Cloning of rat ABCA7 and its preferential expression in platelets

We cloned the full-length cDNA of a rat orthologue of ABCA7 (rABCA7) from rat platelets. The cDNA of rABCA7 is 6510bp in length and encodes a protein of 2170 amino acids. The amino acid sequence of rABCA7 exhibits homology to those of mouse ABCA7 (92.5% identical in amino acid sequence) and human ABCA7 (76.6%). We obtained two clones of monoclonal antibodies against rABCA7 recognizing different epitopes. Analysis of CHO cells stably expressing rABCA7 by confocal laser-scanning microscopy indicated that rABCA7 is mainly located in the plasma membrane. Western blot analysis of rat tissues revealed that rABCA7 was preferentially expressed in platelets and that its apparent molecular mass was 250kDa. This is the first report of the tissue distribution of rABCA7 at the protein level and is the first reported case of ABC transporters being expressed in platelets, suggesting their important role in platelet function.     

Identification of functional domains of the Escherichia coli SeqA protein

The Escherichia coli SeqA protein, a negative regulator of chromosomal DNA replication, prevents the overinitiation of replication within one cell cycle by binding to hemimethylated G-mA-T-C sequences in the replication origin, oriC. In addition to the hemimethylated DNA-binding activity, the SeqA protein has a self-association activity, which is also considered to be essential for its regulatory function in replication initiation. To study the functional domains responsible for the DNA-binding and self-association activities, we performed a deletion analysis of the SeqA protein and found that the N-terminal (amino acid residues 1-59) and the C-terminal (amino acid residues 71-181) regions form structurally distinct domains. The N-terminal domain, which is not involved in DNA binding, has the self-association activity. In contrast, the C-terminal domain, which lacks the self-association activity, specifically binds to the hemimethylated G-mA-T-C sequence. Therefore, two essential SeqA activities, self-association and DNA-binding, are independently performed by the structurally distinct N-terminal and C-terminal domains, respectively.     

Distribution of interstitial telomere-like repeats and their adjacent sequences in a dioecious plant, <Emphasis Type="Italic">Silene latifolia</Emphasis>

The dioecious plant Silene latifolia has large, heteromorphic X and Y sex chromosomes that are thought to be derived from rearrangements of autosomes. To reveal the origin of the sex chromosomes in S. latifolia, we isolated and characterized telomere-homologous sequences from intra-chromosomal regions (interstitial telomere-like repeats; ITRs) and ITR-adjacent sequences (IASs). Nine genomic DNA fragments with degenerate 84- to 175-bp ITRs were isolated from a genomic library and total genome of male plants. Comparing the nucleotide sequences, the IASs of the nine ITRs were classified into seven elements (IAS-a, IAS-b, IAS-c, IAS-d, IAS-e, IAS-f, and IAS-g) by sequence similarity. The ITRs were grouped into two classes (class-I and -II ITRs) according to the classification of IASs. The class-I ITRs were sub-grouped into three subclasses (subclasses-IA, -IB, and -IC ITRs) based on the arrangement of IAS elements. By contrast, the class-II ITR was located between two different IASs (IAS-f and IAS-g). Genomic Southern analyses showed that both the male and female genomes contained six (IAS-f) to 153 (IAS-d) copies of each IAS per haploid genome. Fluorescence in situ hybridization analyses showed that one IAS element, IAS-d, was distributed in the interstitial and proximal regions of the sex chromosomes of S. latifolia. The distribution of IAS-d is important evidence for past telomere-mediated chromosome rearrangements during the evolution of the sex chromosomes of S. latifolia.     

Solution structure of the DFF-C domain of DFF45/ICAD. A structural basis for the regulation of apoptotic DNA fragmentation

DFF45/ICAD has dual functions in the final stage of apoptosis, by acting as both a folding chaperone and a DNase inhibitor of DFF40/CAD. Here, we present the solution structure of the C-terminal domain of DFF45, which is essential for its chaperone-like activity. The structure of this domain (DFF-C) consists of four alpha helices, which are folded in a novel helix-packing arrangement. The 3D structure reveals a large cluster of negatively charged residues on the molecular surface of DFF-C. This observation suggests that charge complementation plays an important role in the interaction of DFF-C with the positively charged catalytic domain of DFF40, and thus for the chaperone activity of DFF45. The structure of DFF-C also provides a rationale for the loss of the chaperone activity in DFF35, a short isoform of DFF45. Indeed, in DFF35, the amino acid sequence is truncated in the middle of the second alpha helix constituting the structure of DFF-C, and thus both the hydrophobic core and the cluster of negative charges are disrupted.     

Homologous pairing and ring and filament structure formation activities of the human Xrcc2*Rad51D complex

The Xrcc2 and Rad51D/Rad51L3 proteins, which belong to the Rad51 paralogs, are required for homologous recombinational repair (HRR) in vertebrates. The Xrcc2 and Rad51D/Rad51L3 genes, whose products interact with each other, have essential roles in ensuring normal embryonic development. In the present study, we coexpressed the human Xrcc2 and Rad51D/Rad51L3 proteins (Xrcc2 and Rad51D, respectively) in Escherichia coli, and purified the Xrcc2*Rad51D complex to homogeneity. The Xrcc2 small middle dotRad51D complex catalyzed homologous pairing between single-stranded and double-stranded DNA, similar to the function of the Xrcc3*Rad51C complex, which is another complex of the Rad51 paralogs. An electron microscopic analysis showed that Xrcc2*Rad51D formed a multimeric ring structure in the absence of DNA. In the presence of ssDNA, Xrcc2*Rad51D formed a filamentous structure, which is commonly observed among the human homologous pairing proteins, Rad51, Rad52, and Xrcc3*Rad51C.     

Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions

We describe microarrays of oligosaccharides as neoglycolipids and their robust display on nitrocellulose. The arrays are obtained from glycoproteins, glycolipids, proteoglycans, polysaccharides, whole organs, or from chemically synthesized oligosaccharides. We show that carbohydrate-recognizing proteins single out their ligands not only in arrays of homogeneous oligosaccharides but also in arrays of heterogeneous oligosaccharides. Initial applications have revealed new findings, including: (i) among O-glycans in brain, a relative abundance of the Lewis(x) sequence based on N-acetyllactosamine recognized by anti-L5, and a paucity of the Lewis(x) sequence based on poly-N-acetyllactosamine recognized by anti-SSEA-1; (ii) insights into chondroitin sulfate oligosaccharides recognized by an antiserum and an antibody (CS-56) to chondroitin sulfates; and (iii) binding of the cytokine interferon-gamma (IFN-gamma) and the chemokine RANTES to sulfated sequences such as HNK-1, sulfo-Lewis(x), and sulfo-Lewis(a), in addition to glycosaminoglycans. The approach opens the way for discovering new carbohydrate-recognizing proteins in the proteome and for mapping the repertoire of carbohydrate recognition structures in the glycome.     

Phylogenetics and evolution of the eastern Asian-eastern North American disjunct aphid tribe,Hormaphidini (Hemiptera: Aphididae)

A conspicuous biogeographic pattern of the Northern Hemisphere is the disjunct occurrence of related taxa on different continents. Perhaps best studied in plants, this pattern includes disjunct distributions of genera in eastern Asia and eastern North America. Such continental disjunctions are thought to be the remnants of a mostly continuously distributed, mixed mesophytic forest dating to the Miocene, which subsequently became fragmented by geological and climatic changes. Some highly host-specific insects, namely aphids, live on descendants of the mixed mesophytic forest taxa and exhibit the same disjunct distributions as that of their host plants. We estimated the phylogeny of Hormaphidini aphids, which host-alternate between witch-hazel (Hamamelis; an eastern Asian-eastern North American disjunct genus) and birch (Betula). Based on partial nuclear elongation factor 1alpha and mitochondrial tRNA leucine/cytochrome oxidase II sequences, trees inferred from maximum-parsimony and maximum-likelihood showed strong support for two monophyletic genera (Hamamelistes and Hormaphis), each containing a clade of Japanese and a clade of North American species. The estimated divergence dates of Asian and North American clades in both genera was 20-30 million years ago, consistent with the idea that aphids may have experienced the same vicariance events as those of their host plants.     

Trehalose augments osteoprotegerin production in the FHs74Int human intestinal epithelial cell line

The disaccharide trehalose has been shown to inhibit both bone loss in ovariectomized mice and excessive osteoclastogenesis in lipopolysaccharide-injected mice. However, the mechanism of osteoclastogenesis inhibition by oral administration of trehalose is still unclear. We report here for the first time that a human intestinal epithelial cell line, FHs74Int, also produces osteoprotegerin (OPG) and that trehalose augments OPG production by this cell line. Thus, these results suggest that trehalose promotes the production of OPG by intestinal epithelial cells, which then acts on bone marrow cells, resulting in the suppression of osteoclastogenesis.     

Molecular diversity of skin mucus lectins in fish

Among lectins in the skin mucus of fish, primary structures of four different types of lectin have been determined. Congerin from the conger eel Conger myriaster and AJL-1 from the Japanese eel Anguilla japonica were identified as galectin, characterized by its specific binding to β-galactoside. Eel has additionally a unique lectin, AJL-2, which has a highly conserved sequence of C-type lectins but displays Ca²⁺-independent activity. This is rational because the lectin exerts its function on the cutaneous surface, which is exposed to a Ca²⁺ scarce environment when the eel is in fresh water. The third type lectin is pufflectin, a mannose specific lectin in the skin mucus of pufferfish Takifugu rubripes. This lectin showed no sequence similarity with any known animal lectins but, surprisingly, shares sequence homology with mannose-binding lectins of monocotyledonous plants. The fourth lectin was found in the ponyfish Leiognathus nuchalis and exhibits homology with rhamnose-binding lectins known in eggs of some fish species. These lectins, except ponyfish lectin, showed agglutination of certain bacteria. In addition, pufflectin was found to bind to a parasitic trematode, Heterobothrium okamotoi. Taken together, these results demonstrate that skin mucus lectins in fish have wide molecular diversity.