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41.
42.
Evidence of compromised blood-spinal cord barrier in early and late symptomatic SOD1 mice modeling ALS 总被引:1,自引:0,他引:1
Garbuzova-Davis S Saporta S Haller E Kolomey I Bennett SP Potter H Sanberg PR 《PloS one》2007,2(11):e1205
Background
The blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), and blood-cerebrospinal fluid barrier (BCSFB) control cerebral/spinal cord homeostasis by selective transport of molecules and cells from the systemic compartment. In the spinal cord and brain of both ALS patients and animal models, infiltration of T-cell lymphocytes, monocyte-derived macrophages and dendritic cells, and IgG deposits have been observed that may have a critical role in motor neuron damage. Additionally, increased levels of albumin and IgG have been found in the cerebrospinal fluid in ALS patients. These findings suggest altered barrier permeability in ALS. Recently, we showed disruption of the BBB and BSCB in areas of motor neuron degeneration in the brain and spinal cord in G93A SOD1 mice modeling ALS at both early and late stages of disease using electron microscopy. Examination of capillary ultrastructure revealed endothelial cell degeneration, which, along with astrocyte alteration, compromised the BBB and BSCB. However, the effect of these alterations upon barrier function in ALS is still unclear. The aim of this study was to determine the functional competence of the BSCB in G93A mice at different stages of disease.Methodology/Principal Findings
Evans Blue (EB) dye was intravenously injected into ALS mice at early or late stage disease. Vascular leakage and the condition of basement membranes, endothelial cells, and astrocytes were investigated in cervical and lumbar spinal cords using immunohistochemistry. Results showed EB leakage in spinal cord microvessels from all G93A mice, indicating dysfunction in endothelia and basement membranes and confirming our previous ultrastructural findings on BSCB disruption. Additionally, downregulation of Glut-1 and CD146 expressions in the endothelial cells of the BSCB were found which may relate to vascular leakage.Conclusions/Significance
Results suggest that the BSCB is compromised in areas of motor neuron degeneration in ALS mice at both early and late stages of the disease. 相似文献43.
Shkoda A Werner T Daniel H Gunckel M Rogler G Haller D 《Journal of proteome research》2007,6(3):1114-1125
The loss of intestinal epithelial cell (IEC) function is a critical component in the initiation and perpetuation of chronic intestinal inflammation in the genetically susceptible host. We applied proteome analysis (PA) to characterize changes in the protein expression profile of primary IEC from patients with Crohn's disease (CD) and ulcerative colitis (UC). Surgical specimens from 18 patients with active CD (N = 6), UC (N = 6), and colonic cancer (N = 6) were used to purify primary IEC from ileal and colonic tissues. Changes in protein expression were identified using 2D-gel electrophoreses (2D SDS-PAGE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS) as well as Western blot analysis. PA of primary IEC from inflamed ileal tissue of CD patients and colonic tissue of UC patients identified 21 protein spots with at least 2-fold changes in steady-state expression levels compared to the noninflamed tissue of control patients. Statistical significance was achieved for 9 proteins including the Rho-GDP dissociation inhibitor alpha that was up-regulated in CD and UC patients. Additionally, 40 proteins with significantly altered expression levels were identified in IEC from inflamed compared to noninflamed tissue regions of single UC (N = 2) patients. The most significant change was detected for programmed cell death protein 8 (7.4-fold increase) and annexin 2A (7.7-fold increase). PA in primary IEC from IBD patients revealed significant expression changes of proteins that are associated with signal transduction, stress response as well as energy metabolism. The induction of Rho GDI alpha expression may be associated with the destruction of IEC homeostasis under condition of chronic intestinal inflammation. 相似文献
44.
Zuniga FA Shi G Haller JF Rubashkin A Flynn DR Iserovich P Fischbarg J 《The Journal of biological chemistry》2001,276(48):44970-44975
The human facilitative transporter Glut1 is the major glucose transporter present in all human cells, has a central role in metabolism, and is an archetype of the superfamily of major protein facilitators. Here we describe a three-dimensional structure of Glut1 based on helical packing schemes proposed for lactose permease and Glut1 and predictions of secondary structure, and refined using energy minimization, molecular dynamics simulations, and quality and environmental scores. The Ramachandran scores and the stereochemical quality of the structure obtained were as good as those for the known structures of the KcsA K(+) channel and aquaporin 1. We found two channels in Glut1. One of them traverses the structure completely, and is lined by many residues known to be solvent-accessible. Since it is delimited by the QLS motif and by several well conserved residues, it may serve as the substrate transport pathway. To validate our structure, we determined the distance between these channels and all the residues for which mutations are known. From the locations of sugar transporter signatures, motifs, and residues important to the transport function, we find that this Glut1 structure is consistent with mutagenesis and biochemical studies. It also accounts for functional deficits in seven pathogenic mutants. 相似文献
45.
Zhu A Haller S Li H Chaudhuri A Blancher A Suyama K 《The Journal of biological chemistry》1999,274(9):5731-5737
The human D antigens, one of the most clinically important blood groups, are presented by RhD protein with a putative 12 transmembrane topology. To understand the molecular basis for the complex antigenic profile of RhD protein, we expressed a series of RhD fusion proteins using different portions of Duffy protein as a tag in erythroleukemic K562 cells. Because the reactivity of monoclonal anti-RhD antibody, LOR15C9, depends mainly on the sequence coded by exon 7 of RhD, we altered DNA sequence corresponding to the amino acid residues 323-331(A) and 350-354(B) in the exon 7. The mutation in region B resulted in a severe reduction in LOR15C9 binding by flow cytometry analysis, suggesting that region B may play an important role in constituting antigen epitopes recognized by LOR15C9. On the other hand, a slight decrease in the antibody binding was observed for the region A mutant, suggesting that the intracellularly located region A may elicit a long distance effect on the formation of exofacial antigen epitopes. In addition, using various monoclonal antibodies against RhD, we compared the antigenic profile of expressed RhD fusion protein with that of endogenous RhD in K562 cells as well as in erythrocytes. 相似文献
46.
Loogväli EL Roostalu U Malyarchuk BA Derenko MV Kivisild T Metspalu E Tambets K Reidla M Tolk HV Parik J Pennarun E Laos S Lunkina A Golubenko M Barac L Pericic M Balanovsky OP Gusar V Khusnutdinova EK Stepanov V Puzyrev V Rudan P Balanovska EV Grechanina E Richard C Moisan JP Chaventré A Anagnou NP Pappa KI Michalodimitrakis EN Claustres M Gölge M Mikerezi I Usanga E Villems R 《Molecular biology and evolution》2004,21(11):2012-2021
It has been often stated that the overall pattern of human maternal lineages in Europe is largely uniform. Yet this uniformity may also result from an insufficient depth and width of the phylogenetic analysis, in particular of the predominant western Eurasian haplogroup (Hg) H that comprises nearly a half of the European mitochondrial DNA (mtDNA) pool. Making use of the coding sequence information from 267 mtDNA Hg H sequences, we have analyzed 830 mtDNA genomes, from 11 European, Near and Middle Eastern, Central Asian, and Altaian populations. In addition to the seven previously specified subhaplogroups, we define fifteen novel subclades of Hg H present in the extant human populations of western Eurasia. The refinement of the phylogenetic resolution has allowed us to resolve a large number of homoplasies in phylogenetic trees of Hg H based on the first hypervariable segment (HVS-I) of mtDNA. As many as 50 out of 125 polymorphic positions in HVS-I were found to be mutated in more than one subcluster of Hg H. The phylogeographic analysis revealed that sub-Hgs H1*, H1b, H1f, H2a, H3, H6a, H6b, and H8 demonstrate distinct phylogeographic patterns. The monophyletic subhaplogroups of Hg H provide means for further progress in the understanding of the (pre)historic movements of women in Eurasia and for the understanding of the present-day genetic diversity of western Eurasians in general. 相似文献
47.
Missorting of LaCrosse virus nucleocapsid protein by the interferon-induced MxA GTPase involves smooth ER membranes 总被引:6,自引:0,他引:6
Reichelt M Stertz S Krijnse-Locker J Haller O Kochs G 《Traffic (Copenhagen, Denmark)》2004,5(10):772-784
The interferon-induced human MxA protein belongs to the class of dynamin-like, large guanosine-5'-triphosphatases that are involved in intracellular vesicle trafficking and organelle homeostasis. MxA shares many properties with the other members of this protein superfamily, including the propensity to self-assemble and to associate with lipid membranes. However, MxA is unique in that it has antiviral activity and inhibits the replication of several RNA viruses. Here, we determined the role of membranes for the antiviral function of MxA using LaCrosse-bunyavirus (LACV). We show that MxA does not affect trafficking and sorting of viral glycoproteins but binds and mislocates the viral nucleocapsid (N) protein into membrane-associated, large perinuclear complexes. We further demonstrate that MxA localizes to a subcompartment of the smooth endoplasmic reticulum where the viral N protein accumulates. In infected MxA-expressing cells, oligomeric MxA/N complexes are formed in close association with COP-I-positive vesicular-tubular membranes. Our results suggest that this membrane compartment is the preferred place where MxA and N interact, leading to efficient sequestration and missorting of an essential viral component. 相似文献
48.
Floxed allele for conditional inactivation of the GABAB(1) gene 总被引:3,自引:0,他引:3
Haller C Casanova E Müller M Vacher CM Vigot R Doll T Barbieri S Gassmann M Bettler B 《Genesis (New York, N.Y. : 2000)》2004,40(3):125-130
GABA(B) receptors are the G-protein-coupled receptors for the neurotransmitter GABA. GABA(B) receptors are broadly expressed in the nervous system. Their complete absence in mice causes premature lethality or--when mice are viable--epilepsy, impaired memory, hyperalgesia, hypothermia, and hyperactivity. A spatially and temporally restricted loss of GABA(B) function would allow addressing how the absence of GABA(B) receptors leads to these diverse phenotypes. To permit a conditional gene inactivation, we flanked critical exons of the GABA(B(1)) gene with lox511 sites. GABA(B(1)) (lox511/lox511) mice exhibit normal levels of GABA(B(1)) protein, are fertile, and do not display any behavioral phenotype. We crossed GABA(B(1)) (lox511/lox511) with Cre-deleter mice to produce mice with an unrestricted GABA(B) receptor elimination. These GABA(B(1)) (-/-) mice no longer synthesize GABA(B(1)) protein and exhibit the expected behavioral abnormalities. The conditional GABA(B(1)) allele described here is therefore suitable for generating mice with a site- and time-specific loss of GABA(B) function. 相似文献
49.
Addition of poly(ethylene glycol) to bioactive proteins (PEGylation) improves their plasma half-life, enhances stability against proteolytic cleavage, and may also decrease protein immunogenicity. Characteristically, PEGylation usually involves a reaction to available lysine amino groups, some of which may be within or near a bioactive site. Thus, most protocols are nonspecific and result in a loss of protein activity. We report herein a strategy for site-specific PEGylation of a thrombomodulin (TM) derivative at the C terminus. A truncated TM mutant consisting of epidermal growth factor (EGF)-like domains 4-6 was expressed in Escherichia coli with a C-terminal azido-methionine. The TM mutant was site-specifically conjugated to a methyl-PEG-triarylphosphine compound via the Staudinger reaction. Enzymatic activity of the TM construct before and after PEGylation was unchanged, which confirms the utility of this site-specific PEGylation scheme. 相似文献
50.
Tang RS Schickli JH MacPhail M Fernandes F Bicha L Spaete J Fouchier RA Osterhaus AD Spaete R Haller AA 《Journal of virology》2003,77(20):10819-10828