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The design of potent indoleamine 2,3-dioxygenase 1 (IDOl) enzyme inhibitors targeting immunopathic neuroinflammation has emerged as an area of interest for the treatment of Alzheimer's disease (AD); additionally, recent findings on the clinical benefits of antibodies preventing [beta]-amyloid (Aft) aggregation have renewed efforts to discover small molecule anti-aggregants targeting proteopathic protein misfolding. Exploiting an endogenous tryptophan-like scaffold, we describe the design and synthesis of small-molecule inhibitors of both immunopathic and proteopathic processes, thus presenting the possibility of single therapeutics acting simultaneously on multiple AD pathogeneses. Specifically, investigations on compounds that inhibit both IDOl (in human recombinant enzyme, transfected HEK293 cells, and interferon-[gamma] stimulated human microglia assays) and Aft aggregation (in thioflavin-T and biotinylated-A[beta] oligomeric assays) are presented. Five compounds have been identified with high potency against both targets, identifying (E)-3-styryl indoles as useful tool compounds for developing Alzheimer's therapeutics. Brain penetration of these compounds via passive diffusion or active transport was predicted using Blood-Brain Barrier Score and Brain Exposure Efficiency Score calculations, respectively; the effects of efflux (pgp, BCRP), and influx (OCT1, OCT2) transporters were similarly predicted. Structure-activity relationships were rationalised with molecular docking and molecular dynamics simulations, which also provide insights for future lead compound optimisation. Key words: Alzheimer's disease, tryptophan, IDO-1, amyloid-[beta], neuroinflammation, neuroimmunology La conception de puissants inhibiteurs de l'indoleamine 2,3 dioxygenase 1 (IDOl) ciblant la neuro-inflammation immunopathique constitue desormais un nouveau champ d'interet pour le traitement de la maladie d'Alzheimer (alzheimer). En outre, des donnees recentes sur les bienfaits cliniques des anticorps empechant l'agregation de la beta-amyloide (Aft) ont ravive les efforts visant a decouvrir de petites molecules antiagregantes qui ciblent le mauvais repliement proteopathique des proteines. En exploitant une structure endogene de type tryptophane, nous avons concu et synthetise des inhibiteurs a petite molecule qui ciblent a la fois les processus immunopathiques et proteopathiques, ouvrant ainsi la voie a des monotherapies agissant simultanement sur plusieurs processus pathogenes de l'alzheimer. En particulier, nous presentons des experiences portant sur des composes qui inhibent a la fois IDOl (dans des essais avec des enzymes recombinantes humaines, des cellules HEK293 transferees et des microglies humaines stimulees par 1'interferon [gamma]) et l'agregation de l'A[beta] (dans des essais de liaison avec la thioflavine-T et d'inhibition de l'oligomerisation de l'A[beta] biotinylee). Nous avons identifie cinq composes presentant une forte affinite pour les deux cibles, ce qui nous a permis de definir les (E) 3 styrylindoles comme des composes pouvant servir d'outils pour le developpement d'agents therapeutiques contre l'alzheimer. La penetration cerebrale de ces composes par diffusion passive ou transport actif a ete estimee a l'aide de calculs de l'indice penetration de la barriere hematoencephalique (<< Score BBB >>) et de l'indice d'efficacite de l'exposition cerebrale (<< Score BEE >>), respectivement. Les effets des transporteurs d'efflux (P gp, BCRP) et d'influx (OCT1, OCT2) ont ete egalement ete estimes par calculs. Nous avons rationalise les relations structure-activite a l'aide de simulations d'amarrage moleculaire et de dynamique moleculaire, qui fournissent egalement des informations pour roptimisation d'eventuels composes tetes de serie. [Traduit par la Redaction] Mots-cles : maladie d'Alzheimer, tryptophane, IDOl, beta-amyloide, neuro-inflammation, neuro-immunologie, Introduction The neuropathology of Alzheimer's disease (AD) involves the brain's innate and adaptive immune systems, resulting in a complex inflammatory state that involves not only aggregation of [beta]-amyloid (Aft) and [...]

Context.--Amyloidosis is an uncommon but important entity. A protein-based classification of amyloidosis defines the underlying disease process, directing clinical management and providing prognostic information. However, in routine surgical pathology there often is no attempt to classify amyloid other than staining to determine light chain-associated amyloidosis. Systemic and localized amyloidosis vary with respect to frequency of organ involvement by different amyloid types, and most amyloid proteins have commercial antibodies available for identification. Objective.--To provide a guide for the likelihood of amyloid type by organ system. Data Sources.--Literature review based on PubMed searches containing the word amyloid, specifically addressing the prevalence and significance of amyloid proteins in each organ system other than the brain, and the authors' practice experience. Conclusions.--In patients with amyloidosis, determination of the responsible protein is critical for appropriate patient care. In large subspecialty practices and reference laboratories with experience in using and analyzing relevant immunohistochemistry, most amyloid proteins can be identified with an organ-specific algorithm. Referring to an organ-based algorithm may be helpful in providing clinicians with a more specific differential diagnosis regarding amyloid type to help guide clinical evaluation and treatment. When the protein cannot be characterized, mass spectrometry can be performed to definitively classify the amyloid type. (Arch Pathol Lab Med. 2020;144:379-387; doi: 10.5858/ arpa.2018-0509-RA), Amyloidosis is defined as tissue deposition of protein fibrils that form insoluble [beta]-pleated sheets. It has a reported prevalence of 20 per million with an incidence of 10 to 13 [...]

Ovarian cancer and triple-negative breast cancer are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins called 'don't eat me' signals--including CD47.sup.1, programmed cell death ligand 1 (PD-L1).sup.2 and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M).sup.3. Monoclonal antibodies that antagonize the interaction of 'don't eat me' signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers.sup.4,5. However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown 'don't eat me' signals. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a promising target for cancer immunotherapy. We demonstrate a role for tumour-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which is expressed by tumour-associated macrophages. We find that many tumours overexpress CD24 and that tumour-associated macrophages express high levels of Siglec-10. Genetic ablation of either CD24 or Siglec-10, as well as blockade of the CD24-Siglec-10 interaction using monoclonal antibodies, robustly augment the phagocytosis of all CD24-expressing human tumours that we tested. Genetic ablation and therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumour growth in vivo and an increase in survival time. These data reveal CD24 as a highly expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24 blockade in cancer immunotherapy. CD24 interacts with the tumour-associated-macrophage receptor Siglec-10 to inhibit the macrophage-mediated clearance of cancer cells, revealing a new 'don't eat me' signal as a potential target for cancer immunotherapy., Author(s): Amira A. Barkal [sup.1] [sup.2] [sup.3] [sup.4] , Rachel E. Brewer [sup.1] [sup.2] [sup.3] , Maxim Markovic [sup.1] [sup.2] [sup.3] , Mark Kowarsky [sup.5] , Sammy A. Barkal [sup.1] [...]

The genus Ebolavirns comprises 5 virus species: Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), Bundibugyo ebolavirus (BDBV), Tai' Forest ebolavirus (TAFV), and Reston ebolavirus (RESTV). The genus Marburgvirus comprises 1 species, [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2009.02.041 Byline: C. Mark Fletcher (1), Michael J. Coyne (1), Otto F. Villa (2), Maria Chatzidaki-Livanis (1), Laurie E. Comstock (1) Keywords: MICROBIO Abstract: The Bacteroides are a numerically dominant genus of the human intestinal microbiota. These organisms harbor a rare bacterial pathway for incorporation of exogenous fucose into capsular polysaccharides and glycoproteins. The infrequency of glycoprotein synthesis by bacteria prompted a more detailed analysis of this process. Here, we demonstrate that Bacteroides fragilis has a general O-glycosylation system. The proteins targeted for glycosylation include those predicted to be involved in protein folding, protein-protein interactions, peptide degradation as well as surface lipoproteins. Protein glycosylation is central to the physiology of B. fragilis and is necessary for the organism to competitively colonize the mammalian intestine. We provide evidence that general O-glycosylation systems are conserved among intestinal Bacteroides species and likely contribute to the predominance of Bacteroides in the human intestine. Author Affiliation: (1) Channing Laboratory, Brigham & Women's Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA (2) Pulmonary Critical Care and Sleep Division, Department of Medicine, Mount Sinai School of Medicine, 1468 Madison Avenue, New York, NY 10029, USA Article History: Received 8 October 2008; Revised 31 December 2008; Accepted 19 February 2009 Article Note: (miscellaneous) Published: April 16, 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.01.003 Byline: Liwei Huang (a), Yongbing Pu (a), Wen Yang Hu (a), Lynn Birch (a), Douglas Luccio-Camelo (a), Terry Yamaguchi (b), Gail S. Prins (a) Keywords: Prostate; Prostate gland; Prostate development; Wnt5a; Estradiol Abstract: The Wnt genes encode a large family of secreted glycoproteins that play important roles in controlling tissue patterning, cell fate and proliferation during development. Currently, little is known regarding the role(s) of Wnt genes during prostate gland development. The present study examines the role of the noncanonical Wnt5a during prostate gland development in rat and murine models. In the rat prostate, Wnt5a mRNA is expressed by distal mesenchyme during the budding stage and localizes to periductal mesenchymal cells with an increasing proximal-to-distal gradient during branching morphogenesis. Wnt5a protein is secreted and localizes to periductal stroma, extracellular matrix and epithelial cells in the distal ducts. While Wnt5a expression is high during active morphogenesis in all prostate lobes, ventral prostate (VP) expression declines rapidly following morphogenesis while dorsal (DP) and lateral lobe (LP) expression remains high into adulthood. Steroids modulate prostatic Wnt5a expression during early development with testosterone suppressing Wnt5a and neonatal estrogen increasing expression. In vivo and ex vivo analyses of developing mouse and rat prostates were used to assess the functional roles of Wnt5a. Wnt5a.sup.-/- murine prostates rescued by organ culture exhibit disturbances in bud position and directed outgrowth leading to large bulbous sacs in place of elongating ducts. In contrast, epithelial cell proliferation, ductal elongation and branchpoint formation are suppressed in newborn rat prostates cultured with exogenous Wnt5a protein. While renal grafts of Wnt5a.sup.-/- murine prostates revealed that Wnt5a is not essential for cyto- and functional differentiation, a role in luminal cell polarity and lumenization of the ducts was indicated. Wnt5a suppresses prostatic Shh expression while Shh stimulates Wnt5a expression in a lobe-specific manner during early development indicating that Wnt5a participates in cross-talk with other members of the gene regulatory network that control prostate development. Although Wnt5a does not influence prostatic expression of other Wnt morphogens, it suppresses Wif-1 expression and can thus indirectly modulate Wnt signaling. In summary, the present finds demonstrate that Wnt5a is essential for normal prostate development where it regulates bud outgrowth, ductal elongation, branching, cell polarity and lumenization. These findings contribute to the growing body of knowledge on regulatory mechanisms involved in prostate gland development which are key to understanding abnormal growth processes associated with aging. Author Affiliation: (a) Department of Urology, College of Medicine, University of Illinois at Chicago, Chicago, IL 60614, USA (b) National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Article History: Received 22 August 2008; Revised 23 December 2008; Accepted 2 January 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.11.033 Byline: Kazuhiro Kawamura (a)(c), Yinghui Ye (a)(d), Cheng Guang Liang (c), Nanami Kawamura (a)(b), Maarten Sollewijn Gelpke (e), Rami Rauch (c), Toshinobu Tanaka (a), Aaron J.W. Hsueh (c) Keywords: Germinal vesicle breakdown; Endothelin-1; Endothelin receptor type A; LH; MAPK; Oocyte Abstract: Mammalian oocytes remain dormant in the diplotene stage of prophase I until the resumption of meiosis characterized by germinal vesicle breakdown (GVBD) following the preovulatory gonadotropin stimulation. Based on genome-wide analysis of peri-ovulatory DNA microarray to identify paracrine hormone-receptor pairs, we found increases in ovarian transcripts for endothelin-1 and endothelin receptor type A (EDNRA) in response to the preovulatory luteinizing hormone (LH)/human chorionic gonadotropin (hCG) stimulation. Immunohistochemical analyses demonstrated localization of EDNRA in granulosa and cumulus cells. In cultured preovulatory follicles, treatment with endothelin-1 promoted oocyte GVBD. The stimulatory effect of endothelin-1 was blocked by cotreatment with antagonists for the type A, but not related type B, receptor. The stimulatory effect of hCG on GVBD was partially blocked by the same antagonist. The endothelin-1 promotion of GVBD was found to be mediated by the MAPK/ERK pathway but not by the inhibitory G protein. Studies using cumulus-oocyte complexes and denuded oocytes demonstrated that the endothelin-1 actions are mediated by cumulus cells. Furthermore, intrabursal administration with endothelin-1 induced oocyte GVBD in preovulatory follicles. Our findings demonstrate a paracrine role of endothelin-1 in the induction of the resumption of meiosis and provide further understanding on the molecular mechanisms underlying the nuclear maturation of oocytes induced by the preovulatory LH surge. Author Affiliation: (a) Department of Obstetrics and Gynecology, Akita University School of Medicine, Akita 010-8543, Japan (b) Dermatology and Plastic Surgery, Akita University School of Medicine, Akita 010-8543, Japan (c) Divison of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305-5317, USA (d) Department of Reproductive Endocrinology, Women's Hospital, Zhejiang University School of Medicine, Zhejiang 310-006, China (e) Molecular Design and Informatics, Schering-Plough Corporation, Oss, P.O. Box 20, 5340 BH, The Netherlands Article History: Received 13 August 2008; Revised 5 November 2008; Accepted 24 November 2008

Mitochondrial dysfunction is a prominent feature of Alzheimer disease but the underlying mechanism is unclear. In this study, we investigated the effect of amyloid precursor protein (APP) and amyloid [beta] on mitochondrial dynamics in neurons. Confocal and electron microscopic analysis demonstrated that [approximately equal to]40% M17 cells overexpressing WT APP (APPwt M17 cells) and more than 80% M17 cells overexpressing APPswe mutant (APPswe M17 cells) displayed alterations in mitochondrial morphology and distribution. Specifically, mitochondria exhibited a fragmented structure and an abnormal distribution accumulating around the perinuclear area. These mitochondrial changes were abolished by treatment with [beta]-site APP-cleaving enzyme inhibitor IV. From a functional perspective, APP overexpression affected mitochondria at multiple levels, including elevating reactive oxygen species levels, decreasing mitochondrial membrane potential, and reducing ATP production, and also caused neuronal dysfunction such as differentiation deficiency upon retinoic acid treatment. At the molecular level, levels of dynamin-like protein 1 and OPAl were significantly decreased whereas levels of Fis1 were significantly increased in APPwt and APPswe M17 cells. Notably, overexpression of dynamin-like protein 1 in these cells rescued the abnormal mitochondrial distribution and differentiation deficiency, but failed to rescue mitochondrial fragmentation and functional parameters, whereas overexpression of OPAl rescued mitochondrial fragmentation and functional parameters, but failed to restore normal mitochondrial distribution. Overexpression of APP or A[beta]-derived diffusible ligand treatment also led to mitochondrial fragmentation and reduced mitochondrial coverage in neuronal processes in differentiated primary hippocampal neurons. Based on these data, we concluded that APP, through amyloid [beta] production, causes an imbalance of mitochondrial fission/fusion that results in mitochondrial fragmentation and abnormal distribution, which contributes to mitochondrial and neuronal dysfunction. amyloid precursor protein | DLP1 | mitochondrial fragmentation | OPAl | perinuclear accumulation

Author(s): Ping Zhu [sup.1] , Jun Liu [sup.1] , Julian Bess Jr [sup.2] , Elena Chertova [sup.2] , Jeffrey D. Lifson [sup.2] , Henry Grisé [sup.1] , Gilad A. Ofek [...], Envelope glycoprotein (Env) spikes on AIDS retroviruses initiate infection of host cells and are therefore targets for vaccine development. Though crystal structures for partial Env subunits are known, the structure and distribution of native Env spikes on virions is obscure. We applied cryoelectron microscopy tomography to define ultrastructural details of spikes. Virions of wild-type human immunodeficiency virus 1 (HIV-1) and a mutant simian immunodeficiency virus (SIV) had ~14 and ~73 spikes per particle, respectively, with some clustering of HIV-1 spikes. Three-dimensional averaging showed that the surface glycoprotein (gp120) &apos;head&apos; of each subunit of the trimeric SIV spike contains a primary mass, with two secondary lobes. The transmembrane glycoprotein &apos;stalk&apos; of each trimer is composed of three independent legs that project obliquely from the trimer head, tripod-like. Reconciling available atomic structures with the three-dimensional whole spike density map yields insights into the orientation of Env spike structural elements and possible structural bases of their functions. AIDS virus structure Cryoelectron microscopy tomography has revealed the protein spikes on the surface of HIV-1 in unprecedented detail. These are the spikes that allow the virus to bind and fuse with human immune cells so effectively. Each virion has an average of 14 spikes; some of the spikes are clustered (pictured here), a feature that has implications for the mechanisms of viral assembly, infectivity and neutralization. And there was a surprise. The spike stalk was assumed to consist of three rods tightly bound together with the head of the spike on the top. In fact it is split into three legs, in a tripod-like pattern that maximizes contact with the viral membrane.

Current design of genetically engineered viruses for selective destruction of cancer cells is based on the observation that attenuated viruses replicate better in tumor cells than in normal cells. The ideal virus, however, is one that can infect only cancer cells by virtue of altered host range. Such a virus can be made more robust than the highly attenuated viruses used in clinical trials. Earlier, we reported the construction of a recombinant herpes simplex virus 1 (R5111) in which the capacity to bind heparan sulfate was disabled and which contained a chimeric IL-13-glycoprotein D that enabled the virus to infect cells expressing the IL-13[alpha]2 receptor (IL-13R[alpha]2) commonly found on the surface of malignant glioblastomas or high-grade astrocytomas. In the earlier report, we showed that the recombinant R5111 was able to enter and infect cells via the interaction of the chimeric glycoprotein D with IL-13R[alpha]2 but that the virus retained the capacity to bind and replicate in cells expressing the natural viral receptors HveA or nectin-1. Here, we report the construction of a recombinant virus (R5141) that can only enter and replicate in cells that express the IL-13R[alpha]2. The recombinant R5141 does not depend on endocytosis to infect cells. It does not infect cells expressing HveA or nectin-1 receptors or cells expressing IL-13R[alpha]2 that had been exposed to soluble IL-13 before infection. The studies described here show that the host range of herpes simplex viruses can be altered by genetic manipulation to specifically target cancer cells. glycoprotein D | HveA | malignant glioblastoma | nectin-1

A demonstration that PrP23-144 amyloids from different species adopt distinct secondary structures and morphologies, and that one or two residues in a critical region control these structural differences is presented. Evidence is provided that protein conformations are transmitted in PrP amyloids strains, establishing a foundation for a structural basis of mammalian prion transmission barriers.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/S0012-1606(02)00124-0 Byline: Wiebke Herzog (a), Xianchun Zeng (a), Zsolt Lele (a), Carmen Sonntag (a), Jing-Wen Ting (b), Chi-Yao Chang (b), Matthias Hammerschmidt (a) Abstract: Formation of the adenohypophysis in mammalian embryos occurs via an invagination of the oral ectoderm to form Rathke's pouch, which becomes exposed to opposing dorsoventral gradients of signaling proteins governing specification of the different hormone-producing pituitary cell types. One signal promoting pituitary cell proliferation and differentiation to ventral cell types is Sonic hedgehog (Shh) from the oral ectoderm. To study pituitary formation and patterning in zebrafish, we cloned four cDNAs encoding different pituitary hormones, prolactin (prl), proopiomelancortin (pomc), thyroid stimulating hormone (tsh), and growth hormone (gh), and analyzed their expression patterns relative to that of the pituitary marker lim3. prl and pomc start to be expressed at the lateral edges of the lim3 expression domain, before pituitary cells move into the head. This indicates that patterning of the pituitary anlage and terminal differentiation of pituitary cells starts while cells are still organized in a placodal fashion at the anterior edge of the developing brain. Following the expression pattern of prl and pomc during development, we show that no pituitary-specific invagination equivalent to Rathke's pouch formation takes place. Rather, pituitary cells move inwards together with stomodeal cells during oral cavity formation, with medial cells of the placode ending up posterior and lateral cells ending up anterior, resulting in an anterior-posterior, rather than a dorsoventral, patterning of the adenohypophysis. Carrying out loss- and gain-of-function experiments, we show that Shh from the ventral diencephalon plays a crucial role during induction, patterning, and growth of the zebrafish adenohypophysis. The phenotypes are very similar to those obtained upon pituitary-specific inactivation or overexpression of Shh in mouse embryo, suggesting that the role of Shh during pituitary development has been largely conserved between fish and mice, despite the different modes of pituitary formation in the two vertebrate classes. Author Affiliation: (a) Max-Planck Institute for Immunobiology, Stuebeweg 51, D-79108 Freiburg, Germany (b) Institute of Zoology, Academica Sinica, Taipei, Taiwan, ROC Article History: Received 5 June 2002; Revised 25 July 2002; Accepted 26 July 2002

Myelin-associated glycoprotein (MAG) binds to the nerve cell surface and inhibits nerve regeneration. The nerve cell surface ligand(s) for MAG are not established, although sialic acid-bearing glycans have been implicated. We identify the nerve cell surface gangliosides GD1a and GT1b as specific functional ligands for MAG-mediated inhibition of neurite outgrowth from primary rat cerebellar granule neurons. MAG-mediated neurite outgrowth inhibition is attenuated by (i) neuraminidase treatment of the neurons; (ii) blocking neuronal ganglioside biosynthesis; (iii) genetically modifying the terminal structures of nerve cell surface gangliosides; and (iv) adding highly specific IgG-class antiganglioside mAbs. Furthermore, neurite outgrowth inhibition is mimicked by highly multivalent clustering of GD1a or GT1b by using precomplexed antiganglioside Abs. These data implicate the nerve cell surface gangliosides GD1a and GT1b as functional MAG ligands and suggest that the first step in MAG inhibition is multivalent ganglioside clustering.

Multiprotein complexes are key determinants of Golgi apparatus structure and its capacity for intracellular transport and glycoprotein modification. Three complexes that have previously been partially characterized include (a) the Golgi transport complex (GTC), identified in an in vitro membrane transport assay, (b) the IdICp complex, identified in analyses of CHO cell mutants with defects in Golgi-associated glycosylation reactions, and (c) the mammalian Sec34 complex, identified by homology to yeast Sec34p, implicated in vesicular transport. We show that these three complexes are identical and rename them the conserved oligomeric Golgi (COG) complex. The COG complex comprises four previously characterized proteins (Cog1/IdIBp, Cog2/IdICp, Cog3/Sec34, and Cog5/GTC-90), three homologues of yeast Sec34/35 complex subunits (Cog4, -6, and -8), and a previously unidentified Golgi-associated protein (Cog7). EM of Id1B and Id1C mutants established that COG is required for normal Golgi morphology. 'Deep etch' EM of purified COG revealed an ~37-nm-long structure comprised of two similarly sized globular domains connected by smaller extensions. Consideration of biochemical and genetic data for mammalian COG and its yeast homologue suggests a model for the subunit distribution within this complex, which plays critical roles in Golgi structure and function.

The human multidrug resistance P-glycoprotein uses ATP to transport a wide variety of structurally unrelated cytotoxic compounds out of the cell. In this study, we used cysteine-scanning mutagenesis and cross-linking studies to identify residues that are exposed to the drug-binding site upon vanadate trapping. In the absence of nucleotides, C222(TM4) was cross-linked to C868(TM10) and C872(TM10); C306(TM 5) was cross-linked to C868(TM10), C872(TM10), C945(TM11), C982(TM12), and C984(TM12); and C339(TM6) was cross-linked to C868(TM10), C872(TM10), C942(TM11), C982(TM12), and C985(TM12). These cysteines are in the middle of the predicted transmembrane (TM) segments and form the drug-binding site. Cross-linking between 332C(TM6) and cysteines introduced at the extracellular side of other TM segments was also done. In the absence of nucleotides, residues 332C and 856C on the extracellular side of TMs 6 and 10, respectively, were cross-linked with a 13-A cross-linker (MSM, 3,6-dioxaoctane-1,8-diyl bismethanethiosulfonate). ATP plus vanadate inhibited cross-linking between 332C(TM6) and 856C(TM10) as well as those in the drug-binding site. Instead, vanadate trapping promoted cross-linking between 332C(TM6) and 976C(TM12) with a 10-A cross-linker (M6M, 1,6-hexanediyl bismethanethiosulfonate). When ATP hydrolysis was allowed to proceed, then 332C(TM12) could form a disulfide bond with 975C(TM12). The cross-linking pattern of 332C(TM6) with residues in TM10 and TM12 indicates that the drug-binding site undergoes dynamic and relatively large conformational changes, and that different residues are exposed to the drug-binding site during the resting phase, upon vanadate trapping and at the completion of the catalytic cycle.

Myelin-associated glycoprotein (MAG) is expressed in periaxonal membranes of myelinating glia where it is believed to function in glia-axon interactions by binding to a component of the axolemma. Experiments involving Western blot overlay and coimmunoprecipitation demonstrated that MAG binds to a phosphorylated neuronal isoform of microtubule-associated protein 1B (MAP1B) expressed in dorsal root ganglion neurons (DRGNs) and axolemma-enriched fractions from myelinated axons of brain, but not to the isoform of MAP1B expressed by glial cells. The expression of some MAP1B as a neuronal plasma membrane glycoprotein (Tanner, S.L., R. Franzen, H. Jaffe, and R.H. Quarles. 2000. J. Neurochem. 75:553-562.), further documented here by its immunostaining without cell permeabilization, is consistent with it being a binding partner for MAG on the axonal surface. Binding sites for a MAG-Fc chimera on DRGNs colocalized with MAP1B on neuronal varicosities, and MAG and MAP1 B also colocalized in the periaxonal region of myelinated axons. In addition, expression of the phosphorylated isoform of MAP1B was increased significantly when DRGNs were cocultured with MAG-transfected COS cells. The interaction of MAG with MAP1B is relevant to the known role of MAG in affecting the cytoskeletal structure and stability of myelinated axons.

By screening a chemical library for the compounds protecting cells from adriamycin (Adr), a series of small molecules was isolated that interfered with the accumulation of Adr in mouse fibroblasts by enhancing efflux of the drug. Isolated compounds also stimulated efflux of Rhodamine 123 (Rho-123), another substrate of multidrug transporters. Stimulation of drug efflux was detectable in the cells expressing P-glycoprotein (P-gp), but not in their P-gp-negative variants, and was completely reversible by the P-gp inhibitors. A dramatic stimulation of P-gp activity against Adr and Rho-123 by the identified compounds was accompanied by suppression of P-gp-mediated efflux of other substrates, such as Taxol (paclitaxel) or Hoechst 33342, indicating that they act as modulators of substrate specificity of P-gp. Consistently, P-gp modulators dramatically altered the pattern of cross-resistance of P-gp-expressing cells to different P-gp substrates: an increase in resistance to Adr, daunorubicin, and etoposide was accompanied by cell sensitization to Vinca alkaloids, gramicidin D, and Taxol with no effect on cell sensitivity to colchicine, actinomycin D, puromycin, and colcemid, as well as to several non-P-gp substrates. The relative effect of P-gp modulators against different substrates varied among the isolated compounds that can be used as fine tools for analyzing mechanisms of drug selectivity of P-gp. These results raise the possibility of a rational control over cell sensitivity to drugs and toxins through modulation of P-gp activity by small molecules. chemical library | efflux

Research has been conducted on the hyperthermophilic archaeon Pyrococcus furiosus which utilizes cellobiose and laminarin. The purification of the cellobiose binding protein from P. furiosus membranes to homogeneity as a glycoprotein is described and the results are presented.

Amino acid sequence analysis of purified fimbriae glycoprotein from Streptococcus salivarius reveals that the protein is filamentous in structure and the internal sequence is composed of a repeated motif of two amino acids alternating with two modified residues. Data indicate that the protein is resistant to dissociation.