Seed germination ecology of the threatened endemic Iberian <Emphasis Type="Italic">Delphinium fissum</Emphasis> subsp. <Emphasis Type="Italic">sordidum</Emphasis> (Ranunculaceae)

Seeds of Delphinium fissum subsp. sordidum are physiologically dormant at maturity, with underdeveloped embryos; thus they have morphophysiological dormancy (MPD). The aims of this study were to determine the requirements for embryo growth, dormancy break and germination, to characterise the type of seed dormancy and to evaluate the effects of light, seed age, pollination mechanism, and inter-annual and inter-population variability on germinative ability. After 3 months of incubation at 5°C (cold stratification) in darkness conditions, the mean embryo length increased from 5.6 to 2.07 mm, with 76% of seeds germinating. Conversely, embryos of seeds incubated during 3 months at 20/7 or 28/14°C hardly grew and no germination was recorded. Since cold stratification was the only requirement for the loss of MPD, and both dry storage in laboratory conditions and warm stratification prior to cold stratification shortened the cold stratification period required for germination, it could be concluded that D. fissum subsp. sordidum seeds have intermediate complex MPD. Cold stratification and incubation in darkness conditions promoted higher germination percentages than those in light. In addition, germinative ability increased with seed age up to 8 months (reaching 96% at 5°C in darkness), showed a pronounced inter-annual and inter-population variability, as well as a significant decrease in seeds coming from pollination by geitonogamy. High temperatures (25/10 or 28/14°C) induced seeds to secondary dormancy, so seedling emergence in the greenhouse was restricted to February–March. The requirements for dormancy break and germination reflect an adaptation to trigger germination in late winter. This study is the first one to document a gradual increase in germination percentage with seed age for plant species with intermediate complex MPD.     

Scarface,a secreted serine protease‐like protein,regulates polarized localization of laminin A at the basement membrane of the Drosophila embryo

Cell–matrix interactions brought about by the activity of integrins and laminins maintain the polarized architecture of epithelia and mediate morphogenetic interactions between apposing tissues. Although the polarized localization of laminins at the basement membrane is a crucial step in these processes, little is known about how this polarized distribution is achieved. Here, in Drosophila, we analyse the role of the secreted serine protease‐like protein Scarface in germ‐band retraction and dorsal closure—morphogenetic processes that rely on the activity of integrins and laminins. We present evidence that scarface is regulated by c‐Jun amino‐terminal kinase and that scarface mutant embryos show defects in these morphogenetic processes. Anomalous accumulation of laminin A on the apical surface of epithelial cells was observed in these embryos before a loss of epithelial polarity was induced. We propose that Scarface has a key role in regulating the polarized localization of laminin A in this developmental context.     

Using real-time impedance-based assays to monitor the effects of fibroblast-derived media on the adhesion,proliferation, migration and invasion of colon cancer cells

Increasing our knowledge of the mechanisms regulating cell proliferation, migration and invasion are central to understanding tumour progression and metastasis. The local tumour microenvironment contributes to the transformed phenotype in cancer by providing specific environmental cues that alter the cells behaviour and promotes metastasis. Fibroblasts have a strong association with cancer and in recent times there has been some emphasis in designing novel therapeutic strategies that alter fibroblast behaviour in the tumour microenvironment. Fibroblasts produce growth factors, chemokines and many of the proteins laid down in the ECM (extracellular matrix) that promote angiogenesis, inflammation and tumour progression. In this study, we use a label-free RTCA (real-time cell analysis) platform (xCELLigence) to investigate how media derived from human fibroblasts alters cancer cell behaviour. We used a series of complimentary and novel experimental approaches to show HCT116 cells adhere, proliferate and migrate significantly faster in the presence of media from human fibroblasts. As well as this, we used the xCELLigence CIM-plates system to show that HCT116 cells invade matrigel layers aggressively when migrating towards media derived from human fibroblasts. These data strongly suggest that fibroblasts have the ability to increase the migratory and invasive properties of HCT116 cells. This is the first study that provides real-time data on fibroblast-mediated migration and invasion kinetics of colon cancer cells.     

Interpopulation variability on embryo growth,seed dormancy break,and germination in the endangered Iberian daffodil Narcissus eugeniae (Amaryllidaceae)

The main goal of the study was to assess germination requirements in a threatened daffodil to elaborate a detailed protocol for plant production from seeds, a key tool for conservation. Experiments were carried out both in the laboratory and outdoor conditions. In Pseudonarcissi section, endemic Iberian species of Narcissus studied heretofore have different levels of morphophysiological dormancy (MPD). Embryo length, radicle emergence, and shoot emergence were analyzed to determine the level of MPD. Both interpopulational variability and seed storage duration were also studied. Mean embryo length in fresh seeds was 1.32 mm and the embryo had to grow until it reached at least 2.00 mm to germinate. Embryo growth occurs during warm stratification, after which the radicle emerges when temperatures go down. Seed dormancy was broken in the laboratory at 28/14°C in darkness followed by 15/4°C, but the germination percentage varies depending on the population. In outdoor conditions, seed dispersal occurs in June, the embryo grows during the summer and then the radicle emerges in autumn. The radicle system continues to grow during the winter months, but the shoot does not emerge until the beginning of the spring because it is physiologically dormant and requires a cold period to break dormancy. Early cold temperatures interrupt embryo growth and induce dormancy in seeds with an advanced embryo development. Seeds of N. eugeniae have deep simple epicotyl MPD. In addition, we found that embryo growth and germination were improved by seed storage duration.     

GALNT11 as a new molecular marker in chronic lymphocytic leukemia

Aberrant mucin O-glycosylation often occurs in different cancers and is characterized by immature expression of simple mucin-type carbohydrates. At present, there are some controversial reports about the Tn antigen (GalNAcα-O-Ser/Thr) expression and there is a great lack of information about the [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts)] expression in chronic lymphocytic leukemia (CLL). To gain insight in these issues we evaluated the Tn antigen expression in CLL patient samples using two Tn binding proteins with different fine specificity. We also studied the expression from 14 GalNAc-Ts genes in CLL patients by RT-PCR. Our results have provided additional information about the expression level of the Tn antigen, suggesting that a low density of Tn residues is expressed in CLL cells. We also found that GALNT11 was expressed in CLL cells and normal T cell whereas little or no expression was found in normal B cells. Based on these results, GALNT11 expression was assessed by qPCR in a cohort of 50 CLL patients. We found significant over-expression of GALNT11 in 96% of B–CLL cells when compared to normal B cells. Moreover, we confirmed the expression of this enzyme at the protein level. Finally we found that GALNT11 expression was significantly associated with the mutational status of the immunoglobulin heavy chain variable region (IGHV), [?²(1) = 18.26; P < 0.0001], lipoprotein lipase expression [?²(1) = 13.72; P = 0.0002] and disease prognosis [?²(1) = 15.49; P < 0.0001]. Our evidence suggests that CLL patient samples harbor aberrant O-glycosylation highlighted by Tn antigen expression and that the over-expression of GALNT11 constitutes a new molecular marker for CLL.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

Use of Synthetic Genes for Cloning,Production and Functional Expression of the Bacteriocins Enterocin A and Bacteriocin E 50-52 by Pichia pastoris and Kluyveromyces lactis

The use of synthetic genes may constitute a successful approach for the heterologous production and functional expression of bacterial antimicrobial peptides (bacteriocins) by recombinant yeasts. In this work, synthetic genes with adapted codon usage designed from the mature amino acid sequence of the bacteriocin enterocin A (EntA), produced by Enterococcus faecium T136, and the mature bacteriocin E 50-52 (BacE50-52), produced by E. faecium NRRL B-32746, were synthesized. The synthetic entA and bacE50-52 were cloned into the protein expression vectors pPICZαA and pKLAC2 for transformation of derived vectors into Pichia pastoris X-33 and Kluyveromyces lactis GG799, respectively. The recombinant vectors were linearized and transformed into competent cells selecting for P. pastoris X-33EAS (entA), P. pastoris X-33BE50-52S (bacE50-52), K. lactis GG799EAS (entA), and K. lactis GG799BE50-52S (bacE50-52). P. pastoris X-33EAS and K. lactis GG799EAS, but not P. pastoris X-33BE50-52S and K. lactis GG799BE50-52S, showed antimicrobial activity in their supernatants. However, purification of the supernatants of the producer yeasts permitted recovery of the bacteriocins EntA and BacE50-52. Both purified bacteriocins were active against Gram-positive bacteria such as Listeria monocytogenes but not against Gram-negative bacteria, including Campylobacter jejuni.     

Studies on the synthesis of β-keto esters derived from dipeptides: search for a low-epimerizing method

Summary β-Keto esters derived from dipeptides are prepared by application of common methodologies employed for the synthesis of amino acid-derived β-keto esters; however, epimerization of the C-terminal residue occurred to different extents depending on the method. In imidazolide activated dipeptides, this epimerization is due to the CDI activation step and to the configurational instability of the intermediate imidazolides in different reaction media. Regarding yield and diastereomeric purity, the method of choice proved to be the reaction of dipeptide-derived imidazolide with the potassium salt of malonic half esters in the presence of MgCl₂.     

The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata

We have determined the complete nucleotide sequence of the small- subunit ribosomal RNA genes for the ciliate protozoans Stylonychia pustulata and Oxytricha nova. The sequences are homologous and sufficiently similar that these organisms must be closely related. In a phylogeny inferred from comparisons of several eukaryotic small-subunit ribosomal RNAs, the divergence of the ciliates from the eukaryotic line of descent is seen to coincide with the radiation of the plants, the animals, and the fungi. This radiation is preceded by the divergence of the slime mold, Dictyostelium discoideum.