Jellyfish Bioprospecting in the Mediterranean Sea: Antioxidant and Lysozyme-Like Activities from Aurelia coerulea (Cnidaria, Scyphozoa) Extracts.

Marine invertebrates represent a vast, untapped source of bioactive compounds. Cnidarians are represented by nearly 10,000 species that contain a complex mixture of venoms, collagen, and other bioactive compounds, including enzymes, oligosaccharides, fatty acids, and lipophilic molecules. Due to their high abundance in coastal waters, several jellyfish taxa may be regarded as candidate targets for the discovery of novel lead molecules and biomaterials and as a potential source of food/feed ingredients. The moon jellyfish Aurelia coerulea is one of the most common jellyfish worldwide and is particularly abundant in sheltered coastal lagoons and marinas of the Mediterranean Sea, where it first appeared-as an alien species-in the last century, when Pacific oyster cultivation began. In the present study, the antioxidant and lysozyme antibacterial activities associated with extracts from different medusa compartments-namely the umbrella, oral arms, and secreted mucus-were investigated. Extracts from the oral arms of A. coerulea displayed significant antioxidant activity. Similarly, lysozyme-like activity was the highest in extracts from oral arms. These findings suggest that A. coerulea outbreaks may be used in the search for novel cytolytic and cytotoxic products against marine bacteria. The geographically wide occurrence and the seasonally high abundance of A. coerulea populations in coastal waters envisage and stimulate the search for biotechnological applications of jellyfish biomasses in the pharmaceutical, nutritional, and nutraceutical sectors.

A mouse seminal vesicle-secreted lysozyme c-like protein modulates sperm capacitation.

Lysozyme (LYZ) c-like proteins are primarily present in the testis and epididymis of male reproductive tissues. Here, we report a novel member of the c-type LYZ family, the seminal vesicle-secreted LYZ c-like protein (SVLLP). Three forms of SVLLP were purified from mouse seminal vesicle secretions and characterized as glycoproteins with the same protein core but different N-linked glycans. SVLLP is structurally similar to c-type LYZ proteins. Only one of the 20 invariant residues was altered in the consensus sequence of c-type LYZs; however, the changed residue (N53S) is one of two essential catalytic residues. LYZ activity assays demonstrated that the three glycoforms of SVLLP lacked enzyme activity. SVLLP is primarily expressed in seminal vesicles. Immunohistochemistry revealed that it occurs in the luminal fluid and mucosal epithelium of the seminal vesicles. Testosterone is not the primary regulator for its expression in the seminal vesicle. SVLLP binds to sperm and suppresses bovine serum albumin-induced sperm capacitation, inhibits the acrosome reaction, and blocks sperm-oocyte interactions in vitro, suggesting that SVLLP is a sperm capacitation inhibitor.

Characterization of the interaction between carbon black and three important antioxidant proteins using multi spectroscopy and modeling simulations.

A major user of carbon black is the pigment and dyes industry, where carbon black is incorporated into paints, inks, printers, and plastics. However, little is known about the mechanism underlying the toxicity of carbon black to antioxidant proteins. Carbon black can cause oxidative stress to organisms after they invade into the body. Antioxidant proteins play a key role in keeping the organism from nanoparticle-induced oxidative damage and tend to bind with nanoparticles immediately after their invading into the biological environment, so it is meaningful to elucidate the toxicity of nanoparticles on the antioxidant proteins. In this study, the toxicity of carbon black (SB100) on three different antioxidant proteins (TF (transferrin), SOD (superoxide dismutase), and LYZ (lysozyme)) were investigated. The multi-spectra studies indicated that SB100 interacted with these three proteins and changed their structure in different ways. SB100 changed the microenvironment of fluorophores in SOD and LYZ by quenching the fluorescence spectra of the two enzymes, while changed that of TF by increasing the fluorescence intensity of TF. SB100 changed the secondary structure of these three proteins by decreasing the α-helix content of TF and increasing that of SOD and LYZ. Moreover, SB100 changed the hydrophobicity of the three proteins in different ways as well. And SOD exhibits a more severe activity inhibition than LYZ after exposed to SB100. In summary, SB100 caused different structural and functional changes to these three antioxidant enzymes.

[Influence of Different Type of Surfactant on Bacteriolytic Activity of Lysozyme].

The influence ofvarious surfactants (anionic sodium dodecyl sulfate, SDS, cationic dodecyltrimethylarnmonium bromide, DTAB, and zwitterionic cocoamidopropylbetaine, CAPB) on the activity of the chicken egg lysozyme is investigated. Lysis of Gram-positive bacteria by the enzyme was carried out at pH 7.2 and ionic strength of 0.15 M. It was found that at low SDS and DTAB concentrations (less than 1 x 10(-5) M) the bacteriolytic activity increases by 30-140%. At higher concentrations (1 x 10(-5) - 1 x 10(4) M) the activity returns to the level observed in the absence of the surfactants. The elevated activity correlated with the formation of hydrophobic lysozyme-surfactant complexes. Introduction of CAPB at concentrations above 1 x 10(-5) M sig, nificantly diminished the bacteriolytic activity due to CAPB induced aggregation of lysozyme.

Conserved C272/278 in b domain regulate the function of PDI-P5 to make lysozymes trypsin-resistant forms via significant intermolecular disulfide cross-linking.

Protein disulfide isomerase-P5 (P5) is thought to have important functions as an oxidoreductase, however, molecular functions of P5 have not been fully elucidated. We have reported that P5 has insulin reductase activity and inhibits lysozyme refolding by formation of lysozyme multimers with hypermolecular mass inactivated by intermolecular disulfides (hyLYS) in oxidative refolding of reduced denatured lysozyme. To explore the role of each domain of P5, we investigated the effects of domain deletion and Cys-Ala mutants of P5 on insulin reduction and the oxidative refolding of the lysozyme. The mutants of catalytic cysteines, C36/39A, C171/174A, and C36/39/171/174A inhibited the lysozyme refolding almost similarly to P5, and even b domain without catalytic cysteines showed moderate inhibitory effect, suggesting that the b domain played a key role in the inhibition. Western blotting analysis of the refolding products indicated that the catalytic cysteines in both the a and a' domains cross-linked lysozyme comparably to form hyLYS resistant to trypsin, in which b domain was suggested to capture lysozyme for the significant sulfhydryl oxidation. The mutant of the conserved cysteines in b domain, C272/278A, did not form hyLYS, however, showed predominant reductase activity, implying that P5 functioned as a potent sulfhydryl oxidase and a predominant reductase depending on the circumstance around C272/278. These results provide new insight into the molecular basis of P5 function.

Cysteine inhibits amyloid fibrillation of lysozyme and directs the formation of small worm-like aggregates through non-covalent interactions.

In this article, we discuss the effects of amino acids on amyloid aggregation of lysozyme. l-cysteine (Cys) dramatically inhibited fibrillation of lysozyme, whereas other amino acids (including l-arginine) did not. In the presence of Cys, the aggregation pathway of lysozyme shifted from fibrillation to the formation of the small worm-like aggregates with unfolding. The interaction between Cys and lysozyme was observed to be non-covalent, suggesting that the thiophilic interaction between the thiol group on the side chain of Cys and the core sequence of lysozyme significantly contributes to the inhibition of amyloid aggregation. These findings provide a new basis for the design of a biocompatible additive to prevent amyloid fibrillation.

Mab_3168c, a putative acetyltransferase, enhances adherence, intracellular survival and antimicrobial resistance of Mycobacterium abscessus.

Mycobacterium abscessus is a non-tuberculous mycobacterium. It can cause diseases in both immunosuppressed and immunocompetent patients and is highly resistant to multiple antimicrobial agents. M. abscessus displays two different colony morphology types: smooth and rough morphotypes. Cells with a rough morphotype are more virulent. The purpose of this study was to identify genes responsible for M. abscessus morphotype switching. With transposon mutagenesis, a mutant with a Tn5 inserted into the promoter region of the mab_3168c gene was found to switch its colonies from a rough to a smooth morphotype. This mutant had a higher sliding motility but a lower ability to form biofilms, aggregate in culture, and survive inside macrophages. Results of bioinformatic analyses suggest that the putative Mab_3168c protein is a member of the GCN5-related N-acetyltransferase superfamily. This prediction was supported by the demonstration that the mab_3168c gene conferred M. abscessus and M. smegmatis cells resistance to amikacin. The multiple roles of mab_3168c suggest that it could be a potential target for development of therapeutic regimens to treat diseases caused by M. abscessus.

Role of 15-hydroxyeicosatetraenoic acid in hemozoin-induced lysozyme release from human adherent monocytes.

Natural hemozoin (nHZ), a lipid-bound ferriprotoporphyrin IX crystal produced by Plasmodium parasites after hemoglobin catabolism, seriously compromises the functions of human monocytes, and 15-hydroxyeicosatetraenoic acid (15-HETE) and 4-hydroxynonenal (4-HNE), two nHZ lipoperoxidation products, have been related to such a functional impairment. nHZ was recently shown to promote inflammation-mediated lysozyme release from human monocytes through p38 mitogen-activated protein kinase- (MAPK)- and nuclear factor (NF)-κB-dependent mechanisms. This study aimed at identifying the molecule of nHZ lipid moiety that was responsible for these effects. Results showed that 15-HETE mimicked nHZ effects on lysozyme release, whereas 4-HNE did not. 15-HETE-enhanced lysozyme release was abrogated by anti-TNF-α and anti-IL-1ß-blocking antibodies and mimicked by recombinant cytokines; on the contrary, MIP-1α/CCL3 was not involved as a soluble mediator of 15-HETE effects. Moreover, 15-HETE early activated p38 MAPK and NF-κB pathways by inducing p38 MAPK phosphorylation; cytosolic I-κBα phosphorylation and degradation; NF-κB nuclear translocation and DNA-binding. Inhibition of both routes through chemical inhibitors (SB203580, quercetin, artemisinin, and parthenolide) prevented 15-HETE-dependent lysozyme release. Collectively, these data suggest that 15-HETE plays a major role in nHZ-enhanced monocyte degranulation.

Protease-activated receptor 2 mediates mucus secretion in the airway submucosal gland.

Protease-activated receptor 2 (PAR2), a G protein-coupled receptor expressed in airway epithelia and smooth muscle, plays an important role in airway inflammation. In this study, we demonstrated that activation of PAR2 induces mucus secretion from the human airway gland and examined the underlying mechanism using the porcine and murine airway glands. The mucosa with underlying submucosal glands were dissected from the cartilage of tissues, pinned with the mucosal side up at the gas/bath solution interface of a physiological chamber, and covered with oil so that secretions from individual glands could be visualized as spherical bubbles in the oil. Secretion rates were determined by optical monitoring of the bubble diameter. The Ca(2+)-sensitive dye Fura2-AM was used to determine intracellular Ca(2+) concentration ([Ca(2+)](i)) by means of spectrofluorometry. Stimulation of human tracheal mucosa with PAR2-activating peptide (PAR2-AP) elevated intracellular Ca(2+) and induced glandular secretion equal to approximately 30% of the carbachol response in the human airway. Porcine gland tissue was more sensitive to PAR2-AP, and this response was dependent on Ca(2+) and anion secretion. When the mouse trachea were exposed to PAR2-AP, large amounts of secretion were observed in both wild type and ΔF508 cystic fibrosis transmembrane conductance regulator mutant mice but there is no secretion from PAR-2 knock out mice. In conclusion, PAR2-AP is an agonist for mucus secretion from the airway gland that is Ca(2+)-dependent and cystic fibrosis transmembrane conductance regulator-independent.

[The estimation of systemic chemotherapy treatment administered in breast cancer on lysozyme activity in tears--preliminary report]. / Ocena wplywu systemowej chemioterapii stosowanej w leczeniu raka sutka na aktywnosc lizozymu zawartego we lzach--doniesienie wstepne.

PURPOSE: Estimation of cytostatics influence used in breast cancer treatment on lysozyme activity in human tears depend on time of treatment. MATERIAL AND METHODS: 8 women were treated at the base of chemotherapy schema: docetaxel with doxorubicin and 4 women treated with schema CMF: cyclophosphamide, methotrexate, 5-fluorouracil. Lysozyme activity in tears was assessed by measurement of diameter zone of Micrococcus lysodeicticus growth inhibition. RESULTS: It was revealed that both chemotherapy schema caused statistically significant reduction of diameter zone of M. lysodeicticus growth inhibition, after first and second course of chemotherapy treatment. After second chemotherapy course CMF schema induced loss of lysozyme activity in patient's tears (zero mm of M. lysodeicticus diameter zone growth inhibition). CONCLUSIONS: Systemic chemotherapy administered in breast cancer induce reduction of lysozyme activity in tears, that may cause higher morbidity of ocular surface infections caused by Gram-positive bacteria.

A time-course study of immune response in Japanese flounder Paralichthys olivaceus exposed to heavy oil.

PURPOSE: The immunotoxicities of oil and its components on fish immunities have been investigated, but there is little literature on the recovery of the fish from the immune suppression. Therefore, the recovery of Japanese flounder Paralichthys olivaceus from an immunosuppressive effect due to heavy oil (HO) exposure was investigated in this study. METHODS: Fish were exposed to HO at a concentration of 0.385 g/L for 2 days, while control fish received no exposure. Seven fish were sampled at 0, 3, 7, and 14 days post-exposure. The respiratory rate was measured everyday as an indicator of the acute effect of HO exposure. Fish serum was collected and used for antibacterial activity assay against Edwardsiella tarda. Expression changes of respiratory and immune-related genes were evaluated by real-time PCR. RESULTS AND DISCUSSION: The respiratory rate was significantly increased in the HO-exposed group until 4 days post-exposure. A respiratory-related gene, ß-hemoglobin, was also significantly downregulated in the spleen both at 0 and 7 days post-exposure and kidney at 3 days post-exposure in HO-exposed fish. Immunotoxicity, including suppression of antibacterial activities and downregulation of the IgM gene, was observed in HO-exposed fish until 3 days post-exposure, but not after that time. From these results, we conclude that the fish likely return to normal status around 1 week.

Activation of c-Myb transcription factor is critical for PMA-induced lysozyme expression in airway epithelial cells.

Lysozyme is a major component of airway epithelial secretions, acts as cationic anti-microbial protein for innate immunity. Although lysozyme plays an important role in airway defense and is a key component of airway secretions under inflammatory conditions, little is understood about the regulation of its expression and the associated signaling pathway. We wanted to examine whether Phorbol 12-myristate 13-acetate (PMA), one of PKC activators, treatment of the airway epithelial cell line NCI-H292 increases lysozyme gene expression. In this study, we sought to determine which signal molecules are involved in PMA-induced lysozyme gene expression. We found that PKC and mitogen-activating protein/ERK2 kinase are essential for PMA-induced lysozyme expression and also mediate the PMA-induced activation of c-Myb protein. We identified a proximal region of the lysozyme promoter essential for promoter activity containing c-Myb transcription factor binding site. Additionally, by site-directed promoter mutagenesis, we identified that c-Myb preferred the CAA motif of the -85/-73 region of the lysozyme promoter. Finally, we showed that overexpression of c-Myb without PMA treatment increased the lysozyme promoter activity and protein expression. From these results, we conclude that PMA induces overexpression of lysozyme via ERK1/2 MAP kinase-c-Myb signaling pathways in NCI-H292 cells.

Genistein and hematological malignancies.

Genistein is an isoflavanoid from soybeans and promising cancer chemotherapeutic agent. Genistein exposure varies widely because of cultural differences in diet. Hypothetically, this could account for differential cancer risk across ethnic populations. Genistein inhibits the growth of many different cancer cell lines by increasing apoptosis, inducing cell cycle delays, and modulating intracellular signaling pathways. Data from recent studies suggest that the therapeutic potential of genistein extends to cancers that affect blood, bone marrow, and lymph nodes. The objective of this paper is to provide background information on genistein, and discuss its potential as a therapeutic agent for treating hematological malignancies.

Investigating the influences of redox buffer compositions on the amyloid fibrillogenesis of hen egg-white lysozyme.

More than twenty different human proteins have been found to fold abnormally resulting in the formation of pathological deposits and several lethal degenerative diseases. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained rather elusive. The present study is aimed at exploring the effect of the ratio of cysteine and cystine in the buffer on the fibrillation of hen egg-white lysozyme. Our results revealed that the inhibition of lysozyme amyloid formation by cysteine in the redox buffer followed a concentration-dependent fashion. Cystine, the oxidized form of cysteine, nevertheless, did not influence the final level of fibrillation although it lengthened the lag period of fibril formation. Moreover, the effect of the ratio of cysteine to cystine in the buffer on the fibrillogenesis of hen lysozyme was found to be greatly associated with the formation of mixed disulfide derivatives. Finally, a possible reaction mechanism was proposed to explain our experimental results. Our study shows that the concentration of mixed disulfide derivative was inversely correlated with the level of lysozyme fibrillogenesis. The results from this work may aid in comprehending the molecular mechanism(s) of amyloid fibrillogenesis for disulfide bonded proteins and the development of effective therapeutics for amyloidogenic diseases.

Effects of Cistus-tea on bacterial colonization and enzyme activities of the in situ pellicle.

OBJECTIVES: Polyphenols are expected to have antibacterial properties. Cistus is a tea rich in polyphenols. The aim of the present in situ study was to investigate the effect of Cistus-tea on the pellicle and on the initial oral biofilm. METHODS: For in situ pellicle formation and initial biofilm formation, bovine enamel slabs were fixed on maxillary splints and carried by four subjects at buccal sites for up to 2 h. Bacteria present in 120-min pellicles were determined with DAPI-staining and fluorescence in situ hybridization with and without a 10 min rinse with Cistus-tea performed 1 min after incorporation of the slabs. In addition, amylase, lysozyme, glucosyltransferase and peroxidase activities immobilised in the pellicle layer were measured before and after rinsing for 10 min with Cistus-tea. RESULTS: The amount of bacteria detected in the 120-min biofilm was reduced significantly, if a 10 min rinse with Cistus-tea was performed one min after insertion of the enamel slabs. DAPI-staining yielded 13.2+/-3.5 for controls and 6.5+/-1.1 x 10(4) bacteria/cm(2), if a rinse with Cistus-tea was applied. Lysozyme, amylase and glucosyltransferase activities immobilised in the pellicle were not affected following a rinse with Cistus-tea. However, peroxidase activity was reduced significantly. CONCLUSIONS: Cistus-tea may be used to reduce the initial bacterial adhesion in the oral cavity.

Effects of dietary beta-1, 3 glucan on innate immune response of large yellow croaker, Pseudosciaena crocea.

The present study was conducted to investigate the effects of dietary beta-1, 3 glucan on the innate immune response and protection against Vibrio harveyi infection in large yellow croaker, Pseudosciaena crocea. A basal diet was supplemented with 0% (control), 0.09% (low) and 0.18% (high) beta-1, 3 glucan to formulate three experimental diets. Each diet was randomly allocated to triplicate groups of fish in floating sea cages (1.5 x 1.5 x 2.0m), and each cage was stocked with 100 fish (initial average weight 9.75+/-0.35 g). Fish were fed twice daily (05:00 and 17:00) to apparent satiation for 8 weeks. The results of 8 weeks feeding trial showed that low glucan supplementation (0.09%) significantly enhanced fish growth, whereas high supplementation (0.18%) did not. The serum lysozyme activity was significantly increased with the increase of dietary glucan (P < 0.05), and fish fed the diet with high glucan had significantly higher lysozyme activity compared with low glucan. There were no significant differences in alternative complement pathway (ACP) activity between fish fed diets with and without supplementation of glucan. The phagocytosis percentage (PP) and respiratory burst activity in fish fed the diet with 0.09% glucan were significantly higher than those in fish fed with the control diet (P < 0.05), but both immunological parameters significantly decreased in fish fed the diet with high supplementation compared with low supplementation and no significant difference was observed between the control and high supplementation groups. The challenge experiment showed that fish fed the diet with low glucan had significantly lower cumulative mortality compared with the control and high glucan groups (P < 0.05), but no significant difference was observed between the control and high supplementation groups. These results suggested that low glucan could enhance growth and innate immunity of large yellow croaker with an 8-week oral administration, but higher supplementation did not influence growth, or further improve immunity of large yellow croaker.

Effects of 5-aza-2'-deoxycytidine on proliferation, differentiation and p15/INK4b regulation of human hematopoietic progenitor cells.

The demethylating agents 5-azacytidine and 5-aza-2'-deoxycytidine (DAC) have been shown to induce differentiation and inhibit growth of leukemic myeloid cells at low concentrations. However, the effect of DAC in changing the differentiation and proliferation behavior of normal human myeloid progenitors has rarely been investigated. Therefore, we established an in vitro model of normal hematopoietic differentiation, using CD34+ cells from mobilized peripheral blood, to study proliferation and colony formation, expression of several myeloid maturation markers and of the inhibitor of cyclin-dependent kinases p15/INK4b. Upon DAC treatment, cell growth was significantly decreased in a dose-dependent manner, without an increase in cytotoxicity. DAC treatment also resulted in a substantial increase of lysozyme-positive cells, which could be enhanced by G-CSF, a modest increase of myeloperoxidase+ and CD15+ cells, as well as an increase of colony-forming cells (CFU-GM) compared to control cells. p15/INK4b protein expression was strongly upregulated upon myeloid maturation, and additional DAC treatment did not change p15 expression or the methylation status of the p15 promoter at the noncytotoxic concentrations used. Taken together, these data indicate a role of DAC in changing myeloid progenitor cell expansion and differentiation. This model appears suitable also for global analyses of multiple differentially methylated genes.

The effects of polycyclic aromatic hydrocarbons on the immune system of fish: a review.

Polycyclic aromatic hydrocarbons are an important class of environmental pollutants that are known to be carcinogenic and immunotoxic. This review summarizes the diverse literature on the effects of these pollutants on innate and acquired immunity in fish and the mechanism of PAH-induced immunotoxicity. Among innate immune parameters, many authors have focused on macrophage activities in fish exposed to polycyclic aromatic hydrocarbons. Macrophage respiratory burst appears especially sensitive to polycyclic aromatic hydrocarbons. Among acquired immune parameters, lymphocyte proliferation appears highly sensitive to polycyclic aromatic hydrocarbon exposure. However, the effects of polycyclic aromatic hydrocarbons on both specific and non-specific immunity are contradictory and depend on the mode of exposure, the dose used or the species studied. In contrast to mammals, fewer studies have been done in fish to determine the mechanism of polycyclic aromatic hydrocarbon-induced toxicity. This phenomenon seems to implicate different intracellular mechanisms such as metabolism by cytochrome P4501A, binding to the Ah-receptor, or increased intracellular calcium. Advances in basic knowledge of fish immunity should lead to improvements in monitoring fish health and predicting the impact of polycyclic aromatic hydrocarbons on fish populations, which is a fundamental ecotoxicological goal.

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80°C) and under high pressure conditions at low temperature (3.75 kbar, -13°C). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

Effects of prolactin and growth hormone on plasma levels of lysozyme and ceruloplasmin in rainbow trout.

In vivo and in vitro effects of prolactin (PRL) and growth hormone (GH) on plasma levels of lysozyme and ceruloplasmin were examined in the rainbow trout (Oncorhynchus mykiss). Hypophysectomy had no effect on the plasma lysozyme level. Implantation of PRL- or GH-containing cholesterol pellets increased the lysozyme level in a dose-related manner. After hypophysectomy and sham operation, plasma ceruloplasmin was elevated above the level in intact fish, suggesting inflammation caused by the surgery. PRL or GH treatment significantly attenuated the increased level of ceruloplasmin in the operated fish. Expression of lysozyme mRNA was detected in the leucocytes isolated from the peripheral blood by RT-PCR. In vitro administration of PRL or GH showed no effect on the proliferation of isolated leucocytes or on the total protein content; however, lysozyme activity in the medium increased in a dose-related manner. These results suggest that PRL and GH directly stimulate lysozyme production without affecting the proliferation of leucocytes, and the attenuated ceruloplasmin level increased in response to inflammation.