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Roosing S.,Howard Hughes Medical Institute | Hofree M.,University of California at San Diego | Kim S.,New York University | Kim S.,Howard Hughes Medical Institute | And 42 more authors.
eLife | Year: 2015

Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome wide siRNA screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies. © 2015, eLife Sciences Publications Ltd. All rights reserved.


Srinivas R.,Indian Institute of Chemical Technology | Srinivas R.,University of North Carolina at Chapel Hill | Karmali P.P.,Indian Institute of Chemical Technology | Karmali P.P.,Burnham Institute | And 10 more authors.
Journal of Medicinal Chemistry | Year: 2010

Mannosylated cationic vectors have been previously used for delivering DNA vaccines to antigen presenting cells (APCs) via mannose receptors expressed on the cell surface of APCs. Here we show that cationic amphiphiles containing mannose-mimicking quinic acid and shikimic acid headgroups deliver genes to APCs via mannose receptor. Cationic amphiphile with shikimic acid headgroup was more efficacious than its mannosyl counterpart in combating mouse tumor growth by dendritic cell (the most professional APC) based genetic immunization. © 2010 American Chemical Society.


Zheng W.H.,University of California at San Diego | Zheng W.H.,Medical College of Wisconsin | Vastermark A.,University of California at San Diego | Shlykov M.A.,University of California at San Diego | And 6 more authors.
BMC Microbiology | Year: 2013

Background: The ATP-Binding Cassette (ABC) functional superfamily includes integral transmembrane exporters that have evolved three times independently, forming three families termed ABC1, ABC2 and ABC3, upon which monophyletic ATPases have been superimposed for energy-coupling purposes [e.g., J Membr Biol 231(1):1-10, 2009]. The goal of the work reported in this communication was to understand how the integral membrane constituents of ABC uptake transporters with different numbers of predicted or established transmembrane segments (TMSs) evolved. In a few cases, high resolution 3-dimensional structures were available, and in these cases, their structures plus primary sequence analyses allowed us to predict evolutionary pathways of origin. Results: All of the 35 currently recognized families of ABC uptake proteins except for one (family 21) were shown to be homologous using quantitative statistical methods. These methods involved using established programs that compare native protein sequences with each other, after having compared each sequence with thousands of its own shuffled sequences, to gain evidence for homology. Topological analyses suggested that these porters contain numbers of TMSs ranging from four or five to twenty. Intragenic duplication events occurred multiple times during the evolution of these porters. They originated from a simple primordial protein containing 3 TMSs which duplicated to 6 TMSs, and then produced porters of the various topologies via insertions, deletions and further duplications. Except for family 21 which proved to be related to ABC1 exporters, they are all related to members of the previously identified ABC2 exporter family. Duplications that occurred in addition to the primordial 3 → 6 duplication included 5 → 10, 6 → 12 and 10 → 20 TMSs. In one case, protein topologies were uncertain as different programs gave discrepant predictions. It could not be concluded with certainty whether a 4 TMS ancestral protein or a 5 TMS ancestral protein duplicated to give an 8 or a 10 TMS protein. Evidence is presented suggesting but not proving that the 2TMS repeat unit in ABC1 porters derived from the two central TMSs of ABC2 porters. These results provide structural information and plausible evolutionary pathways for the appearance of most integral membrane constituents of ABC uptake transport systems. Conclusions: Almost all integral membrane uptake porters of the ABC superfamily belong to the ABC2 family, previously established for exporters. Most of these proteins can have 5, 6, 10, 12 or 20 TMSs per polypeptide chain. Evolutionary pathways for their appearance are proposed. © 2013 Zheng et al.; licensee BioMed Central Ltd.


Hatakeyama S.,Hirosaki University | Kyan A.,Hirosaki University | Yamamoto H.,Hirosaki University | Okamoto A.,Hirosaki University | And 10 more authors.
International Journal of Cancer | Year: 2010

We studied orchiectomy specimens from 130 patients immuhistochemically with testicular germ cell tumor (TGCT) using anticore 2 N- acetylglucosaminyltransferase-1 (C2GnT-1) antibody. The incidence of C2GnT-1 positivity in stage I disease (29.5%, 21/71) was significantly lower than that in higher stages (84.7%, 50/59) (P < 0.001, χ2 test). This significant difference was also found when the cases were divided into seminoma and NSGCT according to histopathological classification. Kaplan-Meier plots and the log rank test showed that in the patients with stage I seminoma, C2GnT-1-positive cases had a higher risk for recurrence (P < 0.001). This was also the case with the patients with stage I NSGCT (P < 0.001). To determine whether C2GnT-1 promotes aggressive behavior of cancer cells, a C2GnT-1-negative human TGCT cell line, JKT-1, was stably transfected with a mammalian expression vector containing C2GnT-1 cDNA. In vitro assays revealed that JKT-1-C2 cells are more invasive than mock transfectants, although there are no differences in proliferation activity. When orthotopically inoculated into athymic nude mice, JKT-1-C2 cells produced larger testicular tumors extending to the retroperitoneum with mesenteric metastasis, while mock transfectants produced small tumors without metastasis (P < 0.01, Mann-Whitney's U-test). When injected via the tail vein, JKT-1-C2 cells produced a number of metastatic lung foci. In contrast, mock transfectants produced a small number of nodules (p < 0.01, Mann-Whitney's U-test). These results strongly suggest that C2GnT-1 enhances the metastatic potential of TGCT and may be a reliable biomarker for aggressive potential of TGCT. © 2009 UICC.


Hetherington J.,University College London | Hetherington J.,AMEE UK Ltd. | Sumner T.,University College London | Sumner T.,Institute for Animal Health | And 13 more authors.
Journal of the Royal Society Interface | Year: 2012

A computational model of the glucagon/insulin-driven liver glucohomeostasis function, focusing on the buffering of glucose into glycogen, has been developed. The model exemplifies an 'engineering' approach to modelling in systems biology, and was produced by linking together seven component models of separate aspects of the physiology. The component models use a variety of modelling paradigms and degrees of simplification. Model parameters were determined by an iterative hybrid of fitting to high-scale physiological data, and determination from small-scale in vitro experiments or molecular biological techniques. The component models were not originally designed for inclusion within such a composite model, but were integrated, with modification, using our published modelling software and computational frameworks. This approach facilitates the development of large and complex composite models, although, inevitably, some compromises must be made when composing the individual models. Composite models of this form have not previously been demonstrated. © 2011 The Royal Society.


Ye F.,University of California at San Diego | Hu G.,Florida State University | Taylor D.,Florida State University | Ratnikov B.,University of California at San Diego | And 6 more authors.
Journal of Cell Biology | Year: 2010

Increased affinity of integrins for the extracellular matrix (activation) regulates cell adhesion and migration, extracellular matrix assembly, and mechanotransduction. Major uncertainties concern the sufficiency of talin for activation, whether conformational change without clustering leads to activation, and whether mechanical force is required for molecular extension. Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin αIIbβ3. Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins. Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin αIIbβ3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling. © 2010 Ye et al.


Xiang Z.,University of Florida | Proneth B.,University of Florida | Dirain M.L.,University of Florida | Litherland S.A.,Burnham Institute | Haskell-Luevano C.,University of Florida
Biochemistry | Year: 2010

The melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic biomarker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and nonobese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [α-, β-, and γ2-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87?132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-dPhe-Arg-Trp-NH2 (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4Rs and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface expression by flow cytometry. The F51L, I69T, and A219V hMC4Rs possessed full agonist activity and significantly decreased endogenous agonist ligand potency. At the E61K, D90N, Y157S, and C271R hMC4Rs, all agonist ligands examined were only partially efficacious in generating a maximal signaling response (partial agonists) and possessed significantly decreased endogenous agonist ligand potency. Only the A219V, G238D, and S295P hMC4Rs possessed significantly decreased AGRP(87?132) antagonist potency. These data provide new information for use in GPCR computational development as well as insights into MC4R structure ad function. © 2010 American Chemical Society.


Sun X.,University of British Columbia | Sun X.,Shandong University | Wu Y.,University of British Columbia | Chen B.,University of British Columbia | And 8 more authors.
Journal of Biological Chemistry | Year: 2011

Individuals with Down syndrome (DS) will inevitably develop Alzheimer disease (AD) neuropathology sometime after middle age, which may be attributable to genes triplicated in individuals with DS. The characteristics of AD neuropathology include neuritic plaques, neurofibrillary tangles, and neuronal loss in various brain regions. The mechanism underlying neurodegeneration in AD and DS remains elusive. Regulator of calcineurin 1 (RCAN1) has been implicated in the pathogenesis of DS. Our data show that RCAN1 expression is elevated in the cortex of DS and AD patients. RCAN1 expression can be activated by the stress hormone dexamethasone. A functional glucocorticoid response element was identified in the RCAN1 isoform 1 (RCAN1-1) promoter region, which is able to mediate the up-regulation of RCAN1 expression. Here we show that overexpression of RCAN1-1 in primary neurons activates caspase-9 and caspase-3 and subsequently induces neuronal apoptosis. Furthermore, we found that the neurotoxicity of RCAN1-1 is inhibited by knock-out of caspase-3 in caspase-3-/- neurons. Our study provides a novel mechanism by which RCAN1 functions as a mediator of stress- and Aβ-induced neuronal death, and overexpression of RCAN1 due to an extra copy of the RCAN1 gene on chromosome 21 contributes to AD pathogenesis in DS. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.


Sumner T.,University College London | Sumner T.,Institute for Animal Health | Hetherington J.,University College London | Seymour R.M.,University College London | And 12 more authors.
Journal of the Royal Society Interface | Year: 2012

Using a composite model of the glucose homeostasis system, consisting of seven interconnected submodels, we enumerate the possible behaviours of the model in response to variation of liver insulin sensitivity and dietary glucose variability. The model can reproduce published experimental manipulations of the glucose homeostasis system and clearly illustrates several important properties of glucose homeostasis-boundedness in model parameters of the region of efficient homeostasis, existence of an insulin sensitivity that allows effective homeostatic control and the importance of transient and oscillatory behaviour in characterizing homeostatic failure. Bifurcation analysis shows that the appearance of a stable limit cycle can be identified. © 2012 The Royal Society.


Staflin K.,Scripps Research Institute | Krueger J.S.,Scripps Research Institute | Hachmann J.,Scripps Research Institute | Forsyth J.S.,Scripps Research Institute | And 7 more authors.
Clinical and Experimental Metastasis | Year: 2010

Advanced metastatic disease is difficult to manage and specific therapeutic targets are rare. We showed earlier that metastatic breast cancer cells use the activated conformer of adhesion receptor integrin αvβ3 for dissemination. We now investigated if targeting this form of the receptor can impact advanced metastatic disease, and we analyzed the mechanisms involved. Treatment of advanced multi-organ metastasis in SCID mice with patient-derived scFv antibodies specific for activated integrin αvβ3 caused stagnation and regression of metastatic growth. The antibodies specifically localized to tumor lesions in vivo and inhibited αvβ3 ligand binding at nanomolar levels in vitro. At the cellular level, the scFs associated rapidly with high affinity αvβ3 and dissociated extremely slowly. Thus, the scFvs occupy the receptor on metastatic tumor cells for prolonged periods of time, allowing for inhibition of established cell interaction with natural αvβ3 ligands. Potential apoptosis inducing effects of the antibodies through interaction with caspase-3 were studied as potential additional mechanism of treatment response. However, in contrast to a previous concept, neither the RGD-containing ligand mimetic scFvs nor RGD peptides bound or activated caspase-3 at the cellular or molecular level. This indicates that the treatment effects seen in the animal model are primarily due to antibody interference with αvβ3 ligation. Inhibition of advanced metastatic disease by treatment with cancer patient derived single chain antibodies against the activated conformer of integrin αvβ3 identifies this form of the receptor as a suitable target for therapy. © 2010 Springer Science+Business Media B.V.

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