Next-generation sequencing: challenges in reproductive genetics

Deep impact: sequencing embryo biopsy specimens at increasing depth

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

Electrocardiographic features of patients with COVID-19: One year of unexpected manifestations

Born Large for Gestational Age: Bigger Is Not Always Better

Celiac Disease: Advances in Treatment via Gluten Modification

The multipotentiality and self-renewal ability of stem cells are controlled by intrinsic genetic pathways that are subject to regulation by extrinsic signals emanating from the stem cell niche. The stem cell niche provides a microenvironment composed of cellular structures or extracellular matrix in which stem cells are maintained as undifferentiated (1Li L. Xie T. Annu. Rev. Cell Dev. Biol. 2005; 21: 605-631Crossref PubMed Scopus (942) Google Scholar, 2Lin H. Nat. Rev. Genet. 2002; 3: 931-940Crossref PubMed Scopus (305) Google Scholar, 3Scadden D.T. Nature. 2006; 441: 1075-1079Crossref PubMed Scopus (1508) Google Scholar, 4Spradling A. Drummond-Barbosa D. Kai T. Nature. 2001; 414: 98-104Crossref PubMed Scopus (1215) Google Scholar, 5Watt F.M. Hogan B.L. Science. 2000; 287: 1427-1430Crossref PubMed Scopus (1474) Google Scholar, 6Fuchs E. Tumbar T. Guasch G. Cell. 2004; 116: 769-778Abstract Full Text Full Text PDF PubMed Scopus (1481) Google Scholar). The concept of "the stem cell niche" was first proposed in studies of the HSC 2The abbreviations used are:HSChematopoietic stem cellGSCgerm line stem cellBMbone marrowCARCXCL12-abundant reticularCBscystoblastsESCescort stem cellCPscyst progenitorsAng-1angiopoietin-1.2The abbreviations used are:HSChematopoietic stem cellGSCgerm line stem cellBMbone marrowCARCXCL12-abundant reticularCBscystoblastsESCescort stem cellCPscyst progenitorsAng-1angiopoietin-1. (7Schofield R. Blood Cells. 1978; 4: 7-25PubMed Google Scholar); however, in vivo evidence of its existence was first shown in the Drosophila GSC (8Cox D.N. Chao A. Baker J. Chang L. Qiao D. Lin H. Genes Dev. 1998; 12: 3715-3727Crossref PubMed Scopus (802) Google Scholar, 9King F.J. Lin H. Development (Camb.). 1999; 126: 1833-1844PubMed Google Scholar, 10Xie T. Spradling A.C. Science. 2000; 290: 328-330Crossref PubMed Scopus (620) Google Scholar). Over the past several years, there has been much progress made in identifying stem cell niches in different mammal tissues, including nerves, hair follicles, intestines, teeth, and BM (11Tumbar T. Guasch G. Greco V. Blanpain C. Lowry W.E. Rendl M. Fuchs E. Science. 2004; 303: 359-363Crossref PubMed Scopus (1631) Google Scholar, 12Yen T.H. Wright N.A. Stem Cell Rev. 2006; 2: 203-212Crossref PubMed Scopus (227) Google Scholar, 13Conover J.C. Notti R.Q. Cell Tissue Res. 2008; 331: 211-224Crossref PubMed Scopus (123) Google Scholar, 14Doetsch F. Curr. Opin. Genet. Dev. 2003; 13: 543-550Crossref PubMed Scopus (529) Google Scholar, 15Ohshima H. Nakasone N. Hashimoto E. Sakai H. Nakakura-Ohshima K. Harada H. Arch. Oral Biol. 2005; 50: 153-157Crossref PubMed Scopus (81) Google Scholar, 16Wilson A. Trumpp A. Nat. Rev. Immunol. 2006; 6: 93-106Crossref PubMed Scopus (1050) Google Scholar). In this review, our focus is on comparing Drosophila GSC niches and mouse HSC niches (two of the best characterized niches). By such comparison, we hope to provide some common principles of stem cell niches that will be useful in other tissue stem cell niche studies. hematopoietic stem cell germ line stem cell bone marrow CXCL12-abundant reticular cystoblasts escort stem cell cyst progenitors angiopoietin-1. hematopoietic stem cell germ line stem cell bone marrow CXCL12-abundant reticular cystoblasts escort stem cell cyst progenitors angiopoietin-1. In recent years, remarkable progress has been made in the identification and characterization of the stem cell niches in invertebrate systems (10Xie T. Spradling A.C. Science. 2000; 290: 328-330Crossref PubMed Scopus (620) Google Scholar, 17Tulina N. Matunis E. Science. 2001; 294: 2546-2549Crossref PubMed Scopus (506) Google Scholar, 18Kiger A.A. Jones D.L. Schulz C. Rogers M.B. Fuller M.T. Science. 2001; 294: 2542-2545Crossref PubMed Scopus (538) Google Scholar, 19Lin H. Spradling A.C. Dev. Genet. 1995; 16: 6-12Crossref PubMed Scopus (180) Google Scholar, 20Crittenden S.L. Bernstein D.S. Bachorik J.L. Thompson B.E. Gallegos M. Petcherski A.G. Moulder G. Barstead R. Wickens M. Kimble J. Nature. 2002; 417: 660-663Crossref PubMed Scopus (337) Google Scholar, 21Mandal L. Martinez-Agosto J.A. Evans C.J. Hartenstein V. Banerjee U. Nature. 2007; 446: 320-324Crossref PubMed Scopus (220) Google Scholar, 22Krzemien J. Dubois L. Makki R. Meister M. Vincent A. Crozatier M. Nature. 2007; 446: 325-328Crossref PubMed Scopus (246) Google Scholar). In studies of GSCs in Drosophila, the ovary and testis provide relatively simple but elegant anatomic structures with few cell types and unique stem cell markers. These advantages facilitated identification of the cellular components of the stem cell niche and definition of the molecular basis of physical interaction between stem cells and their niches (23Song X. Xie T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14813-14818Crossref PubMed Scopus (184) Google Scholar, 24Yamashita Y.M. Jones D.L. Fuller M.T. Science. 2003; 301: 1547-1550Crossref PubMed Scopus (595) Google Scholar) and revealed key niche signals involved in stem cell regulation (18Kiger A.A. Jones D.L. Schulz C. Rogers M.B. Fuller M.T. Science. 2001; 294: 2542-2545Crossref PubMed Scopus (538) Google Scholar, 25LaFever L. Drummond-Barbosa D. Science. 2005; 309: 1071-1073Crossref PubMed Scopus (248) Google Scholar, 26Szakmary A. Cox D.N. Wang Z. Lin H. Curr. Biol. 2005; 15: 171-178Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 27Xie T. Spradling A.C. Cell. 1998; 94: 251-260Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar, 28Yamashita Y.M. Fuller M.T. Jones D.L. J. Cell Sci. 2005; 118: 665-672Crossref PubMed Scopus (166) Google Scholar, 29Parisi M.J. Lin H. Cell Res. 1998; 8: 15-21Crossref PubMed Scopus (38) Google Scholar, 30King F.J. Szakmary A. Cox D.N. Lin H. Mol. Cell. 2001; 7: 497-508Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 31Wang Z. Lin H. Science. 2004; 303: 2016-2019Crossref PubMed Scopus (197) Google Scholar). In the murine hematopoietic system, HSCs have been well defined (32Spangrude G.J. Heimfeld S. Weissman I.L. Science. 1988; 241: 58-62Crossref PubMed Scopus (2226) Google Scholar, 33Weissman I.L. Science. 2000; 287: 1442-1446Crossref PubMed Scopus (713) Google Scholar), but identification of the HSC niches is just beginning. This search has been hampered by the complexity of the BM structure and cellular components and by the lack of unique HSC markers or distinctive characteristics of BM stromal cells. Two HSC niches have been proposed in murine BM, an osteoblastic niche and a vascular niche, in which osteoblasts and vascular endothelial cells have been demonstrated as major components, respectively (34Zhang J. Niu C. Ye L. Huang H. He X. Tong W.G. Ross J. Haug J. Johnson T. Feng J.Q. Harris S. Wiedemann L.M. Mishina Y. Li L. Nature. 2003; 425: 836-841Crossref PubMed Scopus (2385) Google Scholar, 35Calvi L.M. Adams G.B. Weibrecht K.W. Weber J.M. Olson D.P. Knight M.C. Martin R.P. Schipani E. Divieti P. Bringhurst F.R. Milner L.A. Kronenberg H.M. Scadden D.T. Nature. 2003; 425: 841-846Crossref PubMed Scopus (2799) Google Scholar, 36Arai F. Hirao A. Ohmura M. Sato H. Matsuoka S. Takubo K. Ito K. Koh G.Y. Suda T. Cell. 2004; 118: 149-161Abstract Full Text Full Text PDF PubMed Scopus (1540) Google Scholar, 37Nilsson S.K. Johnston H.M. Whitty G.A. Williams B. Webb R.J. Denhardt D.T. Bertoncello I. Bendall L.J. Simmons P.J. Haylock D.N. Blood. 2005; 106: 1232-1239Crossref PubMed Scopus (605) Google Scholar, 38Heissig B. Rafii S. Akiyama H. Ohki Y. Sato Y. Rafael T. Zhu Z. Hicklin D.J. Okumura K. Ogawa H. Werb Z. Hattori K. J. Exp. Med. 2005; 202: 739-750Crossref PubMed Scopus (195) Google Scholar, 39Moore K.A. Curr. Opin. Hematol. 2004; 11: 107-111Crossref PubMed Scopus (56) Google Scholar, 40Taichman R.S. Blood. 2005; 105: 2631-2639Crossref PubMed Scopus (469) Google Scholar, 41Kiel M.J. Yilmaz O.H. Iwashita T. Terhorst C. Morrison S.J. Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (2414) Google Scholar). A recent study suggested that a population of reticular cells named CAR cells, which express a high level of CXCL12 (also known as stromal cell-derived factor-1 or pre-B cell growth-stimulating factor), are in contact with HSCs in both osteoblastic and vascular niches (42Sugiyama T. Kohara H. Noda M. Nagasawa T. Immunity. 2006; 25: 977-988Abstract Full Text Full Text PDF PubMed Scopus (1693) Google Scholar). Drosophila ovary and testis provide attractive models for stem cell niche studies. In Drosophila ovary, cap cells, a specific type of somatic cell located at the tip of the germarium in the ovary, function as the niche for GSCs. An E-cadherin/β-catenin-formed cell-cell adhesion junction mediates the physical interaction between GSCs and their niche cells (23Song X. Xie T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14813-14818Crossref PubMed Scopus (184) Google Scholar, 46Song X. Zhu C.H. Doan C. Xie T. Science. 2002; 296: 1855-1857Crossref PubMed Scopus (389) Google Scholar), ensuring stem cell control by niche signals. Normally, asymmetric division of stem cells results in two daughter cells with different fates: one daughter cell attaches to the niche and is maintained as a stem cell (self-renewal), whereas the other daughter cell leaves the niche and develops into a cystoblast (1Li L. Xie T. Annu. Rev. Cell Dev. Biol. 2005; 21: 605-631Crossref PubMed Scopus (942) Google Scholar, 4Spradling A. Drummond-Barbosa D. Kai T. Nature. 2001; 414: 98-104Crossref PubMed Scopus (1215) Google Scholar). CBs undergo four incomplete cell divisions to form an interconnected 2–16-cell germ line cyst. In addition to the GSC, another type of cell called the ESC also attaches to cap cells interspersed between the GSCs. Together with cap cells, ESCs encapsulate GSCs to separate GSCs from their differentiated daughter cells. When the GSCs divide and detach from the niche to produce CBs and cyst cells, the ESCs also proliferate and differentiate to produce more escort cells. The escort cells expand and continue to encapsulate CBs and cysts during the process of cyst formation and are finally replaced by follicle cells after the 16-cell germ line cyst stage (Fig. 1a). Interestingly, the newly formed cysts (prior to the eight-cell cyst stage, while still encapsulated by the escort cell) can revert to the stem cell state under certain circumstances, suggesting that they may still retain (albeit limited) stem cell properties (47Kai T. Spradling A. Nature. 2004; 428: 564-569Crossref PubMed Scopus (267) Google Scholar). A similar niche is also found in Drosophila testis and is composed of hub cells located at the end of the testis (48Le Bras S. Van Doren M. Dev. Biol. 2006; 294: 92-103Crossref PubMed Scopus (96) Google Scholar). Male GSCs surrounding the hub cells are interspersed with CPs (which are counterparts of ESCs in the ovary). Both GSCs and CPs are attached to the hub cells through an adhesion junction. Similar to ESCs in the ovary, proliferation and differentiation of CPs always accompany proliferation and differentiation of GSCs. CPs in the testis produce cyst cells, which encapsulate the gonialblasts (the immediate daughters of male GSCs) during spermatogonium formation (Fig. 1b). The newly formed gonialblasts can also revert to stem cells under certain circumstances, resembling their counterpart cells (CBs and cysts) in the ovary (49Brawley C. Matunis E. Science. 2004; 304: 1331-1334Crossref PubMed Scopus (326) Google Scholar). The role of cap and hub cells in GSC regulation has been well studied, but the contribution of ESCs and their offspring in the ovary and CPs and their progeny in the testis to GSC self-renewal, proliferation, and differentiation is largely unknown. However, it is known that a dialogue between the two types of cells is required for the coordination during organogenesis (30King F.J. Szakmary A. Cox D.N. Lin H. Mol. Cell. 2001; 7: 497-508Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Cap and hub cells provide an attachment point for anchoring GSCs to the niche; they also produce signals that inhibit differentiation but promote self-renewal of stem cells (17Tulina N. Matunis E. Science. 2001; 294: 2546-2549Crossref PubMed Scopus (506) Google Scholar). The stem cell niches in mammals are very complicated because of the complexity of cellular components of mammalian tissues, even though the mechanism for niche regulation is conserved from invertebrate to vertebrate. In mammals, BM tissue is composed of more than eight different hematopoietic cell lineages supported by a network of mesenchymal stromal cells and vascular endothelial cells. The two HSC niches, osteoblastic (34Zhang J. Niu C. Ye L. Huang H. He X. Tong W.G. Ross J. Haug J. Johnson T. Feng J.Q. Harris S. Wiedemann L.M. Mishina Y. Li L. Nature. 2003; 425: 836-841Crossref PubMed Scopus (2385) Google Scholar, 35Calvi L.M. Adams G.B. Weibrecht K.W. Weber J.M. Olson D.P. Knight M.C. Martin R.P. Schipani E. Divieti P. Bringhurst F.R. Milner L.A. Kronenberg H.M. Scadden D.T. Nature. 2003; 425: 841-846Crossref PubMed Scopus (2799) Google Scholar, 36Arai F. Hirao A. Ohmura M. Sato H. Matsuoka S. Takubo K. Ito K. Koh G.Y. Suda T. Cell. 2004; 118: 149-161Abstract Full Text Full Text PDF PubMed Scopus (1540) Google Scholar, 37Nilsson S.K. Johnston H.M. Whitty G.A. Williams B. Webb R.J. Denhardt D.T. Bertoncello I. Bendall L.J. Simmons P.J. Haylock D.N. Blood. 2005; 106: 1232-1239Crossref PubMed Scopus (605) Google Scholar, 38Heissig B. Rafii S. Akiyama H. Ohki Y. Sato Y. Rafael T. Zhu Z. Hicklin D.J. Okumura K. Ogawa H. Werb Z. Hattori K. J. Exp. Med. 2005; 202: 739-750Crossref PubMed Scopus (195) Google Scholar, 39Moore K.A. Curr. Opin. Hematol. 2004; 11: 107-111Crossref PubMed Scopus (56) Google Scholar, 40Taichman R.S. Blood. 2005; 105: 2631-2639Crossref PubMed Scopus (469) Google Scholar, 41Kiel M.J. Yilmaz O.H. Iwashita T. Terhorst C. Morrison S.J. Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (2414) Google Scholar, 50Balduino A. Hurtado S.P. Frazao P. Takiya C.M. Alves L.M. Nasciutti L.E. El-Cheikh M.C. Borojevic R. Cell Tissue Res. 2005; 319: 255-266Crossref PubMed Scopus (90) Google Scholar, 51Arai F. Hirao A. Suda T. Trends Cardiovasc. Med. 2005; 15: 75-79Crossref PubMed Scopus (66) Google Scholar, 52Adams G.B. Chabner K.T. Alley I.R. Olson D.P. Szczepiorkowski Z.M. Poznansky M.C. Kos C.H. Pollak M.R. Brown E.M. Scadden D.T. Nature. 2006; 439: 599-603Crossref PubMed Scopus (600) Google Scholar) and vascular (41Kiel M.J. Yilmaz O.H. Iwashita T. Terhorst C. Morrison S.J. Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (2414) Google Scholar, 53Kopp H.G. Avecilla S.T. Hooper A.T. Rafii S. Physiology. 2005; 20: 349-356Crossref PubMed Scopus (414) Google Scholar), are found in adult BM tissue. The vascular niche might also exist in fetal hematopoietic tissues such as yolk sac, aorta-gonad-mesonephros region, placenta, liver, and spleen (54Tavian M. Peault B. Exp. Hematol. 2005; 33: 1062-1069Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 55Basto D. Trovisco V. Lopes J.M. Martins A. Pardal F. Soares P. Reis R.M. Acta Neuropathol. 2005; 109: 207-210Crossref PubMed Scopus (84) Google Scholar, 56Gekas C. Dieterlen-Lièvre F. Orkin S.H. Millola H.K.A. Dev. Cell. 2005; 8: 365-375Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar) as well as in adult spleen and liver. Both niches may be critical for HSC self-renewal. Cooperation between these two niches might be required for maintaining normal hematopoietic homeostasis and re-establishing hematopoiesis after injury. Accumulated evidence supports the existence of an osteoblastic niche in BM. The concurrence of hematopoiesis and osteogenesis in BM suggests a close relationship between bone-forming cells and hematopoietic cells (57Patt H.M. Maloney M.A. Proc. Soc. Exp. Biol. Med. 1972; 140: 205-207Crossref PubMed Scopus (35) Google Scholar, 58Maloney M.A. Patt H.M. Proc. Soc. Exp. Biol. Med. 1975; 149: 94-97Crossref PubMed Scopus (12) Google Scholar). Early studies demonstrated that endosteal BM is more highly enriched with HSCs/progenitors compared with central BM (59Lord B.I. Testa N.G. Hendry J.H. Blood. 1975; 46: 65-72Crossref PubMed Google Scholar, 60Gong J.K. Science. 1978; 199: 1443-1445Crossref PubMed Scopus (195) Google Scholar). The facts that hematopoiesis recovery after myeloablative injury occurs on the endosteal bone surface (45Heissig B. Hattori K. Dias S. Friedrich M. Ferris B. Hackett N.R. Crystal R.G. Besmer P. Lyden D. Moore M.A. Werb Z. Rafii S. Cell. 2002; 109: 625-637Abstract Full Text Full Text PDF PubMed Scopus (1510) Google Scholar) and that the majority of HSCs/progenitors are distributed in the endosteal region after transplantation (61Nilsson S.K. Johnston H.M. Coverdale J.A. Blood. 2001; 97: 2293-2299Crossref PubMed Scopus (473) Google Scholar) support the importance of the endosteal BM microenvironment in HSC maintenance and regulation. Several genetically modulated mutant mouse models exhibit defects in hematopoiesis as a consequence of abnormal bone formation or remodeling, indicating the important role of osteoblasts in supporting HSCs in BM (62Deguchi K. Yagi H. Inada M. Yoshizaki K. Kishimoto T. Komori T. Biochem. Biophys. Res. Commun. 1999; 255: 352-359Crossref PubMed Scopus (49) Google Scholar, 63Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3630) Google Scholar, 64Gerber H.P. Vu T.H. Ryan A.M. Kowalski J. Werb Z. Ferrara N. Nat. Med. 1999; 5: 623-628Crossref PubMed Scopus (1707) Google Scholar, 65Nilsson S.K. Bertoncello I. Exp. Hematol. 1994; 22: 377-383PubMed Google Scholar, 66Begg S.K. Radley J.M. Pollard J.W. Chisholm O.T. Stanley E.R. Bertoncello I. J. Exp. Med. 1993; 177: 237-242Crossref PubMed Scopus (160) Google Scholar, 67Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.R. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (394) Google Scholar). Significant reduction in the number or functional disruption of osteoblasts leads to significant reduction in BM HSCs, and this often results in extramedullary hematopoiesis in the spleen (68Visnjic D. Kalajzic Z. Rowe D.W. Katavic V. Lorenzo J. Aguila H.L. Blood. 2004; 103: 3258-3264Crossref PubMed Scopus (598) Google Scholar). Additional trabecular bone formation and a corresponding increase in the number of osteoblasts in mice correlate with an increase in the number of BM HSCs (34Zhang J. Niu C. Ye L. Huang H. He X. Tong W.G. Ross J. Haug J. Johnson T. Feng J.Q. Harris S. Wiedemann L.M. Mishina Y. Li L. Nature. 2003; 425: 836-841Crossref PubMed Scopus (2385) Google Scholar, 35Calvi L.M. Adams G.B. Weibrecht K.W. Weber J.M. Olson D.P. Knight M.C. Martin R.P. Schipani E. Divieti P. Bringhurst F.R. Milner L.A. Kronenberg H.M. Scadden D.T. Nature. 2003; 425: 841-846Crossref PubMed Scopus (2799) Google Scholar). Moreover, osteoblastic cells support HSC self-renewal in in vitro culture (40Taichman R.S. Blood. 2005; 105: 2631-2639Crossref PubMed Scopus (469) Google Scholar) and enhance BM engraftment of HSCs in vivo when they are co-transplanted (69El-Badri N.S. Wang B.Y. Cherry Good R.A. Exp. Hematol. 1998; 26: 110-116PubMed Google Scholar) into recipient mice. Direct evidence of osteoblasts as the HSC niche has been provided in studies of several genetically modulated mouse models (35Calvi L.M. Adams G.B. Weibrecht K.W. Weber J.M. Olson D.P. Knight M.C. Martin R.P. Schipani E. Divieti P. Bringhurst F.R. Milner L.A. Kronenberg H.M. Scadden D.T. Nature. 2003; 425: 841-846Crossref PubMed Scopus (2799) Google Scholar, 36Arai F. Hirao A. Ohmura M. Sato H. Matsuoka S. Takubo K. Ito K. Koh G.Y. Suda T. Cell. 2004; 118: 149-161Abstract Full Text Full Text PDF PubMed Scopus (1540) Google Scholar, 68Visnjic D. Kalajzic Z. Rowe D.W. Katavic V. Lorenzo J. Aguila H.L. Blood. 2004; 103: 3258-3264Crossref PubMed Scopus (598) Google Scholar, 70Wilson A. Murphy M.J. Oskarsson T. Kaloulis K. Bettess M.D. Oser G.M. Pasche A.C. Knabenhans C. MacDonald H.R. Trumpp A. Genes Dev. 2004; 18: 2747-2763Crossref PubMed Scopus (587) Google Scholar, 71Katayama Y. Battista M. Kao W.M. Hidalgo A. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). is that osteoblasts function as the HSC niche; we found that a of osteoblasts that express a high level of function as a key of the osteoblastic niche (34Zhang J. Niu C. Ye L. Huang H. He X. Tong W.G. Ross J. Haug J. Johnson T. Feng J.Q. Harris S. Wiedemann L.M. Mishina Y. Li L. Nature. 2003; 425: 836-841Crossref PubMed Scopus (2385) Google Scholar). This is supported by in vitro functional studies that an increase in in cells BM stromal cell by or the ability of cells to support HSCs F. Hirao A. Ohmura M. Sato H. Matsuoka S. Takubo K. Ito K. Koh G.Y. Suda T. Cell. 2004; 118: 149-161Abstract Full Text Full Text PDF PubMed Scopus (1540) Google Scholar). The existence of an osteoblastic niche has also been in several other studies. and A. Murphy M.J. Oskarsson T. Kaloulis K. Bettess M.D. Oser G.M. Pasche A.C. Knabenhans C. MacDonald H.R. Trumpp A. Genes Dev. 2004; 18: 2747-2763Crossref PubMed Scopus (587) Google Scholar) found that the majority of HSCs/progenitors in the and to osteoblastic cells By they demonstrated that HSCs with have and adhesion to osteoblastic niche cells and to from the osteoblastic niche for proliferation, and differentiation A. Murphy M.J. Oskarsson T. Kaloulis K. Bettess M.D. Oser G.M. Pasche A.C. Knabenhans C. MacDonald H.R. Trumpp A. Genes Dev. 2004; 18: 2747-2763Crossref PubMed Scopus (587) Google Scholar). HSCs in the osteoblastic niche are relatively during normal homeostasis but can be as a of bone A. S. A. K. Y. M. R.M. P. A. A. T. Nat. Med. 2006; 12: PubMed Scopus Google Scholar) or Y. Battista M. Kao W.M. Hidalgo A. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). M.J. Morrison S.J. Cell Stem Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar) that HSCs lack however, that is by HSCs, its is when HSCs are in different found that HSCs to of can the hematopoietic in mice but with the more X. J. J. K. Cell Stem in In which function in bone remodeling, have also been shown to HSC and maintenance by osteoblasts A. S. A. K. Y. M. R.M. P. A. A. T. Nat. Med. 2006; 12: PubMed Scopus Google Scholar). However, on the of the a reduction in the number of osteoblasts may HSC function that HSCs in a for osteoblastic mouse models such as mice and mice Y. Battista M. Kao W.M. Hidalgo A. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. G.A. P. Scadden D.T. L.E. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. N.A. L.E. Orkin S.H. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar) have demonstrated that reduction in trabecular bone or number HSC in a number of HSCs in BM and and extramedullary hematopoiesis in the the vascular niche is and the stage for into and In BM, some of the in the matrix of the osteoblastic niche may also be important for HSC and a matrix by is a of the HSC osteoblastic niche that the of the stem cell and may provide a mechanism for stem cell under of niche S. Y. R. C. T. E. T. D. L.M. S.R. Scadden D.T. J. Exp. Med. 2005; PubMed Scopus Google Scholar). In the high of in the BM endosteal region mediates HSC and in the osteoblastic niche by HSCs through a that is highly in HSCs G.B. Chabner K.T. Alley I.R. Olson D.P. Szczepiorkowski Z.M. Poznansky M.C. Kos C.H. Pollak M.R. Brown E.M. Scadden D.T. Nature. 2006; 439: 599-603Crossref PubMed Scopus (600) Google Scholar). endothelial cells from hematopoietic tissue more support hematopoiesis compared with endothelial cells from other H.G. Avecilla S.T. Hooper A.T. Rafii S. Physiology. 2005; 20: 349-356Crossref PubMed Scopus (414) Google Scholar, C. Z. C. H. R. B. L.A. Blood. 1998; PubMed Google Scholar, M.A. A.M. C. P. L.E. T. Mol. Cell. 2003; PubMed Scopus Google Scholar, P. J.K. 2004; 126: Full Text Full Text PDF PubMed Scopus Google Scholar). (41Kiel M.J. Yilmaz O.H. Iwashita T. Terhorst C. Morrison S.J. Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (2414) Google Scholar) have demonstrated that HSCs to the of in BM and spleen in mice after HSC and also to BM during The of a number of HSCs in the vascular niche that the BM and spleen provides a specific niche for proliferation, and differentiation under both and The that HSCs detach from the osteoblastic niche and the of BM to the vascular region, they hematopoiesis during hematopoietic after S.T. Hattori K. B. R. F. K. Dias S. Zhang F. Hackett N.R. Crystal R.G. L. Hicklin D.J. P. D. Lyden D. F. Rafii S. Nat. Med. 2004; PubMed Scopus Google Scholar, 53Kopp H.G. Avecilla S.T. Hooper A.T. Rafii S. Physiology. 2005; 20: 349-356Crossref PubMed Scopus (414) Google Scholar, S. R. F. Moore M.A. 1997; PubMed Scopus Google Scholar), also suggests the existence of a vascular BM HSC niche. However, the that of osteoblasts results in a reduction of HSCs in BM and extramedullary hematopoiesis (68Visnjic D. Kalajzic Z. Rowe D.W. Katavic V. Lorenzo J. Aguila H.L. Blood. 2004; 103: 3258-3264Crossref PubMed Scopus (598) Google Scholar) suggests that the vascular niche may provide a J.M. Li L. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). CXCL12 is a that cell through interaction with its which is by the cells. in including HSC in BM during the of hematopoiesis from fetal to BM, cell and cell and as demonstrated by mutant mice with disruption T. K. T. T. K. Nagasawa T. Immunity. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). important in HSC and HSC of mice (the into the that CXCL12 is by a population of stromal cells BM (42Sugiyama T. Kohara H. Noda M. Nagasawa T. Immunity. 2006; 25: 977-988Abstract Full Text Full Text PDF PubMed Scopus (1693) Google Scholar, K. T. T. B.I. Nagasawa T. Immunity. 2004; 20: Full Text Full Text PDF PubMed Scopus Google Scholar,

Pulmonary Hypertension: From an Orphan Disease to a Public Health Problem

Impaired Autophagy Triggers Chronic Pancreatitis: Lessons From Pancreas-Specific Atg5 Knockout Mice

Quality of Life and Depression in CKD: Improving Hope and Health

Melanoma Resistance: A Bright Future for Academicians and a Challenge for Patient Advocates

Transforming growth factor beta (TGF-beta) and related growth factors are essential regulators of embryogenesis and tissue homeostasis. The signaling pathways mediated by their receptors and Smad proteins are precisely modulated by various means. Xenopus BAMBI (bone morphogenic protein (BMP) and activin membrane-bound inhibitor) has been shown to function as a general negative regulator of TGF-beta/BMP/activin signaling. Here, we provide evidence that human BAMBI (hBAMBI), like its Xenopus homolog, inhibits TGF-beta- and BMP-mediated transcriptional responses as well as TGF-beta-induced R-Smad phosphorylation and cell growth arrest, whereas knockdown of endogenous BAMBI enhances the TGF-beta-induced reporter expression. Mechanistically, in addition to interfering with the complex formation between the type I and type II receptors, hBAMBI cooperates with Smad7 to inhibit TGF-beta signaling. hBAMBI forms a ternary complex with Smad7 and the TGF-beta type I receptor ALK5/TbetaRI and inhibits the interaction between ALK5/TbetaRI and Smad3, thus impairing Smad3 activation. These findings provide a novel insight to understand the molecular mechanism underlying the inhibitory effect of BAMBI on TGF-beta signaling.

Inward rectifier K+ (Kir) channels are activated by phosphatidylinositol-(4,5)-bisphosphate (PIP2), but G protein-gated Kir (KG) channels further require either G protein βγ subunits (Gβγ) or intracellular Na+ for their activation. To reveal the mechanism(s) underlying this regulation, we compared the crystal structures of the cytoplasmic domain of KG channel subunit Kir3.2 obtained in the presence and the absence of Na+. The Na+-free Kir3.2, but not the Na+-plus Kir3.2, possessed an ionic bond connecting the N terminus and the CD loop of the C terminus. Functional analyses revealed that the ionic bond between His-69 on the N terminus and Asp-228 on the CD loop, which are known to be critically involved in Gβγ- and Na+-dependent activation, lowered PIP2 sensitivity. The conservation of these residues within the KG channel family indicates that the ionic bond is a character that maintains the channels in a closed state by controlling the PIP2 sensitivity.

Collagen prolyl 4-hydroxylases (C-P4Hs) have a critical role in collagen synthesis, since 4-hydroxyproline residues are necessary for folding of the triple-helical molecules. Vertebrate C-P4Hs are alpha(2)beta(2) tetramers in which the beta subunit is identical to protein-disulfide isomerase (PDI). Three isoforms of the catalytic alpha subunit, PHY-1, PHY-2, and PHY-3, have been characterized from Caenorhabditis elegans, PHY-1 and PHY-2 being responsible for the hydroxylation of cuticle collagens, whereas PHY-3 is predicted to be involved in collagen synthesis in early embryos. We have characterized transcripts of two additional C. elegans alpha subunit-like genes, Y43F8B.4 and C14E2.4. Three transcripts were generated from Y43F8B.4, and a polypeptide encoded by one of them, named PHY-4.1, assembled into active (PHY-4.1)(2)/(PDI-2)(2) tetramers and PHY-4.1/PDI-2 dimers when coexpressed with C. elegans PDI-2 in insect cells. The C14E2.4 transcript was found to have a frameshift leading to the absence of codons for two residues critical for P4H catalytic activity. Thus, C. elegans has altogether four functional C-P4H alpha subunits, PHY-1, PHY-2, PHY-3, and PHY-4.1. The tetramers and dimers containing recombinant PHY-4.1 had a distinct substrate specificity from the other C-P4Hs in that they hydroxylated poly(l-proline) and certain other proline-rich peptides, including ones that are expressed in the pharynx, in addition to collagen-like peptides. These data and the observed restricted expression of the phy-4.1 transcript and PHY-4.1 polypeptide in the pharyngeal gland cells and the excretory duct suggest that in addition to collagens, PHY-4.1 may hydroxylate additional proline-rich proteins in vivo.

PDZ domains1 are modular protein interaction domains that play a role in protein targeting and protein complex assembly. Once termed Discs-large homology regions (DHRs) or GLGF repeats (after a conserved Gly-Leu-Gly-Phe sequence found within the domain), these domains of ∼90 amino acids are now primarily known by an acronym of the first three PDZ-containing proteins identified: the postsynaptic protein PSD-95/SAP90, theDrosophila septate junction protein Discs-large, and the tight junction protein ZO-1.

Telomere maintenance is essential for protecting chromosome ends. Aberrations in telomere length have been implicated in cancer and aging. Telomere elongation by human telomerase is inhibited in cis by the telomeric protein TRF1 and its associated proteins. However, the link between TRF1 and inhibition of telomerase elongation of telomeres remains elusive because TRF1 has no direct effect on telomerase activity. We have previously identified one Pin2/TRF1-interacting protein, PinX1, that has the unique property of directly binding and inhibiting telomerase catalytic activity (Zhou, X. Z., and Lu, K. P. (2001) Cell 107, 347-359). However, nothing is known about the role of the PinX1-TRF1 interaction in the regulation of telomere maintenance. By identifying functional domains and key amino acid residues in PinX1 and TRF1 responsible for the PinX1-TRF1 interaction, we show that the TRF homology domain of TRF1 interacts with a minimal 20-amino acid sequence of PinX1 via hydrophilic and hydrophobic interactions. Significantly, either disrupting this interaction by mutating the critical Leu-291 residue in PinX1 or knocking down endogenous TRF1 by RNAi abolishes the ability of PinX1 to localize to telomeres and to inhibit telomere elongation in cells even though neither has any effect on telomerase activity per se. Thus, the telomerase inhibitor PinX1 is recruited to telomeres by TRF1 and provides a critical link between TRF1 and telomerase inhibition to prevent telomere elongation and help maintain telomere homeostasis.