Hypermethylation-mediated regulation of CD44 gene expression in human neuroblastoma

The CD44 adhesion receptor is silenced in highly malignant neuroblastomas (NBs) with MYCN amplification. Because its functional expression is associated with decreased tumorigenic properties, CD44 behaves as a tumor suppressor gene in NB and other cancers. Given that the precise mechanisms responsible for CD44 silencing are not elucidated, we investigated whether CD44 expression could be regulated by DNA hypermethylation. The methylation status of CD44 gene promoter and exon 1 regions was analyzed in 12 NB cell lines and 21 clinical samples after bisulfite genomic modification, followed by PCR and single-strand conformation polymorphism analysis and genomic sequencing. The results showed that almost all CD44-negative cell lines displayed hypermethylation in both regions, whereas all CD44-expressing cell lines were unmethylated. These observations correlated with the ability to restore CD44 mRNA and protein expression by treatment of CD44-negative cells with the 5-aza-2'-deoxycytidine demethylating agent. In contrast, no CD44 gene hypermethylation could be detected in 21 NB clinical samples of different stages, irrespective of CD44 expression. Although our results suggest that aberrant methylation of promoter and exon 1 regions is involved in CD44 silencing in NB cell lines, they also indicate that methylation of unidentified regulatory sequences or methylation-independent mechanisms also control the expression of CD44 in primary NB tumors and cell lines. We therefore conclude that CD44 silencing is controlled by complex and tumor cell-specific processes, including gene hypermethylation. Further investigation of other mechanisms and genes involved in CD44 regulation will be needed before demethylation-mediated reactivation of the CD44 gene can be considered as therapeutic strategy for neuroblastoma and perhaps other related cancers.

Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange

The development and optimization of efficient transformation protocols is essential in new citrus breeding programs, not only for rootstock, but also for scion improvement. Transgenic 'Hamlin' sweet orange (Citrus sinensis (L.) Osbeck) plants were obtained by Agrobacterium tumefaciens-mediated transformation of epicotyl segments collected from seedlings germinated in vitro. Factors influencing genetic transformation efficiency were evaluated including seedling incubation conditions, time of inoculation with Agrobacterium and co-culture conditions. Epicotyl segments were adequate explants for transformation, regenerating plants by direct organogenesis. Higher percentage of transformation was obtained with explants collected from seedlings germinated in darkness, transferred to 16 hours photoperiod for 2-3 weeks, and inoculated with Agrobacterium for 15-45 min. The best co-culture condition was the incubation of the explants in darkness, for three days in culture medium supplemented with 100 muM of acetosyringone. Genetic transformation was confirmed by performing beta-glucoronidase (GUS) assays and, subsequently, by PCR amplification for the nptII and GUS genes.

Hog1 bypasses stress-mediated down-regulation of transcription by RNA polymerase II redistribution and chromatin remodeling

Cells are subjected to dramatic changes of gene expression upon environmental changes. Stresscauses a general down-regulation of gene expression together with the induction of a set of stress-responsivegenes. The p38-related stress-activated protein kinase Hog1 is an important regulator of transcription uponosmostress in yeast. Genome-wide localization studies of RNA polymerase II (RNA Pol II) and Hog1 showed that stress induced major changes in RNA Pol II localization, with a shift toward stress-responsive genes relative to housekeeping genes. RNA Pol II relocalization required Hog1, which was also localized to stress-responsive loci. In addition to RNA Pol II-bound genes, Hog1 also localized to RNA polymerase III-bound genes, pointing to a wider role for Hog1 in transcriptional control than initially expected. Interestingly, an increasing association of Hog1 with stressresponsive genes was strongly correlated with chromatin remodeling and increased gene expression. Remarkably, MNase-Seq analysis showed that although chromatin structure was not significantly altered at a genome-wide level in response to stress, there was pronounced chromatin remodeling for those genes that displayed Hog1 association. Hog1 serves to bypass the general down-regulation of gene expression that occurs in response to osmostress, and does so both by targeting RNA Pol II machinery and by inducing chromatin remodeling at stressresponsive loci.

Determination of the Enantiomers of Mianserin and its Metabolites in Plasma by Capillary Electrophoresis After Liquid-Liquid Extraction and On-Column Sample Preconcentration

Capillary electrophoresis has drawn considerable attention in the past few years, particularly in the field of chiral separations because of its high separation efficiency. However, its routine use in therapeutic drug monitoring is hampered by its low sensitivity due to a short optical path. We have developed a capillary zone electrophoresis (CZE) method using 2mM of hydroxypropyl-β-cyclodextrin as a chiral selector, which allows base-to-base separation of the enantiomers of mianserin (MIA), desmethylmianserin (DMIA), and 8-hydroxymianserin (OHMIA). Through the use of an on-column sample concentration step after liquid-liquid extraction from plasma and through the presence of an internal standard, the quantitation limits were found to be 5 ng/mL for each enantiomer of MIA and DMIA and 15 ng/mL for each enantiomer of OHMIA. To our knowledge, this is the first published CE method that allows its use for therapeutic monitoring of antidepressants due to its sensitivity down to the low nanogram range. The variability of the assays, as assessed by the coefficients of variation (CV) measured at two concentrations for each substance, ranged from 2 to 14% for the intraday (eight replicates) and from 5 to 14% for the interday (eight replicates) experiments. The deviations from the theoretical concentrations, which represent the accuracy of the method, were all within 12.5%. A linear response was obtained for all compounds within the range of concentrations used for the calibration curves (10-150 ng/mL for each enantiomer of MIA and DMIA and 20-300 ng/mL for each enantiomer of OHMIA). Good correlations were calculated between [(R) + (S)]-MIA and DMIA concentrations measured in plasma samples of 20 patients by a nonchiral gas chromatography method and CZE, and between the (R)- and (S)-concentrations of MIA and DMIA measured in plasma samples of 37 patients by a previously described chiral high-performance liquid chromatography method and CZE. Finally, no interference was noted from more than 20 other psychotropic drugs. Thus, this method, which is both sensitive and selective, can be routinely used for therapeutic monitoring of the enantiomers of MIA and its metabolites. It could be very useful due to the demonstrated interindividual variability of the stereoselective metabolism of MIA.

Temozolomide-mediated radiation enhancement in glioblastoma: a report on underlying mechanisms.

PURPOSE: In this study, we investigated the mechanisms by which temozolomide enhances radiation response in glioblastoma cells. EXPERIMENTAL DESIGN: Using a panel of four primary human glioblastoma cell lines with heterogeneous O(6)-methylguanine-DNA methyltransferase (MGMT) protein expression, normal human astrocytes, and U87 xenografts, we investigated (a) the relationship of MGMT status with efficacy of temozolomide-based chemoradiation using a panel of in vitro and in vivo assays; (b) underlying mechanisms by which temozolomide enhances radiation effect in glioblastoma cells; and (c) strategies to overcome resistance to radiation + temozolomide. RESULTS: Temozolomide enhances radiation response most effectively in glioblastomas without detectable MGMT expression. On concurrent radiation + temozolomide administration in MGMT-negative glioblastomas, there seems to be decreased double-strand DNA (dsDNA) repair capacity and enhanced dsDNA damage compared either with radiation alone or with sequentially administered temozolomide. Our data suggest that O(6)-benzylguanine can enhance the antitumor effects of concurrent radiation + temozolomide in MGMT-positive cells by enhancing apoptosis and the degree of dsDNA damage. O(6)-Benzylguanine was most effective when administered concurrently with radiation + temozolomide and had less of an effect when administered with temozolomide in the absence of radiation or when administered sequentially with radiation. Our in vivo data using U87 xenografts confirmed our in vitro findings. CONCLUSIONS: The present study shows that temozolomide enhances radiation response most effectively in MGMT-negative glioblastomas by increasing the degree of radiation-induced double-strand DNA damage. In MGMT-positive glioblastomas, depletion of MGMT by the addition of O(6)-benzylguanine significantly enhances the antitumor effect of concurrent radiation + temozolomide. These are among the first data showing mechanisms of synergy between radiation and temozolomide and the effect of MGMT.

Fluorodeoxyuridine mediated cell cycle synchronization in S-phase increases the Auger radiation cell killing with 125I-iododeoxyuridine.

Aim: 125I-iododeoxyuridine is a potential Auger radiation therapy agent. Its incorporation in DNA of proliferating cells is enhanced by fluorodeoxyuridine. Here, we evaluated therapeutic activities of 125I-iododeoxyuridine in an optimized fluorodeoxyuridine pre-treatment inducing S-phase synchronization. Methods: After S-phase synchronization by fluorodeoxyuridine, cells were treated with 125I-iododeoxyuridine. Apoptosis analysis and S-phase synchronization were studied by flow cytometry. Cell survival was determined by colony-forming assay. Based on measured growth parameters, the number of decays per cell that induced killing was extrapolated. Results: Treatment experiments showed that 72 to 91% of synchronized cells were killed after 0.8 and 8 kBq/ml 125I-iododeoxyuridine incubation, respectively. In controls, only 8 to 38% of cells were killed by corresponding 125I-iododeoxyuridine activities alone and even increasing the activity to 80 kBq/ml gave only 42 % killing. Duplicated treatment cycles or repeated fluorodeoxyuridine pre-treatment allowed enhancing cell killing to >95 % at 8 kBq/ml 125I-iododeoxyuridine. About 50 and 160 decays per S-phase cells in controls and S-phase synchronization, respectively, were responsible for the observed cell killing at 0.8 kBq/ml radio-iododeoxyuridine. Conclusion: These data show the successful application of fluorodeoxyuridine that provided increased 125I-iododeoxyuridine Auger radiation cell killing efficacy through S-phase synchronization and high DNA incorporation of radio-iododeoxyuridine.

Polyethylene glycol and polyvinylpirrolidone effect on bacterial rRNA extraction and hybridization from cells exposed to tannins

In order to detect fluctuations in ruminal microbial populations due to forage tannins using 16S ribosomal RNA (rRNA) probes, recovery of intact rRNA is required. The objective of this work was to evaluate the effect of polyethylene glycol (PEG) and polyvinylpirrolidone (PVP) on extraction of bacterial rRNA, in the presence of tannins from tropical legume forages and other sources, that hybridize with oligonucleotide probes. Ruminococcus albus 8 cells were exposed to 8 g/L tannic acid or 1 g/L condensed tannins extracted from Acacia angustissima, banana (Musa sp.) skin, Desmodium ovalifolium, red grape (Vitis vinifera) skin and Inga edulis, or no tannins. Cells were rinsed with Tris buffer pH 7 containing either 8% PEG or 6% PVP prior to cell lysis. Total RNA samples rinsed with either PEG or PVP migrated through denaturing agarose gels. The 16S rRNA bands successfully hybridized with a R. albus species-specific oligonucleotide probe, regardless of tannin source. The effect of rinsing buffers on the density of 16S rRNA bands, as well as on the hybridization signals was compared. There were significant effects (P<0.01) when the controls were compared to either buffer treatments due to tannin type, buffer used and the interaction of tannin type and buffer. The significant interaction indicates the influence of tannin type on the parameters evaluated.

Light-regulated interactions with SPA proteins underlie cryptochrome-mediated gene expression.

Cryptochromes are a class of photosensory receptors that control important processes in animals and plants primarily by regulating gene expression. How photon absorption by cryptochromes leads to changes in gene expression has remained largely elusive. Three recent studies, including Lian and colleagues (pp. 1023-1028) and Liu and colleagues (pp. 1029-1034) in this issue of Genes & Development, demonstrate that the interaction of light-activated Arabidopsis cryptochromes with a class of regulatory components of E3 ubiquitin ligase complexes leads to environmentally controlled abundance of transcriptional regulators.

Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.

Time-Dependent Specificity of Immunopathologic (C4d-CD68) and Histologic Criteria of Antibody-Mediated Rejection for Donor-Specific Antibodies and Allograft Dysfunction in Heart Transplantation.

BACKGROUND: In heart transplantation, antibody-mediated rejection (AMR) is diagnosed and graded on the basis of immunopathologic (C4d-CD68) and histopathologic criteria found on endomyocardial biopsies (EMB). Because some pathologic AMR (pAMR) grades may be associated with clinical AMR, and because humoral responses may be affected by the intensity of immunosuppression during the first posttransplantation year, we investigated the incidence and positive predictive values (PPV) of C4d-CD68 and pAMR grades for clinical AMR as a function of time. METHODS: All 564 EMB from 40 adult heart recipients were graded for pAMR during the first posttransplantation year. Clinical AMR was diagnosed by simultaneous occurrence of pAMR on EMB, donor specific antibodies and allograft dysfunction. RESULTS: One patient demonstrated clinical AMR at postoperative day 7 and one at 6 months (1-year incidence 5%). C4d-CD68 was found on 4,7% EMB with a "decrescendo" pattern over time (7% during the first 4 months vs. 1.2% during the last 8 months; P < 0.05). Histopathologic criteria of AMR occurred on 10.3% EMB with no particular time pattern. Only the infrequent (1.4%) pAMR2 grade (simultaneous histopathologic and immunopathologic markers) was predictive for clinical AMR, particularly after the initial postoperative period (first 4 months and last 8 months PPV = 33%-100%; P < 0.05). CONCLUSION: In the first posttransplantation year, AMR immunopathologic and histopathologic markers were relatively frequent, but only their simultaneous occurrence (pAMR2) was predictive of clinical AMR. Furthermore, posttransplantation time may modulate the occurrence of C4d-CD68 on EMB and thus the incidence of pAMR2 and its relevance to the diagnosis of clinical AMR.

Cell-mediated immunity to predict cytomegalovirus disease in high-risk solid organ transplant recipients.

Late-onset cytomegalovirus (CMV) disease commonly occurs after discontinuation of antiviral prophylaxis. We determined the utility of testing CD8+ T-cell response against CMV as a predictor of late-onset CMV disease after a standard course of antiviral prophylaxis. Transplant patients at high-risk for CMV disease were enrolled. CD8+ T-cell-mediated immunity (CMI) was tested using the QuantiFERON-CMV assay at baseline, 1, 2 and 3 months posttransplant by measurement of interferon-gamma response to whole blood stimulation with a 21-peptide pool. The primary outcome was the ability of CMI testing to predict CMV disease in the first 6 months posttransplant. There were 108 evaluable patients (D+/R+ n = 39; D-/R+ n = 34; D+/R- n = 35) of whom 18 (16.7%) developed symptomatic CMV disease. At the end of prophylaxis, CMI was detectable in 38/108 (35.2%) patients (cutoff 0.1 IU/mL interferon-gamma). CMV disease occurred in 2/38 (5.3%) patients with a detectable interferon-gamma response versus 16/70 (22.9%) patients with a negative response; p = 0.038. In the subgroup of D+/R- patients, CMV disease occurred in 1/10 (10.0%) patients with a detectable interferon-gamma response (cutoff 0.1 IU/mL) versus 10/25 (40.0%) patients with a negative CMI, p = 0.12. Monitoring of CMI may be useful for predicting late-onset CMV disease.

Bump time-frequency toolbox: a toolbox for time-frequency oscillatory bursts extraction in electrophysiological signals

Background: oscillatory activity, which can be separated in background and oscillatory burst pattern activities, is supposed to be representative of local synchronies of neural assemblies. Oscillatory burst events should consequently play a specific functional role, distinct from background EEG activity – especially for cognitive tasks (e.g. working memory tasks), binding mechanisms and perceptual dynamics (e.g. visual binding), or in clinical contexts (e.g. effects of brain disorders). However extracting oscillatory events in single trials, with a reliable and consistent method, is not a simple task. Results: in this work we propose a user-friendly stand-alone toolbox, which models in a reasonable time a bump time-frequency model from the wavelet representations of a set of signals. The software is provided with a Matlab toolbox which can compute wavelet representations before calling automatically the stand-alone application. Conclusion: The tool is publicly available as a freeware at the address: http:// www.bsp.brain.riken.jp/bumptoolbox/toolbox_home.html

IL-12-mediated STAT4 signaling and TCR signal strength cooperate in the induction of CD40L in human and mouse CD8(+) T cells.

CD40L is one of the key molecules bridging the activation of specific T cells and the maturation of professional and nonprofessional antigen-presenting cells including B cells. CD4(+) T cells have been regarded as the major T-cell subset that expresses CD40L upon cognate activation; however, we demonstrate here that a putative CD8(+) helper T-cell subset expressing CD40L is induced in human and murine CD8(+) T cells in vitro and in mice immunized with antigen-pulsed dendritic cells. IL-12 and STAT4-mediated signaling was the major instructive cytokine signal boosting the ability of CD8(+) T cells to express CD40L both in vitro and in vivo. Additionally, TCR signaling strength modulated CD40L expression in CD8(+) T cells after primary differentiation in vitro as well as in vivo. The induction of CD40L in CD8(+) T cells regulated by IL-12 and TCR signaling may enable CD8(+) T cells to respond autonomously of CD4(+) T cells. Thus, we propose that under proinflammatory conditions, a self-sustaining positive feedback loop could facilitate the efficient priming of T cells stimulated by high affinity peptide displaying APCs.