LHX6 is a sensitive methylation marker in head and neck carcinomas

Head and neck cancer remains a morbid and often fatal disease and at the present time few effective molecular markers have been identified. The purpose of the present work was to identify new molecular markers for head and neck squamous cell carcinoma (HNSCC). We applied methylation-sensitive arbitrarily primed PCR (MS/APPCR) to isolate sequences differentially methylated in HNSCC. The most frequently hypermethylated fragment we found maps close to a cytosine guanine dinucleotide (CpG) island on chromosome 9q33.2, and hypermethylation of this CpG island was associated with transcriptional silencing of an alternative transcript of the LHX6 gene. Using combined bisulfite restriction analysis (COBRA), hypermethylation of this fragment was detected in 13 of 14 (92.8%) HNSCC cell lines studied and 21 of 32 (65.6%) primary tumors, whereas little or no methylation was seen in 10 normal oral mucosa samples. We extended this investigation to other cancer cell lines and methylation was found in those derived from colon, breast, leukemia and lung, and methylation was also found in 12/14 primary colon tumors. These findings suggest that differentially methylated (DIME)-6 hypermethylation is a good cancer marker in HNSCC as well as in other kinds of neoplasias and confirm the importance of searching for markers of epigenetic dysregulation in cancer.

The copper resistance operon copAB from Xanthomonas axonopodis pathovar citri: gene inactivation results in copper sensitivity

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker and the completion of the Xac genome sequence has opened up the possibility of investigating basic cellular mechanisms at the genomic level. Copper compounds have been extensively used in agriculture to control plant diseases. The copA and copB genes, identified by annotation of the Xac genome, encode homologues of proteins involved in copper resistance. A gene expression assay by Northern blotting revealed that copA and copB are expressed as a unique transcript specifically induced by copper. Synthesis of the gene products was also induced by copper, reaching a maximum level at 4 h after addition of copper to the culture medium. CopA was a cytosolic protein and CopB was detected in the cytoplasmic membrane. The gene encoding CopA was disrupted by the insertion of a transposon, leading to mutant strains that were unable to grow in culture medium containing copper, even at the lowest CUSO4 concentration tested (0.25 mM), whereas the wild-type strain was able to grow in the presence of 1 mM copper. Cell suspensions of the wild-type and mutant strains in different copper concentrations were inoculated in lemon leaves to analyse their ability to induce citrus canker symptoms. Cells of mutant strains showed higher sensitivity than the wild-type strain in the presence of copper, i.e. they were not able to induce citrus canker symptoms at high copper concentrations and exhibited a more retarded growth in planta.

cAMP signaling pathway controls glycogen metabolism in Neurospora crassa by regulating the glycogen synthase gene expression and phosphorylation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effect of acute heat stress on heat shock protein 70 messenger RNA and on heat shock protein expression in the liver of broilers

1. The synthesis of heat shock protein 70 (Hsp70) mRNA and the expression of Hsp70 in the liver of broiler chickens submitted to acute heat stress (35 degrees C for 5 h) was investigated.2. Hsp70 expression was detected by SDS-PAGE and Western blot analysis using a polyclonal antiserum against Hsp70 of Blastocladiella emersonii. The specific signal of Hsp70 mRNA was analysed by Northern blot using as probe a Hsp70 cDNA of B. emersonii.3. An increase in the amount of Hsp70 was detected from the first up to the fifth hour of acute heat exposure. This increase in the amount of Hsp70 was accompanied by an increase in Hsp70 mRNA which peaked at 3 h.4. This study shows that the heat induced increase in Hsp70 mRNA and protein in broiler liver, in vivo, are time dependent, similar to that in mammals.

Clarifications on breakpoints in HSAX and BTAX by comparative mapping of F9, HPRT, and XIST in cattle

The coagulation factor IX gene (179), the hypoxanthine phosphoribosyl transferase 1 gene (HPRT1), and the X-inactive specific transcript gene (XIST) were physically assigned in cattle to analyze chromosomal breakpoints on BTAX recently identified by radiation hybrid (RH) mapping experiments. Whereas the FISH assignment of XIST indicates a similar location on the q-arm of the human and cattle X chromosomes, the locus of HPRT1 supported the assumption of a chromosome rearrangement between the distal half of the q-arm of HSAX and the p-arm of BTAX identified by RH mapping. F9 previously located on the Cl-arm of BTAX was assigned to the p-arm of BTAX using RH mapping and FISH. The suggested new position of F9 close to HPRT I supports the homology between HSAXq and BTAXp. The F9 locus corresponds with the gene order found in the homologous human chromosome segment. XIST was assigned on BTAXq23, HPRT1 and F9 were mapped to BTAXp22, and the verification of the location of F9 in a 5000 rad cattle-hamster whole genome radiation hybrid panel linked the gene to markers URB10 and HPRT1. Copyright (C) 2003 S. Karger AG, Basel.

The putative telomerase reverse transcriptase component of Leishmania amazonensis: gene cloning and characterization

The Leishmania amazonensis telomerase gene was cloned by a polymerase chain reaction-based strategy using primers designed from a Leishmania major sequence that shared similarities with conserved telomerase motifs. The genes from three other species were cloned for comparative purposes. A ClustalW multiple-sequence alignment demonstrated that the Leishmania telomerases show greater homology with each other than with the proteins of other kinetoplastids and eukaryotes. Characterization experiments indicated that the putative Leishmania telomerase gene was probably in single copy and located in the largest chromosomes. A single messenger ribonucleic acid transcript was found in promastigotes. Phylogenetic analysis suggested that Leishmania telomerase might represent a liaison between the oldest and the newest branches of telomerases.

The contribution of transposable elements to Bos taurus gene structure

In an effort to identify the contribution of TEs to bovine genome evolution, the abundance, distribution and insertional orientation of TEs were examined in all bovine nuclear genes identified in sequence build 2.1 (released October 11, 2005). Exons, introns and promoter segments (3 kb upstream the transcription initiation sites) were screened with the RepeatMasker program. Most of the genes analyzed contained TE insertions, with an average of 18 insertions/gene. The majority of TE insertions identified were classified as retrotransposons and the remainder classified as DNA transposons. TEs were inserted into exons and promoter segments infrequently, while insertion into intron sequences was strikingly more abundant. The contribution of TEs to exon sequence is of great interest because TE insertions can directly influence the phenotype by altering protein sequences. We report six cases where the entire exon sequences of bovine genes are apparently derived from TEs and one of them, the insertion of Charlie into a bovine transcript similar to the zinc finger 452 gene is analyzed in detail. The great similarity of the TE-cassette sequence to the ZNF452 protein and phylogenetic relationship strongly suggests the occurrence of Charlie 10 DNA exaptation in the mammalian zinc finger 452 gene. (c) 2006 Published by Elsevier B.V.

Expression of heat shock protein in broiler embryo tissues after acute cold or heat stress

This study evaluated the expression of heat shock protein 70 kD (hsp70) in broiler chicken embryos subjected to cold (Experiment 1) or high incubation temperature (Experiment 11). In each experiment, fertile eggs were distributed in three incubators kept at 37.8degreesC. At day 13 (D13), D16, and D19 of incubation, the embryos were subjected to acute cold (32degreesC) or heat (40degreesC) for 4-6 hr. Immediately after cold or heat exposure, samples from the liver, heart, breast muscle, brain, and lungs of 40 embryos were taken per age and treatment (control or stressed embryos), A tissue pool from 10 embryos was used as 1 replication. The levels of hsp70 in each tissue sample was quantified by Western blot analysis. The data were analyzed in a 3 x 2 factorial arrangement of treatments with four replications. hsp70 was detected in all embryo tissues, and the brain contained 2- to 5-times more hsp70 protein compared to the other tissues in either cold or heat stressed embryos. hsp70 increases were observed in the heart and breast muscle of cold stressed embryos at D16 and D19, respectively. Heat stressed embryos showed an increase of hsp70 in the heart at D13 and D19, and in the lung at D19 of incubation. Younger embryos had higher hsp70 synthesis than older embryos, irrespective of the type of thermal stressor. The results indicate that the expression of hsp70 in broiler chicken embryos is affected by cold and heat distress, and is tissue- and age-dependent. (C) 2004 Wiley-Liss, Inc.

The generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags

Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define approximate to23,500 genes, of which only approximate to1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that <1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers.

NtWBC1, an ABC transporter gene specifically expressed in tobacco reproductive organs

To identify genes specifically or predominantly expressed in the stigmas/styles and to establish their possible function in the reproductive process of plants, a tobacco stigma/style cDNA library was constructed and differentially screened, resulting in the isolation of several cDNA clones. The molecular characterization of one of these clones is described here. After sequencing the cDNA and the isolated genomic clone, it was determined that the corresponding gene encodes a protein containing an ATP-binding cassette, characteristic of ABC transporters. This gene, designated as NtWBC1 (Nicotiana tabacum ABC transporter of the White-Brown Complex subfamily), encodes a protein that contains the typical structure of the 'half-transporters' of the White subfamily. To establish the spatial expression pattern of the NtWBC1 gene, northern blot and real-time RT-PCR analyses with total RNA from roots, stems, leaves, sepals, petals, stamens, stigmas/styles, ovaries, and seeds were performed. The result revealed a transcript of 2.5 kb present at high levels in stigmas and styles and a smaller transcript (2.3 kb) present at a lower level in stamens. NtWBC1 expression is developmentally regulated in stigmas/styles, with mRNA accumulation increasing toward anthesis. In situ hybridization experiments demonstrated that NtWBC1 is expressed in the stigmatic secretory zone and in anthers, at the stomium region and at the vascular bundle. NtWBC1 is the first ABC transporter gene with specific expression in plant reproductive organs to be identified and its expression pattern suggests important role(s) in the reproductive process.

Expression and variability of molecular chaperones in the sugarcane expressome

Molecular chaperones perform folding assistance in newly synthesized polypeptides preventing aggregation processes, recovering proteins from aggregates, among other important cellular functions. Thus their study presents great biotechnological importance. The present work discusses the mining for chaperone-related sequences within the sugarcane EST genome project database, which resulted in approximately 300 different sequences. Since molecular chaperones are highly conserved in most organisms studied so far, the number of sequences related to these proteins in sugarcane was very similar to the number found in the Arabidopsis thaliana genome. The Hsp70 family was the main molecular chaperone system present in the sugarcane expressome. However, many other relevant molecular chaperones systems were also present. A digital RNA blot analysis showed that 5'ESTs from all molecular chaperones were found in every sugarcane library, despite their heterogeneous expression profiles. The results presented here suggest the importance of molecular chaperones to polypeptide metabolism in sugarcane cells, based on their abundance and variability. Finally, these data have being used to guide more in deep analysis, permitting the choice of specific targets to study. (c) 2006 Elsevier GmbH. All rights reserved.

The Paracoccidioides brasiliensis gp70 antigen is encoded by a putative member of the flavoproteins monooxygenase family

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Localization and hormonal regulation of endometrial matrix metalloproteinase-26 in the rhesus macaque

The current understanding of hormonal regulation of matrix metalloproteinase-26 (MMP-26) in the primate endometrium is incomplete. The goal of this work was to clarify estrogen and progesterone regulation of MMP-26 in the endometrium of ovariectomized, hormone-treated rhesus macaques.Ovariectomized rhesus macaques (n 66) were treated with estradiol (E-2), E-2 plus progesterone, E-2 followed by progesterone alone or no hormone. Endometrium was collected from the hormone-treated animals during the early, mid- and late proliferative and secretory phases of the artificial menstrual cycle. MMP-26 expression was quantified by real-time PCR, and MMP-26 transcript and protein were localized by in situ hybridization and immunohistochemistry and correlated with estrogen receptor 1 and progesterone receptor (PGR).MMP-26 was localized to glandular epithelium and was undetectable in the endometrial stroma and vasculature. MMP-26 transcript levels were minimal in the hormone-deprived macaques and treatment with E-2 alone did not affect MMP-26 levels. Treatment with progesterone both in the presence and absence of E-2 stimulated MMP-26 expression in the early and mid-secretory phases (P 0.001). MMP-26 expression preceded decidualization of endometrial stroma. MMP-26 levels then declined to baseline in the late secretory phase (P 0.01) despite continued E-2 plus progesterone treatment. Loss of detectable MMP-26 expression in the late secretory phase was correlated with late secretory phase loss of glandular epithelial PGR.Endometrial MMP-26 expression is dependent on the presence of progesterone in the early secretory phase and then gradually becomes refractory to progesterone stimulation in the late secretory phase. In the macaque, MMP-26 is a marker of the pre-decidual, secretory endometrium. During the second half of the late secretory phase, and during decidualization, MMP-26 loses its response to progesterone concurrent with the loss of epithelial PGR. The decline in MMP-26 levels between the mid- and late secretory phases may play a role in the receptive window for embryo implantation.

Selective Ablation of the Androgen Receptor in Mouse Sertoli Cells Affects Sertoli Cell Maturation, Barrier Formation and Cytoskeletal Development

The observation that mice with a selective ablation of the androgen receptor (AR) in Sertoli cells (SC) (SCARKO mice) display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli) and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin). Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2). It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development.

Evaluation of developmental changes in bovine in vitro produced embryos following exposure to bovine Herpesvirus type 5

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)