text:
Get notified about updates to this publication
Page 1
Fuhrmann et al. Molecular Neurodegeneration 2010, 5:25http://www.molecularneurodegeneration.com/content/5/1/25Open AccessRESEARCH ARTICLESolving a 50 year mystery of a missing OPA1 mutation: more insights from the first family diagnosed with autosomal dominant optic atrophyBioMed Central© 2010 Fuhrmann et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Com-mons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduc-tion in any medium, provided the original work is properly cited.Research articleNico Fuhrmann1, Simone Schimpf1, York Kamenisch2, Beate Leo-Kottler3, Christiane Alexander4, Georg Auburger5, Eberhart Zrenner3, Bernd Wissinger1 and Marcel V Alavi*1AbstractBackground: Up to the 1950s, there was an ongoing debate about the diversity of hereditary optic neuropathies, in particular as to whether all inherited optic atrophies can be ascribed to Leber's hereditary optic neuropathy (LHON) or represent different disease entities. In 1954 W. Jaeger published a detailed clinical and genealogical investigation of a large family with explicit autosomal dominant segregation of optic atrophy thus proving the existence of a discrete disease different from LHON, which is nowadays known as autosomal dominant optic atrophy (ADOA). Since the year 2000 ADOA is associated with genomic mutations in the OPA1 gene, which codes for a protein that is imported into mitochondria where it is required for mitochondrial fusion. Interestingly enough, the underlying mutation in this family has not been identified since then.Results: We have reinvestigated this family with the aim to identify the mutation and to further clarify the underlying pathomechanism. Patients showed a classical non-syndromic ADOA. The long term deterioration in vision in the two teenagers examined 50 years later is of particular note 5/20 to 6/120. Multiplex ligation probe amplification revealed a duplication of the OPA1 exons 7-9 which was confirmed by long distance PCR and cDNA analysis, resulting in an in-frame duplication of 102 amino acids. Segregation was verified in 53 available members of the updated pedigree and a penetrance of 88% was calculated. Fibroblast cultures from skin biopsies were established to assess the mitochondrial network integrity and to qualitatively and quantitatively study the consequences of the mutation on transcript and protein level. Fibroblast cultures demonstrated a fragmented mitochondrial network. Processing of the OPA1 protein was altered. There was no correlation of the OPA1 transcript levels and the OPA1 protein levels in the fibroblasts. Intriguingly an overall decrease of mitochondrial proteins was observed in patients' fibroblasts, while the OPA1 transcript levels were elevated.Conclusions: The thorough study of this family provides a detailed clinical picture accompanied by a molecular investigation of patients' fibroblasts. Our data show a classic OPA1-associated non-syndromic ADOA segregating in this family. Cell biological findings suggest that OPA1 is regulated by post-translational mechanisms and we would like to hypothesize that loss of OPA1 function might lead to impaired mitochondrial quality control. With the clinical, genetic and cell biological characterisation of a family described already more than 50 years ago, we span more than half a century of research in optic neuropathies.BackgroundDuring the first half of the last century, there was a con-troversial debate about the clinical and etiological unityof hereditary optic neuropathies. Some ophthalmologistsfavored the idea that the majority of cases are part of themanifestation spectrum of the Optic Atrophy describedby Theodor Leber [1], that we now know as Leber'shereditary optic neuropathy (LHON) [2-4]. Othersargued that there are different disease entities and sug-gested discriminating different forms of optic atrophy [5-7]. In 1954, Wolfgang Jaeger reported his genealogical* Correspondence: email@example.com Molecular Genetics Laboratory, Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tuebingen, GermanyFull list of author information is available at the end of the article
Page 2
Fuhrmann et al. Molecular Neurodegeneration 2010, 5:25http://www.molecularneurodegeneration.com/content/5/1/25Page 2 of 13and clinical findings in an extended German family span-ning five generations. In this pedigree he could clearlydemonstrate that optic atrophy is inherited as a dominanttrait including male-to-male transmission. In addition, hepointed out the presence of blue-yellow color vision dis-turbances in the affected subjects in this family that con-trasts to the red-green defect typically present in familieswith LHON [8]. These features, together with a thoroughreview of prior clinical reports, enabled him to establishautosomal dominant optic atrophy (ADOA) as a distinctdisease entity.Nowadays, ADOA is a well established disease entityand considered the most frequent hereditary optic atro-phy besides LHON. ADOA is clinically characterized by ajuvenile onset with a progressive, bilateral reduction ofvisual acuity, a cecocentral scotoma, temporal pallor ofthe optic disc and tritanopia as the most typical type ofcolor vision defect [9,10]. There is considerable intra- andinterfamilial variability in progression of the disease aswell as in the severity of the visual impairments, rangingfrom very mildly affected subjects to legally blind patients[11-13]. Moreover, asymptomatic carriers have beenreported in many pedigrees demonstrating reduced pen-etrance [14-16]. Histopathologic investigations haveshown a loss of retinal ganglion cells (RGCs) and thinningof the nerve fiber layer [17,18] which later could be con-firmed in an ADOA mouse model [19]. A major locus forADOA was mapped to chromosome 3q28-3q29 by link-age analysis [20] and subsequently the disease-causinggene OPA1 was identified by our group and an indepen-dent group [21,22]. Besides OPA1, two further gene locifor ADOA, OPA4 [23] and OPA5 [24] have been mappedbut the underlying genes remain unknown so far. In addi-tion, a small number of families have been reported withOPA3 mutations that cause ADOA associated with earlyonset cataract [25]. Given the large number of reports offamilies with OPA1 mutations, the other loci seem to playonly a minor role. There are more than 200 OPA1 muta-tions listed in the eOPA1 database http://lbbma.univ-angers.fr/eOPA1[26]. Most of these mutations have beenidentified by conventional, qualitative genetic screeningtechniques that are insensitive for genomic alterations(i.e. gross deletions or duplications). Only one family witha complete OPA1 gene deletion has been reported in theliterature [27] and just recently we have been able to dem-onstrate that genomic rearrangements may constitute aconsiderable proportion of causal OPA1 gene mutations[28].The OPA1 protein is a nuclear encoded, dynamin-related GTPase which is imported into mitochondria andanchored to the inner membrane of the organelle [29].Together with mitofusin 1 and 2 it plays a major role inmitochondrial fusion and therefore is important for themaintenance of the mitochondrial network morphologyand dynamics [30,31]. Furthermore, OPA1 has beenlinked to mitochondrial cristae stability and its remodel-ing in apoptosis [32-34]. How specific OPA1 mutantsaffect these principal functions has only been sparselyinvestigated and why mutations in OPA1 cause a mostlyselective degeneration of RGCs is unknown. On the otherhand there are case reports of syndromic forms of ADOAthat include extra-ocular (sensorineural deafness, ataxia, axonal sensory motor-polyneuropathy, chronic progressive external ophthal-moplegia, mitochondrial myopathy) which have beenassociated with multiple mitochondrial DNA (mtDNA)deletions [35,36]. However, mtDNA deletions can notonly be found in muscles from patients with syndromicADOA, but seem to be present also in muscle biopsiesfrom patients with non-syndromic ADOA [37].Here we report the identification and re-evaluation ofthe large family described by W. Jaeger in 1954. Usingcopy-number sensitive techniques, we have been able tofinally identify the disease causing mutation in this fam-ily. We provide an update of the pedigree, a clinical followup of a branch of this family as well as a qualitative andquantitative investigation of mutated OPA1 transcriptsand OPA1 protein in fibroblast cell lines established fromtwo patients of this family.neurological featuresResultsThe original ADOA family described in 1954 was re-con-tacted with the help of Prof. W. Jaeger (now deceased)and most of the blood samples and medical records werecollected in a field study in the mid-90s. The pedigreewas updated through several personal interviews withfamily members, the latest in 2008 (Figure 1). The recon-structed pedigree now comprises a total of 216 familymembers of whom 57 are affected or were reported tohave been affected. In comparison with the pedigreereported in 1954 we noted 19 additional family membersaffected by ADOA. DNA samples of 22 affected and 31unaffected family members were collected for this studyand fibroblast cultures for functional investigations wereestablished from skin biopsies of two affected subjects(Figure 1, V-24 and V-26).Long-term follow-up of patients revealed a typical non-syndromic ADOATwo affected brothers who were already described in theoriginal report underwent a full ophthalmological re-examination more than 50 years after their initial clinicaldescription (Figure 1A, V-24 and V-26). Patient A (V-24),now aged 69 years and patient B (V-26), now 66 years old,were first examined at the age of 15 and the age of 11,respectively. W. Jaeger reported that both complained ofreduced vision in school and already experienced modestloss of visual acuity at this age. Since then visual acuity
Page 3
Fuhrmann et al. Molecular Neurodegeneration 2010, 5:25http://www.molecularneurodegeneration.com/content/5/1/25Page 3 of 13Figure 1 Pedigree of the investigated ADOA family and segregation analysis of the OPA1 exon 7-9 duplication. (A) Updated pedigree of the family. The gray area indicates the original pedigree as described by W. Jaeger 1954. Only persons of whom DNA samples were available are indexed. (B) Segregation analysis applying a PCR assay specific for the OPA1 exon 7-9 duplication. A fragment covering exon 19 sequences was co-amplified as internal control. Subjects are numbered according to the pedigree. Ntc - non template control.12341245789181920212223456121516172640414345505152545556575860616366162124264445464748496059??A16 21 24 26 44 45 46 47 48 49 59 6045612 15 16 17 26 40 41 43 4550 51 52 54 55 56 57 58 60 61 63 66124578918 19 20 21 22 231234ntcV-VI-VI-VII-VIII-Intron 9-6Exon 19B
Page 4
Fuhrmann et al. Molecular Neurodegeneration 2010, 5:25http://www.molecularneurodegeneration.com/content/5/1/25Page 4 of 13dropped in both patients from 5/25 and 5/20, respec-tively, to each 6/120 in both eyes. A slow course of diseaseprogression was also confirmed by self-reports. Neitherof them remembered periods of fast loss of visual acuity.Yet both patients have now developed cecocentral sco-toma and an optic atrophy progressed to a complete pal-lor of the optic nerve head, especially of the temporal partof the disc (Figure 2). In comparison with the results offormer color vision tests described in Jaeger's originalpaper, both patients reported further deterioration oftheir color vision. Using Ishihara's test charts they bothcould formerly identify at least 5 of the test plates. At therecent examination both patients did not see even the ref-erence plate of the test. Using Stilling-Hertel test charts,patient A could previously identify 11, patient B 13 of theplates. At the recent examination patient A could onlyidentify 2 and patient B 7 plates. Over the period of 54years, the patients' ability to discriminate between colorshas severely diminished.In addition patient A suffered from glaucoma; theintraocular pressure (IOP) was 21/25 mmHG (R/L) with-out medication, while IOP was still in the normal range inpatient B (R/L 18/19 mmHG). Patient B received a stentin 2006. Otherwise the medical history was unremarkableand neither of the patients had diabetes mellitus or hear-ing impairments, typical symptoms in syndromic formsof optic atrophy. In conclusion both patients show a clearand classical non-syndromic ADOA.Mutation analysis showed a duplication of exons 7 to 9 in the OPA1 geneAn initial microsatellite marker analysis revealed signifi-cant linkage with the OPA1 locus (C. Alexander and G.Auburger, unpublished results). However, subsequentscreening of the OPA1 gene by means of DNA sequenc-ing of the coding exons and flanking intron sequencesfrom PCR-amplified genomic DNA did not reveal anyputative pathogenic mutation. We therefore applied Mul-tiplex Ligation Probe Amplification (MLPA) to analysefor copy number variations and genomic rearrangements.Indeed, MLPA showed a significant signal increase forthe OPA1 exon 8 and 9 probe amplicons in the indexpatient in three independent experiments (Figure 3A).Using oligonucleotides placed both - forward and reverse- in exon 8 for a long distance PCR we were able toamplify a specific PCR product of approximately 8 kb insamples of affected individuals only (Figure 3B). Primerwalking revealed the presence of a recombined sequencethat fuses the 5' part of intron 9 (breakpoint atnp194840568, human genome assembly march 2006)with the 3' part of intron 6 (np194832822) of the OPA1gene (Figure 3C). From this we conclude that exons 7-9plus the flanking intron sequences are duplicated in thisfamily. This mutation was also found in two other ADOAfamilies [28]. In silico analysis revealed Type I transposonelements adjacent to both breakpoints (not shown). Wedeveloped a PCR assay that is specific for this duplicationand performed a segregation analysis in all family mem-bers of whom a DNA sample was available (Figure 1B).We found that all affected family members carry theduplication. In addition, three asymptomatic familymembers (Figure 1B; V-47, VI-15, VII-5) were mutationcarriers. Taking into account that 22 of the 25 mutationcarriers are affected we calculate a penetrance of 88% inthis family.The mitochondrial network is fragmented in patients' fibroblastsSince OPA1 is necessary for mitochondrial fusion [30,31],we morphometrically assessed the mitochondrial net-work in fibroblasts of the two patients after mitotrackerstaining and confocal imaging. In comparison with wildtype control cells we found a significantly decreased pro-portion of cells with a tubular mitochondrial networkwhen cultured in standard glucose medium (Figure 4A&4B). Replacement of glucose by galactose in the growthmedium to avoid glycolysis reinforced the decrease incells with tubular mitochondrial network (not shown).These results demonstrate that mutant cells are clearlyimpaired in their ability to fuse mitochondria. We testedalso the integrity of the mtDNA in these cell lines by theamplification of a 12.5 kb fragment, but did not noticeany differences from controls (Figure 4C).Qualitative and quantitative cDNA analyses revealed an in-frame duplication, a reduced expression level of mutant transcripts and an overall increase of OPA1 transcripts in fibroblastsTo study the consequences of this mutation we analysedcDNA from patients' fibroblast cell lines. Thereby wefound minor fragments that may represent aberrantlyspliced mutant transcripts. Subcloning and sequencing ofthe major fragment of increased length revealed that thiscDNA species had the additional exons correctly inte-grated as a duplication of 306 bp (c.678-984dup306). Thisduplication results in an in-frame duplication of 102amino acids (p.L227_K328dup102). To test if there is animbalance between the transcripts of the mutated and theWT allele in our patients we quantified allelic transcriptlevels by pyrosequencing as described previously [38].Analyses of the OPA1 exon 21 polymorphism (c.2109C >T) revealed a mean percentage ratio for the two alleles of48.7% to 51.3% (S.D. = 1.2). This shows that both allelesare transcribed similar. We also quantified differentOPA1 exon-exon junctions in relation to GAPDH by realtime PCR to verify this result. If both alleles are tran-scribed similarly one would expect a ratio of exon 9-7 (x)to exon 6-7 (y) to exon 7-8 (z) of one-to-two-to-three (x :
Page 5
Fuhrmann et al. Molecular Neurodegeneration 2010, 5:25http://www.molecularneurodegeneration.com/content/5/1/25Page 5 of 13Figure 2 Clinical picture of two patients that had been examined already by W. Jäger. (A) Funduscopy of patient B (V-26) showed pallor of the optic disc with temporal prominence. (B) Visual field of patient A (V-24) showed a clear central scotoma. (C) Patient B showed a decentered central scotoma (excentric retinal locus of fixation, note conjugate displacement of the blind spot).A ABCRARALALA
Page 6
Fuhrmann et al. Molecular Neurodegeneration 2010, 5:25http://www.molecularneurodegeneration.com/content/5/1/25Page 6 of 13y : z = 1 : 2 : 3; see Figure 5A &5B, line 1 for an overview).However, we found a ratio x : y : z of 0.60 (S.D. = 0.05) : 2 :2.85 (S.D. = 0.25) in patient A and a ratio x : y : z of 0.62(S.D. = 0.07) : 2 : 2.24 (S.D. = 0.18) for patient B, respec-tively (Figure 5B, line 2 and 3). Controls displayed a ratioy : z of 2 : 1.98 (S.D. = 0.14) thus proving the reliability ofthis method (Figure 5B, lane 4). This inconsistency in theresults of both assays can be interpreted in a way that afraction of the transcripts derived from the mutant allelemay be spliced correctly (i.e. eliminating the 3 duplicatedexons and presenting as wild type) or spliced aberrantly,or both. Of note, the overall steady-state levels of OPA1transcripts in patients' fibroblasts was more than two-fold increased in comparison to the control fibroblasts(Figure 5C).Qualitative and quantitative protein analysis reveals reduced levels of OPA1 and altered processing of the different OPA1 isoformsFor a qualitative and quantitative analysis of OPA1 pro-tein we used total cell lysates from fibroblast cell lines ofpatients V-24 and V-26 and four control fibroblast celllines derived from healthy individuals. The duplicatedexons 7-9 of the mutant OPA1 allele result in an in-frameduplication of 102 amino acid residues within the GTPasedomain of the OPA1 protein (p.L227_K328dup102), thatis expected to increase its molecular weight by 11.8 kDa.We consistently found a reduction of all OPA1 proteinisoforms in both patients' cell lines in relation to actin(Figure 6A). The different OPA1 isoforms were assignedaccording to their size as described before [39]. Still weare aware that the assignment is rather hypothetical andFigure 3 Identification and characterization of the exon 7-9 duplication in OPA1 by MLPA. (A) Typical read out of the MLPA experiment of index patient (VIII-4) depicting a significant increase for the exon 8 and 9 probes. (B) Long distance PCR amplification with forward and reverse primer lo-cated both in exon 8 yielded a fragment of approximately 8 kb in affected family members only (line 1: patient A, line 2: patient B, line 3: not affected control, line 4: no template control). (C) Sequencing of the amplicon shown in (B) reveals the genomic breakpoint and demonstrates that exon 7 is duplicated as well. The numbers above the arrows give the genomic position (human genome assembly, build 36.3), black capital letters indicate re-tained sequences of intron 9-10 and intron 6-7, respectively, while small red letters show the deleted sequence portion. The underlined 3 nucleotides can be found as reminiscence of the deletion event.B...TGAGTGGTGATAAcaa...tacaaaaaattagctgIntron 6-7gttgggtatac...gcaGGCGTGGTGGCAGG...Intron 9-10T G A G T G G TG A T A A G C A G G C G T G G TG G C A G G194840568194832822patient AA12348 kbp1,41,611,2atioRa0,60,80,20,40OPA1 Exon 01AOPA1 Exon 01BOPA1 Exon 02AOPA1 Exon 02BOPA1 Exon 05AOPA1 Exon 05BOPA1 Exon 06Exon 08OPA1Exon 09OPA1Exon 10OPA1Exon 13OPA1Exon 14OPA1Exon 17OPA1Exon 18OPA1Exon 20OPA1Exon 21OPA1Exon 22OPA1Exon 23OPA1Exon 24OPA1Exon 26OPA1Exon 27OPA1Intron 28OPA1Exon 29OPA1RDS Exon 01RDS Exon 02ARDS Exon 02BExon 03RDSVMD2 Exon 01Exon 02VMD2Exon 03VMD2Exon 04VMD2Exon 05VMD2Exon 06VMD2Exon 07VMD2Exon 08VMD2Exon 09VMD2Exon 10VMD2Exon 11VMD2ControlControlControlControlC
Page 7
Fuhrmann et al. Molecular Neurodegeneration 2010, 5:25http://www.molecularneurodegeneration.com/content/5/1/25Page 7 of 13additional experiments would be necessary to define pre-cisely the single bands. However, according to its size theP/M band could represent either OPA1 precursor ormutant protein or both. Densitometric analysis of the dif-ferent isoforms revealed that the S3 and S4 isoforms werereduced to approx. 50% of their levels in controls whileL1, L2 and S5 were reduced not that strong (Figure 6B).The overall OPA1 protein level (excluding the precursor/mutant form) was reduced to approx. 66%. The observedchanges in the relative abundance of the different OPA1isoforms implicate altered proteolytic processing ofOPA1 in mitochondria (e.g. reduced processing when L1and S3 constitutes splice variant 7 and L2, S4 and S5 con-stitute splice variants 1 and 5 as suggested before [39,40]).Since we found a reduction of all OPA1 isoforms inrelation to actin, we assessed several other mitochondrialproteins (Tom40, CoxIX, ATPase β, Hsp60) in total celllysates by westernblot analyses and subsequent densito-metric measurements to clarify whether other mitochon-drial proteins are reduced in their abundance as well. Incomparison to four control fibroblast cell lines we found areduction of all tested mitochondrial proteins of 20 to55% consistent in both patients' fibroblast cell lines (Fig-ure 6C &6Ds). This finding shows that together withOPA1 other mitochondrial proteins seem to be reducedas well.DiscussionHere we report the identification of a large intragenicduplication in the OPA1 gene in a historic family withADOA that was first described in 1954 and that was deci-sive for the definition of ADOA as a separate diseaseentity [8]. The updated pedigree now comprises a total of216 family members of whom 57 are affected or werereported to suffer from ADOA. The family thus consti-tutes probably one of the largest reported in the ADOAliterature and may enable further investigations of sec-ondary factors that modulate disease expression and pen-etrance. Based on our genotyping results we calculated apenetrance of 88% in this family. This is in line with a pre-vious thorough study of several Australian families withADOA that reports also a penetrance of 88% and 82.5%for the fully ascertained sibships, respectively [14,15].Notably, all unaffected mutation carriers in the familypresented herein are females and we previously reportedtwo more unaffected female mutation carriers in otherfamilies, one with the same exon 7-9 duplication muta-tion and one with a complete deletion of the OPA1 gene[28]. All of these unaffected mutation carriers haveaffected siblings or children which share by definition thesame mtDNA haplogroup. This shows that the mtDNAbackground does not play a major role in governing pene-trance in ADOA and we would like to speculate insteadabout an x-chromosomal inherited QTL or environmen-tal factors. In summary, the studied family presents awell-defined clinical picture for non-syndromic ADOA.There is no evidence of associated extra-ocular symp-toms, either in the thorough clinical investigations by W.Jaeger or in our recent re-examinations.Figure 4 Assessment of the mitochondrial network morphology. (A) Representative micrographs of cells with different mitochondrial network morphology. The different categories are indicated. Arrow heads accentuate tubular structures in the mitochondrial network classified as "intermedi-ate". Scale bar, 20 μm. (B) Cells have been classified by two independent qualified persons in a blinded experiment with a third person taking the micrographs (n > 100 for each group). The number of cells that exhibit tubular mitochondrial network morphology is reduced in patients' fibroblasts compared to a control. (C) Amplification of a 12.5 kb mtDNA fragment did not reveal any specific mtDNA deletions for patients or controls. Ntc - non template control.cells [%]20406080100controlpatient Apatient B0fragmentedintermediatetubularBAtubularintermediatefragmented12.520.51234Ccontrolpatient Apatient Bntc
Page 8
Fuhrmann et al. Molecular Neurodegeneration 2010, 5:25http://www.molecularneurodegeneration.com/content/5/1/25Page 8 of 13In contrast to other inherited blinding diseases, inADOA the affected gene is expressed ubiquitously[21,41]. With limitations, this offers the possibility tostudy the consequences of the disease causing mutationon cellular level in any tissue or cell line derived frompatients. For the study presented here, we have estab-lished two fibroblast cell lines from skin biopsies of thetwo affected brothers at the age of 69 and 66, respectivelyand compared them with four different control fibroblastcell lines with the same passage number. Two of the latterwere obtained from young children's circumcision speci-mens whereas the other two derived from skin biopsies ofa middle-aged donor and a donor in his late sixties, whichserved as the primary control. All donors were healthyindividuals. Unfortunately we were not able to obtain skinbiopsies of the unaffected brothers. However, to ourknowledge, donor age has only a minor effect on the rep-licative lifespan of fibroblasts, in contrast to passagenumbers of the cells and/or health status of the donors[42,43], which was both carefully controlled in this study.Donor age shows also only minor effects on mitochon-drial enzyme activity [44]. In agreement with this, wedidn't find any specific mtDNA deletions in patients' celllines or control cells that would refer to a prematureaging effect of the cells.Nevertheless, we found a more fragmented and lesstubular morphology of the mitochondrial network in thepatients' cell lines. Knockout mutants of the OPA1-homologue mgm1/msp1 in yeast show also fragmenta-tion of mitochondria and a reduction of mtDNA content[45,46]. A quite similar mitochondrial phenotype couldalso be observed by down-regulation of OPA1 expressionby RNA interference in HeLa cells, which caused frag-mentation of the mitochondrial network, loss of mito-chondrial membrane potential and disorganization of themitochondrial cristae [33,47]. Spinazzi and colleaguesalso reported fragmentation of the mitochondrial net-work in fibroblasts as well as myotubes from patients thatharbor a deletion in the GTPase domain of OPA1(c1410_144314del38) [48].We found reduced OPA1 protein levels in fibroblastsobtained from patients with non-syndromic ADOA thatharbor a 3-exon-duplication in the GTPase domain ofOPA1 when compared to controls. This is in line withprevious reports on other OPA1 mutations in patientswith ADOA, including the deletion in the GTPasedomain of OPA1 mentioned above, or Opa1 mouse mod-els, which all report reduced OPA1 protein levels, regard-less of the underlying OPA1 mutation [48-52]. Based onthis and the description of different families with non-syndromic ADOA that segregate heterozygous deletionsof the complete OPA1 gene [27,28], haploinsufficiency isbelieved to be a major pathomechanism in OPA1 associ-ated non-syndromic ADOA.On the other hand our data show that there might be anaccumulation of OPA1 transcripts in the patients' cells,which would be in accordance with the observed aug-mentation of the precursor/mutant protein (P/M band),but in contrast to the reduction of the other OPA1 iso-forms in these cells. Previously, we have reported reducedOpa1 protein levels in various tissues obtained fromOpa1 mutant mice. In these mice, the Opa1 transcriptlevels were not altered compared to littermate controls[49]. Taken together, there seems to be no correlation ofthe transcript levels and the protein levels for OPA1,which suggests that OPA1 is regulated on protein levels.Figure 5 Quantification of the different OPA1 transcripts derived from the mutant and the WT allele. (A) A clarifying scheme about the strategy for the relative quantification with boxes indicating the different amplicons of exon/exon junctions and their relative ratios if one assumes that both transcripts are equally abundant. (B) One would expect a ratio of exon 7-8 to exon 6-7 to exon 9-7 of 3 to 2 to 1 when transcripts of both alleles are equally abundant (expected); pa-tients clearly presented with reduced levels of exon 7-8 and 9-7 ampl-icons indicating an imbalance in favor of the transcripts derived from the WT allele (patient A & patient B); controls show no difference in the quantification of the exon 7-8 and 6-7 amplicons, proving that the method works very accurate. (C) The readout of a typical quantification of the exon 6-7 junction in relation to GAPDH shows a significant up regulation of OPA1 in patients' fibroblasts.exon6-7exon 6-7exon 7-8exon 9-7exon7-8exon9-7ABexpectedpatient Apatient Bcontrol123n.d.Ex9Ex6Ex7Ex8Ex10Ex9Ex6Ex7Ex8Ex10 Ex9Ex7Ex8WTc.678-984dup3066-77-86-77-87-89-7relative copiespatient Apatient Bcontrolcontrol1510Crelative copies
Page 9
Fuhrmann et al. Molecular Neurodegeneration 2010, 5:25http://www.molecularneurodegeneration.com/content/5/1/25Page 9 of 13This is supported by a study on OPA1 in conjunctionwith heart failure that also found no correlation of OPA1transcript and OPA1 protein levels [53]. In conclusion,this suggests that this 102 amino acid duplication in theGTPase domain of OPA1 leads to altered OPA1 functionrather than haploinsufficiency. Taking into account thatloss of one complete OPA1 allele leads to a comparableclinical picture (i.e. non-syndromic ADOA) [27,28], onemight speculate that OPA1 function is reduced by thisparticular mutation. Then why and how are OPA1 proteinlevels reduced in ADOA? The ubiquitine-proteasomepathway is not involved in the degradation of OPA1 ascould be shown in Opa1 mutant mice [49].Recently, it has been proposed that mitochondrial fis-sion and selective fusion serves as mitochondrial qualitycontrol. Experiments with photo labeled mitochondriarevealed that selective fusion separates functional fromdysfunctional mitochondria [54-56]. Dysfunctional mito-chondria display a reduced membrane potential ΔΨ [56],which results in increased proteolytic cleavage of OPA1[57-62]. As a consequence dysfunctional mitochondriaare not capable to fuse anymore and are degraded byautophagy [54,56]. According to this, a change in OPA1function which reduces mitochondrial capability to fuseshould also result in increased mitochondrial degradationby autophagy. Indeed, increase in autophagy has beenobserved in the optic nerve of Opa1 mutant mice [63].Figure 6 Reduced OPA1 protein amount and altered processing in fibroblasts from patients with ADOA. (A) Quantitative western blot against OPA1 (upper panel) and actin (lower panel) of the indicated probes. Arrows indicate the corresponding bands; P/M: precursor/mutant protein, L1 to S5 bands according to Duvezin-Caubet et al. 2007 [39]. (B) Densitometric evaluation of the different OPA1 isoforms from the western blot shown in A as mean ± S.D. from patients vs. control. OPA1 protein levels are reduced in the patients' cell lines and the unequal reduction of the different corre-sponding OPA1 isoforms suggest altered proteolytic cleavage of OPA1. (C) Quantitative western blot against indicated mitochondrial proteins in total cell lysates of the indicated probes. (D) Densitometric quantification of the western blot shown in C as mean ± S.D. from patients vs. controls. The analysis shows that all tested mitochondrial proteins are less abundant in patients' cell lines compared to controls.average [%]Opa1Tom40CoxIVATPaseHsp60204060801000ABActinOpa1Tom40CoxIVATPaseHsp60patient Apatient Bcontrol 3control 1control 4control 2L1 L2S3S4S5 P/M10050average [%]CDL1L2S3S4S5P/M110 kDa85 kDa50 kDaα- ctinAα-Opa1patient Apatient Bcontrol 1patientscontrolpatientscontrols
Page 10
Fuhrmann et al. Molecular Neurodegeneration 2010, 5:25http://www.molecularneurodegeneration.com/content/5/1/25Page 10 of 13This might be an explanation for the discrepancy ofOPA1 transcript and OPA1 protein levels. In addition,this would be in agreement with the observed fragmentedmitochondrial network, the reduced proteolytic process-ing and the reduction of OPA1 and other mitochondrialproteins in the cell lines from patients with ADOA foundin this study. Altered mitochondrial quality control wouldalso explain mitochondrial dysfunction and/or the accu-mulation of mtDNA deletions as described for patientswith ADOA [37].Still it needs to be shown, if loss of OPA1 functionimpairs mitochondrial function, which leads to more dys-functional mitochondria and subsequent removal ofthese dysfunctional mitochondria, or if loss of OPA1function impairs mitochondrial quality control directly,which then leads to increased clearance of apparentlyfunctional mitochondria, too.MethodsPatients and SubjectsWe included 53 family members in this study who havebeen recruited in a field study in the mid-nineties. Anupdate of the pedigree was obtained from recent personalinterviews with various family members. Two of thepatients underwent a full ophthalmological examinationat the University Ophthalmic Hospital in Tuebingen in2008. Informed consent was obtained for blood samplesand skin biopsies. The study was performed in accor-dance to the tenets of the Declaration of Helsinki and wasapproved by the local ethics committee.Cell culture and staining of the mitochondrial networkFibroblast cell lines were established from skin biopsiesfrom patients and compared to 4 different healthy controlcell lines with the same passage number. All cell lineswere free of mycoplasma contaminations. Cells werepropagated in minimal essential medium supplementedwith 10% fetal calf serum (FCS) and antibiotics understandard conditions. For assessment of the mitochondrialnetwork structure, cells were cultured for 24 hours in glu-cose-free DMEM supplemented with either 10% FCS and0.45% (w/v) glucose or 10% FCS, 5 mM galactose and 5mM pyruvate prior to staining with 10 nM Mitotrackerfor 45 min at 37°C in the corresponding medium. Themorphology of the mitochondrial network was qualita-tively assessed by two independent people who did notknow either the condition or the origin of the pictureswhich had been taken by a third person with a fluores-cence microscope (AXIO Imager Z1 with ApopTome,Zeiss, Germany).Isolation of DNA and RNAGenomic DNA was extracted from venous blood samplesapplying standard salting-out procedure, or from culti-vated fibroblasts applying High Pure PCR Template Prep-aration Kit (Roche, Mannheim, Germany). Total RNA wasisolated from whole blood drawn in PAXgene tubes usingthe PAXgene blood RNA kit (Qiagen, Hilden, Germany).RNA from fibroblasts was obtained using the RNeasy Kit(Qiagen) and subsequently treated with DNAse (NewEngland Biolabs, Frankfurt, Germany). RNA was reversetranscribed using either the Long Range 2 Step RT-PCRKit (Qiagen) or the Super Script III First-Strand SynthesisSystem for RT-PCR (Invitrogen, Carlsbad, USA) witholigo-dT-primers or random hexamers.Multiplex ligation-dependent probe amplification (MLPA)MLPA reactions were performed using the P97 and P229kit from MRC-Holland (Amsterdam, The Netherlands)following the manufacturers' instructions and applying anexactly defined amount of sample DNA (100 ng). Reac-tions were performed in triplicates and compared withparallel processed controls. The amplified MLPA prod-ucts were separated on a 3100 Capillary Sequencer(Applied Biosystems, Darmstadt, Germany) and analysedusing the Coffalyser spreadsheet (control probe analysis,MRC-Holland).Breakpoint identification and segregation analysisThe exon 7-9 duplication was confirmed by long distancePCR, carried out with the TaKaRa LA Taq™ kit (TAKARABIO INC., Shiga, Japan) applying both forward andreverse oligonucleotides placed in Exon 8, Exon 8L: 5'-GAT GTT CTC TCT GAT TAT GAT GCC-3' and Exon8R: 5'-CAG ATG ATC TTG CGT ATT ATA ACT GG-3'.The duplication breakpoint was identified by directsequencing of the ExoSAP (USB, Staufen, Germany) puri-fied long distance PCR product applying a primer walk-ing strategy. Sequencing was done employing BigDyeTerminator Chemistry 1.1 (Applied Biosystems) andproducts separated on an ABI 3100 DNA Sequencer. Seg-regation analysis was done applying a duplication specificPCR assay with oligonucleotides: Dupl-Ex7-9-L: 5'-TTCTTG ACA GGT TTG ATA TGG AGA-3' and Dupl-Ex7-9-R: 5'-AAT TAA TGC ATG TCA GTG TCA CCT-3'. Inthis case PCR was performed using standard Taq poly-merase and a short extension time of not more than 30sec. Numbering of exons and introns was based on OPA1transcript variant 1 (NM_015560).Real-Time PCRReal Time PCR was performed using cDNA generatedfrom total fibroblast RNA with the following oligonucle-otides: RT-Ex6-7-f: 5'-AAG AAC TTC TGC ACA CTCAGT TGA A-3' & RT-Ex6-7-r: 5'-TTC TAA TCG TTCCAA GAT TCT CTG ATA C-3'; RT-Ex7-8-f: 5'-GGCATT CAT CAT AGA AAG CTT AAG AA-3' & RT-Ex7-8-r: 5'-AAG AAC TTC AGA ATA CAT GTC AAT CAAAG-3'; RT-Ex9-7-f: 5'-GGA GAT GAT GAC ACG TTC
Page 11
Fuhrmann et al. Molecular Neurodegeneration 2010, 5:25http://www.molecularneurodegeneration.com/content/5/1/25Page 11 of 13TCC A-3' & RT-Ex9-7-r: 5'-TTC CAA GAT TCT CTGATA CTT CAA CTT AA-3'. Assays were carried out withthe Power SYBR Green PCR Master Mix on a real time7500 PCR instrument (Applied Biosystems). Relative copynumbers were calculated applying the ΔΔCt method withGAPDH as reference. The pyrosequencing assay has beendescribed in details before [38].Amplification of mtDNAA 12.5 kb mtDNA fragment covering most of the codingregion was amplified using TaKaRa LA Taq reagents(TAKARA BIO INC., Shiga, Japan) under recommendedcycling PCR conditions and applying the followingprimer pair: MIT-LR1F: 5'-ACA ACC CTT CGC TGACGC CAT A-3'; MIT-LR2R: 5'-GGT GGT ACC CAAATC TGC TTC C-3'.Quantitative westernblot analysisFor quantitative Westernblot analysis 15 μg total proteinsolubilized in RIPA was separated on NuPAGE 4-12%Bis-Tris Gels (Invitrogen). Blots were processed forimmunodecoration with antibodies against OPA1 (BD-Transduction, LA, CA, USA), Tom40 (a kind gift of D.Rapaport, Tuebingen), CoxIV (Abcam, Cambridge, MA,USA), ATPase β (BD-Transduction), Hsp60 (Stressgen,Victoria, Canada) or actin (Chemicon, Temecula, CA,USA) applying the ECL chemiluminescence system(Pierce, Rockford, IL, USA). According bands were quan-tified using ImageJ http://rsb.info.nih.gov/ij/. All proteinlevels were normalized to actin levels as loading control.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsNF performed all genetic analyses including real time experiments; he assistedin the study design, interpreted the data and wrote the paper. SS contributedto the cDNA analysis. YK extracted patients' and control fibroblasts propagatedthem and provided antibodies. BLK performed all ophthalmological examina-tions. CA and GA did the field study and provided patients' DNA samples. EZcritically read through the paper. BW critically read through the paper. MVAdesigned the study, performed all western blot analyses as well as the mito-chondrial network analyses, he interpreted the data and wrote the paper. Allauthors read and approved the final manuscript.AcknowledgementsIn memoriam Prof.Wolfgang Jäger. We are indebted to the members of this family for their friendly cooperation and support of this study. We furthermore thank Tao Wei for help with the morphological assessment of the mitochon-drial network in stained fibroblasts. We appreciate helpful discussion and com-ments from Jörg Tatzelt and Konstanze Winklhofer. This work was supported in part by the Friedrich-Spicker-Stiftung, Essen, Germany and the Fritz-Thyssen-Stiftung Köln, Germany. We thank Judy Howell for carefully reading through the manuscript.Author Details1Molecular Genetics Laboratory, Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tuebingen, Germany, 2Department of Dermatology, Eberhard Karls University Tuebingen, Germany, 3Centre for Ophthalmology, Institute for Ophthalmic Research, University of Tuebingen, Germany, 4Department of Neurodegeneration, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany and 5Section Molecular Neurogenetics, Department of Neurology, Goethe University, Frankfurt am Main, GermanyReferences1.Leber T: Über hereditäre und congenital-angelegte Sehnervenleiden. Graefe's Archive for clinical and experimental ophthalmology 1871, 17:249-291.2.Cibis P: Beitrag zur Vererbung der familiären Sehnervenatrophie (Lebersche Krankheit). Klin Mbl Augenheilk 1939, 102:.3. Friemann W: Erbliche Opticusatrophie bei Frauen. Graefe's Archive for Clinical and Experimental Ophthalmology 1949, 149:.4.Stählin S: Gibt es eine erbliche Sehnervenatrophie auβer der Leberschen Atrophie? Arch Augenheilk 1931, 104:.5. Isayama H: Über eine Art von erheblicher Sehnervenatrophie. Acta Soc ophthalm jap 1934, 38:.6.Kawakami R: Beiträge zur Vererbung der familiären Sehnervenatrophie. Graefe's Archive for Clinical and Experimental Ophthalmology 1926, 116:.7.Lodberg CV, Lund A: Hereditery Optic Atrophy With Dominant Transmission. Three Danish Families. Acta Ophthalmol (Copenh) 1950, 28:.8.Jaeger W: Hereditary optic atrophy with dominant transmission; with special reference to the associated color-sense disorder. Albrecht Von Graefes Arch Ophthalmol 1954, 155:457-484.9.Jaeger W: Hereditary optic atrophies in childhood. J Genet Hum 1966, 15:312-321.10. Lorenz B: Hereditary optic atrophy. Ophthalmologe 1994, 91:831-850.11. Caldwell JB, Howard RO, Riggs LA: Dominant juvenile optic atrophy. A study in two families and review of hereditary disease in childhood. Arch Ophthalmol 1971, 85:133-147.12. Kline LB, Glaser JS: Dominant optic atrophy. The clinical profile. Arch Ophthalmol 1979, 97:1680-1686.13. Roggeveen HC, de Winter AP, Went LN: Studies in dominant optic atrophy. Ophthalmic Paediatr Genet 1985, 5:103-109.14. Cohn AC, Toomes C, Potter C, Towns KV, Hewitt AW, Inglehearn CF, Craig JE, Mackey DA: Autosomal dominant optic atrophy: penetrance and expressivity in patients with OPA1 mutations. Am J Ophthalmol 2007, 143:656-662.15. Toomes C, Marchbank NJ, Mackey DA, Craig JE, Newbury-Ecob RA, Bennett CP, Vize CJ, Desai SP, Black GC, Patel N, Teimory M, Markham AF, Inglehearn CF, Churchill AJ: Spectrum, frequency and penetrance of OPA1 mutations in dominant optic atrophy. Hum Mol Genet 2001, 10:1369-1378.16. Li Y, Deng T, Tong Y, Peng S, Dong B, He D: Identification of two novel OPA1 mutations in Chinese families with autosomal dominant optic atrophy. Mol Vis 2008, 14:2451-2457.17. Johnston PB, Gaster RN, Smith VC, Tripathi RC: A clinicopathologic study of autosomal dominant optic atrophy. Am J Ophthalmol 1979, 88:868-875.18. Kjer P, Jensen OA, Klinken L: Histopathology of eye, optic nerve and brain in a case of dominant optic atrophy. Acta Ophthalmol (Copenh) 1983, 61:300-312.19. Heiduschka P, Schnichels S, Fuhrmann N, Hofmeister S, Schraermeyer U, Wissinger B, Alavi MV: Electrophysiological and histologic assessment of retinal ganglion cell fate in a mouse model for OPA1-associated autosomal dominant optic atrophy. Invest Ophthalmol Vis Sci 2010, 51:1424-1431.20. Eiberg H, Kjer B, Kjer P, Rosenberg T: Dominant optic atrophy (OPA1) mapped to chromosome 3q region. I. Linkage analysis. Hum Mol Genet 1994, 3:977-980.21. Alexander C, Votruba M, Pesch UE, Thiselton DL, Mayer S, Moore A, Rodriguez M, Kellner U, Leo-Kottler B, Auburger G, Bhattacharya SS, Wissinger B: OPA1, encoding a dynamin-related GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28. Nat Genet 2000, 26:211-215.22. Delettre C, Lenaers G, Griffoin JM, Gigarel N, Lorenzo C, Belenguer P, Pelloquin L, Grosgeorge J, Turc-Carel C, Perret E, Astarie-Dequeker C, Lasquellec L, Arnaud B, Ducommun B, Kaplan J, Hamel CP: Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy. Nat Genet 2000, 26:207-210.23. Kerrison JB, Arnould VJ, Ferraz Sallum JM, Vagefi MR, Barmada MM, Li Y, Zhu D, Maumenee IH: Genetic heterogeneity of dominant optic Received: 9 August 2009 Accepted: 14 June 2010 Published: 14 June 2010This article is available from: http://www.molecularneurodegeneration.com/content/5/1/25© 2010 Fuhrmann et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Molecular Neurodegeneration 2010, 5:25
Page 12
Fuhrmann et al. Molecular Neurodegeneration 2010, 5:25http://www.molecularneurodegeneration.com/content/5/1/25Page 12 of 13atrophy, Kjer type: Identification of a second locus on chromosome 18q12.2-12.3. Arch Ophthalmol 1999, 117:805-810.24. Barbet F, Hakiki S, Orssaud C, Gerber S, Perrault I, Hanein S, Ducroq D, Dufier JL, Munnich A, Kaplan J, Rozet JM: A third locus for dominant optic atrophy on chromosome 22q. J Med Genet 2005, 42:e1.25. Reynier P, Amati-Bonneau P, Verny C, Olichon A, Simard G, Guichet A, Bonnemains C, Malecaze F, Malinge MC, Pelletier JB, Calvas P, Dollfus H, Belenguer P, Malthièry Y, Lenaers G, Bonneau D: OPA3 gene mutations responsible for autosomal dominant optic atrophy and cataract. J Med Genet 2004, 41:e110.26. Ferre M, Amati-Bonneau P, Tourmen Y, Malthiery Y, Reynier P: eOPA1: an online database for OPA1 mutations. Hum Mutat 2005, 25:423-428.27. Marchbank NJ, Craig JE, Leek JP, Toohey M, Churchill AJ, Markham AF, Mackey DA, Toomes C, Inglehearn CF: Deletion of the OPA1 gene in a dominant optic atrophy family: evidence that haploinsufficiency is the cause of disease. J Med Genet 2002, 39:e47.28. Fuhrmann N, Alavi MV, Bitoun P, Woernle S, Auburger G, Leo-Kottler B, Yu-Wai-Man P, Chinnery P, Wissinger B: Genomic rearrangements in OPA1 are frequent in patients with autosomal dominant optic atrophy. J Med Genet 2009, 46:136-144.29. Olichon A, Emorine LJ, Descoins E, Pelloquin L, Brichese L, Gas N, Guillou E, Delettre C, Valette A, Hamel CP, Ducommun B, Lenaers G, Belenguer P: The human dynamin-related protein OPA1 is anchored to the mitochondrial inner membrane facing the inter-membrane space. FEBS Lett 2002, 523:171-176.30. Chen H, Detmer SA, Ewald AJ, Griffin EE, Fraser SE, Chan DC: Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development. J Cell Biol 2003, 160:189-200.31. Cipolat S, Martins de Brito O, Dal Zilio B, Scorrano L: OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proc Natl Acad Sci USA 2004, 101:15927-15932.32. Frezza C, Cipolat S, Martins de Brito O, Micaroni M, Beznoussenko GV, Rudka T, Bartoli D, Polishuck RS, Danial NN, De Strooper B, Scorrano L: OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion. Cell 2006, 126:177-189.33. Olichon A, Baricault L, Gas N, Guillou E, Valette A, Belenguer P, Lenaers G: Loss of OPA1 perturbates the mitochondrial inner membrane structure and integrity, leading to cytochrome c release and apoptosis. J Biol Chem 2003, 278:7743-7746.34. Cipolat S, Rudka T, Hartmann D, Costa V, Serneels L, Craessaerts K, Metzger K, Frezza C, Annaert W, D'Adamio L, Derks C, Dejaegere T, Pellegrini L, D'Hooge R, Scorrano L, De Strooper B: Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling. Cell 2006, 126:163-175.35. Amati-Bonneau P, Valentino ML, Reynier P, Gallardo ME, Bornstein B, Boissière A, Campos Y, Rivera H, de la Aleja JG, Carroccia R, Iommarini L, Labauge P, Figarella-Branger D, Marcorelles P, Furby A, Beauvais K, Letournel F, Liguori R, La Morgia C, Montagna P, Liguori M, Zanna C, Rugolo M, Cossarizza A, Wissinger B, Verny C, Schwarzenbacher R, Martín MA, Arenas J, Ayuso C, Garesse R, Lenaers G, Bonneau D, Carelli V: OPA1 mutations induce mitochondrial DNA instability and optic atrophy 'plus' phenotypes. Brain 2008, 131:338-351.36. Hudson G, Amati-Bonneau P, Blakely EL, Stewart JD, He L, Schaefer AM, Griffiths PG, Ahlqvist K, Suomalainen A, Reynier P, McFarland R, Turnbull DM, Chinnery PF, Taylor RW: Mutation of OPA1 causes dominant optic atrophy with external ophthalmoplegia, ataxia, deafness and multiple mitochondrial DNA deletions: a novel disorder of mtDNA maintenance. Brain 2008, 131:329-337.37. Yu-Wai-Man P, Griffiths PG, Gorman GS, Lourenco CM, Wright AF, Auer-Grumbach M, Toscano A, Musumeci O, Valentino ML, Caporali L, Lamperti C, Tallaksen CM, Duffey P, Miller J, Whittaker RG, Baker MR, Jackson MJ, Clarke MP, Dhillon B, Czermin B, Stewart JD, Hudson G, Reynier P, Bonneau D, Marques W Jr, Lenaers G, McFarland R, Taylor RW, Turnbull DM, Votruba M, Zeviani M, Carelli V, Bindoff LA, Horvath R, Amati-Bonneau P, Chinnery PF: Multi-system neurological disease is common in patients with OPA1 mutations. Brain 2010, 133:771-786.38. Schimpf S, Fuhrmann N, Schaich S, Wissinger B: Comprehensive cDNA study and quantitative transcript analysis of mutant OPA1 transcripts containing premature termination codons. Hum Mutat 2007.39. Duvezin-Caubet S, Koppen M, Wagener J, Zick M, Israel L, Bernacchia A, Jagasia R, Rugarli EI, Imhof A, Neupert W, Langer T, Reichert AS: OPA1 processing reconstituted in yeast depends on the subunit composition of the m-AAA protease in mitochondria. Mol Biol Cell 2007, 18:3582-3590.40. Olichon A, Elachouri G, Baricault L, Delettre C, Belenguer P, Lenaers G: OPA1 alternate splicing uncouples an evolutionary conserved function in mitochondrial fusion from a vertebrate restricted function in apoptosis. Cell Death Differ 2007, 14:682-692.41. Bette S, Schlaszus H, Wissinger B, Meyermann R, Mittelbronn M: OPA1, associated with autosomal dominant optic atrophy, is widely expressed in the human brain. Acta Neuropathol (Berl) 2005, 109:393-399.42. Goldstein S, Moerman EJ, Soeldner JS, Gleason RE, Barnett DM: Chronologic and physiologic age affect replicative life-span of fibroblasts from diabetic, prediabetic, and normal donors. Science 1978, 199:781-782.43. Cristofalo VJ, Allen RG, Pignolo RJ, Martin BG, Beck JC: Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation. Proc Natl Acad Sci USA 1998, 95:10614-10619.44. Schroeder F, Goetz I, Roberts E: Age-related alterations in cultured human fibroblast membrane structure and function. Mech Ageing Dev 1984, 25:365-389.45. Sesaki H, Southard SM, Yaffe MP, Jensen RE: Mgm1p, a dynamin-related GTPase, is essential for fusion of the mitochondrial outer membrane. Mol Biol Cell 2003, 14:2342-2356.46. Wong ED, Wagner JA, Scott SV, Okreglak V, Holewinske TJ, Cassidy-Stone A, Nunnari J: The intramitochondrial dynamin-related GTPase, Mgm1p, is a component of a protein complex that mediates mitochondrial fusion. J Cell Biol 2003, 160:303-311.47. Lee YJ, Jeong SY, Karbowski M, Smith CL, Youle RJ: Roles of the mammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opa1 in apoptosis. Mol Biol Cell 2004, 15:5001-5011.48. Spinazzi M, Cazzola S, Bortolozzi M, Baracca A, Loro E, Casarin A, Solaini G, Sgarbi G, Casalena G, Cenacchi G, Malena A, Frezza C, Carrara F, Angelini C, Scorrano L, Salviati L, Vergani L: A novel deletion in the GTPase domain of OPA1 causes defects in mitochondrial morphology and distribution, but not in function. Hum Mol Genet 2008, 17:3291-3302.49. Alavi MV, Bette S, Schimpf S, Schuettauf F, Schraermeyer U, Wehrl HF, Ruttiger L, Beck SC, Tonagel F, Pichler BJ, Knipper M, Peters T, Laufs J, Wissinger B: A splice site mutation in the murine Opa1 gene features pathology of autosomal dominant optic atrophy. Brain 2007, 130:1029-1042.50. Davies VJ, Hollins AJ, Piechota MJ, Yip W, Davies JR, White KE, Nicols PP, Boulton ME, Votruba M: Opa1 deficiency in a mouse model of autosomal dominant optic atrophy impairs mitochondrial morphology, optic nerve structure and visual function. Hum Mol Genet 2007, 16:1307-1318.51. Olichon A, Landes T, Arnauné-Pelloquin L, Emorine LJ, Mils V, Guichet A, Delettre C, Hamel C, Amati-Bonneau P, Bonneau D, Reynier P, Lenaers G, Belenguer P: Effects of OPA1 mutations on mitochondrial morphology and apoptosis: relevance to ADOA pathogenesis. J Cell Physiol 2007, 211:423-430.52. Zanna C, Ghelli A, Porcelli AM, Karbowski M, Youle RJ, Schimpf S, Wissinger B, Pinti M, Cossarizza A, Vidoni S, Valentino ML, Rugolo M, Carelli V: OPA1 mutations associated with dominant optic atrophy impair oxidative phosphorylation and mitochondrial fusion. Brain 2008, 131:352-367.53. Chen L, Gong Q, Stice JP, Knowlton AA: Mitochondrial OPA1, apoptosis, and heart failure. Cardiovasc Res 2009, 84:91-99.54. Mouli PK, Twig G, Shirihai OS: Frequency and selectivity of mitochondrial fusion are key to its quality maintenance function. Biophys J 2009, 96:3509-3518.55. Twig G, Hyde B, Shirihai OS: Mitochondrial fusion, fission and autophagy as a quality control axis: the bioenergetic view. Biochim Biophys Acta 2008, 1777:1092-1097.56. Twig G, Elorza A, Molina AJ, Mohamed H, Wikstrom JD, Walzer G, Stiles L, Haigh SE, Katz S, Las G, Alroy J, Wu M, Py BF, Yuan J, Deeney JT, Corkey BE, Shirihai OS: Fission and selective fusion govern mitochondrial segregation and elimination by autophagy. Embo J 2008, 27:433-446.57. Ehses S, Raschke I, Mancuso G, Bernacchia A, Geimer S, Tondera D, Martinou JC, Westermann B, Rugarli EI, Langer T: Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1. J Cell Biol 2009, 187:1023-1036.58. Head B, Griparic L, Amiri M, Gandre-Babbe S, van der Bliek AM: Inducible proteolytic inactivation of OPA1 mediated by the OMA1 protease in mammalian cells. J Cell Biol 2009, 187:959-966.
Page 13
Fuhrmann et al. Molecular Neurodegeneration 2010, 5:25http://www.molecularneurodegeneration.com/content/5/1/25Page 13 of 1359. Duvezin-Caubet S, Jagasia R, Wagener J, Hofmann S, Trifunovic A, Hansson A, Chomyn A, Bauer MF, Attardi G, Larsson NG, Neupert W, Reichert AS: Proteolytic processing of OPA1 links mitochondrial dysfunction to alterations in mitochondrial morphology. J Biol Chem 2006, 281:37972-37979.60. Griparic L, Kanazawa T, van der Bliek AM: Regulation of the mitochondrial dynamin-like protein Opa1 by proteolytic cleavage. J Cell Biol 2007, 178:757-764.61. Ishihara N, Fujita Y, Oka T, Mihara K: Regulation of mitochondrial morphology through proteolytic cleavage of OPA1. Embo J 2006, 25:2966-2977.62. Song Z, Chen H, Fiket M, Alexander C, Chan DC: OPA1 processing controls mitochondrial fusion and is regulated by mRNA splicing, membrane potential, and Yme1L. J Cell Biol 2007, 178:749-755.63. White KE, Davies VJ, Hogan VE, Piechota MJ, Nichols PP, Turnbull DM, Votruba M: OPA1 deficiency associated with increased autophagy in retinal ganglion cells in a murine model of dominant optic atrophy. Invest Ophthalmol Vis Sci 2009, 50:2567-2571.doi: 10.1186/1750-1326-5-25Cite this article as: Fuhrmann et al., Solving a 50 year mystery of a missing OPA1 mutation: more insights from the first family diagnosed with auto-somal dominant optic atrophy Molecular Neurodegeneration 2010, 5:25


text:
use the following search parameters to narrow your results:
e.g. subreddit:aww site:imgur.com dog
subreddit:aww site:imgur.com dog
see the search faq for details.
advanced search: by author, subreddit...
A subreddit for folks who live in the Valley of the Sun to post job openings or job inquiries.
Please include a [HIRING] or [FOR HIRE] tag with your post. Also, please indicate where in the greater Phoenix metropolitan area you are located.
Let's get to work!
This is an archived post. You won't be able to vote or comment.
[FOR HIRE] Commercial and residential electrician. (self.phxjobs)
submitted 3 years ago by woofimaction
We install ceiling fans, switches, receptacles, outdoor and indoor lighting, bathroom lighting, bathroom exhaust fans, washer, dryer and stove receptacles, doorbells, motion sensor lights, rewire houses, install panels and subpanels, licensed with the company Arizona Construction Electronics, 10 years of electrical experience. PM me if you're interested.
there doesn't seem to be anything here
Use of this site constitutes acceptance of our User Agreement and Privacy Policy (updated). © 2016 reddit inc. All rights reserved.
REDDIT and the ALIEN Logo are registered trademarks of reddit inc.
π Rendered by PID 9623 on app-15 at 2016-02-06 01:03:44.854818+00:00 running 32fec41 country code: US.

subset: commoncrawl


text:
Galp CEO May Not Be Reappointed for Second Term, Diario Reports
Galp Energia SGPS SA Chief Executive Officer Manuel Ferreira de Oliveira may not be reappointed to the post for the next term, Diario Economico reported, citing unidentified people close to two of Galp's main shareholders.
Eni SpA of Italy, which owns a third of Galp, is not available to support the reappointment of Ferreira de Oliveira, the Portuguese newspaper said.
Esperaza, a venture that groups Angolan oil company Sonangol SA and Angolan investor Isabel dos Santos, is also not available to support naming the chief executive for another term, according to Diario. Esperaza owns a stake in the Amorim Energia BV venture, which holds 33 percent of Galp.


text:
Definitions for centerfielder
This page provides all possible meanings and translations of the word centerfielder
Princeton's WordNet
center fielder, centerfielder(noun)
the person who plays center field
A player in the centerfield position.
Find a translation for the centerfielder definition in other languages:
Select another language:
Discuss these centerfielder definitions with the community:
Word of the Day
Would you like us to send you a FREE new word definition delivered to your inbox daily?
Use the citation below to add this definition to your bibliography:
"centerfielder." Definitions.net. STANDS4 LLC, 2016. Web. 10 Feb. 2016. <http://www.definitions.net/definition/centerfielder>.


text:
Senior Member
Last Activity: Yesterday 11:46 PM
About Me
- About K.D.M.
- Service provider
- Netherlands - T-Mobile (Ben)
- City
- Wageningen
- Home country
- Netherlands
- Siemens C25 - (1999)
Samsung A500 - (2002)
Samsung E700 - (2004)
Nokia N80 - (2006)
Nokia N95 - (2007)
Nokia N95 8GB - (2008)
Sony Xperia X10 mini PRO - (CM 7.1) (september 2010)
LG Optimus 2X (GB 2.3.7 - CM 7.1) - (april 2011)
Galaxy Nexus Yakju Rooted (Parandoi Android 4.2.2) - (dec 2011)
Now im running a Silver HTC-One on Stock-Sense 4.1.2 - (June 2013)
- Siemens C25 - (1999)
Most Thanked
Post Summary
http://www.sxc.hu/pic/m/s/st/stevetaint/654009_jelly_beans_2.jpg :)
32 gig version €560,- in holland... 6 weeks of waiting... http://www.belsimpel.nl/Samsung/Samsung-Galaxy-Nexus-i9250-Black#volledige-specificaties
Root. And flash a stable rom. Then its verry safe. The downside is that when u drop it, the screen cracks really fast. The + side is that it is not that difficult to replace the gorilla glass by yourself. There is a nice vid of it on youtube. Se...
I use process monitor. I use. The widget to monitor my cpu frequentie and its temperature. It works good.. i got my phone on 1ghz and 900Mv. Its fast enough Sent from my LG-P990 using XDA App
Sadly the LG is not recognized by smartflash whatsoever. Plugging the phone in while holding voldown yields no results, nothing on screen and nothing smartflash can see. is the phone irreversibly bricked? Hade the same problemen. I just install...


text:
9 results
SORT BY Relevancy
Spirit Week: The Julin Jean Show
in Film
Spirit Week Continues: Spirit Camp Cheerleader Julin Jean will be on the show to discuss her films
Interview with Jess Julin Ibsen, Executive Vice President and CTO, Networks, TDC A/S, Denmark
in Technology
Listen here to an exclusive interview with Jess Julin Ibsen, TDC A/S Denmark, on 'Successful deployment of FTTH to support high quality services'
Connie Ragen Green interviews Kristina Julin-Stringer
in Business
Kristina is an entrepreneur whose site is http://www.AccidentalSmallBusinessOwner.com. She will talk about starting a business when you are over age 50.
A killer Camp for Cheerleading
in Movies
Kerry Beyer, Julin Jean and possibly others stop by to discuss his indie feature Spirit Camp. PLus the usual convention schedule, upcoming releases my musings.
2011 Fight for Air Climb with Heather from the American Lung Assocation
in Sports
Over 600 people will be climbing the Bank of America Tower located in down town Jacksonville.
With over 42 floors and 838 steps.
The American Lung Association offers this opportunity to lose weight and get healthy.
The main event is held on Feb 5th 2011.
Visit www.FightforAirClimb.org to learn about the event.
From A to B: How to move your family toward healthier eating
in Food
Ashley Julin, an Armed Forces Veteran, photographer and juicing expert, and Joanna Faillace, Certified Biblical Health Coach, Author & Speaker and Host of Cable TV's Super-Naturally Healthy Cooking Show. Eating healthy can be a challenge. Knowing which foods are safe to eat and making balanced choices for perfect health can be a science. Learn how to choose and prepare nutritious, fat-reducing, and sickness preventative foods.
Spirit Camp
Director Kerry Beyer with actresses Julin and Roxy Vandiver come on to talk about the movie. A great slasher film, one of the better ones in a long while.
Horror Movie Fans Indie Radio with Parrish Randall
in Movies
Tonight, independent film-maker Shane Cole of Masked Films joins us to talk about making feature films the indie way. As well, Melanie House and Julin from "Sweatshop" as well as a host of other Texas features join us to talk about the slashing, smashing Texas terror flick directed by Stacy Davidson and more!!!
Welding For Dummies
in Friends
Welding For Dummies
Today's Best Podcasts
Join Host Live Chats
- Lenny Melnick RotoExperts (9 chatters)
- Tracey and Friends (2 chatters)


text:
Does she annoy the hell out of anyone besides me? Is it the huge head? Huge mouth? Or the annoying way she says italian words? All of the above for me.
Posted 01/03/2013
She reminds me of all the other food network people, she tries too hard. I wish they would relax instead of trying to be cheerful and smiley all the time because it comes off fake and a little creepy. The girl that cooks all the spanish food is just like Giada, too.
Posted 01/03/2013
Giada and Jade are the same name. The former is the Italian version.
DD: Emmaline, 2011
DD: Emmaline, 2011
DS: Henry, 2014


text:
BHS Baseball Award Winners
The 2013 BHS varsity baseball award winners are, in front, Dillon Mullen: 4-Year Letterman, Most Improved; Bobby Cornett: Captain, MVP, Pitching Award, NSC Honorable Mention; and Wes Burkholder: Most Improved, 4-Year Letterman. In back are Bryce Cassel: Coaches Award, NSC Honorable Mention; Drew Coffel: Offense Award, Defense Award, All NSC; Eric Knepper: Captain, Offense Award, All NSC; Tyler Coffel: Captain, Coaches Award, All NSC; and Jacob Bennitt: Pitching Award, All NSC.
You Might Also Like


text:
Local events
Liberty Lake Community Memorial Day Salute and Breakfast
Presented by Liberty Lake Centennial Rotary Club. Funds raised at this event will support Honor Flight. Breakfast served 8-10 a.m. with all you-can-eat pancakes, ham, eggs and coffee. Veterans eat free. at 9 a.m. there will be a dedication ceremony for the Fallen Heroes Circuit Course in memory of U.S. Air Force Capt. Pinckney who was killed along with two airmen when their refueling plane went down over Kyrgyzstan in May 2013 and was stationed at Fairchild AFB. Dress is casual/semi-casual.


text:
Nauticam releases NA-XT1 Housing
Nauticam is shipping a housing for the Fujifilm X-T1 mirrorless camera. Many of the camera's lenses feature manual aperture control, and Nauticam has designed the housing around this requirement.
Nauticam is shipping a housing for the Fujifilm X-T1 mirrorless camera. Many of the camera's lenses feature manual aperture control, and Nauticam has designed the housing around this requirement.
Nauticam has announced their housing for the Nikon D810 SLR. The NA-D810 features access to the camera's Function (Fn) and Preview (Pv) buttons, which can be programmed for other functions, as well as a thumb lever giving access to the camera's Info button.
Nauticam has announced the imminent release of their housing for the Sony DSC RX100 III compact camera. It features the Nauticam vacuum system electronics and two bulkheads. Strobe triggering is available via fiber optic cable.
Nauticam has announced their new housing for the Panasonic Lumix DMC-GH4 camera. The NA-GH4 is somewhat customized for video use, with a specific thumb operated button for ISO adjustment, a lever for white balance and access to both control dials. The company has also announced an upgrade kit for owners of their GH3 housing.
Nauticam has released details of their new housing for the Sony a6000 mirrorless camera. The NA-a6000 features one-handed control of both command dials, and the option of rear button AF actuation. In addition, the housing sports a redesigned (and colored) video record button and the option of switching from EVF to LCD and vica versa.
Nauticam USA has announced Nauticam's housing for the Nikon D4s SLR. Released in conjunction with the new housing is Nauticam's Flash Trigger which allows (manual) strobe triggering via fiber optic with the D4. The new housing is also fitted with the electronics for the company's vacuum system and features improved focus knobs with a higher gear ratio.
Nauticam has "officially" announced their housing for the Olympus OM-D E-M10 mirrorless camera. It is a smaller housing than the one for the E-M1, and features a port lock similar to that of the Nauticam SLR housings. It is supplied with electronics for their vacuum system, and the housing allows access to both LCD and EVF as required. It has ports for fiber optic triggering of strobes.
Nauticam has announced details of their housing for the Sony A5000 mirrorless camera. The housing provides access to the camera's power zoom lever, giving lens zoom function without lens rings. Other features include the port lock lever from the E-M1 housing and built-in vacuum leak detection system.
Nauticam is shipping their housing for the Panasonic DMC-GX7 mirrorless camera. The NA-GX7 is shipping with their vacuum leak detection system and has "one-handed" access to both command dials, video record and EV adjustment. Nauticam also offer port support the majority of lens options for underwater use.
Nauticam has released details of their new housing for the Sony A7 and A7R cameras. The NA-7 has a switch that allows the user to choose either LCD or EVF and a Nikonos 5 pin bulkhead. Perhaps most interestingly, Nauticam are producing a Nikonos adaptor, allowing the use of the Sea & Sea 12mm fisheye or Nikonos 15mm lenses.


text:
Contact Us
Educational Communications
College of Agricultural & Life Sciences
Phone: (208) 885-7982
Fax: (208) 885-9046
Physical Address:
College of Agricultural & Life Sciences
University of Idaho 606 Rayburn St., Rm 10
Moscow, ID 83844-2332
Mailing Address:
Educational Communications
College of Agricultural & Life Sciences
University of Idaho
875 Perimeter Drive MS 2332
Moscow, ID 83844-2332
CALS Educational Communications
Magazine - Programs & People
Winter 2014Idaho's dairy industry ranks No. 3 nationally in milk production. University of Idaho Extension plays a big role in helping Idaho dairies operate efficiently and care for their herds. Kids, camps and gardens combine through the 4-H Youth Development program to yield good citizens. Garbanzo beans evolved from a good rotation crop to a moneymaker thanks to Americans' changing tastes and forward thinking by College of Agricultural and Life Sciences researchers and cooperating farmers. Other stories include CALS new dean John Foltz, an oilseed bonanza and even an offer for free seeds to celebrate the University of Idaho's 125th anniversary and UI Extension's centennial in 2014.
» To Magazine
Winter 2013
Winter 2012
Winter 2011


text:
The Brazilian Journal of Probability and Statistics is an official
publication of the Brazilian Statistical Association and is supported
by the Institute of Mathematical Statistics (IMS). Starting in 2012,
the Journal will publish four issues a year, in February, May, August
and December. The Journal publishes papers in applied probability,
applied statistics, computational statistics, mathematical statistics,
probability theory and stochastic processes.
Proceedings of XIX Brazilian School of Probability to be published at Brazilian Journal of Probability and Statistics seeks high quality research papers in applied or theoretical probability and stochastic processes. (Read more...)
web site contact: email@example.com


text:
1 Kings 1:9King James Version (KJV)
9 And Adonijah slew sheep and oxen and fat cattle by the stone of Zoheleth, which is by Enrogel, and called all his brethren the king's sons, and all the men of Judah the king's servants:
King James Version (KJV)
Already have a redemption code? Redeem it here.
Already redeemed? Refresh the page.
This is a content preview. Buy this title to see more.


text:
$1.99Maria Montessori was an Italian physician and educator best known for the philosophy of education that bears her name, and her writing on scientific pedagogy. Her educational method is in use today in public and private schools throughout the world. In 1947 she returned to India and gave courses in Adyar and Ahmedabad. These courses led to the book The Absorbent Mind, in which Montessori described
$2.36Charles Darwin's Origin of Species (publ. 1859) is a pivotal work in scientific literature and arguably the pivotal work in evolutionary biology. The book's full title is On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life. It introduced the theory that populations evolve over the course of generations through a process of natural
$0.99Meditations is the title of a series of personal writings by Marcus Aurelius setting forth his ideas on Stoic philosophy.Marcus Aurelius wrote the 12 books of the Meditations in Greek as a source for his own guidance and self-improvement. It is possible that large portions of the work were written in Sirmium, where he spent much time planning military campaigns from 170 to 180. We know that some
$5.99This book was written by English writer R. Kipling. The story of Kim, the orphaned son of an Irish soldier and a poor Irish mother who are both dead. Living a vagabond existence in India, under British rule in the late 19th century, Kim makes a living by begging on the streets of Lahore. Kim works occasionally for Mahbub Ali, a Pashtun horse trader who is one of the native agents of the British
The Way To Wealth (Mobi Classics)Benjamin Franklin compiled these pearls of wisdom on wealth and personal finance from Poor Richard's Almanack. Today is 250th anniversary since the book fist appeared as a pamphlet in 1758. Some proverbs from Benjamin Franklin's Way to Wealth: "There are no gains, without pains" "One today is worth two tomorrows" "Time is money" "A life of leisure and a life of laziness are two things" "Get what
$1.99"Cranford" is a short story written by Elizabeth Gaskell in 1851. The protagonist is the young lady Mary Smith who narrates in first person the daily lives of some single and middle aged ladies who live in Cranford, a small provincial town. The protagonists appear as holders of those uses, customs and tradition and the modernity of the nineteenth century was already wiping out. The group of
The Mis-Education of the NegroThe Mis-Education of the Negro is one of the most important books on education ever written. Carter G. Woodson shows us the weakness of Euro-centric based curriculums that fail to include African American history and culture. This system mis-educates the African American student, failing to prepare them for success and to give them an adequate sense of who they are within the system that they must
$5.99More's "Utopia" was written in Latin, and is in two parts, of which the second, describing the place ([Greek text]—or Nusquama, as he called it sometimes in his letters—"Nowhere"), was probably written towards the close of 1515; the first part, introductory, early in 1516. The book was first printed at Louvain, late in 1516, under the editorship of Erasmus, Peter Giles, and other of More's friends
How the Digital Age Stupefies Young Americans and Jeopardizes Our Future(Or, Don 't Trust Anyone Under 30)This shocking, surprisingly entertaining romp into the intellectual nether regions of today's underthirty set reveals the disturbing and, ultimately, incontrovertible truth: cyberculture is turning us into a society of know-nothings.The Dumbest Generation is a dire report on the intellectual life of young adults and a timely warning of its impact on American democracy and culture. For decades,
$8.99Experience and Education is the best concise statement on education ever published by John Dewey, the man acknowledged to be the pre-eminent educational theorist of the twentieth century. Written more than two decades after Democracy and Education (Dewey's most comprehensive statement of his position in educational philosophy), this book demonstrates how Dewey reformulated his ideas as a result of
$0.99The primary ineluctable facts of the birth and death of each one of the constituent members in a social group determine the necessity of education. On one hand, there is the contrast between the immaturity of the new-born members of the group - its future sole representatives - and the maturity of the adult members who possess the knowledge and customs of the group. On the other hand, there is the


text:
MLP FiM custom: Derpy the Steampunk Vampire Hunter by vulpinedesignsWatch
©2012-2016 vulpinedesigns
Rather pleased with this one! Derpy with folded wings, hat, cape, goggles, steam-tank on her back, metal anti-fang collar, hoof-mounted stake cannon on each fore-hoof, holy-water bottles strapped to one hind-hoof, and a long stake strapped to the other. The cape has a gear pattern on the corners as well as Celestia's cutie-mark - I figures that would serve the same purpose as a cross against vamponies!
Blindbag, lots and lots of putty, acrylic paint and lacquer.
Ebay link, usual £0.99 start![link]
Blindbag, lots and lots of putty, acrylic paint and lacquer.
Ebay link, usual £0.99 start![link]
So cute
Looks like I gotta break out the Greenstuff again and buy up Ponies! I've been in a long slump on painting what I normally do, but this will definitely help me jump right on in! Absolutely love it!
Lol, thanks - will look forward to seeing your works! Ponies are just so darn inspiring...I was into wargaming miniatures too before taking up ponies (nowhere near your level though!)
Ah well, thanks,but I really don't think I'm any good at all, I've just been painting for a while and posted the absolute best I've done. However, I'm just about done with my Thunder 'Jack, should have finished him four months ago...
thats a really good cloak.
Thanks, it was quite tricky to paint! There's copper wire inside the putty to hold the shape!
Completely, and totally, awesome.
Nice idea
Steampunk vampire hunter? I haven't heard that before!
The details are fantastic! Great work, as usual.
The details are fantastic! Great work, as usual.


text:
For me, the Latin American Studies minor was the perfect intersection of my Political Science major and Spanish minor. I've always been intrigued by Latin American politics, the culture, the history, and the Spanish language, thus LAS seemed like the completion to this trifecta of my studies at Providence College. The Latin American Studies minor also encouraged and inspired my Political Science Honors Thesis titled "Behind the Smokescreen: An Examination of US Involvement in Colombia".
As I apply to law schools for the fall of 2016, I anticipate on studying International Law and Human Rights Law, with a continued focus on Latin America.
For me, the Latin American Studies minor coincided strongly with my interests in the Spanish language and the current conditions of people living in Latin America. Through the minor I have come to a greater understanding of the disparities in Latin American countries and the connection those often have to their relations with the United States.
As a Peace Corps Volunteer in El Salvador, I find that what I learned about politics, globalization, and the past and present of Latin America has contributed to my understanding of the situation here.
Latin America is America! I chose Latin American Studies because it has no limits. The skills you develop in LAS are extraordinarily transferable in the business world, as I can attest two years into a consulting career that demands regular communication in Spanish and sensitivity to the diversity of Latin culture. LAS at PC connects students with excellent professors and mentors in addition to providing access to the incredible wealth of Catholic and Dominican scholarship across the Western Hemisphere.
The reason I chose to minor in LAS was because I found it to be the perfect unifier for the different things I had been studying. It allowed me to explore a part of the world that has always fascinated me. The minor, and my interest in the region, was a big part of my choice to study abroad in Buenos Aires, Argentina.
My semester in Buenos Aires tied together so many of the things that I had been studying at PC. I think my LAS classes really prepared me for that experience, and have help shaped what I want to do now that I've graduated. Maintaining my Spanish ability is incredibly important to me, and I hope to return to Latin America as soon as I possibly can.
To fulfill requirements for both my major [Global Studies] and minor, I studied abroad in Cusco, Perú, and had the opportunity to visit Chile. From exploring Machu Picchu and other Incan ruins, to visiting a cemetery in Lima filled with victims of Sendero Luminoso, to roaming the interactive exhibits at the Chilean Museum of Memory and Human Rights in Santiago, things I had learned in LAS courses came rushing back to me.
Being in places, seeing artifacts, and even meeting people that I had learned about in Providence was a very grounding experience. As an American who wants to continue to travel and learn about the world, it is important to learn about the history and culture of new places, while also recognizing the complex relationships certain areas have with my own home country. Taking courses in Latin American Studies at PC was only one way to do this, but it certainly influenced my ever-transforming identity as a global citizen.
In our globalized world, we can no longer call ourselves well-rounded students without exploring the realities and complexities that lie outside of our own national borders. The Latin American Studies minor pushed me to study abroad in unconventional, yet remarkable, places and gave me the tools to see myself as a global citizen first and an American citizen second.
As a recent graduate already living as an ex-pat in Spain, I can attest to the fact that the Latin American Studies minor has prepared me with cultural competency for life abroad.
After spending a semester in Costa Rica and time in Mexico, I realized there was so much to learn about our neighbors in the Americas. The LAS minor allowed me to explore Latin American culture and history through a variety of fields of study.
Being part of the LAS program encouraged me to pursue my current position as an intern for a health care organization in Guatemala. The minor opened up opportunities and helped shape my path after Providence College.
Rev. David T. Orique, O.P., Ph.D.Latin American Studies Program DirectorAssistant Professor of firstname.lastname@example.com Center for the Humanities 113
What does it mean to be a Catholic and Dominican college? We invite you to explore this question and the distinctive mission of Providence College.
About Providence College's Catholic and Dominican Identity


text:
Dr. Donald F. Smith, known colloquially as "The History Guy", grew up in Canada and attended the Ontario Veterinary College with the intent of practicing bovine medicine. After graduating in 1974, he enrolled in a one-year internship at the University of Pennsylvania's New Bolton Center, remaining there for a large animal surgical residency.
Donald's first faculty position was at Cornell where he practiced and taught large animal surgery, then departed for the mid-west in 1982 to join the small group of faculty who established the School of Veterinary Medicine at the University of Wisconsin-Madison.
Returning to Cornell five years later as department chair and later as associate dean, Donald helped establish the new veterinary curriculum that was introduced in 1993. As dean at Cornell (1997-2007), he reorganized the college's department structure and led the resurgence in research and expansion of clinical programs.
Since rejoining the teaching faculty, Donald has established a vibrant research and educational program in veterinary history and public policy. His collection of first-person interviews with veterinarians who began their careers during the 1930s and 1940s is a national veterinary treasure, and his popular Veterinary Legacy blog attracts followers from around the world.
Don is the author of the 50th anniversary book of the Association of American Veterinary Medical Colleges due to be released in March 2016. He is also coauthoring with Julie Kumble a book on leadership in the veterinary profession.
Donald is a diplomate of the American College of Veterinary Surgeons and a member of the National Academy of Practices. He and his wife, Doris, live in Ithaca, NY. They have three grown children.


text:
Gwen Stefani by simonhayagWatch
©2009-2016 simonhayag
This is soo cool!
It is really good! I love the belt buckle so much!
Coolio, do you think gwen would make a perfect harley quinn?
Aweosme job man! I miss her where the fuck is she now?
that is my girl!!!!
Totally awesome! No Doubt rocks!
This is really nice, the only thing i suggest is the waist looks a little to bold. if you know what i mean. Idk, everyones a critic
i really like it, but she looks scary skinny.
I love it! I'm a huge No Doubt fan. great work
....this shit is BANANAS!!
I love this so much!
this almost looks like a photo, very well done!
very cool. Can i suggest one celebrity?
No doubt this is amazing!!!
loved them all
awesome !!
she looks a little out of proportion, but in that kinda cool way, like bratz dolls or something, or anime style. lol i have the real picture on my wall. XD
Oh, wow. This is great!
The stomach is very much so the sexy. Nicely done. =]
Love, love, love this!
Gwennn I love this, its so cool and it really captures her
This is so creepy (the body proportions) and amazing at the same time. O':
I like it a lot. :B
I like it a lot. :B
This screams Gwen.
I love it man. the clearity of the belt made the whole picture come to life in a strong and pleasing way.
very cool!
Oh my god This looks so real...
what cool art work! I love your style. The way you capture all the nuances of each persons face is incredible! I'm a huge fan


text:
Originally posted by captain terrific
one year break...ooh...not a good choise, most people who take one year breaks usually never continue school after that.....
This content community relies on user-generated content from our member contributors. The opinions of our members are not those of site ownership who maintains strict editorial agnosticism and simply provides a collaborative venue for free expression.
All content copyright 2016, The Above Network, LLC.


text:
Squib Rights marches
12,603pages on
this wiki
this wiki
|Squib Rights marches
|Event information
Squibs gather in favour of Squib Rights, but the march is interrupted by Pure-blood riots.
The Squib Rights marches were an action by a mass group of Squibs in favour of the betterment of the rights of Squibs[2] that took place in 1968 or 1969, during Eugenia Jenkins's term as Minister for Magic.[1] This demonstration was, presumably, unsuccessful, as a group of extremist Pure-bloods broke out in riot while it was taking place.[2]
Behind the scenes
- The Wizards' Ordinary Magic and Basic Aptitude Test that was once posted on J. K. Rowling's Official Site gave, in one of its questions, the option of these marches happening during the term of Artemisia Lufkin, Grogan Stump, Faris Spavin, Nobby Leach, or Cornelius Fudge. Pottermore, however, revealed the correct answer to be none of these, as they actually took place during Eugenia Jenkins's term. What the correct answer was supposed to be on the W.O.M.B.A.T. is unknown.
- The Squib Rights marches can be the Wizarding-World equivalent to the Civil Rights marches of the Muggle world that occuredd around the same period in history.
- J.K. Rowling's Official Site (First mentioned)
- Pottermore (Mentioned only)
Notes and references
- ↑ 1.0 1.1 Pottermore - New from J.K. Rowling: "Ministers for Magic"
- ↑ 2.0 2.1 Sixth question of the Third W.O.M.B.A.T. at J.K. Rowling's Official Site.


text:
Archive - Jun 2012
June 22nd
Warriors Notes: Could Deng or Dion Be Fits for the Warriors?
On Fri, 06/22/2012 - 11:47am.
Contra Costa Times
Green's versatility improves his NBA stock
On Fri, 06/22/2012 - 11:46am.
FS South
Related Players:
2012 Draft Study Results
By Mike_Misek
Fri, 06/22/2012 - 10:24am
Royce White, a statistical anomaly
North Carolina's Harrison Barnes auditions for Charlotte Bobcats with NBA draft a week away
On Fri, 06/22/2012 - 8:16am.
Detroit Free Press
Related Players:
Phoenix Suns' draft back-up plan on tap
On Fri, 06/22/2012 - 8:11am.
The Arizona Republic
Trade could create way for Cavs to draft Kidd-Gilchrist
On Fri, 06/22/2012 - 8:05am.
Akron Beacon Journal
Related Players:
With his height no longer an issue, Bradley Beal's stature is growing prior to June 28 NBA Draft
On Fri, 06/22/2012 - 8:02am.
Cleveland Plain Dealer
Related Players:
June 21st
It's official: Duke's Andre Dawkins will redshirt next season
On Fri, 06/22/2012 - 6:10am.
Related Players:
The Polarizing Andre Drummond
By Eric_Yearian
Fri, 06/22/2012 - 4:22am
Each year around draft time, there seems to be one prospect that shows all kinds of promise but elicits polarized responses from teams and analysts. This year, that prospect is Connecticut's Andre Drummond.


text:
To make reservations by phone, dial freephone 0800 221 222 in the UK, 1 800 409 929 in Ireland, or view our regional telephone reservation numbers.
You will be greeted at the Porte-Cochère Entrance within seconds by our friendly associates.
Kessler Ballroom Meeting
The Kessler Ballroom can hold classroom-style meetings for up to 120 people.
Kessler Ballroom
Host elegant banquets in our 2,200-square-foot, turn-of-the-century, European-inspired Kessler Ballroom.


text:
Main Office
414 W. Fifth Street
Royal Oak MI 48067
Since 1957, Findling Law has been committed to helping without passing judgment and without compromising your principles.
Our lawyers are leaders in the industry. Every lawyer is required to participate in continuing legal education in excess of that required under law. We live, breath and teach family law.
We have gained a reputation for solving complex problems with creative solutions. We love to handle cases other firms decline.
* Nolo has confirmed that every member attorney has a valid license and is in good standing with the state agency that licenses lawyers. Any past disbarments and suspensions (with possible exceptions for minor violations or nonpayment of dues, in our discretion) will be indicated accordingly in the badge. Member attorneys are required to notify Nolo immediately if they become the subject of any disciplinary action by any state licensing agency.


text:
by day Dennis Fields is a normal kid. but by.. other times of day, he is GRASSMAN! using his mostly plant themed super powers, he fights evil and such!!
I like it, mind if use this as inspiration for a character I've been creating? just haven't found the proper design implements I've wanted but this is gonna help me
sure. it itself is based off of other things ive seen. share the inspiration!
Not the Grassman I was looking for, but I like it.
Slick design. He'd be a great fit in EnterVoid!
whats entervoid
[link] , kind of where the ideas OCTs got started. It's where you enter a character, do an intro comic, and draw comics based on that character. You can also challenge other artists to comic "battles". After each upload and match, you're given constructive feedback to help you improve from the committee.
hey do you have any refs from Grassman/Dennis Fields? like you have for Vale? cuz I have nothing to go off from if I want to draw him. I don't even know how tall he is or how old he is
Must.. DO.. FAN ART..
Must.. DO.. FAN ART..
*o* its wonderful! i love the design and the pose :-Q__ you're an expert with the anatomy!
Thanx but I can't take full credit for the pose. It's based off a spiderman pose I saw.. I am, however, proud of the design :]
thanx.he is my pride and joy!
haha this is the first time I see this picture, I must say I had not expected this! though I was wondering how a normal high school kid was gonna last in a fighting tournament! now it all makes sense
Sweet! A superhero OC!
thanx tho i borrowed heavily from spider-man, im quite proud of Grassman's costume
He's like adorable and BA at the same time!! I love him!
haha XD Its so on XD
Remind me of Kamen Rider Amazon a bit


text:
10-Day Festival Celebrating Sacramento's Unique Architecture Oct 10 - 19
The 10-day Sacramento Architecture festival is a super fun festival, filled with events like an architecture bike tour, a pub crawl, drawing safari for kids and farm-to-fork tour!
Appreciate the River City with Bike Tours, Drawing Safaris, Building Tours or a Pub Crawl.
The American Institute Of Architects, Central Valley Chapter (AIACV), announces the second annual Central Valley Region Architecture Festival.


text:
See other Acapulco properties
Villas La Lupita
Cap. Ant�n de Alaminos #232, Fraccionamiento Magallanes, Acapulco, Mexico, Distance from city centre: 2288km
Real Reviews
Real reviews from fellow travellers
These are trusted reviews from guests who have actually stayed here. We hope you find them helpful.
Note: we only give a property a rating once it has received a minimum of 3 reviews.


text:
Kindly featured by:
Everybody loves SQUARES!!! (In colors)Enjoy these wonderful square photos that surely deserve some more attention! )
10000 dreams by claire-norienWalking in a Dream by JacquelineBarkla
King by JillAuville:thumb208460207:head over heels by bye-bye-omoide
:thumb209365564::thumb207509431:Warmth by Lain-AwakeAtNight
:thumb210374751::thumb207410605:Hope by VexingArt
and how will you find your way by Writtomy Memories by MarioDellagiovannaYou Only Live Once by arrryen
Bending Life by lwc71vanity fair II by Artemisia-dreami can breathe by RickHaigh
Buttersweet by saenjainaStrawberry cake by Lalla-MiraEnd of Autumn by JacquelineBarkla
Horse by beorangeq0123 - around imagination by SlevinAaron:thumb210641413:
Comfortable feelin' by Lain-AwakeAtNightCaffeine in my vein by laura242Light Summer Flower by NorbertKocsis
Sensual by jussta. r i b b o n . p e a r l s . by minor004...PinkTourmaline... by minor004
:thumb210562751:May is pink 19 by martaraffpodoju by illusionality
:thumb210629014:simplicity by Moon-Willow... EVEN MEMORY FADES AWAY by lalitkala
:thumb209214501:awake my soul by miezeTatzePandora Box by Zwoing
:thumb211016310:Sprinke, sprinke by IHaveSeenTheRainrazzy by illusionality
Water Drops 84 by ovidiupopdaisy water drop by efeline:thumb210132882:
la bonne vie by QueenSquidletCharming The Flames by Lain-AwakeAtNight:thum
some more triple s
Bien vu !
Bien vu !
Beautiful, saturated colors. Your photos remind me again and again that the world looks different from how I remember it!
Thank you, B.
But why would the world look so different as you remember it ? You certainly have seen luggage carts before ;-?
Have a nice and restful weekend, dear friend
But why would the world look so different as you remember it ? You certainly have seen luggage carts before ;-?
Have a nice and restful weekend, dear friend
I think that you, the photographer, by showing me what you want me to see, show me something I have not seen before. I know I go around each day thinking about what am I doing next, what am I having for lunch, am I behind today-- all with barely looking at what is around me. I look just enough to keep from tripping!
Well, I believe a lot comes from my scientific education as a geologist. Trying to notice things which haven't been noticed and find relationships and models for the origin is an essential part of geological research in the field ...
this one can't be downgraded!! Great shot!!
I'm smiling to see this again and read the comment stream. Anxiety or amore!
I faved it before but I'm not sure I appreciated the three dimensionality as much as I should have. Really nice shot.
I faved it before but I'm not sure I appreciated the three dimensionality as much as I should have. Really nice shot.
Ha - thanks, Richard. I always have a critical view at my pictures, asking myself how I would things today. And so there comes an update ever once in a while
A comme Amour what else ?
It seems that Yvette is in a different mood here
amore - amore - amore, of course
amore, amore, amore ...
Very cleverly made!
hearts, diamonds... I can't name the third red one... ;D
is there any third red one
Genial. Wie machst Du das nur!
Very good!
I love blue & red....


text:
Dismal Demand for Diesel Fuel [View article]May 7, 2009. 01:30 PM | 0 Likes |Link to Comment
Natural Gas Is Heading to 1997 Levels, Should Stay There Awhile [View article]Apr 27, 2009. 01:13 PM | 3 Likes |Link to Comment
Though Newer Sources of Power Are Being Built, Coal Remains a Cheap BTU [View article]Apr 23, 2009. 11:54 AM | 1 Like |Link to Comment
10 Green Energy Gambles for 2009: 3 Month Update [View article]
On Apr 14 09:27 AM billp37 wrote:
> "Why did my generation fail to develop wind and solar? Because our
> energy choices are ruthlessly ruled, not by political judgments,
> but by the immutable laws of thermodynamics. In engineer-speak, turning
> diffused sources of energy such as photons in sunlight or the kinetic
> energy in wind requires massive investment to concentrate that energy
> into a form that's usable on any meaningful scale. ...
> Now, I was told back in the 1970s the same that you're being told
> today: that wind and solar power are 'alternatives' to fossil fuels.
> A more honest description would be 'supplements'. Taken together,
> wind and solar power today account for just one-sixth of 1% of America's
> annual energy consumption today. Let me repeat that statistic - one-sixth
> of one percent -- .0016. "
> home.comcast.net/~bpayne37/pnmelectric...
>Apr 14, 2009. 09:51 AM | 1 Like |Link to Comment
What's Happening with Inflation? [View article]
Hyperinflation (uncontrolled or unpredictable) leads to bad investment decisions, similar to 1978. Hopefully this is not a repeat.Apr 7, 2009. 09:07 AM | 1 Like |Link to Comment
Li-ion Batteries and How Cheap Beat Cool in the Chevy Volt [View article]
Mobile recharge companies to the rescue.Mar 23, 2009. 02:36 PM | 2 Likes |Link to Comment
What's the Better Solution - Inflation or Deflation? [View article]Feb 20, 2009. 09:13 AM | 0 Likes |Link to Comment
What's the Better Solution - Inflation or Deflation? [View article]
It is not possible for the US to repay in equivalent value the debts it owes, so as long as the US knows how to create inflation, and at the same time delude the capital holders that deflation is a possibility, the inflation tax on unproductive capital will continue.Feb 19, 2009. 11:12 AM | 0 Likes |Link to Comment
Ethanol Industry: Down but Not Out [View article]Feb 4, 2009. 08:51 PM | 0 Likes |Link to Comment
Why a Carbon Tax Would Lower Living Standards [View article]Feb 4, 2009. 08:47 PM | 1 Like |Link to Comment
Chu: We Need Clean Coal, Carbon Capture [View article]Jan 14, 2009. 01:15 PM | 1 Like |Link to Comment
BTW a clip from the Nuclear Enegy Institute website. These folks do this for a living. If it didn't make good business sense to operate them, they wouldn't have restarted them.
"Nuclear power is the lowest cost producer of baseload electricity. Average nuclear production costs have declined more than 30 percent in the last 10 years, to an average of 1.7 cents per kilowatt-hour. This includes the costs of operating and maintaining the plant, purchasing nuclear fuel, and paying for the management of used fuel. Electricity generated from nuclear power also has tremendous forward price stability because only a small part of production costs are fuel costs. Fuel accounts for 80 percent to 90 percent of the cost of electricity produced by fossil fuel-fired generation, making electricity from fossil plants highly susceptible to fluctuations in coal and gas prices."
Jan 5, 2009. 05:28 PM | 1 Like |Link to Comment
First, construction costs are in $ per KW, not KWH. and your wind numbers I presume are max raw power in ideal conditions. Second, if one were to presume that your numbers are correct, then FPL would not be building nuclear, but would be building wind. Third, if your numbers were correct, the wind would not need any subsidy. Finally, nuclear is base load value, whereas wind is opportunity energy. different consumer values.
I applaud your enthusiasm, but for your vision to be correct flies in the face of reality world wide. Nuclear is what sustainable nations use for electrical production.Jan 5, 2009. 05:13 PM | 0 Likes |Link to Comment
"Building new wind power is still less than a third the cost of building new nuclear plants"
Yippee! So the electricity from a wind farm is less than 1 cent per KWH. Why bother metering it?
Fat chance, bucko.Jan 5, 2009. 03:41 PM | 1 Like |Link to Comment
Time for America to Fire First Shot in the EV Revolution [View article]Dec 18, 2008. 11:43 AM | 0 Likes |Link to Comment


text:
Bombing Civilians: The Interview
program date:Sun, 08/23/2009
Following up on Jay Thiemeyer's review of Bombing Civilians, the Old Mole's Laurie Mercier talks with one of the book's editors Marilyn Young, Professor of History at New York University. They discuss the origins of this strategy as well as its ongoing use in Afghanistan and elsewhere. You can read an excerpt from her book here.
- Title: Bombing Civilians: The Interview
- Album: August 24, 2009
- Length: 11:51 minutes (6.78 MB)
- Format: MP3 Mono 44kHz 80Kbps (CBR)
- Login or register to post comments
- Download audio file