The high number of the differentially expressed proteins involved in metabolic processes hints that the cell response to protein misfolding stress is accompanied by active metabolic changes. The major metabolic pathways altered are illustrated in Figure 6. Following TTR-L55P expression, we detected an up- regulation of several glycolytic enzymes (Figures 5A and 6), pointing to an increase in glucose catabolism. In addition, the two enzymes that catalyse ethanol formation from pyruvate (PDC1, pyruvate decarboxylase 1 and ADH1, alcohol dehydrogenase) are down-regulated (Figures 5A and 6). This could reflect a shift in the pyruvate fate, from alcoholic fermentation to the TCA cycle and oxidative phosphorylation. Indeed, the TCA cycle enzymes citrate synthase (CISY1) aconitate hydratase (ACON) and isocitrate dehydrogenase (IDHP), which are responsible for the synthesis of a-ketoglutarate from acetyl-CoA, are up-regulated in BTTR-L55P in compassion to control cells. Consistently, an increased Figure 2. 2D-DIGE differential proteinexpression analysis. (A) Representative 2D-DIGE gel image showing the spot map corresponding to the internal standard (Cy2 labeling), which is common to all gels analyzed. Sample preparation and labeling were performed as described in the material and methods section. Indicated spots showed a statistically significant variation of spot volume with 95% confidence level (p,0.05) and a minimal fold variation of 1.3. These spots were removed for subsequent protein identification by mass spectrometry (see Table 1 for code assignment). (B) Example of one protein present in higher abundance only in BTTR-L55P (Ubiquitin-like protein SMT3, spot 1461), one protein with an increase abundance in BTTR-wt and BTTR-L55P (FK506-binding protein, spot 1698) and one protein present in lower abundance both in BTTR-wt and BTTR- L55P (60 S acidic ribosomal protein P0, spot 1643). The spots of interested are encircled and the graphs represent the normalized spot volume. The spot volumes are an average of the 4 biological replicates used for each experimental group. (C) PCA of the 2D-DIGE results. Each data point in the PCA plot represents the global expression values for all spots with a significant ANOVA value (p,0.05). The PCA clustered the 6 individual Cy3 and Cy5-labeled 2D-gel images into three discreet groups differentiated by two principle components PC1 and PC2, explaining a cumulative 82% of all the variation. A separation between the control (carrying the empty plasmid), cells expressing the non-amyloidogenic TTR-wt (BTTR-wt) and the amyloidogenic variant TTR-L55P (BTTR-L55P) is clearly observed. Replicate samples were encircled manually for illustration. (D) 2D-DIGE image analysis of the protein spot identified as human TTR. Figure shows the spot expression map and three-dimensional spot image for each experimental group.
Both AP-2 and KCDT15 may have a role in obesity and diabetes. Experimental evidence is available for the regulatory role of AP-2 in the expression of adipocyte differentiation and insulin signal transduction genes [51,52,53]. AP-2 acts as a repressor in this context, and while SUMOylation is involved in AP-2 repressor activity  it has not been studied in this context. As to KCTD15, genome wide association studies (GWAS) have indicated a connection to obesity . A recent review  suggests that KCTD15 affects obesity through regulation of AP-2, mediated by SUMOylation. The authors propose that UBC9, the SUMO conjugating enzyme, SUMOylates KCTD15, and KCTD15-SUMO acts as connector between UBC9 and AP-2 to mediate AP-2 SUMOylation. Specifically, SUMOylation of KCTD15 would facilitate SUMO conjugation to lysine 10 (K10) in the AP-2 activation domain to repress AP-2 function [24,55]. Our work raises two questions regarding this model; (1) as shown in this report, SUMOylation of KCTD15 is not required for its inhibition of AP-2 activity. (2) AP-2a has multiple isoforms . The longer isoform, 1a, includes K10, but shorter isoforms such as 1b do not . We showed previously that KCTD15 blocks the activity of AP-2a1a and 1b, as well as AP-2b and AP-2c with high efficiency . These observations appear to argue against the hypothesis of Williams et al., with the proviso that in both cases the context of inhibition was reporter activation and NC formation rather than regulation of obesity-related genes; thus the model could hold in the obesity context. Nevertheless, our observations that KCTD15 inhibition of AP-2 is based on binding to the activation domain with a specific requirement for proline 59 , together with the present finding that SUMOylation is not
The first intron of MPZ shares little sequence conservation between mammals and zebrafish (Fig. 1A); indeed, it is not possible to generate a sequence alignment between mammalian species and zebrafish in this region (data not shown). This observation is surprising given that human MPZ-MCS3 is functional in developing zebrafish. Such a discrepancy has been encountered with other non-coding genomic segments [29,30], and it has been proposed that current algorithms for detecting sequence conser- vation between evolutionarily diverse species are not sufficiently sensitive to identify all functional non-coding sequences . There are two possible explanations for the surprising lack of sequence conservation in light of our finding that human MPZ- MCS3 directs reporter-gene expressionin developing zebrafish. First, there may be functional conservation between the orthologous regions in mammals and zebrafish, with sequence conservation falling below a threshold required for computational detection; it is notable that there are a number of SOX10-binding consensus sequences within this genomic region in zebrafish (data not shown). Second, MPZ-MCS3 may be functional in mammals, but the orthologous region may not be functional in non- mammalian vertebrates (e.g., zebrafish); thus, MPZ-MCS3 might be a mammalian lineage-specific transcriptional regulatory element. Each of these possibilities could be investigated further by analyzing overlapping segments of the first intron of the zebrafish mpz gene for enhancer activity in appropriate cell culture and invivo model systems.
Although the mutation of the Ebox motif leads to a robust decrease of E1a transcript expression (between 70 and 89%), both tissue morphology and GATA4 protein levels appeared surpris- ingly unaffected in homozygous Gata4 EboxKO gonads. This indicates that the Gata4 E1b transcript variant, for which expression levels were either unaffected or slightly increased in homozygous Gata4 EboxKO mutant tissues, is sufficient to ensure appropriate production of a normal and functional GATA4 protein. Such outcome is in total accordance with our prior work, where we showed by polysome analysis that both Gata4 E1a and E1b transcripts are actively translated in mouse testes . This observation also raises the intriguing possibility that, at least under certain circumstances, each transcript might be translated with different efficiency. In this regard, it is interesting to note that, among the several different ways by which translation can be regulated, the secondary structure of the 5 9 UTR is known to have a profound influence on translation efficiency . Indeed, it has been previously shown that loose structures at the 59 end promotes whereas tight structures inhibits translation [29,30]. In support of such a model for translation of the Gata4 transcripts, E1a and E1b mRNAs precisely differ only by the extremity of their 59 UTR and this appears to be enough for generating mRNA variants with different predicted secondary structures . Further investiga- tions will ultimately be required in order to understand this potential difference in transcript translation. For example, it would be particularly interesting to determine if E1b transcripts always exhibit preferential translation over the E1a variant or if this happens only when E1a levels are reduced.
) endA1 nupG] (Invitrogen) used for plasmid propagation was grown at 37 uC in Luria Broth supplemented with 100 mg/ml ampicillin. All plasmids and oligonucleotide primers used in this study are listed in supplementary Table S1 and Table S2, respectively. Target gene amplification was performed using High-Fidelity Taq polymerase (Roche) according to the manufacturer’s instructions. PCR products were subcloned into pCR HII-TOPO (Invitrogen) and checked by DNA sequencing using primers M13for and/or M13rev (59-labeled with infra-red dye 800; MWG). For Dpp65 amplification, template JW4303 and primers 5 9pp65epi+39CMVepi were used. To obtain plasmid pPGK-Dpp65, the Dpp65 fragment was inserted into pPGK via EcoRI/BamHI. The gag-ORF, amplified using pTIL05  as template and primers 59L-A ORF1+39L-A ORF1, was cloned as HindIII/BamHI fragment into vector pPGK to give pG. To obtain expression plasmids pGAG/Dpp65 and pGTXG, PCR reactions were carried out using the template/primer combinations JW4303/ 59CMVepi+39CMVepi and pUG36/59T7Xa-GFP+39GFP, respec- tively. Upon subcloning, both fragments were inserted as SacI/ BamHI fragment into pG. Plasmid yGTXG was constructed by introducing the HindIII/BamHI GTXG fragment from pGTXG into YEp352. The Xa gene (encoding the a-subunit of killer toxin K28) was amplified using vector pM28-SL  as template and primers 5 9SpeXal/39altaaBgl. Subsequently, the 59-terminal Spe I/ Bam HI fragment was integrated into yGTXG [Spe I/Bam HI] to give yGTXaD. GTXaD [Hin dIII/Bam HI] was then inserted into pPGK, and the GTXa fusion was completed by inserting the 39- terminal Bgl II/Bam HI fragment of Xa into the Bam HI digested vector pGTXaD. For amplification of the Ce fragment, template JW4303 and primers 59pp65epi+39CMVepi were used. To obtain plasmid pCe, the Ce fragment was inserted into pPGK via Eco RI/ Bam HI. The multiple protease-deficient S. cerevisiae strain S86c [MATa ura3-2 his3 pra1 prb2 prc1 cps1 L-0 M-0] represents a heat- cured, virus-free derivative of strain S86  that was employed for the invivo assembly of hybrid VLPs as well as for the expression of soluble (naked) Dpp65 in the yeast cell cytosol. If not otherwise stated, cells were cultivated in YPD at 30 uC. Yeast cells were transformed by the lithium acetate method  and transformants were selected on synthetic complete medium lacking uracil (Ura-d/ o). Since in a yeast super-killer ski3 mutant (defective in exosome complex components) translation efficacy of the poly(A) 2 transcript of L-A is more effective and dsRNA copy number is significantly increased [43,44], a Dski3 variant of strain BY4741 [MATa his3D1 leu2D0 met15D0 ura3D0 Dski3] (Euroscarf) was used to prepare natural L-A virions.
Here, in this master project, we instead target the cochlear nucleus of mice using precise stereotaxic coordinate injections, damaging the skull as minimum as possible by carefully drilling small holes, and keeping the cerebellum in place. This will cause minimal additional alteration to the nervous system, and it also allows us to target adult mice directly, thereby avoiding tedious surgeries for exposing the DCN or such as in uterus injections (a procedure where the viral construct is directly injected into the mouse otocyst through the uterus, and which Hernandez et al. (2014) state that 38% of injected animals still failed to express genes of interest). Another advantage of precise injections of adequate amounts of viral vectors (instead of using transgenic ChR2 mice for example that may have a lower expression) is the strong expression of ChR2 we see in membrane and neuritis only in the target area. Furthermore, this work is also the first to use direct insertion of fiber optics, to reach and light stimulate the DCN in mice, compared to other studies requiring profound surgery to access the CN. Our direct, and high precision approach minimizes again the overall damage to the nervous system and makes it possible to use low levels of light (5~8mW/mm 2 ) to
B. burgdorferi survival in the mammalian host requires diverse mechanisms that allow the spirochete to resist and evade the host’s immune responses. However, the genetic components of these important properties of the pathogen have yet to be well defined. Here we demonstrate that spirochetes lacking bbk46 establish an initial infection and are seroreactive but are unable to persist in murine tissues following host antibody production. To our knowledge a similar phenotype has been documented for only two other B. burgdorferi genes, the lp28-1 encoded vls antigenic variation locus [27,29,64–66] and the chromosomally encoded lmp-1(bb0210) gene . Moreover, analogous to the bbk46 mutant, the phenotypes of spirochetes lacking a functional vls locus as well as spirochetes lacking lmp-1 have been shown to be dependent on the host immune response as these mutants demonstrate wild-type survival under immune privileged growth conditions and in immunocompromised mice [27,29,65–67]. Although it is clear that the antigenic switching mechanism conferred by the vls locus is essential for B. burgdorferi persistence in the host [65,66], the precise mechanism of vls-dependent immune evasion remains unknown. Similarly the mechanism of lmp-1-dependent protection of B. burgdorferi against the host’s humoral immune response is unknown . VlsE and Lmp-1 are highly antigenic proteins present on the outer surface of the spirochete [67–69]. The BBK46 open reading frame appears to encode a lipoprotein with a predicted signal sequence for outer surface localization; however, recombinant BBK46 protein produced in E. coli was not found to be seroreactive when analyzed by immunoblot using immune sera collected from mice infected with wild-type B. burgdorferi. Future studies are focused on elucidation of the role of bbk46 in the pathogenesis of B. burgdorferi.
relativamente nova e promissora na aquacultura de países Mediterrânicos, para avaliar três alimentos: uma dieta de referência para larvas, contendo 10 % de um HPP amplamente utilizado e disponível comercialmente (CPSP-90, Sopropêche, France) e duas dietas experimentais, contendo diferentes fracções de HPP, diferindo na distribuição do tamanho dos péptidos, a um nível de inclusão de 20 %. Para além de uma avaliação zootécnica (crescimento, sobrevivência), recorreu-se a uma análise clássica de electroforese bidimensional (2-DE) para separação de proteinas do corpo larvar inteiro, junto com espectrometria de massa por MALDI-ToF-ToF para identificação de proteínas cuja expressão fora alterada pelas dietas experimentais. Nos capítulos 3 e 4, utilizando desta vez larvas de uma das espécies mais importantes na aquacultura Mediterranica, a dourada (Sparus aurata), a abordagem proteómica foi feita recorrendo à técnica altamente sensível 2-D DIGE e focou-se no proteoma da carcaça, em vez de corpo inteiro. Adicionalmente, apenas um tipo (fonte, método de hidrólise) de hidrolisado proteico foi comparado, com diferentes níveis de inclusão no alimento (Capítulo 3) e com diferentes fracções de tamanhos de péptidos (Capítulo 4). De modo a trazer informação complementar ao “screening” obtido pela análise proteómica comparativa e compreender melhor como o crescimento e sobrevivência larvar são afectados pelo azoto alimentar, recorreu-se à análise de excreção larvar de amónia e a um ensaio de marcação radioactiva do alimento vivo, Artemia sp., com hidrolisados proteicos radioactivos para estudar a capacidade digestiva das larvas e o seu metabolismo de azoto. No capítulo 5, a análise por proteómica comparativa foi mais apurada ainda, através da utilização do peixe-zebra (Danio rerio) como organismo modelo (para o qual o genoma se encontra inteiramente sequenciado, o que gera uma muito melhor fiabilidade na identificação proteica) e através da redução da complexidade da amostra proteica estudada (isto é, focando o proteoma sarcoplasmático do músculo esquelético epaxial do tronco). Pretendeu-se desta forma estudar os efeitos do perfil de distribuição dos tamanhos peptídicos dos HPP usados nas dietas no crescimento e proteoma do músculo de peixes juvenís. No Capítulo 6, procurou-se aprofundar o conhecimento sobre os efeitos do conteúdo alimentar em lisina no crescimento, através da análise do proteoma do músculo-esquelético epaxial do tronco de peixe-zebras juvenis.
immediately before cell lysis. Cell extracts were collected, incubated with slow rotation for 15 min at 4uC, and centrifuged for 10 min at 3000 rpm and 4 uC to remove the insoluble fraction. Protein content was measured using a BCA assay kit (Pierce- Thermo, Rockford, IL). For Western blots, 50 mM Bond-Breaker TCEP (Thermo Fisher Scientific, Rockford, IL) was added to cell lysates and 30 m g of each lysates were loaded into each lane. Resolved proteins were transferred to PVDF-P membranes Figure 3. Expression of reporter genes in cerebellar cells transduced with lentiviral vectors under various promoters. Representative confocal images of sagittal cerebellar sections from mice 7–14 days following intracerebellar injection of lentiviral vectors with indicated promoters. Dotted lines demarcate the border between cerebellar cortex layers. In low magnification images (B, D, F, G, and J), the line is drawn between Purkinje and granule layers. In high magnification images (A, C, E, H, and I), two lines are drawn to separate the Purkinje layer from the molecular layer and granule layer. A. Widespread GFP expressionin a cerebellar lobe injected with MND-GFP. B. Single confocal section of MND-GFP transduced cerebellum at higher magnification showing absence of GFP expressionin Purkinje neuron somata (asterisks). C. Low magnification of cerebellum injected with MSCV-GFP. D. High magnification of cerebellum injected with MSCV-GFP demonstrating GFP expressionin small cell bodies in the Purkinje layer with radial processes extending to the pial surface, characteristic of Bergmann glia. E. Venus expressionin a cerebellar lobe injected with UBC-Venus. F, G. High magnification of UBC-Venus infected cerebellum shows venus expressionin multiple small cells in the granule layer (F) and a single Purkinje neuron (G). H, I. GFP expressionin cerebellar lobes of two animals injected with PGK-GFP. Several GFP-expressing Purkinje neurons are visible in H, whereas most GFP-expressing cells in I are in the white matter, with a single GFP-positive Purkinje neuron. J. High magnification view of GFP-positive Purkinje neurons from H. Abbreviations: m = molecular layer; p = Purkinje layer; g = granule layer; wm = white matter. Scale bars, 25 mm (B, D, F, G, J), 100 mm (A, C, E, H, I).
Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understand- ing of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequenc- ing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for proteinexpression speciﬁc to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypep- tide TAP can be overproduced in Xac and puriﬁed from the soluble phase of cell extracts. Our results substantiate the use of our vectors for proteinexpressionin Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium invivo.
Figure 4. Replicon vector based trans-complementation. A–C trans-complementation of a glycoprotein. (A) Trans-complementation of the late glycoprotein gO can be facilitated by oriLyt-induced gene expression. NIH3T3 (striped bars) or gO-ori (plain bars) cell lines have been infected with MCMV-wt (white bars) or MCMVDgO (black bars) at an MOI = 0.05 and centrifugal enhancement. At the indicated time points the number of the infectious virus was quantified in the culture supernatants by standard plaque assay. (B) Immunofluorescence microscopy of infected NIH3T3 or gO- ori cells performed 5 days post infection (MOI = 0.05). Infected cells were stained with the anti-IE antibody CHROMA-101. While MCMVDgO is restricted to focal cell-to-cell spread in NIH3T3 fibroblasts, it spreads like wild type in the trans-complementing cell line gO-ori. (C) PCR analysis of virus progeny produced on gO-ori cell line. Viral DNA was isolated from supernatants of the viral growth curve of (A) from day 5, cleared from residual cells, and analyzed by PCR. The virus polymerase gene, M54 served as a positive control for viral infection. The cellular gene lbr, served as a control for the lack of residual genomic DNA. PCR on the m74 gene, encoding gO, showed the presence of the gene in MCMV-wt and the lack of it in MCMVDgO. No uptake and recombination of gO after passage over gO-ori cells could be detected. D–E Trans-complementation of M50, a protein essential for nuclear export of viral capsids. (D) Detection of the M50HA protein (,35 kDa) in cell lysates of NIH3T3, M50-ori t1 and M50-ori t2. The respective cell lines were infected with MCMV-wt at an MOI of 1 and cell lysates harvested 36 h p.i. (E) Growth of MCMVDM50-cherry on complementing and non- complementing cell line. Supernatant of the reconstitution of MCMVDM50-cherry on M50-ori cl.2.1 was serial diluted and used to infect NIH3T3 or M50-ori cells. The trans-complemented virus MCMVDM50-cherry/M50HA could spread in M50-ori cells, but produced only primary infection in NIH3T3.
S4 Fig. HuR only binds to the Bcl2 ARE-rich sequence after B cell activation. A, B, Analysis of HuR-Bcl2 mRNA interaction in HeLa and HEK293 cells. PAR-CLIP data from Lebedeva et al. (Mol. Cell. 2011 Aug 5;43(3)340–52) and Mukherjee et al. (Mol. Cell. 2011 Aug 5;43(3):327–39) was visualised using the UCSC genome browser and hg18 (A) and hg19 (B) respectively. Bcl2 ARE-rich sequence is indicated by a red box. C, Identification in primary B cells of HuR binding sites across the Bcl2 3’UTR. iCLIP data from three independent iCLIP experiments performed using protein extracts from freshly isolated B cells or LPS-activated B cells were visualised using the UCSC genome browser and mm9 genome annotation. Sum data of the three iCLIP experi- ments per condition are also shown. D, Mapped iCLIP data along the Actb gene is shown as ex- perimental control. E, Validation of HuR-Bcl2 mRNA interaction by RNA immunoprecipitation assays. Total protein extracts from freshly isolated splenic B cells or cells treated with LPS for 48 hours were used for HuR:RNA immunoprecipitation using 2 μg of a mouse IgG 1 against HuR
After an experimental period of 13 days all animals were euthanized by an overdose of Ketamin 10% (3mL/kg) and heart’s blood exsanguination to perform femurs excision. Femurs were submitted to standard histological procedures (decalciﬁcation, dehydration, histological slices - 5µm) in silanized slices to immunohistochemistry assays. Cell culture were tripsinazed by tripsin-EDTA 25% (Sigma Chemical Co., St. Louis, MO, USA) for 10 minutes under 5% of CO 2 atmosphere for cells` liberation. Part of these cells, 10 3 , were ﬁxed in
Altogether, the data from Smith et al. (2001) and Palmer et al. (2011) are consistent with a cell-intrinsic stable component of variation inexpression levels of CD5, resulting in persistent differences between CD5 expression levels of different TCR-transgenic strains (based on Palmer et al., 2011), and among the clones of a polyclonal population (based on Smith et al., 2001). While genetic diversity could explain the stable component in the case of the TCR, the exact same reasoning does not apply to CD5, since it is not known to undergo a process of somatic rearrangement. In order to explain the cell-intrinsic stable component of variation in CD5, two non-exclusive models can be considered, having an intrinsic relationship to those previously discussed in the context of the TCR. First, that there is a physical association between CD5 and TCR, which has indeed been reported by early studies based on co-immunoprecipitation experiments in human and rat T cells (Burgess et al., 1992; Osman et al., 1992; Beyers et al., 1992). This model would imply a positive correlation between the levels of CD5 and TCR in a cell population, since the cells having higher TCR levels would be expected to have higher CD5 levels. However, data from studies reporting TCR and CD5 levels of different T cell populations in various cases are discordant, with some studies showing a negative correlation (Kieper et al., 2004), no clear relationship (Kassiotis et al., 2003), or a positive correlation between the levels of the TCR and CD5 (Palmer et al., 2011; Mandl et al., 2013). This may be related to the partial dependence of CD5 levels on the interaction with self peptide-MHC, since these studies have analyzed this relationship in fresh ex vivo cells. The second model is that the cell- intrinsic CD5 levels describing each clone would be defined during T cell development, as proposed by Azzam et al. (1998). This substantiates the basis for the previously discussed model postulating the definition of average TCR levels based on the strength of signals at this same time, which would be consistent with the view of the ζ chain as the rate-limiting sub-unit for assembly of the TCR (Baniyash, 2004).
Our findings in a large cohort of patients that received ADT, support a role for HIF- 1 in the pathophysiology of castration resistance and the HIF1A +1772 C>T polymorphism as a potential pharmacogenomics predictor of the response to ADT. Concordantly, a recent study demonstrated that HIF1a expression contributed both to metastasis and chemo-resistance of castration resistant prostate cancer . A study comparing HIF1A +1772 C>T genotypes between castration-resistant PCa and non-cancer men showed that the T-allele was overrepresented in the cancer group, although it was not associated with survival . Noteworthy, this report presents data from 196 castration-resistant patients using univariate analysis. Another study observed a somatic rare mutation at the same locus in 1/15 androgen-independent prostate tumours, whereas functional studies demonstrated in androgen-independent prostate cancer cells that the T-allele is associated with increased transcriptional activity and proteinexpression . Therefore, we hypothesise that carrying the T-allele, which stabilises HIF-1 protein and upregulates the HIF1A gene expression, may offer a selective advantage to androgen-independent tumour cells through the upregulation of several genes involved in metastasis, angiogenesis, epithelial-to-mesenchymal transition or in other cancer-associated mechanisms [138,85,233-235]. The SNP in HIF1A at locus +1772 represents a germline variant, suggesting a cumulative impact of higher HIF- 1 expression since birth. However, we hypothesise that HIF1A +1772 functional SNP repercussion when combined with hypoxic environmental events or with other genetic risk factors is triggered to higher extent in response to hypoxia-inductive treatments such as ADT. When confirmed in larger and independent samples, additional therapeutic schemes (such as CYP17A1 inhibitors or chemotherapy) could be offered to carriers of the poor responder TT genotype as alternative to ADT. These patients could also be enrolled in clinical trials with drugs that target HIF-1 function (e.g. tasquinimod and other agents that target HIF-1 or its downstream products) [236-239].
Total protein extracts were prepared from the seeds dried at different water contents and from the seeds subsequently imbibed for 10 days. Three samples of five seeds each were ground in liquid nitrogen using a mortar and pestle. Total protein was extracted according to Gallardo et al (2003), with small modifications. Briefly, after ground, the powder was homogenized on ice with 800 µ L of thiourea, urea lysis buffer containing 7M urea, 2M thiourea, 18mM Trizma HCl, 14mM Trizma base, protease inhibitor cocktail Complete Mini (Roche Diagnostics), 12 units DNAse I, 20 µ L RNAse A (20mg/mL), 0.2% (v/v) Triton X-100, 60mM CHAPS and 17.5mM DTT. After 20 minutes, the tubes were centrifuged at 14,000 rpm for 10 minutes at 4ºC. The supernatant containing the total protein extract was submitted to a second clarifying centrifugation as above and supernatant was stored at -20ºC in aliquots of 100 µ L.
study showed for the first time that hESC-derived RPE cells also express MRP1 at both the mRNA and protein levels. MRP1 mRNA expression clearly peaked in the early stages of differentiation in fusiform-shaped cells, and declined thereafter in fully mature cells with a cobblestone morphology. These results are consistent with those of previous studies [6,26,27], although this is the first study to confirm that MRP1 expression fluctuates depending on the maturation status. The fluctuation inexpression was also observed in an efflux pump functional test with calcein- AM. In the functional test, epithelioid cells had higher activity than cobblestone cells, and undifferentiated hESC had no activity at all. Graff and coworkers  as well as Rao and coworkers  previously reported that MRP1 localizes on the apical side of the BBB. In the present study, the localization of MRP1 changed when hESC-RPE cells matured: in fusiform cells, MRP1 was located primarily subcellularly with only faint expressionin cellular projections in the majority of cells; in early cobblestone MRP1 was located intracellularly near the nucleus; and in mature cells with a cobblestone morphology, MRP1 was located on the apical side of the polarized cells. The change in the amount and localization of MRP1 might also reflect the differences in the MRP1 function in native RPE. The overall intensity in immunofluorescence labeling was very low, even in hESC-RPE
Thirty-four consecutive patients with acromegaly who had previously had pituitary surgery and had tissue available (paraffin block and fresh frozen tumor sample) were included in the study. This study was approved by the Ethics Committees of the Clementino Fraga Filho Uni- versity Hospital/Medical School, Federal University of Rio de Janeiro and the Clinics Hospital, Ribeirão Preto Medical School, São Paulo University. All subjects gave written informed con- sent before study entry. Patients underwent pituitary surgery between 2006 and 2011. Bio- chemical diagnosis of acromegaly was based on international criteria [15, 16]. Exclusion criteria included previous known AIP mutations, a family history of pituitary adenoma, pres- ence of features or family history of Carney complex or multiple endocrine neoplasia type 1 or 4 and preoperative therapy with SSA [as treatment may increase AIP expression ]. Tumor invasiveness was determined according to Knosp-Steiner criteria . GH-secreting pituitary tumor samples were obtained during transsphenoidal surgery: part of the sample was processed for routine histopathological and immunohistochemical studies (including anterior pituitary hormones), and part was snap-frozen and stored at -70°C for molecular biology studies. All samples were micro-dissected by an experienced pathologist in order to separate any non-tu- moral tissue and homogenized using a Polytron homogenizer. In addition, five normal human pituitaries were obtained within 10 hours from the time of death at autopsies of subjects who
The natural history of breast cancer is characterized by het- erogeneity within and between patients, since tumors with similar histopathological diagnosis can follow differ- ent clinical courses and show different responses to ther- apy . These cancers are generally considered to result from the accumulation of multiple clonal changes in genes that regulate cell growth and differentiation [3,4]. One of the mechanisms involved in the carcinogenesis process is loss of tumor suppressor gene function, which normally acts as a negative regulator of cell proliferation. Tumor suppressor gene inactivation contributes to car- cinogenesis by conferring certain advantages to growth that lead to tumor progression. It is generally thought that these genes are recessive, requiring mutation or loss of both alleles for functional inactivation. The loss of heter- ozygosity (LOH) in sporadic cancers reflects somatic dele- tions that involve specific chromosomal regions associated with a tumor suppressor gene located within the deleted region .
It has been well established that the Ndi1 enzyme from yeast can be integrated into the oxidative phosphorylation system in the mitochondria of animals and serves as a functional replacement for complex I. Successful use of Ndi1 was reported not only for rodents [4–7,15,21] but also for other species such as Caenorhabditis elegans , Drosophila  and rabbit . Long term expression of Ndi1 in the host mitochondria was indicative of no major issues with the host defense system against this foreign protein. Our earlier work on the assessment of the inflammatory potential of Ndi1 hinted that there was no positive response in the mouse brain where Ndi1 was present . However, because the brain has an atypical immune system that might explain the absence of detectable immune response, it was important to test using tissues such as skeletal muscles that are abundantly encircled by the circulatory network and therefore have a high interaction with the immune system. In the current study, we were able to demonstrate no inflammatory or immune responses in rat muscles, thereby aiding in establishing a safe and effective protocol for introducing the Ndi1 enzyme in mammals.