To study the effect of saturated fatty acid (SFA)-rich dietary vegetable oils on the lipid profile, endogenous antioxidant enzymes and glucose tolerance in type 2 diabetic rats.Type 2 diabetes was induced by administering streptozotocin  in neonatal rats. Twenty-eight-day-old normal (N) and diabetic (D) male Wistar rats were fed for 45 days with a fat-enriched special diet (10%) prepared with coconut oil (CO) – lauric acid-rich SFA, palm oil (PO) – palmitic acid-rich SFA and groundnut oil (GNO) – control (N and D). Lipid profile, endogenous antioxidant enzymes and oral glucose tolerance tests were monitored.D rats fed with CO (D + CO) exhibited a significant decrease in the total cholesterol and non-high-density lipoprotein cholesterol. Besides, they also showed a trend toward improving antioxidant enzymes and glucose tolerance as compared to the D + GNO group, whereas D + PO treatment aggravated the dyslipidemic condition while causing a significant decrease in the superoxide dismutase levels when compared to N rats fed with GNO (N + GNO). D + PO treatment also impaired the glucose tolerance when compared to N + GNO and D + GNO.The type of FA in the dietary oil determines its deleterious or beneficial effects. Lauric acid present in CO may protect against diabetes-induced dyslipidemia. Dietary fatty acids (FAs) play a key role in various pathological processes involved in diabetes mellitus (DM), such as insulin resistance and atherothrombogenic risk.–5 Both D33In-house laboratory bred male Wistar rats of 4 weeks of age (45 ± 3 g) were selected for the study. The animals were housed in polypropylene cages on clean paddy husk beddings and were maintained under controlled temperature of 20° ± 2°C with an alternating 12-h light:dark cycle (light on 6.00-18.00 h). Diet and water were provided ad libitum. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC). Animal ethical guidelines and good laboratory practice guidelines were followed throughout the experimental period. In addition, all the precautions were taken to minimize pain and discomfort to the animals.Groundnut oil (GNO), CO and PO were purchased from the local market.Diet 1: GNO (Control); Diet 2: CO (lauric acid-rich SFA); Diet 3: PO . The diets were prepared according to the modified American Institute of Nutrition formulae (AIN-76).[2Rats were maintained under standard laboratory conditions and fed with the respective diets till the completion of the experiment.th day, the rats were subjected to an oral glucose tolerance test (OGTT). At the end of 1 h of glucose challenge, the rats showing glucose levels 185 ± 15 mg/dl (against sham control glucose level <100 mg/dl) were included in the study. Later, 28-day-old rats were fed with the respective diets till the completion of the experiment.A mild and stable form of diabetes resembling type 2 human DM was produced by intraperitoneal injection of a freshly prepared solution of Streptozotocin (90 mg/kg) to 2-day-old rat pups. On the 2At the end of 45 days, 2.0 ml of blood was withdrawn from the orbital sinus and the serum was separated by centrifuging at 6000 rpm for 15 min. Lipid profile, viz. total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) were estimated by biochemical kits from Ranbaxy, India using a semiautoanalyser, artos from Switzerland. Non-HDL-C and atherogenic index (AI) were calculated according to the following formulae: Non-HDL-C = TC-HDL-C AI = TC-HDL-C/HDL-CA dose of 1 g/kg of glucose solution (50%) was administered orally to animals that were fasted overnight. Blood waAnimals were sacrificed by cervical dislocation. Livers of the animals were perfused with normal saline and were dissected and processed. Ten percent homogenates were prepared in saline (10%w/v) and centrifuged and the supernatant was used for antioxidant enzyme assays. The total protein content, lipid peroxidation (LPO), catalase and superoxide dismutase (SOD) activity were determined using standard procedures.3P-value at < 0.05 was considered as statistically significant. All the data were processed with Graph Pad Prism version 5.00 software.The results were expressed as mean ± SEM (n = 6). The statistical analysis involving four groups was performed by one-way analysis of variance (ANOVA) followed by Dunnett’s test. P < 0.001), HDL-C (P < 0.05) and non-HDL-C (P < 0.01) as compared with the N + GNO (N control) group. However, the D control group had an apparent increase in AI. Supplementation of CO and PO to D rats showed differential effects. While the CO treatment showed an apparent reduction in AI as compared to the N control, the PO treatment significantly enhanced (P < 0.001) the TC, TG and AI as compared to the N control. Moreover, CO administration significantly reversed the D-associated dyslipidemia with respect to the TC (P < 0.01) and non-HDL-C (P < 0.05) levels whereas PO administration increased the D-associated hyperlipemia (P < 0.01) [The D + GNO (D control) group showed a significant increase in the lipid profile, namely TC ( < 0.01) .P < 0.001) and an increase in LPO (P < 0.01) whereas the CO-treated group produced no changes in these two parameters. Furthermore, PO administration did not significantly alter the D-associated changes. The CO seemed to have apparently reversed the D-induced changes in LPO and antioxidant status [D-induced rats showed a significant drop in SOD while they had elevated LPO. However, D control rats did not show significant changes in catalase as compared to the N + GNO group. Among the two SFA-rich dietary fats studied, the D + PO group showed a significant decrease in SOD  than feeding sunflower oil.[Results of OGTT have shown better glucose tolerance with CO whereas PO has impaired glucose tolerance. The observed difference in insulin sensitivity between two SFA-containing oils studied may be related to the TG level. There are reports on the association of elevated serum TGs with insulin resistance. As per Storlien l muscle. Besides,l muscle.21 The dil muscle.14 But, power oil. ThereforThe present study claims the protective role of lauric acid-rich CO against oxidative stress, dyslipidemia and glucose intolerance. PO due to low content of lauric acid and high content of palmitic acid was found to manifest deleterious effects of SFA. Hence, consumption of high amount of lauric acid- and less amount of palmitic acid-containing oils is likely to provide marked improvement in many aspects of the metabolic syndrome via a range of direct and indirect effects on glucose/lipid metabolism in the body against DM and DM-associated complications. It can be concluded that it is not just SFA but also the type of FA (lauric acid) present in the dietary oil used that is important.


Survivin is a member of the inhibitor-of-apoptosis (IAP) family which is widely expressed by many different cancers. Overexpression of survivin is associated with drug resistance in cancer cells, and reduced patient survival after chemotherapy and radiotherapy. Agents that antagonize the function of survivin hold promise for treating many forms of cancer. The purpose of this study was to investigate whether a cell-permeable dominant-negative survivin protein would demonstrate bioactivity against prostate and cervical cancer cells grown in three dimensional culture.E. coli and purified by affinity chromatography. Western blot analysis revealed that dNSurR9-C84A penetrated into 3D-cultured HeLa and DU145 cancer cells, and a cell viability assay revealed it induced cancer cell death. It increased the activities of caspase-9 and caspase-3, and rendered DU145 cells sensitive to TNF-α via by a mechanism involving activation of caspase-8.A dominant-negative survivin (C84A) protein fused to the cell penetrating peptide poly-arginine (R9) was expressed in The results demonstrate that antagonism of survivin function triggers the apoptosis of prostate and cervical cancer cells grown in 3D culture. It renders cancer cells sensitive to the proapoptotic affects of TNF-α, suggesting that survivin blocks the extrinsic pathway of apoptosis. Combination of the biologically active dNSurR9-C84A protein or other survivin antagonists with TNF-α therapy warrants consideration as an approach to cancer therapy. Survivin is a member of the inhibitors of apoptosis (IAP) family. Overexpression of survivin renders cancer cells resistant to anti-cancer therapy including chemotherapy and radiation therapy -5. It caAt the molecular level, survivin is bifunctional in that it is a suppressor of apoptosis and plays a central role in cell division. A study using surface plasmon resonance spectroscopy and immunoprecipitation analysis showed that a recombinant survivin protein was able to bind directly to both caspase-3 and caspase-7 with nanomolar affinity . Targeti2+-binding baculovirus IAP repeat (BIR) domain consisting of a three-stranded anti-parallel β-sheet surrounded by four small α helices that is linked to a 65 A° amphipathic C-terminal α-helix [Progress in the development of survivin inhibitors has been slow despite the fact that survivin plays multiple roles in cancer cell survival, and renders cancers insensitive to chemotherapy. In the past ten years only a few small molecule inhibitors of survivin have been developed and only one survivin inhibitor, YM155, has reached clinical trials -17. Ther α-helix -20. Surv α-helix -23. A Th α-helix -26. ThesHere we created a cell-permeable dominant-negative C84A survivin protein and investigated its biological activity against cancer cells grown in 3D culture.E.coli cells and its expression induced with IPTG. SDS-PAGE analysis revealed an expressed protein of ~42 kDa in size that matched the expected molecular weight of recombinant dominant-negative survivin of 17 kDa fused to GST of 25 kDa  was constructed based on the finding that mutation of Cys84 to Ala in the extreme C-terminal region of the BIR domain of survivin completely abrogates survivin's ability to inhibit apoptosis, transforming it into a dominant-negative inhibitor of survivin function . The 5'-a Figure . Westerna Figure . The dNSa Figure .A preliminary study revealed that dNSurR9-C84A was able to penetrate into human DU145 prostate cancer cells in two dimensional (2D) cultures within 30 min of addition of the protein  survivin protein has been successfully produced, and demonstrated to exert biological activity in being able to kill prostate and cervical cancer cells. Importantly, it was discovered that dNSurR9-C84A antagonizes survivin's ability to suppress TNF-α signaling, rendering prostate cancer cells susceptible to the proapoptotic effects of TNF-α. dNSurR9-C84A is a both a novel tool for probing the function of survivin, and a potential therapeutic agent for augmenting cancer therapy.The cell lines DU145 (human prostate carcinoma), and HeLa  were purchased from the American Type Culture Collection . DU145 cells were cultured in a mixture of Matrigel™ and RPMI-1640  in a 1:1 ratio. HeLa cells were cultured in a mixture of Matrigel and Dulbecco's modified Eagle medium (DMEM)  in a 1:1 ratio. Both the latter media were supplemented with 10% foetal bovine serum, penicillin (100 U/mL), streptomycin (100 μg/mL) and L-glutamine (0.29 mg/mL). Cells were cultured in the semi-solid medium for five days to allow the formation of three dimensional cellular spheres. Antibodies used in this study included a goat anti-GST antibody  and a rabbit anti-human/mouse survivin antibody .NM_009689) and a T-to-G substitution at nucleotide 354 that changes the cysteine residue at amino acid 84 in the extreme C-terminal region of the BIR domain to an alanine was amplified using the sense primer 5'- GGGGATCCATGCGACGACGACGACGACGACGACGACGAGGAGCTCCGGCGCTGCCCCAG-3' (encodes R9) and the antisense primer 5'-GGGATCCTTAGGCAGCCAGC-3'[BamHI, and cloned into the expression vector pGEX-2T  to give the vector pGEX-2T/dNSurR9-C84A. The integrity of the pGEX-2T/dNSurR9-C84A vector was confirmed by DNA sequence analysis.A dominant-negative cell-permeable form of survivin (dNSurR9-C84A) comprising 9 N-terminal arginine residues  fused to the C84A dominant-negative survivin mutant was constructed for the study. Briefly, the cDNA of a pcDNA3 expression plasmid encoding dominant-negative survivin which contains the entire coding region of mouse survivin   at 30°C for 3 h. The bacteria were pelleted and lysed in STE buffer containing 0.1 mg/ml of lysozyme, 10 mM dithiothreitol and 0.7% sarkosyl, and sonicated for 30 sec. The GST-tagged recombinant dNSurR9-C84A protein was purified by glutathione-Sepharose chromatography, and dialyzed twice against PBS.The dNSurR9-C84A expression vector was transformed into DH5α bacteria, and the transformants were cultured at 37°C in LB medium containing 100 mg/L of ampicillin. When the ODCells were lysed with lysis buffer , and proteins were resolved on 12% polyacrylamide SDS gels under reducing conditions. The gels were either stained with Coomassie blue or proteins electrophoretically transferred to Hybond C Extra nitrocellulose membranes . The membranes were blocked overnight at 4°C with 5% non-fat milk powder, incubated with primary antibodies for 90 min at RT, and then with a horseradish peroxidase-conjugated secondary antibody . Immunoreactivity was detected by Enhanced Chemiluminescence (ECL)  and autoradiography.3) were cultured in a semi-solid culture medium comprised of Matrigel™and RPMI for five days to allow the formation of three dimensional cellular spheres. The cellular spheres were treated with test reagents for 36 h. Dispase , a bacillus-derived neutral metalloprotease, was used to recover cells cultured in the Matrigel™. Live cells were resuspended in PBS and cell viability was analyzed with the CellTiter96 MTS cell proliferation assay kit  with measurements being made on a 96-well plate reader (Bio-Tek Instruments Inc.).Cells . Caspase-8 activity was determined with the Caspase-8 Fluorometric Protease Assay Kit . Caspase-9 activity was determined with the Caspase-9 Fluorometric Protease Assay Kit .The activity of caspase-3 in cell lysates was determined with the Apo-ONEIn-Situ Apoptosis Detection kit, Roche Diagnostic, Mannheim, Germany) to detect DNA fragmentation, counter-stained with propidium iodide (PI), and examined by fluorescence microscopy.Cells were stained with the TUNEL reagent -mediated dUTP-biotin nick end labelling.The authors declare that they have no competing interests.CHAC performed all the experiments and drafted the manuscript. XS and JK cosupervised the student. JB contributed to analyses involving confocal microscopy. LC contributed to the purification of the recombinant protein. GWK was the Principle Investigator who directed the work, supervised the student, and revised the manuscript for publication. All authors read and approved the final manuscript.dNSurR9-C84A is rapidly taken up by DU145 cells. DU145 cells were incubated with either PBS (control) or dNSurR9-C84A protein for 30 min. Cells were stained with an anti-GST antibody and counter-stained with DAPI. Cells were examined by standard immunofluorescence microscopy (top 2 rows) or by confocal microscopy (bottom row). Green fluorescence indicates the presence of GST-tagged dNSurR9-C84A in the cytoplasm. The cells were photographed, and the images merged (right-hand panels).Click here for filedNSurR9-C84A inhibits the viability of DU145 and HeLa cells. (A) Cells were incubated with either PBS (control) or increasing concentrations of dNSurR9-C84A for 12 h, and cell viability was assessed. A statistically significant difference in the viability between cells treated with dNSurR9-C84A and PBS is denoted as "*". *p < 0.05. (B) dNSurR9-C84A induces caspase-3/-7 activities in DU145 cells in a dose-dependent fashion. DU145 cells were incubated with increasing concentrations of dNSurR9-C84A for 90 min. The cells were lysed and caspase-3/-7 activity in the cell lysate measured. A statistically significant difference in the caspase activity between cells treated with dNSurR9-C84A and PBS was denoted as "*". *p < 0.05. (C) dNSurR9-C84A induces DNA fragmentation in DU145 cells. Cells were incubated with PBS or dNSurR9-C84A for 3 h, and stained (green) with the TUNEL agent. The permeabilized cells were counter-stained (red) with propidium iodide.Click here for file


The allele-specific silencing of ataxin-3 significantly decreased the severity of the neuropathological abnormalities associated with MJD. These data demonstrate that RNAi has potential for use in MJD treatment and constitute the first proof-of-principle for allele-specific silencing in the central nervous system.Recent studies have demonstrated that RNAi is a promising approach for treating autosomal dominant disorders. However, discrimination between wild-type and mutant transcripts is essential, to preserve wild-type expression and function. A single nucleotide polymorphism (SNP) is present in more than 70% of patients with Machado-Joseph disease (MJD). We investigated whether this SNP could be used to inactivate mutant ataxin-3 selectively. Lentiviral-mediated silencing of mutant human ataxin-3 was demonstrated  The loss of function of wild-type ataxin-3, which has been shown to play a role in ubiquitin-mediated proteolysis Gene silencing by RNA interference (RNAi) has been successfully used to downregulate the expression of mutant genes and rescue phenotypes in various autosomal dominant neurodegenerative diseases, including Huntington's disease (HD) in vivo in a selective manner. We demonstrate the therapeutic efficacy and selectivity of this approach in a rat model of MJD In the present study, we used lentiviral vectors (LV) encoding short-hairpin RNAs (shRNAs) targeting this SNP, to downregulate mutant human ATX3 (MUT-ATX3) G987GG→C987GG) located at the 3′ end of the CAG expansion of the ataxin-3 gene for the specific silencing of mutant human ataxin-3 mRNA but not of the wild-type ataxin-3 mRNA. The wild-type ataxin-3 gene has a G at position 987, whereas the mutant ataxin-3 has a C at this position. We therefore designed two siRNAs corresponding to this SNP. The sequences are identical with the exception of a single nucleotide (G/C polymorphism) at the center of the shRNA molecule: shAtaxMUT(C) and shAtaxWT(G)  promoter, to facilitate the identification of transduced cells  . These sed cells .lacZ confirmed that the decrease in ataxin-3 mRNA levels reflected siRNA efficiency rather than variations in transfection efficiency (data not shown).Quantitative RT-PCR analysis of 293T cells co-transfected with the shAtaxMUT(C) vector and a vector expressing the human mutant ataxin-3(C) gene  demonstrWe then investigated whether this silencing was associated with a decrease in wild-type and mutant ataxin-3 protein levels. Co-transfection with the mutant ataxin-3 plasmid and the specific shAtaxMUT led to significant (∼70%) downregulation, as observed on western blots, whereas co-transfection with the non specific shAtaxWT had limited effect on mutant ataxin-3 protein levels . ConversWe further assessed the functionality and efficacy of our siRNA in a more physiological situation, by analyzing the silencing of endogenous ataxin-3 in human embryonic kidney 293T cells. We transfected human 293T cells with the shAtaxWT(G), shAtaxMUT(C) or shGFP plasmid. The level of endogenous wild-type ataxin-3 was determined by western blotting and found to be lowered by transfection with shAtaxWT(G), but not with shAtaxMUT(C) or shGFP . These din vivo. We assessed the efficacy and selectivity of this approach in the recently developed rat model of MJD substantia nigra  and striatum  two months after injection, in this model.No data are available concerning the allele-specific silencing of polyglutamine disorders striatum. We recently demonstrated that the striatum is affected in MJD, whether in our rat model, transgenic mice or patients lacZ reporter gene present in the vector encoding the small hairpin RNA.We assessed the efficacy and selectivity of allele-specific silencing, by injecting lentiviral vectors encoding human mutant ataxin-3(C), together with the corresponding shRNA (shAtaxMUT(C)), into rat lacZ reporter gene present in the shAtaxMUT(C) vector  and the non specific shAtaxWT(G) or the shGFP control, immunohistochemical analysis of coronal rat brain sections with anti-ataxin-3 (1H9) and β-galactosidase antibodies showed that many neurons expressed both transgenes . In anim2 , A669TG/G669TG  and TAA1118/TAC1118 (Stop to Tyr). Analysis of these polymorphic alleles showed there to be eight different haplotypes. However, the A669-C987-A1118 haplotype was found in 72% of MJD patients from 249 families from different countries, but in only 2% of the normal population It has been shown 987GG (MUT) or G987GG (WT) alleles. These shRNAs were not produced according to available rules for the design of optimal siRNAs in vitro, but not within the context of lentiviral vectors (24). It was therefore difficult to predict the results, particularly in vivo. Our data clearly demonstrate that shAtaxMUT selectively and efficiently silences the human mutant ataxin-3 gene. In vivo, we observed a significant decrease in the formation and accumulation of ataxin-3-positive aggregates (∼50%) and preservation of DARPP-32 neuronal marker expression (∼70%).We developed two lentiviral vectors encoding previously described shRNA sequences in vitro, by the preservation of wild-type ataxin-3 expression upon shAtaxMUT co-expression, and in vivo, by the limited effect of shAtaxWT on mutant ataxin-3 gene expression. It should be noted that shAtaxMUT and shAtaxWT recognize only human ataxin-3 mRNAs, as the targeted region displays limited similarity of sequence to the endogenous rat ataxin-3 messenger RNA. These experiments therefore replicate allele-specific silencing as it would occur in human patients, with the preservation of wild-type ataxin-3 gene expression, an important safety measure given the role of ataxin-3 in proteolysis.The selectivity of the approach was demonstrated C. elegansDrosophila have shown that wild-type ataxin-3 decreases the neurotoxicity of mutant ataxin-3 via a mechanism involving ubiquitin and the proteasome ATAX3 has been shown to act as a polyubiquitin-binding protein, linking its normal function to protein surveillance pathways Our data suggest that the use of an allele-specific shRNA efficiently decreases both mutant ataxin-3 production and MJD-associated neuropathological symptoms. A recent report implicated RNA toxicity in MJD pathology, providing further support for treatment at RNA level in vivo. These data suggest that it is feasible to treat MJD patients by the selective knockdown of mutant ataxin-3.In conclusion, we have generated lentiviral vectors markedly improving MJD-associated neuropathological signs G987GG→C987GG). A shRNA targeting the Green Fluorescent Protein (shGFP) was used as a control. The sequences of the oligomers used were as follows: shAtaxMUT(C987GGGACCTATCTCTCTTGAAGATAGGTCCCGCTGCTGCTGGGGATCTGTGGTCTCATACAGAACCTAGTTTCCAAAAAAGCAGCAG); shAtaxWT(G987GGGACCTATCTCTCTTGAAGATAGGTCCCCCTGCTGCTGGGGATCTGTGGTCTCATACAGAACCTAGTTTCCAAAAAAGCAGCAG); shGFP(CTAGTTTCCAAAAAAAGCTGACCCTGAAGTTCATCTCTTGAATGAACTTCAGGGTCAGCTTGGGGATCTGTGGTCTCATACAGAAC). Each of these oligomers and the primer H1-3F (CACCGAACGCTGACGTCATCAACCCG) were used for PCR with the pBC-H1 plasmid  containing the H1 promoter  as a template. The PCR product was inserted into the pENTR/D-TOPO vector . The H1-shRNA cassette was then transferred, with the LR clonase recombination system, into the SIN-cPPT-PGK-nls-LacZ-LTR-TRE gateway vector (SIN-CWP-nlsLacZ-TRE-Gateway), which contains a tetracycline-regulated operator upstream from the H1 promoter in the 3′LTR. A LacZ reporter gene was also inserted downstream from the PGK promoter, facilitating the identification of infected neurons.We used two shRNAs discriminating between wild-type and mutant human ataxin-3 mRNA molecules. We engineered a small hairpin RNA specifically targeting the single nucleotide polymorphism  (27Q) or mutant ataxin-3(C) (72Q) 2/95% air atmosphere. On the day before transfection, 293T cells were plated in six-well tissue culture dishes  at a density of 700,000 cells per well. The cells were co-transfected by the calcium phosphate method, with SIN-W-PGK-ATX3 72Q  or SIN-PGK-W-ATX3 27Q  and shATX3 or shGFP (5 µg). Six hours later, the medium was removed and replaced with fresh medium. Forty-eight hours after transfection, the cell cultures were washed with cold PBS, treated with trypsin and the cells were collected by centrifugation.HEK 293T cells were cultured in DMEM  supplemented with 10% fetal bovine serum , 2 mM L-glutamine, 4500 mg/l glucose, 25 mM HEPES, 100 U/ml penicillin, and 100 U/ml streptomycin  at 37°C in a 5% COg, 10 min). The pellets were incubated on ice in lysis buffer  for 30 minutes, with vortexing every 10 minutes. Homogenates were centrifuged at 13,000×g for 30 minutes at 4°C and supernatants were stored at −80°C. Protein concentration was determined with the Bradford protein assay . Protein extract (20 µg) was resolved by electrophoresis in a 7.5 % or 12% SDS-polyacrylamide gels. The proteins were transferred onto nitrocellulose membranes  in TG 10×liquid concentrate buffer  containing 192 mM glycine, 25 mM Tris-HCl, and 20% methanol. The membranes were blocked by incubation in 5% nonfat milk powder in 0.1% Tween 20 in Tris-buffered saline  for 1 h at room temperature, and were then incubated overnight with the following primary antibodies diluted in blocking buffer: anti-Myc tag antibody clone 4A6 , anti-β-galactosidase antibody  and anti-tubulin antibody . Blots were washed three times in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), for 20 min each, and incubated with a horseradish peroxidase (HRP)-coupled secondary biotinylated goat anti-mouse or anti-rabbit IgG antibody  for 1 h at room temperature. Blots were washed three times in TBS-T, for 20 minutes each, and binding to antigens was detected by enhanced chemiluminescence . Semi-quantitative analysis was carried out based on the optical density (OD) of scanned films . Specific ODs were normalized with respect to those for tubulin for experiments on total homogenates. The specific OD was then normalized with respect to the amount of tubulin loaded in the corresponding lane of the same gel. A partition ratio was calculated and expressed as a percentage.Cells were collected by centrifugation (1000×GGCTCACTTTGTGCTCAACATTG) and reverse (HATAX-2R: TCTCATCCTCTCCTCCTCATCCAG) primers. An ABI PRISM 7000 thermal cycler was programmed for an initial denaturation step  followed by 40 amplification cycles . The amplification rate for each target was evaluated from the cycle threshold (Ct) numbers obtained with cDNA dilutions, with correction for human β-actin levels , which were assumed to be constant. Differences between control and experimental samples were calculated using the 2−ΔΔCt method LacZ oligos were used as an internal standard for evaluating transfection efficiency . RT-PCR analysis was performed on 3 to 5 samples from 3 independent transfections. Data are expressed as the mean of normalized values, as relative ATX3 mRNA level±SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Fisher's PLSD post-hoc test . Values of p<0.05 were considered significant.Total RNA was extracted, 48 hours after transfection, with Trizol reagent . Real-time quantitative RT-PCR was performed in triplicate, with 0.4% random-primed cDNAs generated from 400 ng total RNA. PCR was carried out in a 20 µl reaction volume containing Platinium SYBR Green pPCR super Mix-UDG , and 10 µM of both forward (HATAX-1F: ad libitum. The experiments were carried out in accordance with the European Community Council directive (86/609/EEC) for the care and use of laboratory animals.Adult male Wistar rats , each weighing ∼200 g were used. The animals were housed in a temperature-controlled room maintained on a 12 h light/12 h dark cycle. Food and water were provided Concentrated viral stocks were thawed on ice and resuspended by repeated pipetting. The rats were anesthetized as previously described Three (n = 2 for shAtaxMUT and shAtaxWT) or 8 weeks  after lentivirus injection, the animals were perfused with a phosphate solution followed by fixation with 4% paraphormaldehide  and 10% picric acid, their brains removed and the entire striatum was sliced, the sections then being stored in 48-well trays , as previously described The immunohistochemical procedure was initiated by endogenous peroxidase quenching by incubation for 1 h at 37°C in 0.1% diphenylhydrazine in PBS. Free-floating sections were incubated for 1 h at room temperature in 0.1% Triton X-100 in PBS (or 0.02% Triton X-100 in PBS for the anti-ubiquitin antibody) supplemented with 10% NGS , and then with the appropriate antibodies—the mouse monoclonal anti-ataxin-3 antibody 1H9 , a rabbit polyclonal anti-ataxin-3 antibody , kindly provided by Dr. Henry L. Paulson , a rabbit polyclonal anti-ubiquitin antibody , a rabbit polyclonal dopamine- and cAMP-regulated phosphoprotein with a molecular mass of 32 kDa  and an anti-β-galactosidase antibody  diluted in 0.1% Triton X-100 in PBS supplemented with 10% NGS. Sections were washed three times and incubated with the corresponding biotinylated secondary antibody  diluted in 0.1% Triton X-100 in PBS supplemented with 10% NGS for 2 h at room temperature. Sections were washed three times and bound antibodies were visualized with the ABC amplification system  and 3,3′-diaminobenzidine tetrahydrochloride  as the substrate. The sections were mounted, dehydrated by two passages through ethanol and toluol solutions, and coverslipped with Eukitt® .Double-staining for ataxin-3/DARPP-32 and ataxin-3/β-galactosidase - Free-floating sections were incubated at room temperature for 1 h in 0.1% Triton X-100 in PBS supplemented with 10% NGS , followed by blocking solution containing the corresponding antibodies: 1H9 Ab (1∶5000) and anti-DARPP-32 (1∶2000) overnight, 4°C; 1H9 (1∶5000) and anti-β-galactosidase . Sections were washed three times and incubated with the corresponding secondary antibodies coupled to fluorophores  diluted in 0.1% Triton X-100 in PBS supplemented with 10% NGS for 2 h at room temperature. The sections were washed three times in PBS and mounted in Fluorsave Reagent  on microscope slides.d(a1+a2+a3 …), where d is distance between serial sections (200 µm or 300 µm), and a1, a2, a3 .are DARPP-32-depleted areas for individual serial sections. The depleted area corresponds to area with a gray-scale value lower than the mean gray-scale value of all pixels measured in the lesioned area. Data are expressed as the area of the evaluated DARPP-32-depleted region.The extent of ataxin-3 lesions in the striatum was analyzed by digitizing 10 DARPP-32-stained sections per animal , selected so as to obtain complete rostrocaudal sampling of the striatum with a slide scanner and by quantifying the area of the lesion with a semiautomated image-analysis program . Sections from throughout the entire striatum were analyzed. The area of the striatum showing a loss of DARPP-32 staining was measured for each animal, with an operator-independent macro. The volume was then estimated with the following formula: volume  = 2 were reliably detected. None of the objects touching one of the X or Y borders of the fields of view were counted, in any of the images. For each animal, the estimated total number of inclusions (Ne) was calculated as Ne = (Ns)/Sf, where Ns is the number of inclusions detected in all sections and Sf is the rostrocaudal sampling fraction (1/8). Inclusions in striatal neurons tended to be round (mean rotundity index>0.90), with an isotropic orientation in the striatum. We therefore corrected the number of raw cell counts using the Abercrombie factor Cell counts and morphometric analysis of ataxin-3 inclusions were performed as previously described, but with minor modifications Premounted sections were stained with cresyl violet for 2 minutes, differentiated in acetate buffer pH 3.8 to 4 , dehydrated by passing twice through ethanol and toluol solutions, and mounted on microscope slides with Eukitt® .We stained striatal sections with FluoroJade-B , an anionic fluorescein derivative that stains neurons undergoing degeneration. The sections were first washed in water and then mounted on silane-coated glass slides, dehydrated, and stained according to the supplier's manual.. Bright-field and fluorescent images were acquired digitally on an Axioskop 2 Plus microscope (Zeiss) with Axiovision software version 4.2. All photographs for comparison were taken under identical image acquisition conditions and uniform adjustments of brightness and contrast were made to all images.post hoc test . Values of p<0.05 were considered statistically significant.The intensity of immunostaining is expressed as mean±SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Fisher's PLSD


Roseobacter clade of aerobic marine proteobacteria, which compose 10–25% of the total marine bacterial community, has been reported to fix CO2, although it has not been determined what pathway is involved. In this study, we report the first metabolic studies on carbohydrate utilization, CO2 assimilation, and amino acid biosynthesis in the phototrophic Roseobacter clade bacterium Roseobacter denitrificans OCh114. We develop a new minimal medium containing defined carbon source(s), in which the requirements of yeast extract reported previously for the growth of R. denitrificans can be replaced by vitamin B12 . Tracer experiments were carried out in R. denitrificans grown in a newly developed minimal medium containing isotopically labeled pyruvate, glucose or bicarbonate as a single carbon source or in combination. Through measurements of 13C-isotopomer labeling patterns in protein-derived amino acids, gene expression profiles, and enzymatic activity assays, we report that: (1) R. denitrificans uses the anaplerotic pathways mainly via the malic enzyme to fix 10–15% of protein carbon from CO2; (2) R. denitrificans employs the Entner-Doudoroff (ED) pathway for carbohydrate metabolism and the non-oxidative pentose phosphate pathway for the biosynthesis of histidine, ATP, and coenzymes; (3) the Embden-Meyerhof-Parnas  pathway is not active and the enzymatic activity of 6-phosphofructokinase (PFK) cannot be detected in R. denitrificans; and (4) isoleucine can be synthesized from both threonine-dependent  and citramalate-dependent  pathways using pyruvate as the sole carbon source.The  The marine Roseobacter clade are potentially major contributors to global CO2 fixation as they make up 10–25% of the total microbial community in some surface ocean ecosystems Roseobacter clade belong to a group known as Aerobic Anoxygenic Phototrophs (AAPs), the only known organisms performing photosynthesis requiring oxygen but not producing oxygen, while other members are non-phototrophic. CO oxidation was confirmed experimentally for the non-phototrophic Roseobacter clade bacterium Silicibacter pomeroyiRoseobacter clade 2 fixation was suggested in several marine AAPs Roseobacter clade with completed genome sequence indicated that the genes encoding ribulose bisphosphate carboxylase/oxygenase (RUBISCO) and phosphoribulokinase required in the Calvin cycle for carbon fixation, as well as genes for other autotrophic CO2 fixation pathways, are missing in these bacteria Roseobacter clade bacteria can fix CO2, if they indeed fix CO2.Two of the most important sources of carbon sinks known in nature are absorption of CO2 fixation in Roseobacter clade Roseobacter clade are known to require organic carbon sources for growth Roseobacter clade bacteria utilize organic carbon and assimilate CO2 will help us understand the bio-energy metabolism, production of bioactive metabolites, and roles of global carbon cycle in these wide-spread marine bacteria.Anaplerotic pathways have been proposed as an alternative mechanism for CORoseobacter denitrificans OCh114 a (BChl a) aerobically. The genomic sequence of R. denitrificansR. denitrificans, we developed a minimal growth medium containing only defined carbon source(s), optimized the growth conditions of R. denitrificans in different defined carbon sources, and used isotopomer assisted metabolite analyses, biochemical approaches, and gene expression profiles to investigate the carbon assimilation, carbohydrate utilization, and amino acid biosynthesis pathways in R. denitrificans.Here, we report metabolic and biochemical studies of R. denitrificans OCh114 cultures grown aerobically in the light (20 W/m2), dark, and day-night cycles (600 were reached in the dark and day-light cycles compared to in the continuous light growth conditions (R. denitrificans OCh114 2 (or HCO3−). Spectral features of the photosynthesis system and light-harvesting complexes for the cultures grown in the minimal medium containing pyruvate or glucose are similar to the cultures grown in the rich medium . No differences in the 13C-isotopomer abundances of the protein-derived amino acids for cultures grown in the minimal medium containing pyruvate were observed under either dark or illuminated conditions . Although it has been recognized that yeast extract contains rich vitamin mixtures, it also includes amino acids and other undefined carbon sources. Ideally, the 13C-isotopic labeling studies require a minimal medium containing only defined carbon source(s), thus it is desirable to optimize the growth conditions by eliminating the yeast extract. We found that vitamin B12  can serve as an alternative to yeast extract for growing R. denitrificans in defined carbon sources, as cultures with OD600≥3 can be reached in our minimal growth medium with glucose as the sole carbon source , and are included in the growth media of many photoautotrophic, photoheterotrophic and chemoheterotrophic bacteria. Requirement for vitamin B12 in the growth of R. denitrificans may partially explain why yeast extract was necessary to be included in the minimal medium containing either pyruvate or glucose.The previous studies of the growth of n source . Vitamin13C]glucose or D-[6-13C]glucose, the isotopomer labeling data of serine (the precursor is 3-phosphoglycerate) and alanine (the precursor is pyruvate) were different (R. denitrificans and need to be considered to account for the isotopomer abundance in these protein-derived amino acids: (1) the Embden-Meyerhof-Parnas (EMP) pathway (glycolysis), by which one [1-13C]glucose molecule is cleaved into two glyceraldehyde-3-phosphate (GAP) molecules: one is [3-13C]-labeled and the other is unlabeled using either D-[1-13C]glucose or D-[6-13C]glucose; (2) the Entner-Doudoroff (ED) pathway 13C]glucose, and one molecule of pyruvate, where the first carbon is labeled using D-[1-13C]glucose  the pathway . As showR. denitrificans predominantly uses the EMP pathway for carbohydrate metabolism, one will expect similar 13C-isotopomer abundance in serine and alanine . However, this is not consistent with the higher M+0 value (more unlabeled carbon) in serine (0.74) than in alanine (0.56) using D-[1-13C]glucose in our studies (R. denitrificans: eda (RD1_2878), encoding 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14), and edd (RD1_2879), encoding phosphogluconate dehydratase (EC 4.2.1.12). If R. denitrificans uses the ED pathway as one of the alternative carbohydrate utilization pathways, [1-13C]glucose is converted into [1-13C]-2-keto-3-deoxy-6-phosphogluconate, which is cleaved into [1-13C]pyruvate and unlabeled GAP, leading to unlabeled 3-PGA  and edd (RD1_2879) genes are expressed and the transcript level of these genes is higher in the minimal medium containing either pyruvate or glucose than in the rich medium, similar to the gene expression profiles of other genes responsible for carbon fixation and carbon metabolism examined in this report  than using D-[1-13C]glucose (0.74), compared to slightly higher M+0 value in alanine using D-[1-13C]glucose (0.56) versus D-[6-13C] glucose (0.48)  PP pathway (The proposed ED pathway was further tested using D-[6- pathway . Our date (0.48) . The M+0 glucose . Based o pathway .13C]glucose. The ED and PP pathways can explain much higher M+0 value in phenylalanine (0.48) and tyrosine (0.53) using D-[1-13C]glucose than the value in these amino acids (0.04) using D-[6-13C]glucose, and more labeled carbon can be incorporated into PEP and E4P using D-[6-13C]glucose , synthesized from 3-PGA, and erythrose-4-phosphate (E4P), the intermediate in the non-oxidative PP pathway, are the precursors of phenylalanine, tyrosine and tryptophan. Labeled E4P in the non-oxidative PP pathway  leads to]glucose  and 4.13C]glucose (N10-formyl-tetrahydrofolate (THF)), and glutamine (the nitrogen source). The 6-phosphogluconate, an intermediate of the ED/oxidative PP pathway, is the precursor of PRPP. In the oxidative PP pathway , and it is unknown if the oxidative PP pathway is complete because the pgd gene in R. denitrificans is not annotated. Thus, the 13C-isotopomer abundance in histidine using D-[1-13C]glucose is possible from the following sources: (1) from the N10-formyl moiety of THF if the oxidative PP pathway is active, (2) from D-ribose-5-phosphate or D-ribulose-5-phosphate derived from the non-oxidative PP pathway, where the labeled carbon is maintained  than other amino acids (0.42–0.89) was detected using D-[1-]glucose . Histidi pathway , the firintained , or both13C-labeled sources are available using D-[6-13C]glucose (i.e. labeled GAP from the ED pathway and labeled fructose-6-phosphate (F6P)), while only F6P is labeled using D-[1-13C]glucose (GAP is not labeled via the ED pathway using D-[1-13C]glucose)  with D-[1-13C]glucose (13C]glucose (0.10) (13C]glucose (0.28). Together, our studies imply that R. denitrificans uses the non-oxidative PP pathway for histidine, ATP, coenzymes, and nucleic acids biosynthesis, as well as supplying GAP for the ED pathway  . Since t]glucose  and for e (0.10)  than usi pathway  and 4.R. denitrificans2. In our studies, no growth of R. denitrificans was observed in the minimal medium with CO2 (or bicarbonate) as the sole carbon source . Using uniformly 13C-labeled D-glucose (D-[U-13C6] glucose)  , our stuglucose) . Also, apyruvate . Similar glucose . TogetheR. denitrificans has an active TCA cycle and metabolic fluxes in the anaplerotic pathways. PEP can be converted into OAA, the precursor of aspartate, asparagine, methionine, threonine, lysine and isoleucine. OAA can be also generated from pyruvate and through the TCA cycle. Using D-[1-13C] glucose, the M+0 value is similar in aspartate (0.58), threonine (0.47), methionine (0.49) and alanine (0.56) but smaller than serine (0.74) (13C-isotopomer abundance in the amino acids derived from OAA is largely from pyruvate (via the anaplerotic pathway) and/or the TCA cycle flux.Our studies imply that e (0.74) , suggest2 and replenish the intermediates of the TCA cycle, and genes encoding these anaplerotic enzymes can be all found in the genome of R. denitrificans OCh114; malic enzyme  and tme (RD1_0421) than for pyc (RD1_3376) and ppc (RD1_4248) (Four anaplerotic enzymes are known to assimilate COD1_4248) . The act13C]pyruvate was used and the 13C-isotopomer abundance in aspartate (from OAA), serine  and alanine (from pyruvate) was analyzed , consistent with low gene expression of ppdK  and that the pathway from OAA to PEP is likely to be active and pyruvate to OAA is not as strong. This is in agreement with the gene expression profile of pckA (RD1_1376) and pyc (RD1_3376). Hence, most of the 13C-labeling in OAA likely comes from the TCA cycle flux , in which the pyruvate to malate pathway  should be rather active, compatible with higher expression of tme (RD1_0421) than pyc (RD1_3376). The proposed anaplerotic flux is summarized in To investigate metabolic fluxes through the anaplerotic pathways, [3-analyzed . Both QR13C-isotopomer abundance in [1-13C]pyruvate cultures showed higher M+0 value in leucine (0.91) and isoleucine (0.86) compared to valine (0.25) and threonine (0.70) -3-isopropylmalate is released as CO2 catalyzed by 3-isopropylmalate dehydrogenase   in the threonine-dependent pathway e (0.70) . Most of.1.1.85) . However.1.1.85) . The res13C-D-erythro-3-methylmalate as CO2  was active in various growth conditions , since the same precursors (pyruvate and acetyl-CoA) are employed in the biosynthesis of both amino acids. Therefore, both threonine- and citramalate-dependent pathways should be contributed to synthesize isoleucine, considering that the difference in 13C-isotopomer abundance between leucine and isoleucine (0.06–0.07) is larger than the instrumental error (∼0.01). Further, the calculated values of all 13C-isotopomer abundance (from M+0 to M+5) of isoleucine indicate that 80% of the in vivo metabolic flux for isoleucine biosynthesis comes from the citramalte-dependent pathway, 20% from the threonine-dependent pathway, when [3-13C]pyruvate is the carbon source  assimilated) Erythrobacter longusAcidiphilium rubrumErythrobacter sp. strains 2 fixation can be detected between R. denitrificans and A. rubrum. In A. rubrum, CO2 fixation is completely inhibited with the glucose-growth culture, and is enhanced 3- to 5-fold in the light (∼3.0 nmoles/mg dry cell weight) 2 incorporation can be detected in R. denitrificans with either pyruvate- or glucose-growth cultures in either light or dark growth, and is not enhanced by light exposure.In this paper, we reported that Roseobacter clade, many of them cannot generate photosynthetic pigments. To the best of our knowledge, no CO2 assimilation data have been reported for these unpigmented strains of the Roseobacter clade. It will be very useful to compare the difference in the CO2 assimilation between R. denitrificans and un-pigmented Roseobacter clade bacteria.Among the wide-spread marine Rhodobacter capsulatusRhodobacter sphaeroidesR. sphaeroides strains, while the 6-phosphogluconate dehydrogenase gene (pgd) in the oxidative PP pathway cannot be annotated.The ED pathway has generally been viewed as an alternative to the EMP pathway. In examining the carbohydrate catabolism of both R. denitrificans, and that the non-oxidative PP pathway contributes to histidine, ATP, coenzyme and nucleic acid biosynthesis, and that no metabolic flux going through the EMP pathway and the oxidative PP pathway. Our experimental evidences are consistent with the genomic information of R. denitrificans, in which the ED pathway and the non-oxidative PP pathway are complete, but the pfk gene, essential for the EMP pathway, and the pgd gene, required for the oxidative PP pathway, are not annotated.Our studies in this report indicate that the ED pathway plays a significant role in glucose metabolism of Roseobacter clade pgd gene is also missing in Silicibacter pomeroyi DSS-3, Silicibacter sp. TM1040 and Dinoroseobacter shibae DFL12, and that the pfk gene is not found in Jannaschia sp. CCS1, Silicibacter pomeroyi DSS-3, and Silicibacter sp. TM1040. Alternatively, Dinoroseobacter shibae DFL12 has all of the genes in the EMP pathway, and Jannaschia sp. CCS1 has complete oxidative PP pathway. It will be interesting to learn carbohydrate metabolisms in these two bacteria.Searching the genomes of the R. denitrificans and possibly some other resourceful heterotrophic marine Roseobacter clade R. denitrificans demands more NADPH than ATP in the cells when the oxidative PP pathway is not active. The oxidative PP pathway, not the non-oxidative PP pathway, can generate two molecules of NADPH from one molecule of glucose, and can produce approximately 60% of the NADPH required inside the cells.The physiological significance for 2O2 into H2O by glutathione peroxidase R. denitrificans since it is an aerobic anoxygenic bacterium that contains highly absorbing pigments that can sensitize reactive oxygen species formation. When the oxidative PP pathway is not active, the ED pathway may be the best option to generate NADPH required by the cells. It was not established whether an anoxygenic photosynthetic bacterium, like R. denitrificans, can produce glutathione, which can be synthesized by oxygenic phototrophs, such as cyanobacteria and higher plants. Note that genes for glutathione biosynthesis , glutathione reductase , glutathione peroxidase , and some glutathione transferases can be found in R. denitrificans genome, suggesting possible roles of glutathione in reducing oxidative stress in R. denitrificans.NADPH is not only the reducing power for synthesizing energy-rich molecules and biofuels, but also prevents oxidative stress by reducing glutathione via glutathione reductase, which converts reactive HR. denitrificans adapting the ED pathway could be that many marine Roseobacter-lineage bacteria live in coastal seawater, an environment enriched with a wider variety of organic acids, and the ED pathway is a better option for digesting the hexuronic acids (e.g. glucuronic acid). This hypothesis may be supported by the broad substrate specificity of KDPG aldolase identified biochemically Roseobacter strains Roseobacter clade, especially the significance of the ED/non-oxidative PP pathway in these physiologically diverse, widespread and high abundance marine microorganisms.The other possibility for E. coli included, that the anaplerotic and metabolic pathways are not as much in demand in the rich medium as in the minimal medium containing defined carbon source, or some pathways may be shut down due to metabolic regulation R. denitrificans, lower copy numbers of the 16S rRNA gene will be expected in the minimal medium with glucose (grow slowest), and highest number in the rich medium grown under dark conditions (grow fastest). Together, both possible scenarios can explain lower transcript level for all of the target genes we tested in the rich medium. Further investigations are required for addressing this important issue.Throughout the QRT-PCR data analysis, we observed that the transcript level for all of the genes tested is higher in cells grown in the minimal medium containing either pyruvate or glucose than in the rich medium . The dis12 can successfully replace the undefined carbon source, yeast extract, for the growth of R. denitrificans in our developed minimal medium containing defined organic carbon source. While it is not known why the undefined carbon source yeast extract is required for R. denitrificans and several other photosynthetic bacteria, it is not clear why exogenous vitamin B12 is required for R. denitrificans, because all of the genes required for vitamin B12 biosynthesis are annotated in R. denitrificans (based on the information in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database). It is also unclear how cobalamin can be utilized by R. denitrificans, since no known cobalamin transporter genes  can be recognized in the genome. Nonetheless, the effect of exogenous vitamin B12 is clear and its inclusion permitted most of the results reported in this paper. More experimental evidences are required for understanding the roles of yeast extract and exogenous vitamin B12 reported herein for enhancing cell growth.In this report, we demonstrate that vitamin B2 fixation in protein carbon through the anaplerotic enzymes by R. denitrificans, supporting the proposal by Swingley et al. R. denitrificans cannot live with CO2 as the sole carbon source, consistent with lack of genes for key enzymes of the Calvin cycle. Missing genes in the Calvin cycle are also common features in the other four Roseobacter clade bacteria with complete sequences Our studies suggest approximately 10–15% COR. denitrificans uses the non-oxidative PP pathway as the other alternative pathway for sugar degradation to make the ribose-5-phosphate for histidine, cofactors, ATP and nucleic acid biosynthesis, as well as to produce GAP for carbon metabolism. In contrast to the oxidative PP pathway, no CO2 is released in the non-oxidative PP pathway, which is an important feature for carbon fixation by autotrophs using the Calvin cycle. In these autotrophs, the non-oxidative PP pathway is part of the Calvin cycle. Although R. denitrificans cannot use the Calvin cycle for fixing CO2, it does have a complete non-oxidative PP pathway and relies on the pathway for converting hexose phosphate into pentose phosphate.Other than the ED pathway, our studies demonstrate that 13C-labeled glucose, pyruvate and sodium bicarbonate were purchased from Cambridge Isotope Laboratories (CIL), Inc .The DNA oligomers in this report are from Integrated DNA Technology (IDT) and were used without further purification. The R. denitrificans OCh114  was grown aerobically (20–30%-filled Erlenmeyer flasks) on either a nutrition-rich medium  (pH 7.5) or a reported minimal medium 600. We used 2% cultures (50-fold dilution) in the late exponential growth phase grown in the rich medium to inoculate the minimal medium containing defined carbon source(s). To minimize the carryover of the rich medium, inoculated cells were centrifuged and supernatant liquid was removed. Cell pellets were washed with the minimal medium (no carbon source included) twice, resuspended in the minimal medium, and then transferred to the minimal medium containing defined carbon source(s).13C-isotopically labeled carbon source, 0.1% pyruvate (labeled on either the first or third carbon position), 0.1% glucose , or 0.2% NaH13CO3 were added. Due to the requirement of a growth medium without including undefined carbon sources for GC/MS studies, ferric citrate 12. The cultures were grown to an exponential phase, representing the pseudo steady-state, and then harvested for analyzing the isotopomer distribution in key metabolites.In the minimal medium supplied with defined R. denitrificans OCh114. cDNA was synthesized from 1 µg RNA and 100 µM random 9mer DNA using Superscript III reverse transcriptase (Invitrogen). The QRT-PCR reactions were performed via ABI 7500 Real-Time PCR System (Applied Biosystems). The primers (shown in −(ΔCt). Three biological replicates, with six technical replicates for each biological sample, and total eighteen technical replicates were performed for validation, and the mean value was reported  (Sigma-Aldrich) with 25 µM NADH in 0.1 M Tris-HCl buffer at pH 8.0 The amount of D-glucose in solution was determined by hexokinase/glucose-6-phosphate dehydrogenase coupling assay with a D-glucose assay kit , and the reduction of NADg for 15 min at 4°C and washed with 20 mM Tris-HCl buffer at pH 8.0. Cell pellet was resuspended in the same buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF). Resuspended cells were disrupted by sonication, and cell debris was removed with centrifugation at 20,000×g for 30 min. Protein concentration in cell extracts was determined by the Bradford assay Enzymatic activities were performed with cell-free crude extracts prepared as follows. Cells were harvested by centrifugation at 5,000×tert-butyl dimethylsilyl)-N-methyl-trifluoroacetamide (Sigma-Aldrich) at 70°C for 1 h. A gas chromatograph (GC)  equipped with a DB5-MS column  and a mass spectrometer  was used for analyzing metabolite labeling profiles. We report four types of MS fragment data: (1) the [M-15]+ group, which loses a methyl group; (2) the [M-57]+ group, containing un-fragmented amino acids; (3) the [M-159]+ group, which contains amino acids losing α carboxyl group; and (4) the [M-302]+ group, containing only the 1st (α carboxyl group) and 2nd carbons in the amino acid backbone (Note that [M-302]+ and [M-15]+ cannot be detected in some amino acids). Three labeling data were used to trace the carbon metabolic route and to identify active pathways. Throughout the article, M+0, M+1, M+2… stands for the value of unlabeled (M+0) and of additional 13C-labeled protein-derived amino acids .The methods for GC/MS are reported previously Figure S1Spectra, culture, and growth curves of R. denitrificans under different growth conditions. The culture of R. denitrificans grown in the rich medium under the day-light cycles (a). The spectrum (b) and growth curve (c) of the cells grown in the rich medium under dark versus under light. The spectrum of the cells grown under dark and day-light cycle is shown in the inset. The formation of the light harvesting antenna complex II antenna complex (807-nm peak) is repressed in the light, while the level of the reaction center-light harvesting complex I (RC-LHI) core complex (872-nm peak) is similar. Data fit of pyruvate uptake with or without NaHCO3 in the defined medium, and the uptake rate is 2.5×10−2±5×10−4 mmole per hour (d).(1.12 MB TIF)Click here for additional data file.Figure S2The non-oxidative pentose phosphate pathway. All of the reaction steps are reversible. Possible 13C-labeling using [1-13C]glucose or [6-13C]glucose is shown in green or red dots, respectively.(0.47 MB TIF)Click here for additional data file.Figure S3Experimental values of the isotopomer abundance of isoleucine (red bar) using [3-13C]pyruvate as the defined carbon source, and predicted contributions of the threonine (back bar) and citramalate (blue bar) pathways for isoleucine biosynthesis in R. denitrificans.(0.62 MB TIF)Click here for additional data file.Table S113C-isotopomer abundances of tert-butyl dimethylsilyl (TBDMS) derivatives of protein-derived amino acids from R. denitrificans OCh114 grown in the defined medium containing different carbon sources. Except in (b), all of the cultures were grown in the dark. The unlabeled molecule is shown as M+0, and additional mass comes from 13C-labeled carbon source. All of the data, except leucine and isoleucine, are [M-57]+ data (tert-butyl group is removed). The [M-15]+ data (methyl group is cleaved) for leucine and isoleucine are shown, because the [M-57]+ data are overlapped in GC-MS.(0.27 MB DOC)Click here for additional data file.Table S2Primers used for QRT-PCR studies reported in this paper(0.04 MB DOC)Click here for additional data file.Table S3a,bThe theoretical and experimental M+0 values in alanine and serine using D-[1-13C] glucose and D-[6-13C]glucose(0.03 MB DOC)Click here for additional data file.


Following entry, uncoating, and reverse transcription, a number of cellular proteins become associated with the Human Immunodeficiency Virus type 1 (HIV-1) pre-integration complex (PIC). With the goal of obtaining reagents for the analysis of the HIV-1 PIC composition and localisation, we have constructed functional integrase (IN) and matrix (MA) proteins that can be biotinylated during virus production and captured using streptavidin-coated beads.Although the labelled C-terminus allows for the sensitive detection of virion-associated IN, it becomes inaccessible in the presence of cellular proteins. This masking is not dependent on the nature of the tag and does not occur with the tagged MA. It was not observed either with an IN mutant unable to interact with LEDGF/p75, or when LEDGF/p75 was depleted from cells.Our observation suggests that a structural rearrangement or oligomerization of the IN protein occurs during the early steps of infection and that this process is related to the presence of LEDGF/p75. Integration of the Human Immunodeficiency Virus (HIV) DNA into the host cell chromosome mediated by the integrase (IN) protein is an obligatory step of the virus life cycle. This endonuclease encoded by the pol gene generates active CA-3'-hydroxyl ends on the viral cDNA and catalyses strand transfer with the chromosomal DNA. IN is also involved in the processing and trafficking of the viral genome throughout the pre-integration phase including reverse transcription and nuclear import -3. The Iin vivo in the presence of Bir A, a biotin ligase from E. coli  was wasE. coTAACGCTGGTGGTTCTTAACACATGGAATTCTGCAACAAC 3'; EcoRI site in italics) and used in a PCR fusion with F1 fragment using oligonucleotides containing respectively AflII and EcoRI sites . The final PCR product was digested with AflII and EcoRI and was swapped for the corresponding fragment in pCMVΔR8.74. For pCMVΔR8.74-MA-BAD, a BstBI unique site was added by PCR to the 3' end of the MA at position 383 of the GAG coding sequence in the pCMVΔR8.74. A BstBI-BAD linker was made by annealing S4  and AS5 (5'-PO4-GCTTAGAACCACCAGCGTTAGAACGCCATTCCATTTTTTGAGAATCCAGAATTTGACGCAGAGAAGAAGCAA) which was ligated with the BstBI digested pCMVΔR8.74. The HA tag was introduced at the 3'-end of the pol gene of pCMVΔR8.74 by PCR using primers S1 and AS4 . The PCR product was digested with AflII and EcoRI and was swapped for the corresponding fragment in the pCMVΔR8.74. The Q168A mutation was introduced in pCMVΔR8.74-IN-BAD by PCR-directed mutagenesis, using the Quick change II site directed mutagenesis kit (Stratagene) and an oligonucleotide which contained GCG in place of the CAG codon in position 501 of the IN ORF (5' GGACAGGTAAGAGATGCGGCTGAACATCTTAAGAC 3'). The HIV-1-derived self-inactivating pRRL-H1shRNALEDGF/p75-PGK-eGFP-WPRE and pRRL-H1shRNActl-PGK-eGFP-WPRE transfer plasmids were constructed from a previously described system  Triton X-100, 1 mM dithiothreitol supplemented with proteases and phosphatases inhibitors cocktail (Roche)) [All transductions were done with vectors that have equivalent TU/PP ratio. For proteins-BAD capture or immunoprecipitations, fifteen million HEK 293 cells were transduced (MOI 50) with IN-BAD or INQ168A-BAD or MA-BAD or IN-HA vectors or mock-transduced. When necessary, azidothymidine (AZT) was added 24 h before transduction at the final concentration of 100 μM. To remove vector excess, cells were washed two times with Phosphate Buffer Saline (PBS) 2 hours post-infection. Six hours later, cells were washed three times with K buffer  in thenpresence of polybrene . After 3 rounds of transduction over a period of 48 h, cells were cultured for 3 weeks and enriched by sorting GFP+ populations using flow cytometry. For the analysis of LEGDF/p75 protein expression, cells protein extracts were prepared from 107 cells that were lysed for 30 mn in K buffer. For Q-PCR, DNA samples were prepared with the Wizard Genomic DNA Extraction Kit (Promega).For shRNA experiments, 107 particles per lane). IN-BAD and IN-WT were revealed on Western blots probed with an anti-IN antibody  or an anti-biotin antibody (Tebu-bio).To analyse the IN biotinylation status, IN-BAD and IN-WT vector preparations were either directly loaded onto an SDS PAGE or lysed 30 mn in K buffer and incubated 2 hours with 20 μl of paramagnetic streptavidin-coated beads before material elution and loading  or p24 or MA (Tebu-bio) or HA (Roche) antibodies were incubated 2 hours with 20 μl of Protein A-coated beads in 100 μl of K buffer and washed three times to remove antibodies excess. 500 μl of cell lysates were incubated overnight with 20 μl of Protein A-coated beads pre-bound to the antibodies or with 20 μl of streptavidin-coated Dynabeads (Invitrogen) for BAD capture and the eluted material was analysed by Western blotting using the appropriate antibody.gag antigen per 106 HEK 293 cells, MOI 10) as described previously [LEDGF/p75-PGK-eGFP-WPRE or the pRRL-H1shRNActl-PGK-eGFP-WPRE was determined by Q-PCR, amplifying from the genomic DNA the Woodchuck post-trancriptional regulatory element (WPRE) sequences of the lentiviral vector in comparison with the human albumin gene as previously described [The kinetics of viral DNA synthesis and integration of IN-BAD or IN-WT vectors were determined by Q-PCR following transduction (30 ng of p24eviously . The numescribed .1/10 of beads of the streptavidin pull downs or the LEDGF/p75 co-immunoprecipitation were diluted in 10 μl of Tris/EDTA buffer and subjected to a PCR using the MH531 and MH532 oligonucleotides  to ampliThe authors declare that they have no competing interests.SBZ has been involved in the supervising of the study, has trained and supervised JP and SB, designed experiments, conducted experiments with SB and JP, interpreted the data, and drafted the paper. SB has provided a substantial technical assistance. JP has carried out the shRNA experiments. AZB has designed and performed BAD constructions. OD has conceived of and supervised the study, and was involved in drafting the manuscript and revising it critically for intellectual content. All authors read and approved the final manuscript.


Plasmodium berghei scavenger receptor-like protein PbSR is essential for mosquito-to-host transmission of the parasite: PbSR knockout parasites produce normal numbers of oocysts that fail to form sporozoites, pointing to a role for PbSR in the oocyst during sporogony. Here, using fluorescent protein tagging and targeted gene disruption, we show that PbSR is synthesized in macrogametocytes, gets targeted to the crystalloids of developing ookinetes and is involved in crystalloid formation. While oocyst sporulation rates of PbSR knockout parasites are highly reduced in parasite-infected mosquitoes, sporulation rates in vitro are not adversely affected, supporting the view that mosquito factors could be involved in the PbSR loss-of-function phenotype. These findings are the first to identify a parasite protein involved with the crystalloid organelle, and suggest a novel protein-trafficking mechanism to deliver PbSR to the oocysts.Crystalloids are transient organelles that form in developing malaria ookinetes and disappear after ookinete-to-oocyst transition. Their origins and functions remain poorly understood. The  Malaria is the most important parasitic infection in people and causes over one million deaths annually  is a modular protein containing several distinct adhesive-type domains implicated in lipid, protein and carbohydrate binding. It is highly conserved among Plasmodium species. In addition to its adhesive domains, the presence of an ER signal peptide and the absence of typical organellar-targeting sequences suggested an extracellular role for PbSR, possibly involving interaction with the vertebrate host or insect vector  gene cassette was introduced conferring resistance against the antimalarial drug pyrimethamine (tgdhfr cassette into the pbsr locus (data not shown). This was confirmed by assessing the integrity of clonal populations of the genetically modified parasite lines by Southern blot analysis of SpeI-digested genomic DNA .A series of genetically modified parasite lines was generated  to assesethamine . After tomic DNA . AccordibSR/EGFP . Conversarasites . These cpbsr promoter-driven reporter study  tag . GFP fluAnalysis of purified PbSR/EGFP gametocytes by Western blot using an anti-GFP antibody failed to detect the full-length PbSR/GFP product (predicted to be 175 kDa in size), but gave rise to a band of 27 kDa corresponding to monomeric GFP . This inBoth parasite lines PbSR/EGFP and mCherry/PbSR/EGFP gave rise to large numbers of sporozoites in parasite-infected mosquitoes, and were efficiently transmitted to naïve mice by infected mosquito bites (data not shown), demonstrating that the fluorescent protein tags did not affect functionality of the PbSR protein. To assess PbSR protein expression in different life stages, we analysed parasite line mCherry/PbSR/EGFP by western blot with anti-RFP antibody. This analysis confirmed that PbSR is present in gametocytes and is also present, at lower levels, in ookinetes . HoweverConfocal microscopic examination of mCherry/PbSR/EGFP parasites revealed green and red fluorescence throughout the cytoplasm of female gametocytes/macrogametes . This loP. berghei, haemozoin-containing vacuoles accumulate around the edges of the crystalloid inclusion bodies , indicating that PbSR was targeted neither to mitochondrial nor to lysosomal organelles. Closer examination of these ookinetes, however, revealed a clear colocalization of the fluorescence with malaria pigment . Malarian bodies  . Similarly, no crystalloids were found when we examined ookinetes of parasite line ΔPbSR/EGFP. These combined results indicate that PbSR is involved in the formation of the crystalloids. Pigment formation was not affected in parasite lines ΔSRCR/EGFP and ΔPbSR/EGFP (data not shown).GFP fluorescence was observed in female gametocytes of parasite line ΔSRCR/EGFP, indicating that the modified PbSR protein was normally expressed. Its localization in gametocytes appeared similar to that in parasites lines PbSR/EGFP and mCherry/PbSR/EGFP (data not shown). However, ookinetes of parasite line ΔSRCR/EGFP lacked the focal spots corresponding to the crystalloid inclusions and instead displayed dispersed green fluorescence , indicatin vitro using purified in vitro cultured ookinetes as starting material. In parasite line PbSR/EGFP, the fluorescent spots observed in ookinetes – corresponding to the crystalloids – typically disappeared within a day of ookinete-to-oocyst transition, which is in agreement with the observations of oocysts in infected mosquitoes. In vitro sporulation of ΔSRCR/EGFP oocysts was readily observed . This observation is consistent with a scenario in which the macrogametocyte is the only life stage where discernible PbSR protein synthesis occurs, which fits well with our PbSR expression data , which is reportedly present in gametocytes, but absent from late oocysts and sporozoites  through 48% Nycodenz cushions, followed by phosphate-buffered saline (PBS) washes. Ookinete cultures were set up overnight from gametocytaemic blood as previously described  and SR-IF-Xba-R (GGGCGGCCGCTCTAGCATATGCACAAATATACATACATATAA) and introduced into NheI/XbaI-digested pCHT1-KO (loxP-prokaryotic promoter cassette was PCR-amplified with primers pLP-DHFR-F2 (ATAGGGCGAATTGGGCTGCAGATAACTTCGTATAGCATACATTATACG) and pLP-DHFR-R (CTGCAGGCATGCAAGCTGATCAACGTCAGGTGGCACTTTTCG) from template provided in the Creator Acceptor Vector Construction Kit (BD Biosciences). This PCR product was introduced into KpnI/HindIII-digested pSR-REP1 via In-Fusion PCR cloning to give plasmid pLP-DHFR/SR. This plasmid acts as the acceptor vector for all PbSR constructs.A 750 bp fragment corresponding to the 3′UTR of the P. berghei dhfr gene was PCR-amplified with primers pDNR-EGFP-F (ACGAAGTTATCAGTCGACTTAAGCTTAGGGGCCCTCATGAGTAAAGGAGAAGAACTTTTCACTGGA) and pDNR-EGFP-R (CCAAACGAATGGTCTAGTAAGCTCGAAATTGAAGGAAAAAACATCATTTG) from plasmid pOB079 (a gift from O. Billker) and introduced into SalI/HindIII-digested pDNR-Dual (BD Biosciences) via In-Fusion PCR cloning to give plasmid pDNR-EGFP.A 1.2 kb fragment containing the enhanced green fluorescent protein (EGFP)-coding sequence plus the downstream 3′UTR from the pbsr gene was PCR-amplified with primers pDNR-PbSR-F (ACGAAGTTATCAGTCGACGGTACCACTCTATATAAGTATTTTTCTTCATCTTCG), containing a unique 5′ KpnI site, and pDNR-PbSR-R (ATGAGGGCCCCTAAGCTTAAGCGTTTCAAAAAGGTAAATGA) from P. berghei gDNA and introduced into SalI/HindIII-digested pDNR-EGFP via In-Fusion PCR cloning to give plasmid pDNR-PbSR/EGFP.A 4.5 kb fragment containing the coding sequence plus the upstream 5′UTR from the ATGGAAAGTTAAGCTTAGGGGCCCTCAT) and ΔSR-R (AAGCTTAACTTTCCATATACGTTCATAAAAAATTGC). These two primers were designed to contain a 16 bp overlap . After PCR amplification , the template DNA was digested with DpnI and the amplified DNA self-ligated via In-Fusion PCR cloning to give plasmid pDNR-ΔSR/EGFP. In this plasmid, the PbSR-coding sequence is removed with the exception of the N-terminal signal peptide. The same approach was used to create plasmid pDNR-ΔSRCR/EGFP with primers ΔSRCR-F (AAAGTAATGCAAGTGCAGAAGAGGAAGG) and ΔSRCR-R (GCACTTGCATTACTTTTTATATCATCAATAGATGTCAAATGA). This plasmid has the two tandem SRCR domains removed from the PbSR-coding sequence.One microgram of plasmid pDNR-PbSR/EGFP served as template in PCR using primers ΔSR-F (AAAGCTCGAGTGGTGCAAAGCGAAAGTTTG) and XhoI-R (CACCACTCGAGCTTTCCATATACGTTCATAAAAAATTGC). This plasmid has an extra XhoI restriction site introduced directly downstream of the predicted N-terminal signal peptide. The RFP variant mCherry sequence (The same approach was used to create plasmid pDNR-PbSR/EGFP/XhoI with primers XhoI-F (loxP site-specific recombination (BD Biosciences) to give the transfection vectors pLP-PbSR/EGFP, pLP-ΔSR/EGFP, pLP-ΔSRCR/EGFP and pLP-mCherry/PbSR/EGFP respectively.The PbSR-specific inserts contained within pDNR-PbSR/EGFP, pDNR-ΔSR/EGFP, pDNR-ΔSRCR/EGFP and pDNR-mCherry/PbSR/EGFP were introduced into pLP-DHFR/SR via Cre-Parasite transfection, pyrimethamine selection and dilution cloning were performed as previously described , were fractionated by electrophoresis through NuPage 4–12% Bis-Tris precast gels (Invitrogen) and transferred to PVDF membrane (Invitrogen) according to the manufacturer's instruction. The equivalent of 50 000 parasites per sample were analysed unless otherwise stated. Membranes were blocked for non-specific binding in PBS supplemented with 0.1% Tween 20 and 5% skimmed milk for 1 h at room temperature. Primary antibodies  diluted 1:5000 were applied to the membrane for 1 h at room temperature. After washing, membranes were incubated for 1 h with secondary antibody  diluted 1:5000 for 1 h at room temperature. After washing, signal was detected by chemilluminescence (Pierce ECL Western blotting substrate).Live or paraformaldehyde-fixed parasite samples were mounted in Vectashield containing DAPI. Images were captured on a Zeiss Axiovert 200M inverted microscope using Zeiss LSM 510 software.Parasites were prepared for electron microscopy by overnight fixation in 2.5% glutaraldehyde/2.5% paraformaldehyde/0.1 M Na cacodylate buffer at 4°C. Samples were post-fixed with 1% osmium tetroxide/0.1 M Na cacodylate buffer, washed with buffer followed by MilliQ water, bloc stained with 3% aqueous uranyl acetate, dehydrated in ascending ethanol concentrations, rinsed briefly in propylene oxide, then embedded and polymerized in Taab epoxy resin. Ultrathin sections were cut and mounted on Pioloform-coated copper grids and stained with lead citrate. Immunogold labelling was carried out as described .


Protein degradation is essential for cell homeostasis. Targeting of proteins for degradation is often achieved by specific protein sequences or posttranslational modifications such as ubiquitination.By using biochemical and genetic tools we have monitored the localization and degradation of endogenous and chimeric proteins in live primary cells by confocal microscopy and ultra-structural analysis. Here we identify an eight amino acid sequence from the C-terminus of the short-lived GTPase RhoB that directs the rapid degradation of both RhoB and chimeric proteins bearing this sequence through a lysosomal pathway. Elucidation of the RhoB degradation pathway unveils a mechanism dependent on protein isoprenylation and palmitoylation that involves sorting of the protein into multivesicular bodies, mediated by the ESCRT machinery. Moreover, RhoB sorting is regulated by late endosome specific lipid dynamics and is altered in human genetic lipid traffic disease.Our findings characterize a short-lived cytosolic protein that is degraded through a lysosomal pathway. In addition, we define a novel motif for protein sorting and rapid degradation, which allows controlling protein levels by means of clinically used drugs. The degradation of cellular components plays a key role in the maintenance of cellular functions. Protein degradation serves regulatory roles in cell cycle and signalling and provides amino acid supply for protein synthesis. The main proteolytic systems in the cell are the proteasome machinery and the lysosomal pathway. Whereas the proteasome has been mainly involved in the rapid degradation of regulatory or misfolded proteins GTPases of the Rho family play crucial roles in the regulation of the actin cytoskeleton, cell survival and gene expression. Among Rho proteins, RhoB displays unique functions in the control of endocytic traffic, influencing the sorting of signalling kinases From a structural point of view, RhoB is highly homologous to RhoA. However, whereas RhoA is retained in the cytosol bound to RhoGDI under resting conditions Here we have explored the cellular pathways involved in RhoB degradation and the structural determinants directing this process. Our results reveal several novel striking features of RhoB: 1) this short-lived protein is degraded through a lysosomal pathway; 2) both, isoprenylation and palmitoylation are required for RhoB degradation; and 3) the last eight amino acids of RhoB, which comprise the posttranslational modification motif, are sufficient to direct the endo-lysosomal degradation of chimeric proteins. Taken together, our observations delineate a novel pathway for protein degradation.4Cl or inhibition of the vacuolar H+-ATPase with bafilomycin A1 . In contrast, Rab7, which is bi-geranylgeranylated, was retained at the limiting membranes . Thus, disruption of late endosome lipid dynamics promotes a selective trapping of RhoB in these structures. The presence of GFP-RhoB in the intraluminal membranes of U18666A-induced dense MVB was confirmed by IEM  but removal of the statin allowed its appearance in cytosolic vesicles (d).As shown above, the levels of the cytosolic protein RhoGDI are not altered by agents modulating lysosomal protein degradation or endosomal traffic and they remain stable after inhibition of protein synthesis . Therefovesicles d.As observed with other -CINCCKVL chimeric proteins, the levels and the half-life of GFP-RhoGDI-8 were consistently lower than those of GFP-RhoGDI and endogenous RhoGDI . InteresRapid protein degradation contributes to the tight control of RhoB levels. The study of the mechanisms responsible for RhoB degradation points to the existence of a degradation pathway  is sufficient to promote a RhoB-like localization and degradation rate when fused to the C-terminal end of several unrelated proteins. Therefore, an interesting aspect of our studies is the potential use of the CINCCKVL sequence to control the localization and cellular levels of proteins of interest. For instance, chimeric proteins bearing this sequence could be potentially used for delivering bioactive components to the endo-lysosomal compartment to modulate lysosomal function, which has broad implications for cell viability, both in carcinogenesis and degenerative processes. Moreover, levels of CICNCKVL-ending proteins can be increased by lysosomal protease inhibitors. This raises the possibility of using these chimeric proteins as reporters of lysosomal proteolytic activity. Importantly, isoprenylation and palmitoylation can act as switches for the lysosomal targeting and degradation of chimeric proteins bearing CINCCKVL C-terminal sequences. Thus, levels of CINCCKVL-ending chimeric proteins can be regulated by commonly used drugs such as simvastatin. Also, given the increasing interest for the clinical use of isoprenylation inhibitors, the potential for use of CINCCKVL contructs for monitoring isoprenylation efficiency in living cells can be hypothesized. Finally, identification of this novel protein degradation sequence may provide valuable tools to study the mechanisms of MVB formation and lysosomal proteolysis in health and disease.BAEC were obtained and cultured as previously described 1 was from A.G. Scientific, Inc. . LTR was from Molecular Probes (Invitrogen). Simvastatin was a gift form Merck, Sharp & Dohme .Antibodies for Western blot were anti-RhoB (sc-180), anti-RhoGDI (sc-360), anti-RhoA (sc-418), and anti-ubiquitin (sc-8017), from Santa Cruz Biotechnology , anti-GFP from Clontech  and anti-tagRFP from Evrogen . Antibodies for IF were anti-RhoB (sc-8048) and anti-RhoGDI (sc-360), from Santa Cruz Biotechnology, and anti-cathepsin B from Calbiochem. Anti-LBPA was from Echelon Biosciences Inc. . Bafilomycin ApEGFP-C1 and pDsRed1-C1 were from Clontech. pTagRFP-C was from Evrogen. Human RhoGDI cDNA was from Origene . The pcDNA3-HA-RhoB plasmid was generously provided by Dr. G.C. Prendergast. Lamp1-GFP was from Prof. Jennifer Lippincott-Schwartz . GFP-Rab7 wt was kindly provided by Prof. Cecilia Bucci . GFP-Vps4 wt and E334Q were the generous gift of Dr. Philip Woodman .CGAGTTGATGC-3′5′-GCTCTAGATCATAGCACCTTGCA, CGAGCCATTCTG-3′5′-GCTCTAGATCATAGCACCTTGCAGCAGTTGAT, and CGAGTTGATCGAGCCATTCTG-3′5′-GCTCTAGATCATAGCACCTTGCA for the C192S, C189S, and C189,192S mutants, respectively. Mutation sites are underlined. Constructs encoding chimeric proteins comprising GFP and various sequences from the RhoB or TC-10 C-terminus, were generated by PCR using pEGFP-C1 as a template. The forward primer was common for all constructs: 5′-GAGTAGAAGCTTATGGTGAGCAAGGGCGAGGAG-3′. Reverse primers were 5′-CCGAATTCTAGAGGACCTTGCACTTGTACAGCTCGTCC-3′, 5′-CCGAATTCTAGAGGACCTTGCAACAGTTGATACACTTGTACAGCTCGTCCATGC-3′ and 5′-CCGAATTCTCACGTAATTAAACAACAGTTGATACACTTGTA CAGCTCGTCCATGC-3′ for generating the GFP-CKVL, GFP-CINCCKVL (GFP-8) and GFP-CINCCLIT (GFP-8T) constructs, respectively. PCR products were digested with HindIII and EcoRI and cloned into pcDNA3.0. The construct encoding GFP plus the last 22 amino acids of RhoB was generated by amplifying GFP with a BssHII site at its 3′ end, using pEGFP-C1 as a template and primers, forward 5′-GAGTAGAAGCTTATGGTGAGCAAGGGCGAGGAG-3′, and reverse 5′-CATCTTGCGCGCGTCTTGTACAGCTCGTCC-3′. The PCR product was subcloned into the HindIII-BssHII sites of pcDNA-RhoB. The BssHII site is located at position 806 of the sequence of the rat RhoB gene (NM_022542). Therefore, this construct contains the C-terminal sequence of RhoB from T175. pRFP-RhoB was generated by cloning HA-RhoB into the BglII-EcoRI sites of pRFP. For generation of the construct encoding tagRFP-CINCCKVL, tagRFP was amplified by PCR using pTagRFP-C as template and oligonucleotides, forward 5′- CCGCTAGCGCTACCGGTCGCCACCATGG-3′ and reverse, 5′- CCGGATCCCTAGAGGACCTTGCAACAGTTGATACAATTAA GTTTGTGCCCCAGTTTGC-3′. The PCR product was subcloned into the NheI-BamHI sites of the pTagRFP-C plasmid. The cDNA of human RhoGDI  was amplified with oligonucleotides introducing restriction sites for SacI or Eco RI: forward primer 5′- CCGAGCTCAAATGGCTGAGCAGGAGCCC-3′, reverse primer 5′- CCGAATTCTCAGTCCTTCCAGTCCTTCTTGATGG-3′. The PCR product was cloned into the SacI-EcoRI sites of the pEGFP-C1 plasmid. For generation of GFP-RhoGDI-CINCCKVL, the same strategy was used except that the oligonucleotide 5′- CCGAATTCCTAGAGGACCTTGCAACAGTTGATACAGTCCTTCCAGTCCTTCTTGATGG-3′ was used as reverse primer.The generation of pEGFP-C1-HA-RhoB wt and C193S mutant has been described Confluent BAEC or NIH-3T3 fibroblasts or COS7 cells at 80% confluence were transfected with Lipofectamine 2000 (Invitrogen) using 1 µg of construct following the manufacturer instructions.SDS-PAGE and Western blot analysis were performed as described Cells were cultured on glass bottom dishes , transfected with fluorescent constructs, treated with the indicated agents and visualized directly on a confocal microscope . For IF cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% SDS, and incubated with primary antibodies and secondary Alexa-Fluor conjugated antibodies (Invitrogen), counterstained with DAPI (Invitrogen) and mounted with Fluoromount-G . Unless otherwise stated, images shown are single Z-section images for co-localization assessment. All experiments were repeated at least three times and representative results are shown.EM analysis was carried out at the EM Facility of Centro de Biología Molecular Severo Ochoa . For conventional EM analysis cell monolayers were fixed in situ with 2% glutaraldehyde in Sörensen phosphate Na/K buffer (pH 7.4). Fixed cells were processed for embedding in Epoxy, TAAB 812 Resin . Subsequently, cells were subjected to postfixation with 1% osmium tetroxide/0.8% potassium ferrocyanide, incubation with 0.15% tanic acid, incubation with 2% uranyl acetate and dehydration in increasing concentrations of ethanol. Infiltration of the resin was accomplished by increasing concentrations of Epon/ethanol. After polymerization of infiltrated samples, ultrathin sections were stained with saturated uranyl acetate and lead citrate and examined at 80 kV in a Jeol JEM-1010  electron microscope. For immuno-electron microscopy cells were processed by freeze substitution. Cells were fixed in situ with 4% paraformaldehyde/0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). Cells were embedded in 10% gelatine, cryoprotected with 30% glycerol and frozen in liquid nitrogen. Freeze substitution was carried out at −85°C in methanol containing 0.5% uranyl acetate in an Automatic Freeze-Substitution system . Samples were later infiltrated with Lowicryl HM20 and polymerized by UV irradiation at −45°C for two days. Ultra thin Lowicryl sections were obtained in a Reichert-Jung Ultracut E ultramicrotome. Samples were examined as above. For immunogold labelling the ultra thin sections were placed sequentially on top of drops of the different solutions: TBS , 0.2 M ammonium chloride in PBS, blocking buffer (TBS +0.1% BSA and 1% gelatine) and rabbit anti-GFP antibody (Invitrogen) diluted 1∶100 in blocking buffer. After washing, grids were incubated with protein A-15 nm gold conjugate . After washing, grids were air dried and counterstained with 2% aqueous uranyl acetate and 1% lead citrate. Grids were examined as above.Protein half-life was evaluated by time-course experiments in the presence of CHX and Western blot analysis. Stability of the various constructs used was assessed by cell transfection followed by tripsinization to split cells in identical aliquots. After attachment to substrate, cells were treated with CHX and construct levels were estimated by Western blot.The degree of colocalization of the different constructs was analyzed with software from Leica and Scion Corporation for image analysis. Results are presented as average values ± SD or SEM, as indicated. Average values were compared by Student's t-test for unpaired observations.Figure S1Co-localization of RhoB and lysosomal proteases. (a) The distribution of RhoB (red) and cathepsin B  in BAEC treated with vehicle or 10 µM chloroquine for 24 h was assessed by IF, nuclei were stained with DAPI (blue); bar, 20 µm. Graphs represent the fluorescence intensity profile along the lines depicted in the images, showing the points of co-localization. Results are representative of three assays. (b) The percentage of cells showing five or more points of co-localization of RhoB and cathepsin B was calculated by monitoring 40 cells per experimental condition from three different experiments and it is shown as average values ± standard error of mean (SEM).(6.04 MB TIF)Click here for additional data file.Figure S2Effect of autophagy modulation on RhoB levels and degradation. (a) MEFs from wt and Atg5(−/−) mice were incubated with CHX to block new protein synthesis and the levels of endogenous RhoB were assessed at the indicated times by Western blot. (b) BAEC were treated in the absence or presence of 10 mM 3-methyladenine (3-MA) for 24 h and RhoB levels were estimated as above. Dotted lines show sites where lanes from the same gel have been cropped. Results are average values ± SEM of three experiments. (c) BAEC were pretreated with 3-MA for 30 min, after which, CHX was added and levels of RhoB were analyzed at the indicated times. Results are average values ± SEM of three experiments.(2.29 MB TIF)Click here for additional data file.Figure S3Effect of lysosomal inhibitors on the size and pattern of RhoB-positive vesicles. (a) BAEC were transfected with GFP-RhoB as in (1.92 MB TIF)Click here for additional data file.Figure S4Effect of U18666A on RhoB stability. BAEC were treated with 10 µM U18666A for 24 h. Levels of endogenous RhoB and of RhoGDI (as control) were assessed by Western blot at the indicated times after CHX addition. Results shown are average values ± SEM of four experiments.(1.76 MB TIF)Click here for additional data file.Figure S5Co-localization of GFP-RhoB with cholesterol in control or NPC fibroblasts. Control or NPC fibroblasts were transfected with GFP-RhoB. Co-localization with cholesterol was evaluated in fixed cells stained with filipin. Bars, 20 µm. Co-localization is shown as average values ± SEM of determinations from three experiments (*p<0.05 vs control).(3.21 MB TIF)Click here for additional data file.Figure S6Levels of RhoGDI in BAEC detected by IF before and after CHX treatment. BAEC were treated as in (4.04 MB TIF)Click here for additional data file.Figure S7Effect of inhibitors of isoprenylation and palmitoylation on the levels of endogenous RhoB protein or of RhoB constructs in different cell types. (a) NIH-3T3 fibroblasts were transiently transfected with GFP or with GFP-RhoB, and construct levels were assessed by Western blot with an anti-GFP antibody after 24 h treatment with simvastatin (Sv) or 2-bromopalmitate (2-BP). (b) NIH-3T3 fibroblasts or BAEC were treated as in (a) and the levels of the indicated proteins were assessed by Western blot. Dotted lines show sites where lanes from the same gel have been cropped. (c) BAEC were transiently transfected with GFP-RhoB, treated with simvastatin or 2-BP for 3 h and visualized by live confocal microscopy. Note that in simvastatin-treated cells, GFP-RhoB can be detected in the nucleus, which is indicative of the presence of non isoprenylated construct. In contrast, in 2-BP-treated cells the appearance of GFP-RhoB is more diffuse than in control cells but the nucleus is excluded, as expected from an isoprenylated construct. Bars, 20 µm.(3.78 MB TIF)Click here for additional data file.Figure S8Cellular distribution and stability of GFP-RhoGDI and GFP-RhoGDI-8. (a) BAEC were transiently transfected with the indicated constructs as in (6.51 MB TIF)Click here for additional data file.Figure S9Hypothetical model for the degradation pathway of RhoB and -CINCCKVL chimeric proteins. Isoprenylated and palmitoylated RhoB is targeted to intracellular vesicles, mainly Rab7-, Lamp1-positive late endosomes, and to the plasma membrane. The association of the ESCRT complexes on the membrane of late endosomes promotes the invagination of the membrane. The ATPase Vps4 promotes the dissociation of the ESCRT proteins which is required for the intraluminal release of the invaginated vesicles. The late endosome-specific lipid LBPA promotes luminal vesicle formation due to its cone-shaped structure and controls endosome cholesterol content. Our results support a hypothesis according to which late-endosome associated RhoB may be delivered to intraluminal vesicles of MVB in a Vps4- and LBPA-dependent fashion, for degradation in acidic compartments. Inhibition of vesicular acidification with chloroquine or of lysosomal proteases with protease inhibitors results in the formation of dilated MVB in which RhoB accumulates. Genetic or pharmacological disruption of late endosome specific lipid dynamics also alters RhoB sorting. The proposed points of regulation of RhoB targeting to the endo-lysosomal pathway are shown in blue boxes. Based on our experimental observations, -CINCCKVL chimeric proteins would follow a similar pathway for rapid protein degradation. Ub, ubiquitin; GFR, growth factor receptor; GF, growth factor.(4.56 MB TIF)Click here for additional data file.


Sixty-one patients had superficial disease and 42 patients had invasive disease. Cancer-specific survival was shorter in those patients with invasive compared with superficial bladder cancer (P<0.001). On univariate analysis, stratified by stage, increased Ki-67 labelling index (P<0.05), increased COX-2 expression (P<0.05), C-reactive protein (P<0.05) and adjuvant therapy (P<0.01) were associated with poorer cancer-specific survival. On multivariate analysis of these significant factors, stratified by stage, only C-reactive protein  and adjuvant therapy  were independently associated with poorer cancer-specific survival. These results would suggest that tumour-based factors such as grade, COX-2 expression or T-lymphocytic infiltration are subordinate to systemic factors such as C-reactive protein in determining survival in patients with transitional cell carcinoma of the urinary bladder.The relationship between the systemic inflammatory response, tumour proliferative activity, T-lymphocytic infiltration, and COX-2 expression and survival was examined in patients with transitional cell carcinoma of the urinary bladder ( Bladder cancer is the fourth most common malignancy in the Western World. In the UK, there are 12 500 new cases each year and 5000 deaths annually . The morIt is now recognised that disease progression is dependent on a complex interaction of the tumour and the host inflammatory response . RecentlThe basis of the independent relationship between an elevated C-reactive protein concentration and poor survival in cancer is not clear. There are a number of possible explanations. Firstly, that an elevated C-reactive protein identifies tumours capable of producing significant amounts of proinflammatory cytokines, in particular interleukin-6 , tumour proliferative activity (Ki-67), T-lymphocyte  infiltration, and COX-2 expression and cancer-specific survival in patients with transitional cell carcinoma of the bladder.A cross-sectional retrospective study of patients with biopsy-proven transitional cell carcinoma and with a measurement of C-reactive protein before transurethral resection of bladder tumour in Glasgow Royal Infirmary between 1992 and 2001 was carried out. Tumours were grouped according to whether they were superficial  or muscle invasive (pT2–pT4). However, patients with pT1G3 were considered as muscle invasive tumours as they are recognised to have a significantly higher progression rate . At this−1. The coefficient of variation, over the range of measurement, was less than 5% as established by routine quality control procedures. C-reactive protein measurement of greater than 10 mg l−1 was considered to indicate the presence of systemic inflammatory response (Routine laboratory measurement of patient's serum for C-reactive protein concentration was performed. The limit of detection of the assay was a C-reactive protein concentration lower than 5 mg lThe Research Ethics Committee of North Glasgow NHS Trust approved the study.μm) were cut and mounted on slides coated with aminopropyltriethoxysilane for the immuno-histochemistry of Ki-67, CD4+ and CD8+ T-lymphocytes and COX-2 expression.Blocks from the primary tumour were fixed in 10% buffered formalin in saline and embedded in paraffin wax. One representative block of tumour was selected for each patient. Serial individual sections  as previously described  as described previously  as previously described . Cases were counted by two observers and the highest score was chosen as the corresponding index.Quantitative analysis of the lymphoid infiltrate was performed using point counting  with a rSemiquantitative analysis of the COX-2 expression was scored using a weighted histoscore method (The observers (MH and RC) were blinded to the clinical outcome of the patient.χ2) test for trend and Spearman's rank correlation as appropriate.Data are presented as median and range. For the purpose of analysis, the tumour Ki-67 labelling index, CD4+ and CD8+ T-lymphocyte counts and COX-2 expression were grouped by tertiles. The relationships between these and other variables were analyzed using the Mantel–Haenszel .Survival analysis was performed using the Cox proportional hazard model. Multivariate survival analysis was performed using stepwise backward procedure to derive a final model of the variables that had a significant independent relationship with survival. To remove a variable from the model, the corresponding n=103) that were grouped according to stage are shown in the P<0.01) and had higher grade tumours (P<0.001), increased Ki-67 labelling index (P<0.001), increased tumour expression of COX-2 (P<0.001) compared with superficial disease. Also, patients with invasive disease had lower tumour infiltration of CD4+ (P<0.05) and CD8+ T-lymphocytes (P<0.01) and had evidence of a systemic inflammatory response (P<0.05). Patients with invasive bladder cancer received more additional therapy (P<0.05). In total, 22 patients had additional therapy ). Cancer-specific survival was shorter in those patients with invasive bladder cancer compared with superficial bladder cancer (P<0.001).The characteristics of patients with bladder cancer (P<0.05), increased COX-2 expression (P<0.05), C-reactive protein (P<0.05) and no adjuvant therapy (P<0.01) were associated with poorer cancer-specific survival  and no adjuvant therapy  was independently associated with poorer cancer-specific survival. When C-reactive protein was excluded from the multivariate analysis, stratified by stage, only Ki-67 labelling index  and adjuvant therapy  were independently associated with poorer cancer-specific survival.On multivariate analysis of these significant factors, stratified by stage, only C-reactive protein , COX-2 expression (P<0.001), CD4+ (P<0.01) and CD8+ (P<0.001) T-lymphocytes, but not with C-reactive protein (P=0.152). The Ki-67 labelling index was directly associated with COX-2 expression (P<0.001), CD4+ (P<0.001) and CD8+ (P<0.01) T-lymphocytes and, and also with C-reactive protein (P=0.039). The tumour COX-2 expression was directly associated with the tumour CD4+ (P<0.01) and CD8+ (P<0.05) T-lymphocytic infiltrate and weakly with C-reactive protein (P=0.068). The tumour CD4+ T-lymphocytic infiltrate was directly associated with CD8+ (P<0.001) but not with C-reactive protein (P=0.556). The tumour CD8+ T-lymphocytic infiltrate was not associated with C-reactive protein (P=0.892).The inter-relationships between the clinicopathological characteristics are shown in In the present study, tumour grade and Ki-67 labelling index were associated with increased tumour COX-2 expression and infiltration by CD4+ and CD8+ T-lymphocytes in patients with transitional cell carcinoma of the bladder. However, on univariate analysis, only increased Ki-67 labelling index and COX-2 expression were significantly associated with poorer cancer-specific survival. However, neither of these tumour-based factors was independently significant when a marker of the systemic inflammatory response (C-reactive protein) was included in the survival analysis. Therefore, the present study examines, for the first time, the relationship between the preoperative systemic inflammatory response and tumour-based factors and suggests that the systemic inflammatory response is more closely related to outcome in patients with transitional cell carcinoma of the bladder.These results are consistent with the superior prognostic value of C-reactive protein compared with tumour T-lymphocytic infiltration in patients with primary operable colorectal cancer  and its −1) are rare (<5%) in the general elderly population in the West of Scotland  and systemic (C-reactive protein) markers of the inflammatory response were associated with poor cancer-specific survival. However, on multivariate analysis only C-reactive protein had independent prognostic value in patients with transitional cell carcinoma of the urinary bladder.