In the emerging field of environmental genomics, direct cloning and sequencing of genomic fragments from complex microbial communities has proven to be a valuable source of new enzymes, expanding the knowledge of basic biological processes. The central problem of this so called metagenome-approach is that the cloned fragments often lack suitable phylogenetic marker genes, rendering the identification of clones that are likely to originate from the same genome difficult or impossible. In such cases, the analysis of intrinsic DNA-signatures like tetranucleotide frequencies can provide valuable hints on fragment affiliation. With this application in mind, the TETRA web-service and the TETRA stand-alone program have been developed, both of which automate the task of comparative tetranucleotide frequency analysis.Availability: TETRA provides a statistical analysis of tetranucleotide usage patterns in genomic fragments, either via a web-service or a stand-alone program. With respect to discriminatory power, such an analysis outperforms the assignment of genomic fragments based on the (G+C)-content, which is a widely-used sequence-based measure for assessing fragment relatedness. While the web-service is restricted to the calculation of correlation coefficients between tetranucleotide usage patterns of submitted DNA sequences, the stand-alone program generates a much more detailed output, comprising all raw data and graphical plots. The stand-alone program is controlled via a graphical user interface and can batch-process a multitude of sequences. Furthermore, it comes with pre-computed tetranucleotide usage patterns for 166 prokaryote chromosomes, providing a useful reference dataset and source for data-mining.Up to now, the analysis of skewed oligonucleotide distributions within DNA sequences is not a commonly used tool within metagenomics. With the TETRA web-service and stand-alone program, the method is now accessible in an easy to use manner for a broad audience. This will hopefully facilitate the interrelation of genomic fragments from metagenome libraries, ultimately leading to new insights into the genetic potentials of yet uncultured microorganisms. At present, a majority of the microbes from natural microbial communities cannot be transferred into pure cultures, either because proper cultivation conditions have yet to be found, or due to currently unidentified fundamental obstacles . For decGammaproteobacteria [The metagenome-approach allows for the first time to circumvent the restrictions imposed by the limited culturability of environmental microorganisms . Now, DNbacteria -6, and tbacteria , respectHowever, the metagenome-approach is not without its limitations and problems. A major constraint is that especially small genomic fragments, as are obtained from libraries that have been constructed with fosmids or cosmids as cloning vectors, often lack suitable phylogenetic marker genes. This leads to the problem that fragments belonging to the same organism cannot be reliably identified as such unless they overlap. In order to nonetheless interrelate such genomic fragments, measures such as the (G+C)-content or BLAST hits and codon usage of the fragment's coding regions are commonly used to assess whether two unlinked fragments from a metagenome library belong to the same organism. These measures, however, can produce ambiguous or even misleading results, and should be supplemented by additional tools that assess the relatedness of the genomic fragments .Since numerous studies have shown that oligonucleotide frequencies within DNA sequences exhibit species-specific patterns -18, compIn order to automate and facilitate such an analysis, the TETRA software suite was developed, comprising both, a web-service and a stand-alone program.et al. (2004) and the TETRA online manual.The algorithms that are used within TETRA have been described elsewhere . In brieThe TETRA web-service  has been REAL Software Inc., Austin, Texas) and is available for Mac OS X. Versions for Linux and Windows are also available, but differ in details regarding their implementation and features. The counting of the tetranucleotides in the current version of TETRA stand-alone program is done by ocount – a self-written C program that has been integrated into the program.The TETRA stand-alone program can be downloaded for free from the TETRA website . The curThe TETRA web-service computes correlation coefficients between tetranucleotide usage patterns of DNA sequences, which can be used as an indicator of sequence relatedness. Details on the in- and output formats is available in the comprehensive online documentation .The stand-alone version of TETRA has many additional features that are not available via the TETRA web-service. Firstly, it comes with pre-computed tetranucleotide usage patterns of all 166 prokaryote chromosomes that were publicly available by June 2004  is computed in less than 10 minutes on a dual 1.8 GHz G5 (IBM PPC 970) computer. Newly computed tetranucleotide usage patterns are displayed within the 'Navigator' window, which is the central place for data management, access to the raw data and the calculation of plots and correlation coefficients Figure . Raw datA detailed documentation of the TETRA stand-alone program and its functions is available via the program's online help system.As has been demonstrated in a previous study , the anaEscherichia sp., Shigella sp., Yersinia sp. or Mesorhizobium sp., Sinorhizobium sp., Bradyrhizobium sp.). However, more distantly related genera or even species with larger evolutionary distances are often not correctly clustered (e.g. Prochlorococcus sp.).While the method works quite well for sequences in the range of 40 kb, it is certainly not suited for the analysis of single-read end-sequences, which are usually shorter than 1 kb. Since the phylogenetic signal within tetranucleotide usage patterns is faint, the method performs weakly for whole genome phylogenetic tree reconstructions. In a whole-genome tree calculated from the pre-computed 166 prokaryotic chromosomes (data not shown), organisms are mostly grouped at the species level and at the level of genera, when these are closely related : Platform independent (web-service); Mac OS X • Programming language: REALbasic• Other requirements: none• License: none• Any restrictions to use by non-academics: noneBLAST – basic local alignment search toolmegx – marine environmental genomicsTETRA was implemented by HT and JW, TL contributed to the TETRA web-service, MB and FOG contributed important ideas regarding implementation, features and tested the programs.


Feline leukemia virus (FeLV) induces degenerative, proliferative and malignant hematologic disorders in its natural host, the domestic cat. FeLV-945 is a viral variant identified as predominant in a cohort of naturally infected animals. FeLV-945 contains a unique sequence motif in the long terminal repeat (LTR) comprised of a single copy of transcriptional enhancer followed by a 21-bp sequence triplicated in tandem. The LTR is precisely conserved among independent cases of multicentric lymphoma, myeloproliferative disease and anemia in animals from the cohort. The 21-bp triplication was previously shown to act as a transcriptional enhancer preferentially in hematopoietic cells and to confer a replicative advantage. The objective of the present study was to examine the molecular mechanism by which the 21-bp triplication exerts its influence and the selective advantage responsible for its precise conservation.Potential binding sites for the transcription factor, c-Myb, were identified across the repeat junctions of the 21-bp triplication. Such sites would not occur in the absence of the repeat; thus, a requirement for c-Myb binding to the repeat junctions of the triplication would exert a selective pressure to conserve its sequence precisely. Electrophoretic mobility shift assays demonstrated specific binding of c-Myb to the 21-bp triplication. Reporter gene assays showed that the triplication-containing LTR is responsive to c-Myb, and that responsiveness requires the presence of both c-Myb binding sites. Results further indicated that c-Myb in complex with the 21-bp triplication recruits the transcriptional co-activator, CBP, a regulator of normal hematopoiesis. FeLV-945 replication was shown to be positively regulated by CBP in a manner dependent on the presence of the 21-bp triplication.Binding sites for c-Myb across the repeat junctions of the 21-bp triplication may account for its precise conservation in the FeLV-945 LTR. c-Myb binding and CBP recruitment to the LTR positively regulated virus production, and thus may be responsible for the replicative advantage conferred by the 21-bp triplication. Considering that CBP is present in hematopoietic cells in limiting amounts, we hypothesize that FeLV-945 replication in bone marrow may influence CBP availability and thereby alter the regulation of CBP-responsive genes, thus contributing to altered hematopoiesis and consequent hematologic disease. The variable clinical outcome of FeLV infection is thought to reflect this genetic diversity .Feline leukemia virus (FeLV) is a simple gammaretrovirus that induces degenerative, proliferative and malignant hematologic disorders in its natural host, the domestic cat. Like other natural retroviruses, FeLV is not a single genomic species but is a genetically complex family of closely related viruses subject to selective pressures in the host. Variant genomes are generated during virus replication as a result of both error-prone reverse transcription and recombination. The consequence of this variation is a diverse population that is continuously shaped iversity ,2. FeLV-iversity ,4, ChandThe 21-bp triplication was previously shown to provide transcriptional enhancer function to the LTR that contains it, and to function preferentially in primitive hematopoietic cells . In K-56Regarding the molecular mechanism by which the 21-bp triplication functions in the context of the LTR, at least two possibilities have been considered. One possibility is that the 21-bp triplication functions to maintain the appropriate spacing in the LTR between the enhancer and the promoter. A spacer function might be particularly relevant in an LTR like FeLV-945 in which the enhancer is not tandemly repeated. Substitution of the 21-bp repeat element with unrelated sequence of the same length, however, was observed to ablate the replicative advantage, thus indicating that the 21-bp triplication does not perform solely a spacer function . An alteAs described above, previous studies suggested that preserving the protein binding sites may confer a selective advantage that accounts for the precise sequence conservation of the 21-bp triplication in the FeLV-945 LTR . In the To evaluate whether c-Myb binding to the 21-bp triplication regulates LTR function, reporter plasmids were constructed in which expression of the firefly luciferase gene was driven by the U3 region of an FeLV LTR containing one, two or three copies of the 21-bp element. Reporter gene constructs were introduced by lipid-mediated transfection into feline embryonic fibroblasts (FEA) along with increasing amounts of a c-Myb expression vector. Fibroblasts were selected because the level of endogenous c-Myb expression in those cells is low or absent ,15. The Previous studies had shown that the 21-bp triplication contributes enhancer function to the LTR in a cell type-specific manner, and that it is significantly more active in K-562 cells as compared to a fibroblast line ,15. WhenSupershift EMSA was used to evaluate whether c-Myb and CBP might be present in the specific complex that forms on the 21-bp triplication. Specific complex formation was observed using a radiolabeled probe representing the 21-bp triplication in the presence of nuclear extracts from either K-562 cells or feline 3201 T-cells. In both cases, mobility of the complex was reduced (supershifted) when reacted with antibody to c-Myb. When the same reaction was performed in the presence of an antibody to CBP, an identical supershift was observed Figure . No compet al., manuscript submitted]. The 21-bp triplication was shown to enhance transcription from the FeLV LTR and to confer a replicative advantage to the virus, at least in part through the specific binding of unidentified nuclear proteins to the repeat motif , it is likely that the tropism of FeLV-945 in vivo included the hematopoietic progenitors in which c-Myb is expressed. Considering this possibility, we hypothesized that c-Myb may act as a transcriptional regulator of FeLV-945. In support of this hypothesis, reporter gene assays showed that an LTR containing the triplication was responsive to c-Myb in a dose-dependent manner  and was maintained in RPMI 1640 medium with 10% FBS. The FEA cell line, a continuous line of feline embryonic fibroblasts, was obtained from Dr. Jennifer Rojko and was grown in Eagle minimal essential culture medium supplemented with 10% fetal bovine serum (FBS), 0.1 mM non essential amino acids and 50 μg/ml gentamicin reagent solution . 3201 is an FeLV-negative thymic lymphoma cell line of feline origin [me sites . The FeLme sites .TAACGGTTCCGTAGTGA) or a CREB consensus binding site (5'-AGAGATTGCCTGACGTCAGAGAGCTAG). Protein-DNA complexes were resolved by 6% polyacrylamide gel electrophoresis in 0.25X TBE buffer . Gels were then dried at 80°C and exposed to radiographic film for varying periods of time. In some reactions, monoclonal antibody (4 μg) to either c-Myb or CBP, or isotype control antibody, was added after the 30-minute incubation period and incubated overnight at 4°C. Complexes were then resolved by 6% polyacrylamide gel electrophoresis as described above. The mouse monoclonal IgG1 antibody to c-Myb was raised against a recombinant protein corresponding to amino acids 500–640 of the human protein . The mouse monoclonal IgG1 antibody to CBP was raised against a peptide corresponding to amino acids 2422–2441 of CBP of human origin .A double-stranded oligonucleotide probe containing the 21-bp triplication and 40 bp of flanking sequence from the FeLV-945 LTR was radiolabeled using the synthetic oligonucleotide GS945 as template (5'- GCTGAAACAGCAGAAGTTTCAAGGCCACTGCCAGCAGTTTCAAGGCCACTGCCAGCAGTTTCAAGGCCACTGCCAGCAGTCTCCAGGCTCCCCAGTTGAC -3'), the filling primer (5'-CTGGTCAACTGGGGAGCCT-3') and the Klenow fragment of DNA polymerase  to complete the duplex. A homologous probe containing only a single copy of the 21-bp element was similarly synthesized using the oligonucleotide GS61E as template (5'-GCTGAAACAGCAGAAGTTTCAAGGCCACTGCCAGCAGTCTCCAGG CTCCCCAGTTGAC-3'). Nuclear extract from K-562 cells was obtained from Active Motif . Nuclear extracts from FEA and 3201 cells were prepared using the Nuclear Extract Kit from Active Motif  according to manufacturer specifications. Nuclear extracts were also prepared from FEA cells following the lipid-mediated transfection  of FL-Myb, a c-Myb expression vector in which full length murine c-Myb cDNA was inserted into the multiple cloning site of pcDNA3.1 . DNA-protein binding reactions included 5 μg of nuclear extract and 2.4 ng of radiolabeled probe in a 20 μl reaction containing 1 mM Tris pH 7.5, 7.5 mM NaCl, 1 mM EDTA, 0.7% glycerol, 0.1 mM DTT and 2 μg poly(dI-dC). Reactions containing nuclear extracts from K-562 cells were incubated at 4°C for 30 minutes. Reactions containing nuclear extracts from 3201 or FEA cells were incubated at 30°C for 30 minutes. In some reactions, unlabeled probe was added to the reaction as a specific competitor, or HindIII/HaeIII-digested bacteriophage lambda DNA was included as non-specific competitor. Some reactions included as competitor a double-stranded oligonucleotide containing a known high-affinity c-Myb consensus binding site  and +/- (5'-GCTGAAACAGCAGAAGTTTCAAGGCCACTGCCAGCAGTTTCAAGGCCACTGCCAGCAGATTCAAGGCCACTGCCAGCAGTCTCCAGGCTCCCCAGTTGAC-3') that contained a point mutation in the first or second binding site, respectively (mutated base indicated by boldface and underline). The indicated point mutation had previously been shown to ablate c-Myb binding [AluI/HincII and substituted into the LTR of FeLV-A/61E. The KpnI/PstI fragment of the resulting recombinant LTR was then excised and cloned into the pGL2-Basic luciferase reporter plasmid. Finally, a luciferase reporter plasmid was developed that contained the isolated 21-bp triplication cloned upstream of the SV40 promoter in pGL2-Promoter . A double-stranded DNA fragment containing the 21-bp triplication was generated by PCR amplification using the oligonucleotide GS945 (described above) as template and primers fw945-kpn1 (5'- GCTCGGTACCAGCTGAAACAGCAGAAGTTTC) and rv945-sac1 (5'- ATGCTGAGCTCAACTGGGGAGCCTGGAGACT). The resulting amplification product was digested with KpnI/SstI and inserted into the multiple cloning site upstream of the SV40 promoter in the reporter plasmid.Luciferase reporter plasmids were constructed to contain the U3 region of an FeLV LTR containing one, two or three copies of the 21-bp element. The U3 region of the FeLV-A/61E LTR, containing one copy of the 21-bp element, was cloned into the firefly luciferase reporter plasmid pGL2-Basic . The LTR was then substituted between  binding . A doubl5 cells were seeded in triplicate into 6-well tissue culture plates. The next day, reporter plasmids (500 ng) were introduced into cultured cells by lipid-mediated transfection  in the presence of pRL-TK (5 ng) in a 100:1 ratio. pRL-TK encodes Renilla luciferase and was used as an internal control for transfection efficiency. Firefly and Renilla luciferase activities were quantified 24 hours later using the Dual-Luciferase Reporter Assay System . Data from triplicate wells were analyzed statistically using one-way ANOVA and Bonferroni post test. Statistical significance was considered as p < 0.05. In some assays, the c-Myb expression vector FL-Myb (described above) was added to the transfection in increasing amounts (50 ng – 500 ng). The 5XMRE plasmid was used in reporter gene assays as a positive control. This plasmid contains five tandem c-Myb binding sites cloned upstream of a luciferase gene .For reporter gene assays, 2 × 105 K-562 cells, uninfected or chronically infected with recombinant FeLVs GA-945L and GA-61EL (described above) were seeded in triplicate into 24-well tissue culture plates. A full-length CBP expression vector  was introduced into the cells by lipid mediated transfection using Lipofectamine 2000 reagent . Culture supernatants were collected three days later and reverse transcriptase activity was quantified as previously described [5 × 10escribed . Data frNone declared.SLF developed reporter gene constructs and performed binding assays, gene expression and virus replication assays. SP identified and initially demonstrated c-Myb binding sites. KRR developed the reporter gene assays. LSL directed the experimental design, implementation and interpretation of data. All authors read and approved the final manuscript.


Idd (insulin dependent diabetes) loci, which have been mapped to small chromosome intervals, for which the orthologous regions in the human genome can be identified. Here, we have conducted re-sequencing and association analysis of six orthologous genes identified in NOD Idd loci: NRAMP1/SLC11A1 (orthologous to Nramp1/Slc11a1 in Idd5.2), FRAP1 (orthologous to Frap1 in Idd9.2), 4-1BB/CD137/TNFRSF9 (orthologous to 4-1bb/Cd137/Tnrfrsf9 in Idd9.3), CD101/IGSF2 (orthologous to Cd101/Igsf2 in Idd10), B2M (orthologous to B2m in Idd13) and VAV3 (orthologous to Vav3 in Idd18).One strategy to help identify susceptibility genes for complex, multifactorial diseases is to map disease loci in a representative animal model of the disorder. The nonobese diabetic (NOD) mouse is a model for human type 1 diabetes. Linkage and congenic strain analyses have identified several NOD mouse Re-sequencing of a total of 110 kb of DNA from 32 or 96 type 1 diabetes cases yielded 220 single nucleotide polymorphisms (SNPs). Sixty-five SNPs, including 54 informative tag SNPs, and a microsatellite were selected and genotyped in up to 1,632 type 1 diabetes families and 1,709 cases and 1,829 controls.P values > 0.2), indicating that common variation in these key candidate genes does not play a major role in type 1 diabetes susceptibility in the European ancestry populations studied.None of the candidate regions showed evidence of association with type 1 diabetes ( Type 1 diabetes is a common, multifactorial disease believed to be caused in a proportion of cases by an autoimmune destruction of pancreatic β-cells by an inflammatory infiltrate comprising T lymphocytes, dendritic cells and macrophages. This process results from a complex interaction between genetic and environmental risk factors. Genetically, it is under the control of the major histocompatibility complex (MHC) [Idd regions controlling type 1 diabetes susceptibility in the NOD mouse could also determine susceptibility in humans, even though this depends on the frequency of susceptibility alleles in human populations, which affects statistical power, and that the correct candidate gene has been chosen from the Idd interval. These Idd intervals might contain many genes, including several involved in the immune response [A murine model of type 1 diabetes, the NOD mouse, spontaneously develops an autoimmune-mediated diabetes that has many similarities to the human disease. It is likely that components of the pathophysiology and genetic predisposition are conserved across species, and indeed two loci have already been shown to affect type 1 diabetes susceptibility in both species, namely the immunoregulatory MHC HLA class II and CTLA-4 genes. The other causative gene(s) in the known response . Neverthresponse ,4.Ian4/Iddm1 [Cblb (KDP rat) [CTLA4 [The central importance of T cell development and function in type 1 diabetes is evident from the susceptibility genes identified so far. The MHC class II genes are important etiologically in two rat models of type 1 diabetes, the Biobreeding (BB) and KDP strains ,6, the Nn4/Iddm1  affects KDP rat)  and CTLA) [CTLA4  (in humaIl2 [Cd101 [Nramp1/Slc11a1 [Idd5.2 [Idd9.2 [Idd9.3 [Idd10 [Idd13 [Idd18 [Nramp1/Slc11a1 from Idd5.2 [Frap1 from Idd9.2 (unpublished); 4-1bb/Cd137/Tnfrsf9 from Idd9.3 [Cd101/Igsf2 from Idd10 [B2m from Idd13 [Vav3 from Idd18 . Table In our selection of candidate genes within NOD congenic intervals, we have, therefore, biased our choice towards immune-related genes such as  [Idd9.2 , Idd9.3  [Idd9.3 , Idd10 [3 [Idd18  we chosem Idd9.3  (unpubliom Idd13  and Vav34-1BB [NRAMP1 (MIM 600266), FRAP1 (MIM 601231), 4-1BB (MIM 602250), CD101 (MIM 604516), B2M (MIM 109700) and VAV3 (MIM 605541) were re-sequenced in 32 or 96 randomly chosen UK white patients with type 1 diabetes to identify SNPs and for the selection of tag SNPs. As LD between 4-1BB SNPs was weak, eight out of nine common SNPs were genotyped  and analysed using single-locus tests.A tag SNP approach to test for association was adopted for all genes, except for 4-1BB , in orde4-1BB . Coding 4-1BB, FRAP1, or B2M were observed. A synonymous change was detected in exon 3 of NRAMP1 (MAF = 0.32) and a non-synonymous SNP (nsSNP) in exon 15 (MAF = 0.02), causing a conservative amino acid change: Asp543Asn (DIL5202/ss23142243). Interestingly, as in the case of its mouse orthologue [CD101 , VAV3β (unique exon 1 and exons 4 to 27) and VAV3.1 (unique exon 18 and exons 19 to 27) yielded six exonic SNPs  (see Table D101 see . Re-sequSNPs see . Two SNPVAV3 and a microsatellite from NRAMP1 were genotyped a priori in the whole family collection (step 1 and 2) and a single nsSNP from CD101 in step 1 families only (DIL3794/rs3754112). The nsSNP DIL3810/ss23142433 in VAV3 was not tested because it was in quite strong LD with DIL3809/ss23142432 (R2 = 0.64), so that only DIL3809/ss23142432 was genotyped. Note that in our tag approach, the two VAV3 nsSNPs (DIL1521/rs7528153 and DIL3809/ss23142432) were chosen deliberately as tag SNPs.Two common nsSNPs  from P values for association between type 1 diabetes and candidate gene tag SNPs all exceeded 0.2, as did the single-locus test P values for 4-1BB SNPs. Consequently, we did not proceed to genotype in step 2 samples for any of the candidate genes (Table VAV3 and CD101 or the microsatellite of NRAMP1 showed evidence of association (Table NRAMP1 microsatellite promoter (GT)n has previously shown linkage and association with autoimmune disease, and allele A2 with infectious disease susceptibility [In a pragmatic, phased genotyping strategy, in step 1, the multi-locus test es Table  and 4. Non Table . Allele tibility -20. The VAV3, which spans almost 400 kb, less than 10% of the genomic region of VAV3 was re-sequenced to identify SNPs. The general importance of intronic and intergenic regulatory sequences as candidates for disease susceptibility is well recognised. Hence, potential unidentified causal variants in introns or flanking regions of the genes may have been missed, and remain a target for future analyses. Despite finding no evidence of association, it remains possible that there exists a common disease variant in one or more of the six candidate genes tested, which either has an effect smaller than would be detected with this study or is in much weaker LD with the tag SNPs than any other SNP known to us [With regards to our association study in humans, intronic and potential regulatory regions were not sequenced in the candidate genes since these cover large genomic regions, which will have to wait for much more extensive polymorphism maps . For exawn to us .NRAMP  and DIL3799/ss23142349 in CD101 . For both SNPs, P values above 0.05 were obtained (P = 0.19 for DIL5202/ss23142243 and P = 0.80 for DIL3799/ss23142349), therefore, making it less likely that these rare variants contribute to susceptibility to type 1 diabetes. Nevertheless, causal variants with MAFs less than 0.01 [Finally, the possibility of one or more rare disease variants in a locus needs to be considered . The besIdd interval may account for disease susceptibility in the NOD mouse, but the human orthologous region may lack this susceptibility variant. The scenario, in which candidate genes in the NOD Idd interval may not necessarily be harbouring a functional, causal variant in their human orthologue genes, was discussed previously [Idd interval may not be the gene causing susceptibility to disease.Taken together, these data make an association between type 1 diabetes and common variation in coding and untranslated regions of the six functional candidate genes in the investigated human-mouse orthologue regions less likely. Several possibilities may account for this. A gene  in an eviously . It is aThe tag SNP maps described here will be useful for association studies of other diseases. They will be integrated into future SNP maps encompassing the entire orthologous regions and all regulatory sequences and genes encoded within them.CD101 and NRAMP1, DIL3799/ss23142349 and DIL5202/ss23142243, a total of 3,704 type 1 diabetes cases and 3,930 controls were used.All family members were white and of European ancestral origin. The type 1 diabetes families comprised two parents and a least one affected child. The 748 type 1 diabetes families used in 'step 1' were as described previously : 472 UK NRAMP1, the 748 type 1 diabetes families described above were used in addition to 343 multiplex/simplex families from the UK, 159 Norwegian simplex families, 322 Romanian simplex families, and 60 multiplex families from the USA totalling the combined DNA sets to 1,632 type 1 diabetes families, as described previously [For 'step 2' genotyping of eviously .Nested PCR products from DNA from 96 or 32 type 1 diabetes patients were sequenced using an Applied Biosystems (ABI) 3700 capillary sequencer , and SNPs identified using the Staden Package .® assay  [NRAMP1 microsatellite was genotyped on an ABI3700 sequencer using fluorescent primers as previously described [SNPs were genotyped using the Invaderison WI)  and TaqMison WI) . The NRAescribed . Full deNRAMP1 , FRAP1 (UO88966), 4-1BB (UO3387), CD101 (Z33642), B2M (BC032589) and VAV3  was performed by importing Ensembl information into a temporary ACeDB database as described in Burren et al. [Annotation of n et al. .Stata statistical system and may be downloaded from our website [P > 0.05).The program for the selection of tag SNPs  and asso website . All genLMM and DJS contributed equally to this work by performing the genetic studies and writing the manuscript. AV, FP, RP, CL, JH, HF, CM, KMH, GC carried out the genetic studies and collated data. JDC performed the statistical analysis and participated in the design of the study. LJS participated in the sequence analysis. NW participated in design and collated data. KSR, CG, C I-T, DAS, DPS and LBP participated in the study design and coordination. JAT, LSW and RCT helped to draft the manuscript. All authors read and approved the final manuscript.NRAMP1. Novel SNPs are denoted by "ss" numbers and previously published SNPs are denoted by "rs" numbers. Minor allele frequencies are based on the sequencing panel of 32 type 1 diabetes subjects. R2 values for non-typed SNPs. UTR, untranslated region.SNPs, including two deletion/insertion polymorphisms, identified in Click here for fileCD101. Novel SNPs are denoted by "ss" numbers and previously published SNPs are denoted by "rs" numbers. Minor allele frequencies are based on the sequencing panel of 96 type 1 diabetes subjects. R2 values for non-typed SNPs. Note that DIL3969 has an allelic R2 < 0.80 due to technical difficulties with the assay. UTR, untranslated region.SNPs identified in Click here for fileVAV3. Novel SNPs are denoted by "ss" numbers and previously published SNPs are denoted by "rs" numbers. Minor allele frequencies are based on the sequencing panel of 96 type 1 diabetes subjects. R2 values for non-typed SNPs. Note that DIL6496 and DIL6488 in block 1, and DIL1526 have allelic R2 values < 0.80, which was due to technical difficulties with those assays. UTR, untranslated region.SNPs, including four in/dels identified in Click here for fileIdd loci, the location of their human orthologous regions, and selected functional candidate gene within the Idd interval. *Estimated number of genes.Supporting published references for Table Click here for file4-1BB. Minor allele frequencies are based on the sequencing panel of 96 type 1 diabetes subjects. Novel SNPs are denoted by "ss" numbers and previously published SNPs are denoted by "rs" numbers. Note that DIL4247/rs6694557 could not be genotyped due to assay technical difficulties. UTR, untranslated region.SNPs identified in Click here for fileFRAP1. Novel SNPs are denoted by "ss" numbers and previously published SNPs are denoted by "rs" numbers. Minor allele frequencies are based on the sequencing panel of 32 type 1 diabetes subjects. R2 values for non-typed SNPs. UTR, untranslated region.SNPs identified in Click here for fileB2M. Novel SNPs are denoted by "ss" numbers and previously published SNPs are denoted by "rs" numbers. Minor allele frequencies are based on the sequencing panel of 32 type 1 diabetes subjects. R2 values for non-typed SNPs. UTR, untranslated region.SNPs identified in Click here for fileGenotyping counts Numbers of attempted subjects for genotyping and of subjects with genotypes.Click here for file


In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans. Although individual approaches fall short, integrating multiple common genetic and biochemical approaches yields a description of mitochondrial proteins that is more than the sum of its parts About 30% of these proteins have evidence of orthologs in humans (MitoP2 database).About half of the expected mitochondrial proteins in humans are known to date, and already a fifth of these known proteins are associated with human Mendelian disorders  and nonfermentable (lactate) substrates for both natural and synthetic culture media, were purified by either density gradient or free-flow electrophoresis. Preparations were separated into mitochondrial membrane and matrix fractions and analyzed separately for protein content. In total, 20 fractions were digested with trypsin and analyzed by reversed phase high resolution liquid chromatography/tandem MS (LC/MS/MS) . In addiThe performance of our proteomic and other systematic approaches in identifying mitochondrial proteins was evaluated against a reference set of 477 proteins classified as mitochondrial localized based on single gene studies. Of the 546 proteins identified by our proteomic approach, 47% were known mitochondrial, covering 54% of the reference set (256/477). Sorting the 546 candidates by the number of experiments in which they were found demonstrated that the probability of identifying a mitochondrial protein correlated with its detection frequency and with the confidence associated with its identification based on the number of peptide tags identified A. A sepaA comparison between fermentable and nonfermentable growth conditions revealed that more proteins were detected under respiration (448) than fermentation (378) conditions C, consisOf the 546 proteins identified by proteomics, 182 proteins are known to localize outside mitochondria, mainly to the cytoplasm, nucleus, endoplasmic reticulum, and plasma membrane D. In addIn the analysis of complex protein mixtures by MS, low abundance of proteins can preclude their identification . This miWe also extended our comparison to the analysis of protein abundance, which was recently determined for about two-thirds of the yeast proteome under fermentation . To visuAnalysis of the overlap between both proteomic datasets B shows tAre there classes of proteins that were not captured by a proteomic analysis, whether integrative or not, but that could be found using different approaches, and vice versa? To address this question, we performed a comparative analysis of functional categories identified by our proteomic dataset in comparison to functional approaches of gene expression analysis and quantitative deletion phenotype screening—datasets which were generated in this study and by A comparison of the distribution of protein enrichments shows that different functional categories are targeted by different approaches. Overall, the proteomic approach and the deletion approach identified about equal numbers of previously known mitochondrial proteins; however, they overlapped for less than 30% of the proteins. These observations suggest that combining complementary approaches and an integrative data analysis could be advantageous for predicting new mitochondrial proteins.To improve our comparison of methods and to generate a high confidence list of mitochondrial proteins, we expanded our comparison to a total of 22 datasets relevant to the study of the mitochondrial proteome that had been collected to date. These included the experimental and computational approaches listed in the introduction, including a very recent proteome analysis  and GFP-Rickettsia prowazekii, believed to be closest to a common ancestor with mitochondria. The details for each dataset are given in the MitoP2 database , differentially regulated in response to the diauxic shift , or diffWe first assessed the performance of each method. For this purpose, the sensitivity and specificity of the different approaches were calculated by comparing each dataset with the reference set of known mitochondrial proteins . This co(R) of observed proteins present in the mitochondrial reference set relative to the total number of proteins detected. Most proteins belonged to more than one combination, and for these proteins multiple R values were calculated. The MitoP2 value was chosen to represent the highest R value calculated for a protein, representing the specificity of the best combination of methods.We next set out to determine whether the information supplied by each one of the different methods could be combined to achieve a predictive power that exceeded that of any single approach. We assessed the overlap among different combinations of the 22 datasets and defined a metric for attaching a numerical value to the likelihood of a protein being mitochondrial. A predictive score (MitoP2 score) was estimated based on the specificity of the best combination of approaches: we calculated for each approach as well as for all possible combinations of approaches, the percentage Three lines of evidence further support the success of this integrative analysis for defining the yeast mitochondrial proteome. First, the enrichment level for known mitochondrial proteins correlated with the level of the MitoP2 score and the number of experiments in which candidates were identified by proteomics: for the high, medium, and low classes see A the medOur use of mitochondria as a model system for an integrative analysis of a subcellular proteome was aided by the large set of reference proteins known and previous experiments performed. All individual systematic approaches were biased to some extent and incomplete. An integration of data sources is therefore essential to go beyond the limitations of any single method and to achieve a more comprehensive view of the mitochondrial organelle. In similar approaches for other organelles and pathways, the use of reference sets to integrate functional genomic approaches and to define parts lists may prove useful.Most of the mitochondrial reference proteins  had MitoP2 scores greater than 90, and since we have no evidence for a bias in the current reference set, the mitochondrial proteome as defined by the integration of 22 datasets is nearing saturation. In fact, our integration can be used to obtain an estimate of the number of mitochondrial proteins in yeast. Since outer membrane proteins are often not protease protected, the import analysis is conservative and allows us to estimate a lower boundary for the number of mitochondrial localized proteins. Considering that 84% of the reference proteins had MitoP2 scores greater than 90, we can predict a lower bound estimate of approximately 700 mitochondrial localized proteins in yeast (594 predicted true positives/0.84). This number is at the lower level of previous estimates and indicates that the mitochondrial organelle may consist of fewer proteins than the 800 anticipated .In order to make a prediction as to which combination of methods may be best applied to study a new system where no prior datasets exist, we performed an analysis of all pairwise combinations of methods. Among the comparisons, the union of proteomics  and subcThe advantage of integrative analysis combining structural and functional approaches is the high coverage of various mitochondrial components and functions. With this approach we were able to detect with high confidence proteins that had dual localization. For example, Met7p, which was assigned a MitoP2 score of 96, has a cytoplasmic and mitochondrial dual localization . Met7p wIt is clear that the current list of mitochondrial proteins is not complete. The addition of further datasets will improve the prediction, as evidenced by the fact that less than 8% of known mitochondrial proteins have a MitoP2 score less than 70. These proteins thus remain rather undefined by the current integration, and further experimentation is needed to capture this class of mitochondrial proteins, consisting in part of three carrier proteins, 12 dual localized proteins, a few small proteins, and 11 mtDNA-encoded proteins (MitoP2 database). Our method of integration serves as one example; other ways of analyzing and integrating the datasets are possible and may reveal more proteins involved in other aspects of the mitochondrial system.Finally, our study has implications for human diseases . To dateSaccharomyces cerevisiae strains were grown aerobically at 30 °C in SC or YP medium, and cells were harvested in logarithmic growth phase (OD600 < 1.3). Mitochondria were isolated by one of two different methods. One method involved differential centrifugation followed by a Nycodenz density gradient . The peak fraction was isolated, shock-frozen in liquid nitrogen, and used for electron microscopy.To assess purity, the preparations were analyzed by electron microscopy. The mitochondrial preparations were fixed with 4% formaldehyde, 2% glutaraldehyde, 4% sucrose, 2 mM calcium acetate, and 50 mM sodium cacodylate (pH 7.2) at 4 °C. The fixed samples were dissected with a scalpel, washed for 1 h in cacodylate buffer with 1% osmium tetroxide, and dehydrated with alcohol in increasing concentrations. After embedding in Araldite, the preparations were cut into 50-nm slices by means of an ultramicrotome  and then analyzed on a Zeiss  EM 10 electron microscope.Reagents used for the preparation of peptide samples were purchased from the indicated suppliers. Ammonium bicarbonate and methanol were from Fisher Scientific . Sodium carbonate, urea, dithiothreitol, and calcium chloride were obtained from Sigma-Aldrich . Thiourea, trifluoroacetic acid, and acetonitrile were from Aldrich Chemical Company . Sequencing-grade, modified porcine trypsin was obtained from Promega . Ammonium formate was obtained from Fluka . CHAPS and bicinchoninic acid (BCA) assay reagents and standards were from Pierce . Purified water was generated using a Barnstead Nanopure Infinity water purification system .Purified mitochondrial samples were disrupted using a Mini Beadbeater-8  for 3 min at 4,500 rpm with 0.1 mm zirconia/silica beads (Biospec Products) in a 0.5-mL, sterile siliconized microcentrifuge tube. The lysed mitochondria, containing membrane and matrix proteins, were removed from the beads through a puncture at the bottom of the microcentrifuge tube, by centrifugation at 16,000 xg for 2 min at 4 °C, and the flow-through was collected in a second microcentrifuge tube. The collected lysate was then centrifuged at 356,000 xg for 10 min at 4 °C to pellet the mitochondrial membranes. The soluble supernatant was used for the study of mitochondrial matrix proteins, and the pellet was retained for identifying mitochondrial membrane proteins.Using a sonication bath , the membrane pellet was resuspended in 50 mM ammonium bicarbonate (pH 7.8) in an ice bath. The resuspended sample was diluted with ice-cold 100 mM sodium carbonate (pH 11.0) and incubated on ice for 10 min. The membranes were then pelleted by ultracentrifugation at 356,000 xg for 10 min at 4 °C. The pelleted membranes were washed using two aliquots of ice-cold water and pelleted again by centrifugation. The BCA protein assay was performed to determine protein concentration.The membrane pellet was resuspended in 7 M urea, 2 M thiourea, 1% CHAPS in 50 mM ammonium bicarbonate (pH 7.8), using vortexing and sonication in an ice bath. Dithiothreitol was added to a final concentration of 9.7 mM in the resuspended sample, and the proteins were then treated with thermal denaturation for 45 min at 60 °C. The denatured and reduced protein sample was then diluted 10-fold with 50 mM ammonium bicarbonate (pH 7.8), and calcium chloride was added to a final sample concentration of 1 mM. Tryptic digestion was performed for 5 h at 37 °C using a 1:50 (w/w) trypsin-to-protein ratio. Snap-freezing the sample in liquid nitrogen quenched the digestion. The tryptic peptides were cleaned using a 1-mL strong cation exchange column  per the manufacturer's instructions. The eluted peptide sample was concentrated by lyophilization and a BCA assay was performed to determine final peptide concentration. The peptide sample was stored at −80 °C until time for LC/MS/MS analysis.The BCA protein assay was performed on the soluble matrix supernatant. The proteins were thermally denatured and reduced using 7 M urea, 2 M thiourea, and 5 mM dithiothreitol and incubating at 60°C for 30 min. The denatured and reduced protein sample was diluted 10-fold with 50 mM ammonium bicarbonate (pH 7.8), and the concentration of calcium chloride was adjusted to a final concentration of 1 mM. The tryptic digestion of the protein sample was performed in the same manner as described above for the membrane protein sample. The tryptic peptides were cleaned using a 1-mL LC-18 SPE column  per the manufacturer's instructions. The eluted peptide sample was concentrated by lyophilization, a BCA protein assay was performed, and the sample was stored at −80 °C until time for LC/MS/MS analysis.oC for the ESI voltage and heated capillary, respectively. The data-dependent tandem MS analysis was conducted using a series of segmented mass/charge (m/z) ranges. A collision energy setting of 45% was employed for the collision-induced dissociation of the three most abundant ions detected in each MS scan. Dynamic exclusion was used to discriminate against previously analyzed ions. Peptides were identified by searching the tandem MS spectra against the complete annotated S. cerevisiae genome database (available at http://www.yeastgenome.org/) using SEQUEST (ThermoFinnigan)  . “MudPITinnigan) . Fully tSome of the samples analyzed by LC/MS/MS were further analyzed by LC/FTICR. In LC/FTICR, tryptic peptides are analyzed using the same high-resolution reversed phase capillary LC described in the previous section, coupled to an electrospray ionization interface with a Fourier transform-ion cyclotron resonance mass spectrometer . We used5 resolution) were processed and deconvoluted using ICR-2LS  to obtain peak lists containing the monoisotopic mass, observed charge, and intensity of the major ions in each spectrum. The masses were calibrated using the masses of internal calibrant peaks infused at the beginning and end of each LC/FTICR analysis. The peak lists for each analysis were then matched against the potential mass and time (PMT) tags defined previously  using VIPER . The matching involved finding the groups of ions in the data, computing a median monoisotopic mass for each group, and then comparing the mass and elution time of the group with the mass and normalized elution time of each peptide in the PMT tag database , resulting in the generation of an accurate mass and time (AMT) tag. Because the PMT tag database consisted only of the peptide tags produced via the previous LC/MS/MS analyses (a PMT tag database for the whole genome does not exist to date), the LC/FTICR analysis could identify only AMT tags which corresponded to previously identified PMT tags from one of the LC/MS/MS runs.The acquired FTICR spectra . Then 4.5 μg of polyA+ mRNA were reverse transcribed to generate single stranded cDNA. Product was fragmented to approximately 50 bp using DNase digestion, biotin end labeled, and hybridized to Affymetrix S98 arrays as described in the Affymetrix user handbook . Hybridizations were normalized and duplicate samples integrated to arrive at an estimate of absolute transcript abundance using the dChip computational package . For genes with multiple probe sets on the array, only the probe set with the highest signal was used. For every gene, we calculated the fold difference between fermentable and nonfermentable growth conditions and considered significant only genes with a 1.2-fold or greater difference (either increased or decreased expression). In the final list we included only genes that showed a consistent direction of expression difference (increase or decrease) in both rich and synthetic media conditions.R. prowazekii, and Encephalitozoon cuniculi genomes has been conducted using the PSI-BLAST algorithm . MitoProt was run in the same way as in Psort was downloaded locally as a perl5 script  identified in each single genome-wide experiment (specificity) or in the overlap of all possible combinations of datasets  was calculated. Most proteins belonged to more than one combination, and for those proteins multiple R values were calculated. For example, proteins identified by two approaches received three R values: the specificity of the first approach alone, the specificity of the second approach alone, and the specificity of the overlap of both approaches. The MitoP2 value represented the highest R value calculated for a protein. The relevancy was checked according to the binomial law. The value gives a lower limit of the specificity of a defined combination because the mitochondrial reference dataset is not complete. For more detailed description, please see the MitoP2 database.To calculate the MitoP2 score, the percentage For T7 polymerase–driven synthesis of preproteins in vitro, the ORFs were amplified from ATG to STOP-codon by PCR, including the T7 RNA polymerase promoter and transcription initiation site within the 5′ primer. Using reticulocyte lysate (Promega), the resulting PCR products were utilized for coupled in vitro transcription/translation reactions to synthesize preproteins in the presence of 35S-radiolabeled methionine. Mitochondria were isolated by differential centrifugation from yeast strain W334 grown on lactate medium and resuspended at 25 °C in import buffer . Import was initiated by adding 1% to 4% (vol/vol) of reticulocyte lysate containing radiolabelled preprotein. After 15 min, samples were placed on ice and subsequently treated with proteinase K (50 μg/mL) or not for 15 min to remove nonimported proteins. Protease was inhibited by the addition of 2 mM PMSF. Mitochondria were reisolated and analyzed by SDS-PAGE and autoradiography. Control experiments were performed in the absence of membrane potential in the presence of 1 μM valinomycin and 20 μM oligomycin.Dataset S1(358 KB TXT).Click here for additional data file.Table S1(34 KB DOC).Click here for additional data file.Table S2(544 KB DOC).Click here for additional data file.Table S3(828 KB DOC).Click here for additional data file.Table S4(224 KB DOC).Click here for additional data file.http://www-deletion.stanford.edu/YDPM/YDPM_index.html.The YDPM database is the supporting online database for the proteomic, expression, and deletion datasets discussed in this paper, providing access to data analysis files, candidate lists, and a search function for individual ORFs. Available at http://ihg.gsf.de/mitop. The database provides annotated ORF information and the MitoP2 scores for the predicted mitochondrial proteins.The MitoP2 database is a mitochondrial proteome database for yeast and human that integrates published datasets and is available at


HIV-1 Tat activates transcription of HIV-1 viral genes by inducing phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Tat can also disturb cellular metabolism by inhibiting proliferation of antigen-specific T lymphocytes and by inducing cellular apoptosis. Tat-induced apoptosis of T-cells is attributed, in part, to the distortion of microtubules polymerization. LIS1 is a microtubule-associated protein that facilitates microtubule polymerization.in vitro. LIS1 also co-immunoprecipitated with Tat expressed in HeLa cells. Further, LIS1 interacted with Tat in a yeast two-hybrid system.We identified here LIS1 as a Tat-interacting protein during extensive biochemical fractionation of T-cell extracts. We found several proteins to co-purify with a Tat-associated RNAPII CTD kinase activity including LIS1, CDK7, cyclin H, and MAT1. Tat interacted with LIS1 but not with CDK7, cyclin H or MAT1 in vitro and in vivo and that this interaction might contribute to the effect of Tat on microtubule formation.Our results indicate that Tat interacts with LIS1  HIV-1 Tat protein is the viral transactivator encoded in the HIV-1 genome of infected cells . Tat stiIn the present paper, we fractionated extracts of Jurkat T-cells using HIV-1 Tat as an affinity bait and RNAPII CTD activity of the Tat-associated proteins as a selection criteria. We identified by mass-spectrometry and immunoblotting components of the partially purified protein fraction and found LIS1, CDK7, cyclin H, and MAT1. We analyzed interaction of Tat with the identified individual proteins and found that Tat interacts with LIS1. We confirmed this finding by co-immunoprecipitating Tat and LIS1 from HeLa cells that were expressing Tat. And we also confirmed binding of Tat to LIS1 in a yeast two-hybrid system. Our results indicate that HIV-1 Tat interacts directly with LIS1, and therefore this interaction might contribute to the effect of Tat on microtubules formation in the cells.4)2SO4 fractionation as described previously  ATP (6000 Ci/mmol) and (35S)-labeled Methionine were purchased from Amersham Pharmacia Biotech . Econo-Pac CHT-II Cartridge (ceramic hydroxyapatite) was from Bio-Rad . Glutathion-agarose was from Sigma . GST-CTD was expressed in Escherichia coli and purified as we described [Escherichia coli and purified on Glutathione-agarose beads as described [Jurkat T-cells were purchased from National Cell Culture Center . DEAE-Sepharose (FF), SP-Sepharose (FF), Hi Trap heparin columns, γ-P ATP  and monoclonal (NT3 2D1.1) antibodies were received from AIDS Research and Reference Reagents Program (NIH). Anti-Flag antibodies were purchased from Sigma . Polyclonal antibodies to CDK7, and PSTAIRE were purchased from Santa Cruz Biochemical . Polyclonal antibody to CDK9 (PITALRE) were purchased from Biodesign Company . Monoclonal antibodies for LIS1 were as described .2, 6 mM EGTA and 2.5 mM dithiothreitol). Tat-associated CTD kinase activity was assayed by incubating the kinase-bound beads with 100 ng GST-CTD in kinase buffer containing 50 μM ATP and 10 μCi of (32p)ATP for 10 min at room temperature. Phosphorylated GST-CTD was resolved on 10% SDS-PAGE and subjected to autoradiography and quantification with PhosphorImager Storm 860 (Molecular Dynamics).Tat-associated kinase activity was assayed as described previously . Briefly5 cells/ml were centrifuged, washed and Dounce-homogenized in Buffer A  supplemented with 0.1% NP-40. The whole cell extract was prepared and fractionated by ammonium sulfate precipitation. Ammonium sulfate was added to 10% saturation to extract nuclei. After centrifugation, the supernatant, containing approximately 10 g of protein, was further fractionated with ammonium sulfate added to 20%, 40% and 80% saturation. The 40% ammonium sulfate fraction (about 3.5 g of protein) was found to contain the major part of Tat-associated CTD kinase activity. This fraction was diluted with Buffer A until the conductivity was equivalent to 50 mM KCl and then loaded on a DEAE-Sepharose column (about 500 mg of protein per 50 ml column). The column was eluted with a linear gradient of KCl (0.1 to 1 M) in Buffer A. Fractions were assayed for Tat-associated CTD kinase activity as described above. A peak of Tat-associated CTD kinase activity was collected, diluted with Buffer A until conductivity was equivalent to 50 mM KCl and loaded on a 10 ml SP-Sepharose column which was eluted with linear gradient of KCl (0.1 to 1 M) in Buffer A. A flow-through fraction containing Tat-associated CTD kinase activity was further fractionated on Hi Trap heparin columns . Fractions were collected and analyzed for the Tat-associated CTD-kinase activity as described above, as well as by immunoblotting. Fractions containing Tat-associated CTD kinase activity TTK were resolved on 12% SDS-PAGE (20 × 20 cm) stained with colloidal Coumassie Blue and subjected to protein microsequencing.Purification of Tat-associated CTD kinase from Jurkat T-cells was carried as previously described . BrieflyThe procedure for peptide sequencing was performed as described previously. Protein bands visible after colloidal Coomassie blue staining and corresponding to the peak of CTD hyperphosphorylating activity after the heparin-agarose column were subjected to in-gel reduction, carboxyamidomethylation and tryptic digestion . Multiple peptide sequences were determined in a single run by microcapillary reverse-phase chromatography directly coupled to a Finnigan LCQ quadrupole ion trap mass spectrometer equipped with a custom nanoelectrospray source. The column was packed in-house with 5 cm of C18 support into a New Objective one-piece 75 um I.D. column terminating in a 15 μm tip. Flow rate was 190 nanoliters/min. The ion trap was programmed to acquire successive sets of three scan modes consisting of full scan MS over alternating ranges of 395–800 m/z or 800–1300 m/z, followed by two data dependent scans on the most abundant ion in those full scans. These data dependent scans allowed the automatic acquisition of a high resolution (zoom) scan to determine charge state and exact mass, and MS/MS spectra for peptide sequence information. MS/MS spectra were acquired with a relative collision energy of 30%, an isolation width of 2.5 Dalton and recurring ions dynamically excluded. Interpretation of the resulting MS/MS spectra of the peptides was facilitated by programs developed in the Harvard Microchemistry Facility and by database correlation with the algorithm SyQuest .Proteins were transcribed/translated as described previously . Briefly2, 6 mM EGTA, 2.5 mM DTT). The pellet was then resuspended in a 30 μl of 1X SDS loading buffer  and heated at 90°C for 3 minutes. The proteins were resolved on SDS Tris-Tricine PAGE, 10%, to detect LIS1, or 12%, to detect Tat, and immunoblotted with anti-LIS1 or anti-Flag antibodies.HeLa cells were infected with adenovirus vector expressing Flag-tagged Tat protein as we previously described . HeLa whMATα, ura3, his3, tryp1, LexAop (x6) -LEU2), auxotrophic for tryptophan (Trp), uracil (Ura), histidine (His), with LEU2 as a reporter gene. Yeast were transformed by electroporation as follow. One colony of the yeast cells was resuspended into 10 ml of appropriate selective media and grown at 30°C overnight. Cells were collected at 3000 rpm for 10 min, washed twice with HEPES/Sorbitol , resuspended in 200 μl of HEPES/Sorbitol and supplemented with 1 μg of a plasmid DNA. The mixture was pulsed with 2500 V in 0.4 cm cuvette, then 1 ml of appropriate selective media was added and cells were shaken at 30°C for 2 hours. The cells were collected by centrifugation, resuspended into 250 μl of HEPES/Sorbitol and plated on appropriate selective plates. EGY48 cells were transformed with pSH18–34 vector containing Lac Z reporter under the control of LexAop(x8) and also URA3 and ampr genes as selection markers. The transformed yeast cells (EGY48-lacZ) were selected on Uracyl deficient media. HIV-1 Tat first exon was subcloned into pLexA in frame with the LexA(1–202), the DNA binding domain to create the bait vector (pLexA-Tat). LIS1 was subcloned into pJG 4–5 (ampr) in frame with the acid activation domain to create pJG-LIS1 carrying hemagglutinin (HA) tag (Trp selectable marker). The EGY48-lacZ yeast cells were transformed with pLexA-Tat vector, and selected for growth on uracyl and histidine deficient media. The Tat expressing yeast cell growing on Uracyl, Histidine deficient plates were then transformed with pJG-LIS1. To detect interaction between Tat and LIS1 interaction, yeast cells were plated on galactose/raffinose-containing plates to allow expression of Tat and LIS1, and production of β-galactosidase was visualized with 5-bromo-4-chloro-3-indolyl-β-D-galactoside  substrate.The parent yeast cells EGY48 (LexA 2H) genotype (The author(s) declare that they have no competing interests.in vitro and in vivo and participated in the writing and assembling of the manuscript. TA carried out yeast two-hybrid assays. YV provided technical help. WSL performed protein sequencing. WT participated in the design and discussion of the study. TS and OR created vectors for expression of LIS1 and participated in the design of the study. SN purified LIS1 containing protein complex for protein sequencing, performed general control and coordination of the study. All authors read and approved the manuscript.NE carried out studies of LIS1 and Tat interaction Analysis of protein composition of DEAE-Sepharose purified fraction of Tat-associated CTD kinase. Fractions from the DEAE-Sepharose column fractionation shown in Fig. Click here for fileHIV-Tat interacts with a 50 kDa protein from purified Tat-associated CTD kinase. GST-fused Tat 1–72, immobilized on glutathione-agarose beads, was incubated without (lane 1), or with fraction 18 (lane 2), fraction 20 (lane 3), fraction 22 (lane 4), or fraction 24 (lane 5) from the heparin-agarose, shown in Fig. Click here for fileEndogenous LIS1 is present in reticulocyte lysates. Individual protein components of Tat-associated complex were translated in reticulocyte lysate. The lysates were resolved on 12% SDS-Tris-Tricine gel and immunoblotted with anti-LIS1 monoclonal antibodies. Lane 1- CDK7; Lane 2-Cyclin H; Lane 3-MAT1; and Lane 4-LIS1-programmed lysate.Click here for file


Many Japanese believe that low-yield cigarettes are less hazardous than regular cigarettes, and many smokers consume low-yield cigarettes to reduce their risks from smoking. We evaluate the association between actual nicotine intake and brand nicotine yield, and the influence of nicotine dependence on this association.The study subjects included 458 Japanese male smokers, aged 51.2 ± 9.9 years, who participated in health check-ups in a hospital in 1998 and 2000. Each subject filled out a self-administered smoking questionnaire and the score of each on the Fagerström Test for Nicotine Dependence was calculated. Urinary cotinine concentration was measured at the time of participation.The geometric mean of urinary cotinine concentration was 535 ng/mgCr for those who smoked brands with the lowest nicotine (0.1 mg on the package), compared with 1010 ng/mgCr for those who smoked brands with the highest . Thus, despite the 11-fold ratio of nicotine yield on the packages, the ratio of urinary cotinine level was less than twofold. Both nicotine yield on the package and nicotine dependence significantly increased urinary cotinine concentration, and the negative interaction between them almost attained statistical significance. Cotinine concentration in heavily dependent smokers was consistently high regardless of the nicotine yield of brands.The nicotine yield of cigarettes measured by machine-smoking does not reliably predict the exposure of smokers. Smokers consuming low-yield nicotine cigarettes did not reduce actual intake of nicotine to the level that might be expected, especially for those heavily dependent on nicotine. Current labeling practices are misleading for the two-third of smokers who are moderately or highly dependent on nicotine. The Tobacco Institute of Japan reported that, in 2001, of the 20 top brands that share 84% of the total cigarettes consumed in Japan, 6.9% had a nicotine yield reported on their package of 0.1 mg, 73.4% had a yield of 0.2–0.8 mg, and 19.7% had a yield of 0.9 mg or higher .There have been many studies examining whether low-yield cigarettes are less hazardous than regular brands. For example, in the 1980s and 90s, the actual intake of nicotine -9, as weIn Japan, the rate of smoking is still high, being >50% among males. These sThe subjects of this study were male smokers who participated in a health check-up at the Kyoto First Red Cross Hospital from July to December in 1998, or from January to February in 2000. The latter subjects were supplementary to the main group, but the two groups exhibited similar demographics. Smokers were recruited using a routine health check-up questionnaire and were defined in this study as those who smoked at least one cigarette per day. Of the 1,579 male participants in the health check-up during the study period, 513 were identified as smokers, and 479 agreed to participate in this study.Each participant filled out a self-administered questionnaire, which was checked during an interview with a physician. This questionnaire included questions determining score on the Fagerström Test of Nicotine Dependence (FTND) . These iAlso, included were questions about the brand(s) of cigarettes smoked, inhalation pattern , attempt to quit smoking, and stage of behavioral change in the quitting process . SmokersAbout three quarter of plasma nicotine is converted to cotinine, which is excreted in the urine. The half-life of nicotine is 30 minutes  and thatStatistical analysis was performed using data from 458 male smokers who completed the FTND question and whose urinary cotinine levels were measured. We compared the characteristics of subjects among three groups categorized by machine-measured nicotine yield . For each group, we calculated mean machine-measured nicotine yield weighted by the number of subjects. Log-transformed data were used for the urinary cotinine concentrations because it was distributed log-normally. Means were compared using Student's t-test or analysis of variance, and proportions were determined using the chi-square test. The effects on urinary cotinine concentration of machine-measured nicotine yield, number of cigarettes consumed per day, and nicotine dependence were analyzed using a regression model, in which urinary cotinine concentration was the dependent variable, and two of the other parameters were independent variables. Main effect and interaction were evaluated by regression coefficients and partial correlation coefficients. The effect of different cigarette brands was also examined. All statistical procedures were performed by SPSS . A P valOf the 458 subjects, 87 19.0%) smoked cigarette brands yielding 0.1 mg nicotine, 223 (48.7%) smoked cigarettes of 0.2–0.8 mg, and 148 (32.3%) smoked cigarettes of 0.9+ mg  were heavily dependent on nicotine (FTND score>= 7), whereas 87 (19.0%) had low dependence (FTND score< = 3). About 60% of all subjects had attempted to quit, with the proportion similar in the high- and low-nicotine dependent groups (p = 0.73). The stage of behavioral change was different (p = 0.001), however, with smokers of cigarettes yielding 0.1 mg of nicotine being at more advanced stages. The geometric mean of urinary cotinine concentration in all subjects was 784 ng/mg creatinine (Cr), with a distribution of 484 ng/mg Cr (mean-SD) to 1264 ng/mg Cr (mean+SD), as determined by back-transformation of log-transformed data . The levels differed significantly between the machine-measured nicotine yield groups (p < 0.001).Urinary cotinine levels did not differ among smokers of individual brands of yielding 0.1 mg of nicotine . After integration of similar brands, geometric cotinine concentration means were 686 ng/mgCr for those who smoked American brands and 460 ng/mgCr for those who smoked Japanese brands (p = 0.19). Urinary cotinine concentrations also did not differ among smokers of individual cigarettes brands yielding 0.2–0.8 mg nicotine . The geometric means were 823 ng/mgCr for smokers of Japanese brands and 724 ng/mgCr for smokers of American brands. Mentholated cigarettes were consumed by only 5 subjects and were therefore not examined.When we assayed the relationship between urinary cotinine concentrations and number of cigarettes consumed per day by machine-measured nicotine yield of cigarettes, we found that cotinine concentrations were related to number of cigarettes consumed per day Figure . The corWhen we assayed the relationship between urinary cotinine concentrations and machine-measured nicotine yield of cigarettes by FTND score Figure , we founThe ratio of mean nicotine yield was 0.45 for cigarettes yielding 0.2–0.8 mg nicotine, and 0.09 for cigarettes yielding 0.1 mg nicotine, compared with the brands yielding 0.9–2.4 mg Table . In contSelf-reported inhalation patterns did not influence the average urinary cotinine concentration (p = 0.54) when the variable of inhalation pattern was added to the above model with nicotine yield and FTND score.In Japan, low nicotine-yield cigarettes seem to be recognized as less hazardous, and smokers likely think that the hazards of smoking are directly proportional to nicotine or tar yield shown on the cigarette packages. This is supported by the results of the present study, which indicate that smokers of low-yield nicotine cigarettes were more advanced behaviorally in wishing to quit. This is additionally supported by circumstantial evidence -5,14-18 We have shown here that smokers of low nicotine cigarettes did not reduce their actual intake of nicotine to the degree that would be expected from the nicotine yield on the packages. Although smokers of cigarettes yielding 0.1 mg nicotine would be expected to ingest one-eleventh of the nicotine ingested by smokers of cigarettes yielding 0.9–2.4 mg nicotine (average of 1.1 mg), the average urinary cotinine concentration of the former group was more than half that of the latter (535 ng/mg Cr vs. 1010 ng/mg Cr). Moreover, smokers of cigarettes yielding 0.2–0.8 mg nicotine (average of 0.5 mg) had about a 25% decrease in urinary cotinine concentration (770 ng/mg Cr) compared with smokers of cigarettes yielding 0.9–2.4 mg nicotine, despite the two-fold reduction expected from the nicotine yield on the packages. These differences were even smaller in smokers who consumed large numbers of cigarettes per day as well as in smokers with heavy nicotine dependence. The number of cigarettes consumed per day is regarded as a component of nicotine dependence and is included in FTND. There were negative interactions between machine-measured nicotine yield and number of cigarettes consumed per day, and between machine-measured nicotine yield and nicotine dependence. In particular, smokers with heavy nicotine dependence tended to have a high urinary cotinine concentration (about 1200 ng/mgCr) despite differences in machine-measured nicotine yield of cigarettes, which may explain this negative interaction.In contrast, the actual nicotine intake of smokers who consumed small numbers of cigarettes and smokers with a low level of dependency was more strongly correlated with the machine-measured nicotine yield of the cigarettes they consumed. That is, those who smoked light cigarettes absorbed a smaller amount of nicotine, but, again, the amount absorbed was not equal to the difference in nicotine yields on the packages. These associations are evident in the ratios of means shown in Table We determined the full FTND score in each of our subjects, although some components of FTND, including the number of cigarettes consumed and the time from awakening until the first cigarette, were measured in the previous study of smoking in Japan . It has Smokers heavily dependent on nicotine obtained no advantage by smoking low-yield cigarettes. Moreover, they may actually increase their risk due to compensatory behavior, for example, by inhaling more carbon monoxide or other harmful substances contained in cigarette smoke. Our results suggest that tobacco industry advertising may have led these smokers, especially those heavily dependent on nicotine, to underestimate the health risks posed by low-yield cigarettes. This is similar to the results of other studies, which suggested that 'Light' or 'Ultra Light' cigarettes could deliver as much tar and nicotine as 'Regular' cigarettes -11.The compensation mechanisms that may keep blood nicotine at a high level include more puffs per cigarette, greater volume per puff, and greater depth of inhalation, all of which may be conscious or unconscious on the part of smokers. In addition, the filters of low-yield cigarettes are sometimes treated with ammonium to increase absorption of nicotine, thus eliminating the need for deep inhalation . These fAnother important mechanism by which smokers of low-yield cigarettes increase their nicotine intake is by blocking the ventilation holes on the filter with their fingers or lips while holding the cigarette and smoking . These hThe tobacco companies have admitted that they have known of the relationship between the nicotine yield reported on the packages and the actual quantities of tobacco smoke components inhaled . Low-yieThe subjects of this study were participants in a so-called 'human dry dock,' a detailed health check-up system for middle-aged and elderly people, which started in Japan in the 1950's and currently enroll about 10 million . Most ofFemale participants were not examined in this study, mostly because smoking rate of females in our health check-up clinic is less than 10% and some participants refused to answer questions about smoking. However, the advertisement of 'light' cigarettes seems to target young people, especially young women , and theIn conclusion, we have shown here that the difference in intake of nicotine into a smoker's body was smaller than the difference in machine-measured nicotine yield among cigarette brands. Smokers consuming cigarettes with a low nicotine yield did not reduce actual intake of nicotine to the level that they expected. This was especially true for smokers with heavy nicotine dependence. This result should be emphasized in public health messages to smokers as well as to young people likely to start smoking.FTND: Fagerström Test of Nicotine Dependence, FTC: Federal Trade CommissionNone declared.AN participated in the design of the study, collected the data, performed the statistical analysis, drafted the manuscript, and was the principal investigator. MNS conceived of the study, participated in its design, and collected the data. KO supervised the data analysis and the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Schizophrenia, a relatively common psychiatric syndrome, affects virtually all brain functions yet has eluded explanation for more than 100 years. Whether by developmental and/or degenerative processes, abnormalities of neurons and their synaptic connections have been the recent focus of attention. However, our inability to fathom the pathophysiology of schizophrenia forces us to challenge our theoretical models and beliefs. A search for a more satisfying model to explain aspects of schizophrenia uncovers clues pointing to genetically mediated CNS microvascular inflammatory disease.A vascular component to a theory of schizophrenia posits that the physiologic abnormalities leading to illness involve disruption of the exquisitely precise regulation of the delivery of energy and oxygen required for normal brain function. The theory further proposes that abnormalities of CNS metabolism arise because genetically modulated inflammatory reactions damage the microvascular system of the brain in reaction to environmental agents, including infections, hypoxia, and physical trauma. Damage may accumulate with repeated exposure to triggering agents resulting in exacerbation and deterioration, or healing with their removal.There are clear examples of genetic polymorphisms in inflammatory regulators leading to exaggerated inflammatory responses. There is also ample evidence that inflammatory vascular disease of the brain can lead to psychosis, often waxing and waning, and exhibiting a fluctuating course, as seen in schizophrenia. Disturbances of CNS blood flow have repeatedly been observed in people with schizophrenia using old and new technologies. To account for the myriad of behavioral and other curious findings in schizophrenia such as minor physical anomalies, or reported decreased rates of rheumatoid arthritis and highly visible nail fold capillaries, we would have to evoke a process that is systemic such as the vascular and immune/inflammatory systems.A vascular-inflammatory theory of schizophrenia brings together environmental and genetic factors in a way that can explain the diversity of symptoms and outcomes observed. If these ideas are confirmed, they would lead in new directions for treatments or preventions by avoiding inducers of inflammation or by way of inflammatory modulating agents, thus preventing exaggerated inflammation and consequent triggering of a psychotic episode in genetically predisposed persons. Effor process -10.degenerative process in developmental terms.Degenerative models imply that after a period of normal development, the organism, or one of its parts, takes a wrongful turn in its trajectory and begins to malfunction. This describes the eventual outcome for all life forms and is a biological restatement of the second law of thermodynamics. Since degeneration is universal, stating that an illness is degenerative is not particularly helpful. What would be helpful is to determine when in the life course the degeneration begins and how the degeneration is initiated and proceeds. Answers to the "when?" and "how?" questions would then describe the Developmental models of schizophrenia implicate abnormalities of early brain development predisposing to future schizophrenia. The proponents of the model further argue that the perturbations of development are limited to the early times of development and are discontinuous. Without this qualifier, developmental models are indistinguishable from degenerative models where the degeneration commences early in the life span. The early abnormalities are not necessarily the cause of schizophrenia, but, instead, create a state of risk for a future episode of schizophrenia. That is, a diathesis or predisposition is not a disease. Consequently, there must be factors later in life that convert the vulnerability to an illness. These additional factors are presumed to damage development in such a way that a predisposition becomes actualized. To gain a complete understanding of the syndrome, we must again return to the question of " what happens?"Following this line of reasoning, the distinction between degenerative and developmental models blurs. In fact, a medical-behavioral condition can be both developmental and degenerative as exemplified by Down syndrome -13. IndiSchizophrenia involves both developmental and degenerative features. From the time of Bleuler  and KraeThe evidence suggesting early developmental perturbations in schizophrenia is compelling. At the same time, there certainly are examples of deterioration reminiscent of Kraepelin's suggestion for some people with schizophrenia. However, deterioration in clinical course may not indicate CNS deterioration. Instead, the decline could be a secondary consequence of an illness that disrupts education, economic achievement, and social functioning leading to a downward spiral in all aspects of adult life. Consistent with an early degenerative process, there are reports of declining cognitive function preceding onset of psychosis . ProponeThe symptoms of schizophrenia are highly variable. Within families  symptoms can vary widely over time, as illustrated by identical quadruplets concordant for schizophrenia . Even wiper se to have a comprehensive view of the illness.The major behavioral symptoms of schizophrenia include alterations in cognition, memory, perception, thought (inferred from language), motor functions, and affect. People with schizophrenia may show abnormal dermatoglyphics and other minor physical anomalies -42. OtheThe fact that the schizophrenia syndrome, as currently defined, is relatively common provides important information about the frequency of causal factors. About 1% of the population will experience schizophrenia during the lifespan. Except for a few rare exceptions, this 1% risk is remarkably constant around the globe regardless of culture, geography, or ethnicity. Men and women are affected equally. These facts mean that the risk factors for schizophrenia must also be common and ubiquitous. Given that the concordance rate for schizophrenia in identical twins  is only Our challenge is to develop a theory of schizophrenia that can plausibly explain an illness that affects all domains of behavior , that has elements of developmental perturbations early in life leaving clues such as minor physical abnormalities, and also has elements of degenerative changes. At the same time, the defect is so subtle that we can't find the cause(s) with our best modern technology. Furthermore, in spite of brain-wide dysfunctions, many individuals with schizophrenia remain sufficiently intact that, with good treatment and a bit of luck, can maintain jobs and function usefully in society. Thus, we need to find frequent and ubiquitous factors that can affect virtually all brain functions as well as creating somatic signs, but they operate in ways that leave these functions only slightly "off kilter" as compared to the complete disruption seen in strokes, or classical degenerative disorders such as Alzheimer, or as seen in Down syndrome where the behavioral pathology is apparent from earliest stages. As we try to explain schizophrenia, we must account for most all of the developmental and degenerative features of schizophrenia.To account for the panoply of signs and symptoms seen in schizophrenia, any complete theory of schizophrenia must include organism wide systems. In addition to the nervous system, the immune system and the vascular system are defensible candidates. Both are invoked in the following theory: Some schizophrenia psychoses are the result of damage to the micro-vascular system in the brain initiated by genetically influenced abnormal inflammatory processes acting in response to ubiquitous environmental factors that trigger inflammatory responses, including infection, trauma, or hypoxia. It is the relative infrequency of the vulnerable genotypes in the population  that resWe wish to emphasize that our hypothesis specifically identifies the microvascular system as the critical site of inflammation. We postulate that the inflamed micro-vessels lose their coupling with astrocytes, leading to disrupted regulation of cerebral blood flow and damage to the blood brain barrier. These disruptions in homeostatic mechanisms then lead to abnormal signal processing. Our focus on inflammation of the vessels differentiates our hypothesis from models of widespread parenchymal inflammation such as seen in psychotic syndromes following, for example, encephalitis lethargica, or paraneoplastic syndromes. Many acute inflammatory disorders of the brain involve inflammation of both the parenchyma and the vasculature. By contrast, we are proposing a chronic, smoldering, inflammation of the vessels alone. And, finally, we distinguish our hypothesis from the theories of schizophrenia implicating direct parenchymal infection of the brain (c.f. ) and alsMany prior debates about inflammation in the brains of people with schizophrenia have focused on the presence of absence of gliosis  cases of syndromal schizophrenia. Instead, we put forward our hypothesis as an attempt to define a psychiatric syndrome in terms of a particular pathophysiology. Following this course may then help refine our nosology  and cause us to recalculate basics 'facts' such as prevalence rates.Neurons derive their energy from oxygen and glucose delivered by the vascular system, plus lactate and glycogen derived from astroglia . The comDevelopmentally, the CNS vascular system originates from capillary endothelial cells that migrate into developing neuro-ectoderm under the influence of trophic factors such as vascular endothelial growth factor (VEGF)  and erytWhen infectious agents give rise to inflammatory vascular disease, the nature of the infectious agent may be less important that an individual's genetically influenced inflammatory response. The concept that infectious disease may have a genetic component is, of course, not new. Many agricultural geneticists make their livings by breeding disease resistance into both plants and animals ,83. One Familial Mediterranean Fever (FMF) ,101 provJust as the CNS blood supply is highly regulated, the inflammatory systems in the brain require 'fine tuning.' Given the limited ability for adult brain to regenerate, and assuming there is little tissue to spare, it would make sense that the brain should be protected from overabundant inflammatory reactions . AstrocyLet us suppose that schizophrenia develops following an infection  but the host's response is determined by genetic factors regulating the nature and degree of inflammation. That infectious agents may be operative in schizophrenia is supported by several of lines of evidence. Summaries can be found in numerous sources ,112-116.The syndrome of schizophrenia is likely to be etiologically heterogeneous and a multitude of CNS disorders can give rise to schizophrenic-like psychoses . The ideInflammatory processes often damage the precise regulation of cerebral blood flow. The wide spectrum of clinical conditions thought to be created, in part, by inflammatory CNS micro-vessel disease include Alzheimer disease where it is thought that inflammatory processed damage the micro-vascular endothelium causing insufficient blood flow leading to oxidative stress, a build up of amyloid, and eventual cell death -135. CerReturning to the early stages of life, we have seen that the development of the neurons and glia are intimately associated with, and dependent on, the parallel development of the CNS vasculature. If the stated theory is correct, and given the developmental perspective of schizophrenia ---early developmental perturbations of the CNS set the stage for later schizophrenia--- we would expect to find support for the idea that inflammatory events early in life affect CNS vascular function. Such is the case. Whether the early insults are traumatic, infectious, or hypoxic; inflammatory process are involved in the attempts to protect and repair by modulating angiogenesis -148. ThuSince the time of Seymour Kety's pioneering efforts ,153, thePost-streptococcal neuropsychiatric syndromes include Syndenham chorea, the PANDAS/obsessive compulsive syndrome, tics including Tourette syndrome, and possibly, ADHD -184. Psyth and 18th centuries starting with Sydenham himself . The inelf ,202,203 An inflammatory reaction of the CNS vascular endothelium (vasculitis) is a common denominator in the both acute and chronic cerebral consequences of rheumatic fever. ,206-209.The post-strep psychopathologies provide a precedent for the hypothesis of this paper by demonstrating that an infectious process can trigger a series of inflammatory reactions that lead to a variety of somatic and psychiatric syndromes, including psychoses where vascular pathology is implicated. The pathogenicity of a strep infection is a function of the strain (genotype) of the bacterium and the genetically mediated inflammatory mechanisms of the host  and illuThe ideas here are not completely new. Eugen Bleuler  remarkedAbnormal behaviors develop as a result of disruptions in astroglial mediated coupling of cerebral blood flow to neuronal metabolic needs. These subtle disruptions are hard to find, as the microvasculature comprises only about 0.1% of the brain and are of a scale more appropriate for electron microscopy. None-the-less, the hemodynamic perturbations have sufficient impact to cause subtle but widespread disruption of the normally harmonious coordination of CNS function leading to a condition variously conceived as a "neurointegrative defect", "synaptThis theory could explain how developmental events such as prenatal infections ,222, andThis theory also captures many of the little oddities observed in schizophrenia. For example, the reported abnormalities of the nail fold capillary beds seen in some people with schizophrenia  are alsoAnother line of evidence compatible with this theory is the observation that genetic linkages for schizophrenia coincide with sites for glial growth factor cell regulators  and, as It may well be that the environmental components of psychiatric illness such as schizophrenia are relatively minor, ubiquitous, or chance events ,247 thatThe speculations about psychoses developing from vascular/inflammatory processes provide direction for future research across many domains. In addition to pursuing direct evidence of altered activities in inflammatory/immune systems in people with psychoses, the inflammatory/vascular theory has implications for epidemiology, genetics, neuroimaging and neuropathology. For the epidemiologist, the challenge will be to detect relatively small signals against a very noisy background. We hypothesize that the triggers for inflammation can be many and varied and are common factors in the environment. Imagine starting with the clinical syndrome of Sydenham chorea and comparing the rates of strep throat in those affected vs. comparison sample of people without Sydenham chorea. Null hypothesis testing with small sample sizes and nearly ubiquitous etiological agents are clearly not adequate. A second epidemiological challenge is to cast a broad enough net to capture the wide variety of possible contributing factors. Rather than taking a one by one approach to exploring the etiological contributions of, say, virus titers, anoxia, physical trauma, the epidemiologist should look for any and all. It would be predicted that individuals with multiple "hits" (e.g. in utero exposure to virus and low Apgar scores and childhood head trauma) would be at greater risk than those exposed to just one event. If in utero inflammatory processes are active in the genesis of schizophrenia we would also predict an increased rate of fetal deaths in families of schizophrenic probands. A third epidemiological opportunity lies in the search for non-psychiatric inflammatory-related disease or traits in people with psychosis. If something is askew in the inflammatory process in schizophrenia, the effects will show up in other parts of the body. Though requiring replication, the association of psychosis with hemolytic anemia in lupus  providesFor geneticists, the proposed theory obviously points to linkage/association studies using inflammation genes; a few examples were cited previously -238. A sThe inflammatory/vascular theory has much to suggest to neuroimaging research especially in the realm of reinterpreting regional perturbations in metabolic activity as primary disturbances of flow regulation rather than intrinsic neuronal metabolic abnormalities. It would be interesting to assess the impact of vasoactive compounds and inflammatory modulators on neuroimaging studies of regional blood flow. Likewise, further pursuit of neuroimaging evidence of disrupted blood brain barrier, as initiated by Dysken, et al , and witThe neuropathology of schizophrenia, focused mostly on the neurons, is notable for inconsistencies in findings  makes it clear that a vascular-inflammatory theory may apply to a wide range of psychotic conditions that may also include psychoses associated with mood disorders. Whereas, the classical genetic studies support the separateness of schizophrenia and mood disorders , there aHowever, finding similar brain changes in a variety of psychotic conditions does not necessarily mean these changes are epiphenomena. Examples from neuropsychiatry teach us that the underlying pathology does not necessarily define the behavioral symptoms. Thus, psychoses with Huntington disease may be affective-like or schizophreniform. Similar pathophysiological mechanisms may underlie a variety of psychotic phenotypes. The evolution of behavioral symptoms for any given pathophysiology may depend on a variety of moderating variables such as an individual's developmental age when the disease process begins, gender, hormones, genetic 'landscape' upon which the disease process unfolds, along with the nature, frequency, and intensity of successive triggers of inflammatory response.A broad spectrum of observations leads to a working hypothesis that schizophrenia and, possibly, other psychiatric syndromes are the result of genetically mediated inflammatory reactions that damage the neuron-glial-capillary triad with resultant loss of ability to fine tune regional brain metabolism. This hypothesis incorporates genetic, epigenetic , and envADHD attention deficit hyperactivity disorderBDNF brain derived neurotropic factorCBF cerebral blood flowCNS central nervous systemDZ dizygoticFMF familial Mediterranean feverMZ monozygoticNGF nerve growth factorNO nitric oxidePANDAS pediatric autoimmune neurological disorder associated with strep.RF rheumatic feverRHD rheumatic heart diseaseVEGF vascular endothelial growth factorThe author(s) declare that they have no competing interests.This article was the joint effort of both authors with input as noted below.The pre-publication history for this paper can be accessed here: