Fatty acid composition of eggs and its relationships to egg and larval viability from domesticated common sole (Solea solea) breeders.

The study of lipids and fatty acids (FAs) has been used in the assessment of egg quality because their composition can influence the fertilization rate, hatching, survival and growth of marine fish larvae. For these reasons, the lipid content (TL) and fatty acid composition of common sole (Solea solea) eggs were measured and correlated to egg and larval viability parameters throughout an entire reproductive season. Seventeen batches of fertile eggs obtained from natural spawning of captive breeders were characterized for the TL, FA profile, hatching rate (HR) and survival rate of larvae (SR) at 0-6 days post-hatching (dph). The egg FA composition reflected the composition of the feed supplied to the broodstock during summer and autumn (before and during vitellogenesis) rather than that supplied during the spawning season. In general, the egg FA profile showed minimal differences among the early-, mid- and late-spawning periods (possibly due to the change of the diet and/or water temperature) indicating that it is possible to obtain a similar egg quality in terms of egg FA profile over 2 months of spawning. Saturated FAs and monounsaturated FAs (MUFA) were positively correlated with HR, while TL, 22 : 6n-3 (DHA), 20 : 4n-6 (ARA), polyunsaturated FAs of the (n-3) series (n-3 PUFA) and polyunsaturated FAs of the (n-6) series were negatively correlated (p ≤ 0.05). MUFA, 20 : 5n-3 (EPA), n-6/n-3 were positively correlated with SR, while DHA, n-3 PUFA, DHA/EPA were negatively correlated (p ≤ 0.05). In conclusion, the feed supplied before and during vitellogenesis has a major role in determining the egg FA profile in common sole. The relationships found between TL and FAs with egg and larval viability parameters differ from many other farmed marine fish species, which may suggest the need for a specific broodstock feed for this species.

A role for neutral sphingomyelinase-mediated ceramide production in T cell receptor-induced apoptosis and mitogen-activated protein kinase-mediated signal transduction.

Studying apoptosis induced by T cell receptor (TCR) cross-linking in the T cell hybridoma, 3DO, we found both neutral sphingomyelinase activation and production of ceramide upon receptor engagement. Pharmacological inhibition of ceramide production by the fungal toxin, fumonisin B1, impaired TCR-induced interleukin (IL)-2 production and programmed cell death. Addition of either exogenous ceramide or bacterial sphingomyelinase reconstituted both responses. Moreover, specific inactivation of neutral sphingomyelinase by antisense RNA inhibited IL-2 production and mitogen-activated protein kinase activation after TCR triggering. These results suggest that ceramide production by activation of neutral sphingomyelinase is an essential component of the TCR signaling machinery.

Effects of feeding low fishmeal diets with increasing soybean meal levels on growth, gut histology and plasma biochemistry of sea bass.

The aquaculture industry depends upon the development of sustainable protein sources to replace fishmeal (FM) in aquafeeds and the products derived from soybeans are some of the most studied plant feedstuffs. A key area of investigation for continuing to improve modern aquafeeds includes the evaluation of varying proportions and combinations of plant ingredients to identify mixtures that are more efficiently utilized by the fish. This study investigated the effects of increasing soybean meal (SBM) by replacing a mix of plant ingredients in low FM (20%) diets on growth, blood biochemistry profile and gut histology on European sea bass. Five isonitrogenous and isolipidic experimental diets were formulated: four diets containing increasing SBM levels (0, 10, 20 and 30%; 0SBM, 10SBM, 20SBM and 30SBM, respectively) with a low content of FM (20%) and one control diet (0% SBM; 35% FM). Diets containing SBM brought to comparable performance and protein utilization, while 0SBM had negative impact on feed conversion rate and protein utilization. Blood parameters suggested an optimal nutritional status under all feeding treatments, even though slightly decreased values were reported at increasing dietary SBM. Histology examination did not show any changes indicative of soy-induced enteritis. We can conclude that for European sea bass: (i) different blends of plant protein did not affect feed intake despite the 20% FM dietary level; (ii) the inclusion of SBM maintains optimal growth and feed utilization in low FM diets; (iii) blood biochemistry profile showed a good nutritional status under all feeding regimes; (iv) no evidence of soy-induced enteritis was reported in any group fed low FM diets. For formulation of practical diets in on-growing of European sea bass, SBM up to 30% can be successfully incorporated into feeds containing low FM inclusion.

Activation of the oxidative burst in human monocytes is associated with inhibition of methionine-dependent methylation of neutral lipids and phospholipids.

Chemotaxis and generation of the oxidative burst by phagocytes are among the biological functions thought to require methylation reaction(s) for their expression. The present study investigated the effect of different stimuli of the oxidative burst on lipid methylation by human elutriated monocytes as measured by methyl group incorporation from [methyl-3H]methionine into both phospholipid and neutral lipid extracts. Normal monocytes, incubated at 37 degrees C for 1 h with 2 microM methionine, incorporated 10.2-fmol/10(6) cells and 73.6-fmol/10(6) cells of methyl groups into neutral lipids and phospholipids, respectively. Stimulators of the respiratory burst, such as the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine, the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate, and the calcium ionophore, A23187, decreased the incorporation of methyl groups into both neutral lipids and phospholipids in a similar manner. Increasing the concentration of methionine in the medium reversed or attenuated the inhibition achieved at lower levels. An inverse relationship existed between the degree of methylation and the extent of stimulation of the oxidative burst, measured as superoxide anion (O-2) release. Stimulated monocytes oxidized methionine to methionine sulfoxide (which cannot act as a methyl-donor), and this was dependent on activation of the respiratory burst. Elimination of the accumulated methionine sulfoxide by replacement of the medium or by prevention of extracellular methionine oxidation by catalase did not effectively restore the normal level of methylation in stimulated cells, and the reduced methylation was not primarily related to a defective methionine uptake by stimulated monocytes. These data suggest that intracellular events related to activation of the respiratory burst are responsible for the decreased lipid methylation in stimulated cells, possibly by their leading to intracellular formation of methionine sulfoxide and by their limiting the availability of methyl-donor. This mechanism may be of potential relevance for the expression of biological functions where methionine-dependent reactions are involved.

Functional independence and interdependence of the Src homology domains of phospholipase C-gamma1 in B-cell receptor signal transduction.

B-cell receptor (BCR)-induced activation of phospholipase C-gamma1 (PLCgamma1) and PLCgamma2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCgamma activation, the mechanism coupling PLCgamma to the BCR remains undefined. The role of PLCgamma1 SH2 and SH3 domains at different steps of BCR-induced PLCgamma1 activation was examined by reconstitution in a PLCgamma-negative B-cell line. PLCgamma1 membrane translocation required a functional SH2 N-terminal [SH2(N)] domain, was decreased by mutation of the SH3 domain, but was unaffected by mutation of the SH2(C) domain. Tyrosine phosphorylation did not require the SH2(C) or SH3 domains but depended exclusively on a functional SH2(N) domain, which mediated the association of PLCgamma1 with the adapter protein, BLNK. Forcing PLCgamma1 to the membrane via a myristoylation signal did not bypass the SH2(N) domain requirement for phosphorylation, indicating that the phosphorylation mediated by this domain is not due to membrane anchoring alone. Mutation of the SH2(N) or the SH2(C) domain abrogated BCR-stimulated phosphoinositide hydrolysis and signaling events, while mutation of the SH3 domain partially decreased signaling. PLCgamma1 SH domains, therefore, have interrelated but distinct roles in BCR-induced PLCgamma1 activation.

Membrane raft-dependent regulation of phospholipase Cgamma-1 activation in T lymphocytes.

Numerous signaling molecules associate with lipid rafts, either constitutively or after engagement of surface receptors. One such molecule, phospholipase Cgamma-1 (PLCgamma1), translocates from the cytosol to lipid rafts during T-cell receptor (TCR) signaling. To investigate the role played by lipid rafts in the activation of this molecule in T cells, an influenza virus hemagglutinin A (HA)-tagged PLCgamma1 was ectopically expressed in Jurkat T cells and targeted to these microdomains by the addition of a dual-acylation signal. Raft-targeted PLCgamma1 was constitutively tyrosine phosphorylated and induced constitutive NF-AT-dependent transcription and interleukin-2 secretion in Jurkat cells. Tyrosine phosphorylation of raft-targeted PLCgamma1 did not require Zap-70 or the interaction with the adapters Lat and Slp-76, molecules that are necessary for TCR signaling. In contrast, the Src family kinase Lck was required. Coexpression in HEK 293T cells of PLCgamma1-HA with Lck or the Tec family kinase Rlk resulted in preferential phosphorylation of raft-targeted PLCgamma1 over wild-type PLCgamma1. These data show that localization of PLCgamma1 in lipid rafts is sufficient for its activation and demonstrate a role for lipid rafts as microdomains that dynamically segregate and integrate PLCgamma1 with other signaling components.

Serum-associated inhibition of neutrophil Fc receptors in cancer patients.

Rosettes with ox erythrocytes coated with purified IgG antibody were used to detect Fc receptors on neutrophils from 60 patients with solid neoplasias and from 55 normal controls. The mean average of the rosettes in the patients was 48.10%, and that in normal controls was 79.42%, with a highly significant difference according to the Wilcoxon test [negative probability, P(w)-4.47 . 10(-5)]. The low proportion of patients' rosettes ws related to the presence of a serum factor, which also inhibited normal neutrophil rosette formation. Patient neutrophils (or normal neutrophils treated with patient sera) recovered their rosetting capacity when cultured in vitro. No correlation was found between low percentages of rosette-forming cells and the level of circulating immune complexes of the individual patients. Additional evidence also supported the finding that IC and the serum factor are probably unrelated.

Lysis of tumor cells by human blood monocytes by a mechanism independent of activation of the oxidative burst.

Tumorilytic human blood monocytes recognize and destroy neoplastic cells by a mechanism that is nonphagocytic and requires cell-to-cell contact. The mechanism of cytolysis subsequent to binding is controversial. Release of reactive oxygen intermediates by activated rodent macrophages has been suggested as an important mechanism for tumor cell lysis in some short-term cytotoxicity assays. We examined whether oxygen intermediates are also responsible for mediating the lysis of adherent human tumor cells in a long-term (72-h) tumoricidal assay. Human blood monocytes were incubated with medium, concanavalin A-stimulated lymphokine [macrophage-activating factor (MAF)], lipopolysaccharide endotoxin, or human recombinant gamma interferon for 24 h prior to the addition of [125I] iododeoxyuridine-labeled A375 melanoma cells. The following evidence indicated that monocyte-mediated tumor cell lysis was independent of superoxide anion (O2-) and H2O2 production: (a) although human blood monocytes incubated for 24 h with gamma interferon produced twice as much O2- as control or MAF-treated monocytes, gamma interferon did not activate monocyte tumoricidal activity unless combined with lipopolysaccharide endotoxin, 0.2 ng/ml or more; (b) incubating the monocytes with 10 nM phorbol myristate acetate for 0.5 h stimulated O2- production but no cytotoxicity; (c) the cytolytic activity of MAF-treated monocytes was not decreased in the presence of catalase or superoxide dismutase; and (d) finally, peripheral blood monocytes were isolated from six patients with chronic granulomatous disease, activated by MAF or lipopolysaccharide endotoxin, and then assayed for tumoricidal activity. While these activated chronic granulomatous disease monocytes did not produce O2- or H2O2, tumor cell lysis was normal in all six patients. Hence, lysis of tumor cells in a 72-h assay is not dependent upon the generation of O2- and/or H2O2 and is intact in chronic granulomatous disease monocytes.

Characterization of purified cryopreserved human monocyte function in assays of superoxide production, accessory cell function, chemotaxis, and in fluorescent cell sorter analysis.

The ability to use highly purified cryopreserved human monocytes in various in vitro assays has a number of practical and theoretical advantages, including convenience and the potential for enhanced reproducibility. Large numbers (up to 1 X 10(9)) human monocytes can be isolated from a single donor in a purified suspension state, by a combination of leukapheresis and counter-current centrifugal elutriation (CCE) technologies. Following short- and long-term periods of cryopreservation, the viability and phagocytic function of these CCE-purified monocytes was unimpaired. Cryopreserved monocytes were similar to fresh cells in their ability to release superoxide anion (O2-), although unstimulated and stimulated O2- release values tended to increase slightly following weeks to months of cryopreservation. In contrast, even short-term cryopreservation diminished both the random migration and chemotactic responses of human monocytes; however, cryopreserved monocytes could be employed in this assay provided the calculated chemotactic ratio (chemotactic migration/spontaneous migration) was used. Cryopreserved monocytes demonstrate 70% of fresh monocyte accessory cell function in pokeweed mitogen-induced lymphocyte proliferation assays. When the binding of OKT3, OKM1, anti-DR, and fluoresceinated pokeweed mitogen to monocytes was analyzed in the fluorescence activator cell sorter (FACS), cryopreserved and fresh monocytes displayed a similar pattern of membrane reactivity.

Differential changes in lipid metabolism of myeloid and lymphoid cell lines induced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA).

Treatment of the myeloid cell lines, U-937 or HL-60, with 10 nM of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), for 24 h increased the rate of incorporation of [3H]glycerol into total chloroform extracts. A proportionally greater labeling of the non-polar lipid (NL) fraction compared to the polar, phospholipid (PL), fraction was observed. Chromatographic analysis showed a 6-fold increase in the labeling of triacylglycerols (TAG), a 2-fold increase in diacylglycerols, and no changes in monoacylglycerols. PL labeling showed a 3-fold increase in phosphatidylcholine (PC). The effect of TPA on TAG labeling was selectively observed in myeloid cell lines. No such a change was found in the lymphoid cell line. MOLT-3, which did respond to TPA with increased PC labeling. Incorporation of [3H]arachidonic acid (AA) into TAG by U-937 cells was selectively increased (2.5-fold) after treatment with TPA for 24 h. Treatment of U-937 cells with TPA in serum-free medium resulted in no increased labeling of TAG. These studies suggest that changes in TAG metabolism may be characteristic of myeloid differentiation and depend on the presence of serum factor(s).

Differential effects of Cbl and 70Z/3 Cbl on T cell receptor-induced phospholipase Cgamma-1 activity.

We demonstrate that the differential effects Cbl and oncogenic 70Z/3 Cbl have on Ca(2+)/Ras-sensitive NF-AT reporters is partially due to their opposing ability to regulate phospholipase Cgamma1 (PLCgamma1) activation as demonstrated by analysis of the activation of an NF-AT reporter construct and PLCgamma1-mediated inositol phospholipid (PI) hydrolysis. Cbl over-expression resulted in reduced T cell receptor-induced PI hydrolysis, in the absence of any effect on PLCgamma1 tyrosine phosphorylation. In contrast, expression of 70Z/3 Cbl led to an increase in basal and OKT3-induced PLCgamma1 phosphorylation and PI hydrolysis. These data indicate that Cbl and 70Z/3 Cbl differentially regulate PLCgamma1 phosphorylation and activation. The implications of these data on the mechanism of Cbl-mediated signaling regulation are discussed.

Specific regions of the extracellular domain of dlk, an EGF-like homeotic protein involved in differentiation, participate in intramolecular interactions.

The level of expression of dlk, an EGF-like protein possessing six EGF-like repeats in its extracellular region, is critical for 3T3-L1 fibroblasts to differentiate into adipocytes in response to IGF1. The mechanism of action of dlk is not well understood, but its localization on the cell membrane suggests that dlk may function as a receptor, as a ligand or as a regulatory protein modulating the binding, the signaling, or the expression of other molecules involved in cell differentiation and growth. In this work, we demonstrate, by using the Yeast Two-Hybrid system, that dlk interacts with itself through specific regions of its extracellular domain. The strongest interactions were observed between specific EGF-like repeats and between a non EFG-like region where unknown proteases act to generate soluble forms of dlk. These observations suggest that the interaction between two membrane dlk molecules belonging to the same or to different cells, or the interaction between soluble and membrane dlk variants, may be important to regulate dlk function.

Inflammatory mediator release from human monocytes via immobilized Fc receptors. Its potential role in adverse reactions to systemic monoclonal antibody therapy.

Human monocytes released superoxide anion, IL-1, and TNF subsequent to binding of their Fc receptor I to murine IgG2a or rabbit IgG. Fc receptor II binding to murine IgG2b or IgG1 had similar consequences. Immobilized murine monoclonal antibodies, IgG2a anti-CD3 (OKT3) or IgG1 anti-CD44 also induced superoxide anion and monokine production. Monocytes bound OKT3 via FcRI and responded to immobilized OKT3 by inflammatory mediator release in the absence of T cells. These results suggest that direct interaction of immunoglobulins with monocytes via FcR may represent an important phase of the pathophysiology of adverse reactions to systemic monoclonal antibodies.

Differential turnover of enzymes involved in human monocyte eicosanoid metabolism. Selective inhibition of cyclooxygenase product formation by cycloheximide in the absence of effects on 5-lipoxygenase or phospholipase A2.

Human monocytes treated with cycloheximide (CHX) demonstrated a concentration- and time-dependent inhibition of prostaglandin E2 (PGE2) synthesis and release in response to stimulation with phorbol myristate acetate, ionomycin, serum-treated zymosan, or concanavalin A. The effect of CHX required preincubation and was largely reversible within 2 hr. Thromboxane A2 release was affected similarly but no comparable effects were observed on labeled arachidonic acid release or leukotriene B4 generation. The PGE2 response was also inhibited by CHX when monocytes were given exogenous arachidonic acid with or without stimulation. CHX pretreatment also comparably decreased the amount of immunoreactive cyclooxygenase in resting and stimulated monocytes. These data indicate that monocyte cyclooxygenase, in contrast to phospholipase A2 or 5-lipoxygenase and their regulatory proteins, turns over rapidly and may be a target for up- or down-regulation by pharmacologic or (potentially) physiologic agents which affect protein synthesis or degradation.

Defective neutrophil mobilization to skin chambers in cancer patients.

The in vivo migration of neutrophils was evaluated in patients affected by epithelial carcinoma using the quantitative skin-chamber technique. The results demonstrated a significant impairment of the patients' neutrophil migration, which was reduced to approximately 4% of that of the controls. Patients' sera were able to inhibit the chemotactic responsiveness of normal neutrophils in vitro. It is therefore suggested that the defective in vivo migration of neutrophils in cancer patients is related to the presence of humoral cell-directed inhibitory activity. This defect of neutrophil function might contribute to the host-defense impairment of carcinoma patients.

Inhibition of release of arachidonic acid, superoxide, and IL-1 from human monocytes by monoclonal anti-HLA class II antibodies: effects at proximal and distal points of inositol phospholipid hydrolysis pathway.

Incubation of human elutriator-purified monocytes with anti-HLA-DR or DQ antibody inhibited the release of arachidonic acid induced by serum-treated zymosan (STZ), a phagocytic stimulus that is known to induce inositol phospholipid hydrolysis and Ca2+ influx. However, only anti-HLA-DR antibody partially inhibited STZ-induced inositol phospholipid hydrolysis and concanavalin-A-induced Ca2+ influx. Incubation with anti-HLA-DR or -DQ antibody inhibited phorbol ester-induced AA release as well as superoxide production and IL-1 release. Inhibition of monocyte function by anti-class II antibodies was not accompanied by cAMP elevation. Furthermore, addition of exogenous db-cAMP and other agents (forskolin, cholera toxin, or 3-isobutyl-1-methyl-xanthine) that increase cAMP levels through different mechanisms, alone or in combination with anti-HLA antibodies, had no inhibitory effect on factor release. Our results demonstrate that perturbation of class II molecules down-modulates cell activation at more than one point of the signal transduction pathway with dominant inhibition distal to inositol phospholipid hydrolysis. They also suggest that the inhibition by anti-HLA class II antibody is probably not mediated via cAMP elevation.

Stimulation of human monocytes by anti-CD3 monoclonal antibody: induction of inflammatory mediator release via immobilization of Fc receptor by adsorbed immunoglobulin and T-lymphocytes.

Human monocytes released superoxide anion, prostaglandin E2, leukotriene B4, IL-1, and TNF when exposed to plastic surfaces coated with murine anti-CD3 monoclonal antibody, OKT 3. Stimulation of mediator release by OKT 3 was dependent on the amount of antibody immobilized onto wells of plastic tissue culture plates. Soluble antibody or antibody adsorbed to monocytes and reacted with an aggregating ("cross-linking") second antibody failed to induce mediator release. Monocytes "armed" with OKT 3 formed rosettes with T cells in a fashion indistinguishable from that seen between monocytes and T cells sensitized with OKT 3. Monocytes with adsorbed OKT 3 antibodies released IL-1 beta and TNF-alpha when exposed to unsensitized T cells, although increased superoxide release could not be detected. OKT 4a, a murine IgG2a antibody that reacts with a different T cell epitope (CD4), failed to induce cytokine release from monocytes when cross-linked by T cells or a CD4+ T cell line, even in the presence of IL-2 or IFN-gamma. These data indicate that certain antibodies bound to Fc receptors (FcR) of monocytes may trigger monocyte function when reacting with cells bearing the appropriate target antigens. FcR-mediated signaling resulting in mediator release may be involved in initiating or regulating the immune response. Furthermore, systemically administered monoclonal antibodies may induce inflammatory responses and their attendant symptomatologies via their interaction with FcR-bearing inflammatory cells.

Functional consequences of phospholipase A2 activation in human monocytes.

Human monocytes release arachidonic acid upon stimulation with a variety of soluble or particulate agents. These include: phorbol esters (i.e., 12-O-tetradecanoate phorbol-13-acetate, TPA), calcium ionophores (ionomycin), serum-treated zymosan (STZ) concanavalin A (Con A), and, to a minor degree, lipopolysaccharides (LPS). Protein Kinase C activation or increased intracellular Ca2+ are common features of the actions of most, if not all, of these stimuli. Prevention of PKC activation by the use of staurosporine or chelation of extracellular calcium by EGTA selectively impaired AA release, indicating that PLA2 may be regulated by either pathway concurrently. The generation of inositol phosphates and diacylglycerol by the action of phospholipase C, notably upon interaction with opsonized particles during phagocytosis, apparently constitutes the physiological correlate of stimulation via these agents. Release of arachidonic acid by the action of PLA2 or other phospholipid hydrolyzing enzymes leads directly to the formation of cyclooxygenase products. In the presence of markedly elevated calcium concentrations, 5-lipoxygenase (LO) is activated as well, leading to the formation and release of leukotrienes. Agents which stimulate AA release also initiate other monocyte functions, including generation of reactive oxygen intermediates and lymphokine release. This observation makes it tempting to implicate PLA2 activation in many aspects of monocyte physiology. However, no correlation with PLA2 activation and either superoxide or lymphokine release was found when multiple stimuli, including TPA, ionomycin, serum-treated zymosan, concanavalin A, or LPS, were compared simultaneously. Instead, our results indicate that PLA2 activation is regulated by the same mechanisms, including PKC activation and increased Ca2+, as are other enzymes which determine expression of monocyte function. Phospholipase A2 (PLA2) hydrolyzes fatty acid from the sn-2 position of a wide variety of phospholipids. Substrates for this (these) enzyme(s) include species which contain a variety of polar head groups (choline, serine, ethanolamine, etc.) and some phospholipids with either linkages in sn-1. In many cell types, including human monocytes, phospholipase A2 commonly acts on substrates containing arachidonic acid (AA). The liberation of free arachidonate is a first step in the metabolism of prostaglandins, hydroxyeicosatetraeinoic acids, (HETE'S), and leukotrienes (Lt's). Monocytes and macrophages have been shown to be rich sources of arachidonate and its metabolites. Some biologic properties of monocytes, notably their role as immunomodulating cells, have been attributed to eicosanoid production and release. Accordingly, much of the interest regarding PLA2 in human monocytes centers on this aspect of their function.(ABSTRACT TRUNCATED AT 400 WORDS)