Molecular cloning and expression of a Toll receptor gene homologue from Zhikong Scallop, Chlamys farreri

Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors, which show homology with the Drosophila Toll protein and play key roles in detecting various non-self substances and then initiating and activating immune system. In this report, the full length of the first bivalve TLR (named as CfToll-1) is presented. CfToll-1 was originally identified as an EST (expressed sequence tag) fragment from a cDNA library of Zhikong scallop (Chlamys farreri). Its complete sequence was obtained by the construction of Genome Walker library and 5' RACE (rapid amplification of cDNA end) techniques. The full length cDNA of CfToll-1 consisted of 4308 nucleotides with a polyA tail, encoding a putative protein of 1198 amino acids with a 5' UTR (untranslated region) of 211 bp and a 3'UTR of 500 bp. The predicted amino acid sequence comprised an extracellular domain with a potential signal peptide, nineteen leucine-rich repeats (LRR), two LRR-C-terminal (LRRCT) motifs, and a LRR-N-terminal (LRRNT), followed by a transmembrane segment of 20 amino acids, and a cytoplasmic region of 138 amino acids containing the Toll/IL-1R domain (TIR). The deduced amino acid sequence of CfToll-1 was homologous to Drosophila melanogaster Tolls (DmTolls) with 23-35% similarity in the full length amino acids sequence and 30-54% in the TIR domain. Phylogenetic analysis of CfToll-1 with other known TLRs revealed that CfToll-1 was closely related to DmTolls. An analysis of the tissue-specific expression of the CfToll-1 gene by Real-time PCR showed that the transcripts were constitutively expressed in tissues of haemocyte, muscle, mantle, heart, gonad and gill. The temporal expressions of CfToll-1 in the mixed primary cultured haemocytes were observed after the haemocytes were treated with 1 mu g ml(-1) and 100 ng ml(-1) lipopolysaccharide (LPS), respectively. The expression of CfToll-1 was up-regulated and increased about 2-fold at 6 h with the treatment of 1 mu g ml(-1) LPS. The expression of CfToll-1 was down-regulated with the treatment of 100 ng ml(-1) LPS. The results indicated that the expression of CfToll-1 could be regulated by LPS, and this regulation was dose-dependent. (c) 2006 Elsevier Ltd. All rights reserved.

A novel C1q-domain-containing protein from Zhikong scallop Chlamys farreri with lipopolysaccharide binding activity

The C1q-domain-containing (C1qDC) proteins are a family of proteins characterized by a globular C1q (gC1q) domain in their C-terminus. They are involved in various processes of vertebrates and supposed to be an important pattern recognition receptor in innate immunity of invertebrates. In this study, a novel member of C1q-domain-containing protein family was identified from Zhikong scallop Chlamys farreri (designated as CfC1qDC) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfC1qDC was of 777 bp, consisting of a T-terminal untranslated region (UTR) of 62 bp and a 3' UTR of 178 bp with a polyadenylation signal sequence AATAAA and a poly (A) tail. The CfC1qDC cDNA encoded a polypeptide of 178 amino acids, including a signal peptide and a C1q-domain of 158 amino acids with the theoretical isoelectric point of 5.19 and the predicted molecular weight of 17.2 kDa. The C1q-domain in CfC1qDC exhibited homology with those in sialic acid binding lectin from mollusks and C1qDC proteins from higher vertebrates. The typical 10 beta-strand jelly-roll folding topology structure of C1q-domain and the residues essential for effective packing of the hydrophobic core were well conserved in CfC1qDC. By fluorescent quantitative real-time PCR, mRNA transcripts of CfC1qDC were mainly detected in kidney, mantle, adductor muscle and gill, and also marginally detectable in hemocytes. In the bacterial challenge experiment, after the scallops were challenged by Listonella anguillarum, there was a significant up-regulation in the relative expression level of CfC1qDC and at 6 h post-injection, the mRNA expression reached the maximum level and was 4.55-fold higher than that of control scallops. Similarly, the expression of CfC1qDC mRNA in mixed primary cultures of hemocytes stimulated by lipopolysaccharides (LPS) was up-regulated and reached the maximum level at 6 h post-stimulation, and then dropped back to the original level gradually. In order to investigate its function, the cDNA fragment encoding the mature peptide of CfC1qDC was recombined and expressed in Escherichia coli BL21 (DE3). The recombinant CfC1qDC protein displayed a significantly strong activity to bind LIDS from E. coli, although no obvious antibacterial or agglutinating activity toward Gram-negative bacteria E. coli JM109, L. anguillarum and Gram-positive bacteria Micrococcus luteus was observed. These results suggested that CfC1qDC was absolutely a novel member of the C1qDC protein family and was involved in the recognition of invading microorganisms probably as a pattern recognition molecule in mollusk. (c) 2008 Elsevier Ltd. All rights reserved.

Interannual horizontal heat advection in the surface mixed layer over the equatorial Pacific Ocean: assimilation versus TAO analysis

The interannual anomalies of horizontal heat advection in the surface mixed layer over the equatorial Pacific Ocean in an assimilation experiment are studied and compared with existing observational analyses. The assimilation builds upon a hindcast study that has produced a good simulation of the observed equatorial currents and optimizes the simulation of the Reynolds sea surface temperature (SST) data. The comparison suggests that the assimilation has improved the simulation of the interannual horizontal heat advection of the surface mixed layer significantly. During periods of interrupted current measurements, the assimilation is shown to produce more meaningful anomalies of the heat advection than the interpolation of the observational data does. The assimilation also shows that the eddy heat flux due to the correlation between high-frequency current and SST variations, which is largely overlooked by the existing observational analyses, is important for the interannual SST balance over the equatorial Pacific. The interannual horizontal heat advection anomalies are found to be sensitive to SST errors where oceanic currents are strong, which is a challenge for ENSO prediction. The study further suggests that the observational analyses of the tropical SST balance based on the TAO and the Reynolds SST data contain significant errors due to the large gradient errors in the Reynolds SST data, which are amplified into the advection anomalies by the large equatorial currents.

Simulation of the ocean surface mixed layer under the wave breaking

A one-dimensional mixed-layer model, including a Mellor-Yamada level 2.5 turbulence closure scheme, was implemented to investigate the dynamical and thermal structures of the ocean surface mixed layer in the northern South China Sea. The turbulent kinetic energy released through wave breaking was incorporated into the model as a source of energy at the ocean surface, and the influence of the breaking waves on the mixed layer was studied. The numerical simulations show that the simulated SST is overestimated in summer without the breaking waves. However, the cooler SST is simulated when the effect of the breaking waves is considered, the corresponding discrepancy with the observed data decreases up to 20% and the MLD calculated averagely deepens 3.8 m. Owing to the wave-enhanced turbulence mixing in the summertime, the stratification at the bottom of the mixed layer was modified and the temperature gradient spread throughout the whole thermocline compared with the concentrated distribution without wave breaking.

Wave breaking on turbulent energy budget in the ocean surface mixed layer

As an important physical process at the air-sea interface, wave movement and breaking have a significant effect on the ocean surface mixed layer (OSML). When breaking waves occur at the ocean surface, turbulent kinetic energy (TKE) is input downwards, and a sublayer is formed near the surface and turbulence vertical mixing is intensively enhanced. A one-dimensional ocean model including the Mellor-Yamada level 2.5 turbulence closure equations was employed in our research on variations in turbulent energy budget within OSML. The influence of wave breaking could be introduced into the model by modifying an existing surface boundary condition of the TKE equation and specifying its input. The vertical diffusion and dissipation of TKE were effectively enhanced in the sublayer when wave breaking was considered. Turbulent energy dissipated in the sublayer was about 92.0% of the total depth-integrated dissipated TKE, which is twice higher than that of non-wave breaking. The shear production of TKE decreased by 3.5% because the mean flow fields tended to be uniform due to wave-enhanced turbulent mixing. As a result, a new local equilibrium between diffusion and dissipation of TKE was reached in the wave-enhanced layer. Below the sublayer, the local equilibrium between shear production and dissipation of TKE agreed with the conclusion drawn from the classical law-of-the-wall (Craig and Banner, 1994).