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 (LKB-Produkter, Bromma, Sweden) and then analyzed on a Zeiss (Oberkochen, Germany) 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 (Fair Lawn, New Jersey, United States). Sodium carbonate, urea, dithiothreitol, and calcium chloride were obtained from Sigma-Aldrich (St. Louis, Missouri, United States). Thiourea, trifluoroacetic acid, and acetonitrile were from Aldrich Chemical Company (Milwaukee, Wisconsin, United States). Sequencing-grade, modified porcine trypsin was obtained from Promega (Madison, Wisconsin, United States). Ammonium formate was obtained from Fluka (St. Louis, Missouri, United States). CHAPS and bicinchoninic acid (BCA) assay reagents and standards were from Pierce (Rockford, Illinois, United States). Purified water was generated using a Barnstead Nanopure Infinity water purification system (Dubuque, Iowa, United States).Purified mitochondrial samples were disrupted using a Mini Beadbeater-8 (Biospec Products, Bartlesville, Oklahoma, United States) 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 (Branson 1510, Danbury, Connecticut, United States), 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 (Discovery DSC-SCX , Supelco, Bellefonte, Pennsylvania, United States) 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 (Reversed Phase Supelclean LC-18 SPE, Supelco) 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 (Wong Lab, Harvard University). 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 (0.3 mg/mL fatty-acid-free BSA, 0.6 M sorbitol, 80 mM KCl, 10 mM magnesium acetate, 2 mM KH2PO4, 2.5 mM EDTA, 2.5 mM MnCl2, 2 mM ATP, 5 mM NADH, and 50 mM HEPES/KOH [pH 7.2]). 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 [4)2SO4 cut contained Tat-associated CTD kinase activity 2SO4 cut was subsequently fractionated on DEAE-Sepharose . EGY48 yeast cells pretransformed with pSH18–34 reporter plasmid (-Ura selection) were further transformed with different combinations of pJG-LIS1 or pJG4–5 empty vector (-Trp selection) and pLexA Tat or pLexA empty vector (-His selection). Colonies grown on-His/-Trp/-Ura media with glucose were plated on Galactose/Raffinose His /-Trp/-Ura plates, to induce LIS1 and Tat production. The plates also contained 5-Bromo-4-Chloro-3-Indolyl-β-D-galactopyranoside  substrate for β-galactosidase. Tat interacted with LIS1 as it was detected by development of blue color upon conversion of X-gal ; cystein-rich region II (residues 22–37); core region III (residues 38–48); basic region IV (residues 49–59); glutamine-rich region V (residues 60–72); and C-terminal region VI ,40. Zhou32P] ATP (6000 Ci/mmol) and (35S)-labeled Methionine were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Econo-Pac CHT-II Cartridge (ceramic hydroxyapatite) was from Bio-Rad (Hercules, CA). Glutathion-agarose was from Sigma (Atlanta, GA). 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 (CELLEX BIOSCIENCES, MN). 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 (Atlanta, GA). Polyclonal antibodies to CDK7, and PSTAIRE were purchased from Santa Cruz Biochemical (Santa Cruz, CA). Polyclonal antibody to CDK9 (PITALRE) were purchased from Biodesign Company (Saco, ME). 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 (50 mM HEPES [pH 7.9], 5 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 10 μg/ml aprotinin and 10% glycerol) 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 (1 ml, three in series). 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 (Promega, Madison, WI). 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 (4% SDS, 10% glycerol, 5% 2-mecarpthaethanol, 0.002% bromophenol blue) 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 (20 mM HEPES pH 7.9, 1 M 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 (0.9–2.4 mg, weighted mean of 1.1 mg). 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 (0.1 mg, 0.2–0.8 mg, 0.9+ mg). 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 (p = 0.71, adjusted for number of cigarettes and nicotine yield). 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 (thought, affect, motor performance, etc), 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:


The most prominent feature of melancholic depression is a near-total loss of the capacity to derive pleasure from activities or other positive stimuli. Additional symptoms can include psychomotor disturbances, anorexia, excessive guilt, and early awakening from sleep. Melancholic patients may exhibit treatment responses and outcomes that differ from those of non-melancholic patients. Pooled data from double-blind, placebo-controlled studies were utilized to compare the efficacy of duloxetine in depressed patients with and without melancholic features.17) total score, HAMD17 subscales (Maier, anxiety, retardation, sleep), the Clinical Global Impression of Severity (CGI-S) and Patient Global Impression of Improvement (PGI-I) scales, and Visual Analog Scales (VAS) for pain.Efficacy data were pooled from 8 double-blind, placebo-controlled clinical trials of duloxetine. The presence of melancholic features (DSM-IV criteria) was determined using results from the Mini International Neuropsychiatric Interview (MINI). Patients (aged ≥ 18 years) meeting DSM-IV criteria for major depressive disorder (MDD) received duloxetine  or placebo  for up to 9 weeks. Efficacy measures included the 17-item Hamilton Rating Scale for Depression . When analyzed by gender, the magnitude of improvement in efficacy outcomes did not differ significantly between duloxetine-treated male and female melancholic patients. In the two studies that assessed duloxetine 60 mg once-daily dosing, duloxetine-treated melancholic patients had significantly greater improvement compared with placebo on HAMD17 total score, CGI-S, PGI-I, 3 of 4 subscales of the HAMD17, and VAS overall pain severity (p < .01). Estimated probabilities of response and remission were significantly greater for melancholic patients receiving duloxetine 60 mg QD compared with placebo . Duloxetine demonstrated significantly greater improvement in depressive symptom severity, compared with placebo, within both melancholic and non-melancholic cohorts (p ≤ .001 for HAMDIn this analysis of pooled data, the efficacy of duloxetine in patients with melancholic features did not differ significantly from that observed in non-melancholic patients. Diagnostic and Statistical Manual of Mental Disorders, 3rdEdition (DSM-III) to describe a subtype of major depressive disorder (MDD). Although revised in DSM-III-R, the current DSM-IV "With Melancholic Features Specifier" represents a return to the older DSM-III definition. According to DSM-IV criteria, the principal diagnostic feature exhibited by patients with melancholic depression is a loss of pleasure in all, or almost all, activities or a lack of reactivity to usually pleasurable stimuli [Although the first appearance of the term "melancholia" dates back to antiquity, its use stimuli . Additio stimuli , althoug stimuli .Melancholia is encountered equally in both genders , but is in vivo immune response [A considerable body of evidence suggests that biological differences exist between melancholic and non-melancholic depression. The hypercortisolism of melancholia has been described as perhaps the best documented finding in biological psychiatry. Many ofresponse ,19.et al concluded that melancholics appear to respond well to treatment of individual episodes but have frequent recurrences, while non-melancholics may respond less rapidly and less completely in any individual episode but show a general pattern of improvement over time [Given the distinct clinical and physiological features associated with melancholia, it is perhaps not surprising that melancholic patients may exhibit treatment responses and outcomes that differ from those of non-melancholic depressed patients. In reviewing a number of long-term studies, Parker ver time . Overallver time .Melancholic patients are less likely to respond to non-specific supportive therapies, such as cognitive behavior therapy or interpersonal psychotherapy, and more often require pharmacotherapy to achieve a successful treatment outcome ,22. WithAlthough a number of studies have investigated the relative efficacy of specific classes of antidepressant medications in melancholic patients, results have been somewhat inconsistent . While tThe antidepressant duloxetine is a balanced and potent dual reuptake inhibitor of 5-HT and norepinephrine (NE). The efficacy of duloxetine in the treatment of MDD has been established in randomized, double-blind, placebo-controlled studies of up to 9 weeks duration . In the 1 and 2, and paroxetine (20 mg QD) in Studies 3, 4, 7, and 8. Study protocols were reviewed and approved by the ethical review board at each center, in accordance with the principles of the Declaration of Helsinki, and all patients provided written informed consent prior to the administration of any study procedures or study drug. The numbers of patients randomized in each study are summarized in Table 1 [4 [5 [6 [All 8 studies included in these analyses were randomized, multicenter, double-blind, placebo-controlled clinical trials and represented all of the double-blind studies included in the New Drug Application reviewed by regulatory agencies for duloxetine's indication in MDD. All studies incorporated double-blind, variable-expected duration placebo lead-in periods to blind patients and investigators to the start of active therapy. Six studies also utilized an active comparator – fluoxetine (20 mg QD) in Studies  Table 1 , 4 40],,1 and 2, 1 [4 [5 , and 6 [[4 [5 [6  have beeDiagnostic and Statistical Manual of Mental Disorders, 4thEdition (DSM-IV) [17)[In all studies, patients were 18 years of age or older and met criteria for MDD as defined by the (DSM-IV) . The dia(DSM-IV) , a stand-IV) [17) total sc-IV) [17) score ≥ Either feature 1 or 2 in Criteria A AND three (or more) features from Criteria B must be present to qualify for melancholic features."A. Either of the following, occurring during the most severe period of the current episode.1. Loss of pleasure in all, or almost all, activities;2. Lack of reactivity to usually pleasurable stimuliB. Three (or more) of the following:1. Distinct quality of depressed mood;2. Depression regularly worse in the morning;3. Early morning awakening ;4. Marked psychomotor retardation or agitation;5. Significant anorexia or weight loss;6. Excessive or inappropriate guilt."Patients were excluded for the following reasons: a current and primary Axis I disorder, other than MDD; an Axis II disorder which could interfere with protocol compliance; lack of response of the current depressive episode to two or more adequate courses of antidepressant therapy; serious medical illness; a serious risk of suicide; a history of substance abuse or dependence within the last year, or a positive urine drug screen.Concomitant medications with primarily central nervous system activity were not permitted, with the exception of episodic use of chloral hydrate or zolpidem for insomnia. Chronic use of prescription analgesic medications was not allowed; episodic use was permitted at the discretion of the physician in charge of the study. Use of anti-hypertensive medications was not permitted unless the patient had been on a stable dose for at least 3 months prior to study entry.Efficacy analyses were performed on three sets of data, obtained using the following pooling strategies:(A) "All Studies" – data from all 8 studies were pooled. Placebo: melancholic , non-melancholic . Duloxetine (40–120 mg/d): melancholic , non-melancholic . Duloxetine was compared with placebo in one set of analyses. In a second set of analyses using data from the 6 SSRI-controlled studies, duloxetine was compared with fluoxetine and paroxetine: Placebo: melancholic , non-melancholic . Duloxetine (40–120 mg/d): melancholic , non-melancholic . SSRI: melancholic , non-melancholic ;1, 4, 5, 6,7, and 8) that demonstrated a significant advantage for duloxetine over placebo on the primary efficacy measure. Placebo: melancholic , non-melancholic . Duloxetine (40–120 mg/d): melancholic , non-melancholic ;(B) "Positive Studies" – data from placebo- and duloxetine-treated patients were pooled from the 6 studies (5 and 6) that compared duloxetine 60 mg once-daily with placebo. Placebo: melancholic , non-melancholic . Duloxetine (60 mg/d): melancholic , non-melancholic .(C) "Focus Studies" – data were pooled from the 2 studies , Maier (Items 1, 2, 7, 8, 9, and 10), retardation (Items 1, 7, 8, and 14), and sleep (Items 4, 5, and 6); the CGI-S scale; and the Patient Global Impression of Improvement (PGI-I) scale [17 total score. Remission was defined as a HAMD17 total score ≤ 7. In Studies 3 – 8, the severity of painful physical symptoms was assessed using Visual Analog Scales (VAS) for pain [In all 8 studies, the primary efficacy outcome was mean change from baseline to endpoint in HAMDI) scale . Responsfor pain .17 total score, CGI-S, and PGI-I were assessed using an analysis of covariance (ANCOVA) with models that included baseline score, treatment, melancholia status (features present Yes/No), investigator, and the treatment-by-melancholia status interaction as independent variables. Hereafter this analysis is referred to as LOCF mean change.Patients with missing melancholia status were not included in the analyses. All other patients with a baseline and at least one postbaseline observation were included in the analyses. Mean changes from baseline to last observation (carried forward) in HAMDThe treatment-by-melancholic status interaction was the main basis upon which differential treatment effects between the subgroups were assessed. Contrasts between duloxetine and placebo within the melancholic and non-melancholic subgroups were used to assess the clinical relevance of treatment effects.17 value, melancholia status, and the two-way and three-way interactions between treatment, visit, and melancholia status. Hereafter this analysis is referred to as MMRM mean change. The categorical longitudinal analyses were similar in concept to the longitudinal mean change analyses, but simplifications were necessary to reduce the computational complexity.Longitudinal mean changes and categorical changes (estimated probabilities) were assessed using a likelihood-based mixed-effects model repeated measures (MMRM) approach. Models for mean changes included treatment, visit, investigator, baseline HAMD17 value, and the treatment-by-visit interaction. A logit link function and a binomial error structure were included to account for the non-linearity of the response and the non-normality of the data, respectively. Hereafter, this analysis is referred to as categorical MMRM. Remission and response rates at last observation were assessed using Fisher's Exact test.The categorical analyses were applied only to patients with melancholic features so that the main effect of melancholic status and its two-way and three-way interactions with visit and treatment could be deleted. Therefore, the model for the categorical analyses included treatment, visit, investigator, baseline HAMD17, CGI-S and PGI-I was applied to all three databases. The focus for the analyses was on all studies and the positive studies with the primary aim of detecting differential effects of duloxetine in patients with and without melancholic features. The MMRM mean change and categorical MMRM analyses were then applied to a wide variety of outcomes from the focus studies in order to gain an in-depth perspective on the efficacy of duloxetine in patients with melancholic features. In addition, LOCF analyses of data from the focus studies were conducted in order to assess robustness of the MMRM results.The LOCF mean change analysis of HAMDBaseline patient demographics are summarized in Table 17 total scores and CGI-S scores were significantly higher (more severe illness) in melancholic patients compared with non-melancholics . There was a marginally significant difference in VAS overall pain severity at baseline , although the clinical relevance of this small difference is questionable.Mean baseline HAMD17 total score, CGI-S and PGI-I (p ≤ .001). Treatment-by-melancholic status interactions were not significant for HAMD17 total score, CGI-S, or PGI-I, in either data set (p > .50 for each comparison). Using pooled data from all studies, the effect size for HAMD17 total score was 0.33 in melancholic patients and 0.32 in non-melancholics. In the six positive studies, the effect size for HAMD17 total score was 0.39 in melancholic patients compared with 0.43 in non-melancholics.Analyses of mean changes from baseline to last observation (LOCF mean change) from all eight studies and the six positive studies are summarized in Table 17 total score, mean change in CGI-S score, and mean endpoint PGI-I score were not statistically significant, indicating that duloxetine's efficacy was similar in male and female melancholic patients  vs. 0.34 .Within the subset of melancholic patients, treatment-by-gender interactions for mean change in HAMDts Table . In pool17 total score were not significant at thresholds of age 55 (p = .777), age 60 (p = .387), or age 65 (p = .540), indicating that the efficacy of duloxetine did not differ between older and younger melancholic patients irrespective of the old/young age threshold.In melancholic patients, treatment-by-age interactions for mean change in HAMD17 total score, mean change in CGI-S score, and mean endpoint PGI-I score were not statistically significant, indicating that the relative efficacy of duloxetine and SSRIs did not differ significantly between melancholic and non-melancholic patients (Table In the 6 studies that included an SSRI comparator, there were no significant differences in baseline-to-endpoint changes in efficacy measures between duloxetine and SSRI treatment groups Table . In addits Table .5 and 6) are summarized in Table 17 total score and HAMD17 Maier subscale compared with those receiving placebo (p < .001). Significant differences from placebo first occurred at Week 1 . On both clinician-rated (CGI-S) and patient-rated (PGI-I) assessments of global improvement, duloxetine-treated melancholic patients had significantly greater mean improvements compared with melancholics receiving placebo (p < .001). Robustness of the MMRM results was confirmed in that significant differences were also observed in LOCF mean change analyses.Analyses of mean changes from baseline to Week 9 (MMRM mean change) for melancholic patients in the two focus studies (Studies Mean changes from baseline (MMRM mean change) for VAS overall pain were also assessed. Figure Estimated probabilities  of remission at Week 9 were significantly higher for melancholic patients treated with duloxetine (60 mg QD) compared with placebo . The estimated probability of response among melancholic patients was 74.7% for duloxetine compared with 42.2% for placebo (p < .001). The advantage of duloxetine over placebo in remission and response rates was also significant in LOCF analyses (p = .032 and p = .002, respectively).17 total score, CGI-S, PGI-I). These results suggest that duloxetine's efficacy does not differ substantially between melancholic and non-melancholic patients. Furthermore, within the group of melancholic patients, duloxetine's treatment effects were similar in male and female patients . One exception was noted in the anxiety subscale of the HAMD17. In the two focus studies, duloxetine did not achieve separation from placebo in patients with melancholic features, but demonstrated efficacy in non-melancholic patients. The reason for the disparity in outcomes between melancholic and non-melancholic patients on the HAMD17 anxiety subscale is unclear.The magnitude of duloxetine's advantage over placebo was generally similar in melancholic and non-melancholic patients. Treatment-by-melancholic status interactions were not significant for any of the three assessed efficacy measures (HAMDi.e. the MINI plus DSM-IV criteria) produced a substantial number of false positive results with regard to melancholic features. It is possible that the use of an alternative melancholia screening tool, such as the CORE scale developed by Parker et al [In previous studies, the prevalence of melancholic features in populations of depressed outpatients has ranged from 16% to 53%. The substantial variation in the reported rates of melancholia may be a result of the numerous criteria used to define melancholic features over the past two decades, including DSM-III, DSM-III-R, DSM-IV, and the Newcastle 1 Depression Rating Scale  (N1) . In the er et al , would her et al . In a wier et al , and wheer et al ,52. Althet al reported that the presence of melancholic features was not associated with lower body mass index [There was no significant difference in age between melancholic and non-melancholic patient groups, despite the fact that melancholic features are more common in older patients. Patients with melancholic features had significantly lower mean body weight at baseline compared to non-melancholics, with a between-group difference of 3.6 kg (8.0 lbs). Since one of the DSM-IV criteria for melancholic features is "significant anorexia or weight loss", this result does not appear surprising. However, given that the melancholic cohort contained a significantly greater proportion of females when compared with the non-melancholic group, the lower body weight among melancholics may be an artifact of gender rather than of MDD subtype. For example, in a recent study of 1694 depressed patients, Berlin ss index .17 score almost two points higher  in the melancholic group. Melancholic patients also had significantly higher baseline CGI-S scores when compared to those without melancholic features. Although melancholic patients had a slightly higher overall pain severity at baseline , the difference only approached statistical significance and its clinical relevance is questionable.As expected, melancholic patients exhibited a greater severity of illness at baseline compared with non-melancholics, with a mean baseline HAMD17, CGI-S or PGI-I scores among both placebo- and duloxetine-treated melancholic patients were slightly larger than those of non-melancholic patients, resulting in a very similar drug-placebo difference in both patient groups. In measures of pain severity there was evidence of greater placebo response among non-melancholic patients; however, duloxetine's advantage over placebo was similar in melancholic and non-melancholic patients, since patients without melancholic features exhibited more robust responses to both drug and placebo, when compared with melancholic patients.A frequent finding in placebo-controlled antidepressant studies is that placebo responses among melancholic patients are smaller than those observed in non-melancholic patients -25,54, aThere does not appear to be a clear consensus as to which class of antidepressant medication is most efficacious for depressed patients exhibiting melancholic features. Some studies have demonstrated advantages for TCAs over SSRIs in the treatment of melancholia -32, and 17 scale has only limited capability to assess improvement in melancholic symptoms. Anhedonia, one of the key features of melancholia, is only assessed indirectly as a part of question 7, while psychomotor agitation is only partially addressed by question 9. Furthermore, items such as diurnal variation in mood are not specifically measured by the HAMD17.Results from the present investigation must be considered in light of several limitations. Firstly, this was a post-hoc analysis of pooled clinical trial data. Although subgroup analyses of efficacy assessments stratified by melancholic status were pre-specified in each protocol, the pooling strategies and some of the analyses conducted in the present investigation were not pre-specified. Secondly, while melancholic features were evaluated as part of the MINI at study entry, specific melancholic features were not an entry requirement and randomization was not stratified by melancholic status. Furthermore, as discussed previously in some detail, the MINI is not an ideal tool for assessing the presence of melancholic features. Thirdly, the HAMD17 total score. Therefore, results from other efficacy measures should be viewed as secondary in nature. Together, these limitations necessitate that the results from the present investigation cannot be considered confirmatory. A prospectively designed clinical trial will be required to confirm the results suggested by the current set of analyses.Fourthly, the group of patients receiving the recommended target dose of duloxetine (60 mg QD) could not be compared head-to-head with SSRIs, as these two studies did not feature active comparator treatment groups. Finally, although duloxetine demonstrated significant advantage over placebo on a number of efficacy measures, the individual studies were powered on the basis of the primary outcome – mean change in HAMDIn these analyses of pooled data, the efficacy of duloxetine in patients with melancholic features did not differ significantly from that observed in non-melancholic patients. Additional prospectively designed clinical trials evaluating duloxetine's efficacy in melancholic patients will be required to substantiate the preliminary findings described here.Drs. Mallinckrodt, Watkin, Liu, Wohlreich, and Raskin are employees of Eli Lilly and Company.CHM proposed the data pooling strategies, designed the statistical analyses, and participated in interpretation of data and drafting of the manuscript. MMW, JR, and JGW participated in interpretation of data and drafting of the manuscript. CL performed the statistical analyses. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The relative role of anti apoptotic (i.e. Bcl-2) or pro-apoptotic (e.g. Bax) proteins in tumor progression is still not completely understood.Bcl-2 and Bax transgenes and the viability of theses cell lines was analyzed in vitro and in vivo.The rat glioma cell line A15A5 was stably transfected with human In vitro, the transfected cell lines (huBax A15A5 and huBcl-2 A15A5) exhibited different sensitivities toward apoptotic stimuli. huBax A15A5 cells were more sensitive and huBcl-2 A15A5 cells more resistant to apoptosis than mock-transfected A15A5 cells (pCMV A15A5). However, in vivo, in syngenic rat BDIX, these cell lines behaved differently, as no tumor growth was observed with huBax A15A5 cells while huBcl-2 A15A5 cells formed large tumors. The immune system appeared to be involved in the rejection of huBax A15A5 cells since i) huBax A15A5 cells were tumorogenic in nude mice, ii) an accumulation of CD8+ T-lymphocytes was observed at the site of injection of huBax A15A5 cells and iii) BDIX rats, which had received huBax A15A5 cells developed an immune protection against pCMV A15A5 and huBcl-2 A15A5 cells.We show that the expression of Bax and Bcl-2 controls the sensitivity of the cancer cells toward the immune system. This sensitization is most likely to be due to an increase in immune induced cell death and/or the amplification of an anti tumour immune response Glioblastoma Multiforme (GBM) are the most common and aggressive tumors of the central nervous system (CNS) . Currentin vitro and in vivo in the latter pathway [Proteins of the BCL-2 family play a major role in the control of apoptosis both  pathway . These p pathway . Inhibit pathway  while, o pathway . We have pathway . Note th pathway . One of  pathway .Although the CNS is usually considered an immune privileged site , specifiBax or Bcl-2 in a rat glioma model and analyzed the effects of these transgenes on the in vitro and in vivo growth of these cell lines.We have tested this hypothesis by establishing cell lines, which stably express transgenes encoding either for human ® assay from Promega (Charbonnières, France) as described previously [Unless specified, all reagents used in this study were from Sigma . Monoclonal anti-human Bax antibody (clone 4F11) was from Immunotech (Villepinte, France) and monoclonal anti-human Bcl-2 antibody (M 0887) was from Dako (Trappes, France); antibodies against rat Bcl-2 or Bax were respectively from Oncogene (Ab5) (Fontenay sous Bois, France) and from Pharmingen (13456E) (Le Pont de Claix, France). The fluorogenic peptide Ac-DEVD-AMC was from Bachem (Voisins les Bretonneux, France) and the lactate dehydrogenase (LDH) activity was measured using the Cytotox 96eviously ,18,21.HelsinkiDeclaration.Experimental research on animals have been conducted according to recommendations of the French National Ethics committee, and are in compliance with the 2 air-humidified atmosphere at 37°C. Plasmids encoding for human Bcl-2 or Bax were subcloned into pRcCMV (Invitrogen) as described by the manufacturer. A15A5 cells were transfected with either 2 μg pCMV vector, pCMV Bcl-2 or pCMV Bax. Plasmid DNA was introduced into 106 cells by electroporation (GenePulser, BioRad, Yvry sur Seine, France) using 200 V/cm and 250 μF. Transfected cells were selected and cloned in a medium containing neomycin (250 μg/ml) for several weeks before clonal dilution.The rat glioma cell line, A15A5, was obtained from the European Collection of Animal Cell Culture . The cell line was maintained in RPMI-1640 (Invitrogen, Cergy-Pontoise, France) supplemented with 10% heat-inactivated FCS (Eurobio, Les Ulis, France), 100 μg/ml streptomycin, 100 U/ml penicillin and 2 mM L-glutamate in a 5% COin vitro cell proliferation of transfected A15A5 cells.Apoptosis was induced by a short UV-treatment. Both untreated and UV-treated cells were cultured for a further 24 h under serum-free conditions. Cell death was also induced with FasL , doxorubicin , staurosporine , Na-Butyrate  or serum deprivation (d-serum) for 3 days. A MTT assay was used according to the manufacturer's instructions (Promega) to determine 5 cells) or tumors established in rats or mice were homogenized vol./vol. in RIPA buffer (PBS containing 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 10 nM PMSF, 10 nM aprotinin, 1 nM Na-orthovanadate). After several passages in a 2 ml glass Dounce homogenizer, the homogenates were centrifuged at 4°C at 13,000 g for 30 min. The resulting supernatants were assayed for protein concentration using the Bradford technique prior to analysis on 15% SDS-PAGE. Western blots were performed as described earlier [A15A5 transfected cells (10 earlier , using pnude mice were purchased from Iffa-Credo (L'Abresle, France) and were housed under standard conditions in our laboratory. huBax and huBcl-2 as well as pCMV A15A5 cells were injected into the brain of BDIX rats weighing between 200 and 240 g. All animal procedures were performed with approved protocols and in accordance with published recommendations for the proper use and care of laboratory animals. Briefly the rats were anaesthetised with an intraperitoneal injection of pentobarbital (50 mg/kg) and positioned in a stereotactic head frame. Aseptic surgical techniques were used to open the scalp in the midline and to expose the frontal and temporalis bones. A 1.0 mm aperture for implanting tumor cells was drilled through the skull. The stereotaxic position of this injection site was 2.5 mm anterior to the bregma and 2.0 mm to the right of midline. 104 tumor cells were implanted stereotactically at a depth of 3.0 mm into the cerebral parenchyma using a 10 μl Hamilton syringe with a 26-gauge needle. The volume injected was 5 μl and the hole was sealed with sterile bone wax. Alternatively, rats were injected sc with 105 cells into the hindlimbs and tumor growth was monitored every week by measuring the volume of the growing tumors. Swiss nude mice were treated similarly except that 104 cells were injected subcutaneously.Inbred BDIX rats and Swiss sc injections of 3.3 × 104 huBax A15A5 cells 15, 10 and 5 days before injection of pCMV or huBcl-2 A15A5 cells. As a control PBS, A15A5 cell oncolysate, or apoptotic bodies derived from Na-Butyrate (NaB)-treated A15A5 cells as previously described [in vitro by a 10 mM NaB treatment in subconfluent A15A5 cultures. When signs of apoptosis were observed under a microscope (changes in cell morphology, detachment from dishes, chromatin condensation as viewed with Hoechst 33342) apoptotic bodies were collected, centrifuged and conserved at -80°C prior to use. Typically, 250 μg apoptotic bodies were mixed with 5 mg/ml BCG and injected subcutaneously three times over 15 days. As a control we used oncolysates obtained after several cycles of rapid freezing/ thawing of A15A5 cells and the lysates was injected together with 5 mg/ml BCG to the animals as above. On day 0, 5 days post-treatment, four groups of rats were sc challenged with 105 pCMV or huBcl-2 A15A5 cells. Tumor growth was evaluated daily over 60 days.In order to evaluate the implication of huBax A15A5 cells preventive anti-tumoral treatment, we designed a protocol consisting of three escribed . Briefly2O2 in methanol for 20 min. The sections were incubated overnight at 4°C with anti-rat CD8 (hybridoma supernatant, Ox 8) diluted 1 in 2 in 1% BSA in PBS, then a secondary antibody coupled to peroxydase was added. The staining was revealed with an AEC substrate.Immunochemical analysis was performed as on 12 μm brain frozen sections. Briefly, sections were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. Endogenous peroxydase activity was inhibited by a treatment with 0,3% H3 pieces, which were incubated in extraction buffer  at room temperature for 45 min and under constant agitation. The cell suspension was filtered through a sterile grid and washed three times with RPMI and maintained overnight in RPMI before analysis. For the phenotypic analysis, monoclonal antibodies obtained from Pharmingen raised against the following molecules were used: CD3 , CD4 (554835, cl ox35), CD8 (554854, cl. Ox 8), CD161 (555006, cl. 10/78), CMH I (22301 D), OX 41/ CD172 (552297) and OX 62 (555010). Monoclonal antibodies raised against granzyme B (GrB, cl. 2C5/F5) was obtained from Chemicon (France) and Fas (AF 126) from R&D Systems (Lille, France). Cells were incubated with the primary antibodies for 30 min at 4°C and washed twice in PBS + 0.1% BSA. For the intracellular detection of GrB, cells were first fixed in 4% paraformaldehyde for 10 min at room temperature, washed with PBS + 0.1% BSA, then permeabilized with 0.1% saponin then incubated with the anti-GrB antibody for 30 min. The secondary antibody was then added for 30 min at 4°C and the cells washed 3 times with PBS + 0.1% BSA. Cells were analyzed on FACScalibur (Becton Dickinson, Le Pont de Claix France) using Cell Quest Pro software. A total of 5000 cells were counted in each experiment.Tumors were resected and minced into 1–2 mmBax or Bcl-2 transgenes, were obtained as described in materials and methods. Two different clones were used in each experiment. The expression of the transgenes was monitored by immunoblot analysis using antibodies specific for human Bcl-2 or Bax and, as shown in figure A15A5 cells transfected with the pCMV vector, human To examine the effect of the different transgene expression to cell death, we studied their sensitivity toward different inducers of apoptosis using both drugs such as doxo (20 ng/ml) and STS (20 μM) or treatments such as d-serum, NaB (10 mM) or a short (1 min) UV irradiation. Cell death was monitored and quantified by measuring the activity of caspase 3 (namely the cleavage of the peptide Ac-DEVD-AMC) and the activity of the cytosolic enzyme LDH released into the culture medium . As shown in figure in vitro response to externally added FasL or by transient transfection of pCMV GrB, huBax A15A5 and huBcl-2 A15A5 cells as described previously for human glioma cells [The immune system exerts its anti-tumoral surveillance mainly through cell death induced by CTLs and NK . These cma cells . As showin vitro  into syngenic BDIX rats as described in materials and methods. The survival rates of the different groups of rats were different as the median survival for the group of rats which had received huBcl-2 A15A5 and pCMV A15A5 cells were respectively 10.2 and 15.6 days, a difference, which was highly significant (P = 0.0086) (figure data not shown). Brain sections from rats injected with huBcl-2 or A15A5 cells were histologically examined at the time of their death or after 30 days for rats injected with huBax A15A5 cells. As illustrated in figure To determine the influence of Bax and Bcl-2 on tumoral growth ) figure . On the ) figure . It shouTo gain information about the kinetics of tumoral growth, cells were subcutaneously (s.c.) injected into the hindlimbs of syngenic BDIX rats and the volume of the tumors evaluated every 10 days. As shown in figure 5) were inoculated s.c. into Swiss nude mice . Tumor growth in the mice was measured for a period of 45 days (figure data not shown). This result showed that the growth of the huBax A15A5 was not due to the induction of rat Bcl-2 expression during tumoral growth in the Swiss nude mice.To test the involvement of the innate immunity in the control of proliferation, the different types of transfected cells  class I molecules. Tumoral cells were dissociated and enzymatically treated as described in materials and methods. We observed a significant increase in double positive cells CD3/CD8 in huBax A15A5 tumors (~17%) compared to huBcl-2 (~4%) and pCMV (~6%) tumors  In x figure . Howeverx figure . Howeverx figure . On the The apoptosis index has been found to be of significant prognostic significance in patients with high-grade astrocytomas . Our stuInterestingly, a rat model of colon carcinoma has been described in which some clones gave rise to tumors that constantly expanded in the animal to eventually formed metastasis (Pro) while others progress for several days before complete regression (Reg) . The disOur results show thus for the first time, in the same type of cells, the adverse effects of Bax and Bcl-2 in the antitumoral role of the immune system.Taken together, our data provide evidence that BCL-2 family members control tumor growth through their sensitivity to immune-induced cell death and enhancement of the immunogenicity of tumor cells.The authors declared that they have no competing interests.ic: intra-cerebral; sc: subcutaneous.Bax: Baxα ; CNS: central nervous system; CTL: cytotoxic T lymphocytes; GBM: Glioblastoma Multiforme; Gwenola Bougras carried out the phenotyping characterization of the cell lines and in vivo experiments with tumors, Pierre Francois Cartron the molecular genetic studies, Fabien Gautier carried out the experimental studies with apoptotic bodies and Stephane Martin the intracerebral implantation of the rat glioma cell lines. Marité LeCabellec participated in immunohistochemical characterization of the tumors. Marc Grégoire and Khaled Meflah participated in the coordination of the study. Francois M. Vallette conceived of the study, and participated in its design and coordination and drafted the manuscript.The pre-publication history for this paper can be accessed here: