Microarray analysis of the changes in the testis transcriptome resulting from deletions of the male-specific region on the mouse chromosome long arm (MSYq) identified novel Y chromosome-encoded transcripts. Ssty gene family. Large deletions of MSYq are associated with sperm head defects for which Ssty deficiency has been presumed to be responsible.The male-specific region of the mouse Y chromosome long arm (MSYq) is comprised largely of repeated DNA, including multiple copies of the spermatid-expressed Ssty, each of these new MSYq gene families has multicopy relatives on the X chromosome. The Sly family encodes a protein with homology to the chromatin-associated proteins XLR and XMR that are encoded by the X chromosomal relatives. The second MSYq gene family was identified because the transcripts hybridized to a microarrayed X chromosome-encoded testis cDNA. The X loci ('Astx') encoding this cDNA had 92-94% sequence identity to over 100 putative Y loci ('Asty') across exons and introns; only low level Asty transcription was detected. More strongly transcribed recombinant loci were identified that included Asty exons 2-4 preceded by Ssty1 exons 1, 2 and part of exon 3. Transcription from the Ssty1 promotor generated spermatid-specific transcripts that, in addition to the variable inclusion of Ssty1 and Asty exons, included additional exons because of the serendipitous presence of splice sites further downstream.In a search for further candidate genes associated with these defects we analyzed changes in the testis transcriptome resulting from MSYq deletions, using testis cDNA microarrays. This approach, aided by accumulating mouse MSYq sequence information, identified transcripts derived from two further spermatid-expressed multicopy MSYq gene families; like We identified further MSYq-encoded transcripts expressed in spermatids and deriving from multicopy Y genes, deficiency of which may underlie the defects in sperm development associated with MSYq deletions. Ssty gene family [Ssty1 and Ssty2, is expressed in the testis during spermiogenesis and, in the absence of other candidates, it had been postulated that Ssty deficiency is responsible for the defective sperm development in MSYq deficient mice.The mammalian Y chromosome seems predisposed to accumulating multiple copies of genes that play a role in spermatogenesis -7. Detere family ,23-25. TIn this study we utilized custom-made testis cDNA microarrays to identify further Y encoded testis transcripts that are absent or reduced in level as a consequence of MSYq deficiencies.RIIIqdel, with deletion of about two-thirds of MSYq; XYTdym1qdelSry, with deletion of about nine tenths of MSYq; and XSxraY* X, in which the only Y specific sequences are provided by the Y short arm derived sex-reversal factor Sxra, and thus lack all of MSYq. These models are hereafter abbreviated as 2/3MSYq-, 9/10MSYq- and MSYq-.Three MSYq-deficient mouse models were used in this study Figure : XYRIIIqTo analyze the testis transcriptomes of these MSYq deficient mice we used microarrayed testis cDNA clones enriched for cDNAs deriving from spermatogenic cells . Total testis RNA from each of the MSYq-deficient mice (labeled with the fluorochrome Cy3) and from matched controls (labeled with Cy5) was hybridized to the array; four technical replicates were obtained for each model. The initial raw data were filtered to select only clones for which fluorescence intensity data were available from at least two of the technical replicates for each model. After this filtering, 14,681 clones remained from the complete set. As expected, scatter plots of these filtered data show that expression of the vast majority of clones is unchanged between the MSYq deficient and control mice Figure .t test probability of under 1% for the comparison between the replicates for the Cy3 and Cy5 fluorescence intensities. Twenty-three clones were identified as substantially reduced by these criteria in one or more of the MSYq deletion models. BLAST  comparisons were used to identify matching cDNAs (if previously identified) and/or the chromosomal locations of the encoding sequences. Sixteen of the 23 proved to be Ssty cDNA clones, thus demonstrating the efficacy of the strategy. Of the remaining seven clones, five proved to be Y encoded Sly cDNAs (see below), one was X encoded and one was autosomally encoded . Sly is a member of a multicopy family, and in December 2004 a total of 65 Sly family members were predicted based on the Y sequence data. There is a related multicopy X gene family that includes Xmr and Xlr [All five of the additional Y encoded clones were found to have homology to regions of the cDNA clone BC049626 previously identified in a large-scale cDNA sequencing project [ and Xlr ,28.Sly transcript in the MSYq deletion mice, we probed a northern blot of total testis RNA from the three MSYq deficient models and their controls with microarray clone MTnH-K10  were as follows: 2/3MSYq-, 47% (37%); 9/10MSYq-, 81% (84%); and MSYq-, 83% (91%). Because Sly has substantial homology to Xmr, which is also strongly transcribed in the testis, we considered that the remaining hybridization in the two models with severe MSYq deficiency could be due to cross-hybridization with Xmr transcripts. We therefore designed primers for reverse transcriptase polymerase chain reaction (RT-PCR) that distinguished between the two transcripts and used these to amplify template from testis cDNAs derived from the three MSYq deficient models and controls , but there was no amplified product from MSYq- testes. Sequencing of this 227 bp product from 2/3MSYq- and 9/10MSYq- testes confirmed that it derived from Sly. Thus, only MSYq- completely lacks Sly transcripts.To confirm the downregulation of the 0 Figure  and subss Figure . A 266 bIn December 2004, a BLAST analysis of the array clone 8832_f_22 against the mouse genome registered 41 hits on the mouse X chromosome and 710 hits on the mouse Y chromosome. The arrayed cDNA clone was apparently X encoded, there being eight putative loci  with four exons that would encode matching cDNAs; the remaining nine hits were from incomplete loci or short sequence fragments. To investigate the coding potential of the Y sequences identified in the initial analysis, a further BLAST analysis was carried out with a complete X locus, and this identified 123 putative Y chromosomal loci  that retain the same intron/exon structure as the X loci, and with 92-94% sequence identity across exons and introns.Asty exon 1/exon 4 primer pair generated products of 437 bp and 622 bp, and sequencing confirmed that these derived from Y chromosomal loci, the smaller product lacking exon 3. We refer to the similar X and Y loci encoding these X and Y transcripts as Astx and Asty (Amplified spermatogenic transcripts X encoded/Y encoded), respectively .The arrayed X encoded cDNA clone shared homology with three previously identified testis cDNA clones - two apparently X encoded  and one Y encoded (AK016790) - and a BLAST of mouse expressed sequence tags (ESTs) identified further related testis transcripts together with others derived from small intestine, aorta and eight cell embryos. The testis cDNAs and ESTs are summarized in Figure Astx/Asty exons 2 and 3 (and part of the intervening intron), proved to have exonic sequence matching Ssty1 exon 1 and part of exon 3, together with further sequence matching another testis cDNA AK015935  that could encode these transcripts .Intriguingly, the Y encoded transcripts AK016790 and BY716467, in addition to the sequence matching 5 Figure . BLAST sAstx and Asty suggested that the arrayed Astx cDNA clone would cross-hybridize with Asty and Asty(rec) RNAs. We designed primers from Astx/Asty exon 4 for RT-PCR that we thought should specifically amplify either Astx or Asty, but further analysis (see below) identified Asty(rec) transcripts that also include exon 4. RT-PCR analysis using these primers showed that Asty and/or Asty(rec), rather than Astx transcripts, are reduced in MSYq deficient mice , together with other larger transcripts with sizes ranging up to more than 9.5 kb. All bands exhibited progressive reduction in intensity with increasing MSYq deficiency  and 1.5 kb; faint bands approximating these sizes are present. In further attempts to determine the origins of these multiple sized transcripts we probed the blot with a probe matching part of exon 6 of the Asty(rec) transcript AK016790  loci.The close homology between e Figure . Probingy Figure , and thu0 Figure  and founSly clone MTnH-K10 and the Asty exon 4 probe to hybridize to Southern blots of DNA samples from the MSYq deficient and control mice. The Sly probe revealed multiple hybridizing bands in control males that were reduced in intensity in 2/3MSYq- males and exhibited no detectable hybridization in 9/10MSYq- or MSYq- males, or in females . In northern blots of RNAs from 12.5 days postpartum (dpp) to 30.5 dpp testes  transcripts were first detectable at 20.5 dpp, suggesting they are restricted to spermatid stages. To confirm spermatid expression we used the same probes for RNA in situ on testis sections, and for Sly  hybridization was at a level not markedly above background, but it did appear to locate to round spermatids, particularly with respect to hybridization within the nucleus; however, there was a similar localization to round spermatids in the sense control ; this transcript will hybridize to the sense control probe. Thus, we suspect that the round spermatid nuclear localization with the antisense probe does reflect the presence of Asty/Asty(rec) transcripts. However, it is apparent from the northern analysis and in situ analysis that Asty/Asty(rec) transcripts are much less abundant than those of Sly, which is consistent with there being five Sly clones but no Asty/Asty(rec) clones on the testis cDNA microarray.Probing a multiple mouse tissue polyA northern blot Figure  with Slys Figure , Sly andy Figure  high levl Figure . We thenAsty exon 4 probe derive predominantly from the Asty(rec) loci that are almost certainly driven by the spermatid specific Ssty1 promotor, we made attempts to determine whether true Asty transcripts are also spermatid specific. For this we used an Asty exon 1-4 primer pair (previously used to provide evidence for Asty transcripts) to amplify testis cDNA samples from 1.5 dpp to adult. Transcripts were weakly detected by these primers at 14.5 and 18.5 dpp, but the predominant expression was at 22.5 dpp onward when there were transcripts of two sizes . The longer transcript was detected from 14.5 dpp onward, and the shorter transcript from 22.5 dpp onward. Because spermatids first appear at about 20 dpp, we conclude that the shorter Asty isoform appears to be spermatid specific, but that the longer one is not spermatid specific.We know these primers can also amplify Sly gene family, 34 have retained coding potential for a protein that is related to three previously described chromatin associated nuclear proteins: XLR and XMR, which are encoded by members of a complex multicopy X gene family [Of the 65 loci of the e family ,30-32, ae family ,34  in exon 4, six of which would encode a protein of 106 amino acids and two would encode a protein with a carboxyl-terminal extension to 122 amino acids  did not match the sequence of this subset of loci with an exon 1 ORF; thus, it appears likely that the Y-linked members of this family no longer produce a functional protein. Assessing whether any of the novel Asty(rec) transcripts have significant protein encoding potential will require detailed characterization of this complex family of transcripts.All eight s Figure . Surpriss Figure . HoweverSsty gene family are the only Y genes present on MSYq that are expressed in the testis during sperm development. Our strategy was to use testis cDNA microarrays to identify transcripts that are reduced or absent in the testes of mice with MSYq deficiencies, and then to determine their chromosomal assignments. This strategy led to the identification of further testis transcripts unrelated to Ssty, which proved also to be encoded by multicopy Y loci on MSYq and expressed in spermatids.The objective of the present study was to try to establish whether members of the Sly  is most closely related to the multicopy Xlr/Xmr gene family, which is located on the X chromosome. It had previously been reported, based on Southern blot evidence, that multiple Xlr related sequences are present on the X and Y chromosomes, but it was concluded that most if not all of these Xlr related sequences were likely to represent nontranscribed pseudogenes [Xmr, was subsequently described that is specifically expressed in the testis [Sly transcripts, encoded by multiple loci on MSYq, are also abundantly expressed in the testis in spermatids.The first of these, udogenes . HoweverThe XLR protein is a thymocyte nuclear protein that has been shown to colocalize with SATB1, a protein that binds to AT-rich sequences at the base of chromatin loops ,36. XMR Sycp3 is the ancestral gene and that some time before the divergence of human and mouse lineages the gene came onto the X chromosome and became multicopy; these copies then evolved rapidly and independently. At some point after the divergence of mouse and rat, one of the X copies was brought on to the Y chromosome, creating a new X-Y homologous subfamily. This subfamily subsequently became massively amplified in copy number on both the X and Y chromosomes. Like Sly, Xmr and its close relatives are highly expressed in spermatids [Xmr, leads us to suspect that amplification of Xmr and Sly is a result of 'genomic conflict' between sex linked meiotic drivers and suppressors [Because SYCP3 is a fundamental component of the synaptonemal complex during meiosis, it is not surprising that it has been identified in a wide range of vertebrates. A comparison of mammalian Cor1 domain proteins Figure  indicateermatids , and ourpressors ,38.Asty, is also encoded by a multicopy Y gene. Asty has a very high degree of homology (92-94% identity across exons and introns) to a multicopy X gene (Astx), suggesting that it may be a recent arrival on the mouse Y chromosome. Intriguingly, BLAST searches with the microarrayed Astx clone sequence failed to detect similar sequences in the human genome, but in the rat there were four X-linked sequences matching the last third of the mouse Astx/Asty loci. Future analysis of these putative rat loci, in particular to determine whether related sequences are present on the rat Y chromosome, may help to delineate the evolutionary history of this gene family. In addition to these Asty loci, we have identified novel 'recombinant' loci, apparently driven by the Ssty1 promotor, that incorporate a subset of Ssty1 and Asty exons, and through alternative splicing and serendipitous splicing of more downstream sequences they have the potential to produce a range of novel transcripts in addition to the previously described transcripts AK016790 and AK015935.The second MSYq encoded spermatid transcript, Astx have retained a similar ORF in exon 4, it is reasonable to predict that they encode a protein. Interestingly, none of the more than 100 putative Asty loci have retained the Astx exon 4 ORF, despite the greater than 92% sequence identity of these loci. Six copies of Asty do have protein encoding potential in exon 1; however, the only Asty transcripts we have thus far identified do not derive from this subset of loci with exon 1 protein encoding potential. Overall, this evidence suggests that Astx is probably translated and that Asty is not. This does not, however, rule out a functional role for Asty, or indeed the more strongly transcribed Asty(rec), in sperm differentiation, especially given the increasing literature on functional RNAs. A particularly pertinent example in the present context is provided by the Stellate/suppressor of Stellate system in Drosophila, in which transcripts encoded by multicopy loci on the Y regulate expression of a protein expressed in spermatids and encoded by related multicopy loci on the X chromosome [Drosophila arose as a consequence of a past postmeiotic genomic conflict [Astx transcripts are present in spermatids  as well as being expressed in spermatocytes .Because all eight copies of romosome , via an conflict ,40 and tconflict . In thisSsty deficiency as to the cause of the abnormalities in sperm shape associated with MSYq deficiencies. Three features suggest that Sly deficiency is the more likely cause. First, and importantly, retention of one or more transcribed Sly copies in 9/10MSYq- males  provideconflict , and becThe highly repetitive nature of the mouse Y chromosome long arm presents formidable challenges for the determination of its functional gene content. Our strategy of using expression array data to highlight transcriptionally active loci among the sea of partial and degenerate gene copies has proved successful in identifying further MSYq encoded transcripts, deficiency of which may contribute to the abnormal sperm head development and function seen in males with MSYq deficiencies. This type of approach is likely to form an important component of future functional analysis of mammalian Y chromosomes and other repetitive chromosomal regions.Ssty expression and sperm abnormalities, together with appropriate age- and strain-matched controls.All mice were produced on a random bred albino MF1 strain (NIMR colony) background. The mice used to provide RNA for microarray analysis were the three MSYq deficient genotypes  removing the testis determinant Sry from the short arm, this deficiency being complemented by an autosomally located Sry transgene. These males are sterile with virtually all of the sperm having grossly distorted heads [Tdym1Sry males are the appropriate controls.These mice have a 129 strain Y chromosome with a deletion removing approximately nine-tenths of MSYq, and also a small deletion ; the Y* X chromosome provides a second PAR, thus allowing fulfillment of the requirement for PAR synapsis [Rbmy gene family, located on the short arm adjacent to the centromere. The males are sterile and have even more severe sperm head defects than do XY-qdelSry males [Rbmy deficiency is unlikely to be a significant cause of the abnormal sperm development [Sxra originated from a YRIII chromosome, the appropriate controls are again XYRIII.In these mice the only Y specific material is provided by the Y short arm derived synapsis . These mry males ,41. Our elopment . BecauseTestes were obtained from two of each of the MSYq deficient males and the two control genotypes, at 2 months of age. Total RNA was isolated using TRI reagent  and cleaned using RNEasy columns , in accordance with to the manufacturers' protocols. Microarray hybridizations and analysis were carried out as described by Ellis and coworkers , except t tests, and fold change filtering were performed using GeneSpring. Full details of the array experiment were submitted to the ArrayExpress database (accession number E-MEXP-251).The arrayed clones consisted of two subtracted adult mouse testis libraries , six tesSly cDNA clone BC049626 [Asty 314 bp exon 4 probe amplified with primers CAGCAAGGAGAGTGGGGAGTA and CAGTGGGATGTTGGTTTCTAATG. Genomic DNA was extracted from tail biopsies and 15 μg (or 4 μg for the control XY on the long exposure Southern blot) was digested with EcoRI, electrophoresed through a 0.8% agarose gel and transferred to a Hybond-N membrane . After UV crosslinking , the membrane was hybridized overnight with 32P-labelled probes either at low stringency , 5 × Denhart's, 0.1% sodium dodecyl sulfate (SDS), 100 μg/ml salmon sperm DNA; two 30 minute washes ) or at high stringency ). The membrane was exposed to X-ray film or Phosphorimager screen overnight.The probes used for Southern analysis were Sly cDNA clones BC049626 [Asty 314 bp exon 4 probe used for northern analysis. An actin probe that recognizes α- and β-actin transcripts [32P-labelled probes in hybridization buffer . After two 60°C washes  the membrane was exposed to X-ray film for 5 hours and subsequently to a Phosphorimager screen to allow quantitation of hybridization using ImageQuant software.The probes used for northern analysis were as follows: nscripts  served aSly/Xmr RT-PCR, 2 μg total RNA was reverse transcribed in a 40 μl reaction using standard procedures. A 2.5 μl aliquot was then added to a 25 μl PCR reaction. RT-PCR for Astx/Asty was performed using the Qiagen OneStep RT-PCR kit, following the manufacturers' instructions. In both cases amplification was for 30 cycles at an annealing temperature of 60°C.RNA samples were treated for DNA contamination using DNAse I amplification grade kit . For the Sly and Xmr, forward primer GTGCGGTTTGGAAGTGT and reverse primer CTCAAGCAGAAGCAGATG; Asty and Astx exon 4, forward Asty primer GRGGAGTAGAACTCATCATC and forward Astx primer GGGGAGTAGAACTCATCTTTA, with common reverse primer CAGGAGATGACTAACATAGCA; Asty exon 1 to exon 4, forward GGCCTTGCTCTTATGTCATC and reverse CGATGATGAGTGACCTAAAGAT; and Astx exon 1/2 (spanning intron 1) to 3/4 (spanning intron 3), forward GCTCCAGAAGACAGAGATAC and reverse AGACTTCAAACCTCATGCAGT.The primers used were as follows: RT-PCR product was purified using the QiaQuick kit (Qiagen) and cloned using the pGEM-T easy vector system I kit (Invitrogen). Sequencing reactions were performed from 5' and 3' ends using standard protocols . Completed reactions were analyzed by the Cambridge Department of Genetics sequencing service using a 3130xl capillary system . Cycling conditions for the sequencing reactions were 96°C, 55°C and 60°C for 10 s, 5 s and 4 minutes, respectively.In situ hybridization using digoxigenin-labeled cRNA probes from Sly clone MtnH_K10 and Asty and Astx exon 4 were used to localize each mRNA on Bouin-fixed paraffin-embedded mouse testis sections using procedures previously described [escribed , with hyin situ hybridization (FISH) was performed basically as described previously for Cot1 RNA FISH [Astx locus encoding the cDNA AK076884 . Hybridization reactions consisted of 100 ng biotin-labeled BAC probe, 3 μg mouse Cot1 DNA and 10 μg salmon sperm DNA, and were carried out overnight at 37°C. Staging of spermatogenic cells was based on DAPI fluorescence morphology, together with immunolabelling for the synaptonemal complex protein SYCP3  and the phosphorylated form of histone H2AX , as previously described [Astx transcripts was provided by X chromosome painting, as previously described [RNA fluorescence RNA FISH  using anescribed ,47. The escribed , and theescribed .Sly-related clones from the microarray (Additional data file Astx clone and for the Asty RT-PCR products (Additional data file Astx transcriptional analysis (Additional data file The following additional data are available with the online version of this paper: a file providing sequence information for the Sly-related clones from the microarrayA file providing sequence information for the Click here for fileAstx clone and for the Asty RT-PCR productsA file providing sequence information for the microarrayed Click here for fileA diagram providing sequence information on the 'recombinant' loci encoding the transcripts AK016790 and AK015935Click here for fileAstx transcriptional analysisImages showing Click here for file


We discovered that the majority of these variants exhibit no reduced catalytic activity per se. Instead, we observed, the dimerization behavior of TPI is influenced by the particular mutations investigated, and by the use of a potential alternative translation initiation site in the TPI gene. Additionally, we demonstrated that the overexpression of the most frequent TPI variant, Glu104Asp, which displays altered dimerization features, results in diminished endogenous TPI levels in mammalian cells. Thus, our results reveal that enzyme deregulation attributable to aberrant dimerization of TPI, rather than direct catalytic inactivation of the enzyme, underlies the pathogenesis of TPI deficiency. Finally, we discovered that yeast cells expressing a TPI variant exhibiting reduced catalytic activity are more resistant against oxidative stress caused by the thiol-oxidizing reagent diamide. This observed advantage might serve to explain the high allelic frequency of TPI null alleles detected among human populations.Triosephosphate isomerase (TPI) deficiency is an autosomal recessive disorder caused by various mutations in the gene encoding the key glycolytic enzyme TPI. A drastic decrease in TPI activity and an increased level of its substrate, dihydroxyacetone phosphate, have been measured in unpurified cell extracts of affected individuals. These observations allowed concluding that the different mutations in the TPI alleles result in catalytically inactive enzymes. However, despite a high occurrence of TPI null alleles within several human populations, the frequency of this disorder is exceptionally rare. In order to address this apparent discrepancy, we generated a yeast model allowing us to perform comparative  Triosephosphate isomerase deficiency has been initially described in 1965 TPI gene encoding aspartic acid instead of glutamic acid within the enzyme (Glu104Asp variant) and accounts for approximately 80% of mutant alleles within Northern European kindreds with clinical TPI deficiency TPI gene encoding leucine instead of phenylalanine within the enzyme (Phe240Leu variant) could have an effect on the substrate binding site. Other mutations, for instance, the pathogenic TPI variants Gly122Arg or Val154Met could not be assigned to defined domains. However, Perry and Mohrenweiser showed that the Gly122Arg TPI variant, which was identified as an electromorphic variant by screening 3,400 persons in a Caucasian population, is a thermolabile enzyme possibly indicating improper folding TPI gene Bioinformatic analyses have predicted that the human pathogenic mutations, which are not restricted to a specific domain or region within the enzyme, could affect the substrate binding site or the dimerization interface of TPI To date, the best-studied family affected with TPI deficiency is a Hungarian family in which two germ-line identical compound heterozygote brothers have inherited a missense mutation at codon 240 encoding the Phe240Leu TPI variant and a nonsense mutation at codon 145 (Glu145TER) leading to a truncated TPI protein According to the above-mentioned data, it is quite obvious that further functional analysis of the different TPI variants is mandatory for comprehending the molecular mechanisms underlying pathogenesis in TPI deficiency.TPI (please refer to the Supporting Information for oligonucleotide sequences). Afterwards, the DNA fragments were purified and subcloned into the BamHI and XhoI sites of the centromeric expression vector p416GPD ; accession number BC007812) and plasmid pACT2-NTint5, which was isolated from a human fetal brain cDNA library (Clontech) for the generation of the different TPI expression plasmids described in this study. Constructs encoding the TPI variants TPI Met1_AAG (TPI Paris) or TPI Phe240Leu, respectively, were produced by PCR using oligonucleotides introducing the respective mutation as indicated in the Supporting Information. To construct expression plasmids encoding the TPI variants Cys41Tyr, Glu104Asp, Gly122Arg and Ile170Val, the diverse mutations were introduced via PCR using the respective oligonucleotide pairs, which overlap in 5′ to 3′ direction as well as in 3′ to 5′ direction. Then, the two resultant PCR fragments were utilized as DNA templates for a subsequent PCR applying oligonucleotides designed for the amplification of the full coding sequence of SalI and NotI sites of the bait plasmid pBTM117c or the prey plasmid pACT4-1b.For the directed yeast two-hybrid analysis, we generated the respective bait and prey constructs encoding the various LexA-TPI or AD-TPI fusion proteins by ligating the different DNA fragments amplified by PCR into the TPI coding sequences were amplified via PCR using the oligonucleotide pair TPI-pEGFPN1-s-Sal1 and TPI-pEGFPN1-as-BamH1, TPIMet1_AAG-pEGFPN1-s-Sal1 and TPI-pEGFPN1-as-BamH1 for introducing a mutation at the start codon or TPISer3_TER-pEGFPN1-s-Sal1 and TPI-pEGFPN1-as-BamH1 for introducing an artificial stop codon. Subsequently, the PCR fragments were purified and subcloned into the SalI and BamHI sites of the expression vector pEGFP-N1 .To create the mammalian expression plasmids encoding an N-terminal fragment of human TPI fused to the green fluorescent protein (GFP), the first 200 bp of the XhoI/NotI sites of the vector pTL-FlagC, respectively. PCR was performed under conditions described earlier The mammalian expression plasmids pTL-Flag-TPI-WT and pTL-Flag-TPI-Glu104Asp were generated by subcloning the corresponding DNA fragments from pACT-TPI-WT or pACT-TPI-Glu104Asp into the a his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) a his3Δ200 trp1–901 leu2–3,11 gal8 canR cyh2R) tpi1 strain MR100 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 LEU2::tpi1) the LEU2 coding sequence was amplified via PCR using the plasmid pACT2 (Clontech) as DNA template and the oligonucleotides LEU2::tpi-s and LEU2::tpi-as that each encompasses a 35 nucleotide region homologous to the TPI1 coding sequence. After transformation of BY4741 cells via the lithium acetate -leu medium (synthetic complete medium without leucine) supplemented with 3% (v/v) ethanol/0.1% (v/v) glucose, because yeast cells deficient for the Tpi1 enzyme are unable to grow on medium solely supplemented with glucose as carbon source TPI1 coding sequence, yeast clones exhibiting no growth on media supplemented with glucose as carbon source have been selected and the correct insertion of the DNA fragment was confirmed by PCR and by western blot analysis using a polyclonal TPI serum  as indicated and selected single colonies on SC-leu-ura synthetic media supplemented with 3% ethanol/0.1% glucose. Then, the obtained transformants were transferred onto media plates containing glucose as carbon source allowing only yeast cells with functional TPI to grow as well as onto plates containing 3% ethanol/0.1% glucose as control. For this analysis, centromeric plasmids were used for cloning purposes, since these plasmids are known to be highly stable in yeast and are generally present as a single copy per yeast cell GPD promoter was chosen for the expression of exogenous TPI. Additionally, to evaluate the biological reliability of our system, we expressed yeast Tpi1 from a centromeric plasmid in the strain MR100 as well. Enzymatic Tpi activity was determined as described below and compared to the enzyme activity of wild-type yeast strain BY4741. No significant differences in Tpi activity were observed in case the Tpi1 enzyme was expressed from the centromeric plasmid or from the genome (data not shown).For the complementation analyses, we transformed the Δtpi1 yeast expressing wild-type or the different pathogenic TPI variants to oxidants was analyzed as described earlier 600 values of 3.0, 1.0, 0.3 and 0.1. Then, 10 µl aliquots of each culture were spotted onto SC plates containing various concentrations of hydrogen peroxide  tert-butyl hydroperoxide , cumene hydroperoxide  and diamide . Sensitivity was determined by comparing the growth between the different strains after plates were incubated for 3 days at 28°C. Aberrant enrichment of reactive oxygen species was determined by dihydroethidium staining of yeast as described earlier Resistance of lacZ reporter gene activity was analyzed using an assay based on o-nitrophenyl-β-D-galactopyranoside (Applichem) as described earlier To investigate the dimerization behavior of the different pathogenic TPI variants, we exploited the yeast two-hybrid system. We generated a number of bait and prey plasmids as described above and in the Supporting Information. These plasmids were used to transform the yeast strain L40ccua to express the different bait and prey proteins in various combinations as indicated in the corresponding figures. Transformants were selected on SC media lacking tryptophan and leucine. To analyze the activity of the reporter genes, yeast clones were spotted onto SC medium lacking tryptophan, leucine, histidine and uracil. Growth of yeast on selective media was analyzed after incubation of plates for 48–72 h at 30°C. The 340 in 5 s intervals for 3 min using a spectrophotometer (Amersham Ultrospec). Afterwards, the enzymatic activity of TPI under steady-state conditions was calculated as average substrate conversion per minute and the activity of the pathogenic TPI variants was compared to the activity of the wild-type enzyme.To measure the catalytic activity of the human TPI variants in yeast, an enzyme-coupled assay based on a protocol developed by Maitra and Lobo was performed 2 atmosphere at 37°C. Transfection was performed using Polyfect (Qiagen) as recommended by the manufacturer. To prepare cell lysates, the cells were washed in PBS and lysed in a buffer composed of 50 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2, 0.5% NP40, 1 mM EDTA, pH 8.8, 25 U/ml benzonase (Merck) and 2.5% protease inhibitors . SDS-PAGE and immunoblotting were performed as described COS1 cells were grown in Dulbecco's modified Eagle medium  supplemented with 5% fetal bovine serum, 50 U/ml penicillin and 50 µg/ml streptomycin (Biochrom) in a humidified 5% COTPI structural coordinates were obtained from Kinoshita et al . Nevertheless, this residual activity of about 30% is sufficient to suppress the growth defect of Δtpi1 yeast cells on glucose medium as presented in Finally, we investigated the catalytic activity of the wild-type and the pathogenic TPI variants in MR100 yeast cells. The  protein . Howeverin vitro data http://www.cbs.dtu.dk/services/NetStart), which is based on an algorithm for the prediction of translational start sites TPI gene was replaced by an AAG (as observed in the TPI Paris family), (ii) an artificial stop codon was placed at codon position 3, and plasmids encoding the (iii) wild-type TPI or (iv) TPI2ndATG as presented in Δtpi1 yeast strain, transformants were plated on SC-ura medium supplemented with 3% ethanol/0.1% glucose. Afterwards, ethanol lysates of the different yeast cultures were prepared and the expression level of the different TPI proteins was analyzed by immunoblotting. Wild-type TPI and TPI2ndATG are highly expressed in yeast as shown in Met1_AAG was also expressed in yeast demonstrating that the second ATG codon in the TPI gene can be used as translational start site. However, no expression of the TPISer3_TER variant was observed in yeast, hence, this observation could be based on the high activity of nonsense-mediated mRNA decay in yeast TPI gene as alternative translational start site in mammalian cell lines, we generated fusion proteins comprising the first 200 nucleotides of human TPI gene containing the mutations described above fused to GFP. Afterwards, COS1 cells were transfected with the respective expression plasmids or with the empty plasmid pEGFP-N1 and incubated for two days to allow expression of the recombinant proteins. Cell lysates were prepared and subsequently analyzed by immunoblotting . Finally, we analyzed the resistance of the different yeast strains to oxidants such as hydrogen peroxide, tert-butyl hydroperoxide, cumene hydroperoxide and diamide. No differences in the growth rates between the different yeast strains was monitored on medium containing different concentrations of hydrogen peroxide, tert-butyl hydroperoxide or cumene hydroperoxide, respectively (data not shown). However, we discovered that treatment of the different yeast strains with the thiol-oxidizing drug diamide had an effect. Strikingly, yeast cells expressing Ile170Val TPI, the variant exhibiting a reduced catalytic activity as shown in Accumulation of reactive oxygen species (ROS) influences chronological and replicative aging in yeast and plays an important role in the pathogenesis of age related disorders in humans TPI null alleles is much higher as the rare incidence of TPI deficiency in vitro measurements of a mutant TPI variant purified from E. coli demonstrated that the purified TPI protein carrying the Phe240Leu mutation exhibited a 6-fold higher activity than expected from the measurements performed with the patient erythrocyte extract in vivo system allowing analysis of the enzymatic activity of wild-type or pathogenic TPI variants inside a cell without additional side effects. Utilizing yeast genetics, we generated a yeast strain deleted for the TPI1 gene enabling us to investigate the enzymatic and functional properties of a number of human TPI variants in vivo. We showed that human wild-type as well as the pathogenic TPI variants Cys41Tyr, Glu104Asp, Gly122Arg, Ile170Val and Phe240Leu, respectively, all suppress the growth defect of Δtpi1 yeast on glucose medium providing the first in vivo evidence that these pathogenic variants exhibit catalytic activity. We corroborated this result by demonstrating that the respective yeast clones are not hypersensitive to lithium chloride, a phenotype caused by inactivation of the Tpi1 enzyme due to inhibition of the inositol biosynthesis pathway per se of all but one pathogenic TPI variant investigated is not affected as a result of the mutation in the TPI gene. Interestingly, the pathogenic TPI variant Ile170Val displayed only about 30% of the wild-type catalytic activity; hence, this activity is sufficient to suppress the growth defect of Δtpi1 yeast on glucose medium.Metabolic studies demonstrated that the activity of the TPI enzyme is reduced to 3–20% of the wild-type value as measured in unpurified erythrocyte or muscle extracts of TPI deficiency patients Since our results seem to be in sharp contrast to the measured TPI activity in unpurified erythrocyte extracts of TPI deficiency patients at disease stage, they clearly make obvious that regulatory events rather than enzyme inactivity are the basis for this enormous reduction in TPI activity. Indeed it has been reported that binding of TPI to an yet unidentified component of red cell membranes can lessen the activity of TPI TPI gene have been observed in human red blood cells TPI gene encoding MET14 can be used as an alternative translation initiation site in yeast as well as in mammalian cells. However, the resulting protein did not suppress the growth defect of Δtpi1 yeast on glucose medium indicating that this variant lacks catalytic activity. This result is not unexpected, since this TPI variant lacks the catalytic lysine and at least three residues of the intersubunit interface. Interestingly, a TPI deficiency patient which has inherited a start codon mutation in combination with the mutation at position 104, has a stronger pathology in comparison to Glu104Asp homozygotes At least three different isoelectric TPI variants encoded by the single lacZ gene as reporter. This allows determination of the relative strength of protein-protein interactions in yeast, however, these values can not be directly correlated with binding constants (e.g. KD) determined by in vitro measurements 2ndATG variant dimerizes with wild-type TPI, although at reduced levels in comparison to the dimerization of wild-type TPI proteins. Finally, we demonstrated that the dimerization between the pathogenic variants also occurred with lower stringency as indicated by the relative activity of the lacZ reporter gene. In the light of these results, it is quite remarkable that the Glu104Asp TPI variant, which displayed the most modified dimerization behavior, is the most common variant observed amongst the affected individuals. Moreover, this variant is the only TPI variant found homozygous among affected individuals; all other mutations occur only in the compound heterozygous state together with the Glu104Asp variant or with an obvious TPI null allele  to 0.02 (African American population) TPI gene resulting in a catalytic inactive TPI enzyme cause homozygote embryonic lethality in mice. The human population studies performed are indicating that this situation is reflected in humans as well TPI alleles of TPI deficiency patients cannot encode catalytically inactive TPI enzymes. As illustrated and demonstrated in TPI null alleles are lethal, since no homozygotes carrying TPI null alleles can be detected in mouse and humans TPI null alleles, for instance, the TPI Paris  or TPI Alfortwille  TPI alleles resulting in catalytically impaired enzymes could confer an advantage to certain stress stimuli or other environmental conditions.In the light of the above-mentioned findings it is quite intriguing that the frequency of heterozygous individuals carrying one inactive Primer Sequences and Plasmids(0.06 MB DOC)Click here for additional data file.


Hlx, a homeobox transcription factor gene that is expressed in mouse intestinal and hepatic mesenchyme, is required for normal embryonic growth of intestine and liver, and the Hlx-/- genotype is embryonic lethal. We hypothesized that Hlx is required for ENS development.Development of the enteric nervous system (ENS) requires interactions between migrating neural crest cells and the nascent gastrointestinal tract that are dependent upon genes expressed by both cell compartments. Hlx+/+ and Hlx-/- mouse embryos by immunostaining of embryo sections for the neural markers PGP9.5 and Phox2b, or by staining for β-galactosidase in whole-mount embryos containing the dopamine β-hydroxylase-nLacZ transgene. In Hlx+/+ embryos, neural crest cells/enteric neurons have moved from the stomach into the intestine by E10.5. By contrast, neural crest cells/enteric neurons remain largely restricted to the lateral stomach mesenchyme of Hlx-/- embryos, with only a few scattered neural crest cells/enteric neurons in the intestine between E10.5–16.5.Enteric neurons were identified in The Hlx homeobox transcription factor is required for early aspects of ENS development. Gastrointestinal (GI) motility requires smooth muscle contraction and relaxation that is regulated in an integrated fashion by the enteric nervous system (ENS), a complex network of ganglia that can function independently of the central nervous system . The ENSThe development and maintenance of the ENS requires cell-cell interactions between the migrating neural crest cells (NCC) and the resident GI epithelial and mesenchymal cells. A number of signaling pathways between mesenchyme and NCC are required for development of the ENS -10. ThesPhox2b, Pax3 and Ncx/Hox11L.1  at 37°C until color was sufficiently developed . Embryos were then washed with ice-cold PBS and stored at 4°C until dissected within 1–2 days.Whole-mount embryos were fixed with 4% paraformaldehyde in PBS for 15–60 minutes and stained by the method of Sanes et al. ,41. ThusDigital photomicrographs of sections and whole-mount embryos or dissected GI tracts were obtained using an Olympus BX41 stage microscope or an Olympus SZX12 zoom stereo microscope, respectively, using a SPOT Insight Color digital camera  and SPOT imaging software for Macintosh.MDB directed the study, analyzed the data, and drafted the manuscript; DTD performed immunohistochemical experiments and embryo staining and assisted with animal breeding; LCW performed immunohistochemical experiments and embryo staining and assisted with animal breeding; AK assisted in the design of the study and performed immunohistochemical experiments; and RPH provided knockout mice and expertise regarding their phenotype. All authors read and approved the final manuscript.


Hox genes code for homeodomain-containing transcription factors that function in cell fate determination and embryonic development. Hox genes are arranged in clusters with up to 14 genes. This archetypical chordate cluster has duplicated several times in vertebrates, once at the origin of vertebrates and once at the origin of gnathostoms, an additional duplication event is associated with the origin of teleosts and the agnanths, suggesting that duplicated Hox cluster genes are involved in the genetic mechanisms behind the diversification of vertebrate body plans, and the origin of morphological novelties. Preservation of duplicate genes is promoted by functional divergence of paralogs, either by subfunction partitioning among paralogs or the acquisition of a novel function by one paralog. But for Hox genes the mechanisms of paralog divergence is unknown, leaving open the role of Hox gene duplication in morphological evolution.dN/dS ratio tests, branch-site dN/dS ratio tests, clade level amino acid conservation/variation patterns, and relative rate ratio tests, to show that the homeodomain of Hox genes was under positive Darwinian selection after cluster duplications.Here, we use several complementary methods, including branch-specific Our results suggest that positive selection acted on the homeodomain immediately after Hox clusters duplications. The location of sites under positive selection in the homeodomain suggests that they are involved in protein-protein interactions. These results further suggest that adaptive evolution actively contributed to Hox-gene homeodomain functions. The homeobox codes for a highly conserved 60 amino acid DNA-binding motif (the homeodomain) found in transcription factors . One claModels of duplicate gene preservation predict functional differentiation of paralogs based on protein sequence or regulatory divergence ,11. AlthHoxA-11 and HoxB-5  [20]. These data suggest that, in the evolution of ray-finned fishes, some duplicate Hox genes have been preserved by functional differentiation through the action of positive Darwinian selection immediately following the gene duplication. This suggests that Hox genes may have also experienced adaptive evolution following the cluster duplications earlier in vertebrate evolution.How paralogus Hox genes have been retained is not known, although evidence suggestive of positive selection after cluster duplication has been identified in Hox7 , Hox5 anDrosophila homolog, Sex combs reduced (Src), in axis determination indicating strong conservation of function over 500 to 600 million years [Hox cluster duplication and gene diversification has been proposed to be one of the genetic mechanisms behind the diversification of vertebrates and body plans and the origin of morphological novelties -23. Thison years , but couon years  and non-on years ,32.dN/dS ratio tests to detect directional selection and identify positive sites, and comparison of clade level polymorphisms/divergence rates. Our results indicate that after cluster duplication positive Darwinian selection acted on the homeodomain of Hox proteins prior to the divergence of the modern gnathostome and bony fish lineages. We find amino acid substitutions at sites that are not involved in structural constraints and are located on the molecular surface where they are available for protein-protein interactions were targets of positive selection. We suggest that the action of positive selection at a subset of sites not constrained by ancestral (plesiomorphic) functions after cluster duplications led to the emergence of novel protein interactions while maintaining ancestral ones. This model can help reconcile the role of Hox genes in morphological diversification and innovation with their extreme sequence conservation.In this paper we investigate the sequence divergence in homeoboxes from the four gnathostome Hox clusters, including genes from basal vertebrates and sarcopterygians like shark and coelacanth, respectively. This is the first study of homeodomain divergence with extensive taxon sampling allowing us to identify the relative phylogenetic age of substitution events in vertebrate phylogeny. We use three different, but complementary, approaches to test for functional divergence among paralogs: comparison of patterns of amino acid sequence conservation/variation among paralog clades, We compiled a database of Hox genes with 4–5 species for each gene  and compared conserved and variable sites between paralog group members to identify if there are characteristic residues that distinguish which cluster a paralog belongs to  under either duplication model. Comparisons between HoxB and HoxA to protoBC under the (B(A(CD))) model and protoAD to protoBC under the ((AD)(BC)) model, θI(B(A(CD))) = 0.138–134 and θI((AD)(BC)) = 0.001, respectively, were not significant. Type-I sites are defined as those with an amino acid that is conserved in one clade but variable in the sister clade, implying that the site is under structural/functional constraints in the first clade that is absent in the variable clade [To test if 'cluster-specific' residues are functionally divergent we estimated the coefficient of functional divergence (θ), which measures the difference in evolutionary rate at amino acid sites between gene clusters. Rejection of the null hypothesis (θ = 0) is strong evidence for altered functional constraints after gene duplication (or speciation) . We founII values are extremely small (θII = 0.001–0.062), highlighting the conservation between homeodomains from different clusters. These results are not unexpected given that this method calculates θ across all sites in an alignment and thus effectively averages site-wise θ values. With only ~4% of sites/cluster showing a pattern of type-II divergence in our concatenated alignment, it is not likely that the ~46 possible type-II sites have θII values high enough to compensate for the extremely low θII values of the over 900 sites with θII effectively equal to zero.Recently, a method has been developed to test for type-II functional divergence . Type-IIdN) to synonymous (dS) substitution rate (dN/dS = ω) with ω = 1, <1, and >1 indicating neutral evolution, purifying selection and directional selection.Several models of molecular evolution have been proposed to account for the preservation of duplicate genes. Although the details of each model can vary (see introduction), they differ in their predictions regarding the pattern of sequence evolution following gene duplication. The neo-functionalization and divergent selection models predict that the nonsynonymous substitution rate will be increased following gene duplication because of positive Darwinian selection in the gene acquiring the new function, while the Dykhuizen-Hartl and DDC models predict and increase in the substitution rate because of relaxed purifying selection. It is possible to distinguish between these models by comparing nonsynonymous , with tree length dN averaging 1.2 substitutions per nonsynonymous site and tree length dS averaging 18 substitutions per synonymous site along the tree. Interestingly, simulation studies [2 made the likelihood ratio test statistic (LRT) extremely conservative such that the type-I error rate is very small. Similarly, the power of the LRT to reject the null hypothesis even when it is false (type-II error) was found to be conservative even at medium to high levels of sequence divergence [dN and dS from this homeodomain dataset are reliable, if conservative.Unlike the functional divergence methods developed by Gu ,37, esti studies  have shovergence . The powvergence , suggestvergence . Our datdN) to sysnonymous (dS) substitution rate ratio [dN/dS ratio for the homeodomains of all paralog groups is much less than 1 (0.0033–0.0359) highlighting the dominant role purifying selection plays on Hox gene evolution. To test if there was an increase in the nonsynonymous substitution rate following Hox cluster duplication we used a two ratios model that estimated separate ω's for post cluster duplication (ωPCD) and post speciation (ωPS) branches. Post cluster duplication branches evolved significantly faster (3–27x) than post speciation branches for 10 of 13 paralog groups; the remaining 3 paralog groups had ωPCD > ωPS but the results were not significant  and post speciation (ωPS) branches was not better than the simple two-ratio model indicating that paralogs experienced similar selective forces after cluster duplication. These results are consistent with previous data from Hox5, Hox6 and Hox7 and indicate there was a period of rapid evolution of the homeodomain after Hox cluster duplication that could have been the result of either positive Darwinian selection or relaxed purifying selection.To estimate the strength and kind of selection acting on Hox gene homeodomains, we used maximum likelihood methods to estimate the nonsysnonymous  the accuracy of dS, and to a lesser extent dN, is greatly reduced. These two limitations of the dN/dS ratio to detect positive selection are particularly important for studying selective forces after Hox cluster duplications since very few sites (less than 15%) changed after duplication and the duplication events are relative ancient , leading to substantial synonymous site divergence. Thus, even though we found evidence of accelerated rates of sequence evolution post cluster duplication, it is unlikely that the dN/dS ratio tests used above would be able to detect positive selection (ω > 1).Although positive selection at the molecular level is most often tested using the dN/dS ratio is the relative rate ratio test of Creevey and McInerney [One complementary method that has been developed to compensate for some of limitations of the cInerney , which icInerney  and McDocInerney . BrieflyUnder neutral evolution the ratio of RI/RV will not be significantly different from SI/SV. Similarly, a period of relaxed purifying selection may increase RI/RV relative to SI/SV, but RI/RV will never be significantly greater than the neutral expectation given by SI/SV since the rate of replacement substitution can only exceed the rate of silent  substitution under positive selection. During an episode of positive selection, advantageous substitutions will become fixed in a lineage and remain invariant in descendent lineages, elevating the ratio of RI/RV relative to the neutral expectation given by SI/SV. Thus, when lineages are identified with a significantly greater RI/RV than SI/SV positive selection is indicated.dN/dS ratio tests and further suggesting that the increase in rates identified from the dN/dS ratio were due to positive selection.Using the relative rate ratio test to examine selective forces after cluster duplications identified that post-duplication lineages under the ((AD)(BC)) and the (B(A(CD))) models had significantly larger RI/RV than SI/SV Figure  and 3, idN/dS model utilized above has been extended to account for variable dN/dS between sites and can detect positive selection at specific sites in specific lineages under appropriate conditions [PCD) branches identified sites under positive selection after cluster duplication  , high moMapping functionally divergent amino acid sites and sites under positive selection in and around helix-2 onto the logo and crystal structure shows that purifying selection has acted to preserve hydrophobic/aliphatic residues critical for the nuclear exportation signal  and posiThe homeodomain serves multiple functions in addition to DNA-binding, including containing nuclear localization and export signals, transcriptional activation and repression domains and other protein-protein interaction sites ,54. ThesNearly all 'cluster-specific' residues map onto the molecular surface of the homeodomain, similar to the paralog group specific sites , suggestThe homeodomain of Hox genes was identified from BLAST searches of the nr database at NCBI. At least four members of each gene from diverse taxa were included in the dataset. The sequences were aligned based on the translated amino acid sequences with Se-Al v2.0, alignments were simple given the high degree of sequence conservation within paralog groups. Regions of ambiguous alignment just outside of the homeodomain but within exon 2 were excluded. Most alignments ranged from 70–82 amino acids. The alignment is available from V.J.L. and has been deposited in TREEBASE.We used codon-based maximum likelihood models of coding sequence evolution implemented in CODEML in the PAML package of programs (version 3.15) to test for lineages and amino acid sites under positive selection. Sites were classified as being under positive selection if they were identified from the Bayes Empirical Bayes (BEB) method with a posterior probability of greater than 0.90. The branching order of the Hox cluster duplications is still debated (refs), but our analyses suggest that the most likely topologies are ((AD)(BC)) and (B(A(CD))) . We used 2 alternate trees to test for selection: ((AD)(BC)) and (B(A(CD))) and found no significant differences between the results of these different topologies. Functional divergence was tested with DIVERGE alpha1.2 (obtained from X. Gu). We also used the relative rate ratio test of Creevey and McInerny  implemenVJL designed and carried out the project, and wrote the manuscript with contributions from JJR and GPW. JJR provided information on protein-protein interaction sites and GPW provided biological insights and guidance during the course of this study.


A recent study has shown that pure neural stem cells can be derived from embryonic stem (ES) cells and primary brain tissue. In the presence of fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF), this population can be continuously expanded in adherent conditions. In analogy to continuously self-renewing ES cells, these cells were termed ‘NS’ cells . While NS cells have been shown to readily generate neurons and astrocytes, their differentiation into oligodendrocytes has remained enigmatic, raising concerns as to whether they truly represent tripotential neural stem cells.Here we provide evidence that NS cells are indeed tripotent. Upon proliferation with FGF2, platelet-derived growth factor (PDGF) and forskolin, followed by differentiation in the presence of thyroid hormone (T3) and ascorbic acid NS cells efficiently generate oligodendrocytes (∼20%) alongside astrocytes (∼40%) and neurons (∼10%). Mature oligodendroglial differentiation was confirmed by transplantation data showing that NS cell-derived oligodendrocytes ensheath host axons in the brain of myelin-deficient rats.bona fide tripotential neural stem cells.In addition to delineating NS cells as a potential donor source for myelin repair, our data strongly support the view that these adherently expandable cells represent  Neural stem cells are defined as clonogenic cells capable of self-renewal and multipotent differentiation into the three principle cell types of the CNS – neurons, astrocytes and oligodendrocytes. They have been isolated from the fetal Survival, proliferation and differentiation of stem cells appear to be regulated by both cell-autonomous and environmental signals In a recent study Conti et al. have reported on niche independent symmetrical self renewal of adherently growing neural stem cells derived from primary CNS tissue and ES cells Upon exposure to serum or BMP4, NS cells differentiate into astrocytes. Culture without EGF followed by FGF2 withdrawal gives rise to cells with immunochemical and electrophysiological properties of mature neurons. Importantly, even after prolonged expansion, NS cells maintain their potential to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. However, the culture conditions used so far did not support the differentiation of NS cells into oligodendrocytes.In the past, growth and differentiation factors suitable for the proliferation and differentiation of oligodendocyte progenitors have been successfully used to derive myelinating oligodendrocytes from ES cells The clonally derived NS cell line NS-5 generated from mouse ES cells was propagated and passaged according to Conti et al. To study whether NS cells can generate oligodendrocytes in vivo, we transplanted them into the brain of 2- to 3-day-old myelin-deficient (md) rats. This mutant carries a point mutation in the PLP gene, which results in severe dysmyelination and oligodendrocyte cell death Taken together, these data demonstrate that the differentiation spectrum of NS cells is not restricted to neurons and astrocytes but extends also to oligodendrocytes. This multipotential differentiation capacity of ES-cell-derived NS cells confirms their status of a tripotent stem cell. Previous studies have shown that tissue- and ES cell-derived oligodendrocyte progenitors can be efficiently proliferated in the presence of FGF2 and PDGF ES cell-derived NS cells were generated and cultured as previously described Cell cultures were fixed with 4% PFA for 10 min at room temperature. After washing in PBS, cells were blocked with 5% normal goat serum in PBS for 15 min and incubated over night in 1% normal goat serum in PBS with the following primary antibodies: O4 , Rip , proteolipid protein (PLP) , GFAP  and ß-III tubulin . For intracellular antigens cells were permeabilized in PBS containing 0.1% Triton X-100. Antigens were visualized using appropriate fluorochrome-conjugated secondary antibodies applied at 1∶250 for 1h . DAPI was used for nuclear counterstaining. Labeled cells were preserved in Vectashield (Vector Laboratories) and analysed using a Zeiss fluorescence microscope. Negative controls for antibody specificity were performed by omitting the primary antibodies. Quantitative analysis was carried out by counting the number of immunoreactive cells per total number of viable cells as determined by DAPI staining. Data for each marker are based on triplicate cultures with ≥20 randomly chosen high power fields quantified for each staining.Cell transplantation into the brain of early postnatal (P2–P3) myelin-deficient rats  was performed as described


Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics.Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2–3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus.These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation. Mammalian cells harbor a complex endocytic pathway that fulfills a variety of functions such as uptake of nutrients, recycling of proteins and lipids back to the plasma membrane, down-regulation of signaling receptors, detection and destruction of pathogens A heterogeneous class of diseases united under the name of lipid storage diseases leads to the pathological accumulation of specific lipids in late endosomes, which are subsequently thought to trigger membrane trafficking defects Using morphological, chemical and functional approaches, we have here analyzed the consequences of cholesterol accumulation using either NPC1 cells or negatively charged amine 3beta-(2-diethylaminoethoxy)-androstenone HCl (U18666A) To study the effect of late endosomal cholesterol accumulation at the organellar level, we used the baby hamster kidney (BHK) cell line for which a well-established subcellular fractionation protocol is available Late endosomes from control and U18666A-treated cells were then isolated. Fractionation properties were not significantly altered by cholesterol accumulation, since the small GTPase Rab7, a late endosomal marker We then analyzed the cholesterol content of purified late endosomes from control and U18666A treated cells by comparing yields  in agreeThe fact that both the amount of cholesterol and the amount of protein increased in late endosomes of U18666A treated cells suggested that either the size or the number of late endosomes had increased. To address this issue, we compared the volume of late endosomes in control and U18666A-treated cells, after quantification by electron microscope using stereological methods. Late endosomes of control and U18666A-treated BHK cells were labeled by fluid phase uptake of horseradish peroxidase (HRP) for 60 min at 37°C followed by a 10 min chase. As shown in These experiments thus show that U18666A-induced cholesterol accumulation in late endosomes is accompanied by a concomitant increase in the size of the compartment. We wondered whether this was also the case for cholesterol accumulation triggered by the expression of deficient NPC1 protein. To compare cells with the same genetic background, we made use of the NPC1 deficient CHO cell line CT43, and its parental line, 25RA We next analyzed the effect of U18666A on the phospholipid composition of late endosomes. Lipids were extracted and the relative proportions of phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), phosphatidyl serine (PS), sphingomyelins (SM) and LBPA were analyzed by 2 dimensional thin layer chromatography We next investigated whether U18666A would affect the composition/abundance of DRMs in late endosomes. The cholesterol profile along the Optiprep gradient was remarkably similar for late endosomes of U18666A treated and control cells, the low buoyant fractions 1 and 2 containing some 40% of the total cholesterol . We nextWe next investigated the effect of U18666A on late endosomal proteins. The total amounts of Lamp-1, Rab 7 and GPI-anchored proteins  were increased upon U18666A treatment, with the exception of flotillin, which was slightly decreased . TherefoWhen analyzing the overall protein profile on Optiprep gradients after detergent solubilization of late endosomes, a decrease in solubilized proteins was observed upon U18666A treatment with a concomitant≈2-fold increase in DRM-associated proteins . This waWe next wondered whether, in addition to being able to recruit more proteins, late endosomal DRMs from U18666A-treated cells had altered properties such as being more resistant to detergent extraction at 37°C instead of 4°C. Whereas this treatment led to solubilization of GPI-anchored proteins in control cells The increased resistance to detergent solubilization of late endosomal GPI-anchored proteins upon U18666A treatment raised the possibility that the dynamics of GPI-domains in intralumenal vesicles were altered—indeed, we have previously shown that GPI-anchored proteins reside mostly in intralumenal membranes ASSP is a mutant of aerolysin that retains its capacity to bind specifically to GPI-anchored proteins but requires GPI-dependent transport to late endosomes to form channels. It indeed contains an engineered disulfide bridge that becomes reduced in late endosomes, allow the toxin to heptamerize and form channels. Channel formation, in the intralumenal vesicles as inferred by the localization of GPI-anchored proteins The anthrax lethal toxin Finally, to further investigate the consequences of late endosomal cholesterol overload, we made use of Vesicular Stomatitis Virus (VSV), which infects cells from endosomes. The acidic endosomal pH triggers fusion of the viral envelope with endosomal membranes, thereby releasing the viral nucleocapsid into the cytoplasm and allowing infection to proceed. Le Blanc et al. Late endosomes are arguably the most complex station of the endocytic pathway due to their ability to communicate with other organelles, i.e. inter organellar trafficking , but also due to the communication between the intralumenal vesicles and the limiting membrane, i.e. intra organellar trafficking Cholesterol accumulation was induced in BHK cells to allow isolation of late endosomes by subcellular fractionation. Lipid analysis revealed that the composition of late endosomes was remarkably similar in control and U18666A treated cells with the exception of a decrease in the percentage of sphingomyelin and an inversion in the PC to PE ratio. Also the fraction of detergent resistant cholesterol was unaltered suggesting that there was in fact no change in the raft to non-raft ratio. The overall amount of cholesterol was however increased as expected from the literature, but it was accompanied by an almost compensating increase in the amount of other lipids and of proteins. Quantitative electron microscopy confirmed that the late endosomal compartment was enlarged in U18666A treated cells. This was due to the cholesterol accumulation and not to some side effect of the drug since a three-fold increase in cytoplasmic volume occupied by late endosomes was also observed in a CHO cell line deficient in NPC1 and in fibroblast from an NPC1 patient.The increase in late endosomal cholesterol however clearly did not just lead to an identical, albeit larger, compartment, as most drastically illustrated by the severity of the NPC phenotype Previous studies have established that late endosomal motility is impaired in NPC and U18666A-treated Pathological increases in late endosomal cholesterol, such as observed in NPC patients, led to a drastic enlargement of the compartment and alteration of its properties. Unexpectedly however, the raft to non-raft membrane ratio was not altered in late endosomes of U18666A treated cells. But, the action of toxins and viruses that target late endosome intralumenal membranes was significantly reduced, supporting the notion that cholesterol accumulation inhibits intra-endosomal membrane dynamics. Such perturbations of late endosomal membrane domains, in particular raft-like membranes, provide a reasonable explanation for altered protein and lipid trafficking through the compartment in cholesterol storage diseases.Monolayer of baby hamster kidney (BHK) and Chinese Hamster Ovary (CHO) Late endosomal fraction was prepared as described DRMs were prepared from late endosomes as described by 3PO4 solution.Lipids of late endosomes and late endosomes fractions obtained with OptiPrep gradient centrifugation were extracted in CHCl3/MeOH and then separated by two-dimensional thin layer chromatography (TLC) for phospholipids analysis For cholesterol analysis, lipids were extracted as above, analyzed on a one-dimensional TLC in heptane/ethylether/acetic acid  and stained with copper. Both cholesterol and phospholipids were quantified by densitometry using the ScanAnalysis software.Proteins were separated by SDS-PAGE using 12.5% acrylamide gels unless stated otherwise and transferred onto a nitrocellulose membrane. Western blots were revealed with SuperSignal Chemiluminescence (Pierce) and quantified by densitometry. Aerolysin overlays were performed as described by Before infection BHK cells grown on glass cover slips were treated during 9 hours with U18666A. Cells were then incubated 1 h at 4°C with VSV (1 MOI) in MEM pH 7.4 followed by 3 h at 37°C, in the presence of the drug. Finally cells were fixed and analyzed by immunofluorescence and infection was quantified by counting the number of cells expressing either newly synthesized VSV-G protein or GFP Cells grown on cover slips were fixed with 3% paraformaldehyde for 20 min at room temperature and saturated with 10% phosphate-buffered saline-fetal calf serum (PBS-FCS) for 20 min. Cholesterol was labeled with 50 µg/ml Filipin in 10% PBS-FCS. For flotillin-1 labeling cell PFA fixed cells were permeabilized with saponin and stained with a polyclonal antiflotillin-1 antibody BHK cells were treated with 3 µg/ml U18666A for 18 hours or were incubated under identical conditions in the absence of the drug. The cells were then incubated with HRP (10 mg/ml) for 60 min at 37°C followed by a further 10 min incubation without HRP to allow the HRP to chase out of early endosomal compartments. The cells were then fixed with 2.5% glutaraldehyde in PBS and processed for peroxidase detection and EM embedding using standard conditions. Digital images of thin sections were captured at random while scanning across the grid in a regular unbiased manner. Images were overlaid with a lattice grid generated in the imaging program . Volume of the HRP-containing compartments was quantitated by point counting using double lattice grids to relate points over HRP-labeled profiles to the cytoplasmic volume using standard methods


HIV-associated immune reconstitution disease (IRD) is the clinical presentation or deterioration of opportunistic infections that results from enhancement of pathogen-specific immune responses among patients responding to antiretroviral treatment (ART). The vast majority of reported cases of IRD have been associated with mycobacterial, chronic viral and invasive fungal infections; such cases result from dysregulated augmentation of cell-mediated type 1 cytokine-secreting host immune responses. However, the spectrum of infections now recognized as associated with IRD is expanding and includes a number of parasitic infections, which may be mediated by different immunopathological mechanisms. These include leishmaniasis , schistosomiasis and strongyloidiasis. Since the major burden of HIV lies in resource-limited countries where access to ART is now rapidly expanding, increased awareness and knowledge of these phenomena is important. Here we review the clinical spectrum and pathogenesis of IRD associated with parasitic infections. HIV-associated morbidity and mortality has dramatically decreased in many high-income countries since the advent of antiretroviral treatment (ART) in the mid 1990s . Robust IRD is not a new phenomenon, but has long been recognized as a complication occurring among patients with severe immunosuppression in whom immune function is rapidly restored. Thus, for example, IRD may occur following withdrawal or rapid dosage reduction of corticosteroid treatment and among patients in whom the blood neutrophil count recovers following cytotoxic chemotherapy or bone marrow transplantation . The patMycobacterium tuberculosis, Mycobacterium avium complex and other non-tuberculous mycobacteria); (b) viruses ; and (c) fungi  (vide infra).The vast majority of cases of IRD associated with ART have been reported from high-income countries and have been associated with a wide range of non-parasitic opportunistic infections, including: (a) bacteria (ma spp.) . HoweverThe majority of people with HIV/AIDS live in resource-limited countries and in June 2005 WHO estimated that 6·5 million people living in such countries were in urgent need of ART . DespiteIRD can be defined as the clinical presentation or deterioration of opportunistic infections that results from enhancement of pathogen-specific immune responses among patients responding to ART. However, diagnosis in practice is not straightforward, being one of clinical judgement based on various lines of indirect evidence. These may include: (a) the clinical manifestation or pattern of progression of an opportunistic infection that is unusual; (b) a temporal relationship with ART initiation; (c) exclusion of alternative explanations; (d) demonstrated efficacy of ART  evidence of improved CD4 cell function (e.g. development of a positive tuberculin skin test); and (f) histopathology consistent with the diagnosis. Competing explanations for these clinical manifestations that should be excluded are the occurrence of opportunistic infections as a result of residual immunodeficiency and inadequate treatment of an opportunistic infection, including that resulting from drug resistance.+CD45RO+ memory cells previously sequestered in lymphoid tissue and a reduction in apoptotic cell death (+CD62L+ cells (Successful ART is associated with a rapid (> 90%) reduction in plasma viral load within the first weeks of ART. The most characteristic immunological feature of HIV infection is depletion of CD4 T cell numbers, and restoration of the CD4 cell subset during ART appears to occur in two principal phases. The initial rapid phase of CD4 cell recovery can usually be detected within the first 1–2 weeks of starting treatment and extends over 2–3 months . Data sull death ,17. Thosll death –21. A slL+ cells .The increase in circulating CD4 cell numbers is also associated with an improvement in effector function, the extent of which is directly related to the degree of viral load suppression and the CD4 cell counts in the longer term . IL-2-meAlthough HIV is principally characterized by CD4 cell depletion, functional deficits in other cells of the innate and acquired immune systems also occur. However, the literature concerning effects of ART on other cell types is much less comprehensive than that concerning CD4 cell reconstitution. Moreover, many of these effects are CD4 cell-dependent, so that attributing ART-induced improvements in immune function to either CD4 cells or to other cell types is difficult.+ T cell counts are also associated with decreased circulating CD3−/CD16+/CD56+ NK cells  decreases during ART ,16,22, p+ CD25+ IL-10-secreting T regulatory cells  cytokine-secreting immune responses  is the clinical form of leishmaniasis that has the most substantial geographical overlap with HIV and typically occurs as an opportunistic infection among HIV-infected individuals with CD4 cell counts < 200 cells/µL . This oveficient .L. infantum VL in southern France describe asymptomatic patients who developed fever and hepatosplenomegaly due to VL within the first 2 weeks of initiating ART  are associated with an intense granulomatous response, causing tissue pathology via a CD4 cell-dependent process ,73. FromLeishmania braziliensis were established in both patients and both responded well to treatment with either amphotericin B or pentavalent antimony . On. On74). Two cases of PKDL presenting as IRD have been reported from Israel  and ItalLeishmania donovani during ART has been reported from the Netherlands .,84.83,84et al. , schistosomiasis and strongyloidiasis. No studies have yet examined the mechanisms of IRD associated with helminth infections, but these may be due to augmentation of appropriate type 2 immune responses. More research is needed to increase our understanding of these emerging phenomena.