Invariant CD3 subunit dimers (CD3␥, CD3␦, and CD3) are the signaling components of the ␣␤ T cell receptor (TCR). The recently solved structure of murine CD3␥ revealed a unique side-to-side interface and central ␤-sheets conjoined between the two C2-set Ig-like ectodomains, with the pairing of the parallel G strands implying a potential concerted piston-type movement for signal transduction. Although CD3␥ and CD3␦ each dimerize with CD3, there are differential CD3 subunit requirements for receptor assembly and signaling among T lineage subpopulations, presumably mandated by structural differences. Here we present the solution structure of the heterodimeric CD3␦ complex. Whereas the CD3 subunit conformation is virtually identical to that in CD3␥, the CD3␦ ectodomain adopts a C1-set Ig fold, with a narrower GFC front face ␤-sheet that is more parallel to the ABED back face than those ␤-sheets in CD3 and CD3␥. The dimer interface between CD3␦ and CD3 is highly conserved among species and of similar character to that in CD3␥. Glycosylation sites in CD3␦ are arranged such that the glycans may point away from the membrane, consistent with a model of TCR assembly that allows the CD3␦ chain to be in close contact with the TCR ␣-chain. This and many other structural and biological features provide a basis for modeling putative TCR͞CD3 extracellular domain associations. The fact that the two clusters of transmembrane helices, namely, the three CD3-CD3␥-TCR␤ segments and the five CD3-CD3␦-TCR␣-CD3-CD3 segments, are presumably centered beneath the G strand-paired CD3 heterodimers has important implications for TCR signaling.single-chain C1-Ig fold ͉ immunoreceptor tyrosine-based activation motif ͉ NMR structure ͉ T cell development T he ␣␤ T cell receptor (TCR) is a multimeric complex composed of an antigen-binding ␣␤ clonotypic heterodimer and the signal-transducing invariant CD3 subunit dimers CD3␥, CD3␦, and CD3 (1-8). Thus, the ␣␤ TCR complex consists of eight polypeptides (5,8,9). Sequence determination and biochemical analyses suggest that each CD3, CD3␥, and CD3␦ subunit contains an extracellular Ig-like domain, a membrane-proximal stalk region, a transmembrane (TM) helix, and a cytoplasmic tail. The interaction between an ␣␤ TCR heterodimer and a specific antigenic peptide bound to an MHC molecule (pMHC) initiates a cascade of downstream signaling events via the immunoreceptor tyrosinebased activation motifs (ITAMs) in the cytoplasmic tails of the associated CD3 subunits (10-12). The various CD3 chains interact differentially with intracellular adaptors and signaling molecules, inducing distinct patterns of cellular protein tyrosine phosphorylation upon activation (11,(13)(14)(15)(16).How recognition of pMHC by a clonotypic ␣␤ heterodimer on the T cell surface evokes intracellular signaling via the adjacent CD3 components remains unknown. However, the solution structure of a heterodimeric murine CD3␥ complex revealed a unique side-to-side hydrophobic interface with conjoined ␤-sheets between the two Ig-like ectodomai...

Although regulation of chondrogenesis and cartilage development by Wnt signaling is well established, the function of Wnt in the maintenance and destruction of cartilage remains largely unknown. Here we investigated the involvement and regulatory mechanisms of Wnt signaling in cartilage destruction. We found that interleukin-1␤, the primary pro-inflammatory cytokine involved in cartilage destruction, induces expression of Wnt-5a and -7a in primary culture articular chondrocytes. The level of ␤-catenin was also increased in chondrocytes of arthritic cartilage, suggesting the association of Wnt/␤-catenin signaling with arthritic cartilage destruction. In addition, our results show that Wnt-7a induces dedifferentiation and inhibits NO-induced apoptosis of primary culture articular chondrocytes. Wnt-7a induces dedifferentiation of articular chondrocytes by stimulating transcriptional activity of ␤-catenin, whereas NO-induced apoptosis is inhibited via the activation of cell survival signaling, such as phosphatidylinositol 3-kinase and Akt, which block apoptotic signaling cascade. Our results collectively suggest that Wnt-7a is associated with cartilage destruction by regulating the maintenance of differentiation status and the apoptosis of articular chondrocytes via different mechanisms.

The mammalian Y chromosome has unique characteristics compared with the autosomes or X chromosomes. Here we report the finished sequence of the chimpanzee Y chromosome (PTRY), including 271 kb of the Y-specific pseudoautosomal region 1 and 12.7 Mb of the male-specific region of the Y chromosome. Greater sequence divergence between the human Y chromosome (HSAY) and PTRY (1.78%) than between their respective whole genomes (1.23%) confirmed the accelerated evolutionary rate of the Y chromosome. Each of the 19 PTRY protein-coding genes analyzed had at least one nonsynonymous substitution, and 11 genes had higher nonsynonymous substitution rates than synonymous ones, suggesting relaxation of selective constraint, positive selection or both. We also identified lineage-specific changes, including deletion of a 200-kb fragment from the pericentromeric region of HSAY, expansion of young Alu families in HSAY and accumulation of young L1 elements and long terminal repeat retrotransposons in PTRY. Reconstruction of the common ancestral Y chromosome reflects the dynamic changes in our genomes in the 5-6 million years since speciation.

The high-level expression plasmid for streptokinase, pSK100, has been constructed. It contains a tac promoter, an ompA signal sequence, a streptokinase structural gene(skc) and a rrnBT1T2 transcription terminator. E. coli JM109 carrying pSK100 produced about 5,000IU of streptokinase per 1 ml of LB-ampicillin media. About 95% of the expressed streptokinase was secreted into the periplasmic and extracellular fractions. The recombinant streptokinase in high yield and purity may be a potential alternative source for the therapeutic agent.

DNA tumor virus-mediated tumorigenic processes typically involve functional inactivation of cellular tumor suppressors pRB and p53 by viral oncoproteins, with concomitant activation of oncogenic transcription factors such as E2Fs. This feature could be exploited to design a treatment for corresponding malignancies. Here, we report a gene therapy strategy for DNA tumor virus-associated cancers using a synthetic, E2F-regulated gene expression system named pESM6. This system contains multimerized E2F-responsive elements in combination with the binding sites for ubiquitous transcription factors Sp1 and CTF/NF1. pESM6 could drive a high-level transgene expression comparable to that of the CMV IE promoter and exert constitutive activity in cells expressing DNA tumor viral oncogenes. In contrast, it was effectively repressed by pRB and thus only minimally active in nontransformed cells. Expression of cytosine deaminase from pESM6 resulted in a highly efficient and specific killing of HPV-transformed fibroblasts (C3) after treatment with the prodrug 5-fluorocytosine. Also, an effective tumor mass reduction was observed when the vector was injected directly into C3 tumors implanted in C57BL/6 mice. pESM6 showed a superior performance throughout these experiments compared to the previously known E2F-regulated gene vector. These results clearly demonstrate the potential usability of pESM6 for the gene therapy of DNA tumor virus-associated cancers.

Streptokinase(SK), a plasminogen activator, is known to have multi‐domain structure. The function of the C‐terminal region of streptokinase was investigated with SK mutants constructed by truncating 26, 33, 37, 40, 41, 46, 47, 70 or 97 amino acid residues from the C‐terminus. The truncated SKs were expressed in E.coli and purified. The 41 residue deletion (SKP373) from the C‐terminus had not effect on the plasminogen activation activity. However, the deletion of 46 amino acid residues (SKP368) resulted in the dramatic reduction of the plasminogen activation efficiency. The result suggests that the C‐terminal peptide from Met369 to Pro373 of SK may play an important role on the plasminogen activation.

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Streptokinase (SK) is a bacterial plasminogen activator of multi‐domain structure. In deletion analysis of the N‐terminal region of SK, the deletion of 20 amino acids (SKΔN20) resulted in the dramatic reduction of plasminogen activator activity compared to deletion of 7 (SK ΔN7) and 13 amino acids (SKΔN13). The incubation time to reach maximal active site generation in an equimolar mixture of SKΔN20 and plasminogen was the same as that for wild‐type SK. To identify the functional residues important in plasminogen activation, several site‐directed mutations were introduced at the region spanning Serl6‐Val20 of SK. The results showed that Vall9 residue is important for the activity of the SK‐plasminogen complex.