6000 years of environmental changes recorded in Blue Lake, South Australia, based on ostracod ecology and valve chemistry

A 4 m long core taken from the freshwater Blue Lake crater near the township of Mount Gambier in southeastern South Australia provided a high-resolution palaeoclimatic record for the last six millennia. Accelerator Mass Spectrometry (AMS) radiocarbon (¹⁴C) dates were obtained from organic plant fibres and biogenic carbonates from the laminated sequence of the core and from a modern water sample. Large discrepancies between the radiocarbon ages determined from plant fibres and biogenic carbonates indicate the presence of a time-variable lacustrine reservoir, which is consistent with what is known of the lake's hydrology.Ostracod assemblages, associated with stable isotope (δ¹³C, δ¹⁸O) analyses and, in combination with Mg/Ca, Sr/Ca and Na/Ca analyses done on ostracod valves, infer salinity, temperature and water level changes in Blue Lake over the last 6 millenia. The influence of local aquifers through time has also been determined from the Na/Ca of ostracod valves. Approximately 900 year cycles are evident in the δ¹³C record from 5.4 ka to 1.8 ka.The history of Blue Lake records an initial period of high hydrological variability around 6 ka, becoming increasingly deeper as groundwater flowed into the basin. By 4 ka, the lake had reached steady state with the lake level fluctuating by as much as 9 m, although significant geochemical variations represent temperature fluctuations until European settlement near the lake in 1839.     

Salinities, not diets, affect strontium/calcium ratios in otoliths of Anguilla japonica

Although otolith Strontium (Sr)/calcium (Ca) ratios have been widely used to reconstruct the past salinity environmental history of anguillid eels, factors affecting the Sr/Ca ratios in otoliths are incompletely understood. Japanese Eel (Anguilla japonica) elvers (mean length 54.7 ± 2.1 mm) were collected in the estuary during their upstream migration and reared at 5 different salinities (0, 5, 15, 25, and 35 psu) and 3 types of feeding conditions (formulated feed, tubifex, and starvation) for 30 days to evaluate the effects of salinity and diets on otolith Sr/Ca ratios. Ca and Sr concentrations in the ambient water significantly increased with salinity (SAL) as [Ca] _water = 15.50SAL − 5.56, and [Sr] _water = 0.21SAL + 0.03, respectively. Sr/Ca ratios in otoliths increased with salinity (SAL) of the rearing water as [(Sr/Ca) × 1000] _otolith = 0.091SAL + 3.790. In diets, Sr/Ca ratios were 4 times higher in tubifex than in formulated feed. However, in otoliths, ANOVA indicated that Sr/Ca ratios did not differ significantly between groups fed on tubifex or formulated feed (p = 0.118). Otolith Sr/Ca ratios were negatively correlated with fish growth rates while the growth rates differed significantly among rearing conditions with different salinities and diets. Partition coefficients of the Sr/Ca ratios from ambient water to fish tissues and otoliths significantly increased with salinity. The Sr/Ca ratios of Japanese Eel otoliths thus were positively correlated with the ambient salinity and decreased with increasing fish growth rate, but was not affected by fish diet.     

CD14, TLR4 and TRAM Show Different Trafficking Dynamics During LPS Stimulation

Toll‐like receptor 4 (TLR4) is responsible for the immediate response to Gram‐negative bacteria and signals via two main pathways by recruitment of distinct pairs of adaptor proteins. Mal‐MyD88 [Mal (MyD88‐adaptor‐like) ‐ MYD88 (Myeloid differentiation primary response gene (88))] is recruited to the plasma membrane to initiate the signaling cascade leading to production of pro‐inflammatory cytokines while TRAM‐TRIF [TRAM (TRIF‐related adaptor molecule)‐TRIF (TIR‐domain‐containing adapter‐inducing interferon‐β)] is recruited to early endosomes to initiate the subsequent production of type I interferons. We have investigated the dynamics of TLR4 and TRAM during lipopolysaccharide (LPS) stimulation. We found that LPS induced a CD14‐dependent immobile fraction of TLR4 in the plasma membrane. Total internal reflection fluorescence microscopy (TIRF) revealed that LPS stimulation induced clustering of TLR4 into small punctate structures in the plasma membrane containing CD14/LPS and clathrin, both in HEK293 cells and the macrophage model cell line U373‐CD14. These results suggest that laterally immobilized TLR4 receptor complexes are being formed and prepared for endocytosis. RAB11A was found to be involved in localizing TRAM to the endocytic recycling compartment (ERC) and to early sorting endosomes. Moreover, CD14/LPS but not TRAM was immobilized on RAB11A‐positive endosomes, which indicates that TRAM and CD14/LPS can independently be recruited to endosomes.      

Increase in CpG DNA-induced inflammatory responses by DNA oxidation in macrophages and mice

Unmethylated CpG dinucleotide (CpG motif) is involved in the exacerbation of DNA-associated autoimmune diseases. We investigated the effect of DNA containing 8-hydroxydeoxyguanosine (oxo-dG), a representative DNA biomarker for oxidative stress in the diseases, on CpG motif-dependent inflammatory responses. ODN1668 and ODN1720 were selected as CpG-DNA and non-CpG DNA, respectively. Deoxyguanosine in the CpG motif (G9) or outside the motif (G15) of ODN1668 was substituted with oxo-dG to obtain oxo(G9)-1668 and oxo(G15)-1668, respectively. Oxo(G15)-1668 induced a significantly higher amount of tumor necrosis factor (TNF)-α from RAW264.7 macrophage-like cells than ODN1668, whereas oxo(G9)-1668, oxo(G8)-1720, or oxo(G15)-1720 hardly did. CpG DNA-induced TNF-α production was significantly increased by addition of oxo(G8)-1720 or oxo(G15)-1720, but not of ODN1720. This oxo-dG-containing DNA-induced increase in TNF-α production was also observed in primary cultured macrophages isolated from wild-type mice, but not observed in those from Toll-like receptor (TLR)-9 knockout mice. In addition, TNF-α production by ligands for TLR3, TLR4, or TLR7 was not affected by oxo-dG-containing DNA. Then, the footpad swelling induced by subcutaneous injection of ODN1668 into mice was increased by coinjection with oxo(G8)-1720, but not with ODN1720. These results indicate that oxo-dG-containing DNA increases the CpG motif-dependent inflammatory responses, which would exacerbate DNA-related autoimmune diseases.     

Characterization of bbtTICAM from amphioxus suggests the emergence of a MyD88-independent pathway in basal chordates

The MyD88-independent pathway, one of the two crucial TLR signaling routes, is thought to be a vertebrate innovation. However, a novel Toll/interleukin-1 receptor (TIR) adaptor, designated bbtTICAM, which was identified in the basal chordate amphioxus, links this pathway to invertebrates. The protein architecture of bbtTICAM is similar to that of vertebrate TICAM1 (TIR-containing adaptor molecule-1, also known as TRIF), while phylogenetic analysis based on the TIR domain indicated that bbtTICAM is the oldest ortholog of vertebrate TICAM1 and TICAM2 (TIR-containing adaptor molecule-2, also known as TRAM). Similar to human TICAM1, bbtTICAM activates NF-κB in a MyD88-independent manner by interacting with receptor interacting protein (RIP) via its RHIM motif. Such activation requires bbtTICAM to form homodimers in endosomes, and it may be negatively regulated by amphioxus SARM (sterile α and armadillo motif-containing protein) and TRAF2. However, bbtTICAM did not induce the production of type I interferon. Thus, our study not only presents the ancestral features of vertebrate TICAM1 and TICAM2, but also reveals the evolutionary origin of the MyD88-independent pathway from basal chordates, which will aid in understanding the development of the vertebrate TLR network.     

水牛MyD88cDNA的克隆与原核表达

采用RT-PCR方法从水牛外周血白细胞总RNA中扩增出髓样分化因子88 (mydoid differentiation factor 88,MyD88) cDNA序列,PCR产物分离纯化后,与pMD20-T载体连接,重组质粒经PCR、酶切鉴定后测序,并进行生物信息学分析;构建pET28a-MyD88表达载体,并将其转化至E.coli BL21 (DE3),经IPTG诱导表达后,进行SDS-PAGE、镍柱亲和层析纯化和Western blotting分析.结果显示,克隆到的水牛MyD88 cDNA全长为1 189 bp,含有1个891 bp的开放阅读框,编码296个氨基酸,理论等电点为5.65.经IPTG诱导表达后,得到一个带His·Tag的约39 kD的重组融合蛋白.用抗His单克隆抗体进行Western blotting,得到1条约39 kD特异性抗体结合带,表明水牛MyD88原核表达载体成功构建并表达.本研究为进一步开展水牛MyD88的结构功能分析奠定了基础.     

Effect of Glycosylation on the Catalytic and Conformational Stability of Homologous α-Amylases

summary. A thermostable -amylase from B. licheniformis (BLA) and a mesophilic amylase from B. amyloliquefaciens (BAA) were covalently coupled to oxidized synthetic sucrose polymers (OSP400 and OSP70) and polyglutaraldehyde (PGA) by reductive alkylation to study the effect of neoglycosylation on the activity, kinetic and thermodynamic stability. The catalytic efficiency of the modified enzymes was comparable to that of the native enzyme. Covalent coupling decreased the rate of inactivation at all the temperatures studied, both in the presence and absence of added Ca²⁺. The stability of the native enzyme was found to increase upon modification as observed from the increase in t_1/2 in the absence of Ca²⁺ ions by about 1.5–13.7 times (at 85°C) in the case of BLA and 5.7–8.4 times (at 50°C) for BAA. The highest stability was observed for OSP400 modified enzyme with C_m and T_m values of 0.63 M and 7.92°C for BLA and 0.85 M and 5.3°C for BAA, respectively. The order of stability was OSP400 > OSP70 > PGA > Native for both BLA and BAA. The stability of the modified amylases obtained from the present study were superior compared to most of the single and double mutants obtained by site-directed mutagenesis that were constructed so as to enhance the intrinsic stability of these enzymes.This article is dedicated to Dr. P.V. Sundaram.     

Changes of enzyme activity in lipid signaling pathways related to substrate reordering

The static fluid mosaic model of biological membranes has been progressively complemented by a dynamic membrane model that includes phospholipid reordering in domains that are proposed to extend from nanometers to microns. Kinetic models for lipolytic enzymes have only been developed for homogeneous lipid phases. In this work, we develop a generalization of the well-known surface dilution kinetic theory to cases where, in a same lipid phase, both domain and nondomain phases coexist. Our model also allows understanding the changes in enzymatic activity due to a decrease of free substrate concentration when domains are induced by peptides. This lipid reordering and domain dynamics can affect the activity of lipolytic enzymes, and can provide a simple explanation for how basic peptides, with a strong direct interaction with acidic phospholipids (such as beta-amyloid peptide), may cause a complex modulation of the activities of many important enzymes in lipid signaling pathways.     

TLR4 recognizes Pseudallescheria boydii conidia and purified rhamnomannans

Pseudallescheria boydii (Scedosporium apiospermum) is a saprophytic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections, particularly mycetoma, in immunocompromised and immunocompetent hosts. We have previously shown that highly purified α-glucan from P. boydii activates macrophages through Toll-like receptor TLR2, however, the mechanism of P. boydii recognition by macrophage is largely unknown. In this work, we investigated the role of innate immune receptors in the recognition of P. boydii. Macrophages responded to P. boydii conidia and hyphae with secretion of proinflammatory cytokines. The activation of macrophages by P. boydii conidia required functional MyD88, TLR4, and CD14, whereas stimulation by hyphae was independent of TLR4 and TLR2 signaling. Removal of peptidorhamnomannans from P. boydii conidia abolished induction of cytokines by macrophages. A fraction highly enriched in rhamnomannans was obtained and characterized by NMR, high performance TLC, and GC-MS. Preparation of rhamnomannans derived from P. boydii triggered cytokine release by macrophages, as well as MAPKs phosphorylation and IκBα degradation. Cytokine release induced by P. boydii-derived rhamnomannans was dependent on TLR4 recognition and required the presence of non-reducing end units of rhamnose of the rhamnomannan, but not O-linked oligosaccharides from the peptidorhamnomannan. These results imply that TLR4 recognizes P. boydii conidia and this recognition is at least in part due to rhamnomannans expressed on the surface of P. boydii.