Mechanisms contributing to T cell receptor signaling and assembly revealed by the solution structure of an ectodomain fragment of the CD3 epsilon gamma heterodimer.

The T cell receptor (TCR) consists of genetically diverse disulfide-linked alpha and beta chains in noncovalent association with the invariant CD3 subunits. CD3 epsilon and CD3 gamma are integral components of both the TCR and pre-TCR. Here, we present the solution structure of a heterodimeric CD3 epsilon gamma ectodomain complex. A unique side-to-side hydrophobic interface between the two C2-set immunoglobulin-like domains and parallel pairing of their respective C-terminal beta strands are revealed. Mutational analysis confirms the importance of the distinctive linkage as well as the membrane proximal stalk motif (RxCxxCxE) for domain-domain association. These biochemical and structural analyses offer insights into the modular pairwise association of CD3 invariant chains. More importantly, the findings suggest how the rigidified CD3 elements participate in TCR-based signal transduction.     

Expression of Kv1.2 in microglia and its putative roles in modulating production of proinflammatory cytokines and reactive oxygen species

Microglial cells are endowed with different potassium ion channels but their expression and specific functions have remained to be fully clarified. This study has shown Kv1.2 expression in the amoeboid microglia in the rat brain between 1 (P1) and 10 (P10) days of age. Kv1.2 expression was localized in the ramified microglia at P14 and was hardly detected at P21. In postnatal rats exposed to hypoxia, Kv1.2 immunoreactivity in microglia was markedly enhanced. Quantitative RT-PCR analysis confirmed Kv1.2 mRNA expression in microglial cells in vitro . It was further shown that Kv1.2 and protein expression coupled with that of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) was significantly increased when the cells were subjected to hypoxia. The same increase was observed in cells exposed to adenosine 5'-triphosphate (ATP) and lipopolysaccharide (LPS). Concomitantly, the intracellular potassium concentration decreased significantly. Blockade of Kv1.2 channel with rTityustoxin-Kα (TsTx) resulted in partial recovery of intracellular potassium concentration accompanied by a reduced expression of IL-1β and TNF-α mRNA and protein expression and intracellular reactive oxygen species (ROS) production. We conclude that Kv1.2 in microglia modulates IL-1β and TNF-α expression and ROS production probably by regulating the intracellular potassium concentration.     

Simple sequence repeat analyses of interspecific hybrids and MAALs of <Emphasis Type="Italic">Oryza </Emphasis><Emphasis Type="Italic">officinalis</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Wild rice is a valuable resource for the genetic improvement of cultivated rice (Oryza sativa L., AA genome). Molecular markers are important tools for monitoring gene introgression from wild rice into cultivated rice. In this study, Simple sequence repeat (SSR) markers were used to analyze interspecific hybrids of O. sativa-O. officinalis (CC genome), the backcrossing progenies and the parent plants. Results showed that most of the SSR primers (335 out of 396, 84.6%) developed in cultivated rice successfully amplified products from DNA samples of wild rice O. officinalis. The polymorphism ratio of SSR bands between O. sativa and O. officinalis was as high as 93.9%, indicating differences between the two species with respect to SSRs. When the SSR markers were applied in the interspecific hybrids, only a portion of SSR primers amplified O. officinalis-specific bands in the F(1) hybrid (52.5%), BC(1) (52.5%), and MAALs (37.0%); a number of the bands disappeared. Of the 124 SSR loci that detected officinalis-specific bands in MAAL plants, 96 (77.4%) showed synteny between the A and C-genomes, and 20 (16.1%) showed duplication in the C-genome. Sequencing analysis revealed that indels, substitution and duplication contribute to the diversity of SSR loci between the genomes of O. sativa and O. officinalis.     

An EAL domain protein and cyclic AMP contribute to the interaction between the two quorum sensing systems in Escherichia coli

Quorum sensing (QS) is a bacterial cell-cell communication process by which bacteria communicate using extracellular signals called autoinducers. Two QS systems have been identified in Escherichia coli K-12, including an intact QS system 2 that is stimulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and a partial QS system 1 that consists of SdiA (suppressor of cell division inhibitor) responding to signals generated by other microbial species. The relationship between QS system 1 and system 2 in E. coli, however, remains obscure. Here, we show that an EAL domain protein, encoded by ydiV, and cAMP are involved in the interaction between the two QS systems in E. coli. Expression of sdiA and ydiV is inhibited by glucose. SdiA binds to the ydiV promoter region in a dose-dependent, but nonspecific, manner; extracellular autoinducer 1 from other species stimulates ydiV expression in an sdiA-dependent manner. Furthermore, we discovered that the double sdiA-ydiV mutation, but not the single mutation, causes a 2-fold decrease in intracellular cAMP concentration that leads to the inhibition of QS system 2. These results indicate that signaling pathways that respond to important environmental cues, such as autoinducers and glucose, are linked together for their control in E. coli.     

四川石斛属一新种

本文发表了石斛属一新种, 即昭觉石斛Dendrobium zhaojuense S.C.Sun et L.G.Xu。     

Molecular cloning and characterization of human trabeculin-alpha, a giant protein defining a new family of actin-binding proteins

We describe the molecular cloning and characterization of a novel giant human cytoplasmic protein, trabeculin-alpha (M(r) = 614,000). Analysis of the deduced amino acid sequence reveals homologies with several putative functional domains, including a pair of alpha-actinin-like actin binding domains; regions of homology to plakins at either end of the giant polypeptide; 29 copies of a spectrin-like motif in the central region of the protein; two potential Ca(2+)-binding EF-hand motifs; and a Ser-rich region containing a repeated GSRX motif. With similarities to both plakins and spectrins, trabeculin-alpha appears to have evolved as a hybrid of these two families of proteins. The functionality of the actin binding domains located near the N terminus was confirmed with an F-actin binding assay using glutathione S-transferase fusion proteins comprising amino acids 9-486 of the deduced peptide. Northern and Western blotting and immunofluorescence studies suggest that trabeculin is ubiquitously expressed and is distributed throughout the cytoplasm, though the protein was found to be greatly up-regulated upon differentiation of myoblasts into myotubes. Finally, the presence of cDNAs similar to, yet distinct from, trabeculin-alpha in both human and mouse suggests that trabeculins may form a new subfamily of giant actin-binding/cytoskeletal cross-linking proteins.     

Modeling DNA shuffling.

In vitro evolution is a new, important laboratory method to evolve molecules with desired properties. It has been used in a variety of biological studies and drug development. In this paper, we study one important mutagenesis method used in in vitro evolution experiments called DNA shuffling. We construct a mathematical model for DNA shuffling and study the properties of molecules after DNA shuffling experiments based on this model. The model for DNA shuffling consists of two parts. First we apply the Lander-Waterman model for physical mapping by fingerprinting random clones to model the distribution of regions that can be reassembled through DNA shuffling. Then we present a model for recombination between different DNA species with different mutations. We compare our theoretical results with experimental data. Finally we propose novel applications of the theoretical results to the optimal design of DNA shuffling experiments and to physical mapping using DNA shuffling.     

Scavenging effect of benzophenones on the oxidative stress of skeletal muscle cells.

Benzophenone is an ultraviolet (UV)-absorbing agent that has been used in industry and medicine for more than 30 years. Consumers of cosmetics and sunscreens containing UV-absorbers are exposed to benzophenones on a daily basis, owing to the widespread use of these compounds. However, the efficacy of these compounds as scavengers of oxidative stress is still not well established. In the present study, we investigate the antioxidative capacity of six sunscreen benzophenone compounds. A primary myoblast culture was mixed in vitro with 100 microM menadione. The cytotoxic effect by menadione-induced oxidative stress was monitored by the lucigenin- or luminol-amplified chemiluminescence, methylthiotetrazole (MTT) assay, and the antioxidative effects of various benzophenone compounds were evaluated. The results showed that the addition of menadione can induce oxidative stress on myoblasts by superoxide and hydrogen peroxide production, which can be eradicated by superoxide dismutase (SOD) and catalase, respectively, in a dose-dependent mode. The catalase has a protective effect on the cytotoxicity induced by menadione as measured by the MTT assay, while the SOD does not. The selected benzophenones also have a significant scavenging effect on the menadione-induced cell death on the myoblasts. The ortho-dihydroxyl structure and other hydroxy groups in the same ring have a stronger scavenging effect on the superoxide anion on myoblasts; thus, a stable penoxy radical may be formed. The mechanism of this effect remains to be clarified.     

Improved production of insecticidal proteins in Bacillus thuringiensis strains carrying an additional cry1C gene in its chromosome

A cryIC gene, whose product is active against Spodoptera exigua, was introduced into wildtype Bacillus thuringiensis kurstaki strain YBT1520 using an integrative and thermosensitive vector, pBMB-FLCE, which was developed based on B. thuringiensis transposon Tn4430 harboring a tnpI-tnpA gene. With the mediation of TnpI-TnpA, the cry1C gene was integrated into the chromosome of the host strain. To prevent secondary integration, the integrative vector was eliminated by moving recombinant cultures to 46 degrees C for generations. Two integrative recombinant B. thuringiensis strains BMB1520-E and BMB1520-F were obtained. In recombinant BMB1520-F, the cry1C gene was expressed stably at a significant level and did not reduce the expression of endogenous crystal protein genes. Bioassay results indicated that BMB1520-E and BMB1520-F showed a higher level of activity against S. exigua third-instar larvae than did their parent strains, in addition to the high toxicity to Plutella xylostella third-instar later larvae.     

Receptor activator of NF-kappaB (RANK) cytoplasmic motif, 369PFQEP373, plays a predominant role in osteoclast survival in part by activating Akt/PKB and its downstream effector AFX/FOXO4

Receptor activator of NF-kappaB ligand (RANKL) plays a crucial role in osteoclast differentiation, function, and survival. RANKL exerts its effect by activating its receptor RANK (receptor activator of NF-kappaB), which recruits various intracellular signaling molecules via specific motifs in its cytoplasmic tail. Previously, we identified three RANK cytoplasmic motifs (Motif 1, 369PFQEP373; Motif 2, 559PVQEET564; and Motif 3, 604PVQEQG609) mediating osteoclast formation and function. Here, we investigated RANK cytoplasmic motifs involved in osteoclast survival. Motif 1, in contrast to its minimal role in osteoclast formation and function, plays a predominant role in promoting osteoclast survival. Moreover, whereas Motif 2 and Motif 3 are highly potent in osteoclast formation and function, they exert a moderate effect on osteoclast survival. We also investigated the role of these motifs in activating Akt/protein kinase B (PKB), which has been implicated in RANKL-induced osteoclast survival. Motif 1, but not Motif 2 or Motif 3, is able to stimulate Akt/PKB activation. Because Akt/PKB has been shown to utilize distinct downstream effectors (glycogen synthase kinase-3beta, FKHR/FOXO1a, BAD, and AFX/FOXO4) to regulate cell survival, we next determined which downstream effector(s) is activated by Akt/PKB to promote osteoclast survival. Our data revealed that RANKL only stimulates AFX/FOXO4 phosphorylation, indicating that AFX/FOXO4 is a key downstream target activated by Akt/PKB to modulate osteoclast survival. Taken together, we conclude that Motif 1 plays a predominant role in mediating osteoclast survival in part by activating Akt/PKB and its downstream effector AFX/FOXO4.