Pushing the limits of solubility prediction via quality-oriented data selection.

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.

Guidelines on genetic evaluation and management of Lynch syndrome: A consensus statement by the U.S. Multi-Society Task Force on Colorectal Cancer

Agkistrodon contortrix laticinctus myotoxin is a Lys(49)-phospholipase A(2) (EC 3.1.1.4) isolated from the venom of the serpent A. contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A. contortrix laticinctus myotoxin involves the side chains of Phe(121) and Phe(124) and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118-125. Comparison with other Lys(49)-phospholipase A(2) myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca(2+)-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase "active site" and the C-terminal "myotoxic site," justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.

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). 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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. 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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. 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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. 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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. 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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. 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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. 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Compelling evidence shows a strong correlation between accumulation of neurotoxic β-amyloid (Aβ) peptides and oxidative stress in the brains of patients afflicted with Alzheimer disease (AD). One hypothesis for this correlation involves the direct and harmful interaction of aggregated Aβ peptides with enzymes responsible for maintaining normal, cellular levels of reactive oxygen species (ROS). Identification of specific, destructive interactions of Aβ peptides with cellular anti-oxidant enzymes would represent an important step toward understanding the pathogenicity of Aβ peptides in AD. This report demonstrates that exposure of human neuroblastoma cells to cytotoxic preparations of aggregated Aβ peptides results in significant intracellular co-localization of Aβ with catalase, an anti-oxidant enzyme responsible for catalyzing the degradation of the ROS intermediate hydrogen peroxide (H(2)O(2)). These catalase-Aβ interactions deactivate catalase, resulting in increased cellular levels of H(2)O(2). Furthermore, small molecule inhibitors of catalase-amyloid interactions protect the hydrogen peroxide-degrading activity of catalase in Aβ-rich environments, leading to reduction of the co-localization of catalase and Aβ in cells, inhibition of Aβ-induced increases in cellular levels of H(2)O(2), and reduction of the toxicity of Aβ peptides. These studies, thus, provide evidence for the important role of intracellular catalase-amyloid interactions in Aβ-induced oxidative stress and propose a novel molecular strategy to inhibit such harmful interactions in AD.

Kingdom-Agnostic Metagenomics and the Importance of Complete Characterization of Enteric Microbial Communities

Many eukaryotic transcription factors are bimodal in their regulatory properties and can both repress and activate expression of their target genes. These divergent transcriptional properties are conferred through recruitment of auxiliary proteins, denoted coactivators and corepressors. Repression plays a particularly critical role in the functions of the nuclear receptors, a large family of ligand-regulated transcription factors involved in metazoan development, differentiation, reproduction, and homeostasis. The SMRT corepressor interacts directly with nuclear receptors and serves, in turn, as a platform for the assembly of a larger corepressor complex. We report here that SMRT is expressed in cells by alternative mRNA splicing to yield two distinct variants or isoforms. We designate these isoforms SMRTalpha and SMRTtau and demonstrate that these isoforms have significantly different affinities for different nuclear receptors. These isoforms are evolutionarily conserved and are expressed in a tissue-specific manner. Our results suggest that differential mRNA splicing serves to customize corepressor function in different cells, allowing the transcriptional properties of nuclear receptors to be adapted to different contexts.

Homolytic decomposition of PUFA 2The abbreviations used are: PUFA, polyunsaturated fatty acid; DDE, trans, trans-2,4-decadienal; EDE, 4,5-epoxy-(2E)-decenal; HNE, 4-hydroxy-(2E)-nonenal; HPNE, 4-hydroperoxy-(2E)-nonenal; MDA, malondialdehyde; ONE, 4-oxo-(2E)-nonenal; DODE, 9,12-dioxo-(10E)-dodecenoic acid; MS, mass spectrometry; ROS, reactive oxygen species; COX, cyclooxygenase; LOX, lipoxygenase; GST, glutathione S-transferase; LC-MRM-MS, liquid chromatography-multiple reaction monitoring-mass spectrometry; LA, linoleic acid; HPODE, hydroperoxy-(9Z),(11E)-octadecadienoic acid; AA, arachidonic acid; HPETE, hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; APCI, atmospheric pressure chemical ionization; MS/MS, tandem MS; ESI, electrospray ionization; dGuo, 2′-deoxyguanosine; ∈DNA, etheno-DNA; H∈, heptanone-etheno; dAdo, 2′-deoxyadenosine; dCyd, 2′-deoxycytidine; C∈, carboxynonanone-etheno; GC, gas chromatography. 2The abbreviations used are: PUFA, polyunsaturated fatty acid; DDE, trans, trans-2,4-decadienal; EDE, 4,5-epoxy-(2E)-decenal; HNE, 4-hydroxy-(2E)-nonenal; HPNE, 4-hydroperoxy-(2E)-nonenal; MDA, malondialdehyde; ONE, 4-oxo-(2E)-nonenal; DODE, 9,12-dioxo-(10E)-dodecenoic acid; MS, mass spectrometry; ROS, reactive oxygen species; COX, cyclooxygenase; LOX, lipoxygenase; GST, glutathione S-transferase; LC-MRM-MS, liquid chromatography-multiple reaction monitoring-mass spectrometry; LA, linoleic acid; HPODE, hydroperoxy-(9Z),(11E)-octadecadienoic acid; AA, arachidonic acid; HPETE, hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; APCI, atmospheric pressure chemical ionization; MS/MS, tandem MS; ESI, electrospray ionization; dGuo, 2′-deoxyguanosine; ∈DNA, etheno-DNA; H∈, heptanone-etheno; dAdo, 2′-deoxyadenosine; dCyd, 2′-deoxycytidine; C∈, carboxynonanone-etheno; GC, gas chromatography.-derived lipid hydroperoxides results in formation of the α,β-unsaturated aldehydic bifunctional electrophiles DDE, EDE, HNE, HPNE, MDA (shown as β-hydroxyacrolein), ONE, DODE, and 5,8-dioxo-(10E)-octenoic acid (see Fig. 1) (1Blair I.A. Exp. Gerontol. 2001; 36: 1473-1481Crossref PubMed Scopus (169) Google Scholar, 2Lee S.H. Blair I.A. Trends Cardiovasc. Med. 2001; 11: 148-155Crossref PubMed Scopus (121) Google Scholar, 3Blair, I. A. (2005) in Encyclopedia of Mass Spectrometry (Caprioli, R. M., and Gross, M. L., eds) Vol. 3, pp. 283–307, Elsevier Ltd., OxfordGoogle Scholar). Intracellular formation of the bifunctional electrophiles can then result in the formation of GSH, protein, and DNA adducts (1Blair I.A. Exp. Gerontol. 2001; 36: 1473-1481Crossref PubMed Scopus (169) Google Scholar, 2Lee S.H. Blair I.A. Trends Cardiovasc. Med. 2001; 11: 148-155Crossref PubMed Scopus (121) Google Scholar, 3Blair, I. A. (2005) in Encyclopedia of Mass Spectrometry (Caprioli, R. M., and Gross, M. L., eds) Vol. 3, pp. 283–307, Elsevier Ltd., OxfordGoogle Scholar, 5Marnett L.J. Riggins J.N. West J.D. J. Clin. Investig. 2003; 111: 583-593Crossref PubMed Scopus (393) Google Scholar). The analysis of lipid hydroperoxide-derived DNA adducts can facilitate molecular epidemiology studies by providing insight into the amount of a genotoxin that has reached the DNA of the tissue under study (6Kensler T.W. Qian G.S. Chen J.G. Groopman J.D. Nat. Rev. Cancer. 2003; 3: 321-329Crossref PubMed Scopus (179) Google Scholar, 7Sharma R.A. Farmer P.B. Clin. Cancer Res. 2004; 10: 4901-4912Crossref PubMed Scopus (86) Google Scholar). DNA repair enzymes, such as those involved in base excision repair, are able to excise the DNA adducts so that they can potentially be excreted in the urine (7Sharma R.A. Farmer P.B. Clin. Cancer Res. 2004; 10: 4901-4912Crossref PubMed Scopus (86) Google Scholar). This suggests that non-invasive MS-based techniques could be used to monitor urinary DNA adducts arising from lipid hydroperoxide-mediated DNA damage. Unfortunately, to date, the analysis of urinary DNA adducts of lipid hydroperoxide-derived bifunctional electrophiles has not been particularly successful. ONE-derived DNA adducts, which can arise only from lipid peroxidation, have now been characterized in the tissues of mouse models (8Williams M.V. Lee S.H. Pollack M. Blair I.A. J. Biol. Chem. 2006; 281: 10127-10133Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Therefore, it might eventually be possible to detect these specific lipid hydroperoxide-derived DNA adducts after they have been excised from the DNA and excreted in the urine (see Fig. 2).FIGURE 2Formation of ∈DNA and H∈DNA adducts through homolytic decomposition of lipid hydroperoxides. BER, base excision repair.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Oxidative stress represents one of the major cellular responses to toxic insults, carcinogenic chemicals, and environmental agents such as viruses and mycotoxins (9Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5382) Google Scholar). During oxidative stress, there is a decrease in the amount of intracellular GSH and an increase in GSSG (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar) with a concomitant increase in ROS and reactive nitrogen species (9Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5382) Google Scholar) and an increase in the activity of COX-2 (11Lee S.H. Williams M.V. Dubois R.N. Blair I.A. J. Biol. Chem. 2005; 280: 28337-28346Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and LOXs (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar), involved in lipid peroxidation (1Blair I.A. Exp. Gerontol. 2001; 36: 1473-1481Crossref PubMed Scopus (169) Google Scholar). ROS are generated constantly in vivo by a variety of endogenous processes, including normal mitochondrial aerobic respiration, phagocytosis of bacteria- or virus-containing cells, and peroxisome-mediated degradation of fatty acids (9Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5382) Google Scholar). Increased ROS production occurs in inflammation; during radiation; or during metabolism of hormones, drugs, and environmental toxins. This can overwhelm endogenous protective mechanisms and increase ROS-mediated damage to GSH, proteins, DNA, and lipids. The resulting ROS-derived lipid hydroperoxides can then undergo homolytic decomposition to various bifunctional electrophiles that form DNA adducts with different lipid modifications (1Blair I.A. Exp. Gerontol. 2001; 36: 1473-1481Crossref PubMed Scopus (169) Google Scholar, 2Lee S.H. Blair I.A. Trends Cardiovasc. Med. 2001; 11: 148-155Crossref PubMed Scopus (121) Google Scholar, 3Blair, I. A. (2005) in Encyclopedia of Mass Spectrometry (Caprioli, R. M., and Gross, M. L., eds) Vol. 3, pp. 283–307, Elsevier Ltd., OxfordGoogle Scholar;5Marnett L.J. Riggins J.N. West J.D. J. Clin. Investig. 2003; 111: 583-593Crossref PubMed Scopus (393) Google Scholar). Alternatively, the bifunctional electrophiles can arise from homolytic decomposition of COX- and LOX-derived lipid hydroperoxides (11Lee S.H. Williams M.V. Dubois R.N. Blair I.A. J. Biol. Chem. 2005; 280: 28337-28346Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 12Lee S.H. Rangiah K. Williams M.V. Wehr A.Y. Dubois R.N. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1665-1675Crossref PubMed Scopus (33) Google Scholar, 13Jian W. Lee S.H. Arora J.S. Silva Elipe M.V. Blair I.A. Chem. Res. Toxicol. 2005; 18: 599-610Crossref PubMed Scopus (24) Google Scholar). Intracellular lipid hydroperoxides are detoxified by GSH peroxidase-mediated reduction to lipid alcohols, with GSH providing the reducing equivalents (4Blair I.A. Curr. Drug Metab. 2006; 7: 853-872Crossref PubMed Scopus (101) Google Scholar). Peroxidized phospholipids are detoxified by peroxiredoxin 6 to the corresponding phospholipid alcohols in a GSH-dependent manner (14Manevich Y. Fisher A.B. Free Radic. Biol. Med. 2005; 38: 1422-1432Crossref PubMed Scopus (322) Google Scholar). GSH also readily forms adducts with lipid hydroperoxide-derived bifunctional electrophiles, providing an additional protective pathway (4Blair I.A. Curr. Drug Metab. 2006; 7: 853-872Crossref PubMed Scopus (101) Google Scholar, 12Lee S.H. Rangiah K. Williams M.V. Wehr A.Y. Dubois R.N. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1665-1675Crossref PubMed Scopus (33) Google Scholar, 15Jian W. Lee S.H. Mesaros C. Oe T. Silva Elipe M.V. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1008-1018Crossref PubMed Scopus (44) Google Scholar). Formation of the GSH adducts is generally facilitated by GSTs, and the resulting adducts are exported from cells by ATP-binding and non-ATP-binding cassette transporters (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar). Total GSH and GSSG concentrations are modulated in cells by low extracellular concentrations of lipid hydroperoxide-derived bifunctional electrophiles such as HNE. This can increase GSH concentrations by up-regulation of γ-glutamylcysteine ligase, a key enzyme involved in GSH biosynthesis. In contrast, high concentrations of bifunctional electrophiles deplete GSH (4Blair I.A. Curr. Drug Metab. 2006; 7: 853-872Crossref PubMed Scopus (101) Google Scholar) through a direct GST-mediated reaction as well as by inhibition of enzymes involved in GSH biosynthesis. GSH/GSSG homeostasis plays an important role in maintaining cellular redox status, and changes of the half-cell reduction potential of the 2GSH/GSSG couple correlate with the biological status of the cell (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar). It is difficult to accurately quantify intracellular GSH and GSSG concentrations because of the ease with which free sulfhydryl groups are oxidized during the lysis of cells. To overcome this problem, a stable isotope dilution LC-MRM-MS method was developed using 4-fluoro-7-sulfamoylbenzofurazan derivatization of GSH (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar). Increased lipid peroxidation resulting from transfection of the human 15-LOX-1 gene into a mouse macrophage cell line was found to increase intracellular GSH biosynthesis. This resulted in a lower resting cellular redox potential and provided protection against exogenous lipid hydroperoxide-derived bifunctional electrophiles. Therefore, increased intracellular lipid peroxidation can induce a protective adaptive response (10Zhu P. Oe T. Blair I.A. Rapid Commun. Mass Spectrom. 2008; 22: 432-440Crossref PubMed Scopus (62) Google Scholar). ROS-mediated oxidation of esterified LA-containing lipids and free LA results in the formation of four HPODE isomers as enantiomeric pairs. They are subsequently reduced to the corresponding hydroxyoctadecadienoic acids. Similarly, ROS-mediated oxidation of AA-containing lipids and free AA results in the formation of a complex mixture of HPETEs that are reduced to racemic hydroxyeicosatetraenoic acids (HETEs), including (15S)- and (15R)-HETE (11Lee S.H. Williams M.V. Dubois R.N. Blair I.A. J. Biol. Chem. 2005; 280: 28337-28346Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 12Lee S.H. Rangiah K. Williams M.V. Wehr A.Y. Dubois R.N. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1665-1675Crossref PubMed Scopus (33) Google Scholar, 16Brash A.R. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1137) Google Scholar). Lipid hydroperoxides can also be formed by the action of LOXs (9Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5382) Google Scholar) and COXs (17Porter N.A. Caldwell S.E. Mills K.A. Lipids. 1995; 30: 277-290Crossref PubMed Scopus (996) Google Scholar) on PUFAs. High sensitivity, stable isotope dilution, normal phase, chiral LC-electron capture APCI-MS/MS methodology (18Singh G. Gutierrez A. Xu K. Blair I.A. Anal. Chem. 2000; 72: 3007-3013Crossref PubMed Scopus (180) Google Scholar) makes it possible to separate and quantify all of the enantiomers and regioisomers of the PUFA alcohols derived from LA and AA (19Lee S.H. Williams M.V. Dubois R.N. Blair I.A. Rapid Commun. Mass Spectrom. 2003; 17: 2168-2176Crossref PubMed Scopus (156) Google Scholar, 20Lee S.H. Blair I.A. Methods Enzymol. 2007; 433: 159-174Crossref PubMed Scopus (34) Google Scholar). Eicosanoids and isoprostanes can be quantified at the same time. This method revealed that endogenous (15S)-HPETE (as measured by (15S)-HETE release) was formed in amounts that were similar to prostaglandin E2 in unstimulated rat intestinal epithelial cells stably expressing COX-2 (RIES cells) (21Lee S.H. Oe T. Blair I.A. Science. 2001; 292: 2083-2086Crossref PubMed Scopus (409) Google Scholar). When exogenous AA was added to the RIES cells, the profile of eicosanoids was found to be dependent upon the time of incubation as well as the AA concentration (12Lee S.H. Rangiah K. Williams M.V. Wehr A.Y. Dubois R.N. Blair I.A. Chem. Res. Toxicol. 2007; 20: 1665-1675Crossref PubMed Scopus (33) Google Scholar). Interestingly, COX-2-mediated biosynthesis of prostaglandin E2 and (15S)-HETE was found to increase linearly with the addition of increasing amounts of AA to the cells. From the intercepts on the y axes of the regression lines, it was possible to calculate the amount of each eicosanoid formed from endogenous AA in resting cells. The calculated values were very close to the experimentally determined values. It was also possible to estimate how much endogenous AA (0.56 μm) had been mobilized by phospholipases in the unstimulated RIES cells. The ability to determine how much of an eicosanoid metabolite is formed in the absence of exogenous AA stimulation is particularly useful when endogenous production of the metabolite is below the detection limit of the assay that is being utilized. Studies on DNA adducts resulting from lipid hydroperoxide-derived bifunctional electrophiles have relied heavily on the use of reversed-phase LC-ESI-MS methodology (3Blair, I. A. (2005) in Encyclopedia of Mass Spectrometry (Caprioli, R. M., and Gross, M. L., eds) Vol. 3, pp. 283–307, Elsevier Ltd., OxfordGoogle Scholar). However, many of the bifunctional electrophiles themselves are poorly ionized under ESI conditions. Aldehydic bifunctional electrophiles can be converted to oxime derivatives to improve ESI efficiency. However, this results in syn- and anti-oxime isomers together with extremely complex LC chromatograms (22Lee S.H. Blair I.A. Chem. Res. Toxicol. 2000; 13: 698-702Crossref PubMed Scopus (230) Google Scholar). Therefore, normal phase LC-APCI-MS was employed to quantify the lipid hydroperoxide-derived bifunctional electrophiles (21Lee S.H. Oe T. Blair I.A. Science. 2001; 292: 2083-2086Crossref PubMed Scopus (409) Google Scholar, 23Williams M.V. Lee S.H. Blair I.A. Rapid Commun. Mass Spectrom. 2005; 19: 849-858Crossref PubMed Scopus (30) Google Scholar, 24Lee S.H. Oe T. Arora J.S. Blair I.A. J. Mass Spectrom. 2005; 40: 661-668Crossref PubMed Scopus (26) Google Scholar). HNE was more readily ionized than the other bifunctional electrophiles, so its APCI response was almost an order of magnitude more intense compared with the other bifunctional electrophiles. HPNE, trans-EDE, and ONE were the major products identified from the Fe(II)-mediated homolytic decomposition of (13S)-HPODE (24Lee S.H. Oe T. Arora J.S. Blair I.A. J. Mass Spectrom. 2005; 40: 661-668Crossref PubMed Scopus (26) Google Scholar). The possible formation of ONE and HNE from HPNE was confirmed by treating HPNE with increasing concentrations of Fe(II). Vitamin C was more than twice as efficient at initiating the decomposition of 13-HPODE to bifunctional electrophiles compared with transition metal ions. It also induced the conversion of HPNE to ONE and HNE. Normal phase LC-APCI-MS methodology also showed that ONE was a major product from both Fe(II)-and vitamin C-mediated homolytic decomposition of (15S)-HPETE (23Williams M.V. Lee S.H. Blair I.A. Rapid Commun. Mass Spectrom. 2005; 19: 849-858Crossref PubMed Scopus (30) Google Scholar). A number of DNA adducts arising from lipid hydroperoxide-derived bifunctional electrophiles (Fig. 1) have been identified by LC-MS (3Blair, I. A. (2005) in Encyclopedia of Mass Spectrometry (Caprioli, R. M., and Gross, M. L., eds) Vol. 3, pp. 283–307, Elsevier Ltd., OxfordGoogle Scholar). The reaction between (13S)-HPODE and dGuo was shown to result in the formation of a single ∈DNA adduct, H∈dGuo (Fig. 2) (25Rindgen D. Nakajima M. Wehrli S. Xu K. Blair I.A. Chem. Res. Toxicol. 1999; 12: 1195-1204Crossref PubMed Scopus (136) Google Scholar). The initially formed ethano adducts arose from highly regioselective nucleophilic addition of N-2 of the dGuo to the C-1 aldehyde of ONE, followed by reaction of N-1 at C-2 of the resulting α,β-unsaturated ketone. H∈dAdo DNA adducts were also detected in the reaction between dAdo and 13-HPODE-derived ONE (Fig. 2) (26Rindgen D. Lee S.H. Nakajima M. Blair I.A. Chem. Res. Toxicol. 2000; 13: 846-852Crossref PubMed Scopus (70) Google Scholar, 27Lee S.H. Rindgen D. Bible Jr., R.H. Hajdu E. Blair I.A. Chem. Res. Toxicol. 2000; 13: 565-574Crossref PubMed Scopus (103) Google Scholar). Furthermore, the reaction between ONE and dCyd resulted in formation of H∈dCyd (Fig. 2) as a major product (28Pollack M. Oe T. Lee S.H. Silva Elipe M.V. Arison B.H. Blair I.A. Chem. Res. Toxicol. 2003; 16: 893-900Crossref PubMed Scopus (79) Google Scholar). The endogenous formation of unsubstituted ∈DNA adducts was suggested originally to arise from lipid hydroperoxide-mediated epoxidation of HNE to 2,3-epoxy-4-hydroxynonanal and subsequent reaction of this bifunctional electrophile with DNA bases (29Sodum R.S. Chung F.L. Cancer Res. 1991; 51: 137-143PubMed Google Scholar). Subsequently, it was suggested that this reaction was unlikely to occur in cells because of the relatively high pKa of the lipid hydroperoxides (30Douki T. Odin F. Caillat S. Favier A. Cadet J. Free Radic. Biol. Med. 2004; 37: 62-70Crossref PubMed Scopus (78) Google Scholar). Unsubstituted etheno adducts were also formed from the reaction of lipid hydroperoxide-derived DDE with dAdo or dGuo in the presence of peroxides (31Loureiro A.P. Di M.P. Gomes O.F. Medeiros M.H. Chem. Res. Toxicol. 2000; 13: 601-609Crossref PubMed Scopus (74) Google Scholar). This pathway of endogenous ∈DNA adduct formation would require intracellular epoxidation of DDE to occur more rapidly than its detoxification by GSTs and aldoketoreductases (32Jian W. Arora J.S. Oe T. Shuvaev V.V. Blair I.A. Free Radic. Biol. Med. 2005; 39: 1162-1176Crossref PubMed Scopus (46) Google Scholar). Unsubstituted etheno adducts were formed when DNA bases were treated with EDE and HPNE (Fig. 2) (33Lee S.H. Oe T. Blair I.A. Chem. Res. Toxicol. 2002; 15: 300-304Crossref PubMed Scopus (74) Google Scholar, 34Lee S.H. Arora J.A. Oe T. Blair I.A. Chem. Res. Toxicol. 2005; 18: 780-786Crossref PubMed Scopus (61) Google Scholar). However, HPNE was much more reactive to DNA bases compared with EDE. Based on this increased reactivity, it appears that HPNE is most likely the major lipid hydroperoxide-derived bifunctional electrophile responsible for the formation of unsubstituted ∈DNA adducts (Fig. 2) (34Lee S.H. Arora J.A. Oe T. Blair I.A. Chem. Res. Toxicol. 2005; 18: 780-786Crossref PubMed Scopus (61) Google Scholar). Previous studies have shown that environmental chemicals such as vinyl chloride, vinyl fluoride, and chloroethylene oxide form the same unsubstituted ∈DNA adducts (Fig. 2) (35Barbin A. Friesen M. O'Neill I.K. Croisy A. Bartsch H. Chem. Biol. Interact. 1986; 59: 43-54Crossref PubMed Scopus (13) Google Scholar, 36Swenberg, J. A., Bogdanffy, M. S., Ham, A., Holt, S., Kim, A., Morinello, E. J., Ranasinghe, A., Scheller, N., and Upton, P. B. (1999) in Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis (Singer, B., and Bartsch, H., eds) Volume 150, pp. 29–43, IARC Science Publications, Scholar). Therefore, unsubstituted ∈DNA adducts not arise from lipid It has been that a of 13-HPODE would to the formation of a of K. Lipid Res. 2003; PubMed Scopus Google Scholar). is to rapidly form HPNE, which in is converted to ONE and HNE (23Williams M.V. Lee S.H. Blair I.A. Rapid Commun. 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West J.D. J. Clin. Investig. 2003; 111: 583-593Crossref PubMed Scopus (393) Google Scholar, M. J.D. Blair I.A. L.J. Science. PubMed Scopus Google Scholar, M. L.J. Blair I.A. Biol. Mass Spectrom. PubMed Scopus (65) Google Scholar, M. L.J. Blair I.A. J. Mass Spectrom. 1995; 30: Scopus Google Scholar, Blair I.A. L.J. 17: PubMed Scopus Google Scholar, M. Blair I.A. L.J. Chem. Res. Toxicol. 10: PubMed Scopus Google Scholar, M. L.J. J. Mass Spectrom. 2004; 39: PubMed Scopus Google Scholar, L.J. 2000; PubMed Scopus Google Scholar). However, the adduct it forms with dGuo in vivo from base that are generated from ROS-mediated damage to the of DNA K. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). can also be formed from MDA during biosynthesis. is not a specific of lipid hydroperoxide-mediated DNA adduct it be a useful of endogenous DNA damage resulting from oxidative has been detected in the DNA of M. J.D. Blair I.A. L.J. Science. 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The mammalian oligosaccharyltransferase (OST) is an oligomeric complex composed of three membrane proteins of the endoplasmic reticulum: ribophorin I (RI), ribophorin II (RII), and OST48. In addition, sequence homology between the Ost2 subunit of the yeast OST complex and Dad1 (defender against apoptotic death) suggests that Dad1 may represent a fourth subunit of the mammalian OST complex. In attempts to elucidate the structural organization of this complex, we have studied the interactions among its subunits. Using the yeast two-hybrid system, we have shown that the luminal domains of RI and RII (RIL and RIIL, respectively) interacted with the luminal domain of OST48 (OST48L), but no direct interaction was observed between RIL and RIIL. These results were confirmed by biochemical assays. Deletion analyses using the yeast two-hybrid system showed that subdomain of RIL or RIIL adjacent to the respective transmembrane domains interacted with OST48L. Of the three equal length subdomains of OST48L, the one at the N terminus and the one next to the transmembrane domain interacted with RIL. None of these three subdomains of OST48L interacted with RIIL. The yeast two-hybrid assay also revealed affinity between the cytoplasmically located N-terminal region of Dad1 and the short cytoplasmic tail of OST48, thus placing Dad1 firmly into the OST complex. In addition, we found a homotypic interaction between the cytoplasmic domains of RI, which may play a role in the formation of the oligomeric array formed by components of the translocation machinery.

Activated lymphocytes synthesize and secrete substantial amounts of the beta-chemokines macrophage inflammatory protein (MIP)-1 alpha/CCL3 and MIP-1 beta/CCL4, both of which inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). The native form of MIP-1 beta secreted by activated human peripheral blood lymphocytes (MIP-1 beta(3-69)) lacks the two NH(2)-terminal amino acids of the full-length protein. This truncated form of MIP-1 beta has now been affinity-purified from the culture supernatant of such cells, and its structure has been confirmed by mass spectrometry. Functional studies of the purified protein revealed that MIP-1 beta(3-69) retains the abilities to induce down-modulation of surface expression of the chemokine receptor CCR5 and to inhibit the CCR5-mediated entry of HIV-1 in T cells. Characterization of the chemokine receptor specificity of MIP-1 beta(3-69) showed that the truncated protein not only shares the ability of intact MIP-1 beta to induce Ca(2+) signaling through CCR5, but unlike the full-length protein, it also triggers a Ca(2+) response via CCR1 and CCR2b. These results demonstrate that NH(2)-terminally truncated MIP-1 beta functions as a chemokine agonist with expanded receptor reactivity, which may represent an important mechanism for regulation of immune cell recruitment during inflammatory and antiviral responses.

Nucleosome Transactions on the Promoters of the YeastGAL and PHO Genes

Enzyme replacement therapy (ERT) has been shown to be effective at reducing the accumulation of undegraded substrates in lysosomal storage diseases. Most ERT studies have been performed with recombinant proteins that are mixtures of phosphorylated and non-phosphorylated enzyme. Because different cell types use different receptors to take up phosphorylated or non-phosphorylated enzyme, it is difficult to determine which form of enzyme contributed to the clinical response. Here we compare the uptake, distribution, and efficacy of highly phosphorylated and non-phosphorylated beta-glucuronidase (GUSB) in the MPS VII mouse. Highly phosphorylated murine GUSB was efficiently taken up by a wide range of tissues. In contrast, non-phosphorylated murine GUSB was taken up primarily by tissues of the reticuloendothelial (RE) system. Although the tissue distribution was different, the half-lives of both enzymes in any particular tissue were similar. Both preparations of enzyme were capable of preventing the accumulation of lysosomal storage in cell types they targeted. An important difference in clinical efficacy emerged in that phosphorylated GUSB was more efficient than non-phosphorylated enzyme at preventing the hearing loss associated with this disease. These data suggest that both forms of enzyme contribute to the clinical responses of ERT in MPS VII mice but that enzyme preparations containing phosphorylated GUSB are more broadly effective than non-phosphorylated enzyme.

Dynamic modulation of ion channel expression, localization, and/or function drives plasticity in intrinsic neuronal excitability. Voltage-gated Kv2.1 potassium channels are constitutively maintained in a highly phosphorylated state in neurons. Increased neuronal activity triggers rapid calcineurin-dependent dephosphorylation, loss of channel clustering, and hyperpolarizing shifts in voltage-dependent activation that homeostatically suppress neuronal excitability. These changes are reversible, such that rephosphorylation occurs after removal of excitatory stimuli. Here, we show that cyclin-dependent kinase 5 (CDK5), a Pro-directed Ser/Thr protein kinase, directly phosphorylates Kv2.1, and determines the constitutive level of Kv2.1 phosphorylation, the rapid increase in Kv2.1 phosphorylation upon acute blockade of neuronal activity, and the recovery of Kv2.1 phosphorylation after stimulus-induced dephosphorylation. We also demonstrate that although the phosphorylation state of Kv2.1 is also shaped by the activity of the PP1 protein phosphatase, the regulation of Kv2.1 phosphorylation by CDK5 is not mediated through the previously described regulation of PP1 activity by CDK5. Together, these studies support a novel role for CDK5 in regulating Kv2.1 channels through direct phosphorylation.

Born Large for Gestational Age: Bigger Is Not Always Better

The Commensal Microbiota and Enteropathogens in the Pathogenesis of Inflammatory Bowel Diseases