Changes in pH and the Production of Organic Acids During Colonization of Tomato Petioles by Botrytis cinerea

After inoculation of petiole stumps of tomato plants with a tomato isolate of B. cinerea, a transition zone between water-soaked and apparently healthy tissue became clearly visible. Hyphal colonization occurred up to approximately 2 mm beyond this zone. In the colonized tissue the pH values were lower than in the healthy tissue. In the region with most of the tips of the advancing hyphae, however, pH values were slightly, but consistently, higher. In the colonized tissue concentrations of oxalic, citric and succinic acid were higher than in the tissue of healthy, non-inoculated petioles. In vitro this isolate of B. cinerea produced citric, malic and succinic acid. Oxalic acid, however, could not be detected. In media enriched, with citric or malic acid, mycelial production was higher than in media without this enrichment.     

INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence

The CDKN2A tumour suppressor locus encodes two distinct proteins, p16(INK4a) and p14(ARF), both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary fibroblasts from a member of a melanoma-prone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16(INK4a) deficient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14(ARF). Although they have a finite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage-independent colonies that have essentially diploid karyotypes and functional p53. We find that in human fibroblasts, ARF is not induced demonstrably by RAS, pointing to significant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.     

The Dutch N-cascade in the European perspective

The Netherlands is "well known" for its nitrogen problems; it has one of the highest reactive nitrogen (Nr) emission densities in the world. It is a small country at the delta of several large European rivers. Ever since the industrial revolution, there has been a growing excess of nutrients and related emissions into the atmosphere (ammonia, nitrogen oxides and nitrous oxide)and into groundwater and surface water (nitrate), leading to a large range of cascading environmental impacts. Vehicular traffic, sewage and animal husbandry are the main sources of oxidized and reduced forms of Nr. This paper provides an overview of the origin and fate of nitrogen in the Netherlands, the various reported impacts of nitrogen, the Dutch and European policies to reduce nitrogen emissions and related impacts. In addition, ways are presented to go forward to potentially solve the problems in a European perspective. Solutions include the improvement of nitrogen efficiencies in different systems, technological options and education.     

Agrobacterium tumefaciens-Mediated Transformation of Maize Endosperm as a Tool to Study Endosperm Cell Biology

Developing maize (Zea mays) endosperms can be excised from the maternal tissues and undergo tissue/cell-type differentiation under in vitro conditions. We have developed a method to transform in vitro-grown endosperms using Agrobacterium tumefaciens and standard binary vectors. We show that both aleurone and starchy endosperm cells can be successfully transformed using a short cocultivation with A. tumefaciens cells. The highest transformation rates were obtained with the A. tumefaciens EHA101 strain and the pTF101.1 binary vector. The percentage of aleurone cells transformed following this method varied between 10% and 22% whereas up to the eighth layer of starchy endosperm cells underneath the aleurone layer showed transformed cells. Cultured endosperms undergo normal cell type (aleurone and starchy endosperm) differentiation and storage protein accumulation, making them suitable for cell biology and biochemical studies. In addition, transgenic cultured endosperms are able to express and accumulate epitope-tagged storage proteins that can be isolated for biochemical assays or used for immunolabeling techniques.The endosperm is a unique plant tissue that arises from a second fertilization event between a male gamete and the central cell. Its main function is to provide nutrients to the embryo either during seed development or during germination. In cereals, the endosperm consists of three main cell types: the starchy endosperm cells, which constitute the bulk of the endosperm and accumulate large quantities of storage proteins and starch; the epidermal aleurone cells; and the transfer cells, which are in contact with the maternal vascular tissues (Olsen, 2004). The cereal endosperm is important as a model system to study plant development, cell differentiation, programmed cell death, and synthesis, trafficking, and accumulation of storage compounds. In addition, it is a major source of carbohydrate and proteins for human and animal nutrition.In spite of its importance, cell biology studies on the cereal endosperm using modern imaging approaches such as expression of fluorescent subcellular markers are very scarce because: (1) the endosperm is deeply immersed in maternal tissues and therefore, not readily available for imaging analysis and (2) the long time required for transformation and regeneration of stable transgenic plants. Although several approaches for culturing maize (Zea mays) endosperm in vitro have been reported in the past years (Shimamoto et al., 1983), only recently a novel method developed by Odd-Arne Olsen and colleagues (Gruis et al., 2006) has proven to be successful in retaining endosperm tissue and cell type identity in in vitro conditions. Cultures derived from transgenic maize lines in which endosperm cell types are identified by the activity of specific promoters have shown that aleurone and starchy endosperm cell identity continues to be established in vitro (Gruis et al., 2006).Although Agrobacterium tumefaciens is not a natural pathogen of most monocots (Cleene, 1985; Binns and Thomashow, 1988), it has been successfully used to transform many cereals, including maize, wheat (Triticum aestivum), Sorghum, barley (Hordeum vulgare), and rice (Oryza sativa; Grimsley et al., 1989; Gould et al., 1991; Chan et al., 1993; Ishida et al., 1996, 2007; Gurel et al., 2009; Harwood et al., 2009; Hensel et al., 2009). In the case of maize, stable transgenic plants can be obtained by A. tumefaciens-mediated transformation using either super-binary or standard-binary vectors (Frame et al., 2002; Mohanty et al., 2009a, 2009b). However, transformation of isolated maize endosperms have been only possible using transient transformation approaches such as biolistic methods (Torrent et al., 1997; Gruis et al., 2006) and protoplast transfection (Gallie and Young, 1994). Unfortunately, these two methods are not always ideal for cell biology studies. On one hand, biolistic methods often result in high-copy number transgenic events and on the other, protoplasts are usually highly stressed cells not suitable for detailed protein localization studies. A. tumefaciens-mediated transformation methods circumvent these disadvantages by resulting in a low-copy number of transgenes in intact tissues.We have developed a method to transform in vitro-grown endosperms using a brief incubation time with A. tumefaciens cells carrying standard binary vectors. We present here a detailed explanation of the method and quantitative information on the transformation efficiency using different A. tumefaciens strains, culture density, and incubation time. We also provide evidence that the in vitro-differentiated aleurone and starchy endosperm cells are comparable to the corresponding cell types differentiated in planta and therefore, suitable for cell biology studies. In addition, we show that transgenic cultured endosperms are able to express and accumulate epitope-tagged storage proteins that can be isolated for biochemical assays or used for immunolabeling imaging techniques.     

Class III β-tubulin,a novel biomarker in the human melanocyte lineage

It is generally thought that class III β-tubulin expression is limited to cells of the neural lineage and is therefore often used to identify neurons amongst other cell types, both in vivo and in vitro. Melanocytes are derived from the neural crest and share both morphological features and functional characteristics with peripheral neurons. Here, we show that these similarities extend to class III β-tubulin (TUBB3) expression, and that human melanocytes express this protein both in vivo and in vitro. In addition, we studied the expression of class III β-tubulin in two murine melanogenic cell lines and show that expression of this protein starts as melanoblasts mature into melanocytes. Melanin bleaching experiments revealed close proximity between melanin and TUBB3 proteins. In vitro stimulation of primary human melanocytes by α-MSH indicated separate regulatory mechanisms for melanogenesis and to TUBB3 expression. Together, these observations imply that human melanocytes express TUBB3 and that this protein should be recognized as a wider marker for multiple neural crest-derived cells.