Identification and characterization of the EXPA7, EXPA18 and EXT10 genes in Turbinicarpus lophophoroides (Werderm.) Buxb. & Backeb; and their expression analysis in the root under abiotic stress.

Expansin and extensin are proteins involved in resistance to various abiotic stresses by processes of cell wall modification and in the formation and elongation of the hairy root. They are located in several organs of the plant included root epidermis. Turbinicarpus lophophoroides is a cactus model to studies these genes in adventitious and transformed roots. In this study, we identified and characterized the expansin7, expansin18 and extensin10 genes in T. lophophoroides. Bioinformatic analysis indicated that the expansin sequences contained the motifs: HTFYG, HFD, YRR, VPC and YW; and certain conserved cysteine (C) residues. Regarding extensin10, the sequence contains the conserved SPPPP (SP4), YYS and YV motifs. The expression analysis in adventitious and transformed roots under osmotic stress (300 mM mannitol), heat (37 °C) and cold (4 °C); shows a higher expression of TlExpA18 in both roots, a decrease in TlExpA7 in transformed roots and a null expression in TlExt10 in both roots. In addition, a morphological comparison of the maturation/differentiation zone, meristem and cap between adventitious and transformed roots by SEM was performed, finding differences in the quantity and length of the hairy roots and the shape of the root cap. Overall, the study concluded that TlExpA18 and TlExpA7 belong to expansin family and TlExt10 belong to extensin family. The expression characteristics of TlExpA18, TlExpA7 and TlExt10 will facilitate the investigation of its function in stress response and other physiological processes in T. lophophoroides.

Drogas legales en un grupo de adolescentes

El estudio consistió en la descripción y estimación de la prevalencia del consumo de drogas legales en el total de alumnos de 14 años de edad, que cursan noveno grado en el secundario Pedro Fermín Armas, del área Sur de Sancti Spíritus así como determinar la frecuencia del uso de psicotóxicos, motivaciones y lugares de consumo usado por los adolescentes. Fueron encuestados 81 estudiantes. Para la obtención de los datos se utilizó una encuesta confeccionada para dicho estudio, siendo aplicada por los investigadores y con el consentimiento informado de los representantes legales de los adolescentes. Se halló que el 95 por ciento declaró consumir drogas legales y de ellos el 64.3 por ciento eran masculinos. El 92.2 por ciento ingieren alcohol, un 49.4 café y el 22.1 fuman. El mayor número de adolescentes 70.42 bebe solo en fiestas y un 2.82 lo hace diario. Se destacó el uso del cigarro solo en fiestas con un 70.59. De los 38 alumnos que toman café el 60.53 lo hace diario. El lugar de más consumo de alcohol fue la plaza cultural. El 72.84 tenían familiares que ingieren alcohol, un 88.89 ingieren café y un 58 de los familiares fuman. La presión de grupo fue la motivación que predominó. Se observó que el 73.25 conocen los daños que pueden provocar estas drogas Se recomendó ejercer acciones de promoción y prevención de salud en el primer nivel de atención con participación intersectorial para fomentar estilos de vida sanos e incrementar programas de capacitación sobre adicciones en los claustros de profesores y la comunidad

Enzyme-linked immunosorbent assay and Western blot antibody determination in sera from patients diagnosed with different helminthic infections with Anisakis simplex antigen purified by affinity chromatography.

An evaluation of the sensitivity and the specificity of the Anisakis simplex antigens purified by affinity chromatography was performed using sera from patients diagnosed with Anisakis sensitisation and sera from patients previously diagnosed with different helminthic infections. Only the sera of the patients diagnosed with Schistosoma mansoni or Onchocerca volvulus parasitic infections were negative against the A. simplex antigen and its purified fractions (PAK antigen: A. simplex antigen purified using columns prepared with anti-A. simplex rabbit IgG and PAS antigen: PAK antigen purified using columns prepared with anti-Ascaris suum rabbit IgG). However all the sera were positive against the A. suum antigen. In all the sera from the patients diagnosed with Anisakis sensitisation, the antibody levels detected using the purified antigens (PAK and PAS antigens) were lower than the observed using the A. simplex crude extract with the highest diminution in the case of the IgG. When these same sera were tested against the A. simplex crude extract by Western blot, several bands of high molecular masses were observed as well as, intense bands at 60 and/or 40 kDa. A concentration of these last proteins was observed in the PAK and the PAS antigens. When the sensitivity and the specificity determinations were performed, only seven of the 38 patients diagnosed of Anisakis sensitisation were positive, as well as, the sera from the patients diagnosed with parasitisms by Echinococcus granulosus or Fasciola hepatica.

Enzyme-linked immunosorbent assay and Western blot antibody determination in sera from patients diagnosed with different helminthic infections with Anisakis simplex antigen purified by affinity chromatography

An evaluation of the sensitivity and the specificity of the Anisakis simplex antigens purified by affinity chromatography was performed using sera from patients diagnosed with Anisakis sensitisation and sera from patients previously diagnosed with different helminthic infections. Only the sera of the patients diagnosed with Schistosoma mansoni or Onchocerca volvulus parasitic infections were negative against the A. simplex antigen and its purified fractions (PAK antigen: A. simplex antigen purified using columns prepared with anti-A. simplex rabbit IgG and PAS antigen: PAK antigen purified using columns prepared with anti-Ascaris suum rabbit IgG). However all the sera were positive against the A. suum antigen. In all the sera from the patients diagnosed with Anisakis sensitisation, the antibody levels detected using the purified antigens (PAK and PAS antigens) were lower than the observed using the A. simplex crude extract with the highest diminution in the case of the IgG. When these same sera were tested against the A. simplex crude extract by Western blot, several bands of high molecular masses were observed as well as, intense bands at 60 and/or 40 kDa. A concentration of these last proteins was observed in the PAK and the PAS antigens. When the sensitivity and the specificity determinations were performed, only seven of the 38 patients diagnosed of Anisakis sensitisation were positive, as well as, the sera from the patients diagnosed with parasitisms by Echinococcus granulosus or Fasciola hepatica.

Structural and functional analysis of the cAMP binding domain from the regulatory subunit of Mucor rouxii protein kinase A.

The cAMP binding domain of the regulatory subunit (R) of Mucor rouxii protein kinase A was cloned. The deduced amino acid sequence was highly homologous in sequence and in size to the corresponding region in fungal and higher eukaryotic regulatory subunits (47-54%), but particularly homologous (62%) to Blastocladiella emersonii, a fungus classified in a different phylum. Amino acids reported to be important for interaction with cAMP, for cooperativity between the two cAMP binding domains, in the general folding of the domain, and for interaction with the catalytic subunit were conserved in all the fungal sequences. Based on either sequence or functional behavior, the M. rouxii R subunit cannot be classified as being more similar to RI or RII of mammalian systems. The M. rouxii protein sequence was modeled using as template the coordinates of the crystallized bovine regulatory subunit type Ialpha. The quality of the model is good. The two backbones could be perfectly overlapped, except for two loop regions of high divergence. The alpha helix C of domain A, proposed to have a strong interaction with the catalytic subunit, contains a leucine replacing a basic residue (arginine or lysine) commonly found in RI or RII. The domains A and B of the M. rouxii regulatory subunit were overexpressed as fusion proteins with GST. GST domain B protein was inactive. GST domain A was active; the kinetic parameters of affinity toward cAMP analogs, site selectivity, and dissociation kinetics of bound cAMP were analogous to the properties of the domain in the whole regulatory subunit.

Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer.

Alternative mRNA splicing of the fibronectin EDI exon is controlled by a purine-rich exonic splicing enhancer (ESE), postulated as a binding site for SR proteins. By using a transient expression alternative splicing assay combined with promoter swapping, we have demonstrated that the promoter can also control EDI splicing, arguing for coupling between the transcription and splicing machineries. We now report that the SR proteins SF2/ASF and 9G8 stimulate EDI splicing in vivo and that their effect requires an intact EDI ESE. Most importantly, we show that sensitivity to these SR proteins critically depends on the promoter structure, suggesting that the transcription machinery modulates their recruitment to the ESE.

Involvement of fibronectin in the regulation of urokinase production and binding in murine mammary tumor cells.

Tumor invasion and metastasis development is a multistep process involving adhesion molecules as well as tumor proteases. It has been reported that tumor cells lacking fibronectin (FN) expression and engineered to re-express FN showed a marked reduction in metastatic ability. Besides its effects on cell adhesion and migration, FN could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we analyzed the production of urokinase-type plasminogen activator (uPA), and its receptor (uPAR), 2 molecules involved in the invasive phenotype, in cells over-expressing RGD wild-type FN (FNwt clones) or RGD-mutated FN (FN RGD-minus clones). Secreted uPA activity and antigen were significantly up-regulated in FN-expressing clones, although RGD-minus cells secreted approximately 50% less uPA than the FNwt ones. Interestingly, while control and FN RGD-minus clones were able to readily bind uPA to their surface, FNwt clones exhibited impaired uPA binding. Furthermore, treatment of the parental cell line as well as the control and FN-expressing clones with exogenous purified FN or RGD peptides induced up-regulation of uPA production and the reduction of uPA membrane binding, which was associated with lower expression of uPAR. This modulation by FN was found to be dependent on RGD sequence and beta1 integrin. These results strongly suggest a novel activity for the multifunctional glycoprotein FN regarding the regulation of uPA production as well as the capacity of tumor cells to bind uPA.

Expression of RGD minus fibronectin that does not form extracellular matrix fibrils is sufficient to decrease tumor metastasis.

Fibronectin (FN) is a plasma and extracellular matrix (ECM) glycoprotein, the expression of which may modulate the invasive and metastatic abilities of cancer cells. LMM3 is a cell line derived from the highly metastatic mouse mammary adenocarcinoma MM3 and is unable to express FN both at protein and mRNA levels. To study the role of FN in the metastatic process, LMM3 cells were stably transfected with 2 variants (wt and RGD-minus) of a full length human FN cDNA. All analyzed clones secreted recombinant FN and although none assembled FN in the ECM they showed an in vitro reduced migratory ability and an increased adhesive capacity. FN-producing cells were assayed for experimental and spontaneous metastasis. All clones tested showed a significant reduction in the number of experimental lung metastasis when compared with a control clone. Similar trends were observed for spontaneous metastatic ability. Our results indicate that the expression of FN that lacks the well-recognized RGD cell-binding site and that does not form ECM fibrils, is sufficient to decrease the metastatic potential of cancer cells. Our results also suggest that an RGD-independent mechanism may be acting in the prevention of metastasis.

The fibronectin gene as a model for splicing and transcription studies.

The fibronectin (FN) gene has become paradigmatic to illustrate genome evolution by exon shuffling, generation of protein diversity by alternative mRNA splicing, and topological coordination between transcription and splicing. Alternative splicing in three sites of the primary transcript gives rise to multiple FN polypeptides. This process is cell type-, development- and age-regulated. The different FN variants seem to play specific roles in FN dimer secretion, blood clotting, adhesion to lymphoid cells, skin wound healing, atherosclerosis, and liver fibrosis. This review focuses on function assignment to the alternatively spliced segments, as well as on the external signals and cis-acting sequences that control the mechanisms of alternative splicing. We also discuss FN transcriptional regulation in response to viral transformation, growth factors, and cyclic AMP in the light of promoter architecture and its interaction with specific transcription factors. The relevance of FN RNA "tracks" as assembly lines of coordinated transcription and RNA processing is also addressed.

Binding of nuclear factors to a satellite DNA of retroviral origin with marked differences in copy number among species of the rodent Ctenomys.

The major satellite DNA of the subterranean rodent Ctenomys, named RPCS, contains several consensus sequences characteristic of the U3 region of retroviral long terminal repeats (LTRs), such as a polypurine tract, CCAAT boxes, binding sites for the CCAAT/enhancer-binding protein (C/EBP), a TATA box and putative polyadenylation signals. RPCS presents an enormous variation in abundance between species of the same genus: while C. australis or C. talarum have approximately 3 x 10(6) copies per genome, C. opimus has none. A sequence (RPCS-I) with identity to the SV40-enhancer core element, present in all the repeating units of the satellite is specifically protected in DNase I footprintings. Competitions of band-shift assays with different transcription factor binding sites indicate that binding to RPCS-I is specific and involves CCAAT proteins related to NF-1, but not to C/EBP. By the use of quantitative protein/DNA binding assays we determined that, despite of their conspicuous difference in RPCS copy number, C. talarum and C. opimus have equivalent amounts and identical quality of RPCS-binding proteins. These results are consistent with the observation, by in situ hybridization, that RPCS is clustered in heterochromatic regions, where it might have restricted accessibility to transcription factors in vivo. This is the first report of the binding of transcription factors to a satellite DNA of retroviral origin.

The CRE-binding factor ATF-2 facilitates the occupation of the CCAAT box in the fibronectin gene promoter.

The cAMP response element (CRE) and the CCAAT box of the fibronectin gene promoter are separated by only twenty base pairs. A specific factor that binds the CRE interacts cooperatively with the protein which binds to the adjacent CCAAT box, stimulating transcription [1992, J. Biol. Chem. 267, 12767-12774]. Here we show that the CRE factor is an heterodimer between a 43 kDa and the '73 kDa' CRE-binding proteins and we identify the latter as ATF-2 (also named CRE-BPI), a protein implicated in recruiting transcriptional activators to promoters, able to form heterodimers with Jun and for which a sequence-deduced MW of 55 kDa had been previously reported.

Interaction of the -170 cyclic AMP response element with the adjacent CCAAT box in the human fibronectin gene promoter.

In the fibronectin gene promoter the cAMP response element (CRE) and the CCAAT box are separated by only 20 base pairs (bp), i.e. two turns of double helix. Binding of nuclear proteins to these elements, assessed by DNase I footprinting, differs in the different cell types. While in a variety of cells tested (HeLa, granulosa, brain, and adenocarcinoma) only CRE binding activity is observed, liver extracts show both CRE and CCAAT binding activities. Competitions with CRE oligonucleotides were able to prevent the binding of both liver factors, while competitions with CCAAT oligonucleotides only abolished the binding to the CCAAT box. Consistently, the occupation of the CCAAT box was reduced when the distance between the CRE and CCAAT elements was increased in a series of spacing mutants in which DNA fragments of 20, 28, or 44 bp were inserted, and in a construct where the CRE sequence was deleted. Furthermore, the mutants are less efficient than the wild type as templates for in vitro transcription elicited by liver nuclear extracts. Transcriptional activity decreases with the 20- and 28-bp insertions but is partially recovered with the 44-bp insertion. Partial purification of liver CRE- and CCAAT-binding proteins by high performance liquid chromatography on a Mono Q column and recombination of column fractions showed that a novel 73-kDa CRE-binding protein facilitates the association of the CCAAT-binding protein to the CCAAT site of the fibronectin gene.

Inverse correlation between fibronectin mRNA levels and the metastatic potential of two murine mammary adenocarcinomas.

The ability of cells to metastasize is thought to be related with the loss of the capacity to synthesize and secrete fibronectin (FN). We found that primary cultures from 2 murine adenocarcinoma tumors, M3 of moderate metastasizing ability and the highly metastasizing MM3, had a dramatic difference in the immunohistochemical expression of FN. It was shown, as assayed by the S1 mapping technique that, while total FN mRNA was fairly abundant in M3 cells, it was almost undetectable in MM3 cells. This difference is not due to a significant deletion of the FN gene in MM3 cells.

Neurospora crassa cDNA clones coding for a new member of the ras protein family.

A new member of the ras gene family was characterized from Neurospora crassa cDNA libraries. The clone designated NC-ras codes for a polypeptide containing 213 amino acids (Mr 24,000). This polypeptide is 84% homologous to the H-ras-1 domain comprising the first 80 amino acids and 60% homologous to the next 84 residues. The NC-ras polypeptide contains all the well-known sequences involved in the interaction with GTP/GDP, the recognition of the Y13-259 neutralizing antibody, the 'effector site' for interaction with GAP proteins, and the CAAX acylation motif in the COOH-terminal.

Cyclic AMP inhibits fibronectin gene expression in a newly developed granulosa cell line by a mechanism that suppresses cAMP-responsive element-dependent transcriptional activation.

The main role of the ovarian granulosa cells is to nurse the oocyte and to produce estradiol and progesterone upon stimulation by gonadotropins. In fact, follicle-stimulating hormone (FSH) and luteinizing hormone control the expression of several genes during granulosa cell differentiation via cyclic AMP-dependent phosphorylations. Cyclic AMP stimulates transcription of genes that carry the cAMP-responsive element (CRE,5'TGACGTCA3') in their promoters. The fibronectin (FN) gene contains one CRE sequence at position -170. However, gonadotropins and cAMP inhibit FN gene expression in granulosa cells. To study the mechanism of the inhibition we developed a bovine granulosa cell line (BGC-1) that synthesizes estradiol in response to FSH and in which FSH and dibutyryl cAMP specifically decrease FN synthesis and its mRNA levels. The inhibitory effect (a) is not due to an alteration in FN mRNA stability, (b) requires upstream sequences other than CRE, located between positions -510 and -223, that are able to bind granulosa cell nuclear proteins, (c) is entirely dependent on the synthesis of intermediate proteins induced and or phosphorylated by cAMP, and (d) effectively suppresses the CRE-dependent transcriptional activation.