Expression analysis of lipopolysaccharide-and/or concanavalin A/phorbol myristate acetate-stimulated black rockfish peripheral blood leukocytes using a cDNA microarray to identify up-regulated genes

The responses of peripheral blood leucocytes (PBLs) from the black rockfish Sebastes schlegelii stimulated with lipopolysaccharides (LPS) and/or concanavalin A/phorbol myristate acetate (Con A/PMA) were investigated using a cDNA microarray consisting of 5,088 clones. This analysis showed that 254 unique genes were more than twofold upregulated, and they were selected for sequencing. Among the mitogens, 84 genes were more than twofold upregulated in LPS-stimulated PBLs, and 112 genes were induced in Con A/PMA-stimulated PBLs. Moreover, 58 other genes were more than twofold upregulated in PBLs stimulated with both LPS and Con A/PMA. Other genes were not significantly induced. Overall, these results suggest that certain genes in black rockfish have important roles in physiological and pathological processes. Furthermore, the microarray analysis suggested a promising tool for immune mechanisms in teleost fish.     

Identification and expression analysis of an interferon stimulated gene 15 (ISG15) from black rockfish, Sebastes schlegeli

The interferon stimulated gene 15 (ISG15) is strongly induced in many cell types by IFNs, viral infection and double-stranded RNA (poly I:C). The ISG15 homolog cDNA was isolated from the black rockfish poly I:C stimulated leukocyte cDNA library. The black rockfish ISG15 homolog was found to consist of 1070bp encoding 160 amino acid residues. Compared with other known ISG15 peptide sequences, the most conserved regions of the black rockfish ISG15 peptide were found to be the tandem ubiquitin-like domains and a C-terminal LRLRGG conjugating motif, characteristic of mammalian and non-mammalian ISG15 proteins. A phylogenetic analysis based on the deduced amino acid sequence revealed a homologous relationship between the ISG15 sequence of black rockfish and that of Atlantic salmon, Atlantic cod, crucian carp and rainbow trout. The expression of the black rockfish ISG15 molecule was induced in the peripheral blood leukocytes (PBLs) from 1 to 12h following poly I:C stimulation, with a peak at 6h post-stimulation. The black rockfish gene was predominantly expressed in the PBLs and the spleen.     

Microarray analysis of gene expression in peripheral blood leucocytes from rock bream (Oplegnathus fasciatus) after stimulation by LPS, ConA/PMA, and poly I:C

The responses of peripheral blood leucocytes (PBLs) from the rock bream Oplegnathus fasciatus stimulated with lipopolysaccharides (LPS), concanavalin A/phorbol myristate acetate (Con A/PMA), and/or polyriboinosinic polyribocytidylic acid (poly I:C) were investigated using a cDNA microarray consisting of 2,400 clones. A total of 336 unique genes were more than twofold up-regulated: 169 in LPS-stimulated PBLs, 209 in Con A/PMA-stimulated PBLs, and 146 in poly I:C-stimulated PBLs. Moreover, other genes were similarly up-regulated with combinations of stimulants: 85 genes in PBLs stimulated with both LPS and Con A/PMA, 79 in PBLs stimulated with both LPS and poly I:C, and 66 in PBLs stimulated with both Con A/PMA and poly I:C. Furthermore, 42 other genes were more than twofold up-regulated in PBLs stimulated with all tested mitogens. Other genes were not significantly induced. Overall, these results suggest that certain genes in rock bream play important roles in physiological and pathological processes. Furthermore, the microarray analysis showed promise as a tool for investigating immune mechanisms in teleost fish.     

Modulation of the activity of sea bass (Dicentrarchus labrax) head-kidney macrophages by macrophage activating factor(s) and lipopolysaccharide

The aim of this study was to establish the requirements for macrophage activating factor (MAF) production by sea bass head-kidney leucocytes and the kinetics of macrophage activation when exposed to MAF-containing supernatants and/or lipopolysaccharide (LPS), a known macrophage stimulant. MAF activity was found in culture supernatants of total head-kidney leucocytes pulsed with 5 microg ml(-1)Con A, 5 or 10 ng ml(-1)PMA and 100 ng ml(-1)calcium ionophore, or 10 microg ml(-1)Con A alone, as assessed by the capacity to prime macrophages for enhanced production of reactive oxygen intermediates (ROI). Mixed leucocyte cultures from two or eight fish showed higher MAF activity after stimulation, indicating that a mixed leucocyte reaction was also important for MAF production. MAF-induced activation of macrophage cultures was highest at 18 h of exposure and was lost by 72 h except for MAF induced by Con A-stimulation alone. LPS primed macrophages for increased ROI production at early incubation times and down-regulated ROI production after 24 h. LPS had no effect in further stimulating the MAF-induced priming effect on production of ROI and down-regulated the MAF-priming by 48 h. Sea bass head-kidney macrophages did not show increased nitrite production when exposed to MAF and/or LPS, which may be related to their differentiation status.     

Evidence for an age-related attenuation of cerebral microvascular antioxidant response to oxidative stress

Effects of aging and oxidative stress were studied in cerebral microvessels and microvessel-depleted brain from 6-, 18-, and 24-month-old C57Bl/6J mice exposed to normoxia, 24 or 48 h hyperoxia, or 24 h hyperoxia followed by 24 h normoxia. Microvessels lacked smooth muscle and consisted predominantly of endothelium. Following exposure and isolation of microvessel and parenchymal proteins, Western blot analysis was performed for detection of cytosolic thioredoxin 1 (TRx 1) and mitochondrial thioredoxin 2 (TRx 2), protein carbonyl, and mitochondrial superoxide dismutase (MnSOD). Both microvessel and parenchymal TRx 1 levels were increased by hyperoxia; however, the microvascular response was limited and delayed in comparison to that of the parenchymal fraction. Whereas TRx 2 levels in microvessels were increased in older mice, irrespective of exposure condition, hyperoxia per se had little or no apparent effect. Parenchymal cells showed no age-related increase in TRx 2 level under normoxic conditions, but showed increased levels following hyperoxia. Microvessel MnSOD was lower than that in parenchymal cells, but increased with age under normoxia, and also was correlated with the duration of hyperoxia. Although hyperoxia augmented MnSOD levels in young (6 months) and middle-aged (18 months) animals, the response was less pronounced in microvessels from senescent, 24-month-old mice. Unlike microvessels, which showed a sustained age-related increase in MnSOD level under each exposure condition, parenchymal cells from normoxic mice showed no increase, and hyperoxia-induced elevations declined with prolonged 48 h exposure. These results indicate that the microvessel endothelium is (1) subjected to a more intense oxidative environment than neurons and glia and (2) is limited by aging in its ability to respond to oxidative insult.     

Relative roles of T-cell receptor ligands and interleukin-2 in driving T-cell proliferation

Stimulation of T cells by the T-cell receptor (TCR)/CD3 complex results in interleukin-2 (IL-2) synthesis and surface expression of the IL-2 receptor (IL-2R), which in turn drive T-cell proliferation. However, the significance of the requirement of IL-2 in driving T-cell proliferation, when TCR stimulation itself delivers potential mitogenic signals, is unclear. We show that blocking of IL-2 synthesis by Cyclosporin A (CsA) suppressed both the Concanavalin A (Con A)- and phorbol myristate acetate (PMA)/ionomycin-induced proliferation of T cells. The latter is also inhibited by anti-IL-2R. Kinetic studies showed that T-cell proliferation begins to become resistant to CsA inhibition by about 12 h and became largely resistant by 18 h of stimulation. PMA, the protein kinase C activator, enhanced Con A-induced T-cell proliferation if added only within first 12 h of stimulation, and not after that. Given the fact that, in the present study, TCR is downregulated within 2 h of Con A stimulation and T cells entered the S phase of cell cycle by about 18 h of stimulation, the above results suggest that TCR stimulation provides the initial trigger to the resting T cells, which allows the cells to traverse the first two third portions of G1 phase of cell cycle and become proliferation competent. IL-2 action begins afterward, delivering the actual proliferation signal(s), allowing the cells to traverse the rest of G1 phase and enter the S phase of the cell cycle.     

Calcium-dependent eicosanoid metabolism by concanavalin A-stimulated human monocytes in vitro. Synergism with phorbol ester indicates separate regulation of leukotriene B4 synthesis and release.

Human monocytes obtained by counter-current centrifugal elutriation released arachidonic acid when challenged in vitro with Con A, as well as with other soluble (PMA or ionomycin) or particulate stimuli (serum-treated zymosan). Cyclo-oxygenase metabolites were the principal eicosanoids detected in the supernatants of Con A-stimulated, [3H]arachidonate-labeled monocytes, 5-Lipoxygenase (5-LO) products, such as leukotriene B4 (LTB4), were conspicuously absent. Release of arachidonate and its metabolites in response to Con A was dependent on the presence of extracellular Ca2+, but not Mg2+. In contrast to serum-treated zymosan challenge, which resulted in increased inositol trisphosphate and LTB4 release, Con A-induced inositol phospholipid hydrolysis in monocytes was limited to phosphatidylinositol or phosphatidylinositol monophosphate. Despite an inability to augment LTB4 release, Con A or PMA induced a loss of 5-lipoxygenase from a cytosolic compartment that was similar to that achieved with a calcium ionophore (ionomycin), a potent stimulus for LTB4 generation. When cell-associated LTB4 was evaluated, evidence for increased LTB4 production was obtained in response to either stimulus (PMA greater than Con A). In combination, however, PMA and Con A treatment resulted in monocyte LTB4 release comparable with that observed with the calcium ionophore or STZ. LTB4 release in response to all stimuli tested was inhibited by MK-886, a drug that binds to 5-lipoxygenase-activating protein. These results indicate the following: 1) Phospholipase A2 activation and attendant arachidonic acid release induced by agents that increase intracellular Ca2+ and/or generate diacylglycerol results in increased synthesis and release of PG and increased synthesis of leukotrienes, but not necessarily leukotriene release. 2) 5-LO translocation, which may occur independently of increased intracellular Ca2+, may be necessary for LTB4 generation but is insufficient for its release. 3) 5-Lipoxygenase-activating protein activity is necessary for 5-LO activation and LTB4 release in response to all stimuli investigated here. 4) Phorbol ester, an activator of protein kinase C, may synergize with agents such as Con A (which by themselves induce a minimal intracellular Ca2+ rise), so as to result in the release of LTB4. Thus, Con A may represent a class of surface receptor-aggregating agents that initiates inflammatory changes or immunomodulation associated with liberation of PG and might predispose to release of other inflammatory mediators, such as leukotrienes, in the presence of additional signals including protein kinase activation.     

Draft Genome Sequence of Staphylococcus saprophyticus subsp. saprophyticus M1-1, Isolated from the Gills of a Korean Rockfish, Sebastes schlegeli Hilgendorf, after High Hydrostatic Pressure Processing

A bacterium designated M1-1 was isolated from the gills of a Korean rockfish, Sebastes schlegeli Hilgendorf, after high hydrostatic pressure processing. Studies of 16S rRNA phylogeny and comparative genomics demonstrated that the isolate belongs to Staphylococcus saprophyticus subsp. saprophyticus. Here, we report the draft genome sequence of S. saprophyticus subsp. saprophyticus M1-1 (KACC 16562).     

Cloning and characterization of a caspase gene from black tiger shrimp (Penaeus monodon)-infected with white spot syndrome virus (WSSV)

A black tiger shrimp (Penaeus monodon) caspase cDNA homologue (PmCasp) has been identified from a hemocyte library using a previously identified caspase homologue from the banana shrimp (Penaeus merguiensis) as a probe. The full-length PmCasp was 1202bp with a 954bp open reading frame, encoding 317 amino acids. The deduced protein contained a potential active site (QACRG pentapeptide) conserved in most caspases. It had 83% identity with caspase of P. merguiensis and 30% identity with drICE protein of Drosophila melanogaster, and it exhibited caspase-3 activity in vitro. PmCasp was cloned and expressed in Escherichia coli and a rabbit polyclonal antiserum was produced. In Western blots, the antiserum reacted with purified recombinant PmCasp and with lysates of E. coli containing the expressed plasmid. In crude protein extracts from normal shrimp, the antiserum reacted with 36 and 26kDa bands likely to correspond to inactive pro-caspase and its proteolytic intermediate form, respectively. PmCasp expression was measured in normal shrimp and in white spot syndrome virus (WSSV)-infected shrimp at 24 and 48h post-injection (p.i.) by semi-quantitative RT-PCR, Western blot analysis, and immunohistochemistry. Semi-quantitative RT-PCR analysis revealed up-regulation of PmCasp at 48h p.i. and expression remained high up to the moribund state. These results were supported by Western blot analysis showing increased PmCasp protein levels at 24 and 48h p.i. when compared to normal control shrimp. Immunohistochemical analysis of gills from the WSSV-infected shrimp revealed immunoreactivity localized in the cytoplasm of both normal and apparently apoptotic cells. In summary, a caspase-3 like gene is conserved in P. monodon and is up-regulated after WSSV infection.     

Inhibition of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilisation by phorbol ester in rat cultured vascular smooth muscle cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 microM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+]i, were 7.8 +/- 0.3 M and 1 microM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 microM PMA for either 1 or 24 h did not significantly change the K(D) and Bmax of the BK receptor for binding (control: K(D) = 1.7 +/- 0.2 nM; Bmax = 47.3 +/- 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs.     

Two hepcidin-like antimicrobial peptides in Barramundi Lates calcarifer exhibit differing tissue tropism and are induced in response to lipopolysaccharide

Genes encoding two hepcidin-like antimicrobial peptides were discovered in Barramundi, Lates calcarifer (barramundi, Giant sea perch). Analysis of the coding regions indicated that genes for each hepcidin comprised 3 exons and 2 introns. The deduced amino acid sequences for each molecule resulted in a protein comprising a signal sequence of 24 aa in each case, coupled to a prepropeptide of 75 aa for hepcidin 1 and 78 aa for hepcidin 2. A cleavage site was identified in each prepropetide at amino acid 64 with the cleavage motif--QKR/QS--resulting in mature peptides of 25 and 28 amino acids respectively. Each mature peptide contained 8 conserved cysteine residues and 3 dimensional modeling predicted a β-hairpin and β-sheet structure characteristic of human Liver Expressed Antimicrobial Peptide (LEAP). Analysis of the deduced amino acid sequences by BLAST with phylogenetic supported indicated that hepcidin 1 was a HAMP1-type peptide closely related to hepcidins identified in other Perciformes (Micropterus and Pseudosciaena), whilst hepcidin 2 was a HAMP2-type peptide most similar to a hepcidin previously identified in black rock fish (Sebastes schlegeli). Both hepcidin genes were inducible in barramundi following intraperitoneal injection with lipopolysaccharide, with elevated expression detected in liver and head kidney 3 h post IP injection for hepcidin 1 and in liver only for hepcidin 2. The elevated expression was transient with return to normal levels within 24-48 h. No significant expression of either peptide was detected in spleen, skin or gill following IP injection with LPS.     

Molecular signals in B cell activation. II. IL-2-mediated signals are required in late G1 for transition to S phase after ionomycin and PMA treatment

We report that sustained increase of intracellular calcium ion concentration and protein kinase C (PKC) activation maintained throughout the G1 phase of cell cycle do not provide sufficient signals to cause S-phase entry in rabbit B cells, and that additional signals transduced by IL-2 and IL-2 receptor interaction are essential for G1 to S transition. We have shown earlier that rabbit B cells can be activated to produce IL-2 and express functional IL-2 receptors after treatment with ionomycin and PMA. Herein we have compared the response of rabbit PBLs, which contain about 50% T cells, with those of purified B cells. After activation with ionomycin or PMA, comparable numbers of PBLs and B cells entered the cell cycle; but DNA synthesis by the PBL cultures was three to four times higher than that of cultures of purified B cells. Interestingly, IL-2 production by the PBL cultures was also three to four times higher than in B cell cultures, suggesting an involvement of IL-2 in inducing DNA synthesis in these cells. The hypothesis that IL-2, which is produced in early G1, acts in late G1 and is required for G1 to S transition in B cells was supported by the following observations: (i) IL-2 production by B cells was detected as early as 6 hr after activation and preceded DNA synthesis by at least 24 hr. (ii) B cell blasts in G1 (produced by treatment of resting B cells with ionomycin and PMA) showed DNA synthesis in response to IL-2, but showed very little DNA synthesis in response to restimulation with ionomycin and PMA. (iii) A polyclonal rabbit anti-human IL-2 antibody caused nearly complete inhibition of DNA synthesis by B cells activated by ionomycin and PMA. (iv) A PKC inhibitor, K252b, inhibited DNA synthesis in ionomycin and PMA-stimulated cells if added at the beginning of culture but was not inhibitory if added 16 hr later. We conclude that increased [Ca2+]i and PKC activation are not sufficient signals for G1 to S transition in B cells; entry into S is signaled by IL-2, and IL-2-mediated signal transduction probably does not involve increased [Ca2+]i or PKC activation.