The omega-6 arachidonic fatty acid, but not the omega-3 fatty acids, inhibits osteoblastogenesis and induces adipogenesis of human mesenchymal stem cells: potential implication in osteoporosis.

UNLABELLED: Arachidonic fatty acid (AA) induces adipogenesis in human mesenchymal stem cells cultures, and high concentrations inhibit osteoblastogenesis; whereas eicosapentaenoic and docosahexaenoic fatty acids do not induce adipogenesis and do not inhibit osteoblastogenesis. In mesenchymal stem cells, omega-6 arachidonic polyunsaturated fatty acid promotes the differentiation of adipocytes and inhibits the osteoblast differentiation. While omega-3 fatty acids do not affect the adipogenic differentiation their effects on osteoblastogenesis are less relevant. An increased ratio of omega-3/omega-6 fatty acid consumption can prevent bone mass loss. INTRODUCTION: Consumption of omega-3 may protect against osteoporosis since they may inhibit osteoclastogenesis. However, with aging, MSC in bone marrow are increasingly differentiated into adipocytes, reducing the number of osteoblasts. Products derived from omega-6 and omega-3 metabolism may affect MSC differentiation into osteoblasts and adipocytes. METHODS: Human MSC have been differentiated into osteoblasts or adipocytes in the presence of omega-6 (AA), or omega-3 (DHA and EPA), and osteoblastic and adipocytic markers have been analyzed. RESULTS: AA decreases the expression of osteogenic markers and the osteoprotegerin/receptor activator of nuclear factor kappa ß ligand gene expression ratio (opg/rankl). High concentrations of AA inhibit the mineralization and cause the appearance of adipocytes in MSC differentiating into osteoblasts to a higher extent than DHA or EPA. In MSC differentiated into adipocytes, AA increases adipogenesis, while DHA and EPA do not affect it. AA caused the appearance of adipocytes in undifferentiated MSC. The lipoxygenase gene (alox15b) is induced by omega-3 in MSC induced to osteoblasts, and by omega-6 in MSC induced to adipocytes. CONCLUSIONS: An increase in the intake of omega-3 respect to omega-6 may provide protection against the loss of bone mass, since omega-6 favors the osteoclastic activity by diminishing the opg/rankl gene expression in osteoblasts and promotes MSC differentiation into adipocytes, thus diminishing the production of osteoblasts.

Analysis of genetic diversity of southern Spain fig tree (Ficus carica L.) and reference materials as a tool for breeding and conservation.

The common fig tree (Ficus carica L.) is a Mediterranean crop with problematic cultivar identification. The recovery and conservation of possible local varieties for ecological production requires the previous genetic characterization of the available germplasm. In this context, 42 lines corresponding to 12 local varieties and two caprifigs, in addition to 15 reference samples have been fingerprinted using 21 SSR markers. A total of 77 alleles were revealed, detecting a useful level of genetic variability within the local germplasm pools. UPGMA clustering analysis has revealed the genetic structure and relationships among the local and reference germplasm. Eleven of the local varieties could be identified and defined as obtained clusters, showing that SSR analysis is an efficient method to evaluate the Andalusian fig tree diversity for on-farm conservation.

Changes in the redox status of chickpea roots in response to infection by Fusarium oxysporum f. sp. ciceris: apoplastic antioxidant enzyme activities and expression of oxidative stress-related genes.

Activity levels of oxidative stress-related enzymes in the root apoplast during the interaction of WR315 (resistant) and JG62 (susceptible) chickpeas (Cicer arietinum L.) with the highly virulent race 5 of Fusarium oxysporum f. sp. ciceris were compared. Because this fungus develops asymptomatic infections in the chickpea root cortex in both susceptible and resistant plants, but only intrudes into the root xylem in the susceptible variety, the interactions were compared at three specific stages during disease development in JG62: (i) before symptom development (10 days after inoculation); (ii) at the time of appearance of the first disease symptoms (15-17 days after inoculation) and (iii) when all plants had developed disease symptoms (20-22 days after inoculation). Diamine oxidase (DAO), ascorbate peroxidase (APX), glutathione reductase (GR), guaiacol-dependent peroxidase and superoxide dismutase (SOD), but not catalase (CAT), were found in the apoplast of chickpea roots. In terms of APX activity, infection by the pathogen caused a different response in the incompatible compared to the compatible plant. In the case of GR, SOD and DAO activities, the pathogen caused the same response, but it developed earlier (i.e. GR and SOD) or to higher levels (i.e. DAO) in the incompatible interaction. Expression of apx, cat, sod, lipoxygenase (lox) and actin genes was also analysed in infected roots. Infection by F. oxysporum f. sp. ciceris race 5 only caused a significant change in the root expression of lox and actin genes. This up-regulation was earlier (lox) or higher (actin) in the incompatible than in the compatible interaction. Thus, changes in oxidative metabolism differ in compatible and incompatible interactions in Fusarium wilt of chickpea.

Cryopreserved human bone marrow mononuclear cells as a source of mesenchymal stromal cells: application in osteoporosis research.

BACKGROUND: Mesenchymal stromal cells (MSC) are an invaluable tool for research and therapeutic application regarding degenerative diseases such as osteoporosis. METHODS: Human MSC from cryopreserved mononuclear (c-MSC) cell populations were isolated from bone marrow (BM) and compared with MSC isolated directly from the same BM for immunophenotype, differentiation capacity and Parathormone (PTH) response. RESULTS: c-MSC showed a similar immunophenotype, division and differentiation capacity as standard MSC obtained from the same BM. This capacity was maintained during various culture-growing passages. Treatment with PTH(1-34) from days 6 to 24, after c-MSC induction to osteoblasts and adipocytes, had no significant effect on osteoblastogenesis yet inhibited adipogenesis. This effect was similar in MSC from the same BM. DISCUSSION: We propose cryopreservation of mononuclear cells obtained from BM as a simple and convenient means for routine storage of MSC to be used for therapeutic and research applications.

Tissue factor as an effector of angiogenesis and tumor progression in hematological malignancies.

In the last few years, it has become clear that the processes of tumor angiogenesis, metastasis and invasiveness are highly dependent on components of the blood coagulation cascade. One of the key proteins in coagulation is tissue factor (TF). In addition, TF is also known as a mediator of intracellular signaling events that can alter gene expression patterns and cell behavior. TF significantly participates in tumor-associated angiogenesis and its expression levels have been correlated with the metastatic potential of many types of hematological malignancies. Signaling pathways initiated by both, tissue factor-activated factor VII (TF-FVIIa) protease activation of protein-activated receptors (PARs), and phosphorylation of the TF-cytoplasmic domain, appear to regulate these tumoral functions. Advances in antiangiogenic therapies and preclinical studies with TF-targeted therapeutics are hopeful in the control of tumor growth and metastasis, but continued studies on the regulation of TF are still needed. In the last few years, the use of approaches of functional genomics and proteomics has allowed the discovery of new proteins involved in the origin of the neoplasia and their participation in the development of the disease. This review attempts to establish a cellular and molecular causal link between cancer coagulopathy, angiogenesis and tumor progression in hematological malignancies.

Cloning and molecular characterization of B-hordeins from Hordeum chilense (Roem. et Schult.).

One of the main limitations of cereal breeding is the lack of genetic variability within cultivated crops. Hordeum chilense is a wild relative of Hordeum vulgare, which has been successfully used in the synthesis of amphiploids by crossing with Triticum spp. Among the agronomic traits of these new amphiploids, the allelic variation in the endosperm storage proteins and their influence on breadmaking and malting quality are of special interest. B-hordeins are sulfur rich prolamins, which account for 70-80% of the total hordein fraction in barley. In this work, rapid amplification of cDNA ends by PCR (RACE-PCR) has been used for the cloning of the full-length open reading frame (ORF) of six sequences of B3-hordeins from two lines of H. chilense. Two consensus sequences of 813 and 822 bp for the H1 and H7 lines, respectively, were determined by alignment of all the sequences generated. Between both lines, differences involving single base changes, which could correspond to single nucleotide polymorphisms (SNP), insertions and deletions were observed. Of these differences, only six out of the 13 within the ORF caused a change of amino acid. Two insertions/deletions of 9 and 12 bp were also observed between both lines. The derived amino acid sequences showed a similar structure to the B-hordeins from cultivated barley and other prolamins. The repetitive region is based on the repetition of the motif PQQPFPQQ. The copy number of the B3-hordeins was estimated as a minimum of nine and five copies for the H1 and H7 lines, respectively. The expression profile of the B-hordeins through the developing endosperm is also described in this work. This study of the storage proteins of H. chilense is a useful contribution to the knowledge of the genetic diversity available in wild relatives of cultivated barley. In addition, the origin of the different prolamins can be better understood with an in-depth knowledge of its wild equivalent.

Allele-frequency determination of BsmI and FokI polymorphisms of the VDR gene by quantitative real-time PCR (QRT-PCR) in pooled genomic DNA samples.

The vdr gene is a candidate for osteoporosis susceptibility, with conflicting results in association studies. We have designed and optimized an individual allele-specific and DNA pooling PCR-based methodology to quantitate BsmI and FokI polymorphisms of the vdr gene and studied single-nucleotide polymorphisms (SNPs) from pooled DNA samples. The allele frequency in DNA pooling experiments has been analyzed by kinetic PCR: quantitative real-time PCR (QRT-PCR). A Spanish cohort of 225 healthy postmenopausal women was studied. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DEXA) and quantitative ultrasound calcaneous densitometry. Allele-specific PCR amplification of BsmI and FokI genes showed full concordance with the PCR-RFLP approach. The prevalence of the three BsmI VDR genotypes was 19.1, 44.9 and 36.0% for BB, Bb and bb, respectively. In the case of the FokI locus, the prevalence of genotypes was 40.4, 48.0 and 11.6% for FF, Ff and ff, respectively. No positive correlation was found between polymorphism and BMD. The DNA pooling procedure was validated. No differences were found in allele frequencies and T-score data obtained using the high throughput DNA pooling approach, as compared to known individual frequencies. In our hands, this is a very useful approach to study quantitative (thus polygenic) traits like osteoporosis susceptibility.

Cloning and characterization of a gamma-3 hordein mRNA (cDNA) from Hordeum chilense (Roem. et Schult.).

Hordeum chilense is a wild relative of H. vulgare, cultivated barley, that has been successfully used in the synthesis of amphiploids by crossing with Triticum spp. These amphiploids-named generically x Tritordeum-have been tested under field conditions, and one of them, the hexaploid tritordeum obtained following chromosome doubling of the hybrid H. chilense x T. turgidum, shows traits of interest inherited from the barley parent. Of great interest is the allelic variation observed in the endosperm storage proteins and their influence on the breadmaking and malting quality of tritordeum. We report here two mRNA (cDNA) sequences for a gamma-3 hordein from two accession lines of H. chilense, H1 and H7, and their characterization by quantitative real time (QRT)-PCR in the developing endosperm. Sequences were obtained by rapid amplification of cDNA ends and "edge-to-edge" amplification of open reading frames from cDNA of H. chilense. Eight putative single nucleotide polymorphisms and one codon insertion were identified in the sequences of the H1 and H7 gamma-3 hordeins. The deduced amino acid sequences showed similar features to that of the gamma-3 hordein and gamma-gliadins from barley and wheat, respectively. While the repetitive motif (PQQQPF) is similar to that of the gamma-3 hordein from H. vulgare, there are 19 motif repeats in H. vulgare, whereas H. chilense shows 15 tandem repeats. The transcription of the genes encoding for the gamma-3 hordein were monitored by QRT-PCR: in both lines maximum transcription occurred 12 days after flowering.

Development of cost-effective Hordeum chilense DNA markers: molecular aids for marker-assisted cereal breeding.

Hordeum chilense is a potential source of useful genes for wheat breeding. The use of this wild species to increase genetic variation in wheat will be greatly facilitated by marker-assisted introgression. In recent years, the search for the most suitable DNA marker system for tagging H. chilense genomic regions in a wheat background has lead to the development of RAPD and SCAR markers for this species. RAPDs represent an easy way of quickly generating suitable introgression markers, but their use is limited in heterogeneous wheat genetic backgrounds. SCARs are more specific assays, suitable for automatation or multiplexing. Direct sequencing of RAPD products is a cost-effective approach that reduces labour and costs for SCAR development. The use of SSR and STS primers originally developed for wheat and barley are additional sources of genetic markers. Practical applications of the different marker approaches for obtaining derived introgression products are described.

Transferability of olive microsatellite loci across the genus Olea.

The transferability of microsatellite markers developed for olive cultivars ( Olea europaea L.) has been tested and confirmed in the Olea complex. Thirty two genotypes, belonging to different taxa of the genus Olea, have been analyzed with four olive SSRs. Positive amplifications at all loci were obtained in 13 taxa (at least one accession per species). Sixty seven different alleles have been detected at the four loci analyzed. Polymorphic products have been observed at the inter- and intra-species level. Some SSR loci have shown multiple amplification products in some species. The high number of unique alleles has allowed the unambiguous discrimination of most accessions. Similarity coefficients and relationships among the Olea taxa have been calculated based on SSR amplification results. The reliability of SSRs as markers for intra-species variability evaluation has been confirmed while their use to explore relationships at the inter-species level is discussed, being dependent on the locus analyzed.

Rapid verification of wheat-Hordeum introgressions by direct staining of SCAR, STS, and SSR amplicons.

A range of single tagged site (STS), simple sequence repeat (SSR), and sequence-characterized amplified region (SCAR) markers were screened for their utility in detecting Hordeum vulgare and H. chilense chromosomes in a wheat background. PCR conditions were optimized for specific amplification of the targeted sequences and to avoid cross-species amplification. Two H. vulgare derived STSs, six H. vulgare derived SSRs, and nine H. chilense derived SCARs were usable for the detection of five H. vulgare and three H. chilense chromosomes by direct ethidium bromide staining of the PCR products in test tubes, avoiding the more costly and time-consuming DNA electrophoresis step. The practical application of the method is illustrated by the identification of a monotelosomic substitution of H. vulgare chromosome 6HS in tritordeum and a monosomic addition of H. chilense chromosome 6Hch in durum wheat.

Cross-species amplification of the Hordeum chilense genome using barley sequence-tagged-sites (STSs).

A selection of 51 barley Sequence-Tagged Sites (STSs) were studied for their utility in Hordeum chilense. They included four primer sets from wheat origin and six primer sets from oat origin. Forty-four primer pairs amplified H. chilense products consistently. Five primer pairs were suitable for studying the introgression of H. chilense in wheat because they amplified H. chilense products of distinct size. Six of the STSs showed polymorphism between different H. chilense accessions. The results showed that barley STSs could be useful for the genetic characterization of H. chilense, tritordeums and derived introgression lines.

The BLAST algorithms: practical application in molecular cloning, marker-assisted selection (MAS) and introgression of wheat.

BLAST (Basic Local Alignment Search) analyses of Hordeum chilense RAPD markers have shown DNA sequence similarities with several plant genes. Biologically significant hits were scored for: plastocianin (Hordeum vulgare), alpha-gliadin (Triticum aestivum), Grandel-6 retrotransposon (Zea diploperennis), retrofit (gag/pol) copia-like, transposon-like element (Oryza longistaminata), copia-like retrotransposon Hopscotch polyprotein (gag/pol) (Zea mays) and several retrotransposons/retroelements from other species (Arabidopsis thaliana, Oryza sativa, Pisum sativum and Zea mays). Statistically significant hits also included endochitinase (Brassica napius), ribulose-1,5-bisphosphate carboxylase (Euglena gracilis), piruvate, orthophosphate dikinase (Flaveria trinervia), and an ORF flanked by ARS sequences (Triticum aestivum). Interestingly, it was also found that the currently recommended BLAST algorithm (version 2.0.11; gapped) at did not produce any biologically significant hits, whereas the previous version (1.4.11; ungapped) did generate biologically significant results. Thus, we suggest using the new BLAST 2.0.x when strong homologies are expected in short sequence stretches between closely related species. In other instances (and particularly when searching for lower similarities, yet spanning significantly larger stretches between more distant species), the BLAST 1.4.x could yield additional results. BLAST searches are valuable tools to identify and clone DNA sequences of interest, with applications in Marker-Assisted Selection (MAS) and wheat introgression.

New methods to use fish cytochrome P4501A to assess marine organic pollutants.

A new methodology has been developed to assess cytochrome P4501A expression in two South Atlantic Spanish fish, guilthead seabream (Sparus aurata) and grey mullet (Liza aurata), used as pollution bioindicators. Degenerate oligos were used to amplify by reverse transcription and PCR (RT-PCR) specific cyp1A cDNA sequences, used subsequently to design specific primers to get the full cDNA by rapid amplification of cDNA ends. A new assay has been developed to quantitate cyp1A expression by RT-PCR in an automated DNA sequencer. The effect of beta-naphthoflavone inducing biotransformation has been used to compare three distinct pollution biomarkers: EROD activity, ELISA determination of CYP1A, and 2-aminoanthracene (2-AA) activation. Immunodetection by ELISA or Western blot was inconsistent in S. aurata and L. aurata. EROD activity yielded satisfactory results; the higher induction was observed by bioactivation of 2-AA to mutagens detected with strain BA149 of Salmonella typhimurium, in agreement with the high sensitivity previously described for this biomarker. The present paper summarizes the current status of our research.

In vivo transcription of the Escherichia coli oxyR regulon as a function of growth phase and in response to oxidative stress.

Simultaneous expression of seven genes in Escherichia coli was measured by a reverse transcription-multiplex PCR fluorescence procedure. Genes studied were (i) oxyR (transcriptional regulator); (ii) katG, dps, gorA, and ahpCF (controlled by OxyR); (iii) sodA (controlled by SoxRS); and (iv) trxA (not related to OxyR or SoxRS). Except for trxA, transcription of all genes was activated during the course of growth of wild-type bacteria, though notable variations were observed with respect to both the time and extent of activation. Whereas oxyR, katG, dps, and gorA were activated during exponential growth, ahpCF and sodA were stimulated in stationary phase. Maximal induction ranged from 4.6- to 86.5-fold, for gorA and dps, respectively. Treatment with H2O2 stimulated expression of the genes (katG, dps, ahpCF, and gorA) previously identified as members of the OxyR regulon, except for oxyR itself. Induction by H2O2 was a remarkably rapid and reversible process that took place in an OxyR-dependent and sigmaS-independent manner. NaCl induced expression of the genes controlled by OxyR, including the oxyR locus. This transcriptional up-regulation was preserved in a strain with the DeltaoxyR::kan mutation, but it was abolished (ahpCF) or significantly reduced (oxyR and dps) in a strain with the rpoS::Tn10 mutation, potentially reflecting positive transcriptional regulation of the oxyR regulon by sigmaS. Expression of trxA was not increased either by H2O2 stress or by a shift to high-osmolarity conditions.

In vivo transcription of nrdAB operon and of grxA and fpg genes is triggered in Escherichia coli lacking both thioredoxin and glutaredoxin 1 or thioredoxin and glutathione, respectively.

We have previously described () that Escherichia coli maintains a balanced supply of deoxyribonucleotides by a regulatory mechanism that up-regulates the levels of ribonucleotide reductase with the lack of its main hydrogen donors thioredoxin, glutaredoxin 1, and glutathione (GSH). By using a semi-quantitative reverse transcription/multiplex polymerase chain reaction fluorescent procedure that enables simultaneous analysis of up to seven mRNA species, we now demonstrate that regulation operates at the transcriptional level. Double mutant cells lacking both thioredoxin and glutaredoxin 1 had increased transcription of the nrdAB operon, as compared with the corresponding wild type parent (maximal induction of 10- and 9-fold for mRNA of nrdA and nrdB genes, respectively). Likewise, a dramatic increase of 36-fold in grxA mRNA was observed in bacteria simultaneously deficient in thioredoxin and GSH (the physiological reductant of all glutaredoxins). The increased expression of the grxA gene in trxA gshA double mutant bacteria was mimicked in trxA single mutant cells by depletion of GSH with diethylmaleate (DEM). This induction of grxA transcription was rapid since maximal increase was detected upon 10 min of DEM exposure. Like grxA expression, the basal level of fpg mRNA, encoding formamidopyrimidine-DNA glycosylase, was increased (about 4-fold) in a trxA gshA double mutant strain; this expression was also induced upon exposure to DEM (11-fold maximal induction). These results suggest that transcription of grxA might share common redox regulatory mechanism(s) with that of the fpg gene, involved in the repair of 8-oxoguanine in DNA.

Characterization of tissue factor expression on the human endothelial cell line ECV304.

The endothelial cell line ECV304 is a spontaneously transformed cell line established from human umbilical vein. The characterization of tissue factor (TF) expression by ECV304 cells has been accomplished in this study. ECV304 cells expressed both TF mRNA and antigen (TFag) constitutively. In ECV304 cell lysates, the levels of TFag (1.4+/-0.3 ng of TFag/10[6] cells) were considerably higher than in THP-1 monocytoid cells (0.07+/-0.03 ng of TFag/10[6] cells). TFag was also detected on the ECV304 cell surface by flow cytometric studies. In binding analyses, 3.5+/-0.7 x 10(4) molecules of TF per cell were estimated, similar to the amounts found in ECV304 cell lysates (2.9+/-0.6 x 10(4) molecules/cell), suggesting that all TFag was translocated to the cell surface. Phorbol myristate acetate (PMA) stimulation of ECV304 cells resulted in an increase of TF mRNA levels, which was abrogated when gene transcription was impaired, suggesting a transcriptional regulation of the TF gene by PMA. In contrast, TFag was not elevated by PMA-stimulation, indicating the existence of additional posttranscriptional mechanisms. Thus, ECV304 cells constitute a singular endothelial cell model for exploring the regulation of TF expression.

Automated laser-induced fluorescence DNA sequencing: equalizing signal-to-noise ratios significantly enhances overall performance.

We have significantly optimized the performance of the Perkin-Elmer/Applied Biosystems (PE/ABI) 373Stretch DNA sequencer. Best results were obtained using 19% less ddNTPs and 75% more dNTPs relative to the manufacturer's recommended reaction mixture. Other changes included 4.25% (instead of 4%) acrylamide gels run at 38 W (instead of 40 W). These variations produced a significant equalization of the signal-to-noise profile, resulting in longer reads. The overall accuracy improvement for unedited pGEM 1000-base sequences analyzed by the ABI100 basecaller was 8.2%. High accuracy gains were observed in the 501- to 850-base region (6.0%) and, most significantly, from that point to the end of the sequence (41.6%). A decrease in accuracy was also found for the first 30 bases, which usually correspond to vector sequence. Our study has focused primarily on double-stranded DNA (dsDNA) using the DyeDeoxy Terminator chemistry kit and 48-cm well-to-read (WTR) gels since, in our hands, such a combination represents the quicker, most accurate, versatile, and productive choice for large-scale DNA sequencing. Nevertheless, our findings could be also exploited in other sequencing strategies using ssDNA templates, the DyePrimer chemistry, alternative enzyme preparations, or different WTR lengths on the 373Stretch or other sequencing machines.