Congenic method in the chick limb buds by electroporation

Electroporation is a powerful tool with which to study limb development. Limb development, however, remains an intricate series of events, requiring the precise dissection of developmental processes using relevant transgenes. In this review, we describe the anatomy of the limb field as the basis of targeted electroporation, and specific expression vectors are discussed. We share a useful protocol for electroporation of chick limb buds, and the expression pattern of enhanced green fluorescent protein in the limb buds is used to demonstrate relevant embryonic patterning. Finally, useful trouble-shooting techniques are described.     

植物microRNA对重金属胁迫响应的调控

镉是重要的重金属污染物,它严重影响植物生长,并危害人体健康.植物的重金属耐受机理很复杂,需从多个角度阐述.但参与重金属应答的调节性基因研究较少,重金属响应调控网络并不清楚.microRNA(miRNA)是一类新型的调控基因表达的     

Micropropagation of ‘Yamatoimo’ Chinese yam (Dioscorea opposita) from immature leaves

A micropropagation system for Yamatoimo Chinese yam (Dioscorea opposita Thunb.) was developed. Immature leaves collected from virus-free plants growing in the greenhouse were cultured on MS medium supplemented with 8.9 M benzyladenine (BA), 3% (w/v) sucrose and 0.8% (w/v) agar. After 2–3 months, multiple buds that were clumps of green-colored bulbous structures including adventitious buds and meristematic regions 2–3 mm in diameter were formed on immature leaves. Transplanting clusters of multiple buds to fresh MS medium supplemented with 0.11 M -naphthaleneacetic acid (NAA), 0.89 M BA and 6% (w/v) sucrose was effective for inducing shoot formation, leading to plantlet formation. After 6 months, a large number of microtubers, about 3–7 mm diameter, were obtained.Abbreviations NAA -naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog     

Refined structure of the acetazolamide complex of human carbonic anhydrase II at 1.9 Å

The binding of acetazolamide to human carbonic anhydrase II (HCA II) has been investigated by X-ray crystallography. The atomic positions of the enzyme inhibitor complex have been refined at 1.9 Å resolution using the least squares refinement program package PROLSQ. The crystallographic R-factor is 17.6%. The bound inhibitor is clearly resolved in the active site of the enzyme. The acetazolamide amine group is bound as a fourth ligand to the zinc ion, the other three are all histidine residues. In addition to van der Waals' interactions and the previously described binding of the sulphonamide group, the inhibitor forms a hydrogen bond from the carbonyl oxygen of the acetylamido group to the amino group of Gln 92.     

Dormancy in peach (Prunus persica L.) flower buds

Summary Peach buds (floral and vegetative) were periodically collected from midsummer until the spring flowering and sprouted under continuous light, 100% relative humidity and 20–25°C. Treatments with 200 ppm gibberellin A₃ (GA₃) or chilling (2–4°C for 30 days before planting) were applied. Vegetative buds showed well-defined phenological stages: pre-dormancy, true dormancy, and end of dormancy. Both GA₃ and chilling treatments shortened the sprouting times of vegetative dormant buds close to those in predormancy. Isolated floral buds were irresponsive under all conditions and did not sprout even with the GA₃ or chilling treatments. In a comparative study with buds immediately after collection anatomical analysis demonstrated that vegetative buds were almost completely developed by midsummer/early automn and remained in a resting state until the end of winter. Floral buds developed continuously over the same period. Both types of verticils began to differentiate in midsummer. Sepals and petals developed mainly in late summer, androecious floral parts developed throughout the resting period, while gynoecious floral parts showed differentiation in late winter. The flower was completely formed a few days prior to blossoming. Thus, in isolated peach buds fertile verticils are not sufficiently developed during the resting time to allow sprouting.     

Inhibition of carbonic anhydrase activity modifies the toxicity of doxorubicin and melphalan in tumour cells in vitro

Carbonic anhydrase IX (CA IX) is a hypoxia-regulated enzyme, overexpressed in many types of human cancer. CA IX is involved in pH homeostasis, contributing to extracellular acidification and tumourigenesis. Acidification of the extracellular milieu can impact upon cellular uptake of chemotherapeutic drugs by favouring weak acids (e.g. melphalan), but limiting access of weak bases (e.g. doxorubicin). We investigated whether alterations of CA IX activity affected anti-cancer drug uptake and toxicity. CA inhibitor acetazolamide (AZM) enhanced doxorubicin toxicity but reduced melphalan toxicity in cell lines that highly expressed CA IX under anoxic conditions (HT29 and MDA435 CA9/18). The toxicity changes reflected modification of passive drug uptake. AZM did not alter toxicity or uptake in cells with low CA IX activity (HCT116 and MDA435 EV1). AZM lowered intracellular pH in HT29 and MDA435 CA9/18 cells under anoxic conditions. CA IX activity has chemomodulatory properties and is an attractive target for anti-cancer therapy.     

CARBONIC ANHYDRASE IN THE CHLOROPLAST OF A COCCOLITHOPHORID (PRYMNESIOPHYCEAE)1

The activity and subcellular distribution of carbonic anhydrase in a coccolithophorid alga, CCMP 299, was examined. The enzyme could not be detected in crude cell homogenates but was present at high specific activity (27.5 unit·mg^?1 protein) in chloroplasts (density, 1.14 g·cm^?3) isolated in a sucrose gradient. The carbonic anhydrase activity was sensitive to known inhibitors. Inhibition at 50% (I₅₀) was obtained with concentrations of 4.60 mM and 2.65 mM for acetazolamide and NaN₃, respectively. These levels are more consistent with patterns of inhibition previously observed for chloroplastic (as compared to periplasmic) carbonic anhydrase. In this organism, carbonic anhydrase was localized in the chloroplast stroma. These findings are discussed in terms of the relationship among dissolved inorganic carbon interconversions, photosynthesis, and calcification.     

Morphological and hormonal characterisation of strawberry vitroplants raised through axillary or stipular adventitious shooting

Adventitious stipular bud formation occurred in vitro in many strawberry cultivars during the proliferation phase onmedium containing Knop macronutrients, MS micronutrients, vitamins, aminoacids,2.22 M BAP, 2.46 M IBA and 0.29M GA₃. As described previously for cultivarGorella,cultivar Elsanta also showed adventitious stipular buds developing on theabaxial median zone between the stipule tips. To compare the shoots producedfrom both types of buds, clonal propagation was initiated from stipular budsandfrom axillary buds on the above mentioned medium. Stipular buds were separatedfrom the meristem-tip initiated plantlet and cultivated in the presence of alower BAP concentration (1.33 M) to prevent further stipularbud formation.During proliferation cycles, stipular originated propagules werevery easily distinguished by their specific leaf phenotype and light greencolour in comparison to plantlets cloned for an axillary bud. Theirmultiplication rate and cytokinin content were also higher than for axillarybuds. No significant difference was observed in auxin content.