with substrates with boiling points as high as 206 degrees C.

Multiple enzyme forms of β-mannanase activity fromPolyporus versicolor were puritied to molecular homogeneity by a sequence involving DEAE Bio-Gel A chromatography, gel filtration on Sephadex G-100 and high-performance liquid chromatography using anion exchange and hydrophobic Interaction media. Overall, 7.6% of input activity was recovered in four β-mannanases, A, B, C and 2A, which were purified 112.6-, 165.5-, 143.7-and 19.9-fold respectivety. The β-mannanases were acidic proteins displaying isoelectric points from 3.75 to 4.6, molecular weights in the range of 33,900 to 57,500 and increasing hydrophobicity in the order of C>B>2A>A. Optimal pH and temperature for the hydrotysis of glucomannan by all activities were pH 5.5 and 65°C, respectively. All preparations exhibited activity after 30 min at 65°C, or after protease digestion. Although the response of individual enzymes to selected ions was variable, all β-mannanases were inhibited in decreasing order of Hg(2+)>Cu(2+)>Zn(2+)>Mn(2+). All activities functioned as endomannanases.

The interaction of myelin basic protein (MBP) and proteolipid protein (PLP) was studied using a microtitre well binding assay and the ligand-blot overlay technique. The binding of iodinated PLP to MBP that was immobilized on microtitre wells was saturable and reversible. Its selectivity was investigated by the ligand-blot overlay technique. Iodinated PLP was found to bind MBP but not any other CNS myelin proteins. This interaction was not dependent on the phosphoryl moiety of MBP. Binding of PLP to histone H4 also occurred, but the amount of PLP bound per unit MBP was greater than for this histone.

Characteristics of fatty acylation of myelin proteolipid protein (PLP) in vitro were compared with the corresponding process in vivo. Rapid and efficient separation of labelled PLP from other proteins and lipids was effected by extraction into chloroform/methanol/0.1 N HCl (10/10/1) and chromatography on Sephadex LH-60 in the same solvent. Covalent linkage of [3H]-palmitate to PLP was demonstrated by repetitive chromatography on LH-60, thin layer chromatography, and polyacrylamide gel electrophoresis. Reductive cleavage with sodium borohydride of PLP acylated in vitro or in vivo yielded [3H]-hexadecanol, identifying at least one of the acyl linkages as a thiolester bond. When PLP was acylated with acyl-CoA as the fatty acid donor, the reaction occurred non-enzymatically as supported by the following observations: 1) acylation activity increased with increasing pH above pH 7.5, 2) acylation activity was heat stable, 3) acylation activity was not removed from PLP during purification in organic solvents or in Triton X-100-containing buffers, and 4) acylation of tryptic fragments occurred in the absence of an exogenously added enzyme source. The relevance of in vitro fatty acylation of PLP to that in vivo was confirmed by comparison of proteolytically derived peptide maps that showed that likely the same domain of PLP was acylated in vitro and in vivo.