FBXO25-associated nuclear domains: A novel subnuclear structure

Skp1, Cul1, Rbx1, and the FBXO25 protein form a functional ubiquitin ligase complex. Here, we investigate the cellular distribution of FBXO25 and its colocalization with some nuclear proteins by using immunochemical and biochemical approaches. FBXO25 was monitored with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of the FBXO25 sequence. FBXO25 protein was expressed in all mouse tissues tested except striated muscle, as indicated by immunoblot analysis. Confocal analysis revealed that the endogenous FBXO25 was partially concentrated in a novel dot-like nuclear domain that is distinct from clastosomes and other well-characterized structures. These nuclear compartments contain a high concentration of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1. We propose to name these compartments FBXO25-associated nuclear domains. Interestingly, inhibition of transcription by actinomycin D or heat-shock treatment drastically affected the nuclear organization of FBXO25-containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity of the cell. Also, we present evidences that an FBXO25-dependent ubiquitin ligase activity prevents aggregation of recombinant polyglutamine-containing huntingtin protein in the nucleus of human embryonic kidney 293 cells, suggesting that this protein can be a target for the nuclear FBXO25 mediated ubiquitination.

A score system for quality evaluation of RNA sequence tags: an improvement for gene expression profiling

Background: High-throughput molecular approaches for gene expression profiling, such as Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature Sequencing (MPSS) or Sequencing-by-Synthesis (SBS) represent powerful techniques that provide global transcription profiles of different cell types through sequencing of short fragments of transcripts, denominated sequence tags. These techniques have improved our understanding about the relationships between these expression profiles and cellular phenotypes. Despite this, more reliable datasets are still necessary. In this work, we present a web-based tool named S3T: Score System for Sequence Tags, to index sequenced tags in accordance with their reliability. This is made through a series of evaluations based on a defined rule set. S3T allows the identification/selection of tags, considered more reliable for further gene expression analysis. Results: This methodology was applied to a public SAGE dataset. In order to compare data before and after filtering, a hierarchical clustering analysis was performed in samples from the same type of tissue, in distinct biological conditions, using these two datasets. Our results provide evidences suggesting that it is possible to find more congruous clusters after using S3T scoring system. Conclusion: These results substantiate the proposed application to generate more reliable data. This is a significant contribution for determination of global gene expression profiles. The library analysis with S3T is freely available at http://gdm.fmrp.usp.br/s3t/.S3T source code and datasets can also be downloaded from the aforementioned website.

The CEBPA gene is down-regulated in acute promyelocytic leukemia and its upstream promoter, but not the core promoter, is highly methylated

impairment of CCAAT Enhancer Binding Protein alpha (CEBPA) function is a common finding in acute myeloid leukemia; nevertheless, its relevance for acute promyelocytic leukemia pathogenesis is unclear. We analyzed the expression and assessed the methylation status of the core and upstream promoters of CEBPA in acute promyelocytic leukemia at diagnosis. Patients with acute promyelocytic leukemia (n=18) presented lower levels of CEBPA expression compared to healthy controls (n=5), but higher levels than those in acute myeloid leukemia with t(8;21) (n=9) and with inv(16) (n=5). Regarding the core promoter, we detected no methylation in 39 acute promyelocytic leukemia samples or in 8 samples from controls. In contrast, analysis of the upstream promoter showed methylation in 37 of 39 samples, with 17 patients showing methylation levels over 30%. Our results corroborate data obtained in animal models showing that CEBPA is down-regulated in acute promyelocytic leukemia stem cells and suggest that epigenetic mechanisms may be involved.

Global gene expression profile in myelodysplastic syndromes using SAGE

The molecular pathogenesis of myelodysplastic syndromes (MDS) is poorly understood. In order to expand our knowledge of genetic defects in MDS, we determined the overall profile of genes expressed in bone marrow from patients with refractory anemia with excess blasts ( RAEB) by serial analysis of gene expression ( SAGE). The present report describes a partial transcriptome of RAEB bone marrow derived from 56,694 sequenced tags that provides information about expressed gene products. This is the first attempt to determine an overall profile of gene expression specifically in RAEB at diagnosis using SAGE, which should be useful in the understanding of the physiopathology of MDS and in identifying the genes involved.

Polymorphisms and DNA methylation of gene TP53 associated with extra-axial brain tumors

The p53 tumor suppressor gene is the most frequently mutated gene in human cancer; this gene is mutated in up to 50% of human tumors. It has a critical role in the cell cycle, apoptosis and cell senescence, and it participates in many crucial physiological and pathological processes. Polymorphisms of p53 have been suggested to be associated with genetically determined susceptibility in various types of cancer. Another process involved with the development and progression of tumors is DNA hypermethylation. Aberrant methylation of the promoter is an alternative epigenetic change in genetic mechanisms, leading to tumor suppressor gene inactivation. In the present study, we examined the TP53 Arg72Pro and Pro47Ser polymorphisms using PCR-RFLP and the pattern of methylation of the p53 gene by methylation-specific PCR in 90 extra-axial brain tumor samples. Patients who had the allele Pro of the TP53 Arg72Pro polymorphism had an increased risk of tumor development ( odds ratio, OR = 3.23; confidence interval at 95%, 95% CI = 1.71-6.08; P = 0.003), as did the allele Ser of TP53 Pro47Ser polymorphism (OR = 1.28; 95% CI = 0.03-2.10; P = 0.01). Comparison of overall survival of patients did not show significant differences. In the analysis of DNA methylation, we observed that 37.5% of meningiomas, 30% of schwannomas and 52.6% of metastases were hypermethylated, suggesting that methylation is important for tumor progression. We suggest that TP53 Pro47Ser and Arg72Pro polymorphisms and DNA hypermethylation are involved in susceptibility for developing extra-axial brain tumors.

Differential expression of E-cadherin gene in human neuroepithelial tumors

Cadherins are cell-to-cell adhesion molecules that play an important role in the establishment of adherent-type junctions by mediating calcium-dependent cellular interactions. The CDH1 gene encodes the transmembrane glycoprotein E-cadherin which is important in maintaining homophilic cell-cell adhesion in epithelial tissues. E-cadherin interacts with catenin proteins to maintain tissue architecture. Structural defects or loss of expression of E-cadherin have been reported as a common feature in several human cancer types. This study aimed to evaluate the expression of E-cadherin and their correlation with clinical features in microdissected brain tumor samples from 81 patients, divided into 62 astrocytic tumors grades I to IV and 19 medulloblastomas, and from 5 white matter non-neoplasic brain tissue samples. E-cadherin (CDH1) gene expression was analyzed by quantitative real-time polymerase chain reaction. Mann-Whitney, Kruskal-Wallis, Kaplan-Meir, and log-rank tests were performed for statistical analyses. We observed a decrease in expression among pathological grades of neuroepithelial tumors. Non-neoplasic brain tissue showed a higher expression level of CDH1 gene than did neuroepithelial tumors. Expression of E-cadherin gene was higher in astrocytic than embryonal tumors (P = 0.0168). Low-grade malignancy astrocytomas (grades I-II) showed higher CDH1 expression than did high-grade malignancy astrocytomas (grades III-IV) and medulloblastomas (P < 0.0001). Non-neoplasic brain tissue showed a higher expression level of CDH1 gene than grade I malignancy astrocytomas, considered as benign tumors (P = 0.0473). These results suggest that a decrease in E-cadherin gene expression level in high-grade neuroepithelial tumors may be a hallmark of malignancy in dedifferentiated tumors and that it may be possibly correlated with their progression and dissemination.

Observations on Antricola ticks: Small nymphs feed on mammalian hosts and have a salivary gland structure similar to ixodid ticks

Ticks use bloodmeals as a Source of nutrients and energy to molt and survive until the next meal and to oviposit, in the case of females. However, only the larvae of some tick species are known to feed upon bats females are obligatorily autogenous, and nymphal stages are believed to not feed. We investigated the presence of blood ill a natural population of nymphal Antricola delacruzi ticks collected from bat guano; their ability to feed upon laboratory hosts: and the microscopic structure of both salivary glands and gut. DNA amplification of gut contents of freshly collected material was positive for a mammal in 4 of 11 first instar nymphs, but we were unsuccesful in the amplification of host bloodmeal DNA from late instar nymphs. All early nymphal stages (n = 10) fed oil rabbits. and host DNA was detected and sequenced from gut contents. However, all the large nymphs (n = 10) rejected feeding, and host DNA remained undetected in these ticks. All stages of A. delacruzi have salivary glands similar in morphology to the ixodid agranular Type I salivary gland acini and to granular Type II or Type B acini. All stages of A. delacruzi had a similar gut structure. consisting of digestive cells in the basal portion that contained hematin granules. Neither regenerative nor secretory cell traces were observed in the sections Of gut.

PSA and androgen-related gene (AR, CYP17, and CYP19) polymorphisms and the risk of adenocarcinoma at prostate biopsy

The aim of the present study was to examine the impact of polymorphisms in prostate-specific antigen (PSA) and androgen-related genes (AR, CYP17, and CYP19) on prostate cancer (PCa) risk in selected high-risk patients who underwent prostate biopsy. Blood samples and prostate tissues were obtained for DNA analysis. Single-nucleotide polymorphisms in the 50-untranslated regions (UTRs) of the PSA (substitution A > G at position -158) and CYP17 (substitution T > C at 50-UTR) genes were detected by polymerase chain reaction (PCR)-restriction fragment length polymorphism assays. The CAG and TTTA repeats in the AR and CYP19 genes, respectively, were genotyped by PCR-based GeneScan analysis. Patients with the GG genotype of the PSA gene had a higher risk of PCa than those with the AG or AA genotype (OR = 3.79, p = 0.00138). The AA genotype was associated with lower PSA levels (6.44 +/- 1.64 ng/mL) compared with genotypes having at least one G allele (10.44 +/- 10.06 ng/mL) (p = 0.0687, 95% CI - 0.3146 to 8.315, unpaired t-test). The multivariate analysis confirmed the association between PSA levels and PSA genotypes (AA vs. AG+GG; chi(2) = 0.0482) and CYP19 (short alleles homozygous vs. at least one long allele; chi(2) = 0.0110) genotypes. Genetic instability at the AR locus leading to somatic mosaicism was detected in one PCa patient by comparing the length of AR CAG repeats in matched peripheral blood and prostate biopsy cores. Taken together, these findings suggest that the PSA genotype should be a clinically relevant biomarker to predict the PCa risk.

Lineage divergence detected in the malaria vector Anopheles marajoara (Diptera: Culicidae) in Amazonian Brazil

Background: Cryptic species complexes are common among anophelines. Previous phylogenetic analysis based on the complete mtDNA COI gene sequences detected paraphyly in the Neotropical malaria vector Anopheles marajoara. The ""Folmer region"" detects a single taxon using a 3% divergence threshold. Methods: To test the paraphyletic hypothesis and examine the utility of the Folmer region, genealogical trees based on a concatenated (white + 3' COI sequences) dataset and pairwise differentiation of COI fragments were examined. The population structure and demographic history were based on partial COI sequences for 294 individuals from 14 localities in Amazonian Brazil. 109 individuals from 12 localities were sequenced for the nDNA white gene, and 57 individuals from 11 localities were sequenced for the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2). Results: Distinct A. marajoara lineages were detected by combined genealogical analysis and were also supported among COI haplotypes using a median joining network and AMOVA, with time since divergence during the Pleistocene (< 100,000 ya). COI sequences at the 3' end were more variable, demonstrating significant pairwise differentiation (3.82%) compared to the more moderate 2.92% detected by the Folmer region. Lineage 1 was present in all localities, whereas lineage 2 was restricted mainly to the west. Mismatch distributions for both lineages were bimodal, likely due to multiple colonization events and spatial expansion (similar to 798 - 81,045 ya). There appears to be gene flow within, not between lineages, and a partial barrier was detected near Rio Jari in Amapa state, separating western and eastern populations. In contrast, both nDNA data sets (white gene sequences with or without the retention of the 4th intron, and ITS2 sequences and length) detected a single A. marajoara lineage. Conclusions: Strong support for combined data with significant differentiation detected in the COI and absent in the nDNA suggest that the divergence is recent, and detectable only by the faster evolving mtDNA. A within subgenus threshold of >2% may be more appropriate among sister taxa in cryptic anopheline complexes than the standard 3%. Differences in demographic history and climatic changes may have contributed to mtDNA lineage divergence in A. marajoara.

Sequential analysis of global gene expression profiles in immature and in vitro matured bovine oocytes: potential molecular markers of oocyte maturation

Background: Without intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis. Results: 8489 transcripts were detected across the two oocyte groups, of which similar to 25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of a-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation. Conclusion: Using sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology.

Gene silencing during development of in vitro-produced female bovine embryos

In early development, female embryos (XX) produce twice the transcripts of X-linked genes compared with male embryos (XY). During the course of development, inactivation of the X chromosome equilibrates gene dosage, making the development of female embryos viable. Moreover, the biotechnologies used for producing embryos in vitro seem to work better with male embryos, making it easier for them to reach the blastocyst stage and allow for complete gestation. We investigated the expression of three X-linked genes that are involved in development, XIST, G6PD, and HPRT, and of the transcript interferon-tau, in male and female bovine blastocysts produced by nuclear transfer (NT) and by in vitro fertilization (IVF). Oocytes that had been matured in vitro were enucleated and reconstructed with somatic cells from adult animals at 18 h post-maturation. After fusion (two pulses of 2.25 kv/cm) and chemical activation (5.0 mu M ionomycin for 5 min and 2.0 mM 6-DMAP for 3 h), the oocytesomatic cell units were cultivated in CR2 with a monolayer of granulosa cells at 38.8 degrees C, in a humidified 5% CO(2) atmosphere. IVF embryos were inseminated, after centrifugation in a Percoll gradient, with 2 x 10(6) sperm/mL TALP medium supplemented with BSA and PHE and cultivated under the same conditions as the cloned embryos. We used real-time PCR to analyze the gene expression of individual blastocysts compared to expression of the housekeeping gene, GAPDH. The gene XIST was expressed in female embryos and not in male embryos produced by IVF, though it was expressed at low levels in male embryos produced by NT. Unlike previous reports, we found lower levels of the transcript of G6PD in females than in males, suggesting double silencing or other mechanisms of control of this gene. Female embryos produced by IVF expressed the HPRT gene at a higher level than female embryos produced by NT, suggesting that gene silencing proceeds faster in NT-produced female embryos due to ""inactivation memory"" from the nucleus donor. In conclusion, male and female embryos express different levels of X-chromosome genes and failures of these genes that are essential for development could reduce the viability of females. Nuclear transfer can modify this relation, possibly due to epigenetic memory, leading to frequent failures in nuclear reprogramming.

Imprinted gene expression in in vivo- and in vitro-produced bovine embryos and chorio-allantoic membranes

Cloning by nuclear transfer is often associated with poor results due to abnormal nuclear reprogramming of somatic donor cells and altered gene expression patterns. We investigated the expression patterns of imprinted genes IGF2 and IGF2R in 33- to 36-day bovine embryos and chorio-allantoic membranes derived from in vivo- and in vitro-produced embryos by somatic cell nuclear transfer (SCNT), parthenogenetic activation, and in vitro fertilization (IVF). There was a lower IGF2 expression rate in the SCNT (0.19) and parthenogenetic (0.02) groups when compared to in vivo and IVF embryos (2.01; P < 0.05). In the chorio-allantoic membranes, IGF2 showed a baseline expression pattern (P < 0.05) in parthenotes (0.001) when compared to in vivo, IVF (3.13), and SCNT (0.98) groups. IGF2R was less expressed (P < 0.05) in SCNT chorio-allantoic membranes (0.25) when compared to the in vivo group. The low expression of IGF2 in parthenogenetic embryos and chorio-allantoic membranes confirms its imprinted status in cattle. Alterations in the relative frequency of IGF2 and IGF2R transcripts were observed in SCNT-derived bovine embryos and chorioallantoic membranes, respectively, supporting the hypothesis that abnormalities in the expression of imprinted genes are causes of the low efficiency of SCNT procedures in this species.

The structure and dynamics of Abell 1942

Aims. We present a dynamical analysis of the galaxy cluster Abell 1942 based on a set of 128 velocities obtained at the European Southern Observatory. Methods. Data on individual galaxies are presented and the accuracy of the determined velocities as some properties of the cluster are discussed. We have also made use of publicly available Chandra X-ray data. Results. We obtained an improved mean redshift value z = 0.22513 +/- 0.0008 and velocity dispersion sigma = 908(139)(+147) km s(-1). Our analysis indicates that inside a radius of similar to 1.5 h(70)(-1) Mpc (similar to 7 arcmin) the cluster is well relaxed, without any remarkable features and the X-ray emission traces the galaxy distribution fairly well. Two possible optical substructures are seen at similar to 5 arcmin from the centre in the northwest and the southwest directions, but are not confirmed by the velocity field. These clumps are, however, kinematically bound to the main structure of Abell 1942. X-ray spectroscopic analysis of Chandra data resulted in a temperature kT = 5.5+/-0.5 keV and metal abundance Z = 0.33 +/- 0.15 Z(circle dot). The velocity dispersion corresponding to this temperature using the T(X-sigma) scaling relation is in good agreement with the measured galaxy velocities. Our photometric redshift analysis suggests that the weak lensing signal observed to the south of the cluster and previously attributed to a ""dark clump"" is produced by background sources, possibly distributed as a filamentary structure.

A NEW DIAGNOSTIC OF THE RADIAL DENSITY STRUCTURE OF Be DISKS

We analyze the intrinsic polarization of two classical Be stars in the process of losing their circumstellar disks via a Be to normal B star transition originally reported by Wisniewski et al. During each of five polarimetric outbursts which interrupt these disk-loss events, we find that the ratio of the polarization across the Balmer jump (BJ+/BJ-) versus the V-band polarization traces a distinct loop structure as a function of time. Since the polarization change across the Balmer jump is a tracer of the innermost disk density whereas the V-band polarization is a tracer of the total scattering mass of the disk, we suggest that such correlated loop structures in Balmer jump-V-band polarization diagrams (BJV diagrams) provide a unique diagnostic of the radial distribution of mass within Be disks. We use the three-dimensional Monte Carlo radiation transfer code HDUST to reproduce the observed clockwise loops simply by turning ""on/off"" the mass decretion from the disk. We speculate that counterclockwise loop structures we observe in BJV diagrams might be caused by the mass decretion rate changing between subsequent ""on/off"" sequences. Applying this new diagnostic to a larger sample of Be disk systems will provide insight into the time-dependent nature of each system's stellar decretion rate.