Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. We report here analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and novel families of micro-exon genes that undergo frequent alternate splicing. As the first sequenced flatworm, and a representative of the lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, while the identification of membrane receptors, ion channels and more than 300 proteases, provide new insights into the biology of the life cycle and novel targets. Bioinformatics approaches have identified metabolic chokepoints while a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.

Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.

The endemic cichlid fishes in Lake Victoria are a model system for speciation through adaptive radiation. Although the evolution of the sex-determination system may also play a role in speciation, little is known about the sex-determination system of Lake Victoria cichlids. To understand the evolution of the sex-determination system in these fish, we performed cytogenetic analysis in 11 cichlid species from Lake Victoria. B chromosomes, which are present in addition to standard chromosomes, were found at a high prevalence rate (85%) in these cichlids. In one species, B chromosomes were female-specific. Cross-breeding using females with and without the B chromosomes demonstrated that the presence of the B chromosomes leads to a female-biased sex ratio in this species. Although B chromosomes were believed to be selfish genetic elements with little effect on phenotype and to lack protein-coding genes, the present study provides evidence that B chromosomes have a functional effect on female sex determination. FISH analysis using a BAC clone containing B chromosome DNA suggested that the B chromosomes are derived from sex chromosomes. Determination of the nucleotide sequences of this clone (104.5 kb) revealed the presence of several protein-coding genes in the B chromosome, suggesting that B chromosomes have the potential to contain functional genes. Because some sex chromosomes in amphibians and arthropods are thought to be derived from B chromosomes, the B chromosomes in Lake Victoria cichlids may represent an evolutionary transition toward the generation of sex chromosomes.

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The mammalian amelogenin (AMEL) genes are found on both the X and Y chromosomes (gametologous). Comparison of the genomic AMEL sequences in five primates and three other mammals reveals that the 5 portion of the gametologous AMEL loci began to differentiate in the common ancestor of extant mammals, whereas the 3 portion differentiated independently within species of different mammals. The boundary is marked by a transposon insertion in intron 2 and is shared by all species examined. In addition, 540-kb DNA sequences from the short arm of the human X chromosome are aligned with their Y gametologous sequences. The pattern and extent of sequence differences in the 5 portion of the AMEL loci extend to a proximal region that contains the ZFX locus, and those in the 3 portion extend all the way down to the pseudoautosomal boundary (PAB)1. We concluded that the AMEL locus spans an ancient PAB, and that both the ancient and present PABs were determined by chance events during the evolution of mammals and primates. Sex chromosome differentiation likely took place in a region that contains the male-determining loci by suppressing homologous recombination.chromosomal rearrangement ͉ evolutionary strata ͉ recombination suppression L ahn and Page (1) have proposed that there are four distinct evolutionary strata on the human X chromosome, and that differentiation of the X from the Y chromosome was initiated one stratum at a time. This hypothesis is based on the observation that the average extent of the sequence divergences at synonymous sites between X and Y homologous, or more precisely gametologous, loci is Ϸ10% in stratum 4 in contrast to 30% in stratum 3, 50% in stratum 2, and 100% in stratum 1. Stratum 4 spans Ϸ20 megabases on the short arm region of the X chromosome and is bounded by the amelogenin (AMEL) locus and pseudoautosomal boundary (PAB)1. Among seven loci examined in stratum 4 (1), AMEL has been more extensively studied in animals other than humans (2-7). Notably, primate intron 3 sequences suggest that AMEL on the X chromosome (AMELX) began to differentiate from that on the Y chromosome (AMELY) before the split of Old World and New World monkeys (4). On the other hand, cDNA or amino acid sequences analysis of gametologous AMELs shows greater relatedness within a species than among different mammalian species (5).Recently, Iwase et al. (8) compared human BAC clones that encompass the AMELX and AMELY loci. They found that, although the region downstream from intron 2 exhibits Ϸ10% sequence differences per site, the upstream region exhibits a high level that is similar to stratum 3 (Ͼ 20%; Ϸ30% if multiple-hit substitutions are taken into account). This finding does not contradict previous results (4, 5), because AMEL exons 1 and 2 almost exclusively encode the 5Ј untranslated region and are excluded from comparisons of intron 3 or amino acid sequences. Therefore, Iwase et al. (8) pointed out that the boundary between strata 3 and 4 on the human X chromosome lies in AMEL intron 2. Their preliminary study of ge...

The maintenance of organ homeostasis and the control of an appropriate response to environmental alterations require the intimate coordination of cellular functions and tissue organization. An important component of this coordination could be provided by proteins that can have distinct but linked functions on both sides of the plasma membrane. We present a model that proposes that unconventional secretion provides a mechanism through which single proteins can integrate complex tissue functions.Single genes can exert complex and dynamic effects through a number of different processes that multiply the functions of gene products. Alternative splicing can create many different transcripts from a single gene, which can encode proteins that have diverse, even antagonistic, functions. Post-translational modifications can alter the stability, activity, localization and even the basic functions of proteins. Furthermore, a protein can exist in different subcellular locations. More recently, it has become clear that single proteins can function both inside and outside of the cell. These proteins often lack defined secretory signal sequences and transit the plasma membrane by mechanisms that are distinct from the classical endoplasmic reticulum (ER)-Golgi secretory process. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWhen examples of such proteins are examined individually, the multifunctionality of these proteins and the lack of a signal sequence are puzzling -why should a protein with a wellknown function in one context function in such a distinct way in another? We propose that one reason for this is so that the protein can coordinate the organization and maintenance of a global tissue function. Here, we describe three examples of proteins that have intracellular and extracellular roles, we outline their specific functions in the extracellular and the intracellular space, and we discuss how these functions might be linked. All of these proteins have been reported to transit the plasma membrane through unconventional secretory mechanisms. Therefore, we also discuss the possible relationship between unconventional secretion and the coordination of extracellular and intracellular events, and how this relationship might be used to identify other proteins that share these characteristics. Coordinating tissue organizationIn this section, we discuss epimorphin and syntaxin 2, amphoterin and high mobility group protein B1 (HMGB1), and tissue transglutaminase (TTG; also known as TGM2). Syntaxin 2 might coordinate the morphogenesis of secretory organs (a role that was originally attributed to epimorphin, which is encoded by the same gene) with the control of protein secretion; HMGB1 might link inflammation (a role that was originally attributed to amphoterin, which is encoded by the same gene) with the regulation of gene expression; and TTG affects the delivery of and response to apoptotic signals on both sides of the plasma membrane. Morphogenesis and protein secretionEpimorphin was initiall...

Epimorphin (also known as syntaxin 2) acts as an epithelial morphogen when secreted by stromal cells of the mammary gland, lung, liver, colon, pancreas and other tissues, but the same molecule functions within the cell to mediate membrane fusion. How this molecule, which lacks a signal sequence and contains a transmembrane domain at the C-terminus, translocates across the plasma membrane and is secreted to become a morphogen, and how it initiates morphogenic events is not clear. Here, we show that epimorphin is secreted through a non-classical mechanism, similar to that previously described for secretion of the leaderless protein FGF1, and we identify the key molecular elements responsible for translocation and secretion from the cell. We also show that secreted epimorphin binds to αv-integrin-containing receptors on target epithelial cells, leading to activation of specific downstream signaling pathways and induction of epithelial morphogenesis. These findings provide key insight into how epimorphin functions as an epithelial morphogen.

Early apoptotic Jurkat T cells undergo capping of CD43, and its polylactosaminyl saccharide chains serve as ligands for phagocytosis by macrophages. This suggests the presence of a polylactosaminoglycan-binding receptor on macrophages. Here we show that this receptor is nucleolin, a multifunctional shuttling protein present in nucleus, cytoplasm, and on the surface of some types of cells. Nucleolin was detected at the surface of macrophages, and antinucleolin antibody inhibited the binding of the early apoptotic cells to macrophages. Nucleolin-transfected HEK293 cells expressed nucleolin on the cell surface and bound the early apoptotic cells but not phosphatidylserine-exposing late apoptotic cells. This binding was inhibited by anti-nucleolin antibody, by polylactosamine-containing oligosaccharides, and by anti-CD43 antibody. Deletion of the antibody binding region of nucleolin resulted in loss of the apoptotic cell-binding ability. Moreover, truncated recombinant nucleolin in solution containing this region blocked the apoptotic cell binding to macrophages, and the blocking effect was cancelled by the oligosaccharides. These results indicate that nucleolin is a macrophage receptor for apoptotic cells.Macrophages and other phagocytes recognize and ingest apoptotic cells in tissue, preventing their lysis and subsequent release of harmful or immunogenic intracellular components. Therefore, clearance of apoptotic cells by phagocytes is crucial in the maintenance of tissue turnover and homeostasis. Moreover, it has been suggested that the apoptotic cell-ingested phagocytes play a role in suppression or resolution of inflammation (1-5).Cells undergoing apoptosis display a variety of "eat me" signals, namely cell-surface changes to be recognized by phagocytes. These include externalization of phosphatidylserine (PS),3 as yet little identified alterations of carbohydrates, and unidentified alterations of other membrane components such as intercellular adhesion molecule-3 and "thrombospondin binding sites" (1-5). Among these, the most common and the best-characterized change is externalization of PS, although the mechanism of externalization has not been fully understood (6, 7).In contrast to the poor understanding of the nature of eat me signals, various proteins of phagocyte membrane or in extracellular fluid have been reported as receptors or bridging factors for apoptotic cells. For externalized PS on apoptotic cells, CD36 (8), CD68 (9), CLA-1 (10), LOX-1 (11), and PS receptor (12) have been reported as phagocyte receptors; a serum protein ␤ 2 -glycoprotein I (13), complement component C3bi (14), and milk fat globule-epidermal growth factor-factor 8 (15) have been reported to bridge apoptotic cells and phagocytes through exposed PS. For intercellular adhesion molecule-3 on apoptotic cells, CD14 was suggested to be a macrophage receptor (16). Thrombospondin is also known as a bridging protein between unidentified sites on apoptotic cells and CD36 or vitronectin receptor (␣ v ␤ 3 integrin) on phagocytes (17). In addi...