Diagonal two-dimensional electrophoresis (D-2DE): a new approach to study the effect of osmotic stress induced by polyethylene glycol in durum wheat (Triticum durum Desf.).

Acclimatization to stress is associated with profound changes in proteome composition. The use of plant cell and tissue culture offers a means to investigate the physiological and biochemical processes involved in the adaptation to osmotic stress. We employed a new proteomic approach to further understand the response of calli to dehydration induced by polyethylene glycol (PEG6000). Calli of three durum wheat genotypes Djenah Khetifa, Oued Zenati and Waha were treated with two concentrations of polyethylene glycol to mimic osmotic stress. Changes in protein relative abundance were analyzed using a new electrophoretic approach named diagonal two-dimensional electrophoresis (D-2DE), combined with mass spectrometry. Total proteins were extracted from 30-day-old calli from three durum wheat genotypes that showed contrasting levels of drought stress tolerance in the field. The combination of one-dimensional electrophoresis and D-2DE gave a specific imprint of the protein extracts under osmotic stress, as well as characterizing and identifying individual target proteins. Of the variously expressed proteins, three were selected (globulin, GAPDH and peroxidase) and further analyzed using qRT-PCR at the transcriptome level in order to compare the results with the proteomic data. Western blot analysis was used to further validate the differences in relative abundance pattern. The proteins identified through this technique provide new insights as to how calli respond to osmotic stress. Our method of study provides an original and relevant approach of analyzing the osmotic-responsive mechanisms at the cellular level of durum wheat with agronomic perspectives.

Primary granulocytic sarcoma of the breast: a case report and review of the literature.

Primary granulocytic sarcoma is an uncommon entity that rarely presents in the breast. It is frequently misdiagnosed as carcinoma or lymphoma and a broad panel of immunohistochemical markers including epithelial and haematological markers are essential for an accurate diagnosis. We reviewed all the cases of granulocytic sarcoma reported in the English literature since 1970 and present a new case of granulocytic sarcoma of the breast. Systemic chemotherapy for acute myeloid leukemia (AML) appears to be superior to surgery or radiotherapy. Granulocytic sarcoma should be in the pathologic differential diagnosis of a breast mass.

Wheat transformation with glutenin gene.

The rheological properties of wheat grains are associated with the composition of the starchy endosperm in high molecular weight (HMW) glutenin proteins. The HMW glutenin 1xDy12 subunit gene was co introduced with the screenable bar and the reporter gus marker genes in the commercial spring wheat Minaret cultivar (cv). The gene of interest and the marker genes were carried by two separated plasmids, the pBS10BH1 and the pAhC25 respectively. Seven days old calli initiated from immature embryos were bombarded by use of the PDS-1000/He device. A number of different bombardment and culture conditions were tested. These parameters were evaluated on the basis of GUS transient expression. Among those a 4 degrees C cold pre-treatment of the spikes before immature embryos were excised, the culture medium incorporating activated charcoal, silver nitrate and glucose yielded higher GUS transient expression rate. The selective agent bialaphos was maintained at various stages during culture from induction of somatic embryogenesis to rooting of regenerated plantlets. 137 bialaphos resistant plants were obtained among which 109 were carried to maturity. Transgenic plants were characterized by PCR, Southern and SDS-PAGE analysis of the glutenin content.

Yeast RAS2 affects cell viability, mitotic division and transient gene expression in Nicotiana species.

Overexpression of the budding yeast RAS2 gene in Nicotiana plumbaginifolia cells revealed that RAS2 acted as 'suicide' gene in freshly isolated protoplasts from leaves and blocked cell proliferation in cell suspension-derived protoplasts. Among a series of genes tested (such as npt II, CDC35, PDE2), RAS2 was the only one to block the expression of the cat gene, as measured in a transient gene expression assay. Another ras gene, v-Ha-ras, had similar effects. Furthermore, the RAS2 effect was species-specific and depended on the modulation of hormonal metabolism in the transfected cells, while no differences were noticed between the normal and the activated val19 gene. Transfected plant cells are shown to synthesize a RAS2 protein of the same electrophoretic mobility as the yeast RAS2 product. The results are discussed in the broader context of the evolutionarily conserved ras genes involved in vital cellular functions.