Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kappa B, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kappa B activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1). antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules. (C) 2007 Elsevier Ltd. All rights reserved.

Impact of environmental and innate factors on the food habit of Chinese perch Siniperca chuatsi (Basilewsky) (Percichthyidae)

Laboratory and field investigations were conducted to study the food habit of Chinese perch Siniperca chuatsi (Basilewsky) from first feeding through adult stage. Only fish larvae were consumed by Chinese perch larvae (2-21 days from hatching), and the presence of zooplankton did not have any significant effect on their survival rate. The ability of Chinese perch to feed on zooplankton is clearly limited by some innate factor. Instead of gill rakers, Chinese perch larvae have well-developed sharp teeth at the first feeding stage, and are well adapted to the piscivorous feeding habit unique to the larvae of Chinese perch, e.g. they bite and ingest the tails of other fish larvae. At the first feeding stage (2 days from hatching), daily rations were both very low, either in light or complete darkness. Although early-staged Chinese perch larvae (7-17 days from hatching) could feed in complete darkness, their daily rations were always significantly higher in light than in complete darkness. Late-staged Chinese perch larvae (21 days from hatching) were able to feed in complete darkness as well as in light, similar to the case of Chinese perch yearlings. Chinese perch yearlings (total length, 14-16 cm) consumed prey fish only and refused shrimp when visual cues were available (in light), but they consumed both prey when visual cues were not available (in complete darkness), suggesting that prey consumption by Chinese perch yearlings is affected by their sensory modality in predation. Both prey were found in the stomachs of similar-sized Chinese perch (total length, 14-32 cm) from their natural habitat, suggesting that shrimp are consumed by Chinese perch at night. Prey selection of Chinese perch with a length >38 cm, which consumed only fish in the field, appears to be based upon prey size instead of prey type. These results suggest that although environmental factors (e.g. light intensity) affect prey detection by Chinese perch, this fish is anatomically and behaviourally predisposed to prey on live fish from first feeding. This makes it a difficult fish to cultivate using conventional feeds.

The innate immune response to grass carp hemorrhagic virus (GCHV) in cultured Carassius auratus blastulae (CAB) cells

Virus infection of mammalian cells activates an innate antiviral immune response characterized by production of interferon (IFN) and the subsequent transcriptional upregulation of IFN-stimulated genes (ISGs) by the JAK-STAT signaling pathway. Here, we report that a fish cell line, crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells, can produce IFN activity and then form an antiviral state after infection with UV-inactivated grass carp hemorrhagic virus (GCHV), a double-stranded (ds) RNA virus. From UV-inactivated GCHV-infected CAB cells, 15 pivotal genes were cloned and sequenced, and all of them were shown to be involved in IFN antiviral innate immune response. These IFN system genes include the dsRNA signal sensing factor TLR3, IFN, IFN signal transduction factor STAT1, IFN regulatory factor IRF7, putative IFN antiviral effectors Mx1, Mx2, PKR-like, Viperin, IFI56, and other IFN stimulated genes (ISGs) IFI58, ISG15-1, ISG15-2, USP18, Gig1 and Gig2. The identified fish IFN system genes were highly induced by active GCHV, UV-inactivated GCHV, CAB IFN or poly(I).poly(C), and showed similar expression patterns to mammals. The data indicate that an IFN antiviral innate immune response similar to that in mammals exists in the UV-inactivated GCHV-infected CAB cells, and the IFN response contributes to the formation of an antiviral state probably through JAK-STAT signaling pathway. This study provides strong evidence for existence of IFN antiviral innate immune response in fish, and will assist in elucidating the origin and evolution of vertebrate IFN system. (c) 2006 Elsevier Ltd. All rights reserved.

The effects of CpG-C oligodeoxynucleotides on innate immune responses in Eriocheir sinensis (H. Milne-Edwards, 1853)

CpG oligodeoxynucleotides (ODNs) can stimulate the immune system, and therefore are widely used as a therapeutic vaccination and immune adjuvant in human. In the present study, CpG-C, a combination of A- and B-class ODN, was injected into Chinese mitten crab Eriocheir sinensis at three doses (0.1, 1 and 10 mu g crab-1), and the reactive oxygen species (ROS) levels, activities of total intracellular phenoloxidase (PO) and lysozyme-like activities, the mRNA transcripts of EsproPO, EsCrustin and EsALF were assayed to evaluate its modulating effects on the immune system of crab. The ROS levels in all treated and control groups were significantly increased from 6 to 24 h, except that ROS in 0.1 mu g CpG-C-treated crabs was comparable to that of the blank at 6 h. The PO activity was significantly enhanced and EsproPO transcripts were down-regulated (P < 0.01) at 6 h after the injection of 0.1 mu g CpG-C, with no significant changes in the other dosage treatments. The lysozyme-like activities and EsCrustin transcripts in the CpG-C-treatment groups were significantly higher than those of controls. The mRNA expression of EsALF remained almost constant in all the groups during the treatment. These results collectively suggested that CpG-C could activate the immune responses of E. sinensis, and might be used as a novel immunostimulant for disease control in crabs.

The effects of CpG-C oligodeoxynucleotides on innate immune responses in Eriocheir sinensis (H. Milne-Edwards, 1853)

CpG oligodeoxynucleotides (ODNs) can stimulate the immune system, and therefore are widely used as a therapeutic vaccination and immune adjuvant in human. In the present study, CpG-C, a combination of A- and B-class ODN, was injected into Chinese mitten crab Eriocheir sinensis at three doses (0.1, 1 and 10 mu g crab-1), and the reactive oxygen species (ROS) levels, activities of total intracellular phenoloxidase (PO) and lysozyme-like activities, the mRNA transcripts of EsproPO, EsCrustin and EsALF were assayed to evaluate its modulating effects on the immune system of crab. The ROS levels in all treated and control groups were significantly increased from 6 to 24 h, except that ROS in 0.1 mu g CpG-C-treated crabs was comparable to that of the blank at 6 h. The PO activity was significantly enhanced and EsproPO transcripts were down-regulated (P < 0.01) at 6 h after the injection of 0.1 mu g CpG-C, with no significant changes in the other dosage treatments. The lysozyme-like activities and EsCrustin transcripts in the CpG-C-treatment groups were significantly higher than those of controls. The mRNA expression of EsALF remained almost constant in all the groups during the treatment. These results collectively suggested that CpG-C could activate the immune responses of E. sinensis, and might be used as a novel immunostimulant for disease control in crabs.

A fibrinogen-related protein from bay scallop Argopecten irradians involved in innate immunity as pattern recognition receptor

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like domains. Many members of this family play important roles as pattern recognition receptors in innate immune responses. The cDNA of bay scallop Argopecten irradians FREP (designated as AiFREP) was cloned by rapid amplification of cDNA ends (RACE) method based on the expressed sequence tag (EST). The full-length cDNA of AiFREP was of 990 bp. The open reading frame encoded a polypeptide of 251 amino acids, including a signal sequence and a 213 amino acids fibrinogen-like domain. The fibrinogen-like domain of AiFREP was highly similar to those of mammalian ficolins and other FREPs. The temporal expression of AiFREP mRNA in hemolymph was examined by fluorescent quantitative real-time PCR. The mRNA level of scallops challenged by Listonella anguillarum was significantly up-regulated, peaked to 9.39-fold at 9 h after stimulation, then dropped back to 4.37-fold at 12 h, while there was no significant change in the Micrococcus luteus challenged group in all periods of treatment. The function of AiFREP was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta gami (DE3). The recombinant AiFREP (rAiFREP) agglutinated chicken erythrocytes and human A, B, O-type erythrocytes. The agglutinating activities were calcium-dependent and could be inhibited by acetyl group-containing carbohydrates. rAiFREP also agglutinated Gram-negative bacteria E. coli JM109, L anguillarum and Gram-positive bacteria M. luteus in the presence of calcium ions. These results collectively suggested that AiFREP functions as a pattern recognition receptor in the immune response of bay scallop and contributed to nonself recognition in invertebrates, which would also provide clues for elucidating the evolution of the lectin pathway of the complement system. (C) 2008 Elsevier Ltd. All rights reserved.

Construction and characterization of two bacterial artificial chromosome libraries of Zhikong scallop, Chlamys farreri Jones et Preston, and identification of BAC clones containing the genes involved in its innate immune system

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis, and physical mapping of scallop, two BAC libraries were constructed from nuclear DNA of Zhikong scallop, Chlamys farreri Jones et Preston. The libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99% of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0x haploid genome equivalents. Similarly, the MboI library consists of 7680 clones, with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 5.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping, and large-scale sequencing in the species.

An asymptotic analysis for one dimensional stress wave propagation in damaged viscoelastic media

A regular perturbation technique is suggested to deal with the problem of one dimensional stress wave propagation in viscoelastic media with damage. Based upon the first order asymptotic solution obtained, the characteristics of wave attenuation are studied. In fact, there exist three different time-dependent phenomena featuring the dynamic response of the materials, the first expressing the characteristics of wave propagation, the second indicating the innate effect of visco-elastic matrix and the third coming from the time dependent damage. The comparision of first order asymptotic solution with the numerical results calculated by a finite difference procedure shows that the perturbation expansion technique may offer a useful approach to the problem concerned.

Adaptive evolution of primate TRIM5 alpha, a gene restricting HIV-1 infection

Recent studies showed that nonhuman primate TRIM5 alpha can efficiently block HIV-1 infection in human cell lines. It can also restrict other retroviruses, therefore, suggested as a general defender against retrovirus infection. Here, we present an evolutionary analysis of TRIM5 alpha in primates. Our results demonstrated that TRIM5a has been evolving rapidly in primates, which is likely caused by Darwinian positive selection. The SPRY domain of TRM5 alpha, which may be responsible for recognition of incoming viral capsids showed higher nonsynonymous/synonymous substitution ratios than the non-SPRY domain, indicating that the adaptive evolution of TRIM5a ill primates might be an innate strategy developed in defending retrovirus infection during primate evolution. In addition, the comparative protein sequence analysis suggested that the amino acid substitution pattern at a single site (344R/Q/P) located in the SPRY domain may explain the differences in Susceptibilities of HIV-1 infection in diverse primate species. (c) 2005 Elsevier B.V. All rights reserved.

An anionic antimicrobial peptide from toad Bombina maxima

Amphibian skin is a rich resource of antimicrobial peptides like maximins and maximins H from toad Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises maximin 3 and a novel peptide. named maximin H5. was isolated from a skin cDNA library of B. maxima. The predicted primary structure of maximin H5 is ILGPVLGLVSDTLDDVLGIL-NH2,. Containing three aspartate residues and no basic amino acid residues. maximin H5 is characterized by an anionic property. Different from cationic maximin H peptides. only Gram-positive strain Staphylococcus aureus was sensitive to maximin H5. while the other bacteria] and fungal strains tested ere resistant to it. The presence of metal ions. like Zn2+ and Mg2+, did not increase its antimicrobial potency. Maximin H5 represents the first example of potential anionic antimicrobial peptides from amphibians, The results provide the first evidence that. together kith cationic antimicrobial peptides. anionic antimicrobial peptides may also exist naturally as part of the innate defense system. (C), 2002 Elsevier Science (USA). All rights reserved.

Variety of antimicrobial peptides in the Bombina maxima toad and evidence of their rapid diversification

Antimicrobial peptides secreted by the skin of many amphibians play an important role in innate immunity. From two skin cDNA libraries of two individuals of the Chinese red belly toad (Bombina maxima), we identified 56 different antimicrobial peptide cDNA sequences, each of which encodes a precursor peptide that can give rise to two kinds of antimicrobial peptides, maximin and maximin H. Among these cDNA, we found that the mean number of nucleotide substitution per non-synonymous site in both the maximin and maximin H domains significantly exceed the mean number of nucleotide substitution per synonymous site, whereas the same pattern was not observed in other structural regions, such as the signal and propiece peptide regions, suggesting that these antimicrobial peptide genes have been experiencing rapid diversification driven by Darwinian selection. We cloned and sequenced seven genes amplified from skin or liver genomic DNA. These genes have three exons and share the same gene structure, in which both maximin and maximin H are encoded by the third exon. This suggests that alternative splicing and somatic recombination are less likely to play a role in creating the diversity of maximins and maximin Hs. The gene trees based on different domain regions revealed that domain shuffling or gene conversion among these genes might have happened frequently.

Maximins S, a novel group of antimicrobial peptides from toad Bombina maxima

Amphibian skin secretions are rich in antimicrobial peptides acting as important components of innate defense system against invading microorganisms. A novel type of peptide, designated as maximin S, was deduced by random sequencing of 793 clones from a constructed Bombina maxima skin cDNA library. The putative primary structures of maximin S peptides can be grouped into five species, in which maximin S I has 14 amino acid residues and the rest of maximin S peptides (S2-S5) all have 18 amino acid residues. Unlike most of the amphibian antimicrobial peptides so far identified, the newly characterized four maximin S precursors are composed of maximin S I and different combinations of tandem repeated maximin S2-S5 linked by internal peptides. Except maximin S I, the predicted secondary structures of maximin S2-S5 show a similar amphipathic alpha-helical structure. MALDI-TOF mass spectrometry analysis of partially isolated skin secretions of the toad indicates that most of the deduced maximin S peptides are expressed. Two deduced maximin S peptides (S1, S4) were synthesized and their antimicrobial activities were tested. Maximin S4 only had an antibiotic activity against mycoplasma and had no antibacterial or antifungal activity toward tested strains. Maximin S1 had no activity under the same conditions. (C) 2004 Elsevier Inc. All rights reserved.

Purification, characterization and cloning of two novel tigerinin-like peptides from skin secretions of Fejervarya cancrivora

While investigating the innate defense of brackish water-living amphibian and its comparison with freshwater-living amphibians, two novel 12-residue antimicrobial peptides were purified from the skin secretions of the crab-eating frog, Fejervarya cancrivo

Five novel antimicrobial peptides from skin secretions of the frog, Amolops loloensis

While investigating antimicrobial peptide diversity of Amolops loloensis, five novel antimicrobial peptides belonging to two families were identified from skin secretions of this frog. The first family including two members is esculentin-2-AL (esculentin-2-ALa and -ALb): the second family including three members is temporin-AL (temporin-ALd to -ALf). The family of esculentin-2-AL is composed of 37 amino acid residues (aa); the family of temporin-AL is composed of 16, 13 and 10 aa, respectively. All of these antimicrobial peptides showed antimicrobial activities against tested microorganisms. cDNAs encoding precursors of esculentin-2-ALs and temporin-ALs were cloned from the skin cDNA library of A. loloensis. All the precursors share similar overall structures. There is a typical prohormone processing signal (Lys-Arg) located between the acidic propiece and the mature peptide. The antimicrobial peptide family of esculentin-2 is firstly reported in the genus of Amolops. Combined with previous reports, a total of four antimicrobial peptide families have been identified from the genus of Amolops; three of them are also found in the genus of Rana. These results suggest the possible evolutionary connection between the genera Amolops and Rana. (C) 2009 Elsevier Inc. All rights reserved.

A single amino acid defines cross-species reactivity of tree shrew (Tupaia belangeri) CD1d to human invariant natural killer T (iNKT) cells

P>The non-classical major histocompatibility complex (MHC) class I molecule CD1d presents lipid antigens to invariant natural killer T (iNKT) cells, which are an important part of the innate immune system. CD1d/iNKT systems are highly conserved in evoluti